International Journal of Molecular Sciences

2025

Jump to: 2024, 2023, 2022, 2021, 2020, 2019

19 pages, 4700 KiB

Open AccessArticle

Altered Expression of Epigenetic and Transcriptional Regulators in ERβ Knockout Rat Ovaries During Postnatal Development

by Kevin Vo, Yashica Sharma, V. Praveen Chakravarthi, Ryan Mohamadi, Elizabeth S. Bahadursingh, Amelia Mohamadi, Vinesh Dahiya, Cinthia Y. Rosales, Grace J. Pei, Patrick E. Fields and M. A. Karim Rumi

Int. J. Mol. Sci. 2025, 26(2), 760; https://doi.org/10.3390/ijms26020760 - 17 Jan 2025

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Abstract

We analyzed the transcriptome data of wildtype and estrogen receptor β knockout (Erβ^KO) rat ovaries during the early postnatal period and detected remarkable changes in epigenetic regulators and transcription factors. Compared with postnatal day (PD) 4.5 ovaries, PD 6.5 wildtype [...] Read more.

We analyzed the transcriptome data of wildtype and estrogen receptor β knockout (Erβ^KO) rat ovaries during the early postnatal period and detected remarkable changes in epigenetic regulators and transcription factors. Compared with postnatal day (PD) 4.5 ovaries, PD 6.5 wildtype ovaries possessed 581 differentially expressed downstream transcripts (DEDTs), including 17 differentially expressed epigenetic regulators (DEERs) and 23 differentially expressed transcription factors (DETFs). Subsequently, compared with PD 6.5 ovaries, PD 8.5 wildtype ovaries showed 920 DEDTs, including 24 DEERs and 68 DETFs. The DEDTs, DEERs, and DETFs in wildtype ovaries represented the gene expression during primordial follicle activation and the gradual development of primary follicles of first-wave origin because the second-wave follicles remained dormant during this developmental period. When we compared the transcriptome data of age-matched Erβ^KO ovaries, we observed that PD 6.5 Erβ^KO ovaries had 744 DEDTs compared with PD 4.5 ovaries, including 46 DEERs and 55 DETFs. The loss of ERβ rapidly activated the primordial follicles of both first- and second-wave origin on PD 6.5 and showed a remarkable increase in DEDTs (744 vs. 581). However, compared with PD 6.5 ovaries, PD 8.5 Erβ^KO ovaries showed only 191 DEDTs, including 8 DEERs and 10 DETFs. This finding suggests that the PD 8.5 Erβ^KO ovaries did not undergo remarkable ovarian follicle activation greater than that had already occurred in PD 6.5 Erβ^KO ovaries. The results also showed that the numbers of DEERs and DETFs were associated with increased changes in DEDTs; the greater the number of DEERs or DETFs, the larger the number of DEDTs. In addition to the quantitative differences in DEERs and DETFs between the wildtype and Erβ^KO ovaries, the differentially expressed regulators showed distinct patterns. We identified that 17 transcripts were tied to follicle assembly, 6 to follicle activation, and 12 to steroidogenesis. Our observations indicate that a loss of ERβ dysregulates the epigenetic regulators and transcription factors in Erβ^KO ovaries, which disrupts the downstream genes in ovarian follicles and increases follicle activation. Further studies are required to clarify if ERβ directly or indirectly regulates DEDTs, including DEERs and DETFs, during the neonatal development of rat ovarian follicles. Full article

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2024

Jump to: 2025, 2023, 2022, 2021, 2020, 2019

22 pages, 1337 KiB

Open AccessReview

Review of hTERT-Immortalized Cells: How to Assess Immortality and Confirm Identity

by Maria Shitova, Elena Alpeeva and Ekaterina Vorotelyak

Int. J. Mol. Sci. 2024, 25(23), 13054; https://doi.org/10.3390/ijms252313054 - 4 Dec 2024

Cited by 1 | Viewed by 1199

Abstract

Cell immortalization has an important role in scientific research, as well as increasing significance in the context of cell therapy and biotechnology. Over the years, many immortalized cell lines have been produced using human telomerase reverse transcriptase (hTERT) alone or in a combination [...] Read more.

Cell immortalization has an important role in scientific research, as well as increasing significance in the context of cell therapy and biotechnology. Over the years, many immortalized cell lines have been produced using human telomerase reverse transcriptase (hTERT) alone or in a combination with viral oncogenes. Different hTERT-immortalized cells are commercially available, and numerous papers about obtaining immortalized cell lines have also been published. However, no specific list of characteristics that need to be checked to confirm successful immortalization exists. Most researchers evaluate only a few parameters, while different articles contain various opinions on the assessment of these characteristics. Results also vary significantly between different cell types, which have their own traits depending on their origin and functions. In the current paper, we raise these questions and discuss controversial issues concerning currently available testing methods for immortalization evaluation and the value and the limitations of the approaches. In addition, we propose a protocol for evaluation of hTERT immortalization success consisting of the following important steps: the assessment of the proliferation rate and dividing capacity, cell morphology, phenotype, karyotype stability, telomerase activity, the expression of cell-specific markers, and tumorigenicity. To our opinion, the hTERT expression level, telomere length, and senescence-associated β-galactosidase staining are controversial with regard to the implemented methods, so these parameters may be optional. For all the evaluation steps, we recommend to pay attention to the necessity of comparing the traits of the obtained immortalized and parent cells. Full article

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18 pages, 8143 KiB

Open AccessArticle

Comparative Analysis of PGRP Family in Polymorphic Worker Castes of Solenopsis invicta

by Zhanpeng Zhu, Hongxin Wu, Liangjie Lin, Ao Li, Zehong Kang, Jie Zhang, Fengliang Jin and Xiaoxia Xu

Int. J. Mol. Sci. 2024, 25(22), 12289; https://doi.org/10.3390/ijms252212289 - 15 Nov 2024

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Abstract

Peptidoglycan recognition proteins (PGRPs) are a class of pattern recognition receptors (PRRs) that activate the innate immune system in response to microbial infection by detection of peptidoglycan, a distinct component of bacterial cell walls. Bioinformatic studies have revealed four PGRPs in the red [...] Read more.

Peptidoglycan recognition proteins (PGRPs) are a class of pattern recognition receptors (PRRs) that activate the innate immune system in response to microbial infection by detection of peptidoglycan, a distinct component of bacterial cell walls. Bioinformatic studies have revealed four PGRPs in the red imported fire ant Solenopsis invicta; nonetheless, the mechanism of the immune response of S. invicta induced by pathogens is still poorly understood. The peptidoglycan recognition protein full-length cDNA (designated as SiPGRP-S1/S2/S3/L) from S. invicta was used in this investigation. According to the sequencing analysis, there was a significant degree of homology between the anticipated amino acid sequence of SiPGRPs and other members of the PGRPs superfamily. Molecular docking studies demonstrated that SiPGRPs show strong binding affinity for a variety of PGN substrates. Additionally, tissue distribution analysis indicated that SiPGRPs are primarily expressed in several tissues of naïve larvae, including fat body, hemocytes, head, and thorax, as detected by quantitative real-time PCR (RT-qPCR). Microbial challenges resulted in variable changes in mRNA levels across different tissues. Furthermore, the antibacterial effects of antimicrobial peptides (AMPs) produced by major ants infected with Metarhizium anisopliae were assessed. These AMPs demonstrated inhibitory effects against M. anisopliae, Staphylococcus aureus, and Escherichia coli, with the most pronounced effect observed against E. coli. In conclusion, SiPGRPs act as pattern recognition receptors (PRRs) that identify pathogens and initiate the expression of AMPs in S. invicta, this mechanism contributes to the development of biopesticides designed for the targeted control of invasive agricultural pests. Full article

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28 pages, 7608 KiB

Open AccessArticle

Machine Learning Analysis of RNA-Seq Data Identifies Key Gene Signatures and Pathways in Mpox Virus-Induced Gastrointestinal Complications Using Colon Organoid Models

by Mostafa Rezapour, Aarthi Narayanan and Metin Nafi Gurcan

Int. J. Mol. Sci. 2024, 25(20), 11142; https://doi.org/10.3390/ijms252011142 - 17 Oct 2024

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Abstract

Mpox, caused by the Mpox virus (MPXV), emerged globally in 2022 with the Clade IIb strain, presenting a critical public health challenge. While MPXV is primarily characterized by fever and rash, gastrointestinal (GI) complications, such as diarrhea and proctitis, have also been observed. [...] Read more.

Mpox, caused by the Mpox virus (MPXV), emerged globally in 2022 with the Clade IIb strain, presenting a critical public health challenge. While MPXV is primarily characterized by fever and rash, gastrointestinal (GI) complications, such as diarrhea and proctitis, have also been observed. This study is a reanalysis of GSE219036 without own data and focuses on the impact of MPXV infection on the colon, using human-induced pluripotent stem cell-derived colon organoids as a model. We applied a tailored statistical framework for RNA-seq data, Generalized Linear Models with Quasi-Likelihood F-tests and Relaxed Magnitude–Altitude Scoring (GLMQL-RMAS), to identify differentially expressed genes (DEGs) across MPXV clades: MPXV I (Zr-599 Congo Basin), MPXV IIa (Liberia), and MPXV IIb (2022 MPXV). Through a novel methodology called Cross-RMAS, we ranked genes by integrating statistical significance and biological relevance across all clades. Machine learning analysis using the genes identified by Cross-RMAS, demonstrated 100% accuracy in differentiating between the different MPXV strains and mock samples. Furthermore, our findings reveal that MPXV Clade I induces the most extensive alterations in gene expression, with significant upregulation of stress response genes, such as HSPA6 and FOS, and downregulation of genes involved in cytoskeletal organization and vesicular trafficking, such as PSAP and CFL1. In contrast, Clade IIb shows the least impact on gene expression. Through Gene Ontology (GO) analysis, we identified pathways involved in protein folding, immune response, and epithelial integrity that are disrupted in infected cells, suggesting mechanisms by which MPXV may contribute to GI symptoms. Full article

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19 pages, 3252 KiB

Open AccessArticle

Chromogranin B Protects Human Umbilical Endothelial Cells against Oxidative Stress

by Elena Grossini, Sakthipriyan Venkatesan, Mohammad Mostafa Ola Pour, Daniela Ferrante, Daniela Surico, Rosanna Vaschetto, Vincenzo Cantaluppi and Mario Pirisi

Int. J. Mol. Sci. 2024, 25(19), 10296; https://doi.org/10.3390/ijms251910296 - 25 Sep 2024

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Abstract

Chromogranin B (CgB) is involved in the control of the cardiovascular system through the regulation of catecholamine release. Whether CgB can exert direct actions on the endothelium has not yet been clarified. Here, we aimed to investigate the effects of CgB on cell [...] Read more.

Chromogranin B (CgB) is involved in the control of the cardiovascular system through the regulation of catecholamine release. Whether CgB can exert direct actions on the endothelium has not yet been clarified. Here, we aimed to investigate the effects of CgB on cell viability, mitochondrial membrane potential, reactive oxygen species (ROS), glutathione (GSH), nitric oxide (NO) release, and the cytosolic calcium concentration ([Ca²⁺]c) in human vascular endothelial cells (HUVECs) cultured under both physiological and peroxidative conditions. In HUVECs, experiments were conducted to establish the proper concentration and timing of CgB stimulation. Thereafter, specific assays were used to evaluate the response of HUVECs cultured in physiologic or oxidative stress conditions to CgB in the presence or absence of β-adrenergic receptor agonists and antagonists and intracellular pathways blockers. Analysis of cell viability, mitochondrial membrane potential, and NO release revealed that CgB was able to cause increased effects in HUVECs cultured in physiological conditions. Additionally, the same analyses performed in HUVECs cultured with H₂O₂, showed protective effects exerted by CgB, which was also able to counteract ROS release and maintain GSH levels. Furthermore, CgB played a dual role on the [Ca²⁺]c depending on the physiological or peroxidative cell culturing conditions. In conclusion, our data provide new information about the direct role of CgB in the physiological regulation of endothelial function and highlight its potential as a protective agent against peroxidative conditions, such as those found in cardiovascular diseases. Full article

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12 pages, 1867 KiB

Open AccessArticle

Kinetic Modeling of In Vivo K⁺ Distribution and Fluxes with Stable K⁺ Isotopes: Effects of Dietary K⁺ Restriction

by Jang H. Youn, Stefania Gili, Youngtaek Oh, Alicia A. McDonough and John Higgins

Int. J. Mol. Sci. 2024, 25(17), 9664; https://doi.org/10.3390/ijms25179664 - 6 Sep 2024

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Abstract

Maintaining extracellular potassium (K⁺) within narrow limits, critical for membrane potential and excitability, is accomplished through the internal redistribution of K⁺ between extracellular fluid (ECF) and intracellular fluid (ICF) in concert with the regulation of renal K⁺ output to [...] Read more.

Maintaining extracellular potassium (K⁺) within narrow limits, critical for membrane potential and excitability, is accomplished through the internal redistribution of K⁺ between extracellular fluid (ECF) and intracellular fluid (ICF) in concert with the regulation of renal K⁺ output to balance K⁺ intake. Here we present evidence from high-precision analyses of stable K⁺ isotopes in rats maintained on a control diet that the tissues and organs involved in the internal redistribution of K⁺ differ in their speed of K⁺ exchange with ECF and can be grouped into those that exchange K⁺ with ECF either rapidly or more slowly (“fast” and “slow” pools). After 10 days of K⁺ restriction, a compartmental analysis indicates that the sizes of the ICF K⁺ pools decreased but that this decrease in ICF K⁺ pools was not homogeneous, rather occurring only in the slow pool (15% decrease, p < 0.01), representing skeletal muscles, not in the fast pool. Furthermore, we find that the dietary K⁺ restriction is associated with a decline in the rate constants for K⁺ effluxes from both the “fast” and “slow” ICF pools (p < 0.05 for both). These results suggest that changes in unidentified transport pathways responsible for K⁺ efflux from ICF to ECF play an important role in buffering the internal redistribution of K⁺ between ICF and ECF during K⁺ restriction. Thus, the present study introduces novel stable isotope approaches to separately characterize heterogenous ICF K⁺ pools in vivo and assess K⁺ uptake by individual tissues, methods that provide key new tools to elucidate K⁺ homeostatic mechanisms in vivo. Full article

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18 pages, 3034 KiB

Open AccessArticle

Influence of Anesthetic Regimes on Extracellular Vesicles following Remote Ischemic Preconditioning in Coronary Artery Disease

by Phuong N. V. Pham, Loubna Yahsaly, Crista Ochsenfarth, Bernd Giebel, Romina Schnitzler, Peter Zahn and Ulrich H. Frey

Int. J. Mol. Sci. 2024, 25(17), 9304; https://doi.org/10.3390/ijms25179304 - 28 Aug 2024

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Abstract

Remote ischemic preconditioning (RIPC) reduces ischemia-reperfusion injury in aortocoronary bypass surgery, potentially via extracellular vesicles (EVs) and their micro-RNA content. Clinical data implicate that propofol might inhibit the cardioprotective RIPC effect. This prospective, randomized study investigated the influence of different anesthetic regimes on [...] Read more.

Remote ischemic preconditioning (RIPC) reduces ischemia-reperfusion injury in aortocoronary bypass surgery, potentially via extracellular vesicles (EVs) and their micro-RNA content. Clinical data implicate that propofol might inhibit the cardioprotective RIPC effect. This prospective, randomized study investigated the influence of different anesthetic regimes on RIPC efficacy and EV micro-RNA signatures. We also assessed the impact of propofol on cell protection after hypoxic conditioning and EV-mediated RIPC in vitro. H9c2 rat cardiomyoblasts were subjected to hypoxia, with or without propofol, and subsequent simulated ischemia-reperfusion injury. Apoptosis was measured by flow cytometry. Blood samples of 64 patients receiving anesthetic maintenance with propofol or isoflurane, along with RIPC or sham procedures, were analyzed, and EVs were enriched using a polymer-based method. Propofol administration corresponded with increased Troponin T levels (4669 ± 435.6 pg/mL), suggesting an inhibition of the cardioprotective RIPC effect. RIPC leads to a notable rise in miR-21 concentrations in the group receiving propofol anesthesia (fold change 7.22 ± 6.6). In vitro experiments showed that apoptosis reduction was compromised with propofol and only occurred in an EV-enriched preconditioning medium, not in an EV-depleted medium. Our study could clinically and experimentally confirm propofol inhibition of RIPC protection. Increased miR-21 expression could provide evidence for a possible inhibitory mechanism. Full article

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14 pages, 2801 KiB

Open AccessArticle

H3K9me3 Levels Affect the Proliferation of Bovine Spermatogonial Stem Cells

by Rui Yang, Boyang Zhang, Yueqi Wang, Yan Zhang, Yansen Zhao, Daozhen Jiang, Lanxin Chen, Bo Tang and Xueming Zhang

Int. J. Mol. Sci. 2024, 25(17), 9215; https://doi.org/10.3390/ijms25179215 - 25 Aug 2024

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Abstract

Spermatogonial stem cells (SSCs) possess the characteristics of self-renewal and differentiation, as well as the ability to generate functional sperm. Their unique stemness has broad applications in male infertility treatment and species preservation. In rodents, research on SSCs has been widely reported, but [...] Read more.

Spermatogonial stem cells (SSCs) possess the characteristics of self-renewal and differentiation, as well as the ability to generate functional sperm. Their unique stemness has broad applications in male infertility treatment and species preservation. In rodents, research on SSCs has been widely reported, but progress is slow in large livestock such as cattle and pigs due to long growth cycles, difficult proliferation in vitro, and significant species differences. Previously, we showed that histone 3 (H3) lysine 9 (K9) trimethylation (H3K9me3) is associated with the proliferation of bovine SSCs. Here, we isolated and purified SSCs from calf testicular tissues and investigated the impact of different H3K9me3 levels on the in vitro proliferation of bovine SSCs. The enriched SSCs eventually formed classical stem cell clones in vitro in our feeder-free culture system. These clones expressed glial cell-derived neurotrophic factor family receptor alpha-1 (GFRα1, specific marker for SSCs), NANOG (pluripotency protein), C-KIT (germ cell marker), and strong alkaline phosphatase (AKP) positivity. qRT-PCR analysis further showed that these clones expressed the pluripotency genes NANOG and SOX2, and the SSC-specific marker gene GFRα1. To investigate the dynamic relationship between H3K9me3 levels and SSC proliferation, H3K9me3 levels in bovine SSCs were first downregulated using the methyltransferase inhibitor, chaetocin, or transfection with the siRNA of H3K9 methyltransferase suppressor of variegation 3-9 homologue 1 (SUV39H1). The EDU (5-Ethynyl-2′-deoxyuridine) assay revealed that SSC proliferation was inhibited. Conversely, when H3K9me3 levels in bovine SSCs were upregulated by transfecting lysine demethylase 4D (KDM4D) siRNA, the EDU assay showed a promotion of cell proliferation. In summary, this study established a feeder-free culture system to obtain bovine SSCs and explored its effects on the proliferation of bovine SSCs by regulating H3K9me3 levels, laying the foundation for elucidating the regulatory mechanism underlying histone methylation modification in the proliferation of bovine SSCs. Full article

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23 pages, 17872 KiB

Open AccessArticle

Lysophosphatidic Acid Receptors LPAR5 and LPAR2 Inversely Control Hydroxychloroquine-Evoked Itch and Scratching in Mice

by Caroline Fischer, Yannick Schreiber, Robert Nitsch, Johannes Vogt, Dominique Thomas, Gerd Geisslinger and Irmgard Tegeder

Int. J. Mol. Sci. 2024, 25(15), 8177; https://doi.org/10.3390/ijms25158177 - 26 Jul 2024

Viewed by 1353

Abstract

Lysophosphatidic acids (LPAs) evoke nociception and itch in mice and humans. In this study, we assessed the signaling paths. Hydroxychloroquine was injected intradermally to evoke itch in mice, which evoked an increase of LPAs in the skin and in the thalamus, suggesting that [...] Read more.

Lysophosphatidic acids (LPAs) evoke nociception and itch in mice and humans. In this study, we assessed the signaling paths. Hydroxychloroquine was injected intradermally to evoke itch in mice, which evoked an increase of LPAs in the skin and in the thalamus, suggesting that peripheral and central LPA receptors (LPARs) were involved in HCQ-evoked pruriception. To unravel the signaling paths, we assessed the localization of candidate genes and itching behavior in knockout models addressing LPAR5, LPAR2, autotaxin/ENPP2 and the lysophospholipid phosphatases, as well as the plasticity-related genes Prg1/LPPR4 and Prg2/LPPR3. LacZ reporter studies and RNAscope revealed LPAR5 in neurons of the dorsal root ganglia (DRGs) and in skin keratinocytes, LPAR2 in cortical and thalamic neurons, and Prg1 in neuronal structures of the dorsal horn, thalamus and SSC. HCQ-evoked scratching behavior was reduced in sensory neuron-specific Advillin-LPAR5^−/− mice (peripheral) but increased in LPAR2^−/− and Prg1^−/− mice (central), and it was not affected by deficiency of glial autotaxin (GFAP-ENPP2^−/−) or Prg2 (PRG2^−/−). Heat and mechanical nociception were not affected by any of the genotypes. The behavior suggested that HCQ-mediated itch involves the activation of peripheral LPAR5, which was supported by reduced itch upon treatment with an LPAR5 antagonist and autotaxin inhibitor. Further, HCQ-evoked calcium fluxes were reduced in primary sensory neurons of Advillin-LPAR5^−/− mice. The results suggest that LPA-mediated itch is primarily mediated via peripheral LPAR5, suggesting that a topical LPAR5 blocker might suppress “non-histaminergic” itch. Full article

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16 pages, 3701 KiB

Open AccessArticle

The Biological Effect of Platelet-Rich Plasma on Rotator Cuff Tears: A Prospective Randomized In Vivo Study

by Charalampos Pitsilos, Sofia Karachrysafi, Aikaterini Fragou, Ioannis Gigis, Pericles Papadopoulos and Byron Chalidis

Int. J. Mol. Sci. 2024, 25(14), 7957; https://doi.org/10.3390/ijms25147957 - 21 Jul 2024

Viewed by 1561

Abstract

The positive effect of platelet-rich plasma (PRP) on tendon metabolism has been extensively investigated and proven in vitro. Additionally, in vivo animal studies have correlated the application of PRP with the enhancement of tenocyte anabolic activity in the setting of tendon degeneration. However, [...] Read more.

The positive effect of platelet-rich plasma (PRP) on tendon metabolism has been extensively investigated and proven in vitro. Additionally, in vivo animal studies have correlated the application of PRP with the enhancement of tenocyte anabolic activity in the setting of tendon degeneration. However, less is known about its in vivo effect on human tendon biology. The purpose of the current prospective randomized comparative study was to evaluate the effect of PRP on torn human supraspinatus tendon. Twenty consecutive eligible patients with painful and magnetic resonance imaging (MRI)-confirmed degenerative supraspinatus tendon tears were randomized in a one-to-one ratio into two groups. The patients in the experimental group (n = 10) underwent an ultrasound-guided autologous PRP injection in the subacromial space 6 weeks before the scheduled operation. In the control group (n = 10), no injection was made prior to surgery. Supraspinatus tendon specimens were harvested from the lateral end of the torn tendon during shoulder arthroscopy and were evaluated under optical and electron microscopy. In the control group, a mixed cell population of oval and rounded tenocytes within disorganized collagen and sites of accumulated inflammatory cells was detected. In contrast, the experimental group yielded abundant oval-shaped cells with multiple cytoplasmic processes within mainly parallel collagen fibers and less marked inflammation, simulating the intact tendon structure. These findings indicate that PRP can induce microscopic changes in the ruptured tendon by stimulating the healing process and can facilitate a more effective recovery. Full article

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22 pages, 4456 KiB

Open AccessArticle

GRT-X Stimulates Dorsal Root Ganglia Axonal Growth in Culture via TSPO and Kv7.2/3 Potassium Channel Activation

by Léa El Chemali, Suzan Boutary, Song Liu, Guo-Jun Liu, Ryan J. Middleton, Richard B. Banati, Gregor Bahrenberg, Rainer Rupprecht, Michael Schumacher and Liliane Massaad-Massade

Int. J. Mol. Sci. 2024, 25(13), 7327; https://doi.org/10.3390/ijms25137327 - 3 Jul 2024

Cited by 1 | Viewed by 1401

Abstract

GRT-X, which targets both the mitochondrial translocator protein (TSPO) and the Kv7.2/3 (KCNQ2/3) potassium channels, has been shown to efficiently promote recovery from cervical spine injury. In the present work, we investigate the role of GRT-X and its two targets in the axonal [...] Read more.

GRT-X, which targets both the mitochondrial translocator protein (TSPO) and the Kv7.2/3 (KCNQ2/3) potassium channels, has been shown to efficiently promote recovery from cervical spine injury. In the present work, we investigate the role of GRT-X and its two targets in the axonal growth of dorsal root ganglion (DRG) neurons. Neurite outgrowth was quantified in DRG explant cultures prepared from wild-type C57BL6/J and TSPO-KO mice. TSPO was pharmacologically targeted with the agonist XBD173 and the Kv7 channels with the activator ICA-27243 and the inhibitor XE991. GRT-X efficiently stimulated DRG axonal growth at 4 and 8 days after its single administration. XBD173 also promoted axonal elongation, but only after 8 days and its repeated administration. In contrast, both ICA27243 and XE991 tended to decrease axonal elongation. In dissociated DRG neuron/Schwann cell co-cultures, GRT-X upregulated the expression of genes associated with axonal growth and myelination. In the TSPO-KO DRG cultures, the stimulatory effect of GRT-X on axonal growth was completely lost. However, GRT-X and XBD173 activated neuronal and Schwann cell gene expression after TSPO knockout, indicating the presence of additional targets warranting further investigation. These findings uncover a key role of the dual mode of action of GRT-X in the axonal elongation of DRG neurons. Full article

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61 pages, 4977 KiB

Open AccessReview

Challenges and Opportunities Arising from Host–Botrytis cinerea Interactions to Outline Novel and Sustainable Control Strategies: The Key Role of RNA Interference

by Maria Spada, Claudio Pugliesi, Marco Fambrini and Susanna Pecchia

Int. J. Mol. Sci. 2024, 25(12), 6798; https://doi.org/10.3390/ijms25126798 - 20 Jun 2024

Cited by 2 | Viewed by 2330

Abstract

The necrotrophic plant pathogenic fungus Botrytis cinerea (Pers., 1794), the causative agent of gray mold disease, causes significant losses in agricultural production. Control of this fungal pathogen is quite difficult due to its wide host range and environmental persistence. Currently, the management of [...] Read more.

The necrotrophic plant pathogenic fungus Botrytis cinerea (Pers., 1794), the causative agent of gray mold disease, causes significant losses in agricultural production. Control of this fungal pathogen is quite difficult due to its wide host range and environmental persistence. Currently, the management of the disease is still mainly based on chemicals, which can have harmful effects not only on the environment and on human health but also because they favor the development of strains resistant to fungicides. The flexibility and plasticity of B. cinerea in challenging plant defense mechanisms and its ability to evolve strategies to escape chemicals require the development of new control strategies for successful disease management. In this review, some aspects of the host-pathogen interactions from which novel and sustainable control strategies could be developed (e.g., signaling pathways, molecules involved in plant immune mechanisms, hormones, post-transcriptional gene silencing) were analyzed. New biotechnological tools based on the use of RNA interference (RNAi) are emerging in the crop protection scenario as versatile, sustainable, effective, and environmentally friendly alternatives to the use of chemicals. RNAi-based fungicides are expected to be approved soon, although they will face several challenges before reaching the market. Full article

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18 pages, 1582 KiB

Open AccessArticle

Deficiency of Glucocerebrosidase Activity beyond Gaucher Disease: PSAP and LIMP-2 Dysfunctions

by Eleonora Pavan, Paolo Peruzzo, Silvia Cattarossi, Natascha Bergamin, Andrea Bordugo, Annalisa Sechi, Maurizio Scarpa, Jessica Biasizzo, Fabiana Colucci and Andrea Dardis

Int. J. Mol. Sci. 2024, 25(12), 6615; https://doi.org/10.3390/ijms25126615 - 16 Jun 2024

Cited by 2 | Viewed by 4086

Abstract

Glucocerebrosidase (GCase) is a lysosomal enzyme that catalyzes the breakdown of glucosylceramide in the presence of its activator saposin C (SapC). SapC arises from the proteolytical cleavage of prosaposin (encoded by PSAP gene), which gives rise to four saposins. GCase is targeted to [...] Read more.

Glucocerebrosidase (GCase) is a lysosomal enzyme that catalyzes the breakdown of glucosylceramide in the presence of its activator saposin C (SapC). SapC arises from the proteolytical cleavage of prosaposin (encoded by PSAP gene), which gives rise to four saposins. GCase is targeted to the lysosomes by LIMP-2, encoded by SCARB2 gene. GCase deficiency causes Gaucher Disease (GD), which is mainly due to biallelic pathogenetic variants in the GCase-encoding gene, GBA1. However, impairment of GCase activity can be rarely caused by SapC or LIMP-2 deficiencies. We report a new case of LIMP-2 deficiency and a new case of SapC deficiency (missing all four saposins, PSAP deficiency), and measured common biomarkers of GD and GCase activity. Glucosylsphingosine and chitotriosidase activity in plasma were increased in GCase deficiencies caused by PSAP and GBA1 mutations, whereas SCARB2-linked deficiency showed only Glucosylsphingosine elevation. GCase activity was reduced in fibroblasts and leukocytes: the decrease was sharper in GBA1- and SCARB2-mutant fibroblasts than PSAP-mutant ones; LIMP-2-deficient leukocytes displayed higher residual GCase activity than GBA1-mutant ones. Finally, we demonstrated that GCase mainly undergoes proteasomal degradation in LIMP-2-deficient fibroblasts and lysosomal degradation in PSAP-deficient fibroblasts. Thus, we analyzed the differential biochemical profile of GCase deficiencies due to the ultra-rare PSAP and SCARB2 biallelic pathogenic variants in comparison with the profile observed in GBA1-linked GCase deficiency. Full article

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17 pages, 3956 KiB

Open AccessArticle

Investigating the Free Volumes as Nanospaces in Human Stratum Corneum Lipid Bilayers Using Positron Annihilation Lifetime Spectroscopy (PALS)

by Krystyna Mojsiewicz-Pieńkowska, Dagmara Bazar, Jacek Filipecki and Kordian Chamerski

Int. J. Mol. Sci. 2024, 25(12), 6472; https://doi.org/10.3390/ijms25126472 - 12 Jun 2024

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Abstract

This work is the first one that provides not only evidence for the existence of free volumes in the human stratum corneum but also focuses on comparing these experimental data, obtained through the unique positron annihilation lifetime spectroscopy (PALS) method, with theoretical values [...] Read more.

This work is the first one that provides not only evidence for the existence of free volumes in the human stratum corneum but also focuses on comparing these experimental data, obtained through the unique positron annihilation lifetime spectroscopy (PALS) method, with theoretical values published in earlier works. The mean free volume of 0.269 nm was slightly lower than the theoretical value of 0.4 nm. The lifetime τ₃ (1.83 ns with a coefficient of variation CV of 3.21%) is dependent on the size of open sites in the skin. This information was used to calculate the free volume radius R (0.269 nm with CV 2.14%), free volume size Vf (0.081 nm³ with CV 4.69%), and the intensity I₃ (9.01% with CV 10.94%) to estimate the relative fractional free volume f_v (1.32 a.u. with CV 13.68%) in human skin ex vivo. The relation between the lifetime of o-Ps (τ₃) and the radius of free volume (R) was formulated using the Tao–Eldrup model, which assumes spherical voids and applies to sites with radii smaller than 1 nm. The results indicate that PALS is a powerful tool for confirming the existence of free volumes and determining their size. The studies also focused on describing the probable locations of these nanospaces in SC lipid bilayers. According to the theory, these play an essential role in dynamic processes in biological systems, including the diffusion of low-molecular-weight hydrophobic and moderately hydrophilic molecules. The mechanism of their formation has been determined by the molecular dynamics of the lipid chains. Full article

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20 pages, 5349 KiB

Open AccessArticle

A Pilot Study on Circulating, Cellular, and Tissue Biomarkers in Osteosarcopenic Patients

by Francesca Salamanna, Cesare Faldini, Francesca Veronesi, Veronica Borsari, Alberto Ruffilli, Marco Manzetti, Giovanni Viroli, Matteo Traversari, Laura Marchese, Milena Fini and Gianluca Giavaresi

Int. J. Mol. Sci. 2024, 25(11), 5879; https://doi.org/10.3390/ijms25115879 - 28 May 2024

Cited by 1 | Viewed by 1026

Abstract

Aging comes with the loss of muscle and bone mass, leading to a condition known as osteosarcopenia. Circulating, cellular, and tissue biomarkers research for osteosarcopenia is relatively scarce and, currently, no established biomarkers exist. Here we find that osteosarcopenic patients exhibited elevated basophils [...] Read more.

Aging comes with the loss of muscle and bone mass, leading to a condition known as osteosarcopenia. Circulating, cellular, and tissue biomarkers research for osteosarcopenia is relatively scarce and, currently, no established biomarkers exist. Here we find that osteosarcopenic patients exhibited elevated basophils and TNFα levels, along with decreased aPPT, PT/INR, IL15, alpha-Klotho, DHEA-S, and FGF-2 expression and distinctive bone and muscle tissue micro-architecture and biomarker expressions. They also displayed an increase in osteoclast precursors with a concomitant imbalance towards spontaneous osteoclastogenesis. Similarities were noted with osteopenic and sarcopenic patients, including a lower neutrophil percentage and altered cytokine expression. A linear discriminant analysis (LDA) on models based on selected biomarkers showed a classification accuracy in the range of 61–78%. Collectively, our data provide compelling evidence for novel biomarkers for osteosarcopenia that may hold potential as diagnostic tools to promote healthy aging. Full article

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25 pages, 8494 KiB

Open AccessArticle

Ovarian Cancer Cell-Conditioning Medium Induces Cancer-Associated Fibroblast Phenoconversion through Glucose-Dependent Inhibition of Autophagy

by Alessandra Ferraresi, Carlo Girone, Chinmay Maheshwari, Letizia Vallino, Danny N. Dhanasekaran and Ciro Isidoro

Int. J. Mol. Sci. 2024, 25(11), 5691; https://doi.org/10.3390/ijms25115691 - 23 May 2024

Cited by 1 | Viewed by 1770

Abstract

One aspect of ovarian tumorigenesis which is still poorly understood is the tumor–stroma interaction, which plays a major role in chemoresistance and tumor progression. Cancer-associated fibroblasts (CAFs), the most abundant stromal cell type in the tumor microenvironment, influence tumor growth, metabolism, metastasis, and [...] Read more.

One aspect of ovarian tumorigenesis which is still poorly understood is the tumor–stroma interaction, which plays a major role in chemoresistance and tumor progression. Cancer-associated fibroblasts (CAFs), the most abundant stromal cell type in the tumor microenvironment, influence tumor growth, metabolism, metastasis, and response to therapy, making them attractive targets for anti-cancer treatment. Unraveling the mechanisms involved in CAFs activation and maintenance is therefore crucial for the improvement of therapy efficacy. Here, we report that CAFs phenoconversion relies on the glucose-dependent inhibition of autophagy. We show that ovarian cancer cell-conditioning medium induces a metabolic reprogramming towards the CAF-phenotype that requires the autophagy-dependent glycolytic shift. In fact, 2-deoxy-D-glucose (2DG) strongly hampers such phenoconversion and, most importantly, induces the phenoreversion of CAFs into quiescent fibroblasts. Moreover, pharmacological inhibition (by proline) or autophagy gene knockdown (by siBECN1 or siATG7) promotes, while autophagy induction (by either 2DG or rapamycin) counteracts, the metabolic rewiring induced by the ovarian cancer cell secretome. Notably, the nutraceutical resveratrol (RV), known to inhibit glucose metabolism and to induce autophagy, promotes the phenoreversion of CAFs into normal fibroblasts even in the presence of ovarian cancer cell-conditioning medium. Overall, our data support the view of testing autophagy inducers for targeting the tumor-promoting stroma as an adjuvant strategy to improve therapy success rates, especially for tumors with a highly desmoplastic stroma, like ovarian cancer. Full article

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16 pages, 2411 KiB

Open AccessArticle

The Significant Role of PA28αβ in CD8⁺ T Cell-Mediated Graft Rejection Contrasts with Its Negligible Impact on the Generation of MHC-I Ligands

by Katharina Inholz, Ulrika Bader, Sarah Mundt and Michael Basler

Int. J. Mol. Sci. 2024, 25(11), 5649; https://doi.org/10.3390/ijms25115649 - 22 May 2024

Viewed by 921

Abstract

The proteasome generates the majority of peptides presented on MHC class I molecules. The cleavage pattern of the proteasome has been shown to be changed via the proteasome activator (PA)28 alpha beta (PA28αβ). In particular, several immunogenic peptides have been reported to be [...] Read more.

The proteasome generates the majority of peptides presented on MHC class I molecules. The cleavage pattern of the proteasome has been shown to be changed via the proteasome activator (PA)28 alpha beta (PA28αβ). In particular, several immunogenic peptides have been reported to be PA28αβ-dependent. In contrast, we did not observe a major impact of PA28αβ on the generation of different major histocompatibility complex (MHC) classI ligands. PA28αβ-knockout mice infected with the lymphocytic choriomeningitis virus (LCMV) or vaccinia virus showed a normal cluster of differentiation (CD) 8 response and viral clearance. However, we observed that the adoptive transfer of wild-type cells into PA28αβ-knockout mice led to graft rejection, but not vice versa. Depletion experiments showed that the observed rejection was mediated by CD8⁺ cytotoxic T cells. These data indicate that PA28αβ might be involved in the development of the CD8⁺ T cell repertoire in the thymus. Taken together, our data suggest that PA28αβ is a crucial factor determining T cell selection and, therefore, impacts graft acceptance. Full article

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19 pages, 12199 KiB

Open AccessArticle

A Spatio-Temporal-Dependent Requirement of Sonic Hedgehog in the Early Development of Sclerotome-Derived Vertebrae and Ribs

by Nitza Kahane, Yael Dahan-Barda and Chaya Kalcheim

Int. J. Mol. Sci. 2024, 25(11), 5602; https://doi.org/10.3390/ijms25115602 - 21 May 2024

Viewed by 1017

Abstract

Derived from axial structures, Sonic Hedgehog (Shh) is secreted into the paraxial mesoderm, where it plays crucial roles in sclerotome induction and myotome differentiation. Through conditional loss-of-function in quail embryos, we investigate the timing and impact of Shh activity during early formation of [...] Read more.

Derived from axial structures, Sonic Hedgehog (Shh) is secreted into the paraxial mesoderm, where it plays crucial roles in sclerotome induction and myotome differentiation. Through conditional loss-of-function in quail embryos, we investigate the timing and impact of Shh activity during early formation of sclerotome-derived vertebrae and ribs, and of lateral mesoderm-derived sternum. To this end, Hedgehog interacting protein (Hhip) was electroporated at various times between days 2 and 5. While the vertebral body and rib primordium showed consistent size reduction, rib expansion into the somatopleura remained unaffected, and the sternal bud developed normally. Additionally, we compared these effects with those of locally inhibiting BMP activity. Transfection of Noggin in the lateral mesoderm hindered sternal bud formation. Unlike Hhip, BMP inhibition via Noggin or Smad6 induced myogenic differentiation of the lateral dermomyotome lip, while impeding the growth of the myotome/rib complex into the somatic mesoderm, thus affirming the role of the lateral dermomyotome epithelium in rib guidance. Overall, these findings underscore the continuous requirement for opposing gradients of Shh and BMP activity in the morphogenesis of proximal and distal flank skeletal structures, respectively. Future research should address the implications of these early interactions to the later morphogenesis and function of the musculo-skeletal system and of possible associated malformations. Full article

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19 pages, 618 KiB

Open AccessReview

NLRP3 Inflammasome in Acute and Chronic Liver Diseases

by Katia Sayaf, Sara Battistella and Francesco Paolo Russo

Int. J. Mol. Sci. 2024, 25(8), 4537; https://doi.org/10.3390/ijms25084537 - 20 Apr 2024

Cited by 2 | Viewed by 2594

Abstract

NLRP3 (NOD-, LRR-, and pyrin domain-containing protein 3) is an intracellular complex that upon external stimuli or contact with specific ligands, recruits other components, forming the NLRP3 inflammasome. The NLRP3 inflammasome mainly mediates pyroptosis, a highly inflammatory mode of regulated cell death, as [...] Read more.

NLRP3 (NOD-, LRR-, and pyrin domain-containing protein 3) is an intracellular complex that upon external stimuli or contact with specific ligands, recruits other components, forming the NLRP3 inflammasome. The NLRP3 inflammasome mainly mediates pyroptosis, a highly inflammatory mode of regulated cell death, as well as IL-18 and IL-1β production. Acute and chronic liver diseases are characterized by a massive influx of pro-inflammatory stimuli enriched in reactive oxygen species (ROS) and damage-associated molecular patterns (DAMPs) that promote the assemblage and activation of the NLRP3 inflammasome. As the major cause of inflammatory cytokine storm, the NLRP3 inflammasome exacerbates liver diseases, even though it might exert protective effects in regards to hepatitis C and B virus infection (HCV and HBV). Here, we summarize the current knowledge concerning NLRP3 inflammasome function in both acute and chronic liver disease and in the post liver transplant setting, focusing on the molecular mechanisms involved in NLRP3 activity. Full article

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12 pages, 1572 KiB

Open AccessArticle

Sperm Chromatin Dispersion Test Detects Sperm DNA Fragmentation Mainly Associated with Unviable Spermatozoa and Underestimates the Values with Respect to TUNEL Assay

by Maria Emanuela Ragosta, Giulia Traini, Lara Tamburrino, Selene Degl’Innocenti, Maria Grazia Fino, Sara Dabizzi, Linda Vignozzi, Elisabetta Baldi and Sara Marchiani

Int. J. Mol. Sci. 2024, 25(8), 4481; https://doi.org/10.3390/ijms25084481 - 19 Apr 2024

Cited by 2 | Viewed by 1936

Abstract

Several clinical laboratories assess sperm DNA fragmentation (sDF) in addition to semen analysis in male infertility diagnosis. Among tests evaluating sDF, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) and SCD (Sperm Chromatin Dispersion) are widely used. Our lab developed a modified version [...] Read more.

Several clinical laboratories assess sperm DNA fragmentation (sDF) in addition to semen analysis in male infertility diagnosis. Among tests evaluating sDF, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) and SCD (Sperm Chromatin Dispersion) are widely used. Our lab developed a modified version of TUNEL (TUNEL/PI) able to distinguish two sperm populations (PI Brighter and PI Dimmer) differently associated with sperm viability and reproductive outcomes. The aim of this study was to compare sDF levels detected by SCD and TUNEL/PI in the semen samples from 71 male subjects attending our Andrology Laboratory. Our results demonstrate that SCD is less sensitive in determining sDF compared to TUNEL/PI. The statistically significant positive correlation found between sDF evaluated by SCD and PI Dimmer (consisting of all dead spermatozoa) suggests that SCD mainly detects sDF in unviable spermatozoa. We confirmed that most spermatozoa detected by SCD are unviable by performing SCD after incubation in hypo-osmotic medium to discriminate viable and unviable cells in 52 samples. Such results might explain the lower ability of this test in discriminating couples having successful ART outcomes demonstrated in published metanalyses. Overall, our results indicate that SCD is less sensitive in evaluating sDF for diagnostic purposes. Full article

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21 pages, 1143 KiB

Open AccessReview

A Narrative Review on Adipose Tissue and Overtraining: Shedding Light on the Interplay among Adipokines, Exercise and Overtraining

by Marta Mallardo, Aurora Daniele, Giuseppe Musumeci and Ersilia Nigro

Int. J. Mol. Sci. 2024, 25(7), 4089; https://doi.org/10.3390/ijms25074089 - 6 Apr 2024

Cited by 6 | Viewed by 2785

Abstract

Lifestyle factors, particularly physical inactivity, are closely linked to the onset of numerous metabolic diseases. Adipose tissue (AT) has been extensively studied for various metabolic diseases such as obesity, type 2 diabetes, and immune system dysregulation due to its role in energy metabolism [...] Read more.

Lifestyle factors, particularly physical inactivity, are closely linked to the onset of numerous metabolic diseases. Adipose tissue (AT) has been extensively studied for various metabolic diseases such as obesity, type 2 diabetes, and immune system dysregulation due to its role in energy metabolism and regulation of inflammation. Physical activity is increasingly recognized as a powerful non-pharmacological tool for the treatment of various disorders, as it helps to improve metabolic, immune, and inflammatory functions. However, chronic excessive training has been associated with increased inflammatory markers and oxidative stress, so much so that excessive training overload, combined with inadequate recovery, can lead to the development of overtraining syndrome (OTS). OTS negatively impacts an athlete’s performance capabilities and significantly affects both physical health and mental well-being. However, diagnosing OTS remains challenging as the contributing factors, signs/symptoms, and underlying maladaptive mechanisms are individualized, sport-specific, and unclear. Therefore, identifying potential biomarkers that could assist in preventing and/or diagnosing OTS is an important objective. In this review, we focus on the possibility that the endocrine functions of AT may have significant implications in the etiopathogenesis of OTS. During physical exercise, AT responds dynamically, undergoing remodeling of endocrine functions that influence the production of adipokines involved in regulating major energy and inflammatory processes. In this scenario, we will discuss exercise about its effects on AT activity and metabolism and its relevance to the prevention and/or development of OTS. Furthermore, we will highlight adipokines as potential markers for diagnosing OTS. Full article

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18 pages, 11905 KiB

Open AccessArticle

Machine Learning-Based Characterization and Identification of Tertiary Lymphoid Structures Using Spatial Transcriptomics Data

by Songyun Li, Zhuo Wang, Hsien-Da Huang and Tzong-Yi Lee

Int. J. Mol. Sci. 2024, 25(7), 3887; https://doi.org/10.3390/ijms25073887 - 30 Mar 2024

Cited by 1 | Viewed by 2256

Abstract

Tertiary lymphoid structures (TLSs) are organized aggregates of immune cells in non-lymphoid tissues and are associated with a favorable prognosis in tumors. However, TLS markers remain inconsistent, and the utilization of machine learning techniques for this purpose is limited. To tackle this challenge, [...] Read more.

Tertiary lymphoid structures (TLSs) are organized aggregates of immune cells in non-lymphoid tissues and are associated with a favorable prognosis in tumors. However, TLS markers remain inconsistent, and the utilization of machine learning techniques for this purpose is limited. To tackle this challenge, we began by identifying TLS markers through bioinformatics analysis and machine learning techniques. Subsequently, we leveraged spatial transcriptomic data from Gene Expression Omnibus (GEO) and built two support vector classifier models for TLS prediction: one without feature selection and the other using the marker genes. The comparable performances of these two models confirm the efficacy of the selected markers. The majority of the markers are immunoglobulin genes, demonstrating their importance in the identification of TLSs. Our research has identified the markers of TLSs using machine learning methods and constructed a model to predict TLS location, contributing to the detection of TLS and holding the promising potential to impact cancer treatment strategies. Full article

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15 pages, 2675 KiB

Open AccessArticle

BcABF1 Plays a Role in the Feedback Regulation of Abscisic Acid Signaling via the Direct Activation of BcPYL4 Expression in Pakchoi

by Xiaoxue Yang, Meiyun Wang, Qian Zhou, Xinfeng Xu, Ying Li, Xilin Hou, Dong Xiao and Tongkun Liu

Int. J. Mol. Sci. 2024, 25(7), 3877; https://doi.org/10.3390/ijms25073877 - 30 Mar 2024

Cited by 1 | Viewed by 1079

Abstract

Abscisic acid-responsive element-binding factor 1 (ABF1), a key transcription factor in the ABA signal transduction process, regulates the expression of downstream ABA-responsive genes and is involved in modulating plant responses to abiotic stress and developmental processes. However, there is currently limited [...] Read more.

Abscisic acid-responsive element-binding factor 1 (ABF1), a key transcription factor in the ABA signal transduction process, regulates the expression of downstream ABA-responsive genes and is involved in modulating plant responses to abiotic stress and developmental processes. However, there is currently limited research on the feedback regulation of ABF1 in ABA signaling. This study delves into the function of BcABF1 in Pakchoi. We observed a marked increase in BcABF1 expression in leaves upon ABA induction. The overexpression of BcABF1 not only spurred Arabidopsis growth but also augmented the levels of endogenous IAA. Furthermore, BcABF1 overexpression in Arabidopsis significantly decreased leaf water loss and enhanced the expression of genes associated with drought tolerance in the ABA pathway. Intriguingly, we found that BcABF1 can directly activate BcPYL4 expression, a critical receptor in the ABA pathway. Similar to BcABF1, the overexpression of BcPYL4 in Arabidopsis also reduces leaf water loss and promotes the expression of drought and other ABA-responsive genes. Finally, our findings suggested a novel feedback regulation mechanism within the ABA signaling pathway, wherein BcABF1 positively amplifies the ABA signal by directly binding to and activating the BcPYL4 promoter. Full article

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15 pages, 1420 KiB

Open AccessReview

Retinal Pigment Epithelium Pigment Granules: Norms, Age Relations and Pathology

by Alexander Dontsov and Mikhail Ostrovsky

Int. J. Mol. Sci. 2024, 25(7), 3609; https://doi.org/10.3390/ijms25073609 - 23 Mar 2024

Cited by 4 | Viewed by 2481

Abstract

The retinal pigment epithelium (RPE), which ensures the normal functioning of the neural retina, is a pigmented single-cell layer that separates the retina from the Bruch’s membrane and the choroid. There are three main types of pigment granules in the RPE cells of [...] Read more.

The retinal pigment epithelium (RPE), which ensures the normal functioning of the neural retina, is a pigmented single-cell layer that separates the retina from the Bruch’s membrane and the choroid. There are three main types of pigment granules in the RPE cells of the human eye: lipofuscin granules (LG) containing the fluorescent “age pigment” lipofuscin, melanoprotein granules (melanosomes, melanolysosomes) containing the screening pigment melanin and complex melanolipofuscin granules (MLG) containing both types of pigments simultaneously—melanin and lipofuscin. This review examines the functional role of pigment granules in the aging process and in the development of oxidative stress and associated pathologies in RPE cells. The focus is on the process of light-induced oxidative degradation of pigment granules caused by reactive oxygen species. The reasons leading to increased oxidative stress in RPE cells as a result of the oxidative degradation of pigment granules are considered. A mechanism is proposed to explain the phenomenon of age-related decline in melanin content in RPE cells. The essence of the mechanism is that when the lipofuscin part of the melanolipofuscin granule is exposed to light, reactive oxygen species are formed, which destroy the melanin part. As more melanolipofuscin granules are formed with age and the development of degenerative diseases, the melanin in pigmented epithelial cells ultimately disappears. Full article

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14 pages, 3476 KiB

Open AccessArticle

Leucine-Rich Repeat in Polycystin-1 Suppresses Cystogenesis in a Zebrafish (Danio rerio) Model of Autosomal-Dominant Polycystic Kidney Disease

by Biswajit Padhy, Mohammad Amir, Jian Xie and Chou-Long Huang

Int. J. Mol. Sci. 2024, 25(5), 2886; https://doi.org/10.3390/ijms25052886 - 1 Mar 2024

Viewed by 1460

Abstract

Mutations of PKD1 coding for polycystin-1 (PC1) account for most cases of autosomal-dominant polycystic kidney disease (ADPKD). The extracellular region of PC1 contains many evolutionarily conserved domains for ligand interactions. Among these are the leucine-rich repeats (LRRs) in the far N-terminus of PC1. [...] Read more.

Mutations of PKD1 coding for polycystin-1 (PC1) account for most cases of autosomal-dominant polycystic kidney disease (ADPKD). The extracellular region of PC1 contains many evolutionarily conserved domains for ligand interactions. Among these are the leucine-rich repeats (LRRs) in the far N-terminus of PC1. Using zebrafish (Danio rerio) as an in vivo model system, we explored the role of LRRs in the function of PC1. Zebrafish expresses two human PKD1 paralogs, pkd1a and pkd1b. Knockdown of both genes in zebrafish by morpholino antisense oligonucleotides produced phenotypes of dorsal-axis curvature and pronephric cyst formation. We found that overexpression of LRRs suppressed both phenotypes in pkd1-morphant zebrafish. Purified recombinant LRR domain inhibited proliferation of HEK cells in culture and interacted with the heterotrimeric basement membrane protein laminin-511 (α5β1γ1) in vitro. Mutations of amino acid residues in LRRs structurally predicted to bind laminin-511 disrupted LRR–laminin interaction in vitro and neutralized the ability of LRRs to inhibit cell proliferation and cystogenesis. Our data support the hypothesis that the extracellular region of PC1 plays a role in modulating PC1 interaction with the extracellular matrix and contributes to cystogenesis of PC1 deficiency. Full article

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14 pages, 2766 KiB

Open AccessArticle

Skin Anti-Aging Potential through Whitening and Wrinkle Improvement Using Fermented Oil Derived from Hermetia illucens Larvae

by Dooseon Hwang, Tae-Won Goo, Seung Hun Lee and Eun-Young Yun

Int. J. Mol. Sci. 2024, 25(5), 2736; https://doi.org/10.3390/ijms25052736 - 27 Feb 2024

Cited by 1 | Viewed by 1617

Abstract

As the aging population increases, so has interest among emerging seniors in anti-aging ingredients that enhance functionality by incorporating fermentation with natural materials. In this study, fermentation conditions for enhancing the functionality of Hermetia illucens larvae oil (HIO) were established, and its anti-aging [...] Read more.

