Mutations in Disordered Regions Can Cause Disease by Creating Dileucine Motifs.

Many disease-causing missense mutations affect intrinsically disordered regions (IDRs) of proteins, but the molecular mechanism of their pathogenicity is enigmatic. Here, we employ a peptide-based proteomic screen to investigate the impact of mutations in IDRs on protein-protein interactions. We find that mutations in disordered cytosolic regions of three transmembrane proteins (GLUT1, ITPR1, and CACNA1H) lead to an increased clathrin binding. All three mutations create dileucine motifs known to mediate clathrin-dependent trafficking. Follow-up experiments on GLUT1 (SLC2A1), the glucose transporter causative of GLUT1 deficiency syndrome, revealed that the mutated protein mislocalizes to intracellular compartments. Mutant GLUT1 interacts with adaptor proteins (APs) in vitro, and knocking down AP-2 reverts the cellular mislocalization and restores glucose transport. A systematic analysis of other known disease-causing variants revealed a significant and specific overrepresentation of gained dileucine motifs in structurally disordered cytosolic domains of transmembrane proteins. Thus, several mutations in disordered regions appear to cause "dileucineopathies."

Point-activated ESR1Y541S has a dramatic effect on the development of sexually dimorphic organs.

Mutations in the estrogen receptor α (ERα) occur in endocrine-resistant metastatic breast cancer. However, a major gap persists with the lack of genetically tractable immune competent mouse models to study disease. Hence, we developed a Cre-inducible murine model expressing a point-activated ESR1Y541S (ESR1Y537S in humans) driven by its endogenous promoter. Germline expression of mutant ESR1Y541S reveals dramatic developmental defects in the reproductive organs, mammary glands, and bones of the mice. These observations provide critical insights into the tissue-specific roles of ERα during development and highlights the potential use of our model in further developmental and cancer studies.

Transfer learning to leverage larger datasets for improved prediction of protein stability changes.

Amino acid mutations that lower a protein's thermodynamic stability are implicated in numerous diseases, and engineered proteins with enhanced stability can be important in research and medicine. Computational methods for predicting how mutations perturb protein stability are, therefore, of great interest. Despite recent advancements in protein design using deep learning, in silico prediction of stability changes has remained challenging, in part due to a lack of large, high-quality training datasets for model development. Here, we describe ThermoMPNN, a deep neural network trained to predict stability changes for protein point mutations given an initial structure. In doing so, we demonstrate the utility of a recently released megascale stability dataset for training a robust stability model. We also employ transfer learning to leverage a second, larger dataset by using learned features extracted from ProteinMPNN, a deep neural network trained to predict a protein's amino acid sequence given its three-dimensional structure. We show that our method achieves state-of-the-art performance on established benchmark datasets using a lightweight model architecture that allows for rapid, scalable predictions. Finally, we make ThermoMPNN readily available as a tool for stability prediction and design.

Utilizing codon degeneracy in the design of donor DNA for CRISPR/Cas9-mediated gene editing to streamline the screening process for single amino acid mutations.

Chlamydomonas reinhardtii, a unicellular green alga, has been widely used as a model organism for studies of algal, plant and ciliary biology. The generation of targeted amino acid mutations is often necessary, and this can be achieved using CRISPR/Cas9 induced homology-directed repair to install genomic modifications from exogenous donor DNA. Due to the low gene editing efficiency, the technical challenge lies in identifying the mutant cells. Direct sequencing is not practical, and pre-screening is required. Here, we report a strategy for generating and screening for amino acid point mutations using the CRISPR/Cas9 gene editing system. The strategy is based on designing donor DNA using codon degeneracy, which enables the design of specific primers to facilitate mutant screening by PCR. An in vitro assembled RNP complex, along with a dsDNA donor and an antibiotic resistance marker, was electroporated into wild-type cells, followed by PCR screening. To demonstrate this principle, we have generated the E102K mutation in centrin and the K40R mutation in α-tubulin. The editing efficiencies at the target sites for Centrin, TUA1, TUA2 were 4, 24 and 8% respectively, based on PCR screening. More than 80% of the mutants with the expected size of PCR products were precisely edited, as revealed by DNA sequencing. Subsequently, the precision-edited mutants were biochemically verified. The introduction of codon degeneracy did not affect the gene expression of centrin and α-tubulins. Thus, this approach can be used to facilitate the identification of point mutations, especially in genes with low editing rates.

