Search Results (525)

MicroRNAs are key post-transcriptional regulators of gene expression and their dysregulation is often linked to cancer. Epstein-Barr virus encodes 22 BamHI A Rightward Transcript (BART) miRNAs, which are expressed in nearly all EBV-associated cancers and implicated in viral pathogenesis. To investigate biological targets for BART miRNAs in B cell lymphomas, we performed a meta-analysis of publicly available Ago-CLIP datasets from EBV-positive Burkitt lymphomas (BLs), primary effusion lymphomas (PELs), AIDS-associated diffuse large B cell lymphomas (DLBCLs), and lymphoblastoid cell lines (LCLs). Our analysis focused on comparing targets of EBV BART miRNAs across the different types of transformed B cells. Using reporter assays, we then experimentally validated over 50 functional interactions between BART miRNAs and cellular protein-coding transcripts involved in activities such as B cell differentiation (PRDM1, IRF4, and MYC), cell cycle regulation (UHMK1, CDKN1A, MDM2, and NPAT), apoptosis (MCL1), signaling and intracellular trafficking (GAB1, SOS1, MAPK1, RAB11A, CAV1, and RANBP9), and tumor suppression (CCDC6). Moreover, ectopic BART miRNA expression in several EBV-negative BL cells induced transcriptional changes that may influence molecular signatures of EBV-associated BLs. Collectively, our findings reveal novel, functional interactions for BART miRNAs in lymphomas and provide insights into their roles in these B cell cancers. Full article

Chemical modifications of cellular RNAs play key roles in gene expression and host defense. The cap-adjacent N⁶,2′-O-dimethyladenosine (m⁶Am) is a prevalent modification of vertebrate and viral mRNAs and is catalyzed by the newly discovered N⁶ methyltransferase PCIF1. However, its role in gene expression remains unclear due to conflicting reports on its effects on mRNA stability and translation. In this study, we investigated the impact of siRNA-mediated transient suppression of PCIF1 on global mRNA expression in HeLa cells. We identified a subset of differentially expressed genes (DEGs) that exhibited minimal overlap with previously reported DEGs. Subsequent validation revealed that PCIF1 positively and negatively regulates RAB23 and CNOT6 expression, respectively, at both the mRNA and protein levels. Mechanistic analyses demonstrated that PCIF1 regulates the stability of these target mRNAs rather than their transcription, and rescue experiments confirmed the requirement of PCIF1’s methyltransferase activity for these regulations. Furthermore, MeRIP-qPCR analysis showed that PCIF1 suppression significantly reduced the m⁶A levels of RAB23 and CNOT6 mRNAs. These findings suggest that PCIF1 regulates the stability of specific mRNAs in opposite ways through m⁶A modification, providing new insights into the role of m⁶Am in the regulation of gene expression. Full article

Salinity causes widespread crop loss and prompts plants to adapt through changes in gene expression. In this study, we aimed to investigate the function of the non-tandem CCCH zinc-finger (non-TZF) protein gene AtC3H3 in response to salt stress in Arabidopsis. AtC3H3, a gene from the non-TZF gene family known for its RNA-binding and RNase activities, was up-regulated under osmotic stress, such as high salt and drought. When overexpressed in Arabidopsis, AtC3H3 improved tolerance to salt stress, but not drought stress. The expression of well-known abscisic acid (ABA)-dependent salt stress-responsive genes, namely Responsive to Desiccation 29B (RD29B), RD22, and Responsive to ABA 18 (RAB18), and representative ABA-independent salt stress-responsive genes, namely Dehydration-Responsive Element Binding protein 2A (DREB2A) and DREB2B, was significantly higher in AtC3H3-overexpressing transgenic plants (AtC3H3 OXs) than in wild-type plants (WT) under NaCl treatment, indicating its significance in both ABA-dependent and -independent signal transduction pathways. mRNA-sequencing (mRNA-Seq) analysis using NaCl-treated WT and AtC3H3 OXs revealed no potential target mRNAs for the RNase function of AtC3H3, suggesting that the potential targets of AtC3H3 might be noncoding RNAs and not mRNAs. Through this study, we conclusively demonstrated that AtC3H3 plays a crucial role in salt stress tolerance by influencing the expression of salt stress-responsive genes. These findings offer new insights into plant stress response mechanisms and suggest potential strategies for improving crop resilience to salinity stress. Full article