As the aging population increases, so has interest among emerging seniors in anti-aging ingredients that enhance functionality by incorporating fermentation with natural materials. In this study, fermentation conditions for enhancing the functionality of Hermetia illucens larvae oil (HIO) were established, and its anti-aging potential was evaluated. First, the lipase activity and amount of lipid degradation products of the fermentation strains were measured in order to select Lactobacillus gasseri and Lactiplantibacillus plantarum as the strains with high fermentation ability. A fermentation period of 28 d and a fermentation method that uses only the strain culture medium were established by evaluating the fermentation degree after fermenting HIO with the selected strains. The whitening functionality test results of fermented HIO (FHIO) showed an increase of approximately 20% in extracellular tyrosinase inhibition activity compared with HIO. Additionally, within melanocytes, there was a 12% increase in tyrosinase inhibition activity and a 26% enhancement in melanin production inhibition ability. For wrinkle-improving functionality, it was observed that, for fibroblasts, there was a 10% increase in collagen production, a 9% increase in collagenase inhibition ability, and an 8% increase in elastase inhibition ability. Therefore, FHIO was confirmed to be an effective cosmetic raw material, with high functionality for anti-aging within the senior generation. This is achieved through increased whitening and wrinkle-improving functionality. Full article

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20 pages, 6512 KiB

Open AccessArticle

Drought Stress Induced Different Response Mechanisms in Three Dendrobium Species under Different Photosynthetic Pathways

by Ke Xia, Qiaofen Wu, Yanni Yang, Qiao Liu, Zaihua Wang, Zhiguo Zhao, Jie Li, Jinxiang He, Shengfeng Chai and Shuo Qiu

Int. J. Mol. Sci. 2024, 25(5), 2731; https://doi.org/10.3390/ijms25052731 - 27 Feb 2024

Cited by 1 | Viewed by 1262

Abstract

Many Dendrobium species, which hold a high status and value in traditional Chinese medicine, grow on barks and rocks in the wild, often encountering harsh environments and facing droughts. However, the molecular mechanisms underlying the shift in the photosynthetic pathway induced by drought [...] Read more.

Many Dendrobium species, which hold a high status and value in traditional Chinese medicine, grow on barks and rocks in the wild, often encountering harsh environments and facing droughts. However, the molecular mechanisms underlying the shift in the photosynthetic pathway induced by drought remain unclear. To address this issue, three Dendrobium species with different photosynthetic pathways were selected for sequencing and transcriptome data analysis after drought treatment. The findings included 134.43 GB of sequencing data, with numerous Differentially Expressed Genes (DEGs) exhibiting different response mechanisms under drought stress. Gene Ontology (GO)–KEGG-based enrichment analysis of DEGs revealed that metabolic pathways contributed to drought tolerance and alterations in photosynthetic pathways. Phosphoenolpyruvate Carboxylase (PEPC) was subjected to phylogenetic tree construction, sequence alignment, and domain analysis. Under drought stress, variations were observed in the PEPC gene structure and expression among different Dendrobium species; the upregulation of Dc_gene2609 expression may be caused by dof-miR-384, which resulted in the shift from C₃ photosynthesis to CAM, thereby improving drought tolerance in Dendrobium. This study revealed the expression patterns and roles of PEPC genes in enhancing plant drought tolerance and will provide an important basis for in-depth research on Dendrobium’s adaptation mechanisms in arid environments. Full article

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17 pages, 4924 KiB

Open AccessArticle

Transcriptome-Wide Association Study Reveals Potentially Candidate Genes Responsible for Milk Production Traits in Buffalo

by Kelong Wei, Ying Lu, Xiaoya Ma, Anqian Duan, Xingrong Lu, Hamdy Abdel-Shafy and Tingxian Deng

Int. J. Mol. Sci. 2024, 25(5), 2626; https://doi.org/10.3390/ijms25052626 - 23 Feb 2024

Viewed by 1986

Abstract

Identifying key causal genes is critical for unraveling the genetic basis of complex economic traits, yet it remains a formidable challenge. The advent of large-scale sequencing data and computational algorithms, such as transcriptome-wide association studies (TWASs), offers a promising avenue for identifying potential [...] Read more.

Identifying key causal genes is critical for unraveling the genetic basis of complex economic traits, yet it remains a formidable challenge. The advent of large-scale sequencing data and computational algorithms, such as transcriptome-wide association studies (TWASs), offers a promising avenue for identifying potential causal genes. In this study, we harnessed the power of TWAS to identify genes potentially responsible for milk production traits, including daily milk yield (MY), fat percentage (FP), and protein percentage (PP), within a cohort of 100 buffaloes. Our approach began by generating the genotype and expression profiles for these 100 buffaloes through whole-genome resequencing and RNA sequencing, respectively. Through comprehensive genome-wide association studies (GWAS), we pinpointed a total of seven and four single nucleotide polymorphisms (SNPs) significantly associated with MY and FP traits, respectively. By using TWAS, we identified 55, 71, and 101 genes as significant signals for MY, FP, and PP traits, respectively. To delve deeper, we conducted protein–protein interaction (PPI) analysis, revealing the categorization of these genes into distinct PPI networks. Interestingly, several TWAS-identified genes within the PPI network played a vital role in milk performance. These findings open new avenues for identifying potentially causal genes underlying important traits, thereby offering invaluable insights for genomics and breeding in buffalo populations. Full article

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13 pages, 3910 KiB

Open AccessArticle

The Mechanism of Tigecycline Resistance in Acinetobacter baumannii under Sub-Minimal Inhibitory Concentrations of Tigecycline

by Cunwei Liu, Jia Liu, Qinghui Lu, Ping Wang and Qinghua Zou

Int. J. Mol. Sci. 2024, 25(3), 1819; https://doi.org/10.3390/ijms25031819 - 2 Feb 2024

Cited by 2 | Viewed by 1781

Abstract

The presence of sub-minimal inhibitory concentration (sub-MIC) antibiotics in our environment is widespread, and their ability to induce antibiotic resistance is inevitable. Acinetobacter baumannii, a pathogen known for its strong ability to acquire antibiotic resistance, has recently shown clinical resistance to the [...] Read more.

The presence of sub-minimal inhibitory concentration (sub-MIC) antibiotics in our environment is widespread, and their ability to induce antibiotic resistance is inevitable. Acinetobacter baumannii, a pathogen known for its strong ability to acquire antibiotic resistance, has recently shown clinical resistance to the last-line antibiotic tigecycline. To unravel the complex mechanism of A. baumannii drug resistance, we subjected tigecycline-susceptible, -intermediate, and -mildly-resistant strains to successive increases in sub-MIC tigecycline and ultimately obtained tigecycline-resistant strains. The proteome of both key intermediate and final strains during the selection process was analyzed using nanoLC-MS/MS. Among the more than 2600 proteins detected in all strains, we found that RND efflux pump AdeABC was associated with the adaptability of A. baumannii to tigecycline under sub-MIC pressure. qRT-PCR analysis also revealed higher expression of AdeAB in strains that can quickly acquire tigecycline resistance compared with strains that displayed lower adaptability. To validate our findings, we added an efflux pump inhibitor, carbonyl cyanide m-chlorophenyl hydrazine (CCCP), to the medium and observed its ability to inhibit tigecycline resistance in A. baumannii strains with quick adaptability. This study contributes to a better understanding of the mechanisms underlying tigecycline resistance in A. baumannii under sub-MIC pressure. Full article

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18 pages, 5711 KiB

Open AccessArticle

Genome-Wide Association Study and Prediction of Tassel Weight of Tropical Maize Germplasm in Multi-Parent Population

by Meichen Liu, Yudong Zhang, Ranjan K. Shaw, Xingjie Zhang, Jinfeng Li, Linzhuo Li, Shaoxiong Li, Muhammad Adnan, Fuyan Jiang, Yaqi Bi, Xingfu Yin and Xingming Fan

Int. J. Mol. Sci. 2024, 25(3), 1756; https://doi.org/10.3390/ijms25031756 - 1 Feb 2024

Cited by 2 | Viewed by 1797

Abstract

Tassel weight (TW) is a crucial agronomic trait that significantly affects pollen supply and grain yield development in maize breeding. To improve maize yield and develop new varieties, a comprehensive understanding of the genetic mechanisms underlying tassel weight is essential. In this study, [...] Read more.

Tassel weight (TW) is a crucial agronomic trait that significantly affects pollen supply and grain yield development in maize breeding. To improve maize yield and develop new varieties, a comprehensive understanding of the genetic mechanisms underlying tassel weight is essential. In this study, tropical maize inbred lines, namely CML312, CML373, CML444, and YML46, were selected as female parents and crossed with the elite maize inbred line Ye107, which served as the common male parent, to develop a multi-parent population comprising four F8 recombinant inbred line (RIL) subpopulations. Using 6616 high-quality single nucleotide polymorphism (SNP) markers, we conducted genome-wide association analysis (GWAS) and genomic selection (GS) on 642 F8 RILs in four subpopulations across three different environments. Through GWAS, we identified 16 SNPs that were significantly associated with TW, encompassing two stable loci expressed across multiple environments. Furthermore, within the candidate regions of these SNPs, we discovered four novel candidate genes related to TW, namely Zm00001d044362, Zm00001d011048, Zm00001d011049, and Zm00001d031173 distributed on chromosomes 1, 3, and 8, which have not been previously reported. These genes are involved in processes such as signal transduction, growth and development, protein splicing, and pollen development, all of which play crucial roles in inflorescence meristem development, directly affecting TW. The co-localized SNP, S8_137379725, on chromosome 8 was situated within a 16.569 kb long terminal repeat retrotransposon (LTR-RT), located 22.819 kb upstream and 26.428 kb downstream of the candidate genes (Zm00001d011048 and Zm00001d011049). When comparing three distinct GS models, the BayesB model demonstrated the highest accuracy in predicting TW. This study establishes the theoretical foundation for future research into the genetic mechanisms underlying maize TW and the efficient breeding of high-yielding varieties with desired tassel weight through GS. Full article

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23 pages, 7894 KiB

Open AccessReview

New Discoveries on Protein Recruitment and Regulation during the Early Stages of the DNA Damage Response Pathways

by Kelly L. Waters and Donald E. Spratt

Int. J. Mol. Sci. 2024, 25(3), 1676; https://doi.org/10.3390/ijms25031676 - 30 Jan 2024

Cited by 3 | Viewed by 2182

Abstract

Maintaining genomic stability and properly repairing damaged DNA is essential to staying healthy and preserving cellular homeostasis. The five major pathways involved in repairing eukaryotic DNA include base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), non-homologous end joining (NHEJ), and [...] Read more.

Maintaining genomic stability and properly repairing damaged DNA is essential to staying healthy and preserving cellular homeostasis. The five major pathways involved in repairing eukaryotic DNA include base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), non-homologous end joining (NHEJ), and homologous recombination (HR). When these pathways do not properly repair damaged DNA, genomic stability is compromised and can contribute to diseases such as cancer. It is essential that the causes of DNA damage and the consequent repair pathways are fully understood, yet the initial recruitment and regulation of DNA damage response proteins remains unclear. In this review, the causes of DNA damage, the various mechanisms of DNA damage repair, and the current research regarding the early steps of each major pathway were investigated. Full article

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20 pages, 1852 KiB

Open AccessArticle

SumVg: Total Heritability Explained by All Variants in Genome-Wide Association Studies Based on Summary Statistics with Standard Error Estimates

by Hon-Cheong So, Xiao Xue, Zhijie Ma and Pak-Chung Sham

Int. J. Mol. Sci. 2024, 25(2), 1347; https://doi.org/10.3390/ijms25021347 - 22 Jan 2024

Cited by 1 | Viewed by 1582

Abstract

Genome-wide association studies (GWAS) are commonly employed to study the genetic basis of complex traits/diseases, and a key question is how much heritability could be explained by all single nucleotide polymorphisms (SNPs) in GWAS. One widely used approach that relies on summary statistics [...] Read more.

Genome-wide association studies (GWAS) are commonly employed to study the genetic basis of complex traits/diseases, and a key question is how much heritability could be explained by all single nucleotide polymorphisms (SNPs) in GWAS. One widely used approach that relies on summary statistics only is linkage disequilibrium score regression (LDSC); however, this approach requires certain assumptions about the effects of SNPs (e.g., all SNPs contribute to heritability and each SNP contributes equal variance). More flexible modeling methods may be useful. We previously developed an approach recovering the “true” effect sizes from a set of observed z-statistics with an empirical Bayes approach, using only summary statistics. However, methods for standard error (SE) estimation are not available yet, limiting the interpretation of our results and the applicability of the approach. In this study, we developed several resampling-based approaches to estimate the SE of SNP-based heritability, including two jackknife and three parametric bootstrap methods. The resampling procedures are performed at the SNP level as it is most common to estimate heritability from GWAS summary statistics alone. Simulations showed that the delete-d-jackknife and parametric bootstrap approaches provide good estimates of the SE. In particular, the parametric bootstrap approaches yield the lowest root-mean-squared-error (RMSE) of the true SE. We also explored various methods for constructing confidence intervals (CIs). In addition, we applied our method to estimate the SNP-based heritability of 12 immune-related traits (levels of cytokines and growth factors) to shed light on their genetic architecture. We also implemented the methods to compute the sum of heritability explained and the corresponding SE in an R package SumVg. In conclusion, SumVg may provide a useful alternative tool for calculating SNP heritability and estimating SE/CI, which does not rely on distributional assumptions of SNP effects. Full article

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18 pages, 1756 KiB

Open AccessReview

The Multifaceted Functions of TRPV4 and Calcium Oscillations in Tissue Repair

by Dongsheng Jiang, Ruiji Guo, Ruoxuan Dai, Samuel Knoedler, Jin Tao, Hans-Günther Machens and Yuval Rinkevich

Int. J. Mol. Sci. 2024, 25(2), 1179; https://doi.org/10.3390/ijms25021179 - 18 Jan 2024

Cited by 5 | Viewed by 2718

Abstract

The transient receptor potential vanilloid 4 (TRPV4) specifically functions as a mechanosensitive ion channel and is responsible for conveying changes in physical stimuli such as mechanical stress, osmotic pressure, and temperature. TRPV4 enables the entry of cation ions, particularly calcium ions, into the [...] Read more.

The transient receptor potential vanilloid 4 (TRPV4) specifically functions as a mechanosensitive ion channel and is responsible for conveying changes in physical stimuli such as mechanical stress, osmotic pressure, and temperature. TRPV4 enables the entry of cation ions, particularly calcium ions, into the cell. Activation of TRPV4 channels initiates calcium oscillations, which trigger intracellular signaling pathways involved in a plethora of cellular processes, including tissue repair. Widely expressed throughout the body, TRPV4 can be activated by a wide array of physicochemical stimuli, thus contributing to sensory and physiological functions in multiple organs. This review focuses on how TRPV4 senses environmental cues and thereby initiates and maintains calcium oscillations, critical for responses to organ injury, tissue repair, and fibrosis. We provide a summary of TRPV4-induced calcium oscillations in distinct organ systems, along with the upstream and downstream signaling pathways involved. In addition, we delineate current animal and disease models supporting TRPV4 research and shed light on potential therapeutic targets for modulating TRPV4-induced calcium oscillation to promote tissue repair while reducing tissue fibrosis. Full article

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18 pages, 1158 KiB

Open AccessReview

Sphingosine 1-Phosphate Regulates Obesity and Glucose Homeostasis

by Kazuo Kajita, Isao Ishii, Ichiro Mori, Motochika Asano, Masayuki Fuwa and Hiroyuki Morita

Int. J. Mol. Sci. 2024, 25(2), 932; https://doi.org/10.3390/ijms25020932 - 11 Jan 2024

Cited by 5 | Viewed by 2952

Abstract

One of the major global health and welfare issues is the treatment of obesity and associated metabolic disorders, such as type 2 diabetes mellitus and nonalcoholic fatty liver disease. Obesity, caused by the excessive accumulation of triglycerides in adipose tissues, induces adipocyte dysfunction, [...] Read more.

One of the major global health and welfare issues is the treatment of obesity and associated metabolic disorders, such as type 2 diabetes mellitus and nonalcoholic fatty liver disease. Obesity, caused by the excessive accumulation of triglycerides in adipose tissues, induces adipocyte dysfunction, followed by inflammation, in adipose tissues and lipotoxicity in nonadipose tissues. Several studies have shown that obesity and glucose homeostasis are influenced by sphingolipid mediators, including ceramide and sphingosine 1-phosphate (S1P). Cellular accumulation of ceramide impairs pancreatic β-cell survival, confers insulin resistance in the liver and the skeletal muscle, and deteriorates adipose tissue inflammation via unknown molecular mechanisms. The roles of S1P are more complicated, because there are five cell-surface S1P receptors (S1PRs: S1P_1–5) which have altered functions, different cellular expression patterns, and inapparent intracellular targets. Recent findings, including those by our group, support the notable concept that the pharmacological activation of S1P₁ or S1P₃ improves obesity and associated metabolic disorders, whereas that of S1P₂ has the opposite effect. In addition, the regulation of S1P production by sphingosine kinase (SphK) is an essential factor affecting glucose homeostasis. This review summarizes the current knowledge on SphK/S1P/S1PR signaling in and against obesity, insulin resistance, and associated disorders. Full article

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2023

Jump to: 2025, 2024, 2022, 2021, 2020, 2019

12 pages, 3592 KiB

Open AccessArticle

Activation of Young LINE-1 Elements by CRISPRa

by Bei Tong and Yuhua Sun

Int. J. Mol. Sci. 2024, 25(1), 424; https://doi.org/10.3390/ijms25010424 - 28 Dec 2023

Viewed by 1973

Abstract

Long interspersed element-1 (LINE-1; L1s) are mobile genetic elements that comprise nearly 20% of the human genome. L1s have been shown to have important functions in various biological processes, and their dysfunction is thought to be linked with diseases and cancers. However, the [...] Read more.

Long interspersed element-1 (LINE-1; L1s) are mobile genetic elements that comprise nearly 20% of the human genome. L1s have been shown to have important functions in various biological processes, and their dysfunction is thought to be linked with diseases and cancers. However, the roles of the repetitive elements are largely not understood. While the CRISPR activation (CRISPRa) system based on catalytically deadCas9 (dCas9) is widely used for genome-wide interrogation of gene function and genetic interaction, few studies have been conducted on L1s. Here, we report using the CRISPRa method to efficiently activate L1s in human L02 cells, a derivative of the HeLa cancer cell line. After CRISPRa, the young L1 subfamilies such as L1HS/L1PA1 and L1PA2 are found to be expressed at higher levels than the older L1s. The L1s with high levels of transcription are closer to full-length and are more densely occupied by the YY1 transcription factor. The activated L1s can either be mis-spliced to form chimeric transcripts or act as alternative promoters or enhancers to facilitate the expression of neighboring genes. The method described here can be used for studying the functional roles of young L1s in cultured cells of interest. Full article

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13 pages, 2839 KiB

Open AccessArticle

FHL2 Inhibits SARS-CoV-2 Replication by Enhancing IFN-β Expression through Regulating IRF-3

by Zhiqiang Xu, Mingyao Tian, Qihan Tan, Pengfei Hao, Zihan Gao, Chang Li and Ningyi Jin

Int. J. Mol. Sci. 2024, 25(1), 353; https://doi.org/10.3390/ijms25010353 - 26 Dec 2023

Cited by 1 | Viewed by 1417

Abstract

SARS-CoV-2 triggered the global COVID-19 pandemic, posing a severe threat to public health worldwide. The innate immune response in cells infected by SARS-CoV-2 is primarily orchestrated by type I interferon (IFN), with IFN-β exhibiting a notable inhibitory impact on SARS-CoV-2 replication. FHL2, [...] Read more.

SARS-CoV-2 triggered the global COVID-19 pandemic, posing a severe threat to public health worldwide. The innate immune response in cells infected by SARS-CoV-2 is primarily orchestrated by type I interferon (IFN), with IFN-β exhibiting a notable inhibitory impact on SARS-CoV-2 replication. FHL2, acting as a docking site, facilitates the assembly of multiprotein complexes and regulates the transcription of diverse genes. However, the association between SARS-CoV-2 and FHL2 remains unclear. In this study, we report for the first time that SARS-CoV-2 infection in Caco2 cells results in the upregulation of FHL2 expression, while the virus’s N proteins can enhance FHL2 expression. Notably, the knockdown of FHL2 significantly amplifies SARS-CoV-2 replication in vitro. Conversely, the overexpression of FHL2 leads to a marked reduction in SARS-CoV-2 replication, with the antiviral property of FHL2 being independent of the cell or virus type. Subsequent experiments reveal that FHL2 supports IFN-β transcription by upregulating the expression and phosphorylation of IRF-3, thereby impeding SARS-CoV-2 replication in cells. These findings highlight FHL2 as a potential antiviral target for treating SARS-CoV-2 infections. Full article

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20 pages, 11037 KiB

Open AccessArticle

MiR-199a-3p Regulates the PTPRF/β-Catenin Axis in Hair Follicle Development: Insights into the Pathogenic Mechanism of Alopecia Areata

by Jiankui Wang, Yuhao Ma, Tun Li, Jinnan Li, Xue Yang, Guoying Hua, Ganxian Cai, Han Zhang, Zhexi Liu, Keliang Wu and Xuemei Deng

Int. J. Mol. Sci. 2023, 24(24), 17632; https://doi.org/10.3390/ijms242417632 - 18 Dec 2023

Cited by 2 | Viewed by 1812

Abstract

Alopecia areata is an autoimmune disease characterized by the immune system attacking self hair follicles, mainly in the scalp. There is no complete cure, and the pathogenesis is still not fully understood. Here, sequencing of skin tissues collected from 1-month-old coarse- and fine-wool [...] Read more.

Alopecia areata is an autoimmune disease characterized by the immune system attacking self hair follicles, mainly in the scalp. There is no complete cure, and the pathogenesis is still not fully understood. Here, sequencing of skin tissues collected from 1-month-old coarse- and fine-wool lambs identified miR-199a-3p as the only small RNA significantly overexpressed in the fine-wool group, suggesting a role in hair follicle development. MiR-199a-3p expression was concentrated in the dermal papillae cells of sheep hair follicles, along with enhanced β-catenin expression and the inhibition of PTPRF protein expression. We also successfully constructed a mouse model of alopecia areata by intracutaneous injection with an miR-199a-3p antagomir. Injection of the miR-199a-3p agomir resulted in hair growth and earlier anagen entry. Conversely, local injection with the miR-199a-3p antagomir resulted in suppressed hair growth at the injection site, upregulation of immune system-related genes, and downregulation of hair follicle development-related genes. In vivo and in vitro analyses demonstrated that miR-199a-3p regulates hair follicle development through the PTPRF/β-catenin axis. In conclusion, a mouse model of alopecia areata was successfully established by downregulation of a small RNA, suggesting the potential value of miR-199a-3p in the study of alopecia diseases. The regulatory role of miR-199a-3p in the PTPRF/β-catenin axis was confirmed, further demonstrating the link between alopecia areata and the Wnt-signaling pathway. Full article

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21 pages, 5591 KiB

Open AccessArticle

Mismatch Repair Protein Msh6^Tt Is Necessary for Nuclear Division and Gametogenesis in Tetrahymena thermophila

by Lin Wang, Sitong Yang, Yuhuan Xue, Tao Bo, Jing Xu and Wei Wang

Int. J. Mol. Sci. 2023, 24(24), 17619; https://doi.org/10.3390/ijms242417619 - 18 Dec 2023

Viewed by 1667

Abstract

DNA mismatch repair (MMR) improves replication accuracy by up to three orders of magnitude. The MutS protein in E. coli or its eukaryotic homolog, the MutSα (Msh2-Msh6) complex, recognizes base mismatches and initiates the mismatch repair mechanism. Msh6 is an essential protein for [...] Read more.

DNA mismatch repair (MMR) improves replication accuracy by up to three orders of magnitude. The MutS protein in E. coli or its eukaryotic homolog, the MutSα (Msh2-Msh6) complex, recognizes base mismatches and initiates the mismatch repair mechanism. Msh6 is an essential protein for assembling the heterodimeric complex. However, the function of the Msh6 subunit remains elusive. Tetrahymena undergoes multiple DNA replication and nuclear division processes, including mitosis, amitosis, and meiosis. Here, we found that Msh6^Tt localized in the macronucleus (MAC) and the micronucleus (MIC) during the vegetative growth stage and starvation. During the conjugation stage, Msh6^Tt only localized in MICs and newly developing MACs. MSH6^Tt knockout led to aberrant nuclear division during vegetative growth. The MSH6^TtKO mutants were resistant to treatment with the DNA alkylating agent methyl methanesulfonate (MMS) compared to wild type cells. MSH6^Tt knockout affected micronuclear meiosis and gametogenesis during the conjugation stage. Furthermore, Msh6^Tt interacted with Msh2^Tt and MMR-independent factors. Downregulation of MSH2^Tt expression affected the stability of Msh6^Tt. In addition, MSH6^Tt knockout led to the upregulated expression of several MSH6^Tt homologs at different developmental stages. Msh6^Tt is involved in macronuclear amitosis, micronuclear mitosis, micronuclear meiosis, and gametogenesis in Tetrahymena. Full article

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16 pages, 10406 KiB

Open AccessArticle

Extraciliary OFD1 Is Involved in Melanocyte Survival through Cell Adhesion to ECM via Paxillin

by Nan-Hyung Kim, Chang Hoon Lee and Ai-Young Lee

Int. J. Mol. Sci. 2023, 24(24), 17528; https://doi.org/10.3390/ijms242417528 - 15 Dec 2023

Cited by 1 | Viewed by 1177

Abstract

Primary cilia play a significant role in influencing cell fate, including apoptosis in multiple cell types. In the lesional epidermis of vitiligo patients, a reduced number of ciliated cells was observed. Our study also revealed a downregulation of oral–facial digital syndrome type 1 [...] Read more.

Primary cilia play a significant role in influencing cell fate, including apoptosis in multiple cell types. In the lesional epidermis of vitiligo patients, a reduced number of ciliated cells was observed. Our study also revealed a downregulation of oral–facial digital syndrome type 1 (OFD1) in the affected skin of vitiligo patients. However, it remains unknown whether primary cilia are involved in the control of melanocyte apoptosis. While both intraflagellar transport 88 (IFT88) and retinitis pigmentosa GTPase regulator-interacting protein-1 like (RPGRIP1L) are associated with ciliogenesis in melanocytes, only the knockdown of OFD1, but not IFT88 and RPGRIP1L, resulted in increased melanocyte apoptosis. OFD1 knockdown led to a decrease in the expression of proteins involved in cell–extracellular matrix (ECM) interactions, including paxillin. The OFD1 amino acid residues 601-1012 interacted with paxillin, while the amino acid residues 1-601 were associated with ciliogenesis, suggesting that the OFD1 domains responsible for paxillin binding are distinct from those involved in ciliogenesis. OFD1 knockdown, but not IFT88 knockdown, inhibited melanocyte adhesion to the ECM, a defect that was restored by paxillin overexpression. In summary, our findings indicate that the downregulation of OFD1 induces melanocyte apoptosis, independent of any impairment in ciliogenesis, by reducing melanocyte adhesion to the ECM via paxillin. Full article

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8 pages, 1202 KiB

Open AccessBrief Report

Impact of Bariatric Surgery on Subtilisin/Kexin Type 9 (PCSK9) Gene Expression and Inflammation in the Adipose Tissue of Obese Diabetic Rats

by Adrian H. Heald, Helene A. Fachim, Bilal Bashir, Bethanie Garside, Safwaan Adam, Zohaib Iqbal, Akheel A. Syed, Rachelle Donn, Carel W. Le Roux, Mahmoud Abdelaal, James White and Handrean Soran

Int. J. Mol. Sci. 2023, 24(23), 16978; https://doi.org/10.3390/ijms242316978 - 30 Nov 2023

Viewed by 1620

Abstract

Bariatric surgery improves dyslipidaemia and reduces body weight, but it remains unclear how bariatric surgery modulates gene expression in fat cells to influence the proprotein convertase subtilisin/kexin type 9 (PCSK-9) and low-density lipoprotein receptor (LDLR) gene expression. The expression of the PCSK9/LDLR/tumor necrosis [...] Read more.

Bariatric surgery improves dyslipidaemia and reduces body weight, but it remains unclear how bariatric surgery modulates gene expression in fat cells to influence the proprotein convertase subtilisin/kexin type 9 (PCSK-9) and low-density lipoprotein receptor (LDLR) gene expression. The expression of the PCSK9/LDLR/tumor necrosis factor-alpha (TNFα) gene in adipose tissue was measured in two groups of Zucker Diabetic Sprague Dawley (ZDSD) rats after Roux-en-Y gastric bypass (RYGB) surgery or ‘SHAM’ operation. There was lower PCSK9 (p = 0.02) and higher LDLR gene expression (p = 0.02) in adipose tissue in rats after RYGB. Weight change did not correlate with PCSK9 gene expression (r = −0.5, p = 0.08) or TNFα gene expression (r = −0.4, p = 0.1). TNFα gene expression was positively correlated with PCSK9 gene expression (r = 0.7, p = 0.001) but not correlated with LDLR expression (r = −0.3, p = 0.3). Circulating triglyceride levels were lower in RYGB compared to the SHAM group (1.1 (0.8–1.4) vs. 1.5 (1.0–4.2), p = 0.038) mmol/L with no difference in cholesterol levels. LDLR gene expression was increased post-bariatric surgery with the potential to reduce the number of circulating LDL particles. PCSK9 gene expression and TNFα gene expression were positively correlated after RYGB in ZDSD rats, suggesting that the modulation of pro-inflammatory pathways in adipose tissue after RYGB may partly relate to PCSK9 and LDLR gene expression. Full article

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13 pages, 2168 KiB

Open AccessArticle

Knockout of ovary serine protease Leads to Ovary Deformation and Female Sterility in the Asian Corn Borer, Ostrinia furnacalis

by Porui Zhang, Zuerdong Jialaliding, Junwen Gu, Austin Merchant, Qi Zhang and Xuguo Zhou

Int. J. Mol. Sci. 2023, 24(22), 16311; https://doi.org/10.3390/ijms242216311 - 14 Nov 2023

Cited by 4 | Viewed by 4148

Abstract

Oogenesis in insects is a carefully orchestrated process, facilitating the formation of female gametes, which is regulated by multiple extrinsic and intrinsic factors, including ovary serine protease (Osp). As a member of the serine protease family, Osp is a homolog of Nudel, [...] Read more.

Oogenesis in insects is a carefully orchestrated process, facilitating the formation of female gametes, which is regulated by multiple extrinsic and intrinsic factors, including ovary serine protease (Osp). As a member of the serine protease family, Osp is a homolog of Nudel, a maternally required protease defining embryonic dorsoventral polarity in Drosophila. In this study, we used CRISPR/Cas9-mediated mutagenesis to functionally characterize Osp in the Asian corn borer, Ostrinia furnacalis, a devastating maize pest throughout Asia and Australia. Building on previous knowledge, we hypothesized that knockout of Osp would disrupt embryonic development in O. furnacalis females. To examine this overarching hypothesis, we (1) cloned and characterized Osp from O. furnacalis, (2) designed target sites on exons 1 and 4 to construct a CRISPR/Cas9 mutagenesis system, and (3) documented phenotypic impacts among O. furnacalis Osp mutants. As a result, we (1) examined the temporal-spatial expression profiles of OfOsp, which has an open reading frame of 5648 bp in length and encodes a protein of 1873 amino acids; (2) established O. furnacalis Osp mutants; and (3) documented recessive, female-specific sterility among OfOsp^F mutants, including absent or deformed oviducts and reduced fertility in female but not male mutants. Overall, the combined results support our initial hypothesis that Osp is required for embryonic development, specifically ovarian maturation, in O. furnacalis females. Given its substantial impacts on female sterility, Osp provides a potential target for the Sterile Insect Technique (SIT) to manage Lepidoptera pests in general and the species complex Ostrinia in particular. Full article

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15 pages, 4290 KiB

Open AccessArticle

NXC736 Attenuates Radiation-Induced Lung Fibrosis via Regulating NLRP3/IL-1β Signaling Pathway

by Sang Yeon Kim, Sunjoo Park, Ronglan Cui, Hajeong Lee, Hojung Choi, Mohamed El-Agamy Farh, Hai In Jo, Jae Hee Lee, Hyo Jeong Song, Yoon-Jin Lee, Yun-Sil Lee, Bong Yong Lee and Jaeho Cho

Int. J. Mol. Sci. 2023, 24(22), 16265; https://doi.org/10.3390/ijms242216265 - 13 Nov 2023

Cited by 2 | Viewed by 2366

Abstract

Radiation-induced lung fibrosis (RILF) is a common complication of radiotherapy in lung cancer. However, to date no effective treatment has been developed for this condition. NXC736 is a novel small-molecule compound that inhibits NLRP3, but its effect on RILF is unknown. NLRP3 activation [...] Read more.

Radiation-induced lung fibrosis (RILF) is a common complication of radiotherapy in lung cancer. However, to date no effective treatment has been developed for this condition. NXC736 is a novel small-molecule compound that inhibits NLRP3, but its effect on RILF is unknown. NLRP3 activation is an important trigger for the development of RILF. Thus, we aimed to evaluate the therapeutic effect of NXC736 on lung fibrosis inhibition using a RILF animal model and to elucidate its molecular signaling pathway. The left lungs of mice were irradiated with a single dose of 75 Gy. We observed that NXC736 treatment inhibited collagen deposition and inflammatory cell infiltration in irradiated mouse lung tissues. The damaged lung volume, evaluated by magnetic resonance imaging, was lower in NXC736-treated mice than in irradiated mice. NXC736-treated mice exhibited significant changes in lung function parameters. NXC736 inhibited inflammasome activation by interfering with the NLRP3-ASC-cleaved caspase-1 interaction, thereby reducing the expression of IL-1β and blocking the fibrotic pathway. In addition, NXC736 treatment reduced the expression of epithelial–mesenchymal transition markers such as α-SMA, vimentin, and twist by blocking the Smad 2,3,4 signaling pathway. These data suggested that NXC736 is a potent therapeutic agent against RILF. Full article

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9 pages, 3132 KiB

Open AccessArticle

Study on Saccharide–Glucose Receptor Interactions with the Use of Surface Plasmon Resonance

by Maciej Trzaskowski, Marcin Drozd and Tomasz Ciach

Int. J. Mol. Sci. 2023, 24(22), 16079; https://doi.org/10.3390/ijms242216079 - 8 Nov 2023

Cited by 1 | Viewed by 1337

Abstract

The aim of this study was to investigate the process of attachment of saccharide particles differing in degree of complexity to cell receptors responsible for transport of glucose across the cell membrane (GLUT proteins). This phenomenon is currently considered when designing modern medicines, [...] Read more.

The aim of this study was to investigate the process of attachment of saccharide particles differing in degree of complexity to cell receptors responsible for transport of glucose across the cell membrane (GLUT proteins). This phenomenon is currently considered when designing modern medicines, e.g., peptide drugs to which glucose residues are attached, enabling drugs to cross the barrier of cell membranes and act inside cells. This study aims to help us understand the process of assimilation of polysaccharide nanoparticles by tumour cells. In this study, the interactions between simple saccharides (glucose and sucrose) and dextran nanoparticles with two species of GLUT proteins (GLUT1 and GLUT4) were measured using the surface plasmon resonance technique. We managed to observe the interactions of glucose and sucrose with both applied proteins. The lowest concentration that resulted in the detection of interaction was 4 mM of glucose on GLUT1. Nanoparticles were measured using the same proteins with a detection limit of 40 mM. These results indicate that polysaccharide nanoparticles interact with GLUT proteins. The measured strengths of interactions differ between proteins; thus, this study can suggest which protein is preferable when considering it as a mean of nanoparticle carrier transport. Full article

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12 pages, 897 KiB

Open AccessReview

The Role of BDNF, YBX1, CENPF, ZSCAN4, TEAD4, GLIS1 and USF1 in the Activation of the Embryonic Genome in Bovine Embryos

by Bingnan Liu, Jiaxin Yan, Junjie Li and Wei Xia

Int. J. Mol. Sci. 2023, 24(22), 16019; https://doi.org/10.3390/ijms242216019 - 7 Nov 2023

Viewed by 2110

Abstract

Early embryonic development relies on the maternal RNAs and newly synthesized proteins during oogenesis. Zygotic transcription is an important event occurring at a specific time after fertilization. If no zygotic transcription occurs, the embryo will die because it is unable to meet the [...] Read more.

Early embryonic development relies on the maternal RNAs and newly synthesized proteins during oogenesis. Zygotic transcription is an important event occurring at a specific time after fertilization. If no zygotic transcription occurs, the embryo will die because it is unable to meet the needs of the embryo and continue to grow. During the early stages of embryonic development, the correct transcription, translation, and expression of genes play a crucial role in blastocyst formation and differentiation of cell lineage species formation among mammalian species, and any variation may lead to developmental defects, arrest, or even death. Abnormal expression of some genes may lead to failure of the embryonic zygote genome before activation, such as BDNF and YBX1; Decreased expression of CENPF, ZSCAN4, TEAD4, GLIS1, and USF1 genes can lead to embryonic development failure. This article reviews the results of studies on the timing and mechanism of gene expression of these genes in bovine fertilized eggs/embryos. Full article

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16 pages, 2315 KiB

Open AccessArticle

Compromised Muscle Properties in a Severe Hypophosphatasia Murine Model

by Emily G. Pendleton, Anna S. Nichenko, Jennifer Mcfaline-Figueroa, Christiana J. Raymond-Pope, Albino G. Schifino, Taylor M. Pigg, Ruth P. Barrow, Sarah M. Greising, Jarrod A. Call and Luke J. Mortensen

Int. J. Mol. Sci. 2023, 24(21), 15905; https://doi.org/10.3390/ijms242115905 - 2 Nov 2023

Cited by 1 | Viewed by 2146

Abstract

Hypophosphatasia (HPP) is a rare metabolic bone disorder characterized by low levels of tissue non-specific alkaline phosphatase (TNAP) that causes under-mineralization of the bone, leading to bone deformity and fractures. In addition, patients often present with chronic muscle pain, reduced muscle strength, and [...] Read more.

Hypophosphatasia (HPP) is a rare metabolic bone disorder characterized by low levels of tissue non-specific alkaline phosphatase (TNAP) that causes under-mineralization of the bone, leading to bone deformity and fractures. In addition, patients often present with chronic muscle pain, reduced muscle strength, and an altered gait. In this work, we explored dynamic muscle function in a homozygous TNAP knockout mouse model of severe juvenile onset HPP. We found a reduction in skeletal muscle size and impairment in a range of isolated muscle contractile properties. Using histological methods, we found that the structure of HPP muscles was similar to healthy muscles in fiber size, actin and myosin structures, as well as the α-tubulin and mitochondria networks. However, HPP mice had significantly fewer embryonic and type I fibers than wild type mice, and fewer metabolically active NADH+ muscle fibers. We then used oxygen respirometry to evaluate mitochondrial function and found that complex I and complex II leak respiration were reduced in HPP mice, but that there was no disruption in efficiency of electron transport in complex I or complex II. In summary, the severe HPP mouse model recapitulates the muscle strength impairment phenotypes observed in human patients. Further exploration of the role of alkaline phosphatase in skeletal muscle could provide insight into mechanisms of muscle weakness in HPP. Full article

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24 pages, 4571 KiB

Open AccessArticle

Vacuolar Sugar Transporter TMT2 Plays Crucial Roles in Germination and Seedling Development in Arabidopsis

by Yanting Cao, Jinju Hu, Jinrong Hou, Chenguang Fu, Xingyue Zou, Xuxia Han, Pulian Jia, Chenjie Sun, Yan Xu, Yuhan Xue, Yiming Zou, Xinyue Liu, Xueying Chen, Guoyang Li, Jianing Guo, Min Xu and Aigen Fu

Int. J. Mol. Sci. 2023, 24(21), 15852; https://doi.org/10.3390/ijms242115852 - 1 Nov 2023

Viewed by 1868

Abstract

Vacuolar sugar transporters transport sugar across the tonoplast, are major players in maintaining sugar homeostasis, and therefore play vital roles in plant growth, development, and biomass yield. In this study, we analyzed the physiological roles of the tonoplast monosaccharide transporter 2 (TMT2) in [...] Read more.

Vacuolar sugar transporters transport sugar across the tonoplast, are major players in maintaining sugar homeostasis, and therefore play vital roles in plant growth, development, and biomass yield. In this study, we analyzed the physiological roles of the tonoplast monosaccharide transporter 2 (TMT2) in Arabidopsis. In contrast to the wild type (WT) that produced uniform seedlings, the tmt2 mutant produced three types of offspring: un-germinated seeds (UnG), seedlings that cannot form true leaves (tmt2-S), and seedlings that develop normally (tmt2-L). Sucrose, glucose, and fructose can substantially, but not completely, rescue the abnormal phenotypes of the tmt2 mutant. Abnormal cotyledon development, arrested true leaf development, and abnormal development of shoot apical meristem (SAM) were observed in tmt2-S seedlings. Cotyledons from the WT and tmt2-L seedlings restored the growth of tmt2-S seedlings through micrografting. Moreover, exogenous sugar sustained normal growth of tmt2-S seedlings with cotyledon removed. Finally, we found that the TMT2 deficiency resulted in growth defects, most likely via changing auxin signaling, target of rapamycin (TOR) pathways, and cellular nutrients. This study unveiled the essential functions of TMT2 for seed germination and initial seedling development, ensuring cotyledon function and mobilizing sugars from cotyledons to seedlings. It also expanded the current knowledge on sugar metabolism and signaling. These findings have fundamental implications for enhancing plant biomass production or seed yield in future agriculture. Full article

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17 pages, 4463 KiB

Open AccessArticle

Transcriptome Analysis Reveals the Potential Molecular Mechanisms of Tiller Bud Development in Orchardgrass

by Xiaoheng Xu, Guangyan Feng, Zhongfu Yang, Qiuxu Liu, Gang Nie, Dandan Li, Ting Huang, Linkai Huang and Xinquan Zhang

Int. J. Mol. Sci. 2023, 24(21), 15762; https://doi.org/10.3390/ijms242115762 - 30 Oct 2023

Cited by 2 | Viewed by 1346

Abstract

Tillering is a special type of branching and one of the important contributors to the yield of cereal crops. Strigolactone and sucrose play a vital role in controlling tiller formation, but their mechanism has not been elucidated completely in most crops. Orchardgrass ( [...] Read more.

Tillering is a special type of branching and one of the important contributors to the yield of cereal crops. Strigolactone and sucrose play a vital role in controlling tiller formation, but their mechanism has not been elucidated completely in most crops. Orchardgrass (Dactylis glomerata L.) is an important perennial forage with prominent tillering ability among crops. To date, the mechanism of tillering in orchardgrass is still largely unknown. Therefore, we performed a transcriptome and miRNA analysis to reveal the potential RNA mechanism of tiller formation under strigolactone and sucrose treatment in orchardgrass. Our results found that D3, COL5, NCED1, HXK7, miRNA4393-z, and miRNA531-z could be key factors to control tiller bud development in orchardgrass. In addition, strigolactones might affect the ABA biosynthesis pathway to regulate the tiller bud development of orchardgrass, which may be related to the expression changes in miRNA4393-z, NCED1, and D10. miRNA531-z could be involved in the interaction of strigolactones and sucrose in regulating tillering. These results will be further used to clarify the potential mechanism of tillering for breeding new high-tillering and high-production orchardgrass varieties and beneficial to improving the production and reproduction of crops. Full article

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16 pages, 4940 KiB

Open AccessArticle

The Functionality of IbpA from Acholeplasma laidlawii Is Governed by Dynamic Rearrangement of Its Globular–Fibrillar Quaternary Structure

by Liliya S. Chernova, Innokentii E. Vishnyakov, Janek Börner, Mikhail I. Bogachev, Kai M. Thormann and Airat R. Kayumov

Int. J. Mol. Sci. 2023, 24(20), 15445; https://doi.org/10.3390/ijms242015445 - 22 Oct 2023

Viewed by 1607

Abstract

Small heat shock proteins (sHSPs) represent a first line of stress defense in many bacteria. The primary function of these molecular chaperones involves preventing irreversible protein denaturation and aggregation. In Escherichia coli, fibrillar EcIbpA binds unfolded proteins and keeps them in [...] Read more.

Small heat shock proteins (sHSPs) represent a first line of stress defense in many bacteria. The primary function of these molecular chaperones involves preventing irreversible protein denaturation and aggregation. In Escherichia coli, fibrillar EcIbpA binds unfolded proteins and keeps them in a folding-competent state. Further, its structural homologue EcIbpB induces the transition of EcIbpA to globules, thereby facilitating the substrate transfer to the HSP70-HSP100 system for refolding. The phytopathogenic Acholeplasma laidlawii possesses only a single sHSP, AlIbpA. Here, we demonstrate non-trivial features of the function and regulation of the chaperone-like activity of AlIbpA according to its interaction with other components of the mycoplasma multi-chaperone network. Our results show that the efficiency of the A. laidlawii multi-chaperone system is driven with the ability of AlIbpA to form both globular and fibrillar structures, thus combining functions of both IbpA and IbpB when transferring the substrate proteins to the HSP70-HSP100 system. In contrast to EcIbpA and EcIbpB, AlIbpA appears as an sHSP, in which the competition between the N- and C-terminal domains regulates the shift of the protein quaternary structure between a fibrillar and globular form, thus representing a molecular mechanism of its functional regulation. While the C-terminus of AlIbpA is responsible for fibrils formation and substrate capture, the N-terminus seems to have a similar function to EcIbpB through facilitating further substrate protein disaggregation using HSP70. Moreover, our results indicate that prior to the final disaggregation process, AlIbpA can directly transfer the substrate to HSP100, thereby representing an alternative mechanism in the HSP interaction network. Full article

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21 pages, 5233 KiB

Open AccessArticle

Diverse Regulatory Manners and Potential Roles of lncRNAs in the Developmental Process of Asian Honey Bee (Apis cerana) Larval Guts

by Xiaoxue Fan, Xuze Gao, He Zang, Sijia Guo, Xin Jing, Yiqiong Zhang, Xiaoyu Liu, Peiyuan Zou, Mengjun Chen, Zhijian Huang, Dafu Chen and Rui Guo

Int. J. Mol. Sci. 2023, 24(20), 15399; https://doi.org/10.3390/ijms242015399 - 20 Oct 2023

Cited by 4 | Viewed by 1861

Abstract

Long non-coding RNAs (lncRNAs) are crucial modulators in a variety of biological processes, such as gene expression, development, and immune defense. However, little is known about the function of lncRNAs in the development of Asian honey bee (Apis cerana) larval guts. [...] Read more.

Long non-coding RNAs (lncRNAs) are crucial modulators in a variety of biological processes, such as gene expression, development, and immune defense. However, little is known about the function of lncRNAs in the development of Asian honey bee (Apis cerana) larval guts. Here, on the basis of our previously obtained deep-sequencing data from the 4-, 5-, and 6-day-old larval guts of A. cerana workers (Ac4, Ac5, and Ac6 groups), an in-depth transcriptome-wide investigation was conducted to decipher the expression pattern, regulatory manners, and potential roles of lncRNAs during the developmental process of A. cerana worker larval guts, followed by the verification of the relative expression of differentially expressed lncRNAs (DElncRNAs) and the targeting relationships within a competing endogenous RNA (ceRNA) axis. In the Ac4 vs. Ac5 and Ac5 vs. Ac6 comparison groups, 527 and 498 DElncRNAs were identified, respectively, which is suggestive of the dynamic expression of lncRNAs during the developmental process of larval guts. A cis-acting analysis showed that 330 and 393 neighboring genes of the aforementioned DElncRNAs were respectively involved in 29 and 32 functional terms, such as cellular processes and metabolic processes; these neighboring genes were also respectively engaged in 246 and 246 pathways such as the Hedgehog signaling pathway and the Wnt signaling pathway. Additionally, it was found that 79 and 76 DElncRNAs as potential antisense lncRNAs may, respectively, interact with 72 and 60 sense-strand mRNAs. An investigation of competing endogenous RNA (ceRNA) networks suggested that 75 (155) DElncRNAs in the Ac4 vs. Ac5 (Ac5 vs. Ac6) comparison group could target 7 (5) DEmiRNAs and further bind to 334 (248) DEmRNAs, which can be annotated to 33 (29) functional terms and 186 (210) pathways, including 12 (16) cellular- and humoral-immune pathways (lysosome pathway, necroptosis, MAPK signaling pathway, etc.) and 11 (10) development-associated signaling pathways (Wnt, Hippo, AMPK, etc.). The RT-qPCR detection of five randomly selected DElncRNAs confirmed the reliability of the used sequencing data. Moreover, the results of a dual-luciferase reporter assay were indicative of the binding relationship between MSTRG.11294.1 and miR-6001-y and between miR-6001-y and ncbi_107992440. These results demonstrate that DElncRNAs are likely to modulate the developmental process of larval guts via the regulation of the source genes’ transcription, interaction with mRNAs, and ceRNA networks. Our findings not only yield new insights into the developmental mechanism underlying A. cerana larval guts, but also provide a candidate ceRNA axis for further functional dissection. Full article

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21 pages, 570 KiB

Open AccessArticle

Improved piggyBac Transformation with Capped Transposase mRNA in Pest Insects

by Irina Häcker, Tanja Rehling, Henrik Schlosser, Daniela Mayorga-Ch, Mara Heilig, Ying Yan, Peter A. Armbruster and Marc F. Schetelig

Int. J. Mol. Sci. 2023, 24(20), 15155; https://doi.org/10.3390/ijms242015155 - 13 Oct 2023

Cited by 3 | Viewed by 1923

Abstract

Creating transgenic insects is a key technology in insect genetics and molecular biology. A widely used instrument in insect transgenesis is the piggyBac transposase, resulting in essentially random genomic integrations. In contrast, site-specific recombinases allow the targeted integration of the transgene construct into [...] Read more.

Creating transgenic insects is a key technology in insect genetics and molecular biology. A widely used instrument in insect transgenesis is the piggyBac transposase, resulting in essentially random genomic integrations. In contrast, site-specific recombinases allow the targeted integration of the transgene construct into a specific genomic target site. Both strategies, however, often face limitations due to low transgenesis efficiencies. We aimed to enhance transgenesis efficiencies by utilizing capped mRNA as a source of transposase or recombinase instead of a helper plasmid. A systematic comparison of transgenesis efficiencies in Aedes mosquitoes, as models for hard-to-transform insects, showed that suppling piggyBac transposase as mRNA increased the average transformation efficiency in Aedes aegypti from less than 5% with the plasmid source to about 50% with mRNA. Similar high activity was observed in Ae. albopictus with pBac mRNA. No efficiency differences between plasmid and mRNA were observed in recombination experiments. Furthermore, a hyperactive version of piggyBac transposase delivered as a plasmid did not improve the transformation efficiency in Ae. aegypti or the agricultural pest Drosophila suzukii. We believe that the use of mRNA has strong potential for enhancing piggyBac transformation efficiencies in other mosquitoes and important agricultural pests, such as tephritids. Full article

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14 pages, 2879 KiB

Open AccessArticle

SINEs as Potential Expression Cassettes: Impact of Deletions and Insertions on Polyadenylation and Lifetime of B2 and Ves SINE Transcripts Generated by RNA Polymerase III

by Olga R. Borodulina, Ilia G. Ustyantsev and Dmitri A. Kramerov

Int. J. Mol. Sci. 2023, 24(19), 14600; https://doi.org/10.3390/ijms241914600 - 27 Sep 2023

Cited by 3 | Viewed by 1254

Abstract

Short Interspersed Elements (SINEs) are common in the genomes of most multicellular organisms. They are transcribed by RNA polymerase III from an internal promoter comprising boxes A and B. As transcripts of certain SINEs from mammalian genomes can be polyadenylated, such transcripts should [...] Read more.

Short Interspersed Elements (SINEs) are common in the genomes of most multicellular organisms. They are transcribed by RNA polymerase III from an internal promoter comprising boxes A and B. As transcripts of certain SINEs from mammalian genomes can be polyadenylated, such transcripts should contain the AATAAA sequence as well as those called β- and τ-signals. One of the goals of this work was to evaluate how autonomous and independent other SINE parts are β- and τ-signals. Extended regions outside of β- and τ-signals were deleted from SINEs B2 and Ves and the derived constructs were used to transfect HeLa cells in order to evaluate the relative levels of their transcripts as well as their polyadenylation efficiency. If the deleted regions affected boxes A and B, the 5′-flanking region of the U6 RNA gene with the external promoter was inserted upstream. Such substitution of the internal promoter in B2 completely restored its transcription. Almost all tested deletions/substitutions did not reduce the polyadenylation capacity of the transcripts, indicating a weak dependence of the function of β- and τ-signals on the neighboring sequences. A similar analysis of B2 and Ves constructs containing a 55-bp foreign sequence inserted between β- and τ-signals showed an equal polyadenylation efficiency of their transcripts compared to those of constructs without the insertion. The acquired poly(A)-tails significantly increased the lifetime and thus the cellular level of such transcripts. The data obtained highlight the potential of B2 and Ves SINEs as cassettes for the expression of relatively short sequences for various applications. Full article

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18 pages, 2669 KiB

Open AccessArticle

Gene Expression Profiling Reveals Potential Players of Sex Determination and Asymmetrical Development in Chicken Embryo Gonads

by Huaixi Luo, Hao Zhou, Shengyao Jiang, Chuan He, Ke Xu, Jinmei Ding, Jiajia Liu, Chao Qin, Kangchun Chen, Wenchuan Zhou, Liyuan Wang, Wenhao Yang, Wenqi Zhu and He Meng

Int. J. Mol. Sci. 2023, 24(19), 14597; https://doi.org/10.3390/ijms241914597 - 27 Sep 2023

Cited by 3 | Viewed by 1811

Abstract

Despite the notable progress made in recent years, the understanding of the genetic control of gonadal sex differentiation and asymmetrical ovariogenesis in chicken during embryonic development remains incomplete. This study aimed to identify potential key genes and speculate about the mechanisms associated with [...] Read more.

Despite the notable progress made in recent years, the understanding of the genetic control of gonadal sex differentiation and asymmetrical ovariogenesis in chicken during embryonic development remains incomplete. This study aimed to identify potential key genes and speculate about the mechanisms associated with ovary and testis development via an analysis of the results of PacBio and Illumina transcriptome sequencing of embryonic chicken gonads at the initiation of sexual differentiation (E4.5, E5.5, and E6.5). PacBio sequencing detected 328 and 233 significantly up-regulated transcript isoforms in females and males at E4.5, respectively. Illumina sequencing detected 95, 296 and 445 DEGs at E4.5, E5.5, and E6.5, respectively. Moreover, both sexes showed asymmetrical expression in gonads, and more DEGs were detected on the left side. There were 12 DEGs involved in cell proliferation shared between males and females in the left gonads. GO analysis suggested that coagulation pathways may be involved in the degradation of the right gonad in females and that blood oxygen transport pathways may be involved in preventing the degradation of the right gonad in males. These results provide a comprehensive expression profile of chicken embryo gonads at the initiation of sexual differentiation, which can serve as a theoretical basis for further understanding the mechanism of bird sex determination and its evolutionary process. Full article

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19 pages, 5158 KiB

Open AccessArticle

High Prolactin Concentration Induces Ovarian Granulosa Cell Oxidative Stress, Leading to Apoptosis Mediated by L-PRLR and S-PRLR

by Ruochen Yang, Chunhui Duan, Shuo Zhang, Yunxia Guo, Xinyu Shan, Meijing Chen, Sicong Yue, Yingjie Zhang and Yueqin Liu

Int. J. Mol. Sci. 2023, 24(19), 14407; https://doi.org/10.3390/ijms241914407 - 22 Sep 2023

Cited by 4 | Viewed by 1696

Abstract

High prolactin (PRL) concentration has been shown to induce the apoptosis of ovine ovarian granulosa cells (GCs), but the underlying mechanisms are unclear. This study aimed to investigate the mechanism of apoptosis induced by high PRL concentration in GCs. Trial 1: The optimal [...] Read more.