Cancer-associated point mutations within the extracellular domain of PTPRD affect protein stability and HSPG interaction.

PTPRD, a well-established tumor suppressor gene, encodes the protein tyrosine phosphatase-type D. This protein consists of three immunoglobulin-like (Ig) domains, four to eight fibronectin type 3 (FN) domains, a single transmembrane segment, and two cytoplasmic tandem tyrosine phosphatase domains. PTPRD is known to harbor various cancer-associated point mutations. While it is assumed that PTPRD regulates cellular functions as a tumor suppressor through the tyrosine phosphatase activity in the intracellular region, the function of its extracellular domain (ECD) in cancer is not well understood. In this study, we systematically examined the impact of 92 cancer-associated point mutations within the ECD. We found that 69.6% (64 out of 92) of these mutations suppressed total protein expression and/or plasma membrane localization. Notably, almost all mutations (20 out of 21) within the region between the last FN domain and transmembrane segment affected protein expression and/or localization, highlighting the importance of this region for protein stability. We further found that some mutations within the Ig domains adjacent to the glycosaminoglycan-binding pocket enhanced PTPRD's binding ability to heparan sulfate proteoglycans (HSPGs). This interaction is proposed to suppress phosphatase activity. Our findings therefore suggest that HSPG-mediated attenuation of phosphatase activity may be involved in tumorigenic processes through PTPRD dysregulation.

The massive 340 megabase genome of Anisogramma anomala, a biotrophic ascomycete that causes eastern filbert blight of hazelnut.

BACKGROUND: The ascomycete fungus Anisogramma anomala causes Eastern Filbert Blight (EFB) on hazelnut (Corylus spp.) trees. It is a minor disease on its native host, the American hazelnut (C. americana), but is highly destructive on the commercially important European hazelnut (C. avellana). In North America, EFB has historically limited commercial production of hazelnut to west of the Rocky Mountains. A. anomala is an obligately biotrophic fungus that has not been grown in continuous culture, rendering its study challenging. There is a 15-month latency before symptoms appear on infected hazelnut trees, and only a sexual reproductive stage has been observed. Here we report the sequencing, annotation, and characterization of its genome. RESULTS: The genome of A. anomala was assembled into 108 scaffolds totaling 342,498,352 nt with a GC content of 34.46%. Scaffold N50 was 33.3 Mb and L50 was 5. Nineteen scaffolds with lengths over 1 Mb constituted 99% of the assembly. Telomere sequences were identified on both ends of two scaffolds and on one end of another 10 scaffolds. Flow cytometry estimated the genome size of A. anomala at 370 Mb. The genome exhibits two-speed evolution, with 93% of the assembly as AT-rich regions (32.9% GC) and the other 7% as GC-rich (57.1% GC). The AT-rich regions consist predominantly of repeats with low gene content, while 90% of predicted protein coding genes were identified in GC-rich regions. Copia-like retrotransposons accounted for more than half of the genome. Evidence of repeat-induced point mutation (RIP) was identified throughout the AT-rich regions, and two copies of the rid gene and one of dim-2, the key genes in the RIP mutation pathway, were identified in the genome. Consistent with its homothallic sexual reproduction cycle, both MAT1-1 and MAT1-2 idiomorphs were found. We identified a large suite of genes likely involved in pathogenicity, including 614 carbohydrate active enzymes, 762 secreted proteins and 165 effectors. CONCLUSIONS: This study reveals the genomic structure, composition, and putative gene function of the important pathogen A. anomala. It provides insight into the molecular basis of the pathogen's life cycle and a solid foundation for studying EFB.

Investigation of genotype-phenotype and familial features of Turkish dystrophinopathy patients.