The resilient modulus (M_R) and the backcalculated modulus from the FWD testing (E_FWD) of the unbound layers are critical inputs in the analysis/design of pavements. Several studies have tried to develop a conversion factor between these two parameters, while the nonlinear stress dependency of unbound materials and the pavement strain response are mostly missing from the literature. This study aims to compare the laboratory-measured M_R of recycled aggregate base (RAB) materials and a virgin aggregate base using field-based E_FWD and tries to establish pavement’s responses to loading using vertical strains from both the M_R and E_FWD values of the respective materials as comparability parameters between the two. For this purpose, a control virgin aggregate (VA, limestone) and three types of RAB materials were selected to construct four test sections. The test sections were modeled in layered elastic- and finite-element-based pavement response models to calculate the vertical strains at the mid-depth of the base and top of the subgrade layers. A comparison of the lab-calculated vertical strains using M_R with actual vertical strains in the field from E_FWD showed that there was no relationship between the two stiffness parameters in all tested RABs. The vertical strains, based on the lab M_R, undermined the stiffness of the recycled aggregates in the field. In contrast, the values of E_FWD based on the vertical strains remained close to the M_R strains of limestone (VA) throughout the testing period, establishing an E_FWD vs. M_R relationship (M_R = 0.87 E_FWD). The results also show that fine RCA was a better-performing material over three years. This research not only explores how the hydration process in RABs limits the development of M_R-E_FWD correlations but also underscores the need to consider real-world conditions when assessing their performance. Full article

The role of autophagy goes far beyond the elimination of damaged cellular components and the quality control of proteins. It also cleanses cells from inclusions, including pathogenic viruses, and provides energy-forming components. The liver, which is an organ with increased metabolism, is made up of cells that are particularly vulnerable to damage. Therefore, detoxification of liver cells in the process of autophagy has become a very important issue clinically. The aim of this study was an immunohistochemical evaluation of proteins activated in rat liver cells at different stages of hyperbaric autophagy. The rats used for the study were randomly divided into six equivalent groups—three control groups and three experimental groups. Animals from the experimental groups were subjected to hyperbaric treatment in a hyperbaric chamber, with a pressure of 1.6 ATA for 120 min. They breathed atmospheric air. Rats were decapitated within 5 or 10 days after removal from the chamber. Immunohistochemical reactions with beclin 1, LC3B, RAB7, and HSC73 proteins were carried out on preparations made from liver slices. A three-step labeled streptavidin–biotin detection method of paraffin blocks (LSAB three-step) was used for immunohistochemical research. The results were evaluated using computer programs for morphometric analysis of microscopic images by calculating the mean surface areas occupied by a positive immunohistochemical reaction in individual groups for all antibodies tested. Increased closure of substrates in the autophagosome (beclin 1) induced late endosome transport and accelerated autophagosome maturation process (RAB7). Furthermore, a larger number of autophagosomes (LC3B) was observed in liver cells immediately after the cessation of hyperbaric activity; however, this decreased after 5 days. During this time, chaperone-mediated autophagy (HSC73) was observed on a larger scale. This means that increased macroautophagy induced by hyperbaric treatment weakens with time that has elapsed since the cessation of high pressure, whereas similarly induced chaperone-mediated autophagy intensifies over time. Full article