High prolactin (PRL) concentration has been shown to induce the apoptosis of ovine ovarian granulosa cells (GCs), but the underlying mechanisms are unclear. This study aimed to investigate the mechanism of apoptosis induced by high PRL concentration in GCs. Trial 1: The optimal concentration of glutathion was determined according to the detected cell proliferation. The results showed that the optimal glutathione concentration was 5 μmol/mL. Trial 2: 500 ng/mL PRL was chosen as the high PRL concentration. The GCs were treated with 0 ng/mL PRL (C group), 500 ng/mL PRL (P group) or 500 ng/mL PRL, and 5 μmol/mL glutathione (P-GSH group). The results indicated that the mitochondrial respiratory chain complex (MRCC) I–V, ATP production, total antioxidant capacity (T-AOC), superoxide dismutase (SOD), and thioredoxin peroxidase (TPx) in the C group were higher than those in the P group (p < 0.05), while they were lower than those in the P-GSH group (p < 0.05). Compared to the C group, the P group exhibited elevated levels of reactive oxygen species (ROS) and apoptosis (p < 0.05) and increased expression of ATG7 and ATG5 (p < 0.05). However, MRCC I–V, ATP, SOD, A-TOC, TPx, ROS, and apoptosis were decreased after the addition of glutathione (p < 0.05). The knockdown of either L-PRLR or S-PRLR in P group GCs resulted in a significant reduction (p < 0.05) in MRCC I–V, ATP, T-AOC, SOD and TPx, while the overexpression of either receptor showed an opposite trend (p < 0.05). Our findings suggest that high PRL concentrations induce apoptotic cell death in ovine ovarian GCs by downregulating L-PRLR and S-PRLR, activating oxidative stress and autophagic pathways. Full article

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14 pages, 2155 KiB

Open AccessArticle

Small Organic Compounds Mimicking the Effector Domain of Myristoylated Alanine-Rich C-Kinase Substrate Stimulate Female-Specific Neurite Outgrowth

by Monica Tschang, Suneel Kumar, Wise Young, Melitta Schachner and Thomas Theis

Int. J. Mol. Sci. 2023, 24(18), 14271; https://doi.org/10.3390/ijms241814271 - 19 Sep 2023

Viewed by 1417

Abstract

Myristoylated alanine-rich C-kinase substrate (MARCKS) is a critical member of a signaling cascade that influences disease-relevant neural functions such as neural growth and plasticity. The effector domain (ED) of MARCKS interacts with the extracellular glycan polysialic acid (PSA) through the cell membrane to [...] Read more.

Myristoylated alanine-rich C-kinase substrate (MARCKS) is a critical member of a signaling cascade that influences disease-relevant neural functions such as neural growth and plasticity. The effector domain (ED) of MARCKS interacts with the extracellular glycan polysialic acid (PSA) through the cell membrane to stimulate neurite outgrowth in cell culture. We have shown that a synthetic ED peptide improves functional recovery after spinal cord injury in female but not male mice. However, peptides themselves are unstable in therapeutic applications, so we investigated more pharmacologically relevant small organic compounds that mimic the ED peptide to maximize therapeutic potential. Using competition ELISAs, we screened small organic compound libraries to identify molecules that structurally and functionally mimic the ED peptide of MARCKS. Since we had shown sex-specific effects of MARCKS on spinal cord injury recovery, we assayed neuronal viability as well as neurite outgrowth from cultured cerebellar granule cells of female and male mice separately. We found that epigallocatechin, amiodarone, sertraline, tegaserod, and nonyloxytryptamine bind to a monoclonal antibody against the ED peptide, and compounds stimulate neurite outgrowth in cultured cerebellar granule cells of female mice only. Therefore, a search for compounds that act in males appears warranted. Full article

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21 pages, 9134 KiB

Open AccessArticle

Uremic Toxin-Induced Exosome-like Extracellular Vesicles Contain Enhanced Levels of Sulfated Glycosaminoglycans which Facilitate the Interaction with Very Small Superparamagnetic Iron Oxide Particles

by Christian Freise, Andreas Zappe, Norbert Löwa, Jörg Schnorr, Kevin Pagel, Frank Wiekhorst and Matthias Taupitz

Int. J. Mol. Sci. 2023, 24(18), 14253; https://doi.org/10.3390/ijms241814253 - 18 Sep 2023

Cited by 2 | Viewed by 1677

Abstract

Uremic toxins exert pathophysiological effects on cells and tissues, such as the generation of a pro-calcifying subtype of exosome-like extracellular vesicles (EVs) in vascular cells. Little is known about the effects of the toxins on the surface structure of EVs. Thus, we studied [...] Read more.

Uremic toxins exert pathophysiological effects on cells and tissues, such as the generation of a pro-calcifying subtype of exosome-like extracellular vesicles (EVs) in vascular cells. Little is known about the effects of the toxins on the surface structure of EVs. Thus, we studied the effects of uremic toxins on the abundance of sulfated glycosaminoglycans (GAGs) in EVs, and the implications for binding of ligands such as very small superparamagnetic iron oxide particles (VSOPs) which could be of relevance for radiological EV-imaging. Vascular cells were treated with the uremic toxins NaH₂PO₄ and a mixture of urea and indoxyl sulfate. Uremia in rats was induced by adenine feeding. EVs were isolated from culture supernatants and plasma of rats. By proton T1-relaxometry, magnetic particle spectroscopy, and analysis of genes, proteins, and GAG-contents, we analyzed the roles of GAGs in the ligand binding of EVs. By influencing GAG-associated genes in host cells, uremic toxins induced higher GAG contents in EVs, particularly of sulfated chondroitin sulfate and heparan sulfate chains. EVs with high GAG content interacted stronger with VSOPs compared to control ones. This was confirmed by experiments with GAG-depleted EVs from genetically modified CHO cells and with uremic rat-derived EVs. Mechanistically, uremic toxin-induced PI3K/AKT-signaling and expression of the sulfate transporter SLC26A2 in host cells contributed to high GAG contents in EVs. In conclusion, uremic conditions induce enhanced GAG contents in EVs, which entails a stronger interaction with VSOPs. VSOPs might be suitable for radiological imaging of EVs rich in GAGs. Full article

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16 pages, 2960 KiB

Open AccessArticle

Oncogene-Induced Senescence Is a Crucial Antitumor Defense Mechanism of Human Endometrial Stromal Cells

by Artem L. Toropov, Pavel I. Deryabin, Alla N. Shatrova and Aleksandra V. Borodkina

Int. J. Mol. Sci. 2023, 24(18), 14089; https://doi.org/10.3390/ijms241814089 - 14 Sep 2023

Cited by 4 | Viewed by 2012

Abstract

Being the major cellular component of highly dynamic tissue, endometrial stromal cells (EnSCs) are exposed to cycles of proliferation upon hormonal stimulation, which might pose risks for the accumulation of mutations and malignization. However, endometrial stromal tumors are rare and uncommon. The present [...] Read more.

Being the major cellular component of highly dynamic tissue, endometrial stromal cells (EnSCs) are exposed to cycles of proliferation upon hormonal stimulation, which might pose risks for the accumulation of mutations and malignization. However, endometrial stromal tumors are rare and uncommon. The present study uncovered defense mechanisms that might underlie the resistance of EnSCs against oncogenic transformation. All experiments were performed in vitro using the following methods: FACS, WB, RT-PCR, IF, molecular cloning, lentiviral transduction, and CRISPR/Cas9 genome editing. We revealed that the expression of the mutant HRAS^G12V leads to EnSC senescence. We experimentally confirmed the inability of HRAS^G12V-expressing EnSCs to bypass senescence and resume proliferation, even upon estrogen stimulation. At the molecular level, the induction of oncogene-induced senescence (OIS) was accompanied by activation of the MEK/ERK, PI3K/AKT, p53/p21^WAF/CIP/Rb, and p38/p16^INK4a/Rb pathways; however, inhibiting either pathway did not prevent cell cycle arrest. PTEN loss was established as an additional feature of HRAS^G12V-induced senescence in EnSCs. Using CRISPR-Cas9-mediated PTEN knockout, we identified PTEN loss-induced senescence as a reserve molecular mechanism to prevent the transformation of HRAS^G12V-expressing EnSCs. The present study highlights oncogene-induced senescence as an antitumor defense mechanism of EnSCs controlled by multiple backup molecular pathways. Full article

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16 pages, 2458 KiB

Open AccessArticle

Glioma Stem Cells Are Sensitized to BCL-2 Family Inhibition by Compromising Histone Deacetylases

by Aran Merati, Spandana Kotian, Alexus Acton, William Placzek, Erin Smithberger, Abigail K. Shelton, C. Ryan Miller and Josh L. Stern

Int. J. Mol. Sci. 2023, 24(18), 13688; https://doi.org/10.3390/ijms241813688 - 5 Sep 2023

Cited by 1 | Viewed by 1847

Abstract

Glioblastoma (GBM) remains an incurable disease with an extremely high five-year recurrence rate. We studied apoptosis in glioma stem cells (GSCs) in response to HDAC inhibition (HDACi) combined with MEK1/2 inhibition (MEKi) or BCL-2 family inhibitors. MEKi effectively combined with HDACi to suppress [...] Read more.

Glioblastoma (GBM) remains an incurable disease with an extremely high five-year recurrence rate. We studied apoptosis in glioma stem cells (GSCs) in response to HDAC inhibition (HDACi) combined with MEK1/2 inhibition (MEKi) or BCL-2 family inhibitors. MEKi effectively combined with HDACi to suppress growth, induce cell cycle defects, and apoptosis, as well as to rescue the expression of the pro-apoptotic BH3-only proteins BIM and BMF. A RNAseq analysis of GSCs revealed that HDACi repressed the pro-survival BCL-2 family genes MCL1 and BCL-XL. We therefore replaced MEKi with BCL-2 family inhibitors and observed enhanced apoptosis. Conversely, a ligand for the cancer stem cell receptor CD44 led to reductions in BMF, BIM, and apoptosis. Our data strongly support further testing of HDACi in combination with MEKi or BCL-2 family inhibitors in glioma. Full article

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15 pages, 2928 KiB

Open AccessArticle

LH/hCG Regulation of Circular RNA in Mural Granulosa Cells during the Periovulatory Period in Mice

by V. Praveen Chakravarthi, Wei-Ting Hung, Nanda Kumar Yellapu, Sumedha Gunewardena and Lane K. Christenson

Int. J. Mol. Sci. 2023, 24(17), 13078; https://doi.org/10.3390/ijms241713078 - 23 Aug 2023

Viewed by 1544

Abstract

Ovarian follicles undergo a series of dynamic changes following the ovulatory surge of luteinizing hormone including cumulus expansion, oocyte maturation, ovulation, and luteinization. Post-transcriptional gene regulatory events are critical for mediating LH follicular responses, and among all RNA isoforms, circular RNA (circRNA) is [...] Read more.

Ovarian follicles undergo a series of dynamic changes following the ovulatory surge of luteinizing hormone including cumulus expansion, oocyte maturation, ovulation, and luteinization. Post-transcriptional gene regulatory events are critical for mediating LH follicular responses, and among all RNA isoforms, circular RNA (circRNA) is one of the most abundant forms present in cells, yet they remain the least studied. Functionally, circRNA can act as miRNA sponges, protein sponges/decoys, and regulators of transcription and translation. In the context of ovarian follicular development, the identity and roles of circRNA are relatively unknown. In the present study, high throughput RNA sequencing of granulosa cells immediately prior to and 4-h after the LH/hCG surge identified 42,381 circRNA originating from 7712 genes. A total of 54 circRNA were identified as differentially expressed between 0-h and 4-h time points (Fold Change ± 1.5, FDR ≤ 0.1), among them 42 circRNA were upregulated and 12 circRNA were downregulated. All differentially expressed circRNA between the 0-h and 4-h groups were subjected to circinteractome analysis and identified networks of circRNA-protein and circRNA-miRNA were further subjected to “micro-RNA target filter analysis” in Ingenuity Pathway Analyses, which resulted in the identification of miRNA targeted mRNAs. A comparison of these circRNA target mRNAs with LH-induced mRNAs identified Runx2, Egfr, Areg, Sult1el, Cyp19a1, Cyp11a1, and Hsd17b1 as targets of circKif2, circVcan, circMast4, and circMIIt10. These newly identified LH/hCG-induced circRNA, their target miRNA and protein networks provide new insights into the complex interactions associated with periovulatory follicular development. Full article

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28 pages, 14704 KiB

Open AccessReview

Targeting SARS-CoV-2 Non-Structural Proteins

by Donald Tam, Ana C. Lorenzo-Leal, Luis Ricardo Hernández and Horacio Bach

Int. J. Mol. Sci. 2023, 24(16), 13002; https://doi.org/10.3390/ijms241613002 - 20 Aug 2023

Cited by 15 | Viewed by 3207

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an enveloped respiratory β coronavirus that causes coronavirus disease (COVID-19), leading to a deadly pandemic that has claimed millions of lives worldwide. Like other coronaviruses, the SARS-CoV-2 genome also codes for non-structural proteins (NSPs). These [...] Read more.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an enveloped respiratory β coronavirus that causes coronavirus disease (COVID-19), leading to a deadly pandemic that has claimed millions of lives worldwide. Like other coronaviruses, the SARS-CoV-2 genome also codes for non-structural proteins (NSPs). These NSPs are found within open reading frame 1a (ORF1a) and open reading frame 1ab (ORF1ab) of the SARS-CoV-2 genome and encode NSP1 to NSP11 and NSP12 to NSP16, respectively. This study aimed to collect the available literature regarding NSP inhibitors. In addition, we searched the natural product database looking for similar structures. The results showed that similar structures could be tested as potential inhibitors of the NSPs. Full article

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11 pages, 1604 KiB

Open AccessArticle

Effects of Perfluorooctane Sulfonate on Cerebellar Cells via Inhibition of Type 2 Iodothyronine Deiodinase Activity

by Yuki Fujiwara, Yuhei Miyasaka, Ayane Ninomiya, Wataru Miyazaki, Toshiharu Iwasaki, Winda Ariyani, Izuki Amano and Noriyuki Koibuchi

Int. J. Mol. Sci. 2023, 24(16), 12765; https://doi.org/10.3390/ijms241612765 - 14 Aug 2023

Cited by 2 | Viewed by 1744

Abstract

Perfluorooctane sulfonate (PFOS) has been used in a wide variety of industrial and commercial products. The adverse effects of PFOS on the developing brain are becoming of a great concern. However, the molecular mechanisms of PFOS on brain development have not yet been [...] Read more.

Perfluorooctane sulfonate (PFOS) has been used in a wide variety of industrial and commercial products. The adverse effects of PFOS on the developing brain are becoming of a great concern. However, the molecular mechanisms of PFOS on brain development have not yet been clarified. We investigated the effect of early-life exposure to PFOS on brain development and the mechanism involved. We investigated the change in thyroid hormone (TH)-induced dendrite arborization of Purkinje cells in the primary culture of newborn rat cerebellum. We further examined the mechanism of PFOS on TH signaling by reporter gene assay, quantitative RT-PCR, and type 2 iodothyronine deiodinase (D2) assay. As low as 10⁻⁷ M PFOS suppressed thyroxine (T₄)-, but not triiodothyronine (T₃)-induced dendrite arborization of Purkinje cells. Reporter gene assay showed that PFOS did not affect TRα1- and TRβ1-mediated transcription in CV-1 cells. RT-PCR showed that PFOS suppressed D2 mRNA expression in the absence of T₄ in primary cerebellar cells. D2 activity was also suppressed by PFOS in C6 glioma-derived cells. These results indicate that early-life exposure of PFOS disrupts TH-mediated cerebellar development possibly through the disruption of D2 activity and/or mRNA expression, which may cause cerebellar dysfunction. Full article

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19 pages, 2000 KiB

Open AccessReview

Mechanisms of NURR1 Regulation: Consequences for Its Biological Activity and Involvement in Pathology

by Ángel Juan García-Yagüe and Antonio Cuadrado

Int. J. Mol. Sci. 2023, 24(15), 12280; https://doi.org/10.3390/ijms241512280 - 31 Jul 2023

Cited by 5 | Viewed by 2614

Abstract

NURR1 (Nuclear receptor-related 1 protein or NR4A2) is a nuclear protein receptor transcription factor with an essential role in the development, regulation, and maintenance of dopaminergic neurons and mediates the response to stressful stimuli during the perinatal period in mammalian brain development. The [...] Read more.

NURR1 (Nuclear receptor-related 1 protein or NR4A2) is a nuclear protein receptor transcription factor with an essential role in the development, regulation, and maintenance of dopaminergic neurons and mediates the response to stressful stimuli during the perinatal period in mammalian brain development. The dysregulation of NURR1 activity may play a role in various diseases, including the onset and progression of neurodegenerative diseases, and several other pathologies. NURR1 is regulated by multiple mechanisms, among which phosphorylation by kinases or SUMOylation are the best characterized. Both post-translational modifications can regulate the activity of NURR1, affecting its stability and transcriptional activity. Other non-post-translational regulatory mechanisms include changes in its subcellular distribution or interaction with other protein partners by heterodimerization, also affecting its transcription activity. Here, we summarize the currently known regulatory mechanisms of NURR1 and provide a brief overview of its participation in pathological alterations. Full article

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17 pages, 575 KiB

Open AccessReview

Deregulation of All-Trans Retinoic Acid Signaling and Development in Cancer

by Geoffrey Brown

Int. J. Mol. Sci. 2023, 24(15), 12089; https://doi.org/10.3390/ijms241512089 - 28 Jul 2023

Cited by 12 | Viewed by 2933

Abstract

Cancer stem cells are the root cause of cancer, which, in essence, is a developmental disorder. All-trans retinoic acid (ATRA) signaling via ligand-activation of the retinoic acid receptors (RARs) plays a crucial role in tissue patterning and development during mammalian embryogenesis. In [...] Read more.

Cancer stem cells are the root cause of cancer, which, in essence, is a developmental disorder. All-trans retinoic acid (ATRA) signaling via ligand-activation of the retinoic acid receptors (RARs) plays a crucial role in tissue patterning and development during mammalian embryogenesis. In adults, active RARγ maintains the pool of hematopoietic stem cells, whereas active RARα drives myeloid cell differentiation. Various findings have revealed that ATRA signaling is deregulated in many cancers. The enzymes for ATRA synthesis are downregulated in colorectal, gastric, lung, and oropharyngeal cancers. ATRA levels within breast, ovarian, pancreatic, prostate, and renal cancer cells were lower than within their normal counterpart cells. The importance is that 0.24 nM ATRA activates RARγ (for stem cell stemness), whereas 100 times more is required to activate RARα (for differentiation). Moreover, RARγ is an oncogene regarding overexpression within colorectal, cholangiocarcinoma, hepatocellular, ovarian, pancreatic, and renal cancer cells. The microRNA (miR) 30a-5p downregulates expression of RARγ, and miR-30a/miR-30a-5p is a tumor suppressor for breast, colorectal, gastric, hepatocellular, lung, oropharyngeal, ovarian, pancreatic, prostate, and renal cancer. These complementary findings support the view that perturbations to ATRA signaling play a role in driving the abnormal behavior of cancer stem cells. Targeting ATRA synthesis and RARγ has provided promising approaches to eliminating cancer stem cells because such agents have been shown to drive cell death. Full article

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19 pages, 3581 KiB

Open AccessArticle

Proteomics Reveals How the Tardigrade Damage Suppressor Protein Teaches Transfected Human Cells to Survive UV-C Stress

by Enxhi Shaba, Claudia Landi, Carlotta Marzocchi, Lorenza Vantaggiato, Luca Bini, Claudia Ricci and Silvia Cantara

Int. J. Mol. Sci. 2023, 24(14), 11463; https://doi.org/10.3390/ijms241411463 - 14 Jul 2023

Cited by 2 | Viewed by 3002

Abstract

The genome sequencing of the tardigrade Ramazzottius varieornatus revealed a unique nucleosome-binding protein named damage suppressor (Dsup), which was discovered to be crucial for the extraordinary abilities of tardigrades in surviving extreme stresses, such as UV. Evidence in Dsup-transfected human cells suggests that [...] Read more.

The genome sequencing of the tardigrade Ramazzottius varieornatus revealed a unique nucleosome-binding protein named damage suppressor (Dsup), which was discovered to be crucial for the extraordinary abilities of tardigrades in surviving extreme stresses, such as UV. Evidence in Dsup-transfected human cells suggests that Dsup mediates an overall response in DNA damage signaling, DNA repair, and cell cycle regulation, resulting in an acquired resistance to stress. Given these promising outcomes, our study attempts to provide a wider comprehension of the molecular mechanisms modulated by Dsup in human cells and to explore the Dsup-activated molecular pathways under stress. We performed a differential proteomic analysis of Dsup-transfected and control human cells under basal conditions and at 24 h recovery after exposure to UV-C. We demonstrate via enrichment and network analyses, for the first time, that even in the absence of external stimuli, and more significantly, after stress, Dsup activates mechanisms involved with the unfolded protein response, the mRNA processing and stability, cytoplasmic stress granules, the DNA damage response, and the telomere maintenance. In conclusion, our results shed new light on Dsup-mediated protective mechanisms and increases our knowledge of the molecular machineries of extraordinary protection against UV-C stress. Full article

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16 pages, 5206 KiB

Open AccessArticle

Exploring the Pathophysiologic Cascade Leading to Osteoclastogenic Activation in Gaucher Disease Monocytes Generated via CRISPR/Cas9 Technology

by Maximiliano Emanuel Ormazabal, Eleonora Pavan, Emilio Vaena, Dania Ferino, Jessica Biasizzo, Juan Marcos Mucci, Fabrizio Serra, Adriana Cifù, Maurizio Scarpa, Paula Adriana Rozenfeld and Andrea Elena Dardis

Int. J. Mol. Sci. 2023, 24(13), 11204; https://doi.org/10.3390/ijms241311204 - 7 Jul 2023

Cited by 2 | Viewed by 2061

Abstract

Gaucher disease (GD) is caused by biallelic pathogenic variants in the acid β-glucosidase gene (GBA1), leading to a deficiency in the β-glucocerebrosidase (GCase) enzyme activity resulting in the intracellular accumulation of sphingolipids. Skeletal alterations are one of the most disabling features [...] Read more.

Gaucher disease (GD) is caused by biallelic pathogenic variants in the acid β-glucosidase gene (GBA1), leading to a deficiency in the β-glucocerebrosidase (GCase) enzyme activity resulting in the intracellular accumulation of sphingolipids. Skeletal alterations are one of the most disabling features in GD patients. Although both defective bone formation and increased bone resorption due to osteoblast and osteoclast dysfunction contribute to GD bone pathology, the molecular bases are not fully understood, and bone disease is not completely resolved with currently available specific therapies. For this reason, using editing technology, our group has developed a reliable, isogenic, and easy-to-handle cellular model of GD monocytes (GBAKO-THP1) to facilitate GD pathophysiology studies and high-throughput drug screenings. In this work, we further characterized the model showing an increase in proinflammatory cytokines (Interleukin-1β and Tumor Necrosis Factor-α) release and activation of osteoclastogenesis. Furthermore, our data suggest that GD monocytes would display an increased osteoclastogenic potential, independent of their interaction with the GD microenvironment or other GD cells. Both proinflammatory cytokine production and osteoclastogenesis were restored at least, in part, by treating cells with the recombinant human GCase, a substrate synthase inhibitor, a pharmacological chaperone, and an anti-inflammatory compound. Besides confirming that this model would be suitable to perform high-throughput screening of therapeutic molecules that act via different mechanisms and on different phenotypic features, our data provided insights into the pathogenic cascade, leading to osteoclastogenesis exacerbation and its contribution to bone pathology in GD. Full article

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22 pages, 7347 KiB

Open AccessArticle

Bypass of Abasic Site–Peptide Cross-Links by Human Repair and Translesion DNA Polymerases

by Anna V. Yudkina, Alexander E. Barmatov, Nikita A. Bulgakov, Elizaveta O. Boldinova, Evgeniy S. Shilkin, Alena V. Makarova and Dmitry O. Zharkov

Int. J. Mol. Sci. 2023, 24(13), 10877; https://doi.org/10.3390/ijms241310877 - 29 Jun 2023

Cited by 1 | Viewed by 2044

Abstract

DNA–protein cross-links remain the least-studied type of DNA damage. Recently, their repair was shown to involve proteolysis; however, the fate of the peptide remnant attached to DNA is unclear. Particularly, peptide cross-links could interfere with DNA polymerases. Apurinuic/apyrimidinic (AP) sites, abundant and spontaneously [...] Read more.

DNA–protein cross-links remain the least-studied type of DNA damage. Recently, their repair was shown to involve proteolysis; however, the fate of the peptide remnant attached to DNA is unclear. Particularly, peptide cross-links could interfere with DNA polymerases. Apurinuic/apyrimidinic (AP) sites, abundant and spontaneously arising DNA lesions, readily form cross-links with proteins. Their degradation products (AP site–peptide cross-links, APPXLs) are non-instructive and should be even more problematic for polymerases. Here, we address the ability of human DNA polymerases involved in DNA repair and translesion synthesis (POLβ, POLλ, POLη, POLκ and PrimPOL) to carry out synthesis on templates containing AP sites cross-linked to the N-terminus of a 10-mer peptide (APPXL-I) or to an internal lysine of a 23-mer peptide (APPXL-Y). Generally, APPXLs strongly blocked processive DNA synthesis. The blocking properties of APPXL-I were comparable with those of an AP site, while APPXL-Y constituted a much stronger obstruction. POLη and POLκ demonstrated the highest bypass ability. DNA polymerases mostly used dNTP-stabilized template misalignment to incorporate nucleotides when encountering an APPXL. We conclude that APPXLs are likely highly cytotoxic and mutagenic intermediates of AP site–protein cross-link repair and must be quickly eliminated before replication. Full article

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16 pages, 1807 KiB

Open AccessReview

The Brain Pre-Metastatic Niche: Biological and Technical Advancements

by Maximilian Geissler, Weiyi Jia, Emine Nisanur Kiraz, Ida Kulacz, Xiao Liu, Adrian Rombach, Vincent Prinz, Daniel Jussen, Konstantinos D. Kokkaliaris, Hind Medyouf, Lisa Sevenich, Marcus Czabanka and Thomas Broggini

Int. J. Mol. Sci. 2023, 24(12), 10055; https://doi.org/10.3390/ijms241210055 - 13 Jun 2023

Cited by 5 | Viewed by 3789

Abstract

Metastasis, particularly brain metastasis, continues to puzzle researchers to this day, and exploring its molecular basis promises to break ground in developing new strategies for combatting this deadly cancer. In recent years, the research focus has shifted toward the earliest steps in the [...] Read more.

Metastasis, particularly brain metastasis, continues to puzzle researchers to this day, and exploring its molecular basis promises to break ground in developing new strategies for combatting this deadly cancer. In recent years, the research focus has shifted toward the earliest steps in the formation of metastasis. In this regard, significant progress has been achieved in understanding how the primary tumor affects distant organ sites before the arrival of tumor cells. The term pre-metastatic niche was introduced for this concept and encompasses all influences on sites of future metastases, ranging from immunological modulation and ECM remodeling to the softening of the blood–brain barrier. The mechanisms governing the spread of metastasis to the brain remain elusive. However, we begin to understand these processes by looking at the earliest steps in the formation of metastasis. This review aims to present recent findings on the brain pre-metastatic niche and to discuss existing and emerging methods to further explore the field. We begin by giving an overview of the pre-metastatic and metastatic niches in general before focusing on their manifestations in the brain. To conclude, we reflect on the methods usually employed in this field of research and discuss novel approaches in imaging and sequencing. Full article

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16 pages, 2023 KiB

Open AccessArticle

Entangled Motifs in Membrane Protein Structures

by Leonardo Salicari and Antonio Trovato

Int. J. Mol. Sci. 2023, 24(11), 9193; https://doi.org/10.3390/ijms24119193 - 24 May 2023

Cited by 2 | Viewed by 2010

Abstract

Entangled motifs are found in one-third of protein domain structures, a reference set that contains mostly globular proteins. Their properties suggest a connection with co-translational folding. Here, we wish to investigate the presence and properties of entangled motifs in membrane protein structures. From [...] Read more.

Entangled motifs are found in one-third of protein domain structures, a reference set that contains mostly globular proteins. Their properties suggest a connection with co-translational folding. Here, we wish to investigate the presence and properties of entangled motifs in membrane protein structures. From existing databases, we build a non-redundant data set of membrane protein domains, annotated with the monotopic/transmembrane and peripheral/integral labels. We evaluate the presence of entangled motifs using the Gaussian entanglement indicator. We find that entangled motifs appear in one-fifth of transmembrane and one-fourth of monotopic proteins. Surprisingly, the main features of the distribution of the values of the entanglement indicator are similar to the reference case of general proteins. The distribution is conserved across different organisms. Differences with respect to the reference set emerge when considering the chirality of entangled motifs. Although the same chirality bias is found for single-winding motifs in both membrane and reference proteins, the bias is reversed, strikingly, for double-winding motifs only in the reference set. We speculate that these observations can be rationalized in terms of the constraints exerted on the nascent chain by the co-translational bio-genesis machinery, which is different for membrane and globular proteins. Full article

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17 pages, 1913 KiB

Open AccessArticle

Eosinophil Granule Proteins Involvement in Acute Appendicitis—An Allergic Disease?

by Nuno Carvalho, Elisabete Carolino, Hélder Coelho, Ana Lúcia Barreira, Luísa Moreira, Margarida André, Susana Henriques, Carlos Cardoso, Luis Moita and Paulo Matos Costa

Int. J. Mol. Sci. 2023, 24(10), 9091; https://doi.org/10.3390/ijms24109091 - 22 May 2023

Cited by 5 | Viewed by 2035

Abstract

Several pieces of evidence point to an allergic component as a trigger of acute appendicitis. As the Th2 immune response is characterized by eosinophil mobilization to the target organ and release of their cationic granule proteins, it is reasonable to investigate if the [...] Read more.

Several pieces of evidence point to an allergic component as a trigger of acute appendicitis. As the Th2 immune response is characterized by eosinophil mobilization to the target organ and release of their cationic granule proteins, it is reasonable to investigate if the degranulation of eosinophils could be associated with the local injury. The primary aim of this study is to evaluate the participation of eosinophils granules proteins in acute appendicitis, both at local and systemic levels and the secondary aim is to evaluate the diagnostic accuracy of eosinophils granules proteins for the detection of acute appendicitis, as well as for distinguishing between complicated and uncomplicated acute appendicitis. Eosinophil-derived neurotoxin (EDN), eosinophil cationic protein (ECP) and eosinophil peroxidase (EP) are the most well-known eosinophil granule proteins. From August 2021 to April 2022, we present a prospective single-center study to evaluate the EDN, ECP, and EP concentrations simultaneously in appendicular lavage fluid (ALF) and the serum of 22 patients with acute phlegmonous appendicitis (APA), 24 with acute gangrenous appendicitis (AGA), and 14 normal controls. Concerning EDN, no differences were found between groups. ECP concentrations in ALF and serum were significantly higher in the histologically confirmed acute appendicitis compared to the control groups (p < 0.0001 and p < 0.0001, respectively). In ALF, no differences were found between ECP levels in APA: 38.85 ng/mL (IQR 26.50–51.77) and AGA 51.55 ng/mL (IQR 39.55–70.09) groups (p = 0.176). In the serum, no difference was found between ECP levels at APA: 39 ng/mL (IQR 21.30–56.90) and AGA: 51.30 ng/mL (IQR 20.25–62.59) (p = 0.100). For EP, the concentrations in ALF (p < 0.001) and serum (p < 0.001) were both higher in acute appendicitis compared to the control. In ALF, no difference was found between APA: 240.28 ng/mL (IQR 191.2–341.3) and AGA: 302.5 (IQR 227.7–535.85) (p = 0.236). In the serum, no differences were found between APA: 158.4 ng/mL (IQR 111.09–222.1) and AGA: 235.27 (IQR 192.33–262.51) (p = 0.179). Globally, the ALF concentrations were higher than serum concentrations, reflecting an intense inflammatory local reaction in AA. The optimal ECP cut-off for discriminating between acute appendicitis and the controls was >11.41 ng/mL, with a sensitivity of 93.5%, but with a specificity for identifying appendicitis of 21.4%, good discriminative power (AUC = 0.880). For EP, the optimal cut-off was >93.20 ng/mL, with a sensitivity of 87%, but with a specificity of 14.3% (AUC = 0.901), excellent discriminative power. For the diagnosis of perforated AA, the discriminative power of ECP and EP serum concentrations are weak (AUC = 0.562 and AUC = 0.664, respectively). Concerning the presence of peritonitis, the discriminative power of ECP and EP serum concentrations is acceptable, respectively: AUC = 0.724 and AUC = 0.735. Serum levels of EDN (p = 0.119), ECP (p = 0.586) and EP (p = 0.08) in complicated appendicitis were similar to uncomplicated appendicitis. Serum concentrations of ECP and EP can be added to decision-making AA diagnosis. A Th2-type immune response is present in AA. These data bring forward the role of an allergic reaction in the pathogenesis of acute appendicitis. Full article

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14 pages, 3240 KiB

Open AccessArticle

ROS Consumers or Producers? Interpreting Transcriptomic Data by AlphaFold Modeling Provides Insights into Class III Peroxidase Functions in Response to Biotic and Abiotic Stresses

by James New, Daniel Barsky and Claudia Uhde-Stone

Int. J. Mol. Sci. 2023, 24(9), 8297; https://doi.org/10.3390/ijms24098297 - 5 May 2023

Cited by 2 | Viewed by 1977

Abstract

Participating in both biotic and abiotic stress responses, plant-specific class III peroxidases (PERs) show promise as candidates for crop improvement. The multigenic PER family is known to take part in diverse functions, such as lignin formation and defense against pathogens. Traditionally linked to [...] Read more.

Participating in both biotic and abiotic stress responses, plant-specific class III peroxidases (PERs) show promise as candidates for crop improvement. The multigenic PER family is known to take part in diverse functions, such as lignin formation and defense against pathogens. Traditionally linked to hydrogen peroxide (H₂O₂) consumption, PERs can also produce reactive oxygen species (ROS), essential in tissue development, pathogen defense and stress signaling. The amino acid sequences of both orthologues and paralogues of PERs are highly conserved, but discovering correlations between sequence differences and their functional diversity has proven difficult. By combining meta-analysis of transcriptomic data and sequence alignments, we discovered a correlation between three key amino acid positions and gene expression in response to biotic and abiotic stresses. Phylogenetic analysis revealed evolutionary pressure on these amino acids toward stress responsiveness. Using AlphaFold modeling, we found unique interdomain and protein–heme interactions involving those key amino acids in stress-induced PERs. Plausibly, these structural interactions may act as “gate keepers” by preventing larger substrates from accessing the heme and thereby shifting PER function from consumption to the production of ROS. Full article

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17 pages, 3556 KiB

Open AccessArticle

Residual Humidity in Paraffin-Embedded Tissue Reduces Nucleic Acid Stability

by Peter M. Abuja, Daniela Pabst, Benjamin Bourgeois, Martina Loibner, Christine Ulz, Iris Kufferath, Ulrike Fackelmann, Cornelia Stumptner, Rainer Kraemer, Tobias Madl and Kurt Zatloukal

Int. J. Mol. Sci. 2023, 24(9), 8010; https://doi.org/10.3390/ijms24098010 - 28 Apr 2023

Cited by 1 | Viewed by 2412

Abstract

Molecular diagnostics in healthcare relies increasingly on genomic and transcriptomic methodologies and requires appropriate tissue specimens from which nucleic acids (NA) of sufficiently high quality can be obtained. Besides the duration of ischemia and fixation type, NA quality depends on a variety of [...] Read more.

Molecular diagnostics in healthcare relies increasingly on genomic and transcriptomic methodologies and requires appropriate tissue specimens from which nucleic acids (NA) of sufficiently high quality can be obtained. Besides the duration of ischemia and fixation type, NA quality depends on a variety of other pre-analytical parameters, such as storage conditions and duration. It has been discussed that the improper dehydration of tissue during processing influences the quality of NAs and the shelf life of fixed tissue. Here, we report on establishing a method for determining the amount of residual water in fixed, paraffin-embedded tissue (fixed by neutral buffered formalin or a non-crosslinking fixative) and its correlation to the performance of NAs in quantitative real-time polymerase chain reaction (qRT-PCR) analyses. The amount of residual water depended primarily on the fixative type and the dehydration protocol and, to a lesser extent, on storage conditions and time. Moreover, we found that these parameters were associated with the qRT-PCR performance of extracted NAs. Besides the cross-linking of NAs and the modification of nucleobases by formalin, the hydrolysis of NAs by residual water was found to contribute to reduced qRT-PCR performance. The negative effects of residual water on NA stability are not only important for the design and interpretation of research but must also be taken into account in clinical diagnostics where the reanalysis of archived tissue from a primary tumor may be required (e.g., after disease recurrence). We conclude that improving the shelf life of fixed tissue requires meticulous dehydration and dry storage to minimize the degradative influence of residual water on NAs. Full article

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16 pages, 7174 KiB

Open AccessArticle

The Metabolic Switch of Physical Activity in Non-Obese Insulin Resistant Individuals

by Shamma Almuraikhy, Najeha Anwardeen, Asmma Doudin, Maha Sellami, Alexander Domling, Abdelali Agouni, Asmaa A. Al Thani and Mohamed A. Elrayess

Int. J. Mol. Sci. 2023, 24(9), 7816; https://doi.org/10.3390/ijms24097816 - 25 Apr 2023

Cited by 4 | Viewed by 4377

Abstract

Healthy non-obese insulin resistant (IR) individuals are at higher risk of metabolic syndrome. The metabolic signature of the increased risk was previously determined. Physical activity can lower the risk of insulin resistance, but the underlying metabolic pathways remain to be determined. In this [...] Read more.

Healthy non-obese insulin resistant (IR) individuals are at higher risk of metabolic syndrome. The metabolic signature of the increased risk was previously determined. Physical activity can lower the risk of insulin resistance, but the underlying metabolic pathways remain to be determined. In this study, the common and unique metabolic signatures of insulin sensitive (IS) and IR individuals in active and sedentary individuals were determined. Data from 305 young, aged 20–30, non-obese participants from Qatar biobank, were analyzed. The homeostatic model assessment of insulin resistance (HOMA-IR) and physical activity questionnaires were utilized to classify participants into four groups: Active Insulin Sensitive (ISA, n = 30), Active Insulin Resistant (IRA, n = 20), Sedentary Insulin Sensitive (ISS, n = 21) and Sedentary Insulin Resistant (SIR, n = 23). Differences in the levels of 1000 metabolites between insulin sensitive and insulin resistant individuals in both active and sedentary groups were compared using orthogonal partial least square discriminate analysis (OPLS-DA) and linear models. The study indicated significant differences in fatty acids between individuals with insulin sensitivity and insulin resistance who engaged in physical activity, including monohydroxy, dicarboxylate, medium and long chain, mono and polyunsaturated fatty acids. On the other hand, the sedentary group showed changes in carbohydrates, specifically glucose and pyruvate. Both groups exhibited alterations in 1-carboxyethylphenylalanine. The study revealed different metabolic signature in insulin resistant individuals depending on their physical activity status. Specifically, the active group showed changes in lipid metabolism, while the sedentary group showed alterations in glucose metabolism. These metabolic discrepancies demonstrate the beneficial impact of moderate physical activity on high risk insulin resistant healthy non-obese individuals by flipping their metabolic pathways from glucose based to fat based, ultimately leading to improved health outcomes. The results of this study carry significant implications for the prevention and treatment of metabolic syndrome in non-obese individuals. Full article

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18 pages, 4175 KiB

Open AccessArticle

Analysis of Ku70 S155 Phospho-Specific BioID2 Interactome Identifies Ku Association with TRIP12 in Response to DNA Damage

by Sanna Abbasi, Laila Bayat and Caroline Schild-Poulter

Int. J. Mol. Sci. 2023, 24(8), 7041; https://doi.org/10.3390/ijms24087041 - 11 Apr 2023

Cited by 1 | Viewed by 2458

Abstract

The Ku heterodimer, composed of subunits Ku70 and Ku80, is known for its essential role in repairing double-stranded DNA breaks via non-homologous end joining (NHEJ). We previously identified Ku70 S155 as a novel phosphorylation site within the von Willebrand A-like (vWA) domain of [...] Read more.

The Ku heterodimer, composed of subunits Ku70 and Ku80, is known for its essential role in repairing double-stranded DNA breaks via non-homologous end joining (NHEJ). We previously identified Ku70 S155 as a novel phosphorylation site within the von Willebrand A-like (vWA) domain of Ku70 and documented an altered DNA damage response in cells expressing a Ku70 S155D phosphomimetic mutant. Here, we conducted proximity-dependent biotin identification (BioID2) screening using wild-type Ku70, Ku70 S155D mutant, and Ku70 with a phosphoablative substitution (S155A) to identify Ku70 S155D-specific candidate proteins that may rely on this phosphorylation event. Using the BioID2 screen with multiple filtering approaches, we compared the protein interactor candidate lists for Ku70 S155D and S155A. TRIP12 was exclusive to the Ku70 S155D list, considered a high confidence interactor based on SAINTexpress analysis, and appeared in all three biological replicates of the Ku70 S155D-BioID2 mass spectrometry results. Using proximity ligation assays (PLA), we demonstrated a significantly increased association between Ku70 S155D-HA and TRIP12 compared to wild-type Ku70-HA cells. In addition, we were able to demonstrate a robust PLA signal between endogenous Ku70 and TRIP12 in the presence of double-stranded DNA breaks. Finally, co-immunoprecipitation analyses showed an enhanced interaction between TRIP12 and Ku70 upon treatment with ionizing radiation, suggesting a direct or indirect association in response to DNA damage. Altogether, these results suggest an association between Ku70 phospho-S155 and TRIP12. Full article

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12 pages, 3262 KiB

Open AccessArticle

Vesicular Integral-Membrane Protein 36 Is Involved in the Selective Secretion of Fucosylated Proteins into Bile Duct-like Structures in HepG2 Cells

by Mizuki Muranaka, Shinji Takamatsu, Tsunenori Ouchida, Yuri Kanazawa, Jumpei Kondo, Tsutomu Nakagawa, Yuriko Egashira, Koji Fukagawa, Jianguo Gu, Toru Okamoto, Yoshihiro Kamada and Eiji Miyoshi

Int. J. Mol. Sci. 2023, 24(8), 7037; https://doi.org/10.3390/ijms24087037 - 11 Apr 2023

Cited by 1 | Viewed by 3409

Abstract

Fucosylated proteins are widely used as biomarkers of cancer and inflammation. Fucosylated alpha-fetoprotein (AFP-L3) is a specific biomarker for hepatocellular carcinoma. We previously showed that increases in serum AFP-L3 levels depend on increased expression of fucosylation-regulatory genes and abnormal transport of fucosylated proteins [...] Read more.

Fucosylated proteins are widely used as biomarkers of cancer and inflammation. Fucosylated alpha-fetoprotein (AFP-L3) is a specific biomarker for hepatocellular carcinoma. We previously showed that increases in serum AFP-L3 levels depend on increased expression of fucosylation-regulatory genes and abnormal transport of fucosylated proteins in cancer cells. In normal hepatocytes, fucosylated proteins are selectively secreted in the bile duct but not blood. In cases of cancer cells without cellular polarity, this selective secretion system is destroyed. Here, we aimed to identify cargo proteins involved in the selective secretion of fucosylated proteins, such as AFP-L3, into bile duct-like structures in HepG2 hepatoma cells, which have cellular polarity like, in part, normal hepatocytes. α1-6 Fucosyltransferase (FUT8) is a key enzyme to synthesize core fucose and produce AFP-L3. Firstly, we knocked out the FUT8 gene in HepG2 cells and investigated the effects on the secretion of AFP-L3. AFP-L3 accumulated in bile duct-like structures in HepG2 cells, and this phenomenon was diminished by FUT8 knockout, suggesting that HepG2 cells have cargo proteins for AFP-L3. To identify cargo proteins involved in the secretion of fucosylated proteins in HepG2 cells, immunoprecipitation and the proteomic Strep-tag system experiments followed by mass spectrometry analyses were performed. As a result of proteomic analysis, seven kinds of lectin-like molecules were identified, and we selected vesicular integral membrane protein gene VIP36 as a candidate of the cargo protein that interacts with the α1-6 fucosylation (core fucose) on N-glycan according to bibliographical consideration. Expectedly, the knockout of the VIP36 gene in HepG2 cells suppressed the secretion of AFP-L3 and other fucosylated proteins, such as fucosylated alpha-1 antitrypsin, into bile duct-like structures. We propose that VIP36 could be a cargo protein involved in the apical secretion of fucosylated proteins in HepG2 cells. Full article

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21 pages, 1898 KiB

Open AccessReview

Applications and Advances of Multicellular Tumor Spheroids: Challenges in Their Development and Analysis

by Achilleas G. Mitrakas, Avgi Tsolou, Stylianos Didaskalou, Lito Karkaletsou, Christos Efstathiou, Evgenios Eftalitsidis, Konstantinos Marmanis and Maria Koffa

Int. J. Mol. Sci. 2023, 24(8), 6949; https://doi.org/10.3390/ijms24086949 - 8 Apr 2023

Cited by 13 | Viewed by 4903

Abstract

Biomedical research requires both in vitro and in vivo studies in order to explore disease processes or drug interactions. Foundational investigations have been performed at the cellular level using two-dimensional cultures as the gold-standard method since the early 20th century. However, three-dimensional (3D) [...] Read more.

Biomedical research requires both in vitro and in vivo studies in order to explore disease processes or drug interactions. Foundational investigations have been performed at the cellular level using two-dimensional cultures as the gold-standard method since the early 20th century. However, three-dimensional (3D) cultures have emerged as a new tool for tissue modeling over the last few years, bridging the gap between in vitro and animal model studies. Cancer has been a worldwide challenge for the biomedical community due to its high morbidity and mortality rates. Various methods have been developed to produce multicellular tumor spheroids (MCTSs), including scaffold-free and scaffold-based structures, which usually depend on the demands of the cells used and the related biological question. MCTSs are increasingly utilized in studies involving cancer cell metabolism and cell cycle defects. These studies produce massive amounts of data, which demand elaborate and complex tools for thorough analysis. In this review, we discuss the advantages and disadvantages of several up-to-date methods used to construct MCTSs. In addition, we also present advanced methods for analyzing MCTS features. As MCTSs more closely mimic the in vivo tumor environment, compared to 2D monolayers, they can evolve to be an appealing model for in vitro tumor biology studies. Full article

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31 pages, 11986 KiB

Open AccessArticle

Identification of Orbivirus Non-Structural Protein 5 (NS5), Its Role and Interaction with RNA/DNA in Infected Cells

by Fauziah Mohd Jaafar, Baptiste Monsion, Peter P. C. Mertens and Houssam Attoui

Int. J. Mol. Sci. 2023, 24(7), 6845; https://doi.org/10.3390/ijms24076845 - 6 Apr 2023

Cited by 8 | Viewed by 3037

Abstract

Bioinformatic analyses have predicted that orbiviruses encode an additional, small non-structural protein (NS5) from a secondary open reading frame on genome segment 10. However, this protein has not previously been detected in infected mammalian or insect cells. NS5-specific antibodies were generated in mice [...] Read more.

Bioinformatic analyses have predicted that orbiviruses encode an additional, small non-structural protein (NS5) from a secondary open reading frame on genome segment 10. However, this protein has not previously been detected in infected mammalian or insect cells. NS5-specific antibodies were generated in mice and were used to identify NS5 synthesised in orbivirus-infected BSR cells or cells transfected with NS5 expression plasmids. Confocal microscopy shows that although NS5 accumulates in the nucleus, particularly in the nucleolus, which becomes disrupted, it also appears in the cell cytoplasm, co-localising with mitochondria. NS5 helps to prevent the degradation of ribosomal RNAs during infection and reduces host-cell protein synthesis However, it helps to extend cell viability by supporting viral protein synthesis and virus replication. Pulldown studies showed that NS5 binds to ssRNAs and supercoiled DNAs and demonstrates interactions with ZBP1, suggesting that it modulates host-cell responses. Full article

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16 pages, 680 KiB

Open AccessReview

Update on the Molecular Pathology of Cutaneous Squamous Cell Carcinoma

by Elena-Codruta Cozma, Laura Madalina Banciu, Cristina Soare and Sanda-Maria Cretoiu

Int. J. Mol. Sci. 2023, 24(7), 6646; https://doi.org/10.3390/ijms24076646 - 2 Apr 2023

Cited by 5 | Viewed by 4028

Abstract

Cutaneous squamous cell carcinoma (cSCC) is the second most common skin cancer, originating from keratinocytes of the spinous layer. Numerous risk factors have been discovered for the initiation and growth of this type of cancer, such as exposure to UV and ionizing radiation, [...] Read more.

Cutaneous squamous cell carcinoma (cSCC) is the second most common skin cancer, originating from keratinocytes of the spinous layer. Numerous risk factors have been discovered for the initiation and growth of this type of cancer, such as exposure to UV and ionizing radiation, chemical carcinogens, the presence of immunosuppression states, chronic inflammation, infections with high-risk viral strains, and, last but not least, the presence of diseases associated with genetic alterations. The important socio-economic impact, as well as the difficulty associated with therapy for advanced forms, has made the molecular mechanisms underlying this neoplasia more and more intensively studied, with the intention of achieving a better understanding and advancing the treatment of this pathology. This review aims to provide a brief foray into the molecular, genetic, and epigenetic aspects of this cancer, as well as the treatment methods, ranging from the first used to the latest targeted therapies. Full article

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12 pages, 2890 KiB

Open AccessArticle

Global DNA Adenine Methylation in Caenorhabditis elegans after Multigenerational Exposure to Silver Nanoparticles and Silver Nitrate

by Anye Wamucho, Jason Unrine, John May and Olga Tsyusko

Int. J. Mol. Sci. 2023, 24(7), 6168; https://doi.org/10.3390/ijms24076168 - 24 Mar 2023

Cited by 6 | Viewed by 2474

Abstract

Multigenerational and transgenerational reproductive toxicity in a model nematode Caenorhabditis elegans has been shown previously after exposure to silver nanoparticles (Ag-NPs) and silver ions (AgNO₃). However, there is a limited understanding on the transfer mechanism of the increased reproductive sensitivity to [...] Read more.

Multigenerational and transgenerational reproductive toxicity in a model nematode Caenorhabditis elegans has been shown previously after exposure to silver nanoparticles (Ag-NPs) and silver ions (AgNO₃). However, there is a limited understanding on the transfer mechanism of the increased reproductive sensitivity to subsequent generations. This study examines changes in DNA methylation at epigenetic mark N6-methyl-2′-deoxyadenosine (6mdA) after multigenerational exposure of C. elegans to pristine and transformed-via-sulfidation Ag-NPs and AgNO₃. Levels of 6mdA were measured as 6mdA/dA ratios prior to C. elegans exposure (F₀) after two generations of exposure (F₂) and two generations of rescue (F₄) using high-performance liquid chromatography with tandem mass spectrometry (LC-MS/MS). Although both AgNO₃ and Ag-NPs induced multigenerational reproductive toxicity, only AgNO₃ exposure caused a significant increase in global 6mdA levels after exposures (F₂). However, after two generations of rescue (F₄), the 6mdA levels in AgNO₃ treatment returned to F₀ levels, suggesting other epigenetic modifications may be also involved. No significant changes in global DNA methylation levels were observed after exposure to pristine and sulfidized sAg-NPs. This study demonstrates the involvement of an epigenetic mark in AgNO₃ reproductive toxicity and suggests that AgNO₃ and Ag-NPs may have different toxicity mechanisms. Full article

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22 pages, 2380 KiB

Open AccessReview

Emerging Personalized Opportunities for Enhancing Translational Readthrough in Rare Genetic Diseases and Beyond

by Roland N. Wagner, Michael Wießner, Andreas Friedrich, Johanna Zandanell, Hannelore Breitenbach-Koller and Johann W. Bauer

Int. J. Mol. Sci. 2023, 24(7), 6101; https://doi.org/10.3390/ijms24076101 - 23 Mar 2023

Cited by 6 | Viewed by 3047

Abstract

Nonsense mutations trigger premature translation termination and often give rise to prevalent and rare genetic diseases. Consequently, the pharmacological suppression of an unscheduled stop codon represents an attractive treatment option and is of high clinical relevance. At the molecular level, the ability of [...] Read more.