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are X-linked recessive allelic muscle diseases caused by dystrophin gene mutations. Eight hundred thirty-seven patients admitted between 1997 and 2022 were included in the study. Two hundred twenty patients were analyzed by multiplex PCR (mPCR) alone. Five hundred ninety-five patients were investigated by multiplex ligation-dependent probe amplification (MLPA), and 54 patients were examined by sequencing. Deletion was detected in 60% (132/220) of the cases in the mPCR group only and in 58.3% (347/595) of the cases with MLPA analysis. The rates of deletion and duplication were 87.7% and 12.3%, respectively, in the MLPA analysis. Single exon deletions were the most common mutation type. The introns 43-55 (81.8%) and exons 2-21 (13.1%) regions were detected as hot spots in deletions. It was determined that 89% of the mutations were suitable for exon skipping therapy. The reading frame rule did not hold in 7.6% of D/BMD cases (17/224). We detected twenty-five pathogenic/likely pathogenic variants in sequencing, five of which were novel variants. Nonsense mutation was the most common small mutation (44%). 21% of DMD patients were familial. We detected germline mosaicism in four families (4.3%) in the large rearrangement group and one gonosomal mosaicism in a family with a nonsense mutation. This is the largest study examining genotype and phenotype data in Turkish D/BMD families investigated by MLPA analysis. The reading frame hypothesis is not valid in all cases. Sharing the genotype and phenotype characteristics of these cases in the literature will shed light on the molecular structure of DMD and guide gene therapy research. In genetic counseling, carrier screening in the family and possible gonadal mosaicism should be emphasized.

Predicting protein stability changes upon single-point mutation: a thorough comparison of the available tools on a new dataset.

Predicting the difference in thermodynamic stability between protein variants is crucial for protein design and understanding the genotype-phenotype relationships. So far, several computational tools have been created to address this task. Nevertheless, most of them have been trained or optimized on the same and 'all' available data, making a fair comparison unfeasible. Here, we introduce a novel dataset, collected and manually cleaned from the latest version of the ThermoMutDB database, consisting of 669 variants not included in the most widely used training datasets. The prediction performance and the ability to satisfy the antisymmetry property by considering both direct and reverse variants were evaluated across 21 different tools. The Pearson correlations of the tested tools were in the ranges of 0.21-0.5 and 0-0.45 for the direct and reverse variants, respectively. When both direct and reverse variants are considered, the antisymmetric methods perform better achieving a Pearson correlation in the range of 0.51-0.62. The tested methods seem relatively insensitive to the physiological conditions, performing well also on the variants measured with more extreme pH and temperature values. A common issue with all the tested methods is the compression of the $\Delta \Delta G$ predictions toward zero. Furthermore, the thermodynamic stability of the most significantly stabilizing variants was found to be more challenging to predict. This study is the most extensive comparisons of prediction methods using an entirely novel set of variants never tested before.

Citrate synthase variants improve yield of acetyl-CoA derived 3-hydroxybutyrate in Escherichia coli.

BACKGROUND: The microbial chiral product (R)-3-hydroxybutyrate (3-HB) is a gateway to several industrial and medical compounds. Acetyl-CoA is the key precursor for 3-HB, and several native pathways compete with 3-HB production. The principal competing pathway in wild-type Escherichia coli for acetyl-CoA is mediated by citrate synthase (coded by gltA), which directs over 60% of the acetyl-CoA into the tricarboxylic acid cycle. Eliminating citrate synthase activity (deletion of gltA) prevents growth on glucose as the sole carbon source. In this study, an alternative approach is used to generate an increased yield of 3-HB: citrate synthase activity is reduced but not eliminated by targeted substitutions in the chromosomally expressed enzyme. RESULTS: Five E. coli GltA variants were examined for 3-HB production via heterologous overexpression of a thiolase (phaA) and NADPH-dependent acetoacetyl-CoA reductase (phaB) from Cupriavidus necator. In shake flask studies, four variants showed nearly 5-fold greater 3-HB yield compared to the wild-type, although pyruvate accumulated. Overexpression of either native thioesterases TesB or YciA eliminated pyruvate formation, but diverted acetyl-CoA towards acetate formation. Overexpression of pantothenate kinase similarly decreased pyruvate formation but did not improve 3-HB yield. Controlled batch studies at the 1.25 L scale demonstrated that the GltA[A267T] variant produced the greatest 3-HB titer of 4.9 g/L with a yield of 0.17 g/g. In a phosphate-starved repeated batch process, E. coli ldhA poxB pta-ackA gltA::gltA[A267T] generated 15.9 g/L 3-HB (effective concentration of 21.3 g/L with dilution) with yield of 0.16 g/g from glucose as the sole carbon source. CONCLUSIONS: This study demonstrates that GltA variants offer a means to affect the generation of acetyl-CoA derived products. This approach should benefit a wide range of acetyl-CoA derived biochemical products in E. coli and other microbes. Enhancing substrate affinity of the introduced pathway genes like thiolase towards acetyl-CoA will likely further increase the flux towards 3-HB while reducing pyruvate and acetate accumulation.

