Search Results (1,396)

Endothelin Receptor Type B (EDNRB) is expressed in a variety of cells during embryonic stage, including melanocyte precursors cells. Our previous studies found that 11 bp deletion of EDNRB caused the two-end black (TEB) coat color in Chinese pigs. In this study, we aimed to explore the mutant EDNRB on the formation of TEB coat color in Chinese pigs. We constructed recombinant plasmid for wild and mutant EDNRB and EDN1, respectively, and transfected the recombinant plasmid into mouse B16 melanoma cells in groups. Real-time fluorescent quantitative PCR (RT-qPCR) was performed to detect expression of genes that participate in melanin pathway, including PLCγ, Raf, MITF. Comparing to the wild-type EDNRB cells, expression of the three genes in the cell line expressing mutant EDNRB cells was significantly reduced. We measured the melanin content produced by transfected recombinant granulocytes of wild and mutant EDNRB and found that the amount of melanin in mutant EDNRB cells was significantly lower than that of the wild. Wound-healing assay confirmed that the migration and mobility rate of mutant EDNRB cells were significantly lower than the wild. Co-immunoprecipitation further confirmed that mutant EDNRB could not interact with the EDN1 protein. In conclusion, this study revealed that the 11 bp deletion of EDNRB reduced the melanin production, which may be caused by inhibiting the expression of PLCγ, Raf, and MITF. The mutant EDNRB reduced melanocyte migration and could not interact with the EDN1 protein. We explored the effect of mutant EDNRB in Chinese pigs with TEB coat color, and the results provided a reference for exploring molecular mechanism of mutant EDNRB on the formation of TEB coat color pigs. Full article

Filamin C (FLNC) is a structural protein of muscle fibers. Mutations in the FLNC gene are known to cause myopathies and cardiomyopathies in humans. Here we report the generation by a CRISPR/Cas9 editing system injected into zygote pronuclei of two mouse strains carrying filamin C mutations—one of them (AGA) has a deletion of three nucleotides at position c.7418_7420, causing E>>D substitution and N deletion at positions 2472 and 2473, respectively. The other strain carries a deletion of GA nucleotides at position c.7419_7420, leading to a frameshift and a premature stop codon. Homozygous animals (Flnc^AGA/AGA and Flnc^GA/GA) were embryonically lethal. We determined that Flnc^GA/GA embryos died prior to the E12.5 stage and illustrated delayed development after the E9.5 stage. We performed histological analysis of heart tissue and skeletal muscles of heterozygous strains carrying mutations in different combinations (Flnc^GA/wt, Flnc^AGA/wt, and Flnc^GA/AGA). By performing physiological tests (grip strength and endurance tests), we have shown that heterozygous animals of both strains (Flnc^GA/wt, Flnc^AGA/wt) are functionally indistinguishable from wild-type animals. Interestingly, compound heterozygous mice (Flnc^GA/AGA) are viable, develop normally, reach puberty and it was verified by ECG and Eco-CG that their cardiac muscle is functionally normal. Intriguingly, Flnc^GA/AGA mice demonstrated better results in the grip strength physiological test in comparison to WT animals. We also propose a structural model that explains the complementary interaction of two mutant variants of filamin C. Full article

The CRISPR/Cas9 system offers a powerful tool for gene editing to enhance rice productivity. In this study, we successfully edited eight RR-TZF genes in japonica rice Nipponbare using CRISPR-Cas9 technology, achieving a high editing efficiency of 73.8%. Sequencing revealed predominantly short insertions or deletions near the PAM sequence, along with multi-base deletions often flanked by identical bases. Off-target analysis identified 5 out of 31 predicted sites, suggesting the potential for off-target effects, which can be mitigated by designing gRNAs with more than three base mismatches. Notably, new mutations emerged in the progeny of several gene-edited mutants, indicating inheritable genetic mutagenicity. Phenotypic analysis of homozygous mutants revealed varied agronomic traits, even within the same gene, highlighting the complexity of gene-editing outcomes. These findings underscore the importance of backcrossing to minimize off-target and inheritable mutagenicity effects, ensuring more accurate trait evaluation. This study offers insights into CRISPR/Cas9 mechanisms and uncertain factors and may inform future strategies for rice improvement, prompting further research into CRISPR/Cas9’s precision and long-term impacts. Full article