Nonsense mutations trigger premature translation termination and often give rise to prevalent and rare genetic diseases. Consequently, the pharmacological suppression of an unscheduled stop codon represents an attractive treatment option and is of high clinical relevance. At the molecular level, the ability of the ribosome to continue translation past a stop codon is designated stop codon readthrough (SCR). SCR of disease-causing premature termination codons (PTCs) is minimal but small molecule interventions, such as treatment with aminoglycoside antibiotics, can enhance its frequency. In this review, we summarize the current understanding of translation termination (both at PTCs and at cognate stop codons) and highlight recently discovered pathways that influence its fidelity. We describe the mechanisms involved in the recognition and readthrough of PTCs and report on SCR-inducing compounds currently explored in preclinical research and clinical trials. We conclude by reviewing the ongoing attempts of personalized nonsense suppression therapy in different disease contexts, including the genetic skin condition epidermolysis bullosa. Full article

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22 pages, 1510 KiB

Open AccessReview

Neuroinflammation and Oxidative Stress in the Pathogenesis of Autism Spectrum Disorder

by Noriyoshi Usui, Hikaru Kobayashi and Shoichi Shimada

Int. J. Mol. Sci. 2023, 24(6), 5487; https://doi.org/10.3390/ijms24065487 - 13 Mar 2023

Cited by 58 | Viewed by 12563

Abstract

Autism spectrum disorder (ASD) is a neurodevelopmental disorder (NDD) characterized by impairments in social communication, repetitive behaviors, restricted interests, and hyperesthesia/hypesthesia caused by genetic and/or environmental factors. In recent years, inflammation and oxidative stress have been implicated in the pathogenesis of ASD. In [...] Read more.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder (NDD) characterized by impairments in social communication, repetitive behaviors, restricted interests, and hyperesthesia/hypesthesia caused by genetic and/or environmental factors. In recent years, inflammation and oxidative stress have been implicated in the pathogenesis of ASD. In this review, we discuss the inflammation and oxidative stress in the pathophysiology of ASD, particularly focusing on maternal immune activation (MIA). MIA is a one of the common environmental risk factors for the onset of ASD during pregnancy. It induces an immune reaction in the pregnant mother’s body, resulting in further inflammation and oxidative stress in the placenta and fetal brain. These negative factors cause neurodevelopmental impairments in the developing fetal brain and subsequently cause behavioral symptoms in the offspring. In addition, we also discuss the effects of anti-inflammatory drugs and antioxidants in basic studies on animals and clinical studies of ASD. Our review provides the latest findings and new insights into the involvements of inflammation and oxidative stress in the pathogenesis of ASD. Full article

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21 pages, 15070 KiB

Open AccessArticle

VPg Impact on Ryegrass Mottle Virus Serine-like 3C Protease Proteolysis and Structure

by Gints Kalnins, Rebeka Ludviga, Ieva Kalnciema, Gunta Resevica, Vilija Zeltina, Janis Bogans, Kaspars Tars, Andris Zeltins and Ina Balke

Int. J. Mol. Sci. 2023, 24(6), 5347; https://doi.org/10.3390/ijms24065347 - 10 Mar 2023

Cited by 1 | Viewed by 1669

Abstract

Sobemoviruses encode serine-like 3C proteases (Pro) that participate in the processing and maturation of other virus-encoded proteins. Its cis and trans activity is mediated by the naturally unfolded virus-genome-linked protein (VPg). Nuclear magnetic resonance studies show a Pro–VPg complex interaction and VPg tertiary [...] Read more.

Sobemoviruses encode serine-like 3C proteases (Pro) that participate in the processing and maturation of other virus-encoded proteins. Its cis and trans activity is mediated by the naturally unfolded virus-genome-linked protein (VPg). Nuclear magnetic resonance studies show a Pro–VPg complex interaction and VPg tertiary structure; however, information regarding structural changes of the Pro–VPg complex during interaction is lacking. Here, we solved a full Pro–VPg 3D structure of ryegrass mottle virus (RGMoV) that demonstrates the structural changes in three different conformations due to VPg interaction with Pro. We identified a unique site of VPg interaction with Pro that was not observed in other sobemoviruses, and observed different conformations of the Pro β2 barrel. This is the first report of a full plant Pro crystal structure with its VPg cofactor. We also confirmed the existence of an unusual previously unmapped cleavage site for sobemovirus Pro in the transmembrane domain: E/A. We demonstrated that RGMoV Pro in cis activity is not regulated by VPg and that in trans, VPg can also mediate Pro in free form. Additionally, we observed Ca²⁺ and Zn²⁺ inhibitory effects on the Pro cleavage activity. Full article

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17 pages, 3359 KiB

Open AccessArticle

PCSK9 Inhibitors Reduce PCSK9 and Early Atherogenic Biomarkers in Stimulated Human Coronary Artery Endothelial Cells

by Rahayu Zulkapli, Suhaila Abd Muid, Seok Mui Wang and Hapizah Nawawi

Int. J. Mol. Sci. 2023, 24(6), 5098; https://doi.org/10.3390/ijms24065098 - 7 Mar 2023

Cited by 9 | Viewed by 2775

Abstract

Despite reports on the efficacy of proprotein convertase subtilisin-Kexin type 9 (PCSK9) inhibitors as a potent lipid-lowering agent in various large-scale clinical trials, the anti-atherogenic properties of PCSK9 inhibitors in reducing PCSK9 and atherogenesis biomarkers via the NF-ĸB and eNOS pathway has yet [...] Read more.

Despite reports on the efficacy of proprotein convertase subtilisin-Kexin type 9 (PCSK9) inhibitors as a potent lipid-lowering agent in various large-scale clinical trials, the anti-atherogenic properties of PCSK9 inhibitors in reducing PCSK9 and atherogenesis biomarkers via the NF-ĸB and eNOS pathway has yet to be established. This study aimed to investigate the effects of PCSK9 inhibitors on PCSK9, targeted early atherogenesis biomarkers, and monocyte binding in stimulated human coronary artery endothelial cells (HCAEC). HCAEC were stimulated with lipopolysaccharides (LPS) and incubated with evolocumab and alirocumab. The protein and gene expression of PCSK9, interleukin-6 (IL-6), E-selectin, intercellular adhesion molecule 1 (ICAM-1), nuclear factor kappa B (NF-ĸB) p65, and endothelial nitric oxide synthase (eNOS) were measured using ELISA and QuantiGene plex, respectively. The binding of U937 monocytes to endothelial cell capacity was measured by the Rose Bengal method. The anti-atherogenic effects of evolocumab and alirocumab were contributed to by the downregulation of PCSK9, early atherogenesis biomarkers, and the significant inhibition of monocyte adhesion to the endothelial cells via the NF-ĸB and eNOS pathways. These suggest the beyond cholesterol-lowering beneficial effects of PCSK9 inhibitors in impeding atherogenesis during the initial phase of atherosclerotic plaque development, hence their potential role in preventing atherosclerosis-related complications. Full article

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10 pages, 1047 KiB

Open AccessArticle

Essentiality of the Escherichia coli YgfZ Protein for the In Vivo Thiomethylation of Ribosomal Protein S12 by the RimO Enzyme

by Torben Lund, Maria Yohanna Kulkova, Rosa Jersie-Christensen and Tove Atlung

Int. J. Mol. Sci. 2023, 24(5), 4728; https://doi.org/10.3390/ijms24054728 - 1 Mar 2023

Cited by 1 | Viewed by 2353

Abstract

Enzymes carrying Iron-Sulfur (Fe-S) clusters perform many important cellular functions and their biogenesis require complex protein machinery. In mitochondria, the IBA57 protein is essential and promotes assembly of [4Fe-4S] clusters and their insertion into acceptor proteins. YgfZ is the bacterial homologue of IBA57 [...] Read more.

Enzymes carrying Iron-Sulfur (Fe-S) clusters perform many important cellular functions and their biogenesis require complex protein machinery. In mitochondria, the IBA57 protein is essential and promotes assembly of [4Fe-4S] clusters and their insertion into acceptor proteins. YgfZ is the bacterial homologue of IBA57 but its precise role in Fe-S cluster metabolism is uncharacterized. YgfZ is needed for activity of the radical S-adenosyl methionine [4Fe-4S] cluster enzyme MiaB which thiomethylates some tRNAs. The growth of cells lacking YgfZ is compromised especially at low temperature. The RimO enzyme is homologous to MiaB and thiomethylates a conserved aspartic acid in ribosomal protein S12. To quantitate thiomethylation by RimO, we developed a bottom-up LC-MS² analysis of total cell extracts. We show here that the in vivo activity of RimO is very low in the absence of YgfZ and independent of growth temperature. We discuss these results in relation to the hypotheses relating to the role of the auxiliary 4Fe-4S cluster in the Radical SAM enzymes that make Carbon-Sulfur bonds. Full article

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23 pages, 19931 KiB

Open AccessArticle

Structurally Different Yet Functionally Similar: Aptamers Specific for the Ebola Virus Soluble Glycoprotein and GP1,2 and Their Application in Electrochemical Sensing

by Soma Banerjee, Mahsa Askary Hemmat, Shambhavi Shubham, Agnivo Gosai, Sivaranjani Devarakonda, Nianyu Jiang, Charith Geekiyanage, Jacob A. Dillard, Wendy Maury, Pranav Shrotriya, Monica H. Lamm and Marit Nilsen-Hamilton

Int. J. Mol. Sci. 2023, 24(5), 4627; https://doi.org/10.3390/ijms24054627 - 27 Feb 2023

Cited by 4 | Viewed by 3266

Abstract

The Ebola virus glycoprotein (GP) gene templates several mRNAs that produce either the virion-associated transmembrane protein or one of two secreted glycoproteins. Soluble glycoprotein (sGP) is the predominant product. GP1 and sGP share an amino terminal sequence of 295 amino acids but differ [...] Read more.

The Ebola virus glycoprotein (GP) gene templates several mRNAs that produce either the virion-associated transmembrane protein or one of two secreted glycoproteins. Soluble glycoprotein (sGP) is the predominant product. GP1 and sGP share an amino terminal sequence of 295 amino acids but differ in quaternary structure, with GP1 being a heterohexamer with GP2 and sGP a homodimer. Two structurally different DNA aptamers were selected against sGP that also bound GP1,2. These DNA aptamers were compared with a 2′FY-RNA aptamer for their interactions with the Ebola GP gene products. The three aptamers have almost identical binding isotherms for sGP and GP1,2 in solution and on the virion. They demonstrated high affinity and selectivity for sGP and GP1,2. Furthermore, one aptamer, used as a sensing element in an electrochemical format, detected GP1,2 on pseudotyped virions and sGP with high sensitivity in the presence of serum, including from an Ebola-virus-infected monkey. Our results suggest that the aptamers interact with sGP across the interface between the monomers, which is different from the sites on the protein bound by most antibodies. The remarkable similarity in functional features of three structurally distinct aptamers suggests that aptamers, like antibodies, have preferred binding sites on proteins. Full article

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18 pages, 2292 KiB

Open AccessReview

Circular RNA- and microRNA-Mediated Post-Transcriptional Regulation of Preadipocyte Differentiation in Adipogenesis: From Expression Profiling to Signaling Pathway

by Chiu-Jung Huang and Kong Bung Choo

Int. J. Mol. Sci. 2023, 24(5), 4549; https://doi.org/10.3390/ijms24054549 - 25 Feb 2023

Cited by 5 | Viewed by 2285

Abstract

Adipogenesis is an indispensable cellular process that involves preadipocyte differentiation into mature adipocyte. Dysregulated adipogenesis contributes to obesity, diabetes, vascular conditions and cancer-associated cachexia. This review aims to elucidate the mechanistic details on how circular RNA (circRNA) and microRNA (miRNA) modulate post-transcriptional expression [...] Read more.

Adipogenesis is an indispensable cellular process that involves preadipocyte differentiation into mature adipocyte. Dysregulated adipogenesis contributes to obesity, diabetes, vascular conditions and cancer-associated cachexia. This review aims to elucidate the mechanistic details on how circular RNA (circRNA) and microRNA (miRNA) modulate post-transcriptional expression of targeted mRNA and the impacted downstream signaling and biochemical pathways in adipogenesis. Twelve adipocyte circRNA profiling and comparative datasets from seven species are analyzed using bioinformatics tools and interrogations of public circRNA databases. Twenty-three circRNAs are identified in the literature that are common to two or more of the adipose tissue datasets in different species; these are novel circRNAs that have not been reported in the literature in relation to adipogenesis. Four complete circRNA–miRNA-mediated modulatory pathways are constructed via integration of experimentally validated circRNA–miRNA–mRNA interactions and the downstream signaling and biochemical pathways involved in preadipocyte differentiation via the PPARγ/C/EBPα gateway. Despite the diverse mode of modulation, bioinformatics analysis shows that the circRNA–miRNA–mRNA interacting seed sequences are conserved across species, supporting mandatory regulatory functions in adipogenesis. Understanding the diverse modes of post-transcriptional regulation of adipogenesis may contribute to the development of novel diagnostic and therapeutic strategies for adipogenesis-associated diseases and in improving meat quality in the livestock industries. Full article

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11 pages, 6730 KiB

Open AccessArticle

Imaging and Characterization of Oxidative Protein Modifications in Skin

by Ankush Prasad, Hana Duchová, Renuka Ramalingam Manoharan, Deepak Rathi and Pavel Pospíšil

Int. J. Mol. Sci. 2023, 24(4), 3981; https://doi.org/10.3390/ijms24043981 - 16 Feb 2023

Cited by 4 | Viewed by 2082

Abstract

Skin plays an important role in protection, metabolism, thermoregulation, sensation, and excretion whilst being consistently exposed to environmental aggression, including biotic and abiotic stresses. During the generation of oxidative stress in the skin, the epidermal and dermal cells are generally regarded as the [...] Read more.

Skin plays an important role in protection, metabolism, thermoregulation, sensation, and excretion whilst being consistently exposed to environmental aggression, including biotic and abiotic stresses. During the generation of oxidative stress in the skin, the epidermal and dermal cells are generally regarded as the most affected regions. The participation of reactive oxygen species (ROS) as a result of environmental fluctuations has been experimentally proven by several researchers and is well known to contribute to ultra-weak photon emission via the oxidation of biomolecules (lipids, proteins, and nucleic acids). More recently, ultra-weak photon emission detection techniques have been introduced to investigate the conditions of oxidative stress in various living systems in in vivo, ex vivo and in vitro studies. Research into two-dimensional photon imaging is drawing growing attention because of its application as a non-invasive tool. We monitored spontaneous and stress-induced ultra-weak photon emission under the exogenous application of a Fenton reagent. The results showed a marked difference in the ultra-weak photon emission. Overall, these results suggest that triplet carbonyl (³C=O^∗) and singlet oxygen (¹O₂) are the final emitters. Furthermore, the formation of oxidatively modified protein adducts and protein carbonyl formation upon treatment with hydrogen peroxide (H₂O₂) were observed using an immunoblotting assay. The results from this study broaden our understanding of the mechanism of the generation of ROS in skin layers and the formation/contribution of various excited species can be used as tools to determine the physiological state of the organism. Full article

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13 pages, 2415 KiB

Open AccessArticle

Human Omentin-1 Administration Ameliorates Hypertensive Complications without Affecting Hypertension in Spontaneously Hypertensive Rats

by Yuta Okamura, Ryo Niijima, Satoshi Kameshima, Tomoko Kodama, Kosuke Otani, Muneyoshi Okada and Hideyuki Yamawaki

Int. J. Mol. Sci. 2023, 24(4), 3835; https://doi.org/10.3390/ijms24043835 - 14 Feb 2023

Cited by 2 | Viewed by 1925

Abstract

Hypertension is one of the major risk factors for cardiovascular diseases and is caused by various abnormalities including the contractility of blood vessels. Spontaneously hypertensive rats (SHR), whose systemic blood pressure increases with aging, are a frequently used animal model for investigating essential [...] Read more.

Hypertension is one of the major risk factors for cardiovascular diseases and is caused by various abnormalities including the contractility of blood vessels. Spontaneously hypertensive rats (SHR), whose systemic blood pressure increases with aging, are a frequently used animal model for investigating essential hypertension and related complications in humans due to the damage of several organs. Human omentin-1 is an adipocytokine consisting of 313 amino acids. Serum omentin-1 levels decreased in hypertensive patients compared with normotensive controls. Furthermore, omentin-1 knockout mice showed elevated blood pressure and impaired endothelial vasodilation. Taken together, we hypothesized that adipocytokine, human omentin-1 may improve the hypertension and its complications including heart and renal failure in the aged SHR (65–68-weeks-old). SHR were subcutaneously administered with human omentin-1 (18 μg/kg/day, 2 weeks). Human omentin-1 had no effect on body weight, heart rate, and systolic blood pressure in SHR. The measurement of isometric contraction revealed that human omentin-1 had no influence on the enhanced vasocontractile or impaired vasodilator responses in the isolated thoracic aorta from SHR. On the other hand, human omentin-1 tended to improve left ventricular diastolic failure and renal failure in SHR. In summary, human omentin-1 tended to improve hypertensive complications (heart and renal failure), while it had no influence on the severe hypertension in the aged SHR. The further study of human omentin-1 may lead to the development of therapeutic agents for hypertensive complications. Full article

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20 pages, 3278 KiB

Open AccessArticle

Optimization of Biotinylated RNA or DNA Pull-Down Assays for Detection of Binding Proteins: Examples of IRP1, IRP2, HuR, AUF1, and Nrf2

by Yoshiaki Tsuji

Int. J. Mol. Sci. 2023, 24(4), 3604; https://doi.org/10.3390/ijms24043604 - 10 Feb 2023

Cited by 4 | Viewed by 10439

Abstract

Investigation of RNA- and DNA-binding proteins to a defined regulatory sequence, such as an AU-rich RNA and a DNA enhancer element, is important for understanding gene regulation through their interactions. For in vitro binding studies, an electrophoretic mobility shift assay (EMSA) was widely [...] Read more.

Investigation of RNA- and DNA-binding proteins to a defined regulatory sequence, such as an AU-rich RNA and a DNA enhancer element, is important for understanding gene regulation through their interactions. For in vitro binding studies, an electrophoretic mobility shift assay (EMSA) was widely used in the past. In line with the trend toward using non-radioactive materials in various bioassays, end-labeled biotinylated RNA and DNA oligonucleotides can be more practical probes to study protein–RNA and protein–DNA interactions; thereby, the binding complexes can be pulled down with streptavidin-conjugated resins and identified by Western blotting. However, setting up RNA and DNA pull-down assays with biotinylated probes in optimum protein binding conditions remains challenging. Here, we demonstrate the step-by step optimization of pull-down for IRP (iron-responsive-element-binding protein) with a 5′-biotinylated stem-loop IRE (iron-responsive element) RNA, HuR, and AUF1 with an AU-rich RNA element and Nrf2 binding to an antioxidant-responsive element (ARE) enhancer in the human ferritin H gene. This study was designed to address key technical questions in RNA and DNA pull-down assays: (1) how much RNA and DNA probes we should use; (2) what binding buffer and cell lysis buffer we can use; (3) how to verify the specific interaction; (4) what streptavidin resin (agarose or magnetic beads) works; and (5) what Western blotting results we can expect from varying to optimum conditions. We anticipate that our optimized pull-down conditions can be applicable to other RNA- and DNA-binding proteins along with emerging non-coding small RNA-binding proteins for their in vitro characterization. Full article

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23 pages, 2434 KiB

Open AccessReview

Induced Pluripotent Stem Cell-Derived Organoids: Their Implication in COVID-19 Modeling

by Mária Csöbönyeiová, Martin Klein, Marcela Kuniaková, Ivan Varga and Ľuboš Danišovič

Int. J. Mol. Sci. 2023, 24(4), 3459; https://doi.org/10.3390/ijms24043459 - 9 Feb 2023

Cited by 2 | Viewed by 3810

Abstract

The outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a significant global health issue. This novel virus’s high morbidity and mortality rates have prompted the scientific community to quickly find the best COVID-19 model [...] Read more.

The outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a significant global health issue. This novel virus’s high morbidity and mortality rates have prompted the scientific community to quickly find the best COVID-19 model to investigate all pathological processes underlining its activity and, more importantly, search for optimal drug therapy with minimal toxicity risk. The gold standard in disease modeling involves animal and monolayer culture models; however, these models do not fully reflect the response to human tissues affected by the virus. However, more physiological 3D in vitro culture models, such as spheroids and organoids derived from induced pluripotent stem cells (iPSCs), could serve as promising alternatives. Different iPSC-derived organoids, such as lung, cardiac, brain, intestinal, kidney, liver, nasal, retinal, skin, and pancreatic organoids, have already shown immense potential in COVID-19 modeling. In the present comprehensive review article, we summarize the current knowledge on COVID-19 modeling and drug screening using selected iPSC-derived 3D culture models, including lung, brain, intestinal, cardiac, blood vessels, liver, kidney, and inner ear organoids. Undoubtedly, according to reviewed studies, organoids are the state-of-the-art approach to COVID-19 modeling. Full article

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24 pages, 2405 KiB

Open AccessReview

The Chromatin Landscape around DNA Double-Strand Breaks in Yeast and Its Influence on DNA Repair Pathway Choice

by Chiara Frigerio, Elena Di Nisio, Michela Galli, Chiara Vittoria Colombo, Rodolfo Negri and Michela Clerici

Int. J. Mol. Sci. 2023, 24(4), 3248; https://doi.org/10.3390/ijms24043248 - 7 Feb 2023

Cited by 12 | Viewed by 4305

Abstract

DNA double-strand breaks (DSBs) are harmful DNA lesions, which elicit catastrophic consequences for genome stability if not properly repaired. DSBs can be repaired by either non-homologous end joining (NHEJ) or homologous recombination (HR). The choice between these two pathways depends on which proteins [...] Read more.

DNA double-strand breaks (DSBs) are harmful DNA lesions, which elicit catastrophic consequences for genome stability if not properly repaired. DSBs can be repaired by either non-homologous end joining (NHEJ) or homologous recombination (HR). The choice between these two pathways depends on which proteins bind to the DSB ends and how their action is regulated. NHEJ initiates with the binding of the Ku complex to the DNA ends, while HR is initiated by the nucleolytic degradation of the 5′-ended DNA strands, which requires several DNA nucleases/helicases and generates single-stranded DNA overhangs. DSB repair occurs within a precisely organized chromatin environment, where the DNA is wrapped around histone octamers to form the nucleosomes. Nucleosomes impose a barrier to the DNA end processing and repair machinery. Chromatin organization around a DSB is modified to allow proper DSB repair either by the removal of entire nucleosomes, thanks to the action of chromatin remodeling factors, or by post-translational modifications of histones, thus increasing chromatin flexibility and the accessibility of repair enzymes to the DNA. Here, we review histone post-translational modifications occurring around a DSB in the yeast Saccharomyces cerevisiae and their role in DSB repair, with particular attention to DSB repair pathway choice. Full article

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34 pages, 10155 KiB

Open AccessArticle

Dissociation Mode of the O–H Bond in Betanidin, pK_a-Clusterization Prediction, and Molecular Interactions via Shape Theory and DFT Methods

by Iliana María Ramírez-Velásquez, Álvaro H. Bedoya-Calle, Ederley Vélez and Francisco J. Caro-Lopera

Int. J. Mol. Sci. 2023, 24(3), 2923; https://doi.org/10.3390/ijms24032923 - 2 Feb 2023

Viewed by 1936

Abstract

Betanidin (Bd) is a nitrogenous metabolite with significant bioactive potential influenced by pH. Its free radical scavenging activity and deprotonation pathway are crucial to studying its physicochemical properties. Motivated by the published discrepancies about the best deprotonation routes in Bd, this work explores [...] Read more.

Betanidin (Bd) is a nitrogenous metabolite with significant bioactive potential influenced by pH. Its free radical scavenging activity and deprotonation pathway are crucial to studying its physicochemical properties. Motivated by the published discrepancies about the best deprotonation routes in Bd, this work explores all possible pathways for proton extractions on that molecule, by using the direct approach method based on pK_a. The complete space of exploration is supported by a linear relation with constant slope, where the pK_a is written in terms of the associated deprotonated molecule energy. The deprotonation rounds 1, …, 6 define groups of parallel linear models with constant slope. The intercepts of the models just depend on the protonated energy for each round, and then the pK_a can be trivially ordered and explained by the energy. We use the direct approximation method to obtain the value of pK_a. We predict all possible outcomes based on a linear model of the energy and some related verified assumptions. We also include a new measure of similarity or dissimilarity between the protonated and deprotonated molecules, via a geometric–chemical descriptor called the Riemann–Mulliken distance (RMD). The RMD considers the cartesian coordinates of the atoms, the atomic mass, and the Mulliken charges. After exploring the complete set of permutations, we show that the successive deprotonation process does not inherit the local energy minimum and that the commutativity of the paths does not hold either. The resulting clusterization of pK_a can be explained by the local acid and basic groups of the BD, and the successive deprotonation can be predicted by using the chemical explained linear models, which can avoid unnecessary optimizations. Another part of the research uses our own algorithm based on shape theory to determine the protein’s active site automatically, and molecular dynamics confirmed the results of the molecular docking of Bd in protonated and anionic form with the enzyme aldose reductase (AR). Also, we calculate the descriptors associated with the SET and SPLET mechanisms. Full article

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21 pages, 1842 KiB

Open AccessReview

Reflections on the Biology of Cell Culture Models: Living on the Edge of Oxidative Metabolism in Cancer Cells

by Alba Moran-Alvarez, Pedro Gonzalez-Menendez, Juan C. Mayo and Rosa M. Sainz

Int. J. Mol. Sci. 2023, 24(3), 2717; https://doi.org/10.3390/ijms24032717 - 1 Feb 2023

Cited by 4 | Viewed by 3470

Abstract

Nowadays, the study of cell metabolism is a hot topic in cancer research. Many studies have used 2D conventional cell cultures for their simplicity and the facility to infer mechanisms. However, the limitations of bidimensional cell cultures to recreate architecture, mechanics, and cell [...] Read more.

Nowadays, the study of cell metabolism is a hot topic in cancer research. Many studies have used 2D conventional cell cultures for their simplicity and the facility to infer mechanisms. However, the limitations of bidimensional cell cultures to recreate architecture, mechanics, and cell communication between tumor cells and their environment, have forced the development of other more realistic in vitro methodologies. Therefore, the explosion of 3D culture techniques and the necessity to reduce animal experimentation to a minimum has attracted the attention of researchers in the field of cancer metabolism. Here, we revise the limitations of actual culture models and discuss the utility of several 3D culture techniques to resolve those limitations. Full article

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22 pages, 5468 KiB

Open AccessArticle

PTCHD1 Binds Cholesterol but Not Sonic Hedgehog, Suggesting a Distinct Cellular Function

by Mimmu K. Hiltunen, Alex J. Timmis, Maren Thomsen, Danai S. Gkotsi, Hideo Iwaï, Orquidea M. Ribeiro, Adrian Goldman and Natalia A. Riobo-Del Galdo

Int. J. Mol. Sci. 2023, 24(3), 2682; https://doi.org/10.3390/ijms24032682 - 31 Jan 2023

Cited by 2 | Viewed by 3130

Abstract

Deleterious mutations in the X-linked Patched domain-containing 1 (PTCHD1) gene may account for up to 1% of autism cases. Despite this, the PTCHD1 protein remains poorly understood. Structural similarities to Patched family proteins point to a role in sterol transport, but this hypothesis [...] Read more.

Deleterious mutations in the X-linked Patched domain-containing 1 (PTCHD1) gene may account for up to 1% of autism cases. Despite this, the PTCHD1 protein remains poorly understood. Structural similarities to Patched family proteins point to a role in sterol transport, but this hypothesis has not been verified experimentally. Additionally, PTCHD1 has been suggested to be involved in Hedgehog signalling, but thus far, the experimental results have been conflicting. To enable a variety of biochemical and structural experiments, we developed a method for expressing PTCHD1 in Spodoptera frugiperda cells, solubilising it in glycol-diosgenin, and purifying it to homogeneity. In vitro and in silico experiments show that PTCHD1 function is not interchangeable with Patched 1 (PTCH1) in canonical Hedgehog signalling, since it does not repress Smoothened in Ptch1^−/− mouse embryonic fibroblasts and does not bind Sonic Hedgehog. However, we found that PTCHD1 binds cholesterol similarly to PTCH1. Furthermore, we identified 13 PTCHD1-specific protein interactors through co-immunoprecipitation and demonstrated a link to cell stress responses and RNA stress granule formation. Thus, our results support the notion that despite structural similarities to other Patched family proteins, PTCHD1 may have a distinct cellular function. Full article

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9 pages, 5177 KiB

Open AccessCommunication

Investigation of Glucose–Water Mixtures as a Function of Concentration and Temperature by Infrared Spectroscopy

by Maria Teresa Caccamo and Salvatore Magazù

Int. J. Mol. Sci. 2023, 24(3), 2564; https://doi.org/10.3390/ijms24032564 - 29 Jan 2023

Viewed by 2314

Abstract

The main aim of the present paper is to characterize the hydration properties of glucose and the hydrogen bond network in glucose–water mixtures. For these purposes, temperature scans on ten concentration values of glucose–water mixtures were performed by means of Fourier Transform InfraRed [...] Read more.

The main aim of the present paper is to characterize the hydration properties of glucose and the hydrogen bond network in glucose–water mixtures. For these purposes, temperature scans on ten concentration values of glucose–water mixtures were performed by means of Fourier Transform InfraRed (FTIR) spectroscopy. More specifically, in order to get this information an analysis of the intramolecular OH stretching mode, investigating the 3000–3700 cm⁻¹ spectral range, was performed by means of an innovative approach based on the evaluation of the Spectral Distance (SD). The adopted procedure allows evaluating the glucose hydration number as well as characterizing the temperature behavior of the hydrogen bond network in the glucose–water mixtures. The obtained results for the hydration number are in excellent agreement with literature data and suggest the existence of a particular concentration value for which the hydrogen bond network shows a maximum strength. Full article

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15 pages, 2635 KiB

Open AccessArticle

Novel Insight into the Role of Squalene Epoxidase (SQLE) Gene in Determining Milk Production Traits in Buffalo

by Chao Chen, Xiangwei Hu, Muhammad Jamil Ahmad, Kaifeng Niu, Tingzhu Ye, Aixin Liang and Liguo Yang

Int. J. Mol. Sci. 2023, 24(3), 2436; https://doi.org/10.3390/ijms24032436 - 26 Jan 2023

Cited by 1 | Viewed by 2494

Abstract

Understanding the genetic mechanisms underlying milk production traits contribute to improving the production potential of dairy animals. Squalene epoxidase (SQLE) is one of the rate-limiting enzymes for cholesterol biosynthesis and was highly expressed in the buffalo mammary. The objectives of the [...] Read more.

Understanding the genetic mechanisms underlying milk production traits contribute to improving the production potential of dairy animals. Squalene epoxidase (SQLE) is one of the rate-limiting enzymes for cholesterol biosynthesis and was highly expressed in the buffalo mammary. The objectives of the present study were to detect the polymorphisms within SQLE in buffalo, the genetic effects of these mutations on milk production traits, and to understand the gene regulatory effects on buffalo mammary epithelial cells (BuMECs). A total of five SNPs were identified by sequencing, g.18858G > A loci were significantly associated with fat yield, and g.22834C > T loci were significantly associated with peak milk yield, milk yield, fat yield, and protein yield. Notably, linkage disequilibrium analysis indicated that 2 SNPs (g.18858G > A and g.22834C > T) formed one haplotype block, which was found to be significantly associated with milk fat yield, fat percentage, and protein yield. Furthermore, expression of SQLE was measured in different tissues of buffalo and was found to be higher in the mammary. Knockdown of SQLE gene expression significantly affected the growth of BuMECs, including proliferation, cell cycle, and apoptosis, and significantly downregulated the expression of related genes MYC, PCNA, and P21. In addition, knockdown of the SQLE gene significantly reduces triglyceride concentrations and the signal intensity of oil red O staining. In addition, silencing of SQLE was also found to regulate the synthesis and secretion of β-casein and κ-casein negatively. Furthermore, SQLE knockdown is accompanied by the downregulation of critical genes (RPS6KB1, JAK2, eIF4E, and SREBP1) related to milk fat and protein synthesis. The current study showed the potential of the SQLE gene as a candidate for buffalo milk production traits. It provides a new understanding of the physiological mechanisms underlying buffalo milk production regulation. Full article

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26 pages, 423 KiB

Open AccessPerspective

Understanding How Cells Probe the World: A Preliminary Step towards Modeling Cell Behavior?

by Pierre Bongrand

Int. J. Mol. Sci. 2023, 24(3), 2266; https://doi.org/10.3390/ijms24032266 - 23 Jan 2023

Cited by 3 | Viewed by 2438

Abstract

Cell biologists have long aimed at quantitatively modeling cell function. Recently, the outstanding progress of high-throughput measurement methods and data processing tools has made this a realistic goal. The aim of this paper is twofold: First, to suggest that, while much progress has [...] Read more.

Cell biologists have long aimed at quantitatively modeling cell function. Recently, the outstanding progress of high-throughput measurement methods and data processing tools has made this a realistic goal. The aim of this paper is twofold: First, to suggest that, while much progress has been done in modeling cell states and transitions, current accounts of environmental cues driving these transitions remain insufficient. There is a need to provide an integrated view of the biochemical, topographical and mechanical information processed by cells to take decisions. It might be rewarding in the near future to try to connect cell environmental cues to physiologically relevant outcomes rather than modeling relationships between these cues and internal signaling networks. The second aim of this paper is to review exogenous signals that are sensed by living cells and significantly influence fate decisions. Indeed, in addition to the composition of the surrounding medium, cells are highly sensitive to the properties of neighboring surfaces, including the spatial organization of anchored molecules and substrate mechanical and topographical properties. These properties should thus be included in models of cell behavior. It is also suggested that attempts at cell modeling could strongly benefit from two research lines: (i) trying to decipher the way cells encode the information they retrieve from environment analysis, and (ii) developing more standardized means of assessing the quality of proposed models, as was done in other research domains such as protein structure prediction. Full article

16 pages, 5208 KiB

Open AccessArticle

Exploring the Biological Properties of Zn(II) Bisthiosemicarbazone Helicates

by Sandra Fernández-Fariña, Isabel Velo-Heleno, Rocío Carballido, Miguel Martínez-Calvo, Ramiro Barcia, Òscar Palacios, Mercè Capdevila, Ana M. González-Noya and Rosa Pedrido

Int. J. Mol. Sci. 2023, 24(3), 2246; https://doi.org/10.3390/ijms24032246 - 23 Jan 2023

Cited by 6 | Viewed by 2350

Abstract

The design of artificial helicoidal molecules derived from metal ions with biological properties is one of the objectives within metallosupramolecular chemistry. Herein, we report three zinc helicates derived from a family of bisthiosemicarbazone ligands with different terminal groups, Zn₂(L^Me [...] Read more.

The design of artificial helicoidal molecules derived from metal ions with biological properties is one of the objectives within metallosupramolecular chemistry. Herein, we report three zinc helicates derived from a family of bisthiosemicarbazone ligands with different terminal groups, Zn₂(L^Me)₂∙2H₂O 1, Zn₂(L^Ph)₂∙2H₂O 2 and Zn₂(L^PhNO2)₂3, obtained by an electrochemical methodology. These helicates have been fully characterized by different techniques, including X-ray diffraction. Biological studies of the zinc(II) helicates such as toxicity assays with erythrocytes and interaction studies with proteins and oligonucleotides were performed, demonstrating in all cases low toxicity and an absence of covalent interaction with the proteins and oligonucleotides. The in vitro cytotoxicity of the helicates was tested against MCF-7 (human breast carcinoma), A2780 (human ovarian carcinoma cells), NCI-H460 (human lung carcinoma cells) and MRC-5 (normal human lung fibroblasts), comparing the IC₅₀ values with cisplatin. We will try to demonstrate if the terminal substituent of the ligand precursor exerts any effect in toxicity or in the antitumor activity of the zinc helicates. Full article

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13 pages, 2757 KiB

Open AccessArticle

Single-Molecule Localization Microscopy of Presynaptic Active Zones in Drosophila melanogaster after Rapid Cryofixation

by Achmed Mrestani, Katharina Lichter, Anna-Leena Sirén, Manfred Heckmann, Mila M. Paul and Martin Pauli

Int. J. Mol. Sci. 2023, 24(3), 2128; https://doi.org/10.3390/ijms24032128 - 21 Jan 2023

Viewed by 3254

Abstract

Single-molecule localization microscopy (SMLM) greatly advances structural studies of diverse biological tissues. For example, presynaptic active zone (AZ) nanotopology is resolved in increasing detail. Immunofluorescence imaging of AZ proteins usually relies on epitope preservation using aldehyde-based immunocompetent fixation. Cryofixation techniques, such as high-pressure [...] Read more.

Single-molecule localization microscopy (SMLM) greatly advances structural studies of diverse biological tissues. For example, presynaptic active zone (AZ) nanotopology is resolved in increasing detail. Immunofluorescence imaging of AZ proteins usually relies on epitope preservation using aldehyde-based immunocompetent fixation. Cryofixation techniques, such as high-pressure freezing (HPF) and freeze substitution (FS), are widely used for ultrastructural studies of presynaptic architecture in electron microscopy (EM). HPF/FS demonstrated nearer-to-native preservation of AZ ultrastructure, e.g., by facilitating single filamentous structures. Here, we present a protocol combining the advantages of HPF/FS and direct stochastic optical reconstruction microscopy (dSTORM) to quantify nanotopology of the AZ scaffold protein Bruchpilot (Brp) at neuromuscular junctions (NMJs) of Drosophila melanogaster. Using this standardized model, we tested for preservation of Brp clusters in different FS protocols compared to classical aldehyde fixation. In HPF/FS samples, presynaptic boutons were structurally well preserved with ~22% smaller Brp clusters that allowed quantification of subcluster topology. In summary, we established a standardized near-to-native preparation and immunohistochemistry protocol for SMLM analyses of AZ protein clusters in a defined model synapse. Our protocol could be adapted to study protein arrangements at single-molecule resolution in other intact tissue preparations. Full article

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12 pages, 4380 KiB

Open AccessArticle

BMPR2 Variants Underlie Nonsyndromic Oligodontia

by Jinglei Zheng, Haochen Liu, Miao Yu, Bichen Lin, Kai Sun, Hangbo Liu, Hailan Feng, Yang Liu and Dong Han

Int. J. Mol. Sci. 2023, 24(2), 1648; https://doi.org/10.3390/ijms24021648 - 13 Jan 2023

Viewed by 2423

Abstract

Oligodontia manifests as a congenital reduction in the number of permanent teeth. Despite the major efforts that have been made, the genetic etiology of oligodontia remains largely unknown. Bone morphogenetic protein receptor type 2 (BMPR2) variants have been associated with pulmonary [...] Read more.

Oligodontia manifests as a congenital reduction in the number of permanent teeth. Despite the major efforts that have been made, the genetic etiology of oligodontia remains largely unknown. Bone morphogenetic protein receptor type 2 (BMPR2) variants have been associated with pulmonary arterial hypertension (PAH). However, the genetic significance of BMPR2 in oligodontia has not been previously reported. In the present study, we identified a novel heterozygous variant (c.814C > T; p.Arg272Cys) of BMPR2 in a family with nonsyndromic oligodontia by performing whole-exome sequencing. In addition, we identified two additional heterozygous variants (c.1042G > A; p.Val348Ile and c.1429A > G; p.Lys477Glu) among a cohort of 130 unrelated individuals with nonsyndromic oligodontia by performing Sanger sequencing. Functional analysis demonstrated that the activities of phospho-SMAD1/5/8 were significantly inhibited in BMPR2-knockout 293T cells transfected with variant-expressing plasmids, and were significantly lower in BMPR2 heterozygosity simulation groups than in the wild-type group, indicating that haploinsufficiency may represent the genetic mechanism. RNAscope in situ hybridization revealed that BMPR2 transcripts were highly expressed in the dental papilla and adjacent inner enamel epithelium in mice tooth germs, suggesting that BMPR2 may play important roles in tooth development. Our findings broaden the genetic spectrum of oligodontia and provide clinical and genetic evidence supporting the importance of BMPR2 in nonsyndromic oligodontia. Full article

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16 pages, 2047 KiB

Open AccessArticle

Clostridium butyricum Can Promote Bone Development by Regulating Lymphocyte Function in Layer Pullets

by Mengze Song, Xuesong Zhang, Guijuan Hao, Hai Lin and Shuhong Sun

Int. J. Mol. Sci. 2023, 24(2), 1457; https://doi.org/10.3390/ijms24021457 - 11 Jan 2023

Cited by 2 | Viewed by 2166

Abstract

Bone health problems are a serious threat to laying hens; microbiome-based therapies, which are harmless and inexpensive, may be an effective solution for bone health problems. Here, we examined the impacts of supplementation with Clostridium butyricum (CB) on bone and immune homeostasis in [...] Read more.

Bone health problems are a serious threat to laying hens; microbiome-based therapies, which are harmless and inexpensive, may be an effective solution for bone health problems. Here, we examined the impacts of supplementation with Clostridium butyricum (CB) on bone and immune homeostasis in pullets. The results of in vivo experiments showed that feeding the pullets CB was beneficial to the development of the tibia and upregulated the levels of the bone formation marker alkaline phosphatase and the marker gene runt-related transcription factor 2 (RUNX2). For the immune system, CB treatment significantly upregulated IL-10 expression and significantly increased the proportion of T regulatory (Treg) cells in the spleen and peripheral blood lymphocytes. In the in vitro test, adding CB culture supernatant or butyrate to the osteoblast culture system showed no significant effects on osteoblast bone formation, while adding lymphocyte culture supernatant significantly promoted bone formation. In addition, culture supernatants supplemented with treated lymphocytes (pretreated with CB culture supernatants) stimulated higher levels of bone formation. In sum, the addition of CB improved bone health by modulating cytokine expression and the ratio of Treg cells in the immune systems of layer pullets. Additionally, in vitro CB could promote the bone formation of laying hen osteoblasts through the mediation of lymphocytes. Full article

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24 pages, 4899 KiB

Open AccessArticle

Specific Binding of the α-Component of the Lantibiotic Lichenicidin to the Peptidoglycan Precursor Lipid II Predetermines Its Antimicrobial Activity

by Irina S. Panina, Sergey V. Balandin, Andrey V. Tsarev, Anton O. Chugunov, Andrey A. Tagaev, Ekaterina I. Finkina, Daria V. Antoshina, Elvira V. Sheremeteva, Alexander S. Paramonov, Jasmin Rickmeyer, Gabriele Bierbaum, Roman G. Efremov, Zakhar O. Shenkarev and Tatiana V. Ovchinnikova

Int. J. Mol. Sci. 2023, 24(2), 1332; https://doi.org/10.3390/ijms24021332 - 10 Jan 2023

Cited by 13 | Viewed by 2819

Abstract

To date, a number of lantibiotics have been shown to use lipid II—a highly conserved peptidoglycan precursor in the cytoplasmic membrane of bacteria—as their molecular target. The α-component (Lchα) of the two-component lantibiotic lichenicidin, previously isolated from the Bacillus licheniformis VK21 strain, seems [...] Read more.

To date, a number of lantibiotics have been shown to use lipid II—a highly conserved peptidoglycan precursor in the cytoplasmic membrane of bacteria—as their molecular target. The α-component (Lchα) of the two-component lantibiotic lichenicidin, previously isolated from the Bacillus licheniformis VK21 strain, seems to contain two putative lipid II binding sites in its N-terminal and C-terminal domains. Using NMR spectroscopy in DPC micelles, we obtained convincing evidence that the C-terminal mersacidin-like site is involved in the interaction with lipid II. These data were confirmed by the MD simulations. The contact area of lipid II includes pyrophosphate and disaccharide residues along with the first isoprene units of bactoprenol. MD also showed the potential for the formation of a stable N-terminal nisin-like complex; however, the conditions necessary for its implementation in vitro remain unknown. Overall, our results clarify the picture of two component lantibiotics mechanism of antimicrobial action. Full article

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21 pages, 15408 KiB

Open AccessArticle

Structural Dynamics of the Bacillus subtilis MntR Transcription Factor Is Locked by Mn²⁺ Binding

by Zoe Jelić Matošević, Katarina Radman, Jolene Loubser, Ivo Crnolatac, Ivo Piantanida, Ignacy Cukrowski, Ivana Leščić Ašler and Branimir Bertoša

Int. J. Mol. Sci. 2023, 24(2), 957; https://doi.org/10.3390/ijms24020957 - 4 Jan 2023

Cited by 3 | Viewed by 2437

Abstract

Manganese (II) ions are essential for a variety of bacterial cellular processes. The transcription factor MntR is a metallosensor that regulates Mn²⁺ ion homeostasis in the bacterium Bacillus subtilis. Its DNA-binding affinity is increased by Mn²⁺ ion binding, allowing it [...] Read more.

Manganese (II) ions are essential for a variety of bacterial cellular processes. The transcription factor MntR is a metallosensor that regulates Mn²⁺ ion homeostasis in the bacterium Bacillus subtilis. Its DNA-binding affinity is increased by Mn²⁺ ion binding, allowing it to act as a transcriptional repressor of manganese import systems. Although experimentally well-researched, the molecular mechanism that regulates this process is still a puzzle. Computational simulations supported by circular dichroism (CD), differential scanning calorimetry (DSC) and native gel electrophoresis (native-PAGE) experiments were employed to study MntR structural and dynamical properties in the presence and absence of Mn²⁺ ions. The results of molecular dynamics (MD) simulations revealed that Mn²⁺ ion binding reduces the structural dynamics of the MntR protein and shifts the dynamic equilibrium towards the conformations adequate for DNA binding. Results of CD and DSC measurements support the computational results showing the change in helical content and stability of the MntR protein upon Mn²⁺ ion binding. Further, MD simulations show that Mn²⁺ binding induces polarization of the protein electrostatic potential, increasing the positive electrostatic potential of the DNA-binding helices in particular. In order to provide a deeper understanding of the changes in protein structure and dynamics due to Mn²⁺ binding, a mutant in which Mn²⁺ binding is mimicked by a cysteine bridge was constructed and also studied computationally and experimentally. Full article

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2022

Jump to: 2025, 2024, 2023, 2021, 2020, 2019

21 pages, 2163 KiB

Open AccessReview

SurA-like and Skp-like Proteins as Important Virulence Determinants of the Gram Negative Bacterial Pathogens

by Donata Figaj, Patrycja Ambroziak, Iga Rzepka and Joanna Skórko-Glonek

Int. J. Mol. Sci. 2023, 24(1), 295; https://doi.org/10.3390/ijms24010295 - 24 Dec 2022

Cited by 7 | Viewed by 3297

Abstract

In the Gram-negative bacteria, many important virulence factors reach their destination via two-step export systems, and they must traverse the periplasmic space before reaching the outer membrane. Since these proteins must be maintained in a structure competent for transport into or across the [...] Read more.

In the Gram-negative bacteria, many important virulence factors reach their destination via two-step export systems, and they must traverse the periplasmic space before reaching the outer membrane. Since these proteins must be maintained in a structure competent for transport into or across the membrane, they frequently require the assistance of chaperones. Based on the results obtained for the model bacterium Escherichia coli and related species, it is assumed that in the biogenesis of the outer membrane proteins and the periplasmic transit of secretory proteins, the SurA peptidyl–prolyl isomerase/chaperone plays a leading role, while the Skp chaperone is rather of secondary importance. However, detailed studies carried out on several other Gram-negative pathogens indicate that the importance of individual chaperones in the folding and transport processes depends on the properties of client proteins and is species-specific. Taking into account the importance of SurA functions in bacterial virulence and severity of phenotypes due to surA mutations, this folding factor is considered as a putative therapeutic target to combat microbial infections. In this review, we present recent findings regarding SurA and Skp proteins: their mechanisms of action, involvement in processes related to virulence, and perspectives to use them as therapeutic targets. Full article

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17 pages, 6735 KiB

Open AccessReview

Molecular Regulation of Concomitant Lower Urinary Tract Symptoms and Erectile Dysfunction in Pelvic Ischemia

by Tufan Tarcan, Han-Pil Choi and Kazem M. Azadzoi

Int. J. Mol. Sci. 2022, 23(24), 15988; https://doi.org/10.3390/ijms232415988 - 15 Dec 2022

Cited by 5 | Viewed by 2287

Abstract

Aging correlates with greater incidence of lower urinary tract symptoms (LUTS) and erectile dysfunction (ED) in the male population where the pathophysiological link remains elusive. The incidence of LUTS and ED correlates with the prevalence of vascular risk factors, implying potential role of [...] Read more.

Aging correlates with greater incidence of lower urinary tract symptoms (LUTS) and erectile dysfunction (ED) in the male population where the pathophysiological link remains elusive. The incidence of LUTS and ED correlates with the prevalence of vascular risk factors, implying potential role of arterial disorders in concomitant development of the two conditions. Human studies have revealed lower bladder and prostate blood flow in patients with LUTS suggesting that the severity of LUTS and ED correlates with the severity of vascular disorders. A close link between increased prostatic vascular resistance and greater incidence of LUTS and ED has been documented. Experimental models of atherosclerosis-induced chronic pelvic ischemia (CPI) showed increased contractile reactivity of prostatic and bladder tissues, impairment of penile erectile tissue relaxation, and simultaneous development of detrusor overactivity and ED. In the bladder, short-term ischemia caused overactive contractions while prolonged ischemia provoked degenerative responses and led to underactivity. CPI compromised structural integrity of the bladder, prostatic, and penile erectile tissues. Downstream molecular mechanisms appear to involve cellular stress and survival signaling, receptor modifications, upregulation of cytokines, and impairment of the nitric oxide pathway in cavernosal tissue. These observations may suggest pelvic ischemia as an important contributing factor in LUTS-associated ED. The aim of this narrative review is to discuss the current evidence on CPI as a possible etiologic mechanism underlying LUTS-associated ED. Full article

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16 pages, 3757 KiB

Open AccessArticle

Remarkable Plasticity of Bone Iron Homeostasis in Hibernating Daurian Ground Squirrels (Spermophilus dauricus) May Be Involved in Bone Maintenance

by Yue He, Yong Kong, Rongrong Yin, Huajian Yang, Jie Zhang, Huiping Wang and Yunfang Gao

Int. J. Mol. Sci. 2022, 23(24), 15858; https://doi.org/10.3390/ijms232415858 - 13 Dec 2022

Cited by 3 | Viewed by 1892

Abstract

Iron overload is an independent risk factor for disuse osteoporosis. Hibernating animals are natural models of anti-disuse osteoporosis; however, whether iron metabolism is involved in bone adaptation and maintenance during hibernation is unclear. To investigate this question, Daurian ground squirrels (Spermophilus dauricus [...] Read more.

Iron overload is an independent risk factor for disuse osteoporosis. Hibernating animals are natural models of anti-disuse osteoporosis; however, whether iron metabolism is involved in bone adaptation and maintenance during hibernation is unclear. To investigate this question, Daurian ground squirrels (Spermophilus dauricus) (n = 5–6/group) were used to study changes in bone iron metabolism and its possible role in anti-disuse osteoporosis during hibernation. Iron content in the femur and liver first decreased in the torpor group (vs. summer group, −66.8% and −25.8%, respectively), then recovered in the post-hibernation group, suggesting remarkable plasticity of bone iron content. The expression of ferritin in the femur and hepcidin in the liver also initially decreased in the torpor group (vs. summer group, −28.5% and −38.8%, respectively), then increased in the inter-bout arousal (vs. torpor group, 126.2% and 58.4%, respectively) and post-hibernation groups (vs. torpor group, 153.1% and 27.1%, respectively). In conclusion, bone iron metabolism in hibernating Daurian ground squirrels showed remarkable plasticity, which may be a potential mechanism to avoid disuse bone loss during extended periods of inactivity. However, the specific location of iron during low-iron hibernation and the source of iron in post-hibernation recovery need to be further explored. Full article

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18 pages, 3493 KiB

Open AccessArticle

L-RAPiT: A Cloud-Based Computing Pipeline for the Analysis of Long-Read RNA Sequencing Data

by Theodore M. Nelson, Sankar Ghosh and Thomas S. Postler

Int. J. Mol. Sci. 2022, 23(24), 15851; https://doi.org/10.3390/ijms232415851 - 13 Dec 2022

Cited by 2 | Viewed by 3473

Abstract

Long-read sequencing (LRS) has been adopted to meet a wide variety of research needs, ranging from the construction of novel transcriptome annotations to the rapid identification of emerging virus variants. Amongst other advantages, LRS preserves more information about RNA at the transcript level [...] Read more.

Long-read sequencing (LRS) has been adopted to meet a wide variety of research needs, ranging from the construction of novel transcriptome annotations to the rapid identification of emerging virus variants. Amongst other advantages, LRS preserves more information about RNA at the transcript level than conventional high-throughput sequencing, including far more accurate and quantitative records of splicing patterns. New studies with LRS datasets are being published at an exponential rate, generating a vast reservoir of information that can be leveraged to address a host of different research questions. However, mining such publicly available data in a tailored fashion is currently not easy, as the available software tools typically require familiarity with the command-line interface, which constitutes a significant obstacle to many researchers. Additionally, different research groups utilize different software packages to perform LRS analysis, which often prevents a direct comparison of published results across different studies. To address these challenges, we have developed the Long-Read Analysis Pipeline for Transcriptomics (L-RAPiT), a user-friendly, free pipeline requiring no dedicated computational resources or bioinformatics expertise. L-RAPiT can be implemented directly through Google Colaboratory, a system based on the open-source Jupyter notebook environment, and allows for the direct analysis of transcriptomic reads from Oxford Nanopore and PacBio LRS machines. This new pipeline enables the rapid, convenient, and standardized analysis of publicly available or newly generated LRS datasets. Full article

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11 pages, 2244 KiB

Open AccessArticle

Structural Change from Nonparallel to Parallel G-Quadruplex Structures in Live Cancer Cells Detected in the Lysosomes Using Fluorescence Lifetime Imaging Microscopy

by Ting-Yuan Tseng, Chiung-Lin Wang and Ta-Chau Chang

Int. J. Mol. Sci. 2022, 23(24), 15799; https://doi.org/10.3390/ijms232415799 - 13 Dec 2022

Cited by 1 | Viewed by 1451

Abstract

Time-gated fluorescence lifetime imaging microscopy with the o-BMVC fluorescent probe provides a visualizing method for the study of exogenous G-quadruplexes (G4s) in live cancer cells. Previously, imaging results showed that the parallel G4s are accumulated and that nonparallel G4s are not detected [...] Read more.

Time-gated fluorescence lifetime imaging microscopy with the o-BMVC fluorescent probe provides a visualizing method for the study of exogenous G-quadruplexes (G4s) in live cancer cells. Previously, imaging results showed that the parallel G4s are accumulated and that nonparallel G4s are not detected in the lysosomes of CL1-0 live cells. In this work, the detection of the G4 signals from exogenous GTERT-d(FN) G4s in the lysosomes may involve a structural change in live cells from intramolecular nonparallel G4s to intermolecular parallel G4s. Moreover, the detection of the G4 signals in the lysosomes after the 48 h incubation of HT23 G4s with CL1-0 live cells indicates the occurrence of structural conversion from the nonparallel G4s to the parallel G4s of HT23 in the live cells. In addition, the detection of much stronger G4 signals from ss-GTERT-d(FN) than ss-HT23 in the lysosomes of CL1-0 live cells may be explained by the quick formation of the intermolecular parallel G4s of ss-GTERT-d(FN) and the degradation of ss-HT23 before its intramolecular parallel G4 formation. This work provides a new approach to studying G4-lysosome interactions in live cells. Full article

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18 pages, 5566 KiB

Open AccessArticle

Antimicrobial Peptide LCN2 Inhibited Uropathogenic Escherichia coli Infection in Bladder Cells in a High-Glucose Environment through JAK/STAT Signaling Pathway

by Pei-Chi Chen, Chen-Hsun Ho, Chia-Kwung Fan, Shih-Ping Liu and Po-Ching Cheng

Int. J. Mol. Sci. 2022, 23(24), 15763; https://doi.org/10.3390/ijms232415763 - 12 Dec 2022

Cited by 5 | Viewed by 2249

Abstract

JAK/STAT plays a key role in regulating uropathogenic Escherichia coli (UPEC) infection in urothelial cells, probably via antimicrobial peptide (AMP) production, in diabetic patients with urinary tract infections. Whether multiple pathways regulate AMPs, especially lipid-carrying protein-2 (LCN2), to achieve a vital effect is [...] Read more.