Salmonella enteritidis acquires phage resistance through a point mutation in rfbD but loses some of its environmental adaptability.

Phage therapy holds promise as an alternative to antibiotics for combating multidrug-resistant bacteria. However, host bacteria can quickly produce progeny that are resistant to phage infection. In this study, we investigated the mechanisms of bacterial resistance to phage infection. We found that Rsm1, a mutant strain of Salmonella enteritidis (S. enteritidis) sm140, exhibited resistance to phage Psm140, which was originally capable of lysing its host at sm140. Whole genome sequencing analysis revealed a single nucleotide mutation at position 520 (C → T) in the rfbD gene of Rsm1, resulting in broken lipopolysaccharides (LPS), which is caused by the replacement of CAG coding glutamine with a stop codon TAG. The knockout of rfbD in the sm140ΔrfbD strain caused a subsequent loss of sensitivity toward phages. Furthermore, the reintroduction of rfbD in Rsm1 restored phage sensitivity. Moreover, polymerase chain reaction (PCR) amplification of rfbD in 25 resistant strains revealed a high percentage mutation rate of 64% within the rfbD locus. We assessed the fitness of four bacteria strains and found that the acquisition of phage resistance resulted in slower bacterial growth, faster sedimentation velocity, and increased environmental sensitivity (pH, temperature, and antibiotic sensitivity). In short, bacteria mutants lose some of their abilities while gaining resistance to phage infection, which may be a general survival strategy of bacteria against phages. This study is the first to report phage resistance caused by rfbD mutation, providing a new perspective for the research on phage therapy and drug-resistant mechanisms.

A case of exacerbated encephalopathy with stroke-like episodes and lactic acidosis triggered by metformin in a patient with MELAS.

Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) is a maternally inherited multisystemic disorder caused by mutations in mitochondrial DNA that result in cellular energy deficiency. MELAS affects the most metabolically active organs, including the brain, skeletal muscles, cochlea, retina, heart, kidneys, and pancreas. As a result, about 85% of carriers of m.3243A > G, the most common mutation in MELAS, develop diabetes by the age of 70. Although metformin is the most widely prescribed drug for diabetes, its usefulness in mitochondrial dysfunction remains controversial. Here, we present the case of a 32-year-old Korean patient diagnosed with MELAS who presented with exacerbated stroke-like episodes and lactic acidosis triggered by metformin.

PSMD3 gene mutations cause pathological myopia.

PURPOSE: Genetic factors play a prominent role in the pathogenesis of pathological myopia (PM). However, the exact genetic mechanism of PM remains unclear. This study aimed to determine the candidate mutation of PM in a Chinese family and explore the potential mechanism. METHODS: We performed exome sequencing and Sanger sequencing in a Chinese family and 179 sporadic PM cases. The gene expression in human tissue was investigated by RT-quantitative real-time PCR (RT-qPCR) and immunofluorescence. Cell apoptotic rates were tested by annexin V-APC/7AAD and flow cytometry. Psmd3 knock-in mice with point mutation were generated for measuring myopia-related parameters. RESULTS: We screened a novel PSMD3 variant (c.689T>C; p.F230S) in a Chinese family with PM, and another rare mutation (c.1015C>A; p.L339M) was identified in 179 unrelated cases with PM. RT-qPCR and immunofluorescence confirmed the expression of PSMD3 in human eye tissue. Mutation of PSMD3 decreased the mRNA and protein expression, causing apoptosis of human retinal pigment epithelial cells. In in vivo experiments, the axial length (AL) of mutant mice increased significantly compared with that of wild-type mice (p<0.001). CONCLUSIONS: A new potential pathogenic gene, PSMD3, in a PM family was identified, and it may be involved in the elongation of AL and the development of PM.