To elucidate the mechanism of large-scale transcriptional changes dependent on sulfide in Escherichia coli, a large-scale RNA-sequencing analysis was performed on wild-type and sulfide-responsive transcription factor YgaV deletion mutants grown under three conditions: aerobic, semi-aerobic, and semi-aerobic with sulfide. The resulting dataset from these six conditions was subjected to principal component analysis, which categorized the data into five principal components. Estimation of the typical gene expression regulatory mechanisms in each category suggested the presence of mechanisms that are dependent on sulfide but independent of YgaV, as well as those that depend on YgaV but not on sulfide. In YgaV-dependent transcriptional regulation, YgaV was found to function as both a repressor and an activator. These results support the previous hypothesis that YgaV acts as a global regulator responsible for redox homeostasis. Full article

A flagellar motor can rotate either counterclockwise (CCW) or clockwise (CW), and rotational switching is triggered by conformational changes in FliG, although the molecular mechanism is still unknown. Here, we found that cheY deletion, which locks motor rotation in the CCW direction, restored the motility abolished by the fliG L259Q mutation. We found that the CCW-biased fliG G214S mutation also restored the swimming of the L259Q mutant, but the CW-biased fliG G215A mutation did not. Since the L259 residue participates in forming the FliG hydrophobic core at its C-terminal domain, mutations were introduced into residues structurally closer to L259, and their motility was examined. Two mutants, D251R and L329Q, exhibited CW-biased rotation. Our results suggest that mutations in the hydrophobic core of FliG_C collapse its conformational switching and/or stator interaction; however, the CCW state of the rotor enables rotation even with this disruption. Full article

Nitrogen is an essential nutrient that frequently determines the growth rate of fungi. Nitrate transporter proteins (Nrts) play a crucial role in the cellular absorption of nitrate from the environment. Entomopathogenic fungi (EPF) have shown their potential in the biological control of pests. Thus, comprehending the mechanisms that govern the pathogenicity and stress tolerance of EPF is helpful in improving the effectiveness and practical application of these fungal biocontrol agents. In this study, we utilized homologous recombination to create MaNrtB deletion mutants and complementation strains. We systematically investigated the biological functions of the nitrate transporter protein gene MaNrtB in M. acridum. Our findings revealed that the disruption of MaNrtB resulted in delayed conidial germination without affecting conidial production. Stress tolerance assays demonstrated that the MaNrtB disruption strain was more vulnerable to UV-B irradiation, hyperosmotic stress, and cell wall disturbing agents, yet it exhibited increased heat resistance compared to the wild-type strain. Bioassays on the locust Locusta migratoria manilensis showed that the disruption of MaNrtB impaired the fungal virulence owing to the reduced appressorium formation on the insect cuticle and the attenuated growth in the locust hemolymph. These findings provide new perspectives for understanding the pathogenesis of EPF. Full article