Francisella is a highly infectious gram-negative bacterium that causes tularemia in humans and animals. It can survive and multiply in a variety of cells, including macrophages, dendritic cells, amoebae, and arthropod-derived cells. However, the intracellular life cycle of a bacterium varies depending on the cell type. Shortly after the infection of mammalian cells, the bacterium escapes the phagosome into the cytosol, where it replicates. In contrast, in the amoebae Acanthamoeba castellanii and Hartmannella vermiformis, the bacterium replicates within the membrane-bound vacuole. In recent years, the amoeba Dictyostelium discoideum has emerged as a powerful model to study the intracellular cycle and virulence of many pathogenic bacteria. In this study, we used D. discoideum as a model for the infection and isolation of Francisella novicida-containing vacuoles (FCVs) formed after bacteria invade the amoeba. Our results showed that F. novicida localized in a vacuole after invading D. discoideum. Here, we developed a method to isolate FCV and determined its composition by proteomic analyses. Proteomic analyses revealed 689 proteins, including 13 small GTPases of the Rab family. This is the first evidence of F. novicida-containing vacuoles within amoeba, and this approach will contribute to our understanding of host–pathogen interactions and the process of pathogen vacuole formation, as vacuoles containing bacteria represent direct contact between pathogens and their hosts. Furthermore, this method can be translocated on other amoeba models. Full article

The triple combination therapy for cystic fibrosis (CF), including elexacaftor, tezacaftor and ivacaftor (ETI or Trikafta), has been shown to improve lung function and reduce pulmonary exacerbations, thereby enhancing the quality of life for most CF patients. Recent findings suggest that both the individual components and ETI may have potential off-target effects, highlighting the need to understand how these modulators impact cellular physiology, particularly in cells that do not express CF transmembrane conductance regulator (CFTR). We used HEK293 cells, as a cell model not expressing the CFTR protein, to evaluate the effect of ETI and each of its components on autophagic machinery and on the Rab5/7 components of the Rab pathway. We firstly demonstrate that the single modulators Teza and Iva, and the combinations ET and ETI, increased ROS production in the absence of their target while decreasing it in cells expressing the CFTR ∆F508del. This increase in cellular stress was followed by an increase in the total level of polyubiquitinated proteins as well as the p62 level and LC3II/LC3I ratio. Furthermore, we found that ETI had the opposite effect on Rabs by increasing Rab5 levels while decreasing Rab7. Interestingly, these changes were abolished by the expression of mutated CFTR. Overall, our data suggest that in the absence of their target, both the individual modulators and ETI increased ROS production and halted both autophagic flux and plasma membrane protein recycling. Full article

Glaesserella parasuis cytolethal distending toxin (GpCDT) can induce cell cycle arrest and apoptosis. Our laboratory’s previous work demonstrated that GTPase 4b (Rab4b) is a key host protein implicated in GpCDT-induced cytotoxicity. This study investigated the probable involvement of Rab4b in the process. Our study used CRISPR/Cas9 technology to create a Rab4b-knockout cell line. The results showed greater resistance to GpCDT-induced cell cytotoxicity. In contrast, forced Rab4b overexpression increased GpCDT-induced cytotoxicity. Further immunoprecipitation study reveals that GpCDT may bind with Rab4b. In PK-15 cells, GpCDT is transported to the early endosomes and late endosomes, while after knocking out Rab4b, GpCDT cannot be transported to the early endosome via vesicles. Rab4b appears essential for GpCDT-induced cytotoxicity in PK-15 cells. Full article

Background: Computed tomography to body divergence (CTBD) is one of the main barriers to bronchoscopic techniques for the diagnosis of peripherally located lung nodules. Cone-beam CT (CBCT) guidance is being rapidly adopted to correct for this phenomenon and to potentially increase diagnostic outcomes. In this trial, we hypothesized that the addition of mobile CBCT (m-CBCT) could improve the rate of tool in lesion (TIL) and the diagnostic yield of shape-sensing robotic-assisted bronchoscopy (SS-RAB). Methods: This was a prospective, single-arm study, which enrolled patients with peripheral lung nodules of 1–3 cm and compared the rate of TIL and the diagnostic yield of SS-RAB alone and combined with mCBCT. Results: A total of 67 subjects were enrolled, the median nodule size was 1.7 cm (range, 0.9–3 cm). TIL was achieved in 23 patients (34.3%) with SS-RAB alone, and 66 patients (98.6%) with the addition of mCBCT (p < 0.0001). The diagnostic yield of SS-RAB alone was 29.9% (95% CI, 29.3–42.3%) and it was 86.6% (95% CI, 76–93.7%) with the addition of mCBCT (p < 0.0001). There were no pneumothoraxes or any bronchoscopy-related complications, and the median total dose–area product (DAP) was 50.5 Gy-cm². Conclusions: The addition of mCBCT guidance to SS-RAB allows bronchoscopists to compensate for CTBD, leading to an increase in TIL and diagnostic yield, with acceptable radiation exposure. Full article