The P124A mutation of SRP14 alters its migration on SDS-PAGE without impacting its function.

SRP14 is a crucial protein subunit of the signal recognition particle (SRP), a ribonucleoprotein complex essential for co-translational translocation to the endoplasmic reticulum. During our investigation of SRP14 expression across diverse cell lines, we observe variations in its migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), with some cells exhibiting slower migration and others migrating faster. However, the cause of this phenomenon remains elusive. Our research rules out alternative splicing as the cause and, instead, identifies the presence of a P124A mutation in SRP14 (SRP14 P124A) among the faster-migrating variants, while the slower-migrating variants lack this mutation. Subsequent ectopic expression of wild-type SRP14 P124 or SRP14 WT and SRP14 P124A in various cell lines confirms that the P124A mutation indeed leads to faster migration of SRP14. Further mutagenesis analysis shows that the P117A and A121P mutations within the alanine-rich domain at the C-terminus of SRP14 are responsible for migration alterations on SDS-PAGE, whereas mutations outside this domain, such as P39A, Y27F, and T45A, have no such effect. Furthermore, the ectopic expression of SRP14 WT and SRP14 P124A yields similar outcomes in terms of SRP RNA stability, cell morphology, and cell growth, indicating that SRP14 P124A represents a natural variant of SRP14 and retains comparable functionality. In conclusion, the substitution of proline for alanine in the alanine-rich tail of SRP14 results in faster migration on SDS-PAGE, but has little effect on its function.

Characteristics of fluopicolide-resistance mutants in Phytophthora nicotianae, the pathogen causing black shank disease in tobacco.

Black shank, a devastating disease in tobacco production worldwide, is caused by the oomycete plant pathogen Phytophthora nicotianae. Fluopicolide is a pyridinylmethyl-benzamides fungicide with a unique mechanism of action and has been widely used for controlling a variety of oomycetes such as Plasmopara viticola, Phytophthora infestans, Pseudoperonospora cubensis, P. nicotianae and Bremia lactucae. However, the fluopicolide-resistance risk and molecular basis in P. nicotianae have not been reported. In this study, the sensitivity profile of 141 P. nicotianae strains to fluopicolide was determined, with a mean median effective concentration (EC50) value of 0.12 ± 0.06µg/mL. Five stable fluopicolide-resistant mutants of P. nicotianae were obtained by fungicide adaptation, and the compound fitness index of these resistant mutants were lower than that of their parental isolates. Additionally, cross-resistance tests indicated that the sensitivity of fluopicolide did not correlate with other oomycete fungicides, apart from fluopimomide. DNA sequencing revealed two point mutations, G765E and N769Y, in the PpVHA-a protein in the fluopicolide-resistant mutants. Transformation and expression of PpVHA-a genes carrying G765E and N769Y in the sensitive wild-type isolate confirmed that it was responsible for fluopicolide resistance. These results suggest that P. nicotianae has a low to medium resistance risk to fluopicolide in laboratory and that point mutations, G765E and N769Y, in PpVHA-a are associated with the observed fluopicolide resistance.

Sensitivity analysis and point mutations in BcSDHB confer cyclobutrifluram resistance in Botrytis cinerea from China.

Botrytis cinerea is one of the most destructive pathogens worldwide. It can damage over 200 crops, resulting in significant yield and quality losses. Cyclobutrifluram, a new generation of succinate dehydrogenase inhibitors, exhibits excellent inhibitory activity against B. cinerea. However, the baseline sensitivity and resistance of B. cinerea to cyclobutrifluram remains poorly understood. This study was designed to monitor the sensitivity frequency distribution, assess the resistance risk, and clarify the resistance mechanism of B. cinerea to cyclobutrifluram. The baseline sensitivity of B. cinerea isolates to cyclobutrifluram was 0.89 µg/mL. Cyclobutrifluram-resistant B. cinerea populations are present in the field. Six resistant B. cinerea isolates investigated in this study possessed enhanced compound fitness index compared to the sensitive isolates according to mycelial growth, mycelial dry weight, conidiation, conidial germination rate, and pathogenicity. Cyclobutrifluram exhibited no cross-resistance with tebuconazole, fludioxonil, cyprodinil, or iprodione. Sequence alignment revealed that BcSDHB from cyclobutrifluram-resistant B. cinerea isolates had three single substitutions (P225F, N230I, or H272R). Molecular docking verified that these mutations in BcSDHB conferred cyclobutrifluram resistance in B. cinerea. In conclusion, the resistance risk of B. cinerea to cyclobutrifluram is high, and the point mutations in BcSDHB (P225F, N230I, or H272R) confer cyclobutrifluram resistance in B. cinerea. This study provided important insights into cyclobutrifluram resistance in B. cinerea and offered valuable information for monitoring and managing cyclobutrifluram resistance in the future.