Background: Salmonella Typhimurium poses a substantial health risk to both humans and animals. This study evaluated the potential of using the Salmonella Typhimurium ΔsptP mutant as a live-attenuated vaccine candidate by constructing it through homologous recombination and assessing its key biological properties, including growth characteristics, immunogenicity, and protective efficacy. Methods: We generated the ΔsptP mutant through targeted gene deletion, ensuring the preservation of the bacterial strain’s growth and stability. In vitro and in vivo assays were performed to compare the invasive capabilities between the mutant and the wild-type strains. Specifically, we examined the invasion into RAW264.7 murine macrophages and mice. Furthermore, the virulence of the mutant was evaluated by determining the median lethal dose (LD₅₀). To evaluate immunogenicity and protection, mice were immunized with 2 × 10⁴ CFUs of the ΔsptP mutant, followed by a booster immunization, and then challenged with a virulent strain. Results: The ΔsptP mutant exhibited no significant changes in growth characteristics or genetic stability compared to the wild-type strain. However, it demonstrated a significantly diminished capacity for invasion in both murine macrophages and mice. The LD₅₀ for the mutant was 39.92-fold higher than that of the wild-type, indicating a marked reduction in virulence. Mice immunized with the ΔsptP mutant and administered a booster immunization exhibited 87.5% protection against challenge with a virulent strain, as compared to the PBS control group. Moreover, the mutant induced IgG antibody levels comparable to those induced by the wild-type strain. Conclusions: The ΔsptP mutant of Salmonella Typhimurium exhibits markedly reduced virulence while retaining robust immunogenicity and protective efficacy. These findings suggest that the ΔsptP mutant is a promising candidate for a live-attenuated vaccine, potentially providing an effective strategy to prevent Salmonella Typhimurium infections. Full article

Lysine-specific histone demethylase 2 (Kdm2a) is essential for histone modifications involved in development and associated diseases. Nevertheless, the specific functions of Kdm2a in renal development and pathology remain largely unexplored. This study aimed to elucidate the roles of Kdm2a in sustaining the biological functions of the kidney by generating mutant mice with Kdm2a deletion using the Aqp2-cre/Loxp system. Our findings showed that Kdm2a is widely expressed across various mouse tissues, with particularly high expression in the kidney's cortex and medulla, surpassing that in other tissues. Despite no observable effects on morphology or survival following the conditional knockout of Kdm2a, there was a significant reduction in body weight and bilateral kidney weight compared to controls, most pronounced at the 5-week-old stage (P < 0.05). Post Kdm2a deletion, kidney metabolic functions were impaired, evidenced by altered levels of creatinine, urea, total cholesterol, and low-density lipoprotein. Histological examination revealed that Kdm2a-null kidneys exhibited signs of dysfunction, characterized by macrophage infiltration, fibrosis, inflammatory cell infiltration, and mild thrombosis. Further studies revealed that the expression of chemokine- and pro-inflammatory cytokine-related genes Il-6, Il-8, Tnf-a, and Il-1β was significantly increased in the kidneys of Kdm2a cKO mice compared with controls (P < 0.05). Additionally, the expression of reabsorption-related genes (Aqp-3, Aqp-5, and Aqp-8) was markedly downregulated in Kdm2a-deficient kidneys compared with controls (P < 0.05). Collectively, these findings suggest that Kdm2a is crucial for maintaining kidney function and development, partly through the suppression of inflammation and regulation of gene expression. However, the underlying molecular mechanisms of Kdm2a in kidney development warrant further investigation. Full article

African swine fever virus (ASFV) is a severe threat to the global pig industry, and domestic pigs mostly develop severe clinical manifestations upon viral invasion. Currently, there is no available vaccine against ASFV. Its capsid structural protein p72 is one of the immuno-dominant proteins. In this study, we unexpectedly obtained a p72 mutant protein (p72_∆377–428) which deleted the aa 377–428 within p72 and had stable and high expression in E. coli. Using SWISS-MODEL software, the prediction showed that p72_∆377–428 was quite distinct from the wild-type p72 protein in structure. p72_∆377–428 induced stronger antibody production in mice on day 42 and 56 post immunization and could recognize ASFV-infected swine sera. p72_∆377–428 reduced IFN-γ production in the splenocytes from p72_∆377–428-immunized mice and p72_∆377–428-treated swine macrophages compared to p72. p72_∆377–428 also decreased the production of pro-inflammatory cytokine genes, including IL-1β, IL-6, and IL-12, compared to p72 in mice. Further, we found that p72_∆377–428 reduced the induction of pro-inflammatory cytokine genes by inhibiting AKT phosphorylation and HIF1α expression. Taken together, these findings have implications for immunological function and the corresponding mechanism of ASFV p72, and our study indicates that p72_∆377–428 could serve as a novel candidate for ASFV vaccines and diagnostic reagents. Full article