This study systematically investigated the effects of tea polyphenols on methane (CH₄) production and the rumen epithelial cell transport capability in cattle using both in vitro and animal experiments, employing multi-omics techniques. The in vitro results demonstrated that, compared to the control group, tea polyphenols significantly reduced CH₄ production and the acetate/propionate ratio (p < 0.05). Tea polyphenols reduced CH₄ production by inhibiting the relative abundance of unclassified_d_Archaea methanogens and the protozoa Pseudoentodinium and g__Balantioides. The animal experiments showed that tea polyphenols significantly increased the concentrations of T-AOC and GSH-PX in bovine blood (p < 0.05). In addition, microbial groups such as Rikenellaceae_RC9_gut_group, Ruminococcaceae_NK4A214_group, and Butyrivibrio_2 were significantly enriched in the ruminal fluid of the tea polyphenol group (p < 0.05). The proteomic results indicated significant upregulation of proteins such as COIII, S100A8, FABP1, SLC2A8, and SLC29A1 (p < 0.05) and downregulation of proteins including HBB, RAB4A, RBP4, LOC107131172, HBA, and ZFYVE19 (p < 0.05), with FABP1 showing a positive correlation with propionate concentration, and RAB4A had a negative correlation (p < 0.05). Overall, tea polyphenols modulate the microbial composition within the rumen, inhibiting CH₄ production and enhancing the host’s rumen epithelial cell transport capacity for volatile fatty acids. Full article

Although the Japanese encephalitis virus (JEV) infects various cell types, its receptor molecules are still not clearly understood. In our laboratory’s prior research, Rab4b was identified as a potential host factor that facilitates JEV infection in PK15 cells, utilizing a genome-wide CRISPR/Cas9 knockout library (PK-15-GeCKO). To further explore the effect of Rab4b on JEV replication, we used the Rab4b knockout PK15 cell line using the CRISPR/Cas9 technology and overexpressing the Rab4b PK15 cell line, with IFA, RT–qPCR, and Western blot to study the effect of Rab4b on viral replication in the whole life cycle of the JEV. The results show that the knockout of Rab4b inhibited the replication of the JEV in PK15 cells, and the overexpression of Rab4b promoted the replication of the JEV in PK15 cell lines. Furthermore, we demonstrated for the first time that host factor Rab4b facilitates the adsorption, internalization, assembly, and release of the JEV, thereby promoting JEV replication. This study enriches the regulatory network between the JEV and host factors and lays the experimental foundation for further understanding of the function of the Rab4b protein. Full article

Vibrio cholerae is an important foodborne pathogen. Cholix cytotoxin (Cholix), produced by V. cholerae, is a novel eukaryotic elongation factor 2 (eEF2) adenosine diphosphate ribosyltransferase that causes host cell death by inhibiting protein synthesis. However, the role of Cholix in the infectious diseases caused by V. cholerae remains unclear. Some bacterial cytotoxins are carried by host extracellular vesicles (EVs) and transferred to other cells. In this study, we investigated the effects of EV inhibitors and EV-regulating proteins on Cholix-induced hepatocyte death. We observed that Cholix-induced cell death was significantly enhanced in the presence of EV inhibitors (e.g., dimethyl amiloride, and desipramine) and Rab27a-knockdown cells, but it did not involve a sphingomyelin-dependent pathway. RNA sequencing analysis revealed that desipramine, imipramine, and EV inhibitors promoted the Cholix-activated c-Jun NH2-terminal kinase (JNK) pathway. Furthermore, JNK inhibition decreased desipramine-enhanced Cholix-induced poly (ADP-ribose) polymerase (PARP) cleavage. In addition, suppression of Apaf-1 by small interfering RNA further enhanced Cholix-induced PARP cleavage by desipramine. We identified a novel function of desipramine in which the stimulated JNK pathway promoted a mitochondria-independent cell death pathway by Cholix. Full article