JAK/STAT plays a key role in regulating uropathogenic Escherichia coli (UPEC) infection in urothelial cells, probably via antimicrobial peptide (AMP) production, in diabetic patients with urinary tract infections. Whether multiple pathways regulate AMPs, especially lipid-carrying protein-2 (LCN2), to achieve a vital effect is unknown. We investigated the effects of an LCN2 pretreatment on the regulation of the JAK/STAT pathway in a high-glucose environment using a bladder cell model with GFP-UPEC and phycoerythrin-labeled TLR-4, STAT1, and STAT3. Pretreatment with 5 or 25 μg/mL LCN2 for 24 h dose-dependently suppressed UPEC infections in bladder cells. TLR-4, STAT1, and STAT3 expression were dose-dependently downregulated after LCN2 pretreatment. The LCN2-mediated alleviation of UPEC infection in a high-glucose environment downregulated TLR-4 and the JAK/STAT transduction pathway and decreased the UPEC-induced secretion of exogenous inflammatory interleukin (IL)-6 and IL-8. Our study provides evidence that LCN2 can alleviate UPEC infection in bladder epithelial cells by decreasing JAK/STAT pathway activation in a high-glucose environment. LCN2 dose-dependently inhibits UPEC infection via TLR-4 expression and JAK/STAT pathway modulation. These findings may provide a rationale for targeting LCN2/TLR-4/JAK/STAT regulation in bacterial cystitis treatment. Further studies should explore specific mechanisms by which the LCN2, TLR-4, and JAK/STAT pathways participate in UPEC-induced inflammation to facilitate the development of effective therapies for cystitis. Full article

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13 pages, 3283 KiB

Open AccessArticle

Human Dental Pulp Stem Cells Differentiate into Cementoid-Like-Secreting Cells on Decellularized Teeth Scaffolds

by Manuel Mata, Santiago Peydró, José Javier Martín de Llano, María Sancho-Tello and Carmen Carda

Int. J. Mol. Sci. 2022, 23(24), 15588; https://doi.org/10.3390/ijms232415588 - 9 Dec 2022

Cited by 1 | Viewed by 1815

Abstract

Periodontitis is a common inflammatory disease that in some cases can cause tooth loss. Cementum is a mineralized tissue that forms part of the insertion periodontium and serves to fix the teeth to the alveolar bone. In addition, it acts as a reservoir [...] Read more.

Periodontitis is a common inflammatory disease that in some cases can cause tooth loss. Cementum is a mineralized tissue that forms part of the insertion periodontium and serves to fix the teeth to the alveolar bone. In addition, it acts as a reservoir of different growth and differentiation factors, which regulate the biology of the teeth. Cementogenesis is a complex process that is still under investigation and involves different factors, including dentin sialophosphoprotein (DSPP). In this work we studied the role of surface microtopography in the differentiation of human dental pulp stem cells (hDPSCs) into cementoid-like secreting cells. We cultured hDPSCs on decellularized dental scaffolds on either dentin or cementum surfaces. Cell morphology was evaluated by light and electron microscopy. We also evaluated the DSPP expression by immunohistochemistry. The hDPSCs that was cultured on surfaces with accessible dentinal tubules acquired an odontoblastic phenotype and emitted characteristic processes within the dentinal tubules. These cells synthesized the matrix components of a characteristic reticular connective tissue, with fine collagen fibers and DSPP deposits. The hDPSCs that was cultured on cementum surfaces generated a well-organized tissue consisting of layers of secretory cells and dense fibrous connective tissue with thick bundles of collagen fibers perpendicular to the scaffold surface. Intra- and intercellular deposits of DSPP were also observed. The results presented here reinforce the potential for hDPSCs to differentiate in vitro into cells that secrete a cementoid-like matrix in response to the physical stimuli related to the microtopography of contact surfaces. We also highlight the role of DSPP as a component of the newly formed matrix. Full article

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15 pages, 2611 KiB

Open AccessArticle

INO80 Is Required for the Cell Cycle Control, Survival, and Differentiation of Mouse ESCs by Transcriptional Regulation

by Seonho Yoo, Eun Joo Lee, Nguyen Xuan Thang, Hyeonwoo La, Hyeonji Lee, Chanhyeok Park, Dong Wook Han, Sang Jun Uhm, Hyuk Song, Jeong Tae Do, Youngsok Choi and Kwonho Hong

Int. J. Mol. Sci. 2022, 23(23), 15402; https://doi.org/10.3390/ijms232315402 - 6 Dec 2022

Cited by 6 | Viewed by 2705

Abstract

Precise regulation of the cell cycle of embryonic stem cells (ESCs) is critical for their self-maintenance and differentiation. The cell cycle of ESCs differs from that of somatic cells and is different depending on the cell culture conditions. However, the cell cycle regulation [...] Read more.

Precise regulation of the cell cycle of embryonic stem cells (ESCs) is critical for their self-maintenance and differentiation. The cell cycle of ESCs differs from that of somatic cells and is different depending on the cell culture conditions. However, the cell cycle regulation in ESCs via epigenetic mechanisms remains unclear. Here, we showed that the ATP-dependent chromatin remodeler Ino80 regulates the cell cycle genes in ESCs under primed conditions. Ino80 loss led to a significantly extended length of the G1-phase in ESCs grown under primed culture conditions. Ino80 directly bound to the transcription start site and regulated the expression of cell cycle-related genes. Furthermore, Ino80 loss induced cell apoptosis. However, the regulatory mechanism of Ino80 in differentiating ESC cycle slightly differed; an extended S-phase was detected in differentiating inducible Ino80 knockout ESCs. RNA-seq analysis of differentiating ESCs revealed that the expression of genes associated with organ development cell cycle is persistently altered in Ino80 knockout cells, suggesting that cell cycle regulation by Ino80 is not limited to undifferentiated ESCs. Therefore, our study establishes the function of Ino80 in ESC cycle via transcriptional regulation, at least partly. Moreover, this Ino80 function may be universal to other cell types. Full article

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19 pages, 1785 KiB

Open AccessArticle

Endocannabinoid System Regulation in Female Rats with Recurrent Episodes of Binge Eating

by Mariangela Pucci, Claudio D’Addario, Emanuela Micioni Di Bonaventura, Francesca Mercante, Eugenia Annunzi, Federico Fanti, Manuel Sergi, Luca Botticelli, Giacomo Einaudi, Carlo Cifani and Maria Vittoria Micioni Di Bonaventura

Int. J. Mol. Sci. 2022, 23(23), 15228; https://doi.org/10.3390/ijms232315228 - 3 Dec 2022

Cited by 10 | Viewed by 2124

Abstract

Recurrent Binge Eating (BE) episodes characterize several eating disorders. Here, we attempted to reassemble a condition closer to BE disorder, and we analyzed whether recurrent episodes might evoke molecular alterations in the hypothalamus of rats. The hypothalamus is a brain region which is [...] Read more.

Recurrent Binge Eating (BE) episodes characterize several eating disorders. Here, we attempted to reassemble a condition closer to BE disorder, and we analyzed whether recurrent episodes might evoke molecular alterations in the hypothalamus of rats. The hypothalamus is a brain region which is sensitive to stress and relevant in motivated behaviors, such as food intake. A well-characterized animal model of BE, in which a history of intermittent food restriction and stress induce binge-like palatable food consumption, was used to analyze the transcriptional regulation of the endocannabinoid system (ECS). We detected, in rats showing the BE behavior, an up-regulated gene expression of cannabinoid type-1 receptor (CB1), sn-1-specific diacylglycerol lipase, as well as fatty acid amide hydrolase (Faah) and monoacylglycerol lipase. A selective reduction in DNA methylation was also observed at the promoter of Faah, which is consistent with the changes in the gene expression. Moreover, BE behavior in rats was associated with an increase in anandamide (AEA) levels. Our findings support the relevant role of the ECS in the regulation of food intake in rats subjected to repeated BE episodes, and, in particular, on AEA signaling, acting via CB1 and FAAH modulation. Notably, the epigenetic regulation of the Faah gene might suggest this enzyme as a possible target for developing new therapeutical approaches. Full article

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14 pages, 3037 KiB

Open AccessArticle

Differential Expression of RSK4 Transcript Isoforms in Cancer and Its Clinical Relevance

by Sisi Chen, Michael J. Seckl, Marc P. G. Lorentzen and Olivier E. Pardo

Int. J. Mol. Sci. 2022, 23(23), 14569; https://doi.org/10.3390/ijms232314569 - 23 Nov 2022

Cited by 1 | Viewed by 2380

Abstract

While we previously revealed RSK4 as a therapeutic target in lung and bladder cancers, the wider role of this kinase in other cancers remains controversial. Indeed, other reports instead proposed RSK4 as a tumour suppressor in colorectal and gastric cancers and are contradictory [...] Read more.

While we previously revealed RSK4 as a therapeutic target in lung and bladder cancers, the wider role of this kinase in other cancers remains controversial. Indeed, other reports instead proposed RSK4 as a tumour suppressor in colorectal and gastric cancers and are contradictory in breast malignancies. One explanation for these discrepancies may be the expression of different RSK4 isoforms across cancers. Four RNAs are produced from the RSK4 gene, with two being protein-coding. Here, we analysed the expression of the latter across 30 normal and 33 cancer tissue types from the combined GTEx/TCGA dataset and correlated it with clinical features. This revealed the expression of RSK4 isoforms 1 and 2 to be independent prognostic factors for patient survival, pathological stage, cancer metastasis, recurrence, and immune infiltration in brain, stomach, cervical, and kidney cancers. However, we found that upregulation of either isoform can equally be associated with good or bad prognosis depending on the cancer type, and changes in the expression ratio of isoforms fail to predict clinical outcome. Hence, differential isoform expression alone cannot explain the contradictory roles of RSK4 in cancers, and further research is needed to highlight the underlying mechanisms for the context-dependent function of this kinase. Full article

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18 pages, 3224 KiB

Open AccessArticle

Radiosensitizing Effect of Trabectedin on Human Soft Tissue Sarcoma Cells

by Mauro Loi, Giulia Salvatore, Michele Aquilano, Daniela Greto, Cinzia Talamonti, Viola Salvestrini, Maria Elena Melica, Marianna Valzano, Giulio Francolini, Mariangela Sottili, Costanza Santini, Carlotta Becherini, Domenico Andrea Campanacci, Monica Mangoni and Lorenzo Livi

Int. J. Mol. Sci. 2022, 23(22), 14305; https://doi.org/10.3390/ijms232214305 - 18 Nov 2022

Cited by 2 | Viewed by 2149

Abstract

Trabectedin is used for the treatment of advanced soft tissue sarcomas (STSs). In this study, we evaluated if trabectedin could enhance the efficacy of irradiation (IR) by increasing the intrinsic cell radiosensitivity and modulating tumor micro-environment in fibrosarcoma (HS 93.T), leiomyosarcoma (HS5.T), liposarcoma [...] Read more.

Trabectedin is used for the treatment of advanced soft tissue sarcomas (STSs). In this study, we evaluated if trabectedin could enhance the efficacy of irradiation (IR) by increasing the intrinsic cell radiosensitivity and modulating tumor micro-environment in fibrosarcoma (HS 93.T), leiomyosarcoma (HS5.T), liposarcoma (SW872), and rhabdomyosarcoma (RD) cell lines. A significant reduction in cell surviving fraction (SF) following trabectedin + IR compared to IR alone was observed in liposarcoma and leiomyosarcoma (enhancement ratio at 50%, ER50: 1.45 and 2.35, respectively), whereas an additive effect was shown in rhabdomyosarcoma and fibrosarcoma. Invasive cells’ fraction significantly decreased following trabectedin ± IR compared to IR alone. Differences in cell cycle distribution were observed in leiomyosarcoma and rhabdomyosarcoma treated with trabectedin + IR. In all STS lines, trabectedin + IR resulted in a significantly higher number of γ-H2AX (histone H2AX) foci 30 min compared to the control, trabectedin, or IR alone. Expression of ATM, RAD50, Ang-2, VEGF, and PD-L1 was not significantly altered following trabectedin + IR. In conclusion, trabectedin radiosensitizes STS cells by affecting SF (particularly in leiomyosarcoma and liposarcoma), invasiveness, cell cycle distribution, and γ-H2AX foci formation. Conversely, no synergistic effect was observed on DNA damage repair, neoangiogenesis, and immune system. Full article

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11 pages, 2800 KiB

Open AccessArticle

Polyphosphate Activates von Willebrand Factor Interaction with Glycoprotein Ib in the Absence of Factor VIII In Vitro

by Marcela Montilla, Isabel Atienza-Navarro, Francisco Jose García-Cozar, Carmen Castro, Francisco Javier Rodríguez-Martorell and Felix A. Ruiz

Int. J. Mol. Sci. 2022, 23(22), 14118; https://doi.org/10.3390/ijms232214118 - 15 Nov 2022

Cited by 2 | Viewed by 1895

Abstract

Polyphosphate (polyP), a phosphate polymer released by activated platelets, may modulate various stages of hemostasis by binding to blood proteins. In this context, we previously reported that polyP binds to the von Willebrand factor (VWF). One of the most significant functions of VWF [...] Read more.

Polyphosphate (polyP), a phosphate polymer released by activated platelets, may modulate various stages of hemostasis by binding to blood proteins. In this context, we previously reported that polyP binds to the von Willebrand factor (VWF). One of the most significant functions of VWF is to bind to and protect the blood circulating Factor VIII (FVIII). Therefore, here, we study the role of polyP in the VWF–FVIII complex in vitro and suggest its biological significance. Surface plasmon resonance and electrophoretic mobility assays indicated that polyP binds dynamically to VWF only in the absence of FVIII. Using the VWF Ristocetin Cofactor assay, the most accepted method for studying VWF in platelet adhesion, we found that polyP activates this role of VWF only at low levels of FVIII, such as in plasmas with chemically depleted FVIII and plasmas from severe hemophilia A patients. Moreover, we demonstrated that FVIII competes with polyP in the activation of VWF. Finally, polyP also increases the binding of VWF to platelets in samples from patients with type 2 and type 3 von Willebrand disease. We propose that polyP may be used in designing new therapies to activate VWF when FVIII cannot be used. Full article

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16 pages, 1723 KiB

Open AccessReview

Tumor-Derived Exosomes and Their Role in Breast Cancer Metastasis

by Shaojuan Huang, Ming Dong and Qiang Chen

Int. J. Mol. Sci. 2022, 23(22), 13993; https://doi.org/10.3390/ijms232213993 - 13 Nov 2022

Cited by 14 | Viewed by 4411

Abstract

Breast cancer has been the most common cancer in women worldwide, and metastasis is the leading cause of death from breast cancer. Even though the study of breast cancer metastasis has been extensively carried out, the molecular mechanism is still not fully understood, [...] Read more.

Breast cancer has been the most common cancer in women worldwide, and metastasis is the leading cause of death from breast cancer. Even though the study of breast cancer metastasis has been extensively carried out, the molecular mechanism is still not fully understood, and diagnosis and prognosis need to be improved. Breast cancer metastasis is a complicated process involving multiple physiological changes, and lung, brain, bone and liver are the main metastatic targets. Exosomes are membrane-bound extracellular vesicles that contain secreted cellular constitutes. The biogenesis and functions of exosomes in cancer have been intensively studied, and mounting studies have indicated that exosomes play a crucial role in cancer metastasis. In this review, we summarize recent findings on the role of breast cancer-derived exosomes in metastasis organotropism and discuss the potential promising clinical applications of targeting exosomes as novel strategies for breast cancer diagnosis and therapy. Full article

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20 pages, 2667 KiB

Open AccessArticle

Impact of JH Signaling on Reproductive Physiology of the Classical Insect Model, Rhodnius prolixus

by Jimena Leyria, Ian Orchard and Angela B. Lange

Int. J. Mol. Sci. 2022, 23(22), 13832; https://doi.org/10.3390/ijms232213832 - 10 Nov 2022

Cited by 14 | Viewed by 2479

Abstract

In adult females of several insect species, juvenile hormones (JHs) act as gonadotrophic hormones, regulating egg production. JH binds to its nuclear receptor, Methoprene tolerant (Met), triggering its dimerization with the protein Taiman (Tai). The resulting active complex induces transcription of JH response [...] Read more.

In adult females of several insect species, juvenile hormones (JHs) act as gonadotrophic hormones, regulating egg production. JH binds to its nuclear receptor, Methoprene tolerant (Met), triggering its dimerization with the protein Taiman (Tai). The resulting active complex induces transcription of JH response genes, such as Krüppel homolog 1 (Kr-h1). In this study we report for the first time the participation of the isoform JH III skipped bisepoxide (JHSB₃) and its signaling pathway in the reproductive fitness of the classical insect model Rhodnius prolixus. The topical application of synthetic JHSB₃ increases transcript and protein expression of yolk protein precursors (YPPs), mainly by the fat body but also by the ovaries, the second source of YPPs. These results are also confirmed by ex vivo assays. In contrast, when the JH signaling cascade is impaired via RNA interference by downregulating RhoprMet and RhoprTai mRNA, egg production is inhibited. Although RhoprKr-h1 transcript expression is highly dependent on JHSB₃ signaling, it is not involved in egg production but rather in successful hatching. This research contributes missing pieces of JH action in the insect model in which JH was first postulated almost 100 years ago. Full article

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17 pages, 2292 KiB

Open AccessReview

Neuroprotection and Non-Invasive Brain Stimulation: Facts or Fiction?

by Matteo Guidetti, Alessandro Bertini, Francesco Pirone, Gessica Sala, Paola Signorelli, Carlo Ferrarese, Alberto Priori and Tommaso Bocci

Int. J. Mol. Sci. 2022, 23(22), 13775; https://doi.org/10.3390/ijms232213775 - 9 Nov 2022

Cited by 16 | Viewed by 4174

Abstract

Non-Invasive Brain Stimulation (NIBS) techniques, such as transcranial Direct Current Stimulation (tDCS) and repetitive Magnetic Transcranial Stimulation (rTMS), are well-known non-pharmacological approaches to improve both motor and non-motor symptoms in patients with neurodegenerative disorders. Their use is of particular interest especially for the [...] Read more.

Non-Invasive Brain Stimulation (NIBS) techniques, such as transcranial Direct Current Stimulation (tDCS) and repetitive Magnetic Transcranial Stimulation (rTMS), are well-known non-pharmacological approaches to improve both motor and non-motor symptoms in patients with neurodegenerative disorders. Their use is of particular interest especially for the treatment of cognitive impairment in Alzheimer’s Disease (AD), as well as axial disturbances in Parkinson’s (PD), where conventional pharmacological therapies show very mild and short-lasting effects. However, their ability to interfere with disease progression over time is not well understood; recent evidence suggests that NIBS may have a neuroprotective effect, thus slowing disease progression and modulating the aggregation state of pathological proteins. In this narrative review, we gather current knowledge about neuroprotection and NIBS in neurodegenerative diseases (i.e., PD and AD), just mentioning the few results related to stroke. As further matter of debate, we discuss similarities and differences with Deep Brain Stimulation (DBS)—induced neuroprotective effects, and highlight possible future directions for ongoing clinical studies. Full article

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16 pages, 3187 KiB

Open AccessArticle

Revealing the Changes in Saliva and Serum Proteins of Pigs with Meningitis Caused by Streptococcus Suis: A Proteomic Approach

by María José López-Martínez, Anđelo Beletić, Josipa Kuleš, Dina Rešetar-Maslov, Ivana Rubić, Vladimir Mrljak, Edgar Garcia Manzanilla, Elena Goyena, Silvia Martínez-Subiela, José Joaquín Cerón and Alberto Muñoz-Prieto

Int. J. Mol. Sci. 2022, 23(22), 13700; https://doi.org/10.3390/ijms232213700 - 8 Nov 2022

Cited by 7 | Viewed by 2252

Abstract

Meningitis due to Streptococcus suis causes high mortality and morbidity on pig farms and has increasing zoonotic potential worldwide. Saliva proteome analysis would potentially be useful in elucidating pathophysiological changes and mining for new biomarkers to diagnose and monitor S. suis infection. The [...] Read more.

Meningitis due to Streptococcus suis causes high mortality and morbidity on pig farms and has increasing zoonotic potential worldwide. Saliva proteome analysis would potentially be useful in elucidating pathophysiological changes and mining for new biomarkers to diagnose and monitor S. suis infection. The objective of this study was to investigate the changes in the salivary and serum proteome profile of piglets with meningitis. The LC-MS/MS TMT proteomic approach was used to analyze saliva and serum samples from 20 male piglets: 10 with meningitis and 10 healthy. In saliva, 11 proteins had higher and 10 had lower relative abundance in piglets with meningitis. The proteins with the highest relative abundance were metavinculin (VCL) and desmocollin-2 (DSC2). Adenosine deaminase (ADA) was selected for validation using a spectrophotometric assay and demonstrated excellent performance in the differentiation between healthy and pigs with meningitis due to S. suis. In serum, the most protruding changes occurred for one SERPIN and haptoglobin (HP). In saliva and serum, the highest number of proteins with altered abundance were linked, via the enrichment analysis, with platelet and neutrophil pathways. Overall, meningitis caused by S. suis resulted in specific proteome changes in saliva and serum, reflecting different pathophysiological mechanisms, and marking new potential biomarkers for this infection. Full article

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17 pages, 1650 KiB

Open AccessReview

The Origin, Function, Distribution, Quantification, and Research Advances of Extracellular DNA

by Kaixin Yang, Lishuang Wang, Xinghong Cao, Zhaorui Gu, Guowei Zhao, Mengqu Ran, Yunjun Yan, Jinyong Yan, Li Xu, Chunhui Gao and Min Yang

Int. J. Mol. Sci. 2022, 23(22), 13690; https://doi.org/10.3390/ijms232213690 - 8 Nov 2022

Cited by 8 | Viewed by 2884

Abstract

In nature, DNA is ubiquitous, existing not only inside but also outside of the cells of organisms. Intracellular DNA (iDNA) plays an essential role in different stages of biological growth, and it is defined as the carrier of genetic information. In addition, extracellular [...] Read more.

In nature, DNA is ubiquitous, existing not only inside but also outside of the cells of organisms. Intracellular DNA (iDNA) plays an essential role in different stages of biological growth, and it is defined as the carrier of genetic information. In addition, extracellular DNA (eDNA) is not enclosed in living cells, accounting for a large proportion of total DNA in the environment. Both the lysis-dependent and lysis-independent pathways are involved in eDNA release, and the released DNA has diverse environmental functions. This review provides an insight into the origin as well as the multiple ecological functions of eDNA. Furthermore, the main research advancements of eDNA in the various ecological environments and the various model microorganisms are summarized. Furthermore, the major methods for eDNA extraction and quantification are evaluated. Full article

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12 pages, 1086 KiB

Open AccessArticle

Impact of Melatonin on RAW264.7 Macrophages during Mechanical Strain

by Eva Paddenberg, Anne Forneck, Matthias Widbiller, Martyna Smeda, Jonathan Jantsch, Peter Proff, Christian Kirschneck and Agnes Schröder

Int. J. Mol. Sci. 2022, 23(21), 13397; https://doi.org/10.3390/ijms232113397 - 2 Nov 2022

Cited by 3 | Viewed by 2078

Abstract

The concentration of melatonin is elevated during the night when patients mainly wear removable orthodontic appliances. Next to periodontal ligament fibroblasts and osteoblasts, macrophages react to mechanical strain with an increased expression of inflammatory mediators. Here, we investigated the impact of melatonin on [...] Read more.

The concentration of melatonin is elevated during the night when patients mainly wear removable orthodontic appliances. Next to periodontal ligament fibroblasts and osteoblasts, macrophages react to mechanical strain with an increased expression of inflammatory mediators. Here, we investigated the impact of melatonin on RAW264.7 macrophages exposed to tensile or compressive strain occurring during orthodontic tooth movement in the periodontal ligament. Before exposure to mechanical strain for 4 h, macrophages were pre-incubated with different melatonin concentrations for 24 h, to determine the dependence of melatonin concentration. Afterwards, we performed experiments with and without mechanical strain, the most effective melatonin concentration (25 µM), and the melatonin receptor 2 (MT2) specific antagonist 4P-PDOT. The expression of inflammatory genes and proteins was investigated by RT-qPCR, ELISAs, and immunoblot. Both tensile and compressive strain increased the expression of the investigated inflammatory factors interleukin-1-beta, interleukin-6, tumor necrosis factor alpha, and prostaglandin endoperoxide synthase-2. This effect was inhibited by the addition of melatonin. Incubation with 4P-PDOT blocked this anti-inflammatory effect of melatonin. Melatonin had an anti-inflammatory effect on macrophages exposed to mechanical strain, independent of the type of mechanical strain. As inhibition was possible with 4P-PDOT, the MT2 receptor might be involved in the regulation of the observed effects. Full article

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17 pages, 1696 KiB

Open AccessArticle

The Bottlenecks of Preparing Virus Particles by Size Exclusion for Antibody Generation

by Chi-Hsin Lee, Peng-Nien Huang, Pharaoh Fellow Mwale, Wei-Chu Wang, Sy-Jye Leu, Sung-Nien Tseng, Shin-Ru Shih, Liao-Chun Chiang, Yan-Chiao Mao, Bor-Yu Tsai, Nhlanhla Benedict Dlamini, Tien-Cuong Nguyen, Chen-Hsin Tsai and Yi-Yuan Yang

Int. J. Mol. Sci. 2022, 23(21), 12967; https://doi.org/10.3390/ijms232112967 - 26 Oct 2022

Cited by 2 | Viewed by 2261

Abstract

Enterovirus 71 (EV71) is the major etiological agent contributing to the development of hand-foot-mouth disease (HFMD). There are not any global available vaccines or antibody drugs against EV71 released yet. In this study, we perform the virus immunization in a cost-effective and convenient [...] Read more.

Enterovirus 71 (EV71) is the major etiological agent contributing to the development of hand-foot-mouth disease (HFMD). There are not any global available vaccines or antibody drugs against EV71 released yet. In this study, we perform the virus immunization in a cost-effective and convenient approach by preparing virus particles from size exclusion and immunization of chicken. Polyclonal yolk-immunoglobulin (IgY) was simply purified from egg yolk and monoclonal single-chain variable fragments (scFv) were selected via phage display technology with two scFv libraries containing 6.0 × 10⁶ and 1.3 × 10⁷ transformants. Specific clones were enriched after 5 rounds of bio-panning and four identical genes were classified after the sequence analysis. Moreover, the higher mutation rates were revealed in the CDR regions, especially in the CDR3. IgY showed specific binding activities to both EV71-infected and Coxsackievirus 16-infected cell lysates and high infectivity inhibitory activity of EV71. However, while IgY detected a 37 kDa protein, the selected scFv seemingly detected higher size proteins which could be cell protein instead of EV71 proteins. Despite the highly effective chicken antibody generation, the purity of virus particles prepared by size exclusion is the limitation of this study, and further characterization should be carried out rigorously. Full article

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20 pages, 5846 KiB

Open AccessArticle

Three Heat Shock Protein Genes and Antioxidant Enzymes Protect Pardosa pseudoannulata (Araneae: Lycosidae) from High Temperature Stress

by Di Fu, Jing Liu, Ying-Na Pan, Jia-Yun Zhu, Feng Xiao, Min Liu and Rong Xiao

Int. J. Mol. Sci. 2022, 23(21), 12821; https://doi.org/10.3390/ijms232112821 - 24 Oct 2022

Cited by 6 | Viewed by 2129

Abstract

Pardosa pseudoannulata (P. pseudoannulata) is an essential natural predatory enemy in rice ecosystems. The fluctuating climate may cause them to experience heat stress, whereas heat shock proteins (HSPs) and antioxidant enzymes help resist heat damage. Herein, we cloned and characterized the [...] Read more.

Pardosa pseudoannulata (P. pseudoannulata) is an essential natural predatory enemy in rice ecosystems. The fluctuating climate may cause them to experience heat stress, whereas heat shock proteins (HSPs) and antioxidant enzymes help resist heat damage. Herein, we cloned and characterized the full-length genes PpHSP27, PpHSP60, and PpHSC70 from P. pseudoannulata. Changes in gene expression levels and superoxide dismutase (SOD), catalase (CAT), and glutathione transferase (GST) activities in adult male and female P. pseudoannulata were measured at different stress exposure times and temperatures. We found that the abovementioned HSP genes belong to the sHSP, HSP60, and HSP70 families. The expression of the three HSP genes and the activities of SOD, CAT, and GST were significantly upregulated with the increasing stress temperature and time. The knockdown of the three HSP genes via RNA interference significantly decreased the survival rate of male and female P. pseudoannulata during high temperature stress. Thus, PpHSP27, PpHSP60, and PpHSC70 play an important role in the heat tolerance of P. pseudoannulata, and SOD, CAT, and GST enable recovery heat stress-induced oxidative damage. Their changes and regulation during high temperature stress can improve spiders’ adaptability in the field and enhance the biological control of environmental pests. Full article

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15 pages, 2880 KiB

Open AccessArticle

Reactive Centre Loop Mutagenesis of SerpinB3 to Target TMPRSS2 and Furin: Inhibition of SARS-CoV-2 Cell Entry and Replication

by Saravjeet Singh, Sophie O’Reilly, Hossam Gewaid, Andrew G. Bowie, Virginie Gautier and D. Margaret Worrall

Int. J. Mol. Sci. 2022, 23(20), 12522; https://doi.org/10.3390/ijms232012522 - 19 Oct 2022

Cited by 7 | Viewed by 3720

Abstract

The SARS-CoV-2 virus can utilize host cell proteases to facilitate cell entry, whereby the Spike (S) protein is cleaved at two specific sites to enable membrane fusion. Furin, transmembrane protease serine 2 (TMPRSS2), and cathepsin L (CatL) are the major proteases implicated, and [...] Read more.

The SARS-CoV-2 virus can utilize host cell proteases to facilitate cell entry, whereby the Spike (S) protein is cleaved at two specific sites to enable membrane fusion. Furin, transmembrane protease serine 2 (TMPRSS2), and cathepsin L (CatL) are the major proteases implicated, and are thus targets for anti-viral therapy. The human serpin (serine protease inhibitor) alpha-1 antitrypsin (A1AT) shows inhibitory activity for TMPRSS2, and has previously been found to suppress cell infection with SARS-CoV-2. Here, we have generated modified serpin inhibitors with increased specificity for these cellular proteases. Using SerpinB3 (SCCA-1), a cross-class inhibitor of CatL, as a scaffold, we have designed and produced reactive centre loop (RCL) variants to more specifically target both furin and TMPRSS2. Two further variants were generated by substituting the RCL P7–P1 with the spike protein S1/S2 cleavage site from either SARS-CoV-2 alpha or delta (P681R) sequences. Altered inhibitory specificity of purified recombinant proteins was verified in protease assays, with attenuated CatL inhibition and gain of furin or TMPRSS2 inhibition, as predicted, and modified serpins were shown to block S protein cleavage in vitro. Furthermore, the serpin variants were able to inhibit S-pseudoparticle entry into A549-ACE2-TMPRSS2 cells and suppress SARS-CoV-2 replication in Vero E6 cells expressing TMPRSS2. The construct designed to inhibit TMPRSS2 (B3-TMP) was most potent. It was more effective than A1AT for TMPRSS2 enzyme inhibition (with an eighteen-fold improvement in the second order inhibition rate constant) and for blocking SARS-CoV-2 viral replication. These findings advance the potential for serpin RCL mutagenesis to generate new inhibitors, and may lead to novel anti-viral biological molecules. Full article

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15 pages, 3468 KiB

Open AccessArticle

KDF1 Novel Variant Causes Unique Dental and Oral Epithelial Defects

by Miao Yu, Hangbo Liu, Yang Liu, Jinglei Zheng, Junyi Wu, Kai Sun, Hailan Feng, Haochen Liu and Dong Han

Int. J. Mol. Sci. 2022, 23(20), 12465; https://doi.org/10.3390/ijms232012465 - 18 Oct 2022

Cited by 4 | Viewed by 2301

Abstract

Keratinocyte differentiation factor 1 (KDF1) is a recently identified and rare candidate gene for human tooth agenesis; however, KDF1-related morphological characteristics and pathological changes in dental tissue and the oral epithelium remain largely unknown. Here, we employed whole-exome sequencing (WES) [...] Read more.

Keratinocyte differentiation factor 1 (KDF1) is a recently identified and rare candidate gene for human tooth agenesis; however, KDF1-related morphological characteristics and pathological changes in dental tissue and the oral epithelium remain largely unknown. Here, we employed whole-exome sequencing (WES) and Sanger sequencing to screen for the suspected variants in a cohort of 151 tooth agenesis patients, and we segregated a novel KDF1 heterozygous missense variation, c.920G>C (p.R307P), in a non-syndromic tooth agenesis family. Essential bioinformatics analyses and tertiary structural predictions were performed to analyze the structural changes and functional impacts of the novel KDF1 variant. The subsequent functional assessment using a TOP-flash/FOP-flash luciferase reporter system demonstrated that KDF1 variants suppressed the activation of canonical Wnt signaling in 293T cells. To comprehensively investigate the KDF1-related oral morphological anomalies, we performed scanning electron microscopy and ground section of the lower right lateral deciduous incisor extracted from #285 proband, and histopathological assessment of the gingiva. The phenotypic analyses revealed a series of tooth morphological anomalies related to the KDF1 variant R307P, including a shovel-shaped lingual surface of incisors and cornicione-shaped marginal ridges with anomalous morphological occlusal grooves of premolars and molars. Notably, keratinized gingival epithelium abnormalities were revealed in the proband and characterized by epithelial dyskeratosis with residual nuclei, indistinct stratum granulosum, epithelial hyperproliferation, and impaired epithelial differentiation. Our findings revealed new developmental anomalies in the tooth and gingival epithelium of a non-syndromic tooth agenesis individual with a novel pathogenic KDF1 variant, broadening the phenotypic spectrum of KDF1-related disorders and providing new evidence for the crucial role of KDF1 in regulating human dental and oral epithelial development. Full article

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14 pages, 3226 KiB

Open AccessArticle

Binding of Gamma-Glutamyl Transferase to TLR4 Signalling Allows Tissue Factor Activation in Monocytes

by Chiara Sanguinetti, Valentina Scalise, Tommaso Neri, Alessandro Celi, Vanessa Susini, Maria Franzini and Roberto Pedrinelli

Int. J. Mol. Sci. 2022, 23(20), 12207; https://doi.org/10.3390/ijms232012207 - 13 Oct 2022

Cited by 5 | Viewed by 2133

Abstract

Gamma-glutamyl transferase (GGT) is involved in the progression of atherosclerosis, since its enzymatic activity promotes the generation of reactive oxygen species (ROS). Besides, GGT may act as a prothrombotic factor by inducing tissue factor (TF) expression, independently of its enzymatic activity. The aim [...] Read more.

Gamma-glutamyl transferase (GGT) is involved in the progression of atherosclerosis, since its enzymatic activity promotes the generation of reactive oxygen species (ROS). Besides, GGT may act as a prothrombotic factor by inducing tissue factor (TF) expression, independently of its enzymatic activity. The aim of this study was to assess whether GGT-induced TF stimulation was a consequence of binding to toll-like receptor 4 (TLR4) expressed on monocytes, the precursors of macrophages and foam cells which colocalize with GGT activity within atherosclerotic plaques. Experiments were performed in human peripheral blood mononuclear cells (PBMCs), THP-1 cells (a monocytic cellular model), and HEK293 cells, which were genetically modified to study the activation of TLR4. TF procoagulant activity was assessed by a one-stage clotting time test, and TF protein expression was estimated by western blot. Human recombinant (hr) GGT protein increased TF procoagulant activity and protein expression in both PBMCs and THP-1 cells. The GGT-induced TF stimulation was prevented by cellular pretreatment with TLR4/NF-κB inhibitors (LPS-Rs, CLI-095, and BAY-11-7082), and HEK293 cells lacking TLR4 confirmed that TLR4 is essential for GGT-induced activation of NF-κB. In conclusion, hrGGT induced TF expression in monocytes through a cytokine-like mechanism that involved the activation of TLR4/NF-κB signaling. Full article

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14 pages, 3242 KiB

Open AccessArticle

Mammary-Enriched Transcription Factors Synergize to Activate the Wap Super-Enhancer for Mammary Gland Development

by Uijin Kim, Suyeon Kim, Nahyun Kim and Ha Youn Shin

Int. J. Mol. Sci. 2022, 23(19), 11680; https://doi.org/10.3390/ijms231911680 - 2 Oct 2022

Cited by 3 | Viewed by 2066

Abstract

Super-enhancers are large clusters of enhancers critical for cell-type-specific development. In a previous study, 440 mammary-specific super-enhancers, highly enriched for an active enhancer mark H3K27ac; a mediator MED1; and the mammary-enriched transcription factors ELF5, NFIB, STAT5A, and GR, were identified in the genome [...] Read more.

Super-enhancers are large clusters of enhancers critical for cell-type-specific development. In a previous study, 440 mammary-specific super-enhancers, highly enriched for an active enhancer mark H3K27ac; a mediator MED1; and the mammary-enriched transcription factors ELF5, NFIB, STAT5A, and GR, were identified in the genome of the mammary epithelium of lactating mice. However, the triggering mechanism for mammary-specific super-enhancers and the molecular interactions between key transcription factors have not been clearly elucidated. In this study, we investigated in vivo protein–protein interactions between major transcription factors that activate mammary-specific super-enhancers. In mammary epithelial cells, ELF5 strongly interacted with NFIB while weakly interacting with STAT5A, and it showed modest interactions with MED1 and GR, a pattern unlike that in non-mammary cells. We further investigated the role of key transcription factors in the initial activation of the mammary-specific Wap super-enhancer, using CRISPR-Cas9 genome editing to introduce single or combined mutations at transcription factor binding sites in the pioneer enhancer of the Wap super-enhancer in mice. ELF5 and STAT5A played key roles in igniting Wap super-enhancer activity, but an intact transcription factor complex was required for the full function of the super-enhancer. Our study demonstrates that mammary-enriched transcription factors within a protein complex interact with different intensities and synergize to activate the Wap super-enhancer. These findings provide an important framework for understanding the regulation of cell-type-specific development. Full article

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21 pages, 1955 KiB

Open AccessReview

A Pathophysiological Intersection of Diabetes and Alzheimer’s Disease

by Maša Čater and Sabine M. Hölter

Int. J. Mol. Sci. 2022, 23(19), 11562; https://doi.org/10.3390/ijms231911562 - 30 Sep 2022

Cited by 13 | Viewed by 4206

Abstract

Diabetes is among the most prevalent diseases of the modern world and is strongly linked to an increased risk of numerous neurodegenerative disorders, although the exact pathophysiological mechanisms are not clear yet. Insulin resistance is a serious pathological condition, connecting type 2 diabetes, [...] Read more.

Diabetes is among the most prevalent diseases of the modern world and is strongly linked to an increased risk of numerous neurodegenerative disorders, although the exact pathophysiological mechanisms are not clear yet. Insulin resistance is a serious pathological condition, connecting type 2 diabetes, metabolic syndrome, and obesity. Recently, insulin resistance has been proven to be connected also to cognitive decline and dementias, including the most prevalent form, Alzheimer’s disease. The relationship between diabetes and Alzheimer’s disease regarding pathophysiology is so significant that it has been proposed that some presentations of the condition could be termed type 3 diabetes. Full article

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16 pages, 2150 KiB

Open AccessReview

Roles of Regulatory T Cell-Derived Extracellular Vesicles in Human Diseases

by Can Lin, Jihua Guo and Rong Jia

Int. J. Mol. Sci. 2022, 23(19), 11206; https://doi.org/10.3390/ijms231911206 - 23 Sep 2022

Cited by 18 | Viewed by 2835

Abstract

Regulatory T (Treg) cells play crucial roles in maintaining immune self-tolerance and immune homeostasis, and closely associated with many human diseases. Recently, Treg cells-derived extracellular vesicles (Treg-EVs) have been demonstrated as a novel cell-contact independent inhibitory mechanism of Treg cells. Treg-EVs contain many [...] Read more.

Regulatory T (Treg) cells play crucial roles in maintaining immune self-tolerance and immune homeostasis, and closely associated with many human diseases. Recently, Treg cells-derived extracellular vesicles (Treg-EVs) have been demonstrated as a novel cell-contact independent inhibitory mechanism of Treg cells. Treg-EVs contain many specific biological molecules, which are delivered to target cells and modulate immune responses by inhibiting T cell proliferation, inducing T cell apoptosis, and changing the cytokine expression profiles of target cells. The abnormal quantity or function of Treg-EVs is associated with several types of human diseases or conditions, such as transplant rejection, inflammatory diseases, autoimmune diseases, and cancers. Treg-EVs are promising novel potential targets for disease diagnosis, therapy, and drug transport. Moreover, Treg-EVs possess distinct advantages over Treg cell-based immunotherapies. However, the therapeutic potential of Treg-EVs is limited by some factors, such as the standardized protocol for isolation and purification, large scale production, and drug loading efficiency. In this review, we systematically describe the structure, components, functions, and basic mechanisms of action of Treg-EVs and discuss the emerging roles in pathogenesis and the potential application of Treg-EVs in human diseases. Full article

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16 pages, 4636 KiB

Open AccessArticle

Role of RNAIII in Resistance to Antibiotics and Antimicrobial Agents in Staphylococcus epidermidis Biofilms

by Andrej Minich, Veronika Lišková, Ľubica Kormanová, Ján Krahulec, Júlia Šarkanová, Mária Mikulášová, Zdenko Levarski and Stanislav Stuchlík

Int. J. Mol. Sci. 2022, 23(19), 11094; https://doi.org/10.3390/ijms231911094 - 21 Sep 2022

Cited by 5 | Viewed by 2619

Abstract

Staphylococcus epidermidis is a known opportunistic pathogen and is one of the leading causes of chronic biofilm-associated infections. Biofilm formation is considered as a main strategy to resist antibiotic treatment and help bacteria escape from the human immune system. Understanding the complex mechanisms [...] Read more.

Staphylococcus epidermidis is a known opportunistic pathogen and is one of the leading causes of chronic biofilm-associated infections. Biofilm formation is considered as a main strategy to resist antibiotic treatment and help bacteria escape from the human immune system. Understanding the complex mechanisms in biofilm formation can help find new ways to treat resistant strains and lower the prevalence of nosocomial infections. In order to examine the role of RNAIII regulated by the agr quorum sensing system and to what extent it influences biofilm resistance to antimicrobial agents, deletion mutant S. epidermidis RP62a-ΔRNAIII deficient in repressor domains with a re-maining functional hld gene was created. A deletion strain was used to examine the influence of oxacillin in combination with vanillin on biofilm resistance and cell survival was determined. Utilizing real-time qPCR, confocal laser scanning microscopy (CLSM), and crystal violet staining analyses, we found that the RNAIII-independent controlled phenol soluble modulins (PSMs) and RNAIII effector molecule have a significant role in biofilm resistance to antibiotics and phenolic compounds, and it protects the integrity of biofilms. Moreover, a combination of antibiotic and antimicrobial agents can induce methicillin-resistant S. epidermidis biofilm formation and can lead to exceedingly difficult medical treatment. Full article

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16 pages, 3315 KiB

Open AccessArticle

Unveiling Mycoviromes Using Fungal Transcriptomes

by Yeonhwa Jo, Hoseong Choi, Hyosub Chu and Won Kyong Cho

Int. J. Mol. Sci. 2022, 23(18), 10926; https://doi.org/10.3390/ijms231810926 - 18 Sep 2022

Cited by 8 | Viewed by 2465

Abstract

Viruses infecting fungi are referred to as mycoviruses. Here, we carried out in silico mycovirome studies using public fungal transcriptomes mostly derived from mRNA libraries. We identified 468 virus-associated contigs assigned to 5 orders, 21 families, 26 genera, and 88 species. We assembled [...] Read more.

Viruses infecting fungi are referred to as mycoviruses. Here, we carried out in silico mycovirome studies using public fungal transcriptomes mostly derived from mRNA libraries. We identified 468 virus-associated contigs assigned to 5 orders, 21 families, 26 genera, and 88 species. We assembled 120 viral genomes with diverse RNA and DNA genomes. The phylogenetic tree and genome organization unveiled the possible host origin of mycovirus species and diversity of their genome structures. Most identified mycoviruses originated from fungi; however, some mycoviruses had strong phylogenetic relationships with those from insects and plants. The viral abundance and mutation frequency of mycoviruses were very low; however, the compositions and populations of mycoviruses were very complex. Although coinfection of diverse mycoviruses in the fungi was common in our study, most mycoviromes had a dominant virus species. The compositions and populations of mycoviruses were more complex than we expected. Viromes of Monilinia species revealed that there were strong deviations in the composition of viruses and viral abundance among samples. Viromes of Gigaspora species showed that the chemical strigolactone might promote virus replication and mutations, while symbiosis with endobacteria might suppress virus replication and mutations. This study revealed the diversity and host distribution of mycoviruses. Full article

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15 pages, 7250 KiB

Open AccessArticle

Zrsr2 Is Essential for the Embryonic Development and Splicing of Minor Introns in RNA and Protein Processing Genes in Zebrafish

by Rachel Weinstein, Kevin Bishop, Elizabeth Broadbridge, Kai Yu, Blake Carrington, Abdel Elkahloun, Tao Zhen, Wuhong Pei, Shawn M. Burgess, Paul Liu, Erica Bresciani and Raman Sood

Int. J. Mol. Sci. 2022, 23(18), 10668; https://doi.org/10.3390/ijms231810668 - 14 Sep 2022

Cited by 5 | Viewed by 3202

Abstract

ZRSR2 (zinc finger CCCH-type, RNA binding motif and serine/arginine rich 2) is an essential splicing factor involved in 3′ splice-site recognition as a component of both the major and minor spliceosomes that mediate the splicing of U2-type (major) and U12-type (minor) introns, respectively. [...] Read more.

ZRSR2 (zinc finger CCCH-type, RNA binding motif and serine/arginine rich 2) is an essential splicing factor involved in 3′ splice-site recognition as a component of both the major and minor spliceosomes that mediate the splicing of U2-type (major) and U12-type (minor) introns, respectively. Studies of ZRSR2-depleted cell lines and ZRSR2-mutated patient samples revealed its essential role in the U12-dependent minor spliceosome. However, the role of ZRSR2 during embryonic development is not clear, as its function is compensated for by Zrsr1 in mice. Here, we utilized the zebrafish model to investigate the role of zrsr2 during embryonic development. Using CRISPR/Cas9 technology, we generated a zrsr2-knockout zebrafish line, termed zrsr2^hg129/hg129 (p.Trp167Argfs*9) and examined embryo development in the homozygous mutant embryos. zrsr2^hg129/hg129 embryos displayed multiple developmental defects starting at 4 days post fertilization (dpf) and died after 8 dpf, suggesting that proper Zrsr2 function is required during embryonic development. The global transcriptome analysis of 3 dpf zrsr2^hg129/hg129 embryos revealed that the loss of Zrsr2 results in the downregulation of essential metabolic pathways and the aberrant retention of minor introns in about one-third of all minor intron-containing genes in zebrafish. Overall, our study has demonstrated that the role of Zrsr2 as a component of the minor spliceosome is conserved and critical for proper embryonic development in zebrafish. Full article

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22 pages, 2583 KiB

Open AccessReview

The Complexity and Multiplicity of the Specific cAMP Phosphodiesterase Family: PDE4, Open New Adapted Therapeutic Approaches

by Claire Lugnier

Int. J. Mol. Sci. 2022, 23(18), 10616; https://doi.org/10.3390/ijms231810616 - 13 Sep 2022

Cited by 26 | Viewed by 7165

Abstract

Cyclic nucleotides (cAMP, cGMP) play a major role in normal and pathologic signaling. Beyond receptors, cyclic nucleotide phosphodiesterases; (PDEs) rapidly convert the cyclic nucleotide in its respective 5′-nucleotide to control intracellular cAMP and/or cGMP levels to maintain a normal physiological state. However, in [...] Read more.

Cyclic nucleotides (cAMP, cGMP) play a major role in normal and pathologic signaling. Beyond receptors, cyclic nucleotide phosphodiesterases; (PDEs) rapidly convert the cyclic nucleotide in its respective 5′-nucleotide to control intracellular cAMP and/or cGMP levels to maintain a normal physiological state. However, in many pathologies, dysregulations of various PDEs (PDE1-PDE11) contribute mainly to organs and tissue failures related to uncontrolled phosphorylation cascade. Among these, PDE4 represents the greatest family, since it is constituted by 4 genes with multiple variants differently distributed at tissue, cellular and subcellular levels, allowing different fine-tuned regulations. Since the 1980s, pharmaceutical companies have developed PDE4 inhibitors (PDE4-I) to overcome cardiovascular diseases. Since, they have encountered many undesired problems, (emesis), they focused their research on other PDEs. Today, increases in the knowledge of complex PDE4 regulations in various tissues and pathologies, and the evolution in drug design, resulted in a renewal of PDE4-I development. The present review describes the recent PDE4-I development targeting cardiovascular diseases, obesity, diabetes, ulcerative colitis, and Crohn’s disease, malignancies, fatty liver disease, osteoporosis, depression, as well as COVID-19. Today, the direct therapeutic approach of PDE4 is extended by developing allosteric inhibitors and protein/protein interactions allowing to act on the PDE interactome. Full article

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13 pages, 2407 KiB

Open AccessArticle

In Vitro Characterization of the Physical Interactions between the Long Noncoding RNA TERRA and the Telomeric Proteins TRF1 and TRF2

by Patricia L. Abreu, Yong Woo Lee and Claus M. Azzalin

Int. J. Mol. Sci. 2022, 23(18), 10463; https://doi.org/10.3390/ijms231810463 - 9 Sep 2022

Cited by 5 | Viewed by 2543

Abstract

RNA-protein interactions drive key cellular pathways such as protein translation, nuclear organization and genome stability maintenance. The human telomeric protein TRF2 binds to the long noncoding RNA TERRA through independent domains, including its N-terminal B domain. We previously demonstrated that TRF2 B domain [...] Read more.

RNA-protein interactions drive key cellular pathways such as protein translation, nuclear organization and genome stability maintenance. The human telomeric protein TRF2 binds to the long noncoding RNA TERRA through independent domains, including its N-terminal B domain. We previously demonstrated that TRF2 B domain binding to TERRA supports invasion of TERRA into telomeric double stranded DNA, leading to the formation of telomeric RNA:DNA hybrids. The other telomeric protein TRF1, which also binds to TERRA, suppresses this TRF2-associated activity by preventing TERRA-B domain interactions. Herein, we show that the binding of both TRF1 and TRF2 to TERRA depends on the ability of the latter to form G-quadruplex structures. Moreover, a cluster of arginines within the B domain is largely responsible for its binding to TERRA. On the other side, a patch of glutamates within the N-terminal A domain of TRF1 mainly accounts for the inhibition of TERRA-B domain complex formation. Finally, mouse TRF2 B domain binds to TERRA, similarly to its human counterpart, while mouse TRF1 A domain lacks the inhibitory activity. Our data shed further light on the complex crosstalk between telomeric proteins and RNAs and suggest a lack of functional conservation in mouse. Full article

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25 pages, 7494 KiB

Open AccessArticle

Effect of Delta and Omicron Mutations on the RBD-SD1 Domain of the Spike Protein in SARS-CoV-2 and the Omicron Mutations on RBD-ACE2 Interface Complex

by Wai-Yim Ching, Puja Adhikari, Bahaa Jawad and Rudolf Podgornik

Int. J. Mol. Sci. 2022, 23(17), 10091; https://doi.org/10.3390/ijms231710091 - 3 Sep 2022

Cited by 11 | Viewed by 2870

Abstract

The receptor-binding domain (RBD) is the essential part in the Spike-protein (S-protein) of SARS-CoV-2 virus that directly binds to the human ACE2 receptor, making it a key target for many vaccines and therapies. Therefore, any mutations at this domain could affect the efficacy [...] Read more.

The receptor-binding domain (RBD) is the essential part in the Spike-protein (S-protein) of SARS-CoV-2 virus that directly binds to the human ACE2 receptor, making it a key target for many vaccines and therapies. Therefore, any mutations at this domain could affect the efficacy of these treatments as well as the viral-cell entry mechanism. We introduce ab initio DFT-based computational study that mainly focuses on two parts: (1) Mutations effects of both Delta and Omicron variants in the RBD-SD1 domain. (2) Impact of Omicron RBD mutations on the structure and properties of the RBD-ACE2 interface system. The in-depth analysis is based on the novel concept of amino acid-amino acid bond pair units (AABPU) that reveal the differences between the Delta and/or Omicron mutations and its corresponding wild-type strain in terms of the role played by non-local amino acid interactions, their 3D shapes and sizes, as well as contribution to hydrogen bonding and partial charge distributions. Our results also show that the interaction of Omicron RBD with ACE2 significantly increased its bonding between amino acids at the interface providing information on the implications of penetration of S-protein into ACE2, and thus offering a possible explanation for its high infectivity. Our findings enable us to present, in more conspicuous atomic level detail, the effect of specific mutations that may help in predicting and/or mitigating the next variant of concern. Full article

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25 pages, 6141 KiB

Open AccessArticle

Unusual Cytochrome c552 from Thioalkalivibrio paradoxus: Solution NMR Structure and Interaction with Thiocyanate Dehydrogenase

by Vladimir V. Britikov, Eduard V. Bocharov, Elena V. Britikova, Natalia I. Dergousova, Olga G. Kulikova, Anastasia Y. Solovieva, Nikolai S. Shipkov, Larisa A. Varfolomeeva, Tamara V. Tikhonova, Vladimir I. Timofeev, Eleonora V. Shtykova, Dmitry A. Altukhov, Sergey A. Usanov, Alexander S. Arseniev, Tatiana V. Rakitina and Vladimir O. Popov

Int. J. Mol. Sci. 2022, 23(17), 9969; https://doi.org/10.3390/ijms23179969 - 1 Sep 2022

Cited by 3 | Viewed by 2991

Abstract

The search of a putative physiological electron acceptor for thiocyanate dehydrogenase (TcDH) newly discovered in the thiocyanate-oxidizing bacteria Thioalkalivibrio paradoxus revealed an unusually large, single-heme cytochrome c (CytC552), which was co-purified with TcDH from the periplasm. Recombinant CytC552, produced in Escherichia coli as [...] Read more.