Fusarium sacchari is one of the primary causal agents of Pokkah boeng disease (PBD), an important disease of sugarcane worldwide. The acyl-CoA dehydrogenases (ACADs) constitute a family of flavoenzymes involved in the β-oxidation of fatty acids and amino acid catabolism in mitochondria. However, the role of ACADs in the pathogenesis of F. sacchari is unclear. Here, 14 ACAD-encoding genes (FsACAD-1–FsACAD-14) were identified by screening the entire genome sequence of F. sacchari. The FsACAD genes are distributed across seven chromosomes and were classified into seven clades based on phylogenetic analysis of the protein sequences. In vivo mRNA quantification revealed that the FsACAD genes are differentially expressed during sugarcane infection, and their expression patterns differ significantly in response to the in vitro induction of fatty acids of different classes. Fatty acid utilization assays of the FsACAD-deletion mutants revealed that the FsACADs varied in their preference and ability to break down different fatty acids and amino acids. There was variation in the adverse impact of FsACAD-deletion mutants on fungal traits, including growth, conidiation, stress tolerance, and virulence. These findings provide insights into the roles of FsACADs in F. sacchari, and the identification of FsACADs offers potential new targets for the improved control of PBD. Full article

We generated and characterized knockout mutant lines of the OsPHS3 gene using the CRISPR/Cas9 system. The knockout lines of the OsPHS3 gene showed that 1 bp and 7 bp deletion, early termination codons were used for protein production. Agronomic characteristics of knock-out lines were reduced in plant height, culm diameter, panicle length, seed size and weight, except for the number of tillers. In addition, we analyzed the expression levels of carotenoid biosynthesis genes by qRT-PCR. Among the genes encoding carotenoid metabolic pathway enzymes, the level of transcripts of PSY1, PSY2, PSY3, PDS and ZDS were higher in the KO lines than in the WT line. In contrast, transcription of the ε-LCY, β-LCY and ZEP1 genes were downregulated in the KO lines compared to the WT line. Also, the KO lines decreased carotenoid content and ABA amount compared to WT, while preharvest sprouts increased. These results suggested that they would certainly help explain the molecular mechanisms of PHS in other crops, such as wheat and barley, which are susceptible to PHS. Full article

The pentose phosphate pathway (PPP) is essential for human health and provides, amongst others, the reduction power to cope with oxidative stress. In contrast to the model baker’s yeast, the PPP also contributes to a large extent to glucose metabolism in the milk yeast Kluyveromyces lactis. Yet, the physiological consequences of mutations in genes encoding PPP enzymes in K. lactis have been addressed for only a few. We here embarked on a systematic study of such mutants, deleting ZWF1, SOL4, GND1, RKI1, RPE1, TKL1, TAL1, and SHB17. Interestingly, GND1, RKI1, and TKL1 were found to be essential under standard growth conditions. Epistasis analyses revealed that a lack of Zwf1 rescued the lethality of the gnd1 deletion, indicating that it is caused by the accumulation of 6-phosphogluconate. Moreover, the slow growth of a tal1 null mutant, which lacks fructose-1,6-bisphosphate aldolase, was aggravated by deleting the SHB17 gene encoding sedoheptulose-1,7-bisphosphatase. A mitotically stable tetOFF system was established for conditional expression of TAL1 and TKL1, encoding transaldolase and transketolase in the non-oxidative part of the PPP, and employed in a global proteome analysis upon depletion of the enzymes. Results indicate that fatty acid degradation is upregulated, providing an alternative energy source. In addition, tal1 and tkl1 null mutants were complemented by heterologous expression of the respective genes from baker’s yeast and humans. These data demonstrate the importance of the PPP for basic sugar metabolism and oxidative stress response in K. lactis and the potential of this yeast as a model for the study of PPP enzymes from heterologous sources, including human patients. Full article