Small GTP-binding proteins of the Rab family regulate intracellular vesicle trafficking across many aspects of the transport system. Among these, Rab9 is recognized for its role in controlling the transport system not only around the trans-Golgi network but also around the late endosome. However, the specific functions across different cell types and tissues remain unclear. Here, for the first time, we report that Rab9 negatively regulates morphological changes in the FBD-102b cell line, an oligodendroglial precursor cell line undergoing morphological differentiation. The knockdown of Rab9 led to an increase in cell shape alterations characterized by widespread membrane extensions. These changes were accompanied by increased expression levels of oligodendroglial cell differentiation and myelination marker proteins. Notably, the knockdown of Rab9 was capable of recovering defective cell morphological changes induced by tunicamycin, an inducer of endoplasmic reticulum (ER) stress, which is one of the major causes of oligodendroglial cell diseases such as Pelizaeus–Merzbacher disease (PMD, currently known as hypomyelinating leukodystrophy type 1 [HLD1]). In addition, Rab9 knockdown recovered levels of ER stress marker proteins and differentiation markers. Similar results were obtained in the cases of dithiothreitol (DTT), another chemical ER stress inducer, as well as HLD1-associated proteolipid protein 1 (PLP1) mutant protein. These results indicate a unique role for Rab9 in oligodendroglial cell morphological changes, suggesting its potential as a therapeutic target for mitigating diseases such as HLD1 at the molecular and cellular levels. Full article

This work focused on the decolorization of methyl red (MR) from an aqueous solution utilizing Rumex abyssinicus-derived biochar (RAB). RAB was prepared to involve unit operations such as size reduction, drying, and carbonization. The pyrolysis of the precursor material was carried out at a temperature of 500 °C for two hours. After that, the prepared RAB was characterized by the pH point of zero charge (pHpzc), the Brunauer–Emmett–Teller (BET) method, Scanning Electron Microscopy (SEM) and Fourier-Transform Infrared (FTIR) spectroscopy. On the other hand, a batch adsorption experiment of MR removal onto RAB was conducted, considering four operating parameters: pH, contact time, adsorbent dose, and initial dye concentration. The characterization of the adsorbent material revealed a porous and heterogeneous surface morphology during SEM, a specific surface area of 45.8 m²/g during the BET method, the presence of various functional groups during FTIR, and a pHpzc of 6.2. The batch adsorption experiment analysis results revealed that a maximum removal efficiency of 99.2% was attained at an optimum working condition of pH 6, contact time of 40 min, initial dye concentration of 70 mg/L and adsorbent dosage of 0.2 g/100 mL. Furthermore, Freundlich isotherm (R² = 0.99) and pseudo-second-order kinetics (R² = 0.99) models confirmed the heterogeneous surface interaction and chemisorption nature. Generally, this study highlighted that RAB could be a potential adsorbent for the detoxification of MR-containing industrial effluents. Full article

Endoplasmic reticulum (ER)–endolysosome interactions regulate cholesterol exchange between the ER and the endolysosome. ER–endolysosome membrane contact sites mediate the ER–endolysosome interaction. VAP-ORP1L (vesicle-associated membrane protein-associated protein- OSBP-related protein 1L) interaction forms the major contact site between the ER and the lysosome, which is regulated by Rab7. RILP (Rab7-interacting lysosomal protein) is the downstream effector of Rab7, but its role in the organelle interaction between the ER and the lysosome is not clear. In this study, we found RILP interacts with ORP1L to competitively inhibit the formation of the VAP–ORP1L contact site. Immunofluorescence microscopy revealed that RILP induces late endosome/lysosome clustering, which reduces the contact of endolysosomes with the ER, interfering with the ER–endolysosome interaction. Further examination demonstrated that over-expression of RILP results in the accumulation of cholesterol in the clustered endolysosomes, which triggers cellular autophagy depending on RILP. Our results suggest that RILP interferes with the ER–endolysosome interaction to inhibit cholesterol flow from the endolysosome to the ER, which feedbacks to trigger autophagy. Full article