The search of a putative physiological electron acceptor for thiocyanate dehydrogenase (TcDH) newly discovered in the thiocyanate-oxidizing bacteria Thioalkalivibrio paradoxus revealed an unusually large, single-heme cytochrome c (CytC552), which was co-purified with TcDH from the periplasm. Recombinant CytC552, produced in Escherichia coli as a mature protein without a signal peptide, has spectral properties similar to the endogenous protein and serves as an in vitro electron acceptor in the TcDH-catalyzed reaction. The CytC552 structure determined by NMR spectroscopy reveals significant differences compared to those of the typical class I bacterial cytochromes c: a high solvent accessible surface area for the heme group and so-called “intrinsically disordered” nature of the histidine-rich N- and C-terminal regions. Comparison of the signal splitting in the heteronuclear NMR spectra of oxidized, reduced, and TcDH-bound CytC552 reveals the heme axial methionine fluxionality. The TcDH binding site on the CytC552 surface was mapped using NMR chemical shift perturbations. Putative TcDH-CytC552 complexes were reconstructed by the information-driven docking approach and used for the analysis of effective electron transfer pathways. The best pathway includes the electron hopping through His528 and Tyr164 of TcDH, and His83 of CytC552 to the heme group in accordance with pH-dependence of TcDH activity with CytC552. Full article

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22 pages, 6842 KiB

Open AccessReview

The Role of Protein Arginine Methyltransferases in DNA Damage Response

by Charles Brobbey, Liu Liu, Shasha Yin and Wenjian Gan

Int. J. Mol. Sci. 2022, 23(17), 9780; https://doi.org/10.3390/ijms23179780 - 29 Aug 2022

Cited by 27 | Viewed by 4265

Abstract

In response to DNA damage, cells have developed a sophisticated signaling pathway, consisting of DNA damage sensors, transducers, and effectors, to ensure efficient and proper repair of damaged DNA. During this process, posttranslational modifications (PTMs) are central events that modulate the recruitment, dissociation, [...] Read more.

In response to DNA damage, cells have developed a sophisticated signaling pathway, consisting of DNA damage sensors, transducers, and effectors, to ensure efficient and proper repair of damaged DNA. During this process, posttranslational modifications (PTMs) are central events that modulate the recruitment, dissociation, and activation of DNA repair proteins at damage sites. Emerging evidence reveals that protein arginine methylation is one of the common PTMs and plays critical roles in DNA damage response. Protein arginine methyltransferases (PRMTs) either directly methylate DNA repair proteins or deposit methylation marks on histones to regulate their transcription, RNA splicing, protein stability, interaction with partners, enzymatic activities, and localization. In this review, we summarize the substrates and roles of each PRMTs in DNA damage response and discuss the synergistic anticancer effects of PRMTs and DNA damage pathway inhibitors, providing insight into the significance of arginine methylation in the maintenance of genome integrity and cancer therapies. Full article

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19 pages, 9142 KiB

Open AccessArticle

Ultrastructural Characterization of Human Bronchial Epithelial Cells during SARS-CoV-2 Infection: Morphological Comparison of Wild-Type and CFTR-Modified Cells

by Flavia Merigo, Virginia Lotti, Paolo Bernardi, Anita Conti, Andrea Di Clemente, Marco Ligozzi, Anna Lagni, Claudio Sorio, Andrea Sbarbati and Davide Gibellini

Int. J. Mol. Sci. 2022, 23(17), 9724; https://doi.org/10.3390/ijms23179724 - 27 Aug 2022

Cited by 5 | Viewed by 2542

Abstract

SARS-CoV-2 replicates in host cell cytoplasm. People with cystic fibrosis, considered at risk of developing severe symptoms of COVID-19, instead, tend to show mild symptoms. We, thus, analyzed at the ultrastructural level the morphological effects of SARS-CoV-2 infection on wild-type (WT) and F508del [...] Read more.

SARS-CoV-2 replicates in host cell cytoplasm. People with cystic fibrosis, considered at risk of developing severe symptoms of COVID-19, instead, tend to show mild symptoms. We, thus, analyzed at the ultrastructural level the morphological effects of SARS-CoV-2 infection on wild-type (WT) and F508del (ΔF) CFTR-expressing CFBE41o- cells at early and late time points post infection. We also investigated ACE2 expression through immune-electron microscopy. At early times of infection, WT cells exhibited double-membrane vesicles, representing typical replicative structures, with granular and vesicular content, while at late time points, they contained vesicles with viral particles. ∆F cells exhibited double-membrane vesicles with an irregular shape and degenerative changes and at late time of infection, showed vesicles containing viruses lacking a regular structure and a well-organized distribution. ACE2 was expressed at the plasma membrane and present in the cytoplasm only at early times in WT, while it persisted even at late times of infection in ΔF cells. The autophagosome content also differed between the cells: in WT cells, it comprised vesicles associated with virus-containing structures, while in ΔF cells, it comprised ingested material for lysosomal digestion. Our data suggest that CFTR-modified cells infected with SARS-CoV-2 have impaired organization of normo-conformed replicative structures. Full article

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33 pages, 6833 KiB

Open AccessArticle

mPR-Specific Actions Influence Maintenance of the Blood–Brain Barrier (BBB)

by Johnathan Abou-Fadel, Xiaoting Jiang, Akhil Padarti, Dinesh G. Goswami, Mark Smith, Brian Grajeda, Muaz Bhalli, Alexander Le, Wendy E. Walker and Jun Zhang

Int. J. Mol. Sci. 2022, 23(17), 9684; https://doi.org/10.3390/ijms23179684 - 26 Aug 2022

Cited by 12 | Viewed by 2507

Abstract

Cerebral cavernous malformations (CCMs) are characterized by abnormally dilated intracranial microvascular sinusoids that result in increased susceptibility to hemorrhagic stroke. It has been demonstrated that three CCM proteins (CCM1, CCM2, and CCM3) form the CCM signaling complex (CSC) to mediate angiogenic signaling. Disruption [...] Read more.

Cerebral cavernous malformations (CCMs) are characterized by abnormally dilated intracranial microvascular sinusoids that result in increased susceptibility to hemorrhagic stroke. It has been demonstrated that three CCM proteins (CCM1, CCM2, and CCM3) form the CCM signaling complex (CSC) to mediate angiogenic signaling. Disruption of the CSC will result in hemorrhagic CCMs, a consequence of compromised blood–brain barrier (BBB) integrity. Due to their characteristically incomplete penetrance, the majority of CCM mutation carriers (presumed CCM patients) are largely asymptomatic, but when symptoms occur, the disease has typically reached a clinical stage of focal hemorrhage with irreversible brain damage. We recently reported that the CSC couples both classic (nuclear; nPRs) and nonclassic (membrane; mPRs) progesterone (PRG)-receptors-mediated signaling within the CSC-mPRs-PRG (CmP) signaling network in nPR(−) breast cancer cells. In this report, we demonstrate that depletion of any of the three CCM genes or treatment with mPR-specific PRG actions (PRG/mifepristone) results in the disruption of the CmP signaling network, leading to increased permeability in the nPR(−) endothelial cells (ECs) monolayer in vitro. Finally, utilizing our in vivo hemizygous Ccm mutant mice models, we demonstrate that depletion of any of the three CCM genes, in combination with mPR-specific PRG actions, is also capable of leading to defective homeostasis of PRG in vivo and subsequent BBB disruption, allowing us to identify a specific panel of etiological blood biomarkers associated with BBB disruption. To our knowledge, this is the first report detailing the etiology to predict the occurrence of a disrupted BBB, an indication of early hemorrhagic events. Full article

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14 pages, 5057 KiB

Open AccessArticle

The VEGF Inhibitor Soluble Fms-like Tyrosine Kinase 1 Does Not Promote AKI-to-CKD Transition

by Cleo C. L. van Aanhold, Angela Koudijs, Kyra L. Dijkstra, Ron Wolterbeek, Jan A. Bruijn, Cees van Kooten and Hans J. Baelde

Int. J. Mol. Sci. 2022, 23(17), 9660; https://doi.org/10.3390/ijms23179660 - 26 Aug 2022

Viewed by 2404

Abstract

(1) Background: Soluble Fms-like tyrosine kinase 1 (sFLT1) is an endogenous VEGF inhibitor. sFLT1 has been described as an anti-inflammatory treatment for diabetic nephropathy and heart fibrosis. However, sFLT1 has also been related to peritubular capillary (PTC) loss, which promotes fibrogenesis. Here, we [...] Read more.

(1) Background: Soluble Fms-like tyrosine kinase 1 (sFLT1) is an endogenous VEGF inhibitor. sFLT1 has been described as an anti-inflammatory treatment for diabetic nephropathy and heart fibrosis. However, sFLT1 has also been related to peritubular capillary (PTC) loss, which promotes fibrogenesis. Here, we studied whether transfection with sFlt1 aggravates experimental AKI-to-CKD transition and whether sFLT1 is increased in human kidney fibrosis. (2) Methods: Mice were transfected via electroporation with sFlt1. After confirming transfection efficacy, mice underwent unilateral ischemia/reperfusion injury (IRI) and were sacrificed 28 days later. Kidney histology and RNA were analyzed to study renal fibrosis, PTC damage and inflammation. Renal sFLT1 mRNA expression was measured in CKD biopsies and control kidney tissue. (3) Results: sFlt1 transfection did not aggravate renal fibrosis, PTC loss or macrophage recruitment in IRI mice. In contrast, higher transfection efficiency was correlated with reduced expression of pro-fibrotic and pro-inflammatory markers. In the human samples, sFLT1 mRNA levels were similar in CKD and control kidneys and were not correlated with interstitial fibrosis or PTC loss. (4) Conclusion: As we previously found that sFLT1 has therapeutic potential in diabetic nephropathy, our findings indicate that sFLT1 can be administered at a dose that is therapeutically effective in reducing inflammation, without promoting maladaptive kidney damage. Full article

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11 pages, 1500 KiB

Open AccessCase Report

Evolution of Graves’ Disease during Immune Reconstitution following Nonmyeloablative Haploidentical Peripheral Blood Stem Cell Transplantation in a Boy Carrying Germline SAMD9L and FLT3 Variants

by Peng Peng Ip, Li-Hua Fang, Yi-Ling Shen, Kuan-Chiun Tung, Ming-Tsong Lai, Li-Ying Juan, Liuh-Yow Chen and Rong-Long Chen

Int. J. Mol. Sci. 2022, 23(16), 9494; https://doi.org/10.3390/ijms23169494 - 22 Aug 2022

Cited by 1 | Viewed by 4548

Abstract

Graves’ disease, characterized by hyperthyroidism resulting from loss of immune tolerance to thyroid autoantigens, may be attributable to both genetic and environmental factors. Allogeneic hematopoietic stem cell transplantation (HSCT) represents a means to induce immunotolerance via an artificial immune environment. We present a [...] Read more.

Graves’ disease, characterized by hyperthyroidism resulting from loss of immune tolerance to thyroid autoantigens, may be attributable to both genetic and environmental factors. Allogeneic hematopoietic stem cell transplantation (HSCT) represents a means to induce immunotolerance via an artificial immune environment. We present a male patient with severe aplastic anemia arising from a germline SAMD9L missense mutation who successfully underwent HSCT from his HLA-haploidentical SAMD9L non-mutated father together with nonmyeloablative conditioning and post-transplant cyclophosphamide at 8 years of age. He did not suffer graft-versus-host disease, but Graves’ disease evolved 10 months post-transplant when cyclosporine was discontinued for one month. Reconstitution of peripheral lymphocyte subsets was found to be transiently downregulated shortly after Graves’ disease onset but recovered upon antithyroid treatment. Our investigation revealed the presence of genetic factors associated with Graves’ disease, including HLA-B*46:01 and HLA-DRB1*09:01 haplotypes carried by the asymptomatic donor and germline FLT3 c.2500C>T mutation carried by both the patient and the donor. Given his current euthyroid state with normal hematopoiesis, the patient has returned to normal school life. This rare event of Graves’ disease in a young boy arising from special HSCT circumstances indicates that both the genetic background and the HSCT environment can prompt the evolution of Graves’ disease. Full article

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15 pages, 3191 KiB

Open AccessArticle

Uromodulin Regulates Murine Aquaporin−2 Activity via Thick Ascending Limb–Collecting Duct Cross−Talk during Water Deprivation

by Tomoaki Takata, Shintaro Hamada, Yukari Mae, Takuji Iyama, Ryohei Ogihara, Misako Seno, Kazuomi Nakamura, Miki Takata, Takaaki Sugihara and Hajime Isomoto

Int. J. Mol. Sci. 2022, 23(16), 9410; https://doi.org/10.3390/ijms23169410 - 20 Aug 2022

Cited by 5 | Viewed by 2469

Abstract

Uromodulin, a urinary protein synthesized and secreted from the thick ascending limb (TAL) of the loop of Henle, is associated with hypertension through the activation of sodium reabsorption in the TAL. Uromodulin is a potential target for hypertension treatment via natriuresis. However, its [...] Read more.

Uromodulin, a urinary protein synthesized and secreted from the thick ascending limb (TAL) of the loop of Henle, is associated with hypertension through the activation of sodium reabsorption in the TAL. Uromodulin is a potential target for hypertension treatment via natriuresis. However, its biological function in epithelial cells of the distal nephron segment, particularly the collecting duct, remains unknown. Herein, we examined the regulation of uromodulin production during water deprivation in vivo as well as the effect of uromodulin on the activity of the water channel aquaporin−2 (AQP2) in vitro and in vivo using transgenic mice. Water deprivation upregulated uromodulin production; immunofluorescence experiments revealed uromodulin adhesion on the apical surface of the collecting duct. Furthermore, the activation of AQP2 was attenuated in mice lacking uromodulin. Uromodulin enhanced the phosphorylation and apical trafficking of AQP2 in mouse collecting duct cells treated with the vasopressin analog dDAVP. The uromodulin-induced apical trafficking of AQP2 was attenuated via endocytosis inhibitor treatment, suggesting that uromodulin activates AQP2 through the suppression of endocytosis. This study provides novel insights into the cross−talk between TAL and the collecting duct, and indicates that the modulation of uromodulin is a promising approach for diuresis and hypertension treatment. Full article

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14 pages, 2726 KiB

Open AccessArticle

Insulin Receptor-Related Receptor Regulates the Rate of Early Development in Xenopus laevis

by Daria D. Korotkova, Elena A. Gantsova, Alexander S. Goryashchenko, Fedor M. Eroshkin, Oxana V. Serova, Alexey S. Sokolov, Fedor Sharko, Svetlana V. Zhenilo, Natalia Y. Martynova, Alexander G. Petrenko, Andrey G. Zaraisky and Igor E. Deyev

Int. J. Mol. Sci. 2022, 23(16), 9250; https://doi.org/10.3390/ijms23169250 - 17 Aug 2022

Cited by 4 | Viewed by 2479

Abstract

The orphan insulin receptor-related receptor (IRR) encoded by insrr gene is the third member of the insulin receptor family, also including the insulin receptor (IR) and the insulin-like growth factor receptor (IGF-1R). IRR is the extracellular alkaline medium sensor. In mice, insrr is [...] Read more.

The orphan insulin receptor-related receptor (IRR) encoded by insrr gene is the third member of the insulin receptor family, also including the insulin receptor (IR) and the insulin-like growth factor receptor (IGF-1R). IRR is the extracellular alkaline medium sensor. In mice, insrr is expressed only in small populations of cells in specific tissues, which contain extracorporeal liquids of extreme pH. In particular, IRR regulates the metabolic bicarbonate excess in the kidney. In contrast, the role of IRR during Xenopus laevis embryogenesis is unknown, although insrr is highly expressed in frog embryos. Here, we examined the insrr function during the Xenopus laevis early development by the morpholino-induced knockdown. We demonstrated that insrr downregulation leads to development retardation, which can be restored by the incubation of embryos in an alkaline medium. Using bulk RNA-seq of embryos at the middle neurula stage, we showed that insrr downregulation elicited a general shift of expression towards genes specifically expressed before and at the onset of gastrulation. At the same time, alkali treatment partially restored the expression of the neurula-specific genes. Thus, our results demonstrate the critical role of insrr in the regulation of the early development rate in Xenopus laevis. Full article

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16 pages, 2542 KiB

Open AccessReview

Biochemical Hazards during Three Phases of Assisted Reproductive Technology: Repercussions Associated with Epigenesis and Imprinting

by Yves Menezo, Kay Elder, Patrice Clement, Arthur Clement and Pasquale Patrizio

Int. J. Mol. Sci. 2022, 23(16), 8916; https://doi.org/10.3390/ijms23168916 - 10 Aug 2022

Cited by 6 | Viewed by 2537

Abstract

Medically assisted reproduction, now considered a routine, successful treatment for infertility worldwide, has produced at least 8 million live births. However, a growing body of evidence is pointing toward an increased incidence of epigenetic/imprinting disorders in the offspring, raising concern that the techniques [...] Read more.

Medically assisted reproduction, now considered a routine, successful treatment for infertility worldwide, has produced at least 8 million live births. However, a growing body of evidence is pointing toward an increased incidence of epigenetic/imprinting disorders in the offspring, raising concern that the techniques involved may have an impact on crucial stages of early embryo and fetal development highly vulnerable to epigenetic influence. In this paper, the key role of methylation processes in epigenesis, namely the essential biochemical/metabolic pathways involving folates and one-carbon cycles necessary for correct DNA/histone methylation, is discussed. Furthermore, potential contributors to epigenetics dysregulation during the three phases of assisted reproduction: preparation for and controlled ovarian hyperstimulation (COH); methylation processes during the preimplantation embryo culture stages; the effects of unmetabolized folic acid (UMFA) during embryogenesis on imprinting methyl “tags”, are described. Advances in technology have opened a window into developmental processes that were previously inaccessible to research: it is now clear that ART procedures have the potential to influence DNA methylation in embryonic and fetal life, with an impact on health and disease risk in future generations. Critical re-evaluation of protocols and procedures is now an urgent priority, with a focus on interventions targeted toward improving ART procedures, with special attention to in vitro culture protocols and the effects of excessive folic acid intake. Full article

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17 pages, 2089 KiB

Open AccessReview

New Advances in Using Virus-like Particles and Related Technologies for Eukaryotic Genome Editing Delivery

by Pin Lyu and Baisong Lu

Int. J. Mol. Sci. 2022, 23(15), 8750; https://doi.org/10.3390/ijms23158750 - 6 Aug 2022

Cited by 10 | Viewed by 4487

Abstract

The designer nucleases, including Zinc Finger Nuclease (ZFN), Transcription Activator-Like Effector Nuclease (TALEN), and Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated (CRISPR/Cas), have been widely used for mechanistic studies, animal model generation, and gene therapy development. Clinical trials using designer nucleases to treat genetic [...] Read more.

The designer nucleases, including Zinc Finger Nuclease (ZFN), Transcription Activator-Like Effector Nuclease (TALEN), and Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated (CRISPR/Cas), have been widely used for mechanistic studies, animal model generation, and gene therapy development. Clinical trials using designer nucleases to treat genetic diseases or cancers are showing promising results. Despite rapid progress, potential off-targets and host immune responses are challenges to be addressed for in vivo uses, especially in clinical applications. Short-term expression of the designer nucleases is necessary to reduce both risks. Currently, delivery methods enabling transient expression of designer nucleases are being pursued. Among these, virus-like particles as delivery vehicles for short-term designer nuclease expression have received much attention. This review will summarize recent developments in using virus-like particles (VLPs) for safe delivery of gene editing effectors to complement our last review on the same topic. First, we introduce some background information on how VLPs can be used for safe and efficient CRISPR/Cas9 delivery. Then, we summarize recently developed virus-like particles as genome editing vehicles. Finally, we discuss applications and future directions. Full article

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25 pages, 1939 KiB

Open AccessReview

Brain Energy Metabolism in Ischemic Stroke: Effects of Smoking and Diabetes

by Ali Ehsan Sifat, Saeideh Nozohouri, Sabrina Rahman Archie, Ekram Ahmed Chowdhury and Thomas J. Abbruscato

Int. J. Mol. Sci. 2022, 23(15), 8512; https://doi.org/10.3390/ijms23158512 - 31 Jul 2022

Cited by 30 | Viewed by 6100

Abstract

Proper regulation of energy metabolism in the brain is crucial for maintaining brain activity in physiological and different pathophysiological conditions. Ischemic stroke has a complex pathophysiology which includes perturbations in the brain energy metabolism processes which can contribute to worsening of brain injury [...] Read more.

Proper regulation of energy metabolism in the brain is crucial for maintaining brain activity in physiological and different pathophysiological conditions. Ischemic stroke has a complex pathophysiology which includes perturbations in the brain energy metabolism processes which can contribute to worsening of brain injury and stroke outcome. Smoking and diabetes are common risk factors and comorbid conditions for ischemic stroke which have also been associated with disruptions in brain energy metabolism. Simultaneous presence of these conditions may further alter energy metabolism in the brain leading to a poor clinical prognosis after an ischemic stroke event. In this review, we discuss the possible effects of smoking and/or diabetes on brain glucose utilization and mitochondrial energy metabolism which, when present concurrently, may exacerbate energy metabolism in the ischemic brain. More research is needed to investigate brain glucose utilization and mitochondrial oxidative metabolism in ischemic stroke in the presence of smoking and/or diabetes, which would provide further insights on the pathophysiology of these comorbid conditions and facilitate the development of therapeutic interventions. Full article

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17 pages, 2593 KiB

Open AccessArticle

Integrated CRISPR-Cas9 System-Mediated Knockout of IFN-γ and IFN-γ Receptor 1 in the Vero Cell Line Promotes Viral Susceptibility

by Suyeon Kim, Aleksandra Nowakowska, Young Bong Kim and Ha Youn Shin

Int. J. Mol. Sci. 2022, 23(15), 8217; https://doi.org/10.3390/ijms23158217 - 26 Jul 2022

Cited by 7 | Viewed by 3266

Abstract

The current pandemic and the possible emergence of new viruses urgently require the rapid development of antiviral vaccines and therapeutics. However, some viruses or newly generated variants are difficult to culture in common cell types or exhibit low viral susceptibility in vivo, making [...] Read more.

The current pandemic and the possible emergence of new viruses urgently require the rapid development of antiviral vaccines and therapeutics. However, some viruses or newly generated variants are difficult to culture in common cell types or exhibit low viral susceptibility in vivo, making it difficult to manufacture viral vector-based vaccines and understand host–virus interactions. To address these issues, we established new cell lines deficient in both type I and type II interferon responses, which are essential for host immunity and interference with virus replication. These cell lines were generated by developing an integrated CRISPR-Cas9 system that simultaneously expresses dual-guide RNA cassettes and Cas9 nuclease in a single plasmid. Using this highly efficient gene-editing system, we successfully established three cell lines starting from IFN-α/β-deficient Vero cells, deleting the single interferon-gamma (IFNG) gene, the IFNG receptor 1 (IFNGR1) gene, or both genes. All cell lines clearly showed a decrease in IFN-γ-responsive antiviral gene expression and cytokine production. Moreover, production of IFN-γ-induced cytokines remained low, even after HSV-1 or HCoV-OC43 infection, while expression of the receptor responsible for viral entry increased. Ultimately, knockout of IFN-signaling genes in these cell lines promoted cytopathic effects and increased apoptosis after viral infection up to three-fold. These results indicate that our integrated CRISPR-Cas9-mediated IFNG- and IFNGR1-knockout cell lines promote virus replication and will be useful in viral studies used to design novel vaccines and therapies. Full article

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12 pages, 2507 KiB

Open AccessArticle

Four Novel PAX9 Variants and the PAX9-Related Non-Syndromic Tooth Agenesis Patterns

by Haochen Liu, Hangbo Liu, Lanxin Su, Jinglei Zheng, Hailan Feng, Yang Liu, Miao Yu and Dong Han

Int. J. Mol. Sci. 2022, 23(15), 8142; https://doi.org/10.3390/ijms23158142 - 24 Jul 2022

Cited by 9 | Viewed by 2327

Abstract

The purpose of this research was to investigate and identify PAX9 gene variants in four Chinese families with non-syndromic tooth agenesis. We identified pathogenic gene variants by whole-exome sequencing (WES) and Sanger sequencing and then studied the effects of these variants on function [...] Read more.

The purpose of this research was to investigate and identify PAX9 gene variants in four Chinese families with non-syndromic tooth agenesis. We identified pathogenic gene variants by whole-exome sequencing (WES) and Sanger sequencing and then studied the effects of these variants on function by bioinformatics analysis and in vitro experiments. Four novel PAX9 heterozygous variants were identified: two missense variants (c.191G > T (p.G64V) and c.350T > G (p.V117G)) and two frameshift variants (c.352delC (p.S119Pfs*2) and c.648_649insC(p.Y217Lfs*100)). The bioinformatics analysis showed that these variants might be pathogenic. The tertiary structure analysis showed that these four variants could cause structural damage to PAX9 proteins. In vitro functional studies demonstrated that (1) the p.Y217Lfs*100 variant greatly affects mRNA stability, thereby affecting endogenous expression; (2) the p. S119Pfs* 2 variant impairs the subcellular localization of the nuclear expression of the wild-type PAX9 protein; and (3) the four variants (p.G64V, p.V117G, p.S119Pfs*2, and p.Y217Lfs*100) all significantly affect the downstream transcriptional activity of the BMP4 gene. In addition, we summarized and analyzed tooth missing positions caused by PAX9 variants and found that the maxillary second molar (84.11%) and mandibular second molar (84.11%) were the most affected tooth positions by summarizing and analyzing the PAX9-related non-syndromic tooth agenesis positions. Our results broaden the variant spectrum of the PAX9 gene related to non-syndromic tooth agenesis and provide useful information for future genetic counseling. Full article

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24 pages, 3804 KiB

Open AccessReview

T-Cell Intracellular Antigen 1-Like Protein in Physiology and Pathology

by Beatriz Ramos Velasco and José M. Izquierdo

Int. J. Mol. Sci. 2022, 23(14), 7836; https://doi.org/10.3390/ijms23147836 - 16 Jul 2022

Cited by 11 | Viewed by 3480

Abstract

T-cell intracellular antigen 1 (TIA1)-related/like (TIAR/TIAL1) protein is a multifunctional RNA-binding protein (RBP) involved in regulating many aspects of gene expression, independently or in combination with its paralog TIA1. TIAR was first described in 1992 by Paul Anderson’s lab in relation to the [...] Read more.

T-cell intracellular antigen 1 (TIA1)-related/like (TIAR/TIAL1) protein is a multifunctional RNA-binding protein (RBP) involved in regulating many aspects of gene expression, independently or in combination with its paralog TIA1. TIAR was first described in 1992 by Paul Anderson’s lab in relation to the development of a cell death phenotype in immune system cells, as it possesses nucleolytic activity against cytotoxic lymphocyte target cells. Similar to TIA1, it is characterized by a subcellular nucleo-cytoplasmic localization and ubiquitous expression in the cells of different tissues of higher organisms. In this paper, we review the relevant structural and functional information available about TIAR from a triple perspective (molecular, cellular and pathophysiological), paying special attention to its expression and regulation in cellular events and processes linked to human pathophysiology. Full article

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26 pages, 9771 KiB

Open AccessArticle

Directed Evolution of Phi Class Glutathione Transferases Involved in Multiple-Herbicide Resistance of Grass Weeds and Crops

by Elisavet Ioannou, Anastassios C. Papageorgiou and Nikolaos E. Labrou

Int. J. Mol. Sci. 2022, 23(13), 7469; https://doi.org/10.3390/ijms23137469 - 5 Jul 2022

Cited by 10 | Viewed by 2934

Abstract

The extensive application of herbicides in crop cultivation has indisputably led to the emergence of weed populations characterized by multiple herbicide resistance (MHR). This phenomenon is associated with the enhanced metabolism and detoxifying ability of endogenous enzymes, such as phi class glutathione transferases [...] Read more.

The extensive application of herbicides in crop cultivation has indisputably led to the emergence of weed populations characterized by multiple herbicide resistance (MHR). This phenomenon is associated with the enhanced metabolism and detoxifying ability of endogenous enzymes, such as phi class glutathione transferases (GSTFs). In the present work, a library of mutant GSTFs was created by in vitro directed evolution via DNA shuffling. Selected gstf genes from the weeds Alopecurus myosuroides and Lolium rigidum, and the cereal crops Triticum durum and Hordeum vulgare were recombined to forge a library of novel chimeric GSTFs. The library was activity screened and the best-performing enzyme variants were purified and characterized. The work allowed the identification of enzyme variants that exhibit an eight-fold improvement in their catalytic efficiency, higher thermal stability (8.3 °C) and three-times higher inhibition sensitivity towards the herbicide butachlor. The crystal structures of the best-performing enzyme variants were determined by X-ray crystallography. Structural analysis allowed the identification of specific structural elements that are responsible for k_cat regulation, thermal stability and inhibition potency. These improved novel enzymes hold the potential for utilization in biocatalysis and green biotechnology applications. The results of the present work contribute significantly to our knowledge of the structure and function of phi class plant GSTs and shed light on their involvement in the mechanisms of MHR. Full article

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17 pages, 3447 KiB

Open AccessArticle

Codon Usage Provides Insights into the Adaptive Evolution of Mycoviruses in Their Associated Fungi Host

by Qianqian Wang, Xueliang Lyu, Jiasen Cheng, Yanping Fu, Yang Lin, Assane Hamidou Abdoulaye, Daohong Jiang and Jiatao Xie

Int. J. Mol. Sci. 2022, 23(13), 7441; https://doi.org/10.3390/ijms23137441 - 4 Jul 2022

Cited by 14 | Viewed by 2582

Abstract

Codon usage bias (CUB) could reflect co-evolutionary changes between viruses and hosts in contrast to plant and animal viruses, and the systematic analysis of codon usage among the mycoviruses that infect plant pathogenic fungi is limited. We performed an extensive analysis of codon [...] Read more.

Codon usage bias (CUB) could reflect co-evolutionary changes between viruses and hosts in contrast to plant and animal viruses, and the systematic analysis of codon usage among the mycoviruses that infect plant pathogenic fungi is limited. We performed an extensive analysis of codon usage patterns among 98 characterized RNA mycoviruses from eight phytopathogenic fungi. The GC and GC3s contents of mycoviruses have a wide variation from 29.35% to 64.62% and 24.32% to 97.13%, respectively. Mycoviral CUB is weak, and natural selection plays a major role in the formation of mycoviral codon usage pattern. In this study, we demonstrated that the codon usage of mycoviruses is similar to that of some host genes, especially those involved in RNA biosynthetic process and transcription, suggesting that CUB is a potential evolutionary mechanism that mycoviruses adapt to in their hosts. Full article

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22 pages, 11005 KiB

Open AccessArticle

Relaxin Inhibits the Cardiac Myofibroblast NLRP3 Inflammasome as Part of Its Anti-Fibrotic Actions via the Angiotensin Type 2 and ATP (P2X7) Receptors

by Felipe Tapia Cáceres, Tracey A. Gaspari, Mohammed Akhter Hossain and Chrishan S. Samuel

Int. J. Mol. Sci. 2022, 23(13), 7074; https://doi.org/10.3390/ijms23137074 - 25 Jun 2022

Cited by 11 | Viewed by 2793

Abstract

Chronic NLRP3 inflammasome activation can promote fibrosis through its production of interleukin (IL)-1β and IL-18. Conversely, recombinant human relaxin (RLX) can inhibit the pro-fibrotic interactions between IL-1β, IL-18 and transforming growth factor (TGF)-β1. Here, the broader extent by which RLX targeted the myofibroblast [...] Read more.

Chronic NLRP3 inflammasome activation can promote fibrosis through its production of interleukin (IL)-1β and IL-18. Conversely, recombinant human relaxin (RLX) can inhibit the pro-fibrotic interactions between IL-1β, IL-18 and transforming growth factor (TGF)-β1. Here, the broader extent by which RLX targeted the myofibroblast NLRP3 inflammasome to mediate its anti-fibrotic effects was elucidated. Primary human cardiac fibroblasts (HCFs), stimulated with TGF-β1 (to promote myofibroblast (HCMF) differentiation), LPS (to prime the NLRP3 inflammasome) and ATP (to activate the NLRP3 inflammasome) (T+L+A) or benzoylbenzoyl-ATP (to activate the ATP receptor; P2X7R) (T+L+Bz), co-expressed relaxin family peptide receptor-1 (RXFP1), the angiotensin II type 2 receptor (AT₂R) and P2X7R, and underwent increased protein expression of toll-like receptor (TLR)-4, NLRP3, caspase-1, IL-1β and IL-18. Whilst RLX co-administration to HCMFs significantly prevented the T+L+A- or T+L+Bz-stimulated increase in these end points, the inhibitory effects of RLX were annulled by the pharmacological antagonism of either RXFP1, AT₂R, P2X7R, TLR-4, reactive oxygen species (ROS) or caspase-1. The RLX-induced amelioration of left ventricular inflammation, cardiomyocyte hypertrophy and fibrosis in isoproterenol (ISO)-injured mice, was also attenuated by P2X7R antagonism. Thus, the ability of RLX to ameliorate the myofibroblast NLRP3 inflammasome as part of its anti-fibrotic effects, appeared to involve RXFP1, AT₂R, P2X7R and the inhibition of TLR-4, ROS and caspase-1. Full article

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11 pages, 1451 KiB

Open AccessArticle

Major Histocompatibility Complex Class I Chain-Related α (MICA) STR Polymorphisms in COVID-19 Patients

by Juan Francisco Gutiérrez-Bautista, Alba Martinez-Chamorro, Antonio Rodriguez-Nicolas, Antonio Rosales-Castillo, Pilar Jiménez, Per Anderson, Miguel Ángel López-Ruz, Miguel Ángel López-Nevot and Francisco Ruiz-Cabello

Int. J. Mol. Sci. 2022, 23(13), 6979; https://doi.org/10.3390/ijms23136979 - 23 Jun 2022

Cited by 2 | Viewed by 2432

Abstract

The SARS-CoV-2 disease presents different phenotypes of severity. Comorbidities, age, and being overweight are well established risk factors for severe disease. However, innate immunity plays a key role in the early control of viral infections and may condition the gravity of COVID-19. Natural [...] Read more.

The SARS-CoV-2 disease presents different phenotypes of severity. Comorbidities, age, and being overweight are well established risk factors for severe disease. However, innate immunity plays a key role in the early control of viral infections and may condition the gravity of COVID-19. Natural Killer (NK) cells are part of innate immunity and are important in the control of virus infection by killing infected cells and participating in the development of adaptive immunity. Therefore, we studied the short tandem repeat (STR) transmembrane polymorphisms of the major histocompatibility complex class I chain-related A (MICA), an NKG2D ligand that induces activation of NK cells, among other cells. We compared the alleles and genotypes of MICA in COVID-19 patients versus healthy controls and analyzed their relation to disease severity. Our results indicate that the MICA*A9 allele is related to infection as well as to symptomatic disease but not to severe disease. The MICA*A9 allele may be a risk factor for SARS-CoV-2 infection and symptomatic disease. Full article

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12 pages, 2332 KiB

Open AccessArticle

IKKγ/NEMO Localization into Multivesicular Bodies

by Lisa-Marie Wackernagel, Mohsen Abdi Sarabi, Sönke Weinert, Werner Zuschratter, Karin Richter, Klaus Dieter Fischer, Ruediger C. Braun-Dullaeus and Senad Medunjanin

Int. J. Mol. Sci. 2022, 23(12), 6778; https://doi.org/10.3390/ijms23126778 - 17 Jun 2022

Cited by 3 | Viewed by 2672

Abstract

The NF-κB pathway is central pathway for inflammatory and immune responses, and IKKγ/NEMO is essential for NF-κB activation. In a previous report, we identified the role of glycogen synthase kinase-3β (GSK-3β) in NF-κB activation by regulating IKKγ/NEMO. Here, we show that NEMO phosphorylation [...] Read more.

The NF-κB pathway is central pathway for inflammatory and immune responses, and IKKγ/NEMO is essential for NF-κB activation. In a previous report, we identified the role of glycogen synthase kinase-3β (GSK-3β) in NF-κB activation by regulating IKKγ/NEMO. Here, we show that NEMO phosphorylation by GSK-3β leads to NEMO localization into multivesicular bodies (MVBs). Using the endosome marker Rab5, we observed localization into endosomes. Using siRNA, we identified the AAA-ATPase Vps4A, which is involved in recycling the ESCRT machinery by facilitating its dissociation from endosomal membranes, which is necessary for NEMO stability and NF-κB activation. Co-immunoprecipitation studies of NEMO and mutated NEMO demonstrated its direct interaction with Vps4A, which requires NEMO phosphorylation. The transfection of cells by a mutated and constitutively active form of Vps4A, Vps4A-E233Q, resulted in the formation of large vacuoles and strong augmentation in NEMO expression compared to GFP-Vps4-WT. In addition, the overexpression of the mutated form of Vps4A led to increased NF-κB activation. The treatment of cells with the pharmacologic V-ATPase inhibitor bafilomycin A led to a dramatic downregulation of NEMO and, in this way, inhibited NF-κB signal transduction. These results reveal an unexpected role for GSK-3β and V-ATPase in NF-κB signaling activation. Full article

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22 pages, 2403 KiB

Open AccessReview

Epigenetic Connection of the Calcitonin Gene-Related Peptide and Its Potential in Migraine

by Michal Fila, Anna Sobczuk, Elzbieta Pawlowska and Janusz Blasiak

Int. J. Mol. Sci. 2022, 23(11), 6151; https://doi.org/10.3390/ijms23116151 - 30 May 2022

Cited by 21 | Viewed by 5852

Abstract

The calcitonin gene-related peptide (CGRP) is implicated in the pathogenesis of several pain-related syndromes, including migraine. Targeting CGRP and its receptor by their antagonists and antibodies was a breakthrough in migraine therapy, but the need to improve efficacy and limit the side effects [...] Read more.

The calcitonin gene-related peptide (CGRP) is implicated in the pathogenesis of several pain-related syndromes, including migraine. Targeting CGRP and its receptor by their antagonists and antibodies was a breakthrough in migraine therapy, but the need to improve efficacy and limit the side effects of these drugs justify further studies on the regulation of CGRP in migraine. The expression of the CGRP encoding gene, CALCA, is modulated by epigenetic modifications, including the DNA methylation, histone modification, and effects of micro RNAs (miRNAs), circular RNAs, and long-coding RNAs (lncRNAs). On the other hand, CGRP can change the epigenetic profile of neuronal and glial cells. The promoter of the CALCA gene has two CpG islands that may be specifically methylated in migraine patients. DNA methylation and lncRNAs were shown to play a role in the cell-specific alternative splicing of the CALCA primary transcript. CGRP may be involved in changes in neural cytoarchitecture that are controlled by histone deacetylase 6 (HDAC6) and can be related to migraine. Inhibition of HDAC6 results in reduced cortical-spreading depression and a blockade of the CGRP receptor. CGRP levels are associated with the expression of several miRNAs in plasma, making them useful peripheral markers of migraine. The fundamental role of CGRP in inflammatory pain transmission may be epigenetically regulated. In conclusion, epigenetic connections of CGRP should be further explored for efficient and safe antimigraine therapy. Full article

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16 pages, 3071 KiB

Open AccessArticle

Curvicollide D Isolated from the Fungus Amesia sp. Kills African Trypanosomes by Inhibiting Transcription

by Matilde Ortiz-Gonzalez, Ignacio Pérez-Victoria, Inmaculada Ramirez-Macias, Nuria de Pedro, Angel Linde-Rodriguez, Víctor González-Menéndez, Victoria Sanchez-Martin, Jesús Martín, Ana Soriano-Lerma, Olga Genilloud, Virginia Perez-Carrasco, Francisca Vicente, José Maceira, Carlos A. Rodrígues-Poveda, José María Navarro-Marí, Fernando Reyes, Miguel Soriano and Jose A. Garcia-Salcedo

Int. J. Mol. Sci. 2022, 23(11), 6107; https://doi.org/10.3390/ijms23116107 - 29 May 2022

Cited by 1 | Viewed by 2773

Abstract

Sleeping sickness or African trypanosomiasis is a serious health concern with an added socio-economic impact in sub-Saharan Africa due to direct infection in both humans and their domestic livestock. There is no vaccine available against African trypanosomes and its treatment relies only on [...] Read more.

Sleeping sickness or African trypanosomiasis is a serious health concern with an added socio-economic impact in sub-Saharan Africa due to direct infection in both humans and their domestic livestock. There is no vaccine available against African trypanosomes and its treatment relies only on chemotherapy. Although the current drugs are effective, most of them are far from the modern concept of a drug in terms of toxicity, specificity and therapeutic regime. In a search for new molecules with trypanocidal activity, a high throughput screening of 2000 microbial extracts was performed. Fractionation of one of these extracts, belonging to a culture of the fungus Amesia sp., yielded a new member of the curvicollide family that has been designated as curvicollide D. The new compound showed an inhibitory concentration 50 (IC₅₀) 16-fold lower in Trypanosoma brucei than in human cells. Moreover, it induced cell cycle arrest and disruption of the nucleolar structure. Finally, we showed that curvicollide D binds to DNA and inhibits transcription in African trypanosomes, resulting in cell death. These results constitute the first report on the activity and mode of action of a member of the curvicollide family in T. brucei. Full article

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26 pages, 8406 KiB

Open AccessArticle

Loss of BMP2 and BMP4 Signaling in the Dental Epithelium Causes Defective Enamel Maturation and Aberrant Development of Ameloblasts

by Claes-Göran Reibring, Maha El Shahawy, Kristina Hallberg, Brian D. Harfe, Anders Linde and Amel Gritli-Linde

Int. J. Mol. Sci. 2022, 23(11), 6095; https://doi.org/10.3390/ijms23116095 - 29 May 2022

Viewed by 3377

Abstract

BMP signaling is crucial for differentiation of secretory ameloblasts, the cells that secrete enamel matrix. However, whether BMP signaling is required for differentiation of maturation-stage ameloblasts (MA), which are instrumental for enamel maturation into hard tissue, is hitherto unknown. To address this, we [...] Read more.

BMP signaling is crucial for differentiation of secretory ameloblasts, the cells that secrete enamel matrix. However, whether BMP signaling is required for differentiation of maturation-stage ameloblasts (MA), which are instrumental for enamel maturation into hard tissue, is hitherto unknown. To address this, we used an in vivo genetic approach which revealed that combined deactivation of the Bmp2 and Bmp4 genes in the murine dental epithelium causes development of dysmorphic and dysfunctional MA. These fail to exhibit a ruffled apical plasma membrane and to reabsorb enamel matrix proteins, leading to enamel defects mimicking hypomaturation amelogenesis imperfecta. Furthermore, subsets of mutant MA underwent pathological single or collective cell migration away from the ameloblast layer, forming cysts and/or exuberant tumor-like and gland-like structures. Massive apoptosis in the adjacent stratum intermedium and the abnormal cell-cell contacts and cell-matrix adhesion of MA may contribute to this aberrant behavior. The mutant MA also exhibited severely diminished tissue non-specific alkaline phosphatase activity, revealing that this enzyme’s activity in MA crucially depends on BMP2 and BMP4 inputs. Our findings show that combined BMP2 and BMP4 signaling is crucial for survival of the stratum intermedium and for proper development and function of MA to ensure normal enamel maturation. Full article

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13 pages, 5413 KiB

Open AccessArticle

Determination of IgG1 and IgG3 SARS-CoV-2 Spike Protein and Nucleocapsid Binding—Who Is Binding Who and Why?

by Jason K. Iles, Raminta Zmuidinaite, Christoph Sadee, Anna Gardiner, Jonathan Lacey, Stephen Harding, Gregg Wallis, Roshani Patel, Debra Roblett, Jonathan Heeney, Helen Baxendale and Ray Kruse Iles

Int. J. Mol. Sci. 2022, 23(11), 6050; https://doi.org/10.3390/ijms23116050 - 27 May 2022

Cited by 7 | Viewed by 3476

Abstract

The involvement of immunoglobulin (Ig) G3 in the humoral immune response to SARS-CoV-2 infection has been implicated in the pathogenesis of acute respiratory distress syndrome (ARDS) in COVID-19. The exact molecular mechanism is unknown, but it is thought to involve this IgG subtype’s [...] Read more.

The involvement of immunoglobulin (Ig) G3 in the humoral immune response to SARS-CoV-2 infection has been implicated in the pathogenesis of acute respiratory distress syndrome (ARDS) in COVID-19. The exact molecular mechanism is unknown, but it is thought to involve this IgG subtype’s differential ability to fix, complement and stimulate cytokine release. We examined the binding of convalescent patient antibodies to immobilized nucleocapsids and spike proteins by matrix-assisted laser desorption/ionization–time of flight (MALDI-ToF) mass spectrometry. IgG3 was a major immunoglobulin found in all samples. Differential analysis of the spectral signatures found for the nucleocapsid versus the spike protein demonstrated that the predominant humoral immune response to the nucleocapsid was IgG3, whilst for the spike protein it was IgG1. However, the spike protein displayed a strong affinity for IgG3 itself, as it would bind from control plasma samples, as well as from those previously infected with SARS-CoV-2, similar to the way protein G binds IgG1. Furthermore, detailed spectral analysis indicated that a mass shift consistent with hyper-glycosylation or glycation was a characteristic of the IgG3 captured by the spike protein. Full article

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16 pages, 3545 KiB

Open AccessArticle

Structure-Based Development of SARS-CoV-2 Spike Interactors

by Flavia Squeglia, Maria Romano, Luciana Esposito, Giovanni Barra, Pietro Campiglia, Marina Sala, Maria Carmina Scala, Alessia Ruggiero and Rita Berisio

Int. J. Mol. Sci. 2022, 23(10), 5601; https://doi.org/10.3390/ijms23105601 - 17 May 2022

Cited by 4 | Viewed by 2668

Abstract

Coronaviruses, including SARS-CoV-2 (the etiological agent of the current COVID-19 pandemic), rely on the surface spike glycoprotein to access the host cells, mainly through the interaction of their receptor-binding domain (RBD) with the human angiotensin-converting enzyme 2 (ACE2). Therefore, molecular entities able to [...] Read more.

Coronaviruses, including SARS-CoV-2 (the etiological agent of the current COVID-19 pandemic), rely on the surface spike glycoprotein to access the host cells, mainly through the interaction of their receptor-binding domain (RBD) with the human angiotensin-converting enzyme 2 (ACE2). Therefore, molecular entities able to interfere with the binding of the SARS-CoV-2 spike protein to ACE2 have great potential to inhibit viral entry. Starting from the available structural data on the interaction between SARS-CoV-2 spike protein and the host ACE2 receptor, we engineered a set of soluble and stable spike interactors, here denoted as S-plugs. Starting from the prototype S-plug, we adopted a computational approach by combining stability prediction, associated to single-point mutations, with molecular dynamics to enhance both S-plug thermostability and binding affinity to the spike protein. The best developed molecule, S-plug3, possesses a highly stable α-helical con-formation (with melting temperature Tm of 54 °C) and can interact with the spike RBD and S1 domains with similar low nanomolar affinities. Importantly, S-plug3 exposes the spike RBD to almost the same interface as the human ACE2 receptor, aimed at the recognition of all ACE2-accessing coronaviruses. Consistently, S-plug3 preserves a low nanomolar dissociation constant with the delta B.1.617.2 variant of SARS-CoV-2 spike protein (K_D = 29.2 ± 0.6 nM). Taken together, we provide valid starting data for the development of therapeutical and diagnostic tools against coronaviruses accessing through ACE2. Full article

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16 pages, 2489 KiB

Open AccessArticle

Expression of Oxidative Stress and Inflammation-Related Genes in Nasal Mucosa and Nasal Polyps from Patients with Chronic Rhinosinusitis

by Hrvoje Mihalj, Josip Butković, Stana Tokić, Mario Štefanić, Tomislav Kizivat, Maro Bujak, Mirela Baus Lončar and Martina Mihalj

Int. J. Mol. Sci. 2022, 23(10), 5521; https://doi.org/10.3390/ijms23105521 - 15 May 2022

Cited by 11 | Viewed by 3431

Abstract

Chronic rhinosinusitis (CRS) is a prevalent, multifaceted inflammatory condition affecting the nasal cavity and the paranasal sinuses, frequently accompanied by formation of nasal polyps (CRSwNP). This apparently uniform clinical entity is preceded by heterogeneous changes in cellular and molecular patterns, suggesting the presence [...] Read more.

Chronic rhinosinusitis (CRS) is a prevalent, multifaceted inflammatory condition affecting the nasal cavity and the paranasal sinuses, frequently accompanied by formation of nasal polyps (CRSwNP). This apparently uniform clinical entity is preceded by heterogeneous changes in cellular and molecular patterns, suggesting the presence of multiple CRS endotypes and a diverse etiology. Alterations of the upper airway innate defense mechanisms, including antimicrobial and antioxidant capacity, have been implicated in CRSwNP etiology. The aim of this study was to investigate mRNA expression patterns of antioxidative enzymes, including superoxide dismutase (SOD) and peroxiredoxin-2 (PRDX2), and innate immune system defense players, namely the bactericidal/permeability-increasing fold-containing family A, member 1 (BPIFA1) and PACAP family members, particularly adenylate-cyclase-activating polypeptide receptor 1 (ADCYAP1) in nasal mucosa and nasal polyps from CRSwNP patients. Additional stratification based on age, sex, allergic comorbidity, and disease severity was applied. The results showed that ADCYAP1, BPIFA1, and PRDX2 transcripts are differentially expressed in nasal mucosa and scale with radiologically assessed disease severity in CRSwNP patients. Sinonasal transcriptome is not associated with age, sex, and smoking in CRSwNP. Surgical and postoperative corticosteroid (CS) therapy improves endoscopic appearance of the mucosa, but variably reverses target gene expression patterns in the nasal cavity of CRSwNP patients. Transcriptional cross-correlations analysis revealed an increased level of connectedness among differentially expressed genes under inflammatory conditions and restoration of basic network following CS treatment. Although results of the present study imply a possible engagement of ADCYAP1 and BPIFA1 as biomarkers for CRSwNP, a more profound study taking into account disease severity and CRSwNP endotypes prior to the treatment would provide additional information on their sensitivity. Full article

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19 pages, 5829 KiB

Open AccessArticle

Early Growth Response 1 Strengthens Pol-III-Directed Transcription and Transformed Cell Proliferation by Controlling PTEN/AKT Signalling Activity

by Zhongyu Wu, Liyun Huang, Shasha Zhao, Juan Wang, Cheng Zhang, Xiaoye Song, Qiyue Chen, Jiannan Du, Deen Yu, Xiaomeng Sun, Yue Zhang, Wensheng Deng, Shihua Zhang and Huan Deng

Int. J. Mol. Sci. 2022, 23(9), 4930; https://doi.org/10.3390/ijms23094930 - 29 Apr 2022

Cited by 3 | Viewed by 2234

Abstract

RNA polymerase III (Pol III) products play essential roles in ribosome assembly, protein synthesis, and cell survival. Deregulation of Pol-III-directed transcription is closely associated with tumorigenesis. However, the regulatory pathways or factors controlling Pol-III-directed transcription remain to be investigated. In this study, we [...] Read more.

RNA polymerase III (Pol III) products play essential roles in ribosome assembly, protein synthesis, and cell survival. Deregulation of Pol-III-directed transcription is closely associated with tumorigenesis. However, the regulatory pathways or factors controlling Pol-III-directed transcription remain to be investigated. In this study, we identified a novel role of EGR1 in Pol-III-directed transcription. We found that Filamin A (FLNA) silencing stimulated EGR1 expression at both RNA and protein levels. EGR1 expression positively correlated with Pol III product levels and cell proliferation activity. Mechanistically, EGR1 downregulation dampened the occupancies of Pol III transcription machinery factors at the loci of Pol III target genes. Alteration of EGR1 expression did not affect the expression of p53, c-MYC, and Pol III general transcription factors. Instead, EGR1 activated RhoA expression and inhibited PTEN expression in several transformed cell lines. We found that PTEN silencing, rather than RhoA overexpression, could reverse the inhibition of Pol-III-dependent transcription and cell proliferation caused by EGR1 downregulation. EGR1 could positively regulate AKT phosphorylation levels and is required for the inhibition of Pol-III-directed transcription mediated by FLNA. The findings from this study indicate that EGR1 can promote Pol-III-directed transcription and cell proliferation by controlling the PTEN/AKT signalling pathway. Full article

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17 pages, 4402 KiB

Open AccessArticle

Map of Enteropathogenic Escherichia coli Targets Mitochondria and Triggers DRP-1-Mediated Mitochondrial Fission and Cell Apoptosis in Bovine Mastitis

by Yanan Li, Yaohong Zhu, Bingxin Chu, Ning Liu, Shiyan Chen, Jiufeng Wang and Yunjing Zou

Int. J. Mol. Sci. 2022, 23(9), 4907; https://doi.org/10.3390/ijms23094907 - 28 Apr 2022

Cited by 10 | Viewed by 3034

Abstract

Bovine mastitis seriously affects bovine health and dairy product quality. Escherichia coli is the most important pathogen in the environment and dairy products. Enteropathogenic Escherichia coli (EPEC) is a zoonotic pathogen, which seriously threatens the health of people and dairy cows. We recently [...] Read more.

Bovine mastitis seriously affects bovine health and dairy product quality. Escherichia coli is the most important pathogen in the environment and dairy products. Enteropathogenic Escherichia coli (EPEC) is a zoonotic pathogen, which seriously threatens the health of people and dairy cows. We recently reported that E. coli can induce endogenous apoptosis in bovine mammary epithelial cells. However, the mechanism of EPEC-damaged mitochondria and -induced bovine mastitis is unclear. In this study, we found that EPEC can induce DRP-1-dependent mitochondrial fission and apoptosis. This was verified by the application of Mdivi, a DRP-1 inhibitor. Meanwhile, in order to verify the role of the Map virulence factor in EPEC-induced bovine mastitis, we constructed a map mutant, complementary strain, and recombinant plasmid Map_His. In the present study, we find that Map induced DRP-1-mediated mitochondrial fission, resulting in mitochondrial dysfunction and apoptosis. These inferences were further verified in vivo by establishing a mouse mastitis model. After the map gene was knocked out, breast inflammation and apoptosis in mice were significantly alleviated. All results show that EPEC targets mitochondria by secreting the Map virulence factor to induce DRP-1-mediated mitochondrial fission, mitochondrial dysfunction, and endogenous apoptosis in bovine mastitis. Full article

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25 pages, 9051 KiB

Open AccessArticle

Myo5b Transports Fibronectin-Containing Vesicles and Facilitates FN1 Secretion from Human Pleural Mesothelial Cells

by Tsuyoshi Sakai, Young-yeon Choo, Osamu Sato, Reiko Ikebe, Ann Jeffers, Steven Idell, Torry Tucker and Mitsuo Ikebe

Int. J. Mol. Sci. 2022, 23(9), 4823; https://doi.org/10.3390/ijms23094823 - 27 Apr 2022

Cited by 7 | Viewed by 2510

Abstract

Pleural mesothelial cells (PMCs) play a central role in the progression of pleural fibrosis. As pleural injury progresses to fibrosis, PMCs transition to mesenchymal myofibroblast via mesothelial mesenchymal transition (MesoMT), and produce extracellular matrix (ECM) proteins including collagen and fibronectin (FN1). FN1 plays [...] Read more.