During lytic replication of human cytomegalovirus (HCMV), the most abundant viral transcripts are long non-coding RNAs (lncRNAs). Viral lncRNAs can have a variety of functions, some of which are necessary for viral production and the modulation of host processes during infection. HCMV produces four lncRNAs, Beta2.7 (RNA2.7), RNA4.9, RNA5.0 and RNA1.2. While there has been research on these viral lncRNAs, many of their functions remain uncharacterized. To determine the function of RNA1.2, we explored its requirement during lytic infection by generating viral mutants containing either a full or partial deletion of the RNA1.2 locus. Within permissive fibroblasts, the RNA1.2 deletion mutants showed no defects in viral DNA synthesis, transcript expression, protein production, or generation of viral progeny. Further investigation to identify potential cellular and viral protein binding partners of RNA1.2 was performed using liquid chromatography-mass spectrometry (LC-MS). A significant number of cellular proteins were identified and associated with RNA1.2. Specifically those associated with the innate immune response, mitochondrial processes, and cell cycle regulation. While RNA1.2 is dispensable for lytic replication, these findings suggest it may play a pivotal role in modulating the host response. Full article

Colletotrichum gloeosporioides is a major phytopathogen responsible for anthracnose in Capsicum annuum (pepper) which leads to significant yield losses. At present, the molecular mechanism of C. gloeosporioides pathogenesis is not very clear. In this study, we focused on the MET30 protein and its key WD40 domain, with an emphasis on its role in the biological functions of C. gloeosporioides. Bioinformatics analysis revealed that the MET30 protein contains a conserved F-box domain and multiple WD40 repeats, which interact with other proteins to participate in various cellular processes, including nutrient acquisition, stress responses, and pathogenicity. Gene knockout and complementation experiments demonstrated that deleting the MET30 protein or its WD40 domain significantly reduced the rates of spore production and hyphal growth while increasing tolerance to environmental stresses such as high salinity and oxidative stress. Furthermore, pathogenicity assays revealed that the WD40 domain of the MET30 protein is crucial for regulating fungal pathogenicity, as mutants lacking WD40 domains presented increased virulence on pepper leaves. These findings suggest that the WD40 domain, in synergy with the MET30 protein, regulates the pathogenicity and stress response of C. gloeosporioides, provides new insights into the molecular mechanisms of anthracnose, and offers potential strategies for effective disease control. Full article

Autophagy is a vital cellular pathway in eukaryotic cells, including neurons, where it plays significant roles in neurodevelopment and maintenance. A crucial step in autophagy is the formation of the class III phosphatidylinositol 3-kinase complex 1 (PI3KC3-C1), which is essential for initiating autophagosome biogenesis. Beclin 1 is the key component of PI3KC3-C1, and its interactors have been reported to affect autophagy. The brain-enriched adaptor protein FE65 has been shown to interact with Alzheimer’s disease amyloid precursor protein (APP) to alter the processing of APP. Additionally, FE65 has been implicated in various cellular pathways, including autophagy. We demonstrate here that FE65 positively regulates autophagy. FE65, through its C-terminus, has been shown to interact with Beclin 1. Notably, the overexpression of FE65 enhances Beclin 1-mediated autophagy, whereas this process is attenuated in FE65 knockout cells. Moreover, the stimulatory effect of FE65 on Beclin 1-mediated autophagy is diminished by an FE65 C-terminus deletion mutant that disrupts the FE65–Beclin 1 interaction. Lastly, we have found that the FE65-Beclin 1 interaction modulates the kinase activity of the PI3KC3-C1 complex. Together, we have identified FE65 as a novel Beclin 1 interactor, and this interaction potentiates autophagy. Full article