Pleural mesothelial cells (PMCs) play a central role in the progression of pleural fibrosis. As pleural injury progresses to fibrosis, PMCs transition to mesenchymal myofibroblast via mesothelial mesenchymal transition (MesoMT), and produce extracellular matrix (ECM) proteins including collagen and fibronectin (FN1). FN1 plays an important role in ECM maturation and facilitates ECM-myofibroblast interaction, thus facilitating fibrosis. However, the mechanism of FN1 secretion is poorly understood. We report here that myosin 5b (Myo5b) plays a critical role in the transportation and secretion of FN1 from human pleural mesothelial cells (HPMCs). TGF-β significantly increased the expression and secretion of FN1 from HPMCs and facilitates the close association of Myo5B with FN1 and Rab11b. Moreover, Myo5b directly binds to GTP bound Rab11b (Rab11b-GTP) but not GDP bound Rab11b. Myo5b or Rab11b knockdown via siRNA significantly attenuated the secretion of FN1 without changing FN1 expression. TGF-β also induced Rab11b-GTP formation, and Rab11b-GTP but not Rab11b-GDP significantly activated the actin-activated ATPase activity of Myo5B. Live cell imaging revealed that Myo5b- and FN1-containing vesicles continuously moved together in a single direction. These results support that Myo5b and Rab11b play an important role in FN1 transportation and secretion from HPMCs, and consequently may contribute to the development of pleural fibrosis. Full article

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14 pages, 5108 KiB

Open AccessArticle

The Tbx6 Transcription Factor Dorsocross Mediates Dpp Signaling to Regulate Drosophila Thorax Closure

by Juan Lu, Yingjie Wang, Xiao Wang, Dan Wang, Gert O. Pflugfelder and Jie Shen

Int. J. Mol. Sci. 2022, 23(9), 4543; https://doi.org/10.3390/ijms23094543 - 20 Apr 2022

Cited by 2 | Viewed by 2688

Abstract

Movement and fusion of separate cell populations are critical for several developmental processes, such as neural tube closure in vertebrates or embryonic dorsal closure and pupal thorax closure in Drosophila. Fusion failure results in an opening or groove on the body surface. [...] Read more.

Movement and fusion of separate cell populations are critical for several developmental processes, such as neural tube closure in vertebrates or embryonic dorsal closure and pupal thorax closure in Drosophila. Fusion failure results in an opening or groove on the body surface. Drosophila pupal thorax closure is an established model to investigate the mechanism of tissue closure. Here, we report the identification of T-box transcription factor genes Dorsocross (Doc) as Decapentaplegic (Dpp) targets in the leading edge cells of the notum in the late third instar larval and early pupal stages. Reduction of Doc in the notum region results in a thorax closure defect, similar to that in dpp loss-of-function flies. Nine genes are identified as potential downstream targets of Doc in regulating thorax closure by molecular and genetic screens. Our results reveal a novel function of Doc in Drosophila development. The candidate target genes provide new clues for unravelling the mechanism of collective cell movement. Full article

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16 pages, 1340 KiB

Open AccessReview

Molecular Dynamics and Evolution of Centromeres in the Genus Equus

by Francesca M. Piras, Eleonora Cappelletti, Marco Santagostino, Solomon G. Nergadze, Elena Giulotto and Elena Raimondi

Int. J. Mol. Sci. 2022, 23(8), 4183; https://doi.org/10.3390/ijms23084183 - 10 Apr 2022

Cited by 6 | Viewed by 3246

Abstract

The centromere is the chromosomal locus essential for proper chromosome segregation. While the centromeric function is well conserved and epigenetically specified, centromeric DNA sequences are typically composed of satellite DNA and represent the most rapidly evolving sequences in eukaryotic genomes. The presence of [...] Read more.

The centromere is the chromosomal locus essential for proper chromosome segregation. While the centromeric function is well conserved and epigenetically specified, centromeric DNA sequences are typically composed of satellite DNA and represent the most rapidly evolving sequences in eukaryotic genomes. The presence of satellite sequences at centromeres hampered the comprehensive molecular analysis of these enigmatic loci. The discovery of functional centromeres completely devoid of satellite repetitions and fixed in some animal and plant species represented a turning point in centromere biology, definitively proving the epigenetic nature of the centromere. The first satellite-free centromere, fixed in a vertebrate species, was discovered in the horse. Later, an extraordinary number of satellite-free neocentromeres had been discovered in other species of the genus Equus, which remains the only mammalian genus with numerous satellite-free centromeres described thus far. These neocentromeres arose recently during evolution and are caught in a stage of incomplete maturation. Their presence made the equids a unique model for investigating, at molecular level, the minimal requirements for centromere seeding and evolution. This model system provided new insights on how centromeres are established and transmitted to the progeny and on the role of satellite DNA in different aspects of centromere biology. Full article

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13 pages, 1027 KiB

Open AccessReview

A New Target of Dental Pulp-Derived Stem Cell-Based Therapy on Recipient Bone Marrow Niche in Systemic Lupus Erythematosus

by Soichiro Sonoda and Takayoshi Yamaza

Int. J. Mol. Sci. 2022, 23(7), 3479; https://doi.org/10.3390/ijms23073479 - 23 Mar 2022

Cited by 8 | Viewed by 3368

Abstract

Recent advances in mesenchymal stem/stromal cell (MSC) research have led us to consider the feasibility of MSC-based therapy for various diseases. Human dental pulp-derived MSCs (hDPSCs) have been identified in the dental pulp tissue of deciduous and permanent teeth, and they exhibit properties [...] Read more.

Recent advances in mesenchymal stem/stromal cell (MSC) research have led us to consider the feasibility of MSC-based therapy for various diseases. Human dental pulp-derived MSCs (hDPSCs) have been identified in the dental pulp tissue of deciduous and permanent teeth, and they exhibit properties with self-renewal and in vitro multipotency. Interestingly, hDPSCs exhibit superior immunosuppressive functions toward immune cells, especially T lymphocytes, both in vitro and in vivo. Recently, hDPSCs have been shown to have potent immunomodulatory functions in treating systemic lupus erythematosus (SLE) in the SLE MRL/lpr mouse model. However, the mechanisms underlying the immunosuppressive efficacy of hDPSCs remain unknown. This review aims to introduce a new target of hDPSC-based therapy on the recipient niche function in SLE. Full article

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20 pages, 1261 KiB

Open AccessReview

Cannabinoids: Therapeutic Use in Clinical Practice

by Cristina Pagano, Giovanna Navarra, Laura Coppola, Giorgio Avilia, Maurizio Bifulco and Chiara Laezza

Int. J. Mol. Sci. 2022, 23(6), 3344; https://doi.org/10.3390/ijms23063344 - 19 Mar 2022

Cited by 113 | Viewed by 20738

Abstract

Medical case reports suggest that cannabinoids extracted from Cannabis sativa have therapeutic effects; however, the therapeutic employment is limited due to the psychotropic effect of its major component, Δ9-tetrahydrocannabinol (THC). The new scientific discoveries related to the endocannabinoid system, including new receptors, ligands, [...] Read more.

Medical case reports suggest that cannabinoids extracted from Cannabis sativa have therapeutic effects; however, the therapeutic employment is limited due to the psychotropic effect of its major component, Δ9-tetrahydrocannabinol (THC). The new scientific discoveries related to the endocannabinoid system, including new receptors, ligands, and mediators, allowed the development of new therapeutic targets for the treatment of several pathological disorders minimizing the undesirable psychotropic effects of some constituents of this plant. Today, FDA-approved drugs, such as nabiximols (a mixture of THC and non-psychoactive cannabidiol (CBD)), are employed in alleviating pain and spasticity in multiple sclerosis. Dronabinol and nabilone are used for the treatment of chemotherapy-induced nausea and vomiting in cancer patients. Dronabinol was approved for the treatment of anorexia in patients with AIDS (acquired immune deficiency syndrome). In this review, we highlighted the potential therapeutic efficacy of natural and synthetic cannabinoids and their clinical relevance in cancer, neurodegenerative and dermatological diseases, and viral infections. Full article

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21 pages, 688 KiB

Open AccessReview

Thromboinflammation in Myeloproliferative Neoplasms (MPN)—A Puzzle Still to Be Solved

by Vikas Bhuria, Conny K. Baldauf, Burkhart Schraven and Thomas Fischer

Int. J. Mol. Sci. 2022, 23(6), 3206; https://doi.org/10.3390/ijms23063206 - 16 Mar 2022

Cited by 18 | Viewed by 4839

Abstract

Myeloproliferative neoplasms (MPNs), a group of malignant hematological disorders, occur as a consequence of somatic mutations in the hematopoietic stem cell compartment and show excessive accumulation of mature myeloid cells in the blood. A major cause of morbidity and mortality in these patients [...] Read more.

Myeloproliferative neoplasms (MPNs), a group of malignant hematological disorders, occur as a consequence of somatic mutations in the hematopoietic stem cell compartment and show excessive accumulation of mature myeloid cells in the blood. A major cause of morbidity and mortality in these patients is the marked prothrombotic state leading to venous and arterial thrombosis, including myocardial infarction (MI), deep vein thrombosis (DVT), and strokes. Additionally, many MPN patients suffer from inflammation-mediated constitutional symptoms, such as fever, night sweats, fatigue, and cachexia. The chronic inflammatory syndrome in MPNs is associated with the up-regulation of various inflammatory cytokines in patients and is involved in the formation of the so-called MPN thromboinflammation. JAK2-V617F, the most prevalent mutation in MPNs, has been shown to activate a number of integrins on mature myeloid cells, including granulocytes and erythrocytes, which increase adhesion and drive venous thrombosis in murine knock-in/out models. This review aims to shed light on the current understanding of thromboinflammation, involvement of neutrophils in the prothrombotic state, plausible molecular mechanisms triggering the process of thrombosis, and potential novel therapeutic targets for developing effective strategies to reduce the MPN disease burden. Full article

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17 pages, 4989 KiB

Open AccessArticle

The Splicing of the Mitochondrial Calcium Uniporter Genuine Activator MICU1 Is Driven by RBFOX2 Splicing Factor during Myogenic Differentiation

by Denis Vecellio Reane, Cristina Cerqua, Sabrina Sacconi, Leonardo Salviati, Eva Trevisson and Anna Raffaello

Int. J. Mol. Sci. 2022, 23(5), 2517; https://doi.org/10.3390/ijms23052517 - 24 Feb 2022

Cited by 6 | Viewed by 3077

Abstract

Alternative splicing, the process by which exons within a pre-mRNA transcript are differentially joined or skipped, is crucial in skeletal muscle since it is required both during myogenesis and in post-natal life to reprogram the transcripts of contractile proteins, metabolic enzymes, and transcription [...] Read more.

Alternative splicing, the process by which exons within a pre-mRNA transcript are differentially joined or skipped, is crucial in skeletal muscle since it is required both during myogenesis and in post-natal life to reprogram the transcripts of contractile proteins, metabolic enzymes, and transcription factors in functionally distinct muscle fiber types. The importance of such events is underlined by the numerosity of pathological conditions caused by alternative splicing aberrations. Importantly, many skeletal muscle Ca²⁺ homeostasis genes are also regulated by alternative splicing mechanisms, among which is the Mitochondrial Ca²⁺ Uniporter (MCU) genuine activator MICU1 which regulates MCU opening upon cell stimulation. We have previously shown that murine skeletal muscle MICU1 is subjected to alternative splicing, thereby generating a splice variant—which was named MICU1.1—that confers unique properties to the mitochondrial Ca²⁺ uptake and ensuring sufficient ATP production for muscle contraction. Here we extended the analysis of MICU1 alternative splicing to human tissues, finding two additional splicing variants that were characterized by their ability to regulate mitochondrial Ca²⁺ uptake. Furthermore, we found that MICU1 alternative splicing is induced during myogenesis by the splicing factor RBFOX2. These results highlight the complexity of the alternative splicing mechanisms in skeletal muscle and the regulation of mitochondrial Ca²⁺ among tissues. Full article

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33 pages, 14904 KiB

Open AccessReview

Making Sense of “Nonsense” and More: Challenges and Opportunities in the Genetic Code Expansion, in the World of tRNA Modifications

by Olubodun Michael Lateef, Michael Olawale Akintubosun, Olamide Tosin Olaoba, Sunday Ocholi Samson and Malgorzata Adamczyk

Int. J. Mol. Sci. 2022, 23(2), 938; https://doi.org/10.3390/ijms23020938 - 15 Jan 2022

Cited by 13 | Viewed by 6772

Abstract

The evolutional development of the RNA translation process that leads to protein synthesis based on naturally occurring amino acids has its continuation via synthetic biology, the so-called rational bioengineering. Genetic code expansion (GCE) explores beyond the natural translational processes to further enhance the [...] Read more.

The evolutional development of the RNA translation process that leads to protein synthesis based on naturally occurring amino acids has its continuation via synthetic biology, the so-called rational bioengineering. Genetic code expansion (GCE) explores beyond the natural translational processes to further enhance the structural properties and augment the functionality of a wide range of proteins. Prokaryotic and eukaryotic ribosomal machinery have been proven to accept engineered tRNAs from orthogonal organisms to efficiently incorporate noncanonical amino acids (ncAAs) with rationally designed side chains. These side chains can be reactive or functional groups, which can be extensively utilized in biochemical, biophysical, and cellular studies. Genetic code extension offers the contingency of introducing more than one ncAA into protein through frameshift suppression, multi-site-specific incorporation of ncAAs, thereby increasing the vast number of possible applications. However, different mediating factors reduce the yield and efficiency of ncAA incorporation into synthetic proteins. In this review, we comment on the recent advancements in genetic code expansion to signify the relevance of systems biology in improving ncAA incorporation efficiency. We discuss the emerging impact of tRNA modifications and metabolism in protein design. We also provide examples of the latest successful accomplishments in synthetic protein therapeutics and show how codon expansion has been employed in various scientific and biotechnological applications. Full article

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2021

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23 pages, 4512 KiB

Open AccessArticle

Poly(C)-binding Protein 2 Regulates the p53 Expression via Interactions with the 5′-Terminal Region of p53 mRNA

by Damian M. Janecki, Agata Swiatkowska, Joanna Szpotkowska, Anna Urbanowicz, Martyna Kabacińska, Kamil Szpotkowski and Jerzy Ciesiołka

Int. J. Mol. Sci. 2021, 22(24), 13306; https://doi.org/10.3390/ijms222413306 - 10 Dec 2021

Cited by 8 | Viewed by 2979

Abstract

The p53 protein is one of the major transcriptional factors which guards cell homeostasis. Here, we showed that poly(C)-binding protein 2 (PCBP2) can bind directly to the 5′ terminus of p53 mRNA by means of electrophoretic mobility shift assay. Binding sites of PCBP2 [...] Read more.

The p53 protein is one of the major transcriptional factors which guards cell homeostasis. Here, we showed that poly(C)-binding protein 2 (PCBP2) can bind directly to the 5′ terminus of p53 mRNA by means of electrophoretic mobility shift assay. Binding sites of PCBP2 within this region of p53 mRNA were mapped using Pb2+-induced cleavage and SAXS methods. Strikingly, the downregulation of PCBP2 in HCT116 cells resulted in a lower level of p53 protein under normal and stress conditions. Quantitative analysis of p53 mRNA in PCBP2-downregulated cells revealed a lower level of p53 mRNA under normal conditions suggesting the involvement of PCBP2 in p53 mRNA stabilisation. However, no significant change in p53 mRNA level was observed upon PCBP2 depletion under genotoxic stress. Moreover, a higher level of p53 protein in the presence of rapamycin or doxorubicin and the combination of both antibiotics was noticed in PCBP2-overexpressed cells compared to control cells. These observations indicate the potential involvement of PCBP2 in cap-independent translation of p53 mRNA especially occurring under stress conditions. It has been postulated that the PCBP2 protein is engaged in the enhancement of p53 mRNA stability, probably via interacting with its 3′ end. Our data show that under stress conditions PCBP2 also modulates p53 translation through binding to the 5′ terminus of p53 mRNA. Thus PCBP2 emerges as a double-function factor in the p53 expression. Full article

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19 pages, 7486 KiB

Open AccessArticle

Thermodynamic Studies of Interactions between Sertraline Hydrochloride and Randomly Methylated β-Cyclodextrin Molecules Supported by Circular Dichroism Spectroscopy and Molecular Docking Results

by Sylwia Belica-Pacha, Mateusz Daśko, Vyacheslav Buko, Ilya Zavodnik, Katarzyna Miłowska and Maria Bryszewska

Int. J. Mol. Sci. 2021, 22(22), 12357; https://doi.org/10.3390/ijms222212357 - 16 Nov 2021

Cited by 8 | Viewed by 3194

Abstract

The interaction between sertraline hydrochloride (SRT) and randomly methylated β-cyclodextrin (RMβCD) molecules have been investigated at 298.15 K under atmospheric pressure. The method used—Isothermal Titration Calorimetry (ITC) enabled to determine values of the thermodynamic functions like the enthalpy (ΔH), the entropy (ΔS) and [...] Read more.

The interaction between sertraline hydrochloride (SRT) and randomly methylated β-cyclodextrin (RMβCD) molecules have been investigated at 298.15 K under atmospheric pressure. The method used—Isothermal Titration Calorimetry (ITC) enabled to determine values of the thermodynamic functions like the enthalpy (ΔH), the entropy (ΔS) and the Gibbs free energy (ΔG) of binding for the examined system. Moreover, the stoichiometry coefficient of binding (n) and binding/association constant (K) value have been calculated from the experimental results. The obtained outcome was compared with the data from the literature for other non-ionic βCD derivatives interacting with SRT and the enthalpy-entropy compensation were observed and interpreted. Furthermore, the connection of RMβCD with SRT was characterized by circular dichroism spectroscopy (CD) and complexes of βCD derivatives with SRT were characterized through the computational studies with the use of molecular docking (MD). Full article

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27 pages, 1871 KiB

Open AccessReview

Cytokine Networks in the Pathogenesis of Rheumatoid Arthritis

by Naoki Kondo, Takeshi Kuroda and Daisuke Kobayashi

Int. J. Mol. Sci. 2021, 22(20), 10922; https://doi.org/10.3390/ijms222010922 - 10 Oct 2021

Cited by 247 | Viewed by 15155

Abstract

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic systemic inflammation causing progressive joint damage that can lead to lifelong disability. The pathogenesis of RA involves a complex network of various cytokines and cells that trigger synovial cell proliferation and cause damage [...] Read more.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic systemic inflammation causing progressive joint damage that can lead to lifelong disability. The pathogenesis of RA involves a complex network of various cytokines and cells that trigger synovial cell proliferation and cause damage to both cartilage and bone. Involvement of the cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-6 is central to the pathogenesis of RA, but recent research has revealed that other cytokines such as IL-7, IL-17, IL-21, IL-23, granulocyte macrophage colony-stimulating factor (GM-CSF), IL-1β, IL-18, IL-33, and IL-2 also play a role. Clarification of RA pathology has led to the development of therapeutic agents such as biological disease-modifying anti-rheumatic drugs (DMARDs) and Janus kinase (JAK) inhibitors, and further details of the immunological background to RA are emerging. This review covers existing knowledge regarding the roles of cytokines, related immune cells and the immune system in RA, manipulation of which may offer the potential for even safer and more effective treatments in the future. Full article

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21 pages, 35903 KiB

Open AccessArticle

Rab11-FIP1 and Rab11-FIP5 Regulate pIgR/pIgA Transcytosis through TRIM21-Mediated Polyubiquitination

by Xuxu Fan, Dihan Zhou, Bali Zhao, Huijun Sha, Mengxue Li, Xian Li, Jingyi Yang and Huimin Yan

Int. J. Mol. Sci. 2021, 22(19), 10466; https://doi.org/10.3390/ijms221910466 - 28 Sep 2021

Cited by 7 | Viewed by 3525

Abstract

Polymeric immunoglobulin receptor (pIgR)-mediated polymeric immunoglobulin A (pIgA) transcytosis across mucosal epithelial cells plays an essential role in mucosal immunity. The general trafficking process has been well investigated, yet the elaborate regulatory mechanisms remain enigmatic. We identified a new pIgR interacting protein, the [...] Read more.

Polymeric immunoglobulin receptor (pIgR)-mediated polymeric immunoglobulin A (pIgA) transcytosis across mucosal epithelial cells plays an essential role in mucosal immunity. The general trafficking process has been well investigated, yet the elaborate regulatory mechanisms remain enigmatic. We identified a new pIgR interacting protein, the Rab11 effector Rab11-FIP1. Rab11-FIP1 and Rab11-FIP5 knockdown additively impaired pIgA transcytosis in both polarized and incompletely polarized cells. Moreover, Rab11-FIP1 and Rab11-FIP5 knockdown exhibited more significant inhibitory effects on pIgA transcytosis in incompletely polarized cells than in polarized cells. Interestingly, the trafficking process of pIgA in incompletely polarized cells is distinct from that in polarized cells. In incompletely polarized cells, the endocytic pIgR/pIgA was first transported from the basolateral plasma membrane to the vicinity of the centrosome where Rab11-FIP1 and Rab11-FIP5 bound to it, before the Rab11a-positive endosomes containing pIgR/pIgA, Rab11-FIP1 and Rab11-FIP5 were further transported to the apical plasma membrane via Golgi apparatus. During the trafficking process, TRIM21 mediated the K11-linked polyubiquitination of Rab11-FIP1 and the K6-linked polyubiquitination of Rab11-FIP5 to promote their activation and pIgA transcytosis. This study indicates that polyubiquitinated Rab11-FIP1 and Rab11-FIP5 mediated by TRIM21 cooperatively facilitate pIgA transcytosis and provides new insights into the intracellular trafficking process of pIgA in incompletely polarized cells. Full article

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15 pages, 2648 KiB

Open AccessArticle

Chromatin Dynamics and Gene Expression Response to Heat Exposure in Field-Conditioned versus Laboratory-Cultured Nematostella vectensis

by Eviatar Weizman, Mieka Rinsky, Noa Simon-Blecher, Sarit Lampert-Karako, Orly Yaron, Ann M. Tarrant and Oren Levy

Int. J. Mol. Sci. 2021, 22(14), 7454; https://doi.org/10.3390/ijms22147454 - 12 Jul 2021

Cited by 4 | Viewed by 2997

Abstract

Organisms’ survival is associated with the ability to respond to natural or anthropogenic environmental stressors. Frequently, these responses involve changes in gene regulation and expression, consequently altering physiology, development, or behavior. Here, we present modifications in response to heat exposure that mimics extreme [...] Read more.

Organisms’ survival is associated with the ability to respond to natural or anthropogenic environmental stressors. Frequently, these responses involve changes in gene regulation and expression, consequently altering physiology, development, or behavior. Here, we present modifications in response to heat exposure that mimics extreme summertime field conditions of lab-cultured and field-conditioned Nematostella vectensis. Using ATAC-seq and RNA-seq data, we found that field-conditioned animals had a more concentrated reaction to short-term thermal stress, expressed as enrichment of the DNA repair mechanism pathway. By contrast, lab animals had a more diffuse reaction that involved a larger number of differentially expressed genes and enriched pathways, including amino acid metabolism. Our results demonstrate that pre-conditioning affects the ability to respond efficiently to heat exposure in terms of both chromatin accessibility and gene expression and reinforces the importance of experimentally addressing ecological questions in the field. Full article

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27 pages, 11418 KiB

Open AccessArticle

Different Roles of p62 (SQSTM1) Isoforms in Keratin-Related Protein Aggregation

by Meghana Somlapura, Benjamin Gottschalk, Pooja Lahiri, Iris Kufferath, Daniela Pabst, Thomas Rülicke, Wolfgang F. Graier, Helmut Denk and Kurt Zatloukal

Int. J. Mol. Sci. 2021, 22(12), 6227; https://doi.org/10.3390/ijms22126227 - 9 Jun 2021

Cited by 5 | Viewed by 4395

Abstract

p62/Sequestosome-1 (p62) is a multifunctional adaptor protein and is also a constant component of disease-associated protein aggregates, including Mallory–Denk bodies (MDBs), in steatohepatitis and hepatocellular carcinoma. We investigated the interaction of the two human p62 isoforms, p62-H1 (full-length isoform) and p62-H2 (partly devoid [...] Read more.

p62/Sequestosome-1 (p62) is a multifunctional adaptor protein and is also a constant component of disease-associated protein aggregates, including Mallory–Denk bodies (MDBs), in steatohepatitis and hepatocellular carcinoma. We investigated the interaction of the two human p62 isoforms, p62-H1 (full-length isoform) and p62-H2 (partly devoid of PB1 domain), with keratins 8 and 18, the major components of MDBs. In human liver, p62-H2 is expressed two-fold higher compared to p62-H1 at the mRNA level and is present in slightly but not significantly higher concentrations at the protein level. Co-transfection studies in CHO-K1 cells, PLC/PRF/5 cells as well as p62⁻ total-knockout and wild-type mouse fibroblasts revealed marked differences in the cytoplasmic distribution and aggregation behavior of the two p62 isoforms. Transfection-induced overexpression of p62-H2 generated large cytoplasmic aggregates in PLC/PRF/5 and CHO-K1 cells that mostly co-localized with transfected keratins resembling MDBs or (transfection without keratins) intracytoplasmic hyaline bodies. In fibroblasts, however, transfected p62-H2 was predominantly diffusely distributed in the cytoplasm. Aggregation of p62-H2 and p62ΔSH2 as well as the interaction with K8 (but not with K18) involves acquisition of cross-β-sheet conformation as revealed by staining with luminescent conjugated oligothiophenes. These results indicate the importance of considering p62 isoforms in protein aggregation disease. Full article

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24 pages, 10969 KiB

Open AccessArticle

Protein Sumoylation Is Crucial for Phagocytosis in Entamoeba histolytica Trophozoites

by Mitzi Díaz-Hernández, Rosario Javier-Reyna, Izaid Sotto-Ortega, Guillermina García-Rivera, Sarita Montaño, Abigail Betanzos, Dxinegueela Zanatta and Esther Orozco

Int. J. Mol. Sci. 2021, 22(11), 5709; https://doi.org/10.3390/ijms22115709 - 27 May 2021

Cited by 5 | Viewed by 3426

Abstract

Posttranslational modifications provide Entamoeba histolytica proteins the timing and signaling to intervene during different processes, such as phagocytosis. However, SUMOylation has not been studied in E. histolytica yet. Here, we characterized the E. histolytica SUMO gene, its product (EhSUMO), and the relevance of [...] Read more.

Posttranslational modifications provide Entamoeba histolytica proteins the timing and signaling to intervene during different processes, such as phagocytosis. However, SUMOylation has not been studied in E. histolytica yet. Here, we characterized the E. histolytica SUMO gene, its product (EhSUMO), and the relevance of SUMOylation in phagocytosis. Our results indicated that EhSUMO has an extended N-terminus that differentiates SUMO from ubiquitin. It also presents the GG residues at the C-terminus and the ΨKXE/D binding motif, both involved in target protein contact. Additionally, the E. histolytica genome possesses the enzymes belonging to the SUMOylation-deSUMOylation machinery. Confocal microscopy assays disclosed a remarkable EhSUMO membrane activity with convoluted and changing structures in trophozoites during erythrophagocytosis. SUMOylated proteins appeared in pseudopodia, phagocytic channels, and around the adhered and ingested erythrocytes. Docking analysis predicted interaction of EhSUMO with EhADH (an ALIX family protein), and immunoprecipitation and immunofluorescence assays revealed that the association increased during phagocytosis; whereas the EhVps32 (a protein of the ESCRT-III complex)-EhSUMO interaction appeared stronger since basal conditions. In EhSUMO knocked-down trophozoites, the bizarre membranous structures disappeared, and EhSUMO interaction with EhADH and EhVps32 diminished. Our results evidenced the presence of a SUMO gene in E. histolytica and the SUMOylation relevance during phagocytosis. This is supported by bioinformatics screening of many other proteins of E. histolytica involved in phagocytosis, which present putative SUMOylation sites and the ΨKXE/D binding motif. Full article

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14 pages, 3073 KiB

Open AccessArticle

Role of Genetic Variation in Cytochromes P450 in Breast Cancer Prognosis and Therapy Response

by Viktor Hlaváč, Radka Václavíková, Veronika Brynychová, Pavel Ostašov, Renata Koževnikovová, Katerina Kopečková, David Vrána, Jiří Gatěk and Pavel Souček

Int. J. Mol. Sci. 2021, 22(6), 2826; https://doi.org/10.3390/ijms22062826 - 10 Mar 2021

Cited by 7 | Viewed by 2954

Abstract

Breast cancer is the most frequent cancer in the female population worldwide. The role of germline genetic variability in cytochromes P450 (CYP) in breast cancer prognosis and individualized therapy awaits detailed elucidation. In the present study, we used the next-generation sequencing to assess [...] Read more.

Breast cancer is the most frequent cancer in the female population worldwide. The role of germline genetic variability in cytochromes P450 (CYP) in breast cancer prognosis and individualized therapy awaits detailed elucidation. In the present study, we used the next-generation sequencing to assess associations of germline variants in the coding and regulatory sequences of all human CYP genes with response of the patients to the neoadjuvant cytotoxic chemotherapy and disease-free survival (n = 105). A total of 22 prioritized variants associating with a response or survival in the above evaluation phase were then analyzed by allelic discrimination in the large confirmation set (n = 802). Associations of variants in CYP1B1, CYP4F12, CYP4X1, and TBXAS1 with the response to the neoadjuvant cytotoxic chemotherapy were replicated by the confirmation phase. However, just association of variant rs17102977 in CYP4X1 passed the correction for multiple testing and can be considered clinically and statistically validated. Replicated associations for variants in CYP4X1, CYP24A1, and CYP26B1 with disease-free survival of all patients or patients stratified to subgroups according to therapy type have not passed a false discovery rate test. Although statistically not confirmed by the present study, the role of CYP genes in breast cancer prognosis should not be ruled out. In conclusion, the present study brings replicated association of variant rs17102977 in CYP4X1 with the response of patients to the neoadjuvant cytotoxic chemotherapy and warrants further research of genetic variation CYPs in breast cancer. Full article

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24 pages, 2796 KiB

Open AccessArticle

Robust Detection of Somatic Mosaicism and Repeat Interruptions by Long-Read Targeted Sequencing in Myotonic Dystrophy Type 1

by Antoine Mangin, Laure de Pontual, Yu-Chih Tsai, Laetitia Monteil, Mathilde Nizon, Pierre Boisseau, Sandra Mercier, Janet Ziegle, John Harting, Cheryl Heiner, Geneviève Gourdon and Stéphanie Tomé

Int. J. Mol. Sci. 2021, 22(5), 2616; https://doi.org/10.3390/ijms22052616 - 5 Mar 2021

Cited by 24 | Viewed by 5482

Abstract

Myotonic dystrophy type 1 (DM1) is the most complex and variable trinucleotide repeat disorder caused by an unstable CTG repeat expansion, reaching up to 4000 CTG in the most severe cases. The genetic and clinical variability of DM1 depend on the sex and [...] Read more.

Myotonic dystrophy type 1 (DM1) is the most complex and variable trinucleotide repeat disorder caused by an unstable CTG repeat expansion, reaching up to 4000 CTG in the most severe cases. The genetic and clinical variability of DM1 depend on the sex and age of the transmitting parent, but also on the CTG repeat number, presence of repeat interruptions and/or on the degree of somatic instability. Currently, it is difficult to simultaneously and accurately determine these contributing factors in DM1 patients due to the limitations of gold standard methods used in molecular diagnostics and research laboratories. Our study showed the efficiency of the latest PacBio long-read sequencing technology to sequence large CTG trinucleotides, detect multiple and single repeat interruptions and estimate the levels of somatic mosaicism in DM1 patients carrying complex CTG repeat expansions inaccessible to most methods. Using this innovative approach, we revealed the existence of de novo CCG interruptions associated with CTG stabilization/contraction across generations in a new DM1 family. We also demonstrated that our method is suitable to sequence the DM1 locus and measure somatic mosaicism in DM1 families carrying more than 1000 pure CTG repeats. Better characterization of expanded alleles in DM1 patients can significantly improve prognosis and genetic counseling, not only in DM1 but also for other tandem DNA repeat disorders. Full article

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25 pages, 5078 KiB

Open AccessArticle

A Role for Human DNA Polymerase λ in Alternative Lengthening of Telomeres

by Elisa Mentegari, Federica Bertoletti, Miroslava Kissova, Elisa Zucca, Silvia Galli, Giulia Tagliavini, Anna Garbelli, Antonio Maffia, Silvia Bione, Elena Ferrari, Fabrizio d’Adda di Fagagna, Sofia Francia, Simone Sabbioneda, Liuh-Yow Chen, Joachim Lingner, Valerie Bergoglio, Jean-Sébastien Hoffmann, Ulrich Hübscher, Emmanuele Crespan and Giovanni Maga

Int. J. Mol. Sci. 2021, 22(5), 2365; https://doi.org/10.3390/ijms22052365 - 27 Feb 2021

Cited by 3 | Viewed by 4004

Abstract

Telomerase negative cancer cell types use the Alternative Lengthening of Telomeres (ALT) pathway to elongate telomeres ends. Here, we show that silencing human DNA polymerase (Pol λ) in ALT cells represses ALT activity and induces telomeric stress. In addition, replication stress in the [...] Read more.

Telomerase negative cancer cell types use the Alternative Lengthening of Telomeres (ALT) pathway to elongate telomeres ends. Here, we show that silencing human DNA polymerase (Pol λ) in ALT cells represses ALT activity and induces telomeric stress. In addition, replication stress in the absence of Pol λ, strongly affects the survival of ALT cells. In vitro, Pol λ can promote annealing of even a single G-rich telomeric repeat to its complementary strand and use it to prime DNA synthesis. The noncoding telomeric repeat containing RNA TERRA and replication protein A negatively regulate this activity, while the Protection of Telomeres protein 1 (POT1)/TPP1 heterodimer stimulates Pol λ. Pol λ associates with telomeres and colocalizes with TPP1 in cells. In summary, our data suggest a role of Pol λ in the maintenance of telomeres by the ALT mechanism. Full article

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24 pages, 2731 KiB

Open AccessArticle

CLytA-DAAO Chimeric Enzyme Bound to Magnetic Nanoparticles. A New Therapeutical Approach for Cancer Patients?

by María Fuentes-Baile, Elizabeth Pérez-Valenciano, Pilar García-Morales, Camino de Juan Romero, Daniel Bello-Gil, Víctor M. Barberá, Álvaro Rodríguez-Lescure, Jesús M. Sanz, Cristina Alenda and Miguel Saceda

Int. J. Mol. Sci. 2021, 22(3), 1477; https://doi.org/10.3390/ijms22031477 - 2 Feb 2021

Cited by 15 | Viewed by 3047

Abstract

D-amino acid oxidase (DAAO) is an enzyme that catalyzes the oxidation of D-amino acids generating H₂O₂. The enzymatic chimera formed by DAAO bound to the choline-binding domain of N-acetylmuramoyl-L-alanine amidase (CLytA) induces cytotoxicity in several pancreatic and colorectal carcinoma [...] Read more.

D-amino acid oxidase (DAAO) is an enzyme that catalyzes the oxidation of D-amino acids generating H₂O₂. The enzymatic chimera formed by DAAO bound to the choline-binding domain of N-acetylmuramoyl-L-alanine amidase (CLytA) induces cytotoxicity in several pancreatic and colorectal carcinoma and glioblastoma cell models. In the current work, we determined whether the effect of CLytA-DAAO immobilized in magnetic nanoparticles, gold nanoparticles, and alginate capsules offered some advantages as compared to the free CLytA-DAAO. Results indicate that the immobilization of CLytA-DAAO in magnetic nanoparticles increases the stability of the enzyme, extending its time of action. Besides, we compared the effect induced by CLytA-DAAO with the direct addition of hydrogen peroxide, demonstrating that the progressive generation of reactive oxygen species by CLytA-DAAO is more effective in inducing cytotoxicity than the direct addition of H₂O₂. Furthermore, a pilot study has been initiated in biopsies obtained from pancreatic and colorectal carcinoma and glioblastoma patients to evaluate the expression of the main genes involved in resistance to CLytA-DAAO cytotoxicity. Based on our findings, we propose that CLytA-DAAO immobilized in magnetic nanoparticles could be effective in a high percentage of patients and, therefore, be used as an anti-cancer therapy for pancreatic and colorectal carcinoma and glioblastoma. Full article

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2020

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21 pages, 1814 KiB

Open AccessReview

The Role of the LINC Complex in Sperm Development and Function

by Vera Kmonickova, Michaela Frolikova, Klaus Steger and Katerina Komrskova

Int. J. Mol. Sci. 2020, 21(23), 9058; https://doi.org/10.3390/ijms21239058 - 28 Nov 2020

Cited by 19 | Viewed by 4867

Abstract

The LINC (LInker of Nucleoskeleton and Cytoskeleton) complex is localized within the nuclear envelope and consists of SUN (Sad1/UNc84 homology domain-containing) proteins located in the inner nuclear membrane and KASH (Klarsicht/Anc1/Syne1 homology domain-containing) proteins located in the outer nuclear membrane, hence linking nuclear [...] Read more.

The LINC (LInker of Nucleoskeleton and Cytoskeleton) complex is localized within the nuclear envelope and consists of SUN (Sad1/UNc84 homology domain-containing) proteins located in the inner nuclear membrane and KASH (Klarsicht/Anc1/Syne1 homology domain-containing) proteins located in the outer nuclear membrane, hence linking nuclear with cytoplasmic structures. While the nucleoplasm-facing side acts as a key player for correct pairing of homolog chromosomes and rapid chromosome movements during meiosis, the cytoplasm-facing side plays a pivotal role for sperm head development and proper acrosome formation during spermiogenesis. A further complex present in spermatozoa is involved in head-to-tail coupling. An intact LINC complex is crucial for the production of fertile sperm, as mutations in genes encoding for complex proteins are known to be associated with male subfertility in both mice and men. The present review provides a comprehensive overview on our current knowledge of LINC complex subtypes present in germ cells and its central role for male reproduction. Future studies on distinct LINC complex components are an absolute requirement to improve the diagnosis of idiopathic male factor infertility and the outcome of assisted reproduction. Full article

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13 pages, 2505 KiB

Open AccessArticle

Sustained Exposure of Substance P Causes Tendinopathy

by Seo Yoon Oh, Do Kyung Kim, Soo Hong Han, Hyun Hae Lee, Yunhui Jeong, Minjung Baek, Hyeongkyung Kim, Wooyeol Ahn and Soonchul Lee

Int. J. Mol. Sci. 2020, 21(22), 8633; https://doi.org/10.3390/ijms21228633 - 16 Nov 2020

Cited by 9 | Viewed by 3193

Abstract

Recently, neuromediators such as substance P (SP) have been found to be important factors in tendon homeostasis. Some studies have found SP to be the cause of inflammation and tendinopathy, whereas others have determined it to be a critical component of tendon healing. [...] Read more.

Recently, neuromediators such as substance P (SP) have been found to be important factors in tendon homeostasis. Some studies have found SP to be the cause of inflammation and tendinopathy, whereas others have determined it to be a critical component of tendon healing. As demonstrated by these conflicting findings, the effects of SP on tendinopathy remain unclear. In this study, we hypothesized that the duration of SP exposure determines its effect on the tendons, with repetitive long-term exposure leading to the development of tendinopathy. First, we verified the changes in gene and protein expression using in vitro tenocytes with 10-day exposure to SP. SP and SP + Run groups were injected with SP in their Achilles tendon every other day for 14 days. Achilles tendons were then harvested for biomechanical testing and histological processing. Notably, tendinopathic changes with decreased tensile strength, as observed in the Positive Control, were observed in the Achilles in the SP group compared to the Negative Control. Subsequent histological analysis, including Alcian blue staining, also revealed alterations in the Achilles tendon, which were generally consistent with the findings of tendinopathy in SP and SP + Run groups. Immunohistochemical analysis revealed increased expression of SP in the SP group, similar to the Positive Control. In general, the SP + Run group showed worse tendinopathic changes. These results suggest that sustained exposure to SP may be involved in the development of tendinopathy. Future research on inhibiting SP is warranted to target SP in the treatment of tendinopathy and may be beneficial to patients with tendinopathy. Full article

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19 pages, 6949 KiB

Open AccessReview

Membrane Curvature, Trans-Membrane Area Asymmetry, Budding, Fission and Organelle Geometry

by Alexander A. Mironov, Anna Mironov, Jure Derganc and Galina V. Beznoussenko

Int. J. Mol. Sci. 2020, 21(20), 7594; https://doi.org/10.3390/ijms21207594 - 14 Oct 2020

Cited by 11 | Viewed by 3728

Abstract

In biology, the modern scientific fashion is to mostly study proteins. Much less attention is paid to lipids. However, lipids themselves are extremely important for the formation and functioning of cellular membrane organelles. Here, the role of the geometry of the lipid bilayer [...] Read more.

In biology, the modern scientific fashion is to mostly study proteins. Much less attention is paid to lipids. However, lipids themselves are extremely important for the formation and functioning of cellular membrane organelles. Here, the role of the geometry of the lipid bilayer in regulation of organelle shape is analyzed. It is proposed that during rapid shape transition, the number of lipid heads and their size (i.e., due to the change in lipid head charge) inside lipid leaflets modulates the geometrical properties of organelles, in particular their membrane curvature. Insertion of proteins into a lipid bilayer and the shape of protein trans-membrane domains also affect the trans-membrane asymmetry between surface areas of luminal and cytosol leaflets of the membrane. In the cases where lipid molecules with a specific shape are not predominant, the shape of lipids (cylindrical, conical, or wedge-like) is less important for the regulation of membrane curvature, due to the flexibility of their acyl chains and their high ability to diffuse. Full article

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8 pages, 2198 KiB

Open AccessCommunication

Streamlining Quantitative Analysis of Long RNA Sequencing Reads

by Sebastian Oeck, Alicia I. Tüns, Sebastian Hurst and Alexander Schramm

Int. J. Mol. Sci. 2020, 21(19), 7259; https://doi.org/10.3390/ijms21197259 - 1 Oct 2020

Cited by 1 | Viewed by 3573

Abstract

Transcriptome analyses allow for linking RNA expression profiles to cellular pathways and phenotypes. Despite improvements in sequencing methodology, whole transcriptome analyses are still tedious, especially for methodologies producing long reads. Currently, available data analysis software often lacks cost- and time-efficient workflows. Although kit-based [...] Read more.

Transcriptome analyses allow for linking RNA expression profiles to cellular pathways and phenotypes. Despite improvements in sequencing methodology, whole transcriptome analyses are still tedious, especially for methodologies producing long reads. Currently, available data analysis software often lacks cost- and time-efficient workflows. Although kit-based workflows and benchtop platforms for RNA sequencing provide software options, e.g., cloud-based tools to analyze basecalled reads, quantitative, and easy-to-use solutions for transcriptome analysis, especially for non-human data, are missing. We therefore developed a user-friendly tool, termed Alignator, for rapid analysis of long RNA reads requiring only FASTQ files and an Ensembl cDNA database reference. After successful mapping, Alignator generates quantitative information for each transcript and provides a table in which sequenced and aligned RNA are stored for further comparative analyses. Full article

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18 pages, 2901 KiB

Open AccessArticle

Novel Insights into the Biochemical Mechanism of CK1ε and its Functional Interplay with DDX3X

by Bartolo Bono, Giulia Franco, Valentina Riva, Anna Garbelli and Giovanni Maga

Int. J. Mol. Sci. 2020, 21(17), 6449; https://doi.org/10.3390/ijms21176449 - 3 Sep 2020

Cited by 1 | Viewed by 3342

Abstract

Casein Kinase 1 epsilon (CK1ε) is a member of the serine (Ser)/threonine (Thr) CK1 family, known to have crucial roles in several biological scenarios and, ever more frequently, in pathological contexts, such as cancer. Recently, the human DEAD-box RNA helicase 3 X-linked (DDX3X), [...] Read more.

Casein Kinase 1 epsilon (CK1ε) is a member of the serine (Ser)/threonine (Thr) CK1 family, known to have crucial roles in several biological scenarios and, ever more frequently, in pathological contexts, such as cancer. Recently, the human DEAD-box RNA helicase 3 X-linked (DDX3X), involved in cancer proliferation and viral infections, has been identified as one of CK1ε substrates and its positive regulator in the Wnt/β-catenin network. However, the way by which these two proteins influence each other has not been fully clarified. In order to further investigate their interplay, we defined the kinetic parameters of CK1ε towards its substrates: ATP, casein, Dvl2 and DDX3X. CK1ε affinity for ATP depends on the nature of the substrate: increasing of casein concentrations led to an increase of Km^ATP, while increasing DDX3X reduced it. In literature, DDX3X is described to act as an allosteric activator of CK1ε. However, when we performed kinase reactions combining DDX3X and casein, we did not find a positive effect of DDX3X on casein phosphorylation by CK1ε, while both substrates were phosphorylated in a competitive manner. Moreover, CK1ε positively stimulates DDX3X ATPase activity. Our data provide a more detailed kinetic characterization on the functional interplay of these two proteins. Full article

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11 pages, 1900 KiB

Open AccessArticle

Complement C4 Gene Copy Number Variation Genotyping by High Resolution Melting PCR

by Claudia P. Jaimes-Bernal, Monte Trujillo, Francisco José Márquez and Antonio Caruz

Int. J. Mol. Sci. 2020, 21(17), 6309; https://doi.org/10.3390/ijms21176309 - 31 Aug 2020

Cited by 5 | Viewed by 3628

Abstract

Background: Complement C4 gene copy number variation plays an important role as a determinant of genetic susceptibility to common diseases, such as systemic lupus erythematosus, schizophrenia, rheumatoid arthritis, and infectious diseases. This study aimed to develop an assay for the quantification of copy [...] Read more.

Background: Complement C4 gene copy number variation plays an important role as a determinant of genetic susceptibility to common diseases, such as systemic lupus erythematosus, schizophrenia, rheumatoid arthritis, and infectious diseases. This study aimed to develop an assay for the quantification of copy number variations in the C4 locus. Methods: the assay was based on a gene ratio analysis copy enumeration (GRACE) PCR combined with high resolution melting (HRM) PCR. The test was optimized using samples of a known genotype and validated with 72 DNA samples from healthy blood donors. Results: to validate the assay, standard curves were generated by plotting the C4/RP1 ratio values against copy number variation (CNV) for each gene, using genomic DNA with known C4 CNV. The range of copy numbers in control individuals was comparable to distributions observed in previous studies of European descent. Conclusions: the method herein described significantly simplifies C4 CNV diagnosis to validate the assay. Full article

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19 pages, 1108 KiB

Open AccessReview

Current Approaches and Applications in Avian Genome Editing

by Joonbum Lee, Dong-Hwan Kim and Kichoon Lee

Int. J. Mol. Sci. 2020, 21(11), 3937; https://doi.org/10.3390/ijms21113937 - 30 May 2020

Cited by 23 | Viewed by 5187

Abstract

Advances in genome-editing technologies and sequencing of animal genomes enable researchers to generate genome-edited (GE) livestock as valuable animal models that benefit biological researches and biomedical and agricultural industries. As birds are an important species in biology and agriculture, their genome editing has [...] Read more.

Advances in genome-editing technologies and sequencing of animal genomes enable researchers to generate genome-edited (GE) livestock as valuable animal models that benefit biological researches and biomedical and agricultural industries. As birds are an important species in biology and agriculture, their genome editing has gained significant interest and is mainly performed by using a primordial germ cell (PGC)-mediated method because pronuclear injection is not practical in the avian species. In this method, PGCs can be isolated, cultured, genetically edited in vitro, and injected into a recipient embryo to produce GE offspring. Recently, a couple of GE quail have been generated by using the newly developed adenovirus-mediated method. Without technically required in vitro procedures of the PGC-mediated method, direct injection of adenovirus into the avian blastoderm in the freshly laid eggs resulted in the production of germ-line chimera and GE offspring. As more approaches are available in avian genome editing, avian research in various fields will progress rapidly. In this review, we describe the development of avian genome editing and scientific and industrial applications of GE avian species. Full article

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18 pages, 2452 KiB

Open AccessArticle

Beneficial Effects of Melatonin on Apolipoprotein-E Knockout Mice by Morphological and ¹⁸F-FDG PET/CT Assessments

by Lorenzo Nardo, Rita Rezzani, Luca Facchetti, Gaia Favero, Caterina Franco, Yasser Gaber Abdelhafez, Ramsey Derek Badawi, Michele Guindani, Youngho Seo and Miguel Pampaloni

Int. J. Mol. Sci. 2020, 21(8), 2920; https://doi.org/10.3390/ijms21082920 - 22 Apr 2020

Cited by 5 | Viewed by 3413

Abstract

Atherosclerosis represents one of the main risk factors for the development of cardiovascular diseases. Their etiologies have been studied in recent years in order to better define therapeutic targets for intervention and to identify diagnostic methods. Two different subtypes of macrophages, M1 and [...] Read more.

Atherosclerosis represents one of the main risk factors for the development of cardiovascular diseases. Their etiologies have been studied in recent years in order to better define therapeutic targets for intervention and to identify diagnostic methods. Two different subtypes of macrophages, M1 and M2, have been described in physiological conditions. They can also be found in the atherosclerotic process, where they both have opposite roles in disease progression. Perivascular brown adipose tissue is also involved in inflammation and endothelial damage. In this work, we provide insights into the protective role of melatonin in the atherosclerotic process by morphological and ¹⁸F-FDG-PET/CT analyses. In particular, we examined the effects of melatonin on pathways that are linked to atherosclerosis development. We showed that melatonin, by suppressing M1 activity, reduced inflammation and directed macrophage polarization toward the M2 macrophage subtype. Moreover, melatonin preserved the activity of perivascular brown adipose tissue. In addition, ¹⁸F-FDG uptake is very high in mice treated with melatonin, confirming that other factors may alter ¹⁸F-FDG distribution. In conclusion, we showed that melatonin affects inflammatory pathways that have been linked to atherosclerosis, assessed the relationships of the ¹⁸F-FDG PET/CT parameters with macrophage markers and the production of their cytokines, which that have been defined by morphological evaluations. Full article

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24 pages, 2358 KiB

Open AccessArticle

Insertion of Telomeric Repeats in the Human and Horse Genomes: An Evolutionary Perspective

by Marco Santagostino, Francesca M. Piras, Eleonora Cappelletti, Simone Del Giudice, Ornella Semino, Solomon G. Nergadze and Elena Giulotto

Int. J. Mol. Sci. 2020, 21(8), 2838; https://doi.org/10.3390/ijms21082838 - 18 Apr 2020

Cited by 8 | Viewed by 3872

Abstract

Interstitial telomeric sequences (ITSs) are short stretches of telomeric-like repeats (TTAGGG)n at nonterminal chromosomal sites. We previously demonstrated that, in the genomes of primates and rodents, ITSs were inserted during the repair of DNA double-strand breaks. These conclusions were derived from sequence comparisons [...] Read more.

Interstitial telomeric sequences (ITSs) are short stretches of telomeric-like repeats (TTAGGG)n at nonterminal chromosomal sites. We previously demonstrated that, in the genomes of primates and rodents, ITSs were inserted during the repair of DNA double-strand breaks. These conclusions were derived from sequence comparisons of ITS-containing loci and ITS-less orthologous loci in different species. To our knowledge, insertion polymorphism of ITSs, i.e., the presence of an ITS-containing allele and an ITS-less allele in the same species, has not been described. In this work, we carried out a genome-wide analysis of 2504 human genomic sequences retrieved from the 1000 Genomes Project and a PCR-based analysis of 209 human DNA samples. In spite of the large number of individual genomes analyzed we did not find any evidence of insertion polymorphism in the human population. On the contrary, the analysis of ITS loci in the genome of a single horse individual, the reference genome, allowed us to identify five heterozygous ITS loci, suggesting that insertion polymorphism of ITSs is an important source of genetic variability in this species. Finally, following a comparative sequence analysis of horse ITSs and of their orthologous empty loci in other Perissodactyla, we propose models for the mechanism of ITS insertion during the evolution of this order. Full article

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11 pages, 847 KiB

Open AccessReview

Towards a New Understanding of Decision-Making by Hematopoietic Stem Cells

by Geoffrey Brown

Int. J. Mol. Sci. 2020, 21(7), 2362; https://doi.org/10.3390/ijms21072362 - 29 Mar 2020

Cited by 4 | Viewed by 3880

Abstract

Cells within the hematopoietic stem cell compartment selectively express receptors for cytokines that have a lineage(s) specific role; they include erythropoietin, macrophage colony-stimulating factor, granulocyte colony-stimulating factor, granulocyte/macrophage colony-stimulating factor and the ligand for the fms-like tyrosine kinase 3. These hematopoietic cytokines can [...] Read more.

Cells within the hematopoietic stem cell compartment selectively express receptors for cytokines that have a lineage(s) specific role; they include erythropoietin, macrophage colony-stimulating factor, granulocyte colony-stimulating factor, granulocyte/macrophage colony-stimulating factor and the ligand for the fms-like tyrosine kinase 3. These hematopoietic cytokines can instruct the lineage fate of hematopoietic stem and progenitor cells in addition to ensuring the survival and proliferation of cells that belong to a particular cell lineage(s). Expression of the receptors for macrophage colony-stimulating factor and granulocyte colony-stimulating factor is positively autoregulated and the presence of the cytokine is therefore likely to enforce a lineage bias within hematopoietic stem cells that express these receptors. In addition to the above roles, macrophage colony-stimulating factor and granulocyte/macrophage colony-stimulating factor are powerful chemoattractants. The multiple roles of some hematopoietic cytokines leads us towards modelling hematopoietic stem cell decision-making whereby these cells can ‘choose’ just one lineage fate and migrate to a niche that both reinforces the fate and guarantees the survival and expansion of cells as they develop. Full article

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16 pages, 3361 KiB

Open AccessArticle

Augmenting Vacuolar H⁺-ATPase Function Prevents Cardiomyocytes from Lipid-Overload Induced Dysfunction

by Shujin Wang, Li-Yen Wong, Dietbert Neumann, Yilin Liu, Aomin Sun, Gudrun Antoons, Agnieszka Strzelecka, Jan F.C. Glatz, Miranda Nabben and Joost J.F.P. Luiken

Int. J. Mol. Sci. 2020, 21(4), 1520; https://doi.org/10.3390/ijms21041520 - 23 Feb 2020

Cited by 19 | Viewed by 5387

Abstract

The diabetic heart is characterized by a shift in substrate utilization from glucose to lipids, which may ultimately lead to contractile dysfunction. This substrate shift is facilitated by increased translocation of lipid transporter CD36 (SR-B2) from endosomes to the sarcolemma resulting in increased [...] Read more.

The diabetic heart is characterized by a shift in substrate utilization from glucose to lipids, which may ultimately lead to contractile dysfunction. This substrate shift is facilitated by increased translocation of lipid transporter CD36 (SR-B2) from endosomes to the sarcolemma resulting in increased lipid uptake. We previously showed that endosomal retention of CD36 is dependent on the proper functioning of vacuolar H⁺-ATPase (v-ATPase). Excess lipids trigger CD36 translocation through inhibition of v-ATPase function. Conversely, in yeast, glucose availability is known to enhance v-ATPase function, allowing us to hypothesize that glucose availability, via v-ATPase, may internalize CD36 and restore contractile function in lipid-overloaded cardiomyocytes. Increased glucose availability was achieved through (a) high glucose (25 mM) addition to the culture medium or (b) adenoviral overexpression of protein kinase-D1 (a kinase mediating GLUT4 translocation). In HL-1 cardiomyocytes, adult rat and human cardiomyocytes cultured under high-lipid conditions, each treatment stimulated v-ATPase re-assembly, endosomal acidification, endosomal CD36 retention and prevented myocellular lipid accumulation. Additionally, these treatments preserved insulin-stimulated GLUT4 translocation and glucose uptake as well as contractile force. The present findings reveal v-ATPase functions as a key regulator of cardiomyocyte substrate preference and as a novel potential treatment approach for the diabetic heart. Full article

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17 pages, 2479 KiB

Open AccessArticle

Effect of Cholesterol on the Organic Cation Transporter OCTN1 (SLC22A4)

by Lorena Pochini, Gilda Pappacoda, Michele Galluccio, Francesco Pastore, Mariafrancesca Scalise and Cesare Indiveri

Int. J. Mol. Sci. 2020, 21(3), 1091; https://doi.org/10.3390/ijms21031091 - 6 Feb 2020

Cited by 8 | Viewed by 4114

Abstract

The effect of cholesterol was investigated on the OCTN1 transport activity measured as [¹⁴C]-tetraethylamonium or [³H]-acetylcholine uptake in proteoliposomes reconstituted with native transporter extracted from HeLa cells or the human recombinant OCTN1 over-expressed in E. coli. Removal of [...] Read more.

The effect of cholesterol was investigated on the OCTN1 transport activity measured as [¹⁴C]-tetraethylamonium or [³H]-acetylcholine uptake in proteoliposomes reconstituted with native transporter extracted from HeLa cells or the human recombinant OCTN1 over-expressed in E. coli. Removal of cholesterol from the native transporter by MβCD before reconstitution led to impairment of transport activity. A similar activity impairment was observed after treatment of proteoliposomes harboring the recombinant (cholesterol-free) protein by MβCD, suggesting that the lipid mixture used for reconstitution contained some cholesterol. An enzymatic assay revealed the presence of 10 µg cholesterol/mg total lipids corresponding to 1% cholesterol in the phospholipid mixture used for the proteoliposome preparation. On the other way around, the activity of the recombinant OCTN1 was stimulated by adding the cholesterol analogue, CHS to the proteoliposome preparation. Optimal transport activity was detected in the presence of 83 µg CHS/ mg total lipids for both [¹⁴C]-tetraethylamonium or [³H]-acetylcholine uptake. Kinetic analysis of transport demonstrated that the stimulation of transport activity by CHS consisted in an increase of the Vmax of transport with no changes of the Km. Altogether, the data suggests a direct interaction of cholesterol with the protein. A further support to this interpretation was given by a docking analysis indicating the interaction of cholesterol with some protein sites corresponding to CARC-CRAC motifs. The observed direct interaction of cholesterol with OCTN1 points to a possible direct influence of cholesterol on tumor cells or on acetylcholine transport in neuronal and non-neuronal cells via OCTN1. Full article

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11 pages, 3296 KiB

Open AccessArticle

BMAL1 Modulates Epidermal Healing in a Process Involving the Antioxidative Defense Mechanism

by Ericka J. D. Silveira, Carlos H. V. Nascimento Filho, Veronica Q. Yujra, Liana P. Webber, Rogerio M. Castilho and Cristiane H. Squarize

Int. J. Mol. Sci. 2020, 21(3), 901; https://doi.org/10.3390/ijms21030901 - 30 Jan 2020

Cited by 19 | Viewed by 4532

Abstract

The circadian rhythm regulates the physiology and behavior of living organisms in a time-dependent manner. Clock genes have distinct roles including the control over gene expression mediated by the transcriptional activators CLOCK and BMAL1, and the suppression of gene expression mediated by the [...] Read more.

The circadian rhythm regulates the physiology and behavior of living organisms in a time-dependent manner. Clock genes have distinct roles including the control over gene expression mediated by the transcriptional activators CLOCK and BMAL1, and the suppression of gene expression mediated by the transcriptional repressors PER1/2 and CRY1/2. The balance between gene expression and repression is key to the maintenance of tissue homeostasis that is disrupted in the event of an injury. In the skin, a compromised epithelial barrier triggers a cascade of events that culminate in the mobilization of epithelial cells and stem cells. Recruited epithelial cells migrate towards the wound and reestablish the protective epithelial layer of the skin. Although we have recently demonstrated the involvement of BMAL and the PI3K signaling in wound healing, the role of the circadian clock genes in tissue repair remains poorly understood. Here, we sought to understand the role of BMAL1 on skin healing in response to injury. We found that genetic depletion of BMAL1 resulted in delayed healing of the skin as compared to wild-type control mice. Furthermore, we found that loss of Bmal1 was associated with the accumulation of Reactive Oxygen Species Modulator 1 (ROMO1), a protein responsible for inducing the production of intracellular reactive oxygen species (ROS). The slow healing was associated with ROS and superoxide dismutase (SOD) production, and pharmacological inhibition of the oxidative stress signaling (ROS/SOD) led to cellular proliferation, upregulation of Sirtuin 1 (SIRT1), and rescued the skin healing phenotype of Bmal1^−/− mice. Overall, our study points to BMAL1 as a key player in tissue regeneration and as a critical regulator of ROMO1 and oxidative stress in the skin. Full article

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14 pages, 4411 KiB

Open AccessArticle

Helicase-Like Transcription Factor HLTF and E3 Ubiquitin Ligase SHPRH Confer DNA Damage Tolerance through Direct Interactions with Proliferating Cell Nuclear Antigen (PCNA)

by Mareike Seelinger and Marit Otterlei

Int. J. Mol. Sci. 2020, 21(3), 693; https://doi.org/10.3390/ijms21030693 - 21 Jan 2020

Cited by 21 | Viewed by 4974

Abstract

To prevent replication fork collapse and genome instability under replicative stress, DNA damage tolerance (DDT) mechanisms have evolved. The RAD5 homologs, HLTF (helicase-like transcription factor) and SHPRH (SNF2, histone-linker, PHD and RING finger domain-containing helicase), both ubiquitin ligases, are involved in several DDT [...] Read more.

To prevent replication fork collapse and genome instability under replicative stress, DNA damage tolerance (DDT) mechanisms have evolved. The RAD5 homologs, HLTF (helicase-like transcription factor) and SHPRH (SNF2, histone-linker, PHD and RING finger domain-containing helicase), both ubiquitin ligases, are involved in several DDT mechanisms; DNA translesion synthesis (TLS), fork reversal/remodeling and template switch (TS). Here we show that these two human RAD5 homologs contain functional APIM PCNA interacting motifs. Our results show that both the role of HLTF in TLS in HLTF overexpressing cells, and nuclear localization of SHPRH, are dependent on interaction of HLTF and SHPRH with PCNA. Additionally, we detected multiple changes in the mutation spectra when APIM in overexpressed HLTF or SHPRH were mutated compared to overexpressed wild type proteins. In plasmids from cells overexpressing the APIM mutant version of HLTF, we observed a decrease in C to T transitions, the most common mutation caused by UV irradiation, and an increase in mutations on the transcribed strand. These results strongly suggest that direct binding of HLTF and SHPRH to PCNA is vital for their function in DDT. Full article

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2019

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10 pages, 3287 KiB

Open AccessCommunication

PHLDA1 Does Not Contribute Directly to Heat Shock-Induced Apoptosis of Spermatocytes

by Patryk Janus, Katarzyna Mrowiec, Natalia Vydra, Piotr Widłak, Agnieszka Toma-Jonik, Joanna Korfanty, Ryszard Smolarczyk and Wiesława Widłak

Int. J. Mol. Sci. 2020, 21(1), 267; https://doi.org/10.3390/ijms21010267 - 30 Dec 2019

Cited by 2 | Viewed by 3481

Abstract

Spermatocytes are among the most heat-sensitive cells and the exposure of testes to heat shock results in their Heat Shock Factor 1 (HSF1)-mediated apoptosis. Several lines of evidence suggest that pleckstrin-homology-like domain family A, member 1 (PHLDA1) plays a role in promoting heat [...] Read more.

Spermatocytes are among the most heat-sensitive cells and the exposure of testes to heat shock results in their Heat Shock Factor 1 (HSF1)-mediated apoptosis. Several lines of evidence suggest that pleckstrin-homology-like domain family A, member 1 (PHLDA1) plays a role in promoting heat shock-induced cell death in spermatogenic cells, yet its precise physiological role is not well understood. Aiming to elucidate the hypothetical role of PHLDA1 in HSF1-mediated apoptosis of spermatogenic cells we characterized its expression in mouse testes during normal development and after heat shock. We stated that transcription of Phlda1 is upregulated by heat shock in many adult mouse organs including the testes. Analyzes of the Phlda1 expression during postnatal development indicate that it is expressed in pre-meiotic or somatic cells of the testis. It starts to be transcribed much earlier than spermatocytes are fully developed and its transcripts and protein products do not accumulate further in the later stages. Moreover, neither heat shock nor expression of constitutively active HSF1 results in the accumulation of PHLDA1 protein in meiotic and post-meiotic cells although both conditions induce massive apoptosis of spermatocytes. Furthermore, the overexpression of PHLDA1 in NIH3T3 cells leads to cell detachment, yet classical apoptosis is not observed. Therefore, our findings indicate that PHLDA1 cannot directly contribute to the heat-induced apoptosis of spermatocytes. Instead, PHLDA1 could hypothetically participate in death of spermatocytes indirectly via activation of changes in the somatic or pre-meiotic cells present in the testes. Full article

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10 pages, 1362 KiB

Open AccessReview

Are Leukaemic Stem Cells Restricted to a Single Cell Lineage?

by Geoffrey Brown, Lucía Sánchez and Isidro Sánchez-García

Int. J. Mol. Sci. 2020, 21(1), 45; https://doi.org/10.3390/ijms21010045 - 19 Dec 2019

Cited by 2 | Viewed by 3055

Abstract

Cancer-stem-cell theory states that most, if not all, cancers arise from a stem/uncommitted cell. This theory revolutionised our view to reflect that cancer consists of a hierarchy of cells that mimic normal cell development. Elegant studies of twins who both developed acute lymphoblastic [...] Read more.

Cancer-stem-cell theory states that most, if not all, cancers arise from a stem/uncommitted cell. This theory revolutionised our view to reflect that cancer consists of a hierarchy of cells that mimic normal cell development. Elegant studies of twins who both developed acute lymphoblastic leukaemia in childhood revealed that at least two genomic insults are required for cancer to develop. These ‘hits’ do not appear to confer a growth advantage to cancer cells, nor do cancer cells appear to be better equipped to survive than normal cells. Cancer cells created by investigators by introducing specific genomic insults generally belong to one cell lineage. For example, transgenic mice in which the LIM-only 2 (LMO2, associated with human acute T-lymphoblastic leukaemia) and BCR-ABL^p210 (associated with human chronic myeloid leukaemia) oncogenes were active solely within the haematopoietic stem-cell compartment developed T-lymphocyte and neutrophil lineage-restricted leukaemia, respectively. This recapitulated the human form of these diseases. This ‘hardwiring’ of lineage affiliation, either throughout leukaemic stem cell development or at a particular stage, is different to the behaviour of normal haematopoietic stem cells. While normal cells directly commit to a developmental pathway, they also remain versatile and can develop into a terminally differentiated cell that is not part of the initial lineage. Many cancer stem cells do not have this versatility, and this is an essential difference between normal and cancer stem cells. In this report, we review findings that support this notion. Full article

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16 pages, 6154 KiB

Open AccessArticle

A Paradigm in Immunochemistry, Revealed by Monoclonal Antibodies to Spatially Distinct Epitopes on Syntenin-1

by Ian R. D. Johnson, Alexandra Sorvina, Jessica M. Logan, Courtney R. Moore, Jessica K. Heatlie, Emma J. Parkinson-Lawrence, Stavros Selemidis, John J. O’Leary, Lisa M. Butler and Douglas A. Brooks

Int. J. Mol. Sci. 2019, 20(23), 6035; https://doi.org/10.3390/ijms20236035 - 29 Nov 2019

Cited by 5 | Viewed by 4340

Abstract

Syntenin-1 is an essential multi-functional adaptor protein, which has multiple roles in membrane trafficking and exosome biogenesis, as well as scaffolding interactions with either the actin cytoskeleton or focal adhesions. However, how this functional multiplicity relates to syntenin-1 distribution in different endosome compartments [...] Read more.

Syntenin-1 is an essential multi-functional adaptor protein, which has multiple roles in membrane trafficking and exosome biogenesis, as well as scaffolding interactions with either the actin cytoskeleton or focal adhesions. However, how this functional multiplicity relates to syntenin-1 distribution in different endosome compartments or other intracellular locations and its underlying involvement in cancer pathogenesis have yet to be fully defined. To help facilitate the investigation of syntenin-1 biology, we developed two specific monoclonal antibodies (Synt-2C6 and Synt-3A11) to spatially distinct linear sequence epitopes on syntenin-1, which were each designed to be unique at the six-amino acid level. These antibodies produced very different intracellular staining patterns, with Synt-2C6 detecting endosomes and Synt-3A11 producing a fibrillar staining pattern suggesting a cytoskeletal localisation. Treatment of cells with Nocodazole altered the intracellular localisation of Synt-3A11, which was consistent with the syntenin-1 protein interacting with microtubules. In prostate tissue biopsies, Synt-3A11 defined atrophy and early-stage prostate cancer, whereas Synt-2C6 only showed minimal interaction with atrophic tissue. This highlights a critical need for site-specific antibodies and a knowledge of their reactivity to define differential protein distributions, interactions and functions, which may differ between normal and malignant cells. Full article

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17 pages, 3375 KiB

Open AccessArticle

Caspase-8 Regulates Endoplasmic Reticulum Stress-Induced Necroptosis Independent of the Apoptosis Pathway in Auditory Cells

by Akihiro Kishino, Ken Hayashi, Miyoko Maeda, Toyoharu Jike, Chiaki Hidai, Yasuyuki Nomura and Takeshi Oshima

Int. J. Mol. Sci. 2019, 20(23), 5896; https://doi.org/10.3390/ijms20235896 - 24 Nov 2019

Cited by 38 | Viewed by 5912

Abstract

The aim of this study is to elucidate the detailed mechanism of endoplasmic reticulum (ER) stress-induced auditory cell death based on the function of the initiator caspases and molecular complex of necroptosis. Here, we demonstrated that ER stress initiates not only caspase-9-dependent intrinsic [...] Read more.

The aim of this study is to elucidate the detailed mechanism of endoplasmic reticulum (ER) stress-induced auditory cell death based on the function of the initiator caspases and molecular complex of necroptosis. Here, we demonstrated that ER stress initiates not only caspase-9-dependent intrinsic apoptosis along with caspase-3, but also receptor-interacting serine/threonine kinase (RIPK)1-dependent necroptosis in auditory cells. We observed the ultrastructural characteristics of both apoptosis and necroptosis in tunicamycin-treated cells under transmission electron microscopy (TEM). We demonstrated that ER stress-induced necroptosis was dependent on the induction of RIPK1, negatively regulated by caspase-8 in auditory cells. Our data suggested that ER stress-induced intrinsic apoptosis depends on the induction of caspase-9 along with caspase-3 in auditory cells. The results of this study reveal that necroptosis could exist for the alternative backup cell death route of apoptosis in auditory cells under ER stress. Interestingly, our data results in a surge in the recognition that therapies aimed at the inner ear protection effect by caspase inhibitors like zVAD-fmk might arrest apoptosis but can also have the unanticipated effect of promoting necroptosis. Thus, RIPK1-dependent necroptosis would be a new therapeutic target for the treatment of sensorineural hearing loss due to ER stress. Full article

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18 pages, 1869 KiB

Open AccessArticle

A Single Synonymous Variant (c.354G>A [p.P118P]) in ADAMTS13 Confers Enhanced Specific Activity

by Ryan Hunt, Gaya Hettiarachchi, Upendra Katneni, Nancy Hernandez, David Holcomb, Jacob Kames, Redab Alnifaidy, Brian Lin, Nobuko Hamasaki-Katagiri, Aaron Wesley, Tal Kafri, Christina Morris, Laura Bouché, Maria Panico, Tal Schiller, Juan Ibla, Haim Bar, Amra Ismail, Howard Morris, Anton Komar and Chava Kimchi-Sarfaty Show full author list Hide full author list

Int. J. Mol. Sci. 2019, 20(22), 5734; https://doi.org/10.3390/ijms20225734 - 15 Nov 2019

Cited by 20 | Viewed by 5680

Abstract

Synonymous variants within coding regions may influence protein expression and function. We have previously reported increased protein expression levels ex vivo (~120% in comparison to wild-type) from a synonymous polymorphism variant, c.354G>A [p.P118P], of the ADAMTS13 gene, encoding a plasma protease responsible for [...] Read more.

Synonymous variants within coding regions may influence protein expression and function. We have previously reported increased protein expression levels ex vivo (~120% in comparison to wild-type) from a synonymous polymorphism variant, c.354G>A [p.P118P], of the ADAMTS13 gene, encoding a plasma protease responsible for von Willebrand Factor (VWF) degradation. In the current study, we investigated the potential mechanism(s) behind the increased protein expression levels from this variant and its effect on ADAMTS13 physico-chemical properties. Cell-free assays showed enhanced translation of the c.354G>A variant and the analysis of codon usage characteristics suggested that introduction of the frequently used codon/codon pair(s) may have been potentially responsible for this effect. Limited proteolysis, however, showed no substantial influence of altered translation on protein conformation. Analysis of post-translational modifications also showed no notable differences but identified three previously unreported glycosylation markers. Despite these similarities, p.P118P variant unexpectedly showed higher specific activity. Structural analysis using modeled interactions indicated that subtle conformational changes arising from altered translation kinetics could affect interactions between an exosite of ADAMTS13 and VWF resulting in altered specific activity. This report highlights how a single synonymous nucleotide variation can impact cellular expression and specific activity in the absence of measurable impact on protein structure. Full article

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22 pages, 4572 KiB

Open AccessArticle

EPO and TMBIM3/GRINA Promote the Activation of the Adaptive Arm and Counteract the Terminal Arm of the Unfolded Protein Response after Murine Transient Cerebral Ischemia

by Pardes Habib, Ann-Sophie Stamm, Joerg B. Schulz, Arno Reich, Alexander Slowik, Sandro Capellmann, Michael Huber and Thomas Wilhelm

Int. J. Mol. Sci. 2019, 20(21), 5421; https://doi.org/10.3390/ijms20215421 - 31 Oct 2019

Cited by 14 | Viewed by 4255

Abstract

Ischemic stroke is known to cause the accumulation of misfolded proteins and loss of calcium homeostasis leading to impairment of endoplasmic reticulum (ER) function. The unfolded protein response (UPR) is an ER-located and cytoprotective pathway that aims to resolve ER stress. Transmembrane BAX [...] Read more.

Ischemic stroke is known to cause the accumulation of misfolded proteins and loss of calcium homeostasis leading to impairment of endoplasmic reticulum (ER) function. The unfolded protein response (UPR) is an ER-located and cytoprotective pathway that aims to resolve ER stress. Transmembrane BAX inhibitor-1 motif-containing (TMBIM) protein family member TMBIM3/GRINA is highly expressed in the brain and mostly located at the ER membrane suppressing ER calcium release by inositol-1,4,5-trisphosphate receptors. GRINA confers neuroprotection and is regulated by erythropoietin (EPO) after murine cerebral ischemia. However, the role of GRINA and the impact of EPO treatment on the post-ischemic UPR have not been elucidated yet. We subjected GRINA-deficient (Grina^−/−) and wildtype mice to transient (30 min) middle cerebral artery occlusion (tMCAo) followed by 6 h or 72 h of reperfusion. We administered EPO or saline 0, 24 and 48 h after tMCAo/sham surgery. Oxygen–glucose deprivation (OGD) and pharmacological stimulation of the UPR using Tunicamycin and Thapsigargin were carried out in primary murine cortical mixed cell cultures. Treatment with the PERK-inhibitor GSK-2606414, IRE1a-RNase-inhibitor STF-083010 and EPO was performed 1 h prior to either 1 h, 2 h or 3 h of OGD. We found earlier and larger infarct demarcations in Grina^−/− mice compared to wildtype mice, which was accompanied by a worse neurological outcome and an abolishment of EPO-mediated neuroprotection after ischemic stroke. In addition, GRINA-deficiency increased apoptosis and the activation of the corresponding PERK arm of the UPR after stroke. EPO enhanced the post-ischemic activation of pro-survival IRE1a and counteracted the pro-apoptotic PERK branch of the UPR. Both EPO and the PERK-inhibitor GSK-2606414 reduced cell death and regulated Grina mRNA levels after OGD. In conclusion, GRINA plays a crucial role in post-ischemic UPR and the use of both GSK-2606414 and EPO might lead to neuroprotection. Full article

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17 pages, 4059 KiB

Open AccessArticle

Renal Artery Stenosis Alters Gene Expression in Swine Scattered Tubular-Like Cells

by Arash Aghajani Nargesi, Xiang-Yang Zhu, Yuanhang Liu, Hui Tang, Kyra L. Jordan, Lilach O. Lerman and Alfonso Eirin

Int. J. Mol. Sci. 2019, 20(20), 5069; https://doi.org/10.3390/ijms20205069 - 12 Oct 2019

Cited by 10 | Viewed by 3402

Abstract

Background: Scattered tubular-like cells (STCs) proliferate and differentiate to support neighboring injured renal tubular cells during recovery from insults. Renal artery stenosis (RAS) induces renal ischemia and hypertension and leads to loss of kidney function, but whether RAS alters renal endogenous repair mechanisms, [...] Read more.

Background: Scattered tubular-like cells (STCs) proliferate and differentiate to support neighboring injured renal tubular cells during recovery from insults. Renal artery stenosis (RAS) induces renal ischemia and hypertension and leads to loss of kidney function, but whether RAS alters renal endogenous repair mechanisms, such as STCs, remains unknown. We hypothesize that RAS in swine modifies the messenger RNA (mRNA) profile of STCs, blunting their in vitro reparative capacity. Methods: CD24+/CD133+ STCs were isolated from pig kidneys after 10-weeks of RAS or sham (n = 3 each) and their gene cargo analyzed using high-throughput mRNAseq. Expression profiles for upregulated and downregulated mRNAs in RAS-STCs were functionally interpreted by gene ontology analysis. STC activation was assessed by counting the total number of STCs in pig kidney sections using flow cytometry, whereas cell proliferation was assessed in vitro. Results: Of all expressed genes, 1430 genes were upregulated and 315 downregulated in RAS- versus Normal-STCs. Expression of selected candidate genes followed the same fold change directions as the mRNAseq findings. Genes upregulated in RAS-STCs were involved in cell adhesion, extracellular matrix remodeling, and kidney development, whereas those downregulated in RAS-STCs are related to cell cycle and cytoskeleton. The percentage of STCs from dissociated kidney cells was higher in RAS versus Normal pigs, but their proliferation rate was blunted. Conclusions: Renal ischemia and hypertension in swine induce changes in the mRNA profile of STCs, associated with increased STC activation and impaired proliferation. These observations suggest that RAS may alter the reparative capacity of STCs. Full article

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12 pages, 1325 KiB

Open AccessArticle

Systematic Approach to Developing Splice Modulating Antisense Oligonucleotides

by May T. Aung-Htut, Craig S. McIntosh, Kristin A. Ham, Ianthe L. Pitout, Loren L. Flynn, Kane Greer, Sue Fletcher and Steve D. Wilton

Int. J. Mol. Sci. 2019, 20(20), 5030; https://doi.org/10.3390/ijms20205030 - 11 Oct 2019

Cited by 16 | Viewed by 6087

Abstract

The process of pre-mRNA splicing is a common and fundamental step in the expression of most human genes. Alternative splicing, whereby different splice motifs and sites are recognised in a developmental and/or tissue-specific manner, contributes to genetic plasticity and diversity of gene expression. [...] Read more.

The process of pre-mRNA splicing is a common and fundamental step in the expression of most human genes. Alternative splicing, whereby different splice motifs and sites are recognised in a developmental and/or tissue-specific manner, contributes to genetic plasticity and diversity of gene expression. Redirecting pre-mRNA processing of various genes has now been validated as a viable clinical therapeutic strategy, providing treatments for Duchenne muscular dystrophy (inducing specific exon skipping) and spinal muscular atrophy (promoting exon retention). We have designed and evaluated over 5000 different antisense oligonucleotides to alter splicing of a variety of pre-mRNAs, from the longest known human pre-mRNA to shorter, exon-dense primary gene transcripts. Here, we present our guidelines for designing, evaluating and optimising splice switching antisense oligomers in vitro. These systematic approaches assess several critical factors such as the selection of target splicing motifs, choice of cells, various delivery reagents and crucial aspects of validating assays for the screening of antisense oligonucleotides composed of 2′-O-methyl modified bases on a phosphorothioate backbone. Full article

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12 pages, 1841 KiB

Open AccessArticle

Endoplasmic Reticulum Stress Increases DUSP5 Expression via PERK-CHOP Pathway, Leading to Hepatocyte Death

by Hye Jin Jo, Jin Won Yang, Ji Hye Park, Eul Sig Choi, Chae-Seok Lim, Seoul Lee and Chang Yeob Han

Int. J. Mol. Sci. 2019, 20(18), 4369; https://doi.org/10.3390/ijms20184369 - 5 Sep 2019

Cited by 33 | Viewed by 4899

Abstract

Hepatocyte death is critical for the pathogenesis of liver disease progression, which is closely associated with endoplasmic reticulum (ER) stress responses. However, the molecular basis for ER stress-mediated hepatocyte injury remains largely unknown. This study investigated the effect of ER stress on dual-specificity [...] Read more.

Hepatocyte death is critical for the pathogenesis of liver disease progression, which is closely associated with endoplasmic reticulum (ER) stress responses. However, the molecular basis for ER stress-mediated hepatocyte injury remains largely unknown. This study investigated the effect of ER stress on dual-specificity phosphatase 5 (DUSP5) expression and its role in hepatocyte death. Analysis of Gene Expression Omnibus (GEO) database showed that hepatic DUSP5 levels increased in the patients with liver fibrosis, which was verified in mouse models of liver diseases with ER stress. DUSP5 expression was elevated in both fibrotic and acutely injured liver of mice treated with liver toxicants. Treatment of ER stress inducers enhanced DUSP5 expression in hepatocytes, which was validated in vivo condition. The induction of DUSP5 by ER stress was blocked by either treatment with a chemical inhibitor of the protein kinase RNA-like endoplasmic reticulum kinase (PERK) pathway, or knockdown of C/EBP homologous protein (CHOP), whereas it was not affected by the silencing of IRE1 or ATF6. In addition, DUSP5 overexpression decreased extracellular-signal-regulated kinase (ERK) phosphorylation, but increased cleaved caspase-3 levels. Moreover, the reduction of cell viability under ER stress condition was attenuated by DUSP5 knockdown. In conclusion, DUSP5 expression is elevated in hepatocytes by ER stress through the PERK-CHOP pathway, contributing to hepatocyte death possibly through ERK inhibition. Full article

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13 pages, 2912 KiB

Open AccessArticle

Modulation of Tetraspanin 32 (TSPAN32) Expression in T Cell-Mediated Immune Responses and in Multiple Sclerosis

by Salvo Danilo Lombardo, Emanuela Mazzon, Maria Sofia Basile, Giorgia Campo, Federica Corsico, Mario Presti, Placido Bramanti, Katia Mangano, Maria Cristina Petralia, Ferdinando Nicoletti and Paolo Fagone

Int. J. Mol. Sci. 2019, 20(18), 4323; https://doi.org/10.3390/ijms20184323 - 4 Sep 2019

Cited by 25 | Viewed by 4200

Abstract

Tetraspanins are a conserved family of proteins involved in a number of biological processes including, cell–cell interactions, fertility, cancer metastasis and immune responses. It has previously been shown that TSPAN32 knockout mice have normal hemopoiesis and B-cell responses, but hyperproliferative T cells. Here, [...] Read more.

Tetraspanins are a conserved family of proteins involved in a number of biological processes including, cell–cell interactions, fertility, cancer metastasis and immune responses. It has previously been shown that TSPAN32 knockout mice have normal hemopoiesis and B-cell responses, but hyperproliferative T cells. Here, we show that TSPAN32 is expressed at higher levels in the lymphoid lineage as compared to myeloid cells. In vitro activation of T helper cells via anti-CD3/CD28 is associated with a significant downregulation of TSPAN32. Interestingly, engagement of CD3 is sufficient to modulate TSPAN32 expression, and its effect is potentiated by costimulation with anti-CD28, but not anti-CTLA4, -ICOS nor -PD1. Accordingly, we measured the transcriptomic levels of TSPAN32 in polarized T cells under Th1 and Th2 conditions and TSPAN32 resulted significantly reduced as compared with unstimulated cells. On the other hand, in Treg cells, TSPAN32 underwent minor changes upon activation. The in vitro data were finally translated into the context of multiple sclerosis (MS). Encephalitogenic T cells from Myelin Oligodendrocyte Glycoprotein (MOG)-Induced Experimental Autoimmune Encephalomyelitis (EAE) mice showed significantly lower levels of TSPAN32 and increased levels of CD9, CD53, CD82 and CD151. Similarly, in vitro-activated circulating CD4 T cells from MS patients showed lower levels of TSPAN32 as compared with cells from healthy donors. Overall, these data suggest an immunoregulatory role for TSPAN32 in T helper immune response and may represent a target of future immunoregulatory therapies for T cell-mediated autoimmune diseases. Full article

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39 pages, 3791 KiB

Open AccessReview

Improving Lodging Resistance: Using Wheat and Rice as Classical Examples

by Liaqat Shah, Muhammad Yahya, Syed Mehar Ali Shah, Muhammad Nadeem, Ahmad Ali, Asif Ali, Jing Wang, Muhammad Waheed Riaz, Shamsur Rehman, Weixun Wu, Riaz Muhammad Khan, Adil Abbas, Aamir Riaz, Galal Bakr Anis, Hongqi Si, Haiyang Jiang and Chuanxi Ma

Int. J. Mol. Sci. 2019, 20(17), 4211; https://doi.org/10.3390/ijms20174211 - 28 Aug 2019

Cited by 184 | Viewed by 13842

Abstract

One of the most chronic constraints to crop production is the grain yield reduction near the crop harvest stage by lodging worldwide. This is more prevalent in cereal crops, particularly in wheat and rice. Major factors associated with lodging involve morphological and anatomical [...] Read more.

One of the most chronic constraints to crop production is the grain yield reduction near the crop harvest stage by lodging worldwide. This is more prevalent in cereal crops, particularly in wheat and rice. Major factors associated with lodging involve morphological and anatomical traits along with the chemical composition of the stem. These traits have built up the remarkable relationship in wheat and rice genotypes either prone to lodging or displaying lodging resistance. In this review, we have made a comparison of our conceptual perceptions with foregoing published reports and proposed the fundamental controlling techniques that could be practiced to control the devastating effects of lodging stress. The management of lodging stress is, however, reliant on chemical, agronomical, and genetic factors that are reducing the risk of lodging threat in wheat and rice. But, still, there are many questions remain to be answered to elucidate the complex lodging phenomenon, so agronomists, breeders, physiologists, and molecular biologists require further investigation to address this challenging problem. Full article

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23 pages, 4023 KiB

Open AccessArticle

Compromised Barrier Function in Human Induced Pluripotent Stem-Cell-Derived Retinal Pigment Epithelial Cells from Type 2 Diabetic Patients

by Mostafa Kiamehr, Alexa Klettner, Elisabeth Richert, Ali Koskela, Arto Koistinen, Heli Skottman, Kai Kaarniranta, Katriina Aalto-Setälä and Kati Juuti-Uusitalo

Int. J. Mol. Sci. 2019, 20(15), 3773; https://doi.org/10.3390/ijms20153773 - 1 Aug 2019

Cited by 37 | Viewed by 4786

Abstract

In diabetic patients, high blood glucose induces alterations in retinal function and can lead to visual impairment due to diabetic retinopathy. In immortalized retinal pigment epithelial (RPE) cultures, high glucose concentrations are shown to lead to impairment in epithelial barrier properties. For the [...] Read more.

In diabetic patients, high blood glucose induces alterations in retinal function and can lead to visual impairment due to diabetic retinopathy. In immortalized retinal pigment epithelial (RPE) cultures, high glucose concentrations are shown to lead to impairment in epithelial barrier properties. For the first time, the induced pluripotent stem-cell-derived retinal pigment epithelium (hiPSC-RPE) cell lines derived from type 2 diabetics and healthy control patients were utilized to assess the effects of glucose concentration on the cellular functionality. We show that both type 2 diabetic and healthy control hiPSC-RPE lines differentiate and mature well, both in high and normal glucose concentrations, express RPE specific genes, secrete pigment epithelium derived factor, and form a polarized cell layer. Here, type 2 diabetic hiPSC-RPE cells had a decreased barrier function compared to controls. Added insulin increased the epithelial cell layer tightness in normal glucose concentrations, and the effect was more evident in type 2 diabetics than in healthy control hiPSC-RPE cells. In addition, the preliminary functionality assessments showed that type 2 diabetic hiPSC-RPE cells had attenuated autophagy detected via ubiquitin-binding protein p62/Sequestosome-1 (p62/SQSTM1) accumulation, and lowered pro- matrix metalloproteinase 2 (proMMP2) as well as increased pro-MMP9 secretion. These results suggest that the cellular ability to tolerate stress is possibly decreased in type 2 diabetic RPE cells. Full article

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13 pages, 1413 KiB

Open AccessReview

Mast Cells in Cardiovascular Disease: From Bench to Bedside

by M. A. W. Hermans, J. E. Roeters van Lennep, P. L. A. van Daele and I. Bot

Int. J. Mol. Sci. 2019, 20(14), 3395; https://doi.org/10.3390/ijms20143395 - 10 Jul 2019

Cited by 37 | Viewed by 6002

Abstract

Mast cells are pluripotent leukocytes that reside in the mucosa and connective tissue. Recent studies show an increased prevalence of cardiovascular disease among patients with mastocytosis, which is a hematological disease that is characterized by the accumulation of mast cells due to clonal [...] Read more.

Mast cells are pluripotent leukocytes that reside in the mucosa and connective tissue. Recent studies show an increased prevalence of cardiovascular disease among patients with mastocytosis, which is a hematological disease that is characterized by the accumulation of mast cells due to clonal proliferation. This association suggests an important role for mast cells in cardiovascular disease. Indeed, the evidence establishing the contribution of mast cells to the development and progression of atherosclerosis is continually increasing. Mast cells may contribute to plaque formation by stimulating the formation of foam cells and causing a pro-inflammatory micro-environment. In addition, these cells are able to promote plaque instability by neo-vessel formation and also by inducing intraplaque hemorrhage. Furthermore, mast cells appear to stimulate the formation of fibrosis after a cardiac infarction. In this review, the available data on the role of mast cells in cardiovascular disease are summarized, containing both in vitro research and animal studies, followed by a discussion of human data on the association between cardiovascular morbidity and diseases in which mast cells are important: Kounis syndrome, mastocytosis and allergy. Full article

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13 pages, 2685 KiB

Open AccessArticle

Calcium Homeostasis Is Modified in Skeletal Muscle Fibers of Small Ankyrin1 Knockout Mice

by Enrico Pierantozzi, Péter Szentesi, Dána Al-Gaadi, Tamás Oláh, Beatrix Dienes, Mónika Sztretye, Daniela Rossi, Vincenzo Sorrentino and László Csernoch

Int. J. Mol. Sci. 2019, 20(13), 3361; https://doi.org/10.3390/ijms20133361 - 9 Jul 2019

Cited by 7 | Viewed by 4418

Abstract

Small Ankyrins (sAnk1) are muscle-specific isoforms generated by the Ank1 gene that participate in the organization of the sarcoplasmic reticulum (SR) of striated muscles. Accordingly, the volume of SR tubules localized around the myofibrils is strongly reduced in skeletal muscle fibers of 4- [...] Read more.

Small Ankyrins (sAnk1) are muscle-specific isoforms generated by the Ank1 gene that participate in the organization of the sarcoplasmic reticulum (SR) of striated muscles. Accordingly, the volume of SR tubules localized around the myofibrils is strongly reduced in skeletal muscle fibers of 4- and 10-month-old sAnk1 knockout (KO) mice, while additional structural alterations only develop with aging. To verify whether the lack of sAnk1 also alters intracellular Ca²⁺ handling, cytosolic Ca²⁺ levels were analyzed in stimulated skeletal muscle fibers from 4- and 10-month-old sAnk1 KO mice. The SR Ca²⁺ content was reduced in sAnk1 KO mice regardless of age. The amplitude of the Ca²⁺ transients induced by depolarizing pulses was decreased in myofibers of sAnk1 KO with respect to wild type (WT) fibers, while their voltage dependence was not affected. Furthermore, analysis of spontaneous Ca²⁺ release events (sparks) on saponin-permeabilized muscle fibers indicated that the frequency of sparks was significantly lower in fibers from 4-month-old KO mice compared to WT. Furthermore, both the amplitude and spatial spread of sparks were significantly smaller in muscle fibers from both 4- and 10-month-old KO mice compared to WT. These data suggest that the absence of sAnk1 results in an impairment of SR Ca²⁺ release, likely as a consequence of a decreased Ca²⁺ store due to the reduction of the SR volume in sAnk1 KO muscle fibers. Full article

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12 pages, 990 KiB

Open AccessArticle

The TAFs of TFIID Bind and Rearrange the Topology of the TATA-Less RPS5 Promoter

by Sarah N. Le, Christopher R. Brown, Stacy Harvey, Hinrich Boeger, Hans Elmlund and Dominika Elmlund

Int. J. Mol. Sci. 2019, 20(13), 3290; https://doi.org/10.3390/ijms20133290 - 4 Jul 2019

Cited by 6 | Viewed by 4527

Abstract

The general transcription factor TFIID is a core promoter selectivity factor that recognizes DNA sequence elements and nucleates the assembly of a pre-initiation complex (PIC). The mechanism by which TFIID recognizes the promoter is poorly understood. The TATA-box binding protein (TBP) is a [...] Read more.

The general transcription factor TFIID is a core promoter selectivity factor that recognizes DNA sequence elements and nucleates the assembly of a pre-initiation complex (PIC). The mechanism by which TFIID recognizes the promoter is poorly understood. The TATA-box binding protein (TBP) is a subunit of the multi-protein TFIID complex believed to be key in this process. We reconstituted transcription from highly purified components on a ribosomal protein gene (RPS5) and discovered that TFIIDΔTBP binds and rearranges the promoter DNA topology independent of TBP. TFIIDΔTBP binds ~200 bp of the promoter and changes the DNA topology to a larger extent than the nucleosome core particle. We show that TBP inhibits the DNA binding activities of TFIIDΔTBP and conclude that the complete TFIID complex may represent an auto-inhibited state. Furthermore, we show that the DNA binding activities of TFIIDΔTBP are required for assembly of a PIC poised to select the correct transcription start site (TSS). Full article

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16 pages, 2403 KiB

Open AccessCommunication

In Vivo Piggybac-Based Gene Delivery towards Murine Pancreatic Parenchyma Confers Sustained Expression of Gene of Interest

by Masahiro Sato, Emi Inada, Issei Saitoh, Shingo Nakamura and Satoshi Watanabe

Int. J. Mol. Sci. 2019, 20(13), 3116; https://doi.org/10.3390/ijms20133116 - 26 Jun 2019

Cited by 4 | Viewed by 5049

Abstract

The pancreas is a glandular organ that functions in the digestive system and endocrine system of vertebrates. The most common disorders involving the pancreas are diabetes, pancreatitis, and pancreatic cancer. In vivo gene delivery targeting the pancreas is important for preventing or curing [...] Read more.

The pancreas is a glandular organ that functions in the digestive system and endocrine system of vertebrates. The most common disorders involving the pancreas are diabetes, pancreatitis, and pancreatic cancer. In vivo gene delivery targeting the pancreas is important for preventing or curing such diseases and for exploring the biological function of genes involved in the pathogenesis of these diseases. Our previous experiments demonstrated that adult murine pancreatic cells can be efficiently transfected by exogenous plasmid DNA following intraparenchymal injection and subsequent in vivo electroporation using tweezer-type electrodes. Unfortunately, the induced gene expression was transient. Transposon-based gene delivery, such as that facilitated by piggyBac (PB), is known to confer stable integration of a gene of interest (GOI) into host chromosomes, resulting in sustained expression of the GOI. In this study, we investigated the use of the PB transposon system to achieve stable gene expression when transferred into murine pancreatic cells using the above-mentioned technique. Expression of the GOI (coding for fluorescent protein) continued for at least 1.5 months post-gene delivery. Splinkerette-PCR-based analysis revealed the presence of the consensus sequence TTAA at the junctional portion between host chromosomes and the transgenes; however, this was not observed in all samples. This plasmid-based PB transposon system enables constitutive expression of the GOI in pancreas for potential therapeutic and biological applications. Full article

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16 pages, 5686 KiB

Open AccessArticle

Immortalized Human hTert/KER-CT Keratinocytes a Model System for Research on Desmosomal Adhesion and Pathogenesis of Pemphigus Vulgaris

by Benedikt Beckert, Francesca Panico, Robert Pollmann, Rüdiger Eming, Antje Banning and Ritva Tikkanen

Int. J. Mol. Sci. 2019, 20(13), 3113; https://doi.org/10.3390/ijms20133113 - 26 Jun 2019

Cited by 16 | Viewed by 6721

Abstract

Pemphigus Vulgaris is an autoimmune disease that results in blister formation in the epidermis and in mucosal tissues due to antibodies recognizing desmosomal cadherins, mainly desmoglein-3 and -1. Studies on the molecular mechanisms of Pemphigus have mainly been carried out using the spontaneously [...] Read more.

Pemphigus Vulgaris is an autoimmune disease that results in blister formation in the epidermis and in mucosal tissues due to antibodies recognizing desmosomal cadherins, mainly desmoglein-3 and -1. Studies on the molecular mechanisms of Pemphigus have mainly been carried out using the spontaneously immortalized human keratinocyte cell line HaCaT or in primary keratinocytes. However, both cell systems have suboptimal features, with HaCaT cells exhibiting a large number of chromosomal aberrations and mutated p53 tumor suppressor, whereas primary keratinocytes are short-lived, heterogeneous and not susceptible to genetic modifications due to their restricted life-span. We have here tested the suitability of the commercially available human keratinocyte cell line hTert/KER-CT as a model system for research on epidermal cell adhesion and Pemphigus pathomechanisms. We here show that hTert cells exhibit a calcium dependent expression of desmosomal cadherins and are well suitable for typical assays used for studies on Pemphigus, such as sequential detergent extraction and Dispase-based dissociation assay. Treatment with Pemphigus auto-antibodies results in loss of monolayer integrity and altered localization of desmoglein-3, as well as loss of colocalization with flotillin-2. Our findings demonstrate that hTert cells are well suitable for studies on epidermal cell adhesion and Pemphigus pathomechanisms. Full article

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14 pages, 4296 KiB

Open AccessArticle

Molecular Cloning and Sexually Dimorphic Expression Analysis of nanos2 in the Sea Urchin, Mesocentrotus nudus

by Jian Zhang, Xiao Han, Jin Wang, Bing-Zheng Liu, Jin-Liang Wei, Wei-Jie Zhang, Zhi-Hui Sun and Ya-Qing Chang

Int. J. Mol. Sci. 2019, 20(11), 2705; https://doi.org/10.3390/ijms20112705 - 1 Jun 2019

Cited by 13 | Viewed by 4246

Abstract

Sea urchin (Mesocentrotus nudus) is an economically important mariculture species in China and the gonads are the solely edible parts to human. The molecular mechanisms of gonad development have attracted increasing attention in recent years. Although the nanos2 gene has been [...] Read more.

Sea urchin (Mesocentrotus nudus) is an economically important mariculture species in China and the gonads are the solely edible parts to human. The molecular mechanisms of gonad development have attracted increasing attention in recent years. Although the nanos2 gene has been identified as a germ cell marker in several invertebrates, little is known about nanos2 in adult sea urchins. Hereinto, we report the characterization of Mnnano2, an M. nudus nanos2 homology gene. Mnnanos2 is a maternal factor and can be detected continuously during embryogenesis and early ontogeny. Real-time quantitative PCR (RT-qPCR) and section in situ hybridization (ISH) analysis revealed a dynamic and sexually dimorphic expression pattern of Mnnano2 in the gonads. Its expression reached the maximal level at Stage 2 along with the gonad development in both ovary and testis. In the ovary, Mnnanos2 is specifically expressed in germ cells. In contrast, Mnnanos2 is expressed in both nutritive phagocytes (NP) cells and male germ cells in testis. Moreover, knocking down of Mnnanos2 by means of RNA interference (RNAi) reduced nanos2 and boule expression but conversely increased the expression of foxl2. Therefore, our data suggest that Mnnanos2 may serve as a female germ cell marker during gametogenesis and provide chances to uncover its function in adult sea urchin. Full article

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14 pages, 1227 KiB

Open AccessReview

Akt Signaling in Macrophage Polarization, Survival, and Atherosclerosis

by MacRae F. Linton, Javid J. Moslehi and Vladimir R. Babaev

Int. J. Mol. Sci. 2019, 20(11), 2703; https://doi.org/10.3390/ijms20112703 - 1 Jun 2019

Cited by 197 | Viewed by 12324

Abstract

The PI3K/Akt pathway plays a crucial role in the survival, proliferation, and migration of macrophages, which may impact the development of atherosclerosis. Changes in Akt isoforms or modulation of the Akt activity levels in macrophages significantly affect their polarization phenotype and consequently atherosclerosis [...] Read more.

The PI3K/Akt pathway plays a crucial role in the survival, proliferation, and migration of macrophages, which may impact the development of atherosclerosis. Changes in Akt isoforms or modulation of the Akt activity levels in macrophages significantly affect their polarization phenotype and consequently atherosclerosis in mice. Moreover, the activity levels of Akt signaling determine the viability of monocytes/macrophages and their resistance to pro-apoptotic stimuli in atherosclerotic lesions. Therefore, elimination of pro-apoptotic factors as well as factors that antagonize or suppress Akt signaling in macrophages increases cell viability, protecting them from apoptosis, and this markedly accelerates atherosclerosis in mice. In contrast, inhibition of Akt signaling by the ablation of Rictor in myeloid cells, which disrupts mTORC2 assembly, significantly decreases the viability and proliferation of blood monocytes and macrophages with the suppression of atherosclerosis. In addition, monocytes and macrophages exhibit a threshold effect for Akt protein levels in their ability to survive. Ablation of two Akt isoforms, preserving only a single Akt isoform in myeloid cells, markedly compromises monocyte and macrophage viability, inducing monocytopenia and diminishing early atherosclerosis. These recent advances in our understanding of Akt signaling in macrophages in atherosclerosis may have significant relevance in the burgeoning field of cardio-oncology, where PI3K/Akt inhibitors being tested in cancer patients can have significant cardiovascular and metabolic ramifications. Full article

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25 pages, 4934 KiB

Open AccessArticle

The Impact of the C-Terminal Region on the Interaction of Topoisomerase II Alpha with Mitotic Chromatin

by Melissa Antoniou-Kourounioti, Michael L. Mimmack, Andrew C.G. Porter and Christine J. Farr

Int. J. Mol. Sci. 2019, 20(5), 1238; https://doi.org/10.3390/ijms20051238 - 12 Mar 2019

Cited by 23 | Viewed by 5315

Abstract

Type II topoisomerase enzymes are essential for resolving DNA topology problems arising through various aspects of DNA metabolism. In vertebrates two isoforms are present, one of which (TOP2A) accumulates on chromatin during mitosis. Moreover, TOP2A targets the mitotic centromere during prophase, persisting there [...] Read more.

Type II topoisomerase enzymes are essential for resolving DNA topology problems arising through various aspects of DNA metabolism. In vertebrates two isoforms are present, one of which (TOP2A) accumulates on chromatin during mitosis. Moreover, TOP2A targets the mitotic centromere during prophase, persisting there until anaphase onset. It is the catalytically-dispensable C-terminal domain of TOP2 that is crucial in determining this isoform-specific behaviour. In this study we show that, in addition to the recently identified chromatin tether domain, several other features of the alpha-C-Terminal Domain (CTD). influence the mitotic localisation of TOP2A. Lysine 1240 is a major SUMOylation target in cycling human cells and the efficiency of this modification appears to be influenced by T1244 and S1247 phosphorylation. Replacement of K1240 by arginine results in fewer cells displaying centromeric TOP2A accumulation during prometaphase-metaphase. The same phenotype is displayed by cells expressing TOP2A in which either of the mitotic phosphorylation sites S1213 or S1247 has been substituted by alanine. Conversely, constitutive modification of TOP2A by fusion to SUMO2 exerts the opposite effect. FRAP analysis of protein mobility indicates that post-translational modification of TOP2A can influence the enzyme’s residence time on mitotic chromatin, as well as its subcellular localisation. Full article

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12 pages, 4073 KiB

Open AccessArticle

Long Non Coding RNA H19: A New Player in Hypoxia-Induced Multiple Myeloma Cell Dissemination

by Chiara Corrado, Viviana Costa, Gianluca Giavaresi, Annalisa Calabrese, Alice Conigliaro and Riccardo Alessandro

Int. J. Mol. Sci. 2019, 20(4), 801; https://doi.org/10.3390/ijms20040801 - 13 Feb 2019

Cited by 26 | Viewed by 4207

Abstract

The long non-coding RNA H19 (lncH19) is broadly transcribed in the first stage of development and silenced in most cells of an adult organism; it appears again in several tumors where, through different molecular mediators, promotes cell proliferation, motility and metastases. LncH19 has [...] Read more.

The long non-coding RNA H19 (lncH19) is broadly transcribed in the first stage of development and silenced in most cells of an adult organism; it appears again in several tumors where, through different molecular mediators, promotes cell proliferation, motility and metastases. LncH19 has been associated with hypoxia-inducible factor 1-alpha (HIF-1α) activation and, in some tumors, it has proved to be necessary and required to sustain hypoxic responses. Here we propose to investigate a putative role for the lncH19 in hypoxia induced multiple myeloma (MM) progression. Transcriptional analysis of MM cell lines (RPMI and MM1.S) exposed to normoxia or hypoxia (1% O₂) was done in order to evaluate lncH19 levels under hypoxic stimulation. Then, to investigate the role of lncH19 in hypoxia mediated MM progression, transcriptional, protein and functional assays have been performed on hypoxia stimulated MM cell lines, silenced or not for lncH19. Our data demonstrated that hypoxic stimulation in MM cell lines induced the overexpression of lncH19, which, in turn, is required for the expression of the hypoxia induced genes involved in MM dissemination, such as C-X-C Motif Chemokine Receptor 4 (CXCR4) and Snail. Moreover, adhesion assays demonstrated that lncH19 silencing abrogates the increased adhesion on stromal cells induced by the hypoxic condition. Finally, Western blot analysis indicated that lncH19 silencing impaired HIF1α nuclear translocation. The LncH19, required for the induction of hypoxic responses in MM cells, could represent a new therapeutic target for MM. Full article

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12 pages, 2130 KiB

Open AccessArticle

Mitochondrial Dysfunction in Skeletal Muscle of a Fibromyalgia Model: The Potential Benefits of Melatonin

by Gaia Favero, Francesca Bonomini, Caterina Franco and Rita Rezzani

Int. J. Mol. Sci. 2019, 20(3), 765; https://doi.org/10.3390/ijms20030765 - 11 Feb 2019

Cited by 37 | Viewed by 12055

Abstract

Fibromyalgia syndrome (FMS) is considered a musculoskeletal disorder associated to other symptoms including chronic pain. Since the hypothesis of FMS etiogenesis is consistent with mitochondrial dysfunction and oxidative stress, we evaluated the pathophysiological correlation among these factors studying some proteins involved in the [...] Read more.

Fibromyalgia syndrome (FMS) is considered a musculoskeletal disorder associated to other symptoms including chronic pain. Since the hypothesis of FMS etiogenesis is consistent with mitochondrial dysfunction and oxidative stress, we evaluated the pathophysiological correlation among these factors studying some proteins involved in the mitochondrial homeostasis. We focused our attention on the roles of peroxisome proliferator activated receptor gamma coactivator-1alpha (PGC-1α), mitofusin2 (Mfn2), and coenzyme Q10 (CoQ10) in reserpine-induced myalgic (RIM) rats that manifest fibromyalgia-like chronic pain symptoms. First, we underlined that RIM rats are a good model for studying the pathophysiology of FMS and moreover, we found that PGC-1α, Mfn2, and CoQ10 are involved in FMS. In fact, their expressions were reduced in gastrocnemius muscle determining an incorrect mitochondrial homeostasis. Today, none of the currently available drugs are fully effective against the symptoms of this disease and they, often, induce several adverse events; hence, many scientists have taken on the challenge of searching for non-pharmacological treatments. Another goal of this study was therefore the evaluation of the potential benefits of melatonin, an endogenous indoleamine having several functions including its potent capacity to induce antioxidant enzymes and to determine the protective or reparative mechanisms in the cells. We observed that melatonin supplementation significantly preserved all the studied parameters, counteracting oxidative stress in RIM rats and confirming that this indoleamine should be taken in consideration for improving health and/or counteract mitochondrial related diseases. Full article

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