Resistance risk, resistance mechanism and the effect on DON production of a new SDHI fungicide cyclobutrifluram in Fusarium graminearum.

Fusarium head blight in wheat is caused by Fusarium graminearum, resulting in significant yield losses and grain contamination with deoxynivalenol (DON), which poses a potential threat to animal health. Cyclobutrifluram, a newly developed succinate dehydrogenase inhibitor, has shown excellent inhibition of Fusarium spp. However, the resistance risk of F. graminearum to cyclobutrifluram and the molecular mechanism of resistance have not been determined. In this study, we established the average EC50 of a range of F. graminearum isolates to cyclobutrifluram to be 0.0110 µg/mL. Six cyclobutrifluram-resistant mutants were obtained using fungicide adaptation. All mutants exhibited impaired fitness relative to their parental isolates. This was evident from measurements of mycelial growth, conidiation, conidial germination, virulence, and DON production. Interestingly, cyclobutrifluram did not seem to affect the DON production of either the sensitive isolates or the resistant mutants. Furthermore, a positive cross-resistance was observed between cyclobutrifluram and pydiflumetofen. These findings suggest that F. graminearum carries a moderate to high risk of developing resistance to cyclobutrifluram. Additionally, point mutations H248Y in FgSdhB and A73V in FgSdhC1 of F. graminearum were observed in the cyclobutrifluram-resistant mutants. Finally, an overexpression transformation assay and molecular docking indicated that FgSdhBH248Y or FgSdhC1A73V could confer resistance of F. graminearum to cyclobutrifluram.

Versatile generation of precise gene edits in bovines using SEGCPN.

BACKGROUND: Gene knockout and knock-in have been widely performed in large farm animals based on genome editing systems. However, many types of precise gene editing, including targeted deletion, gene tagging, and large gene fragment replacement, remain a challenge in large farm animals. RESULTS: Here, we established versatile self-excising gene-targeting technology in combination with programmable nucleases (SEGCPN) to efficiently generate various types of precise gene editing in bovine. First, we used this versatile method to successfully generate bovine embryos with point mutations and 11-bp deletions at the MSTN locus. Second, we successfully generated bulls with EGFP labeling at the SRY locus. Finally, we successfully generated humanized cows in which the endogenous 18-kb α-casein gene was replaced with a 2.6-kb human α-lactalbumin gene. CONCLUSIONS: In summary, our new SEGCPN method offers unlimited possibilities for various types of precise gene editing in large animals for application both in agriculture and disease models.

Molecular Mechanisms and Biological Characteristics of Botrytis cinerea Field Isolate Resistance to Pyrisoxazole in Liaoning Province.

Botrytis cinerea is a broad-host-range necrotrophic phytopathogen responsible for serious diseases in leading crops worldwide. The novel sterol 14α-demethylase inhibitor (DMI) pyrisoxazole was recently registered for the control of tomato gray mold caused by B. cinerea in China. One hundred fifty-seven isolates of B. cinerea were collected from tomato greenhouses in 14 cities of Liaoning Province from 2016 to 2021 and examined for sensitivity to pyrisoxazole, with a mean EC50 value of 0.151 µg/ml. Three highly resistant isolates, XD-5, DG-4, and GQ-3, were screened, and the EC50 values were 0.734, 0.606, and 0.639 µg/ml with corresponding resistance factors of 12.88, 10.63, and 11.21, respectively. Compared with field-sensitive strains, the highly resistant isolate XD-5 exhibited fitness defects in traits, including mycelial growth, conidial production, and pathogenicity, but DG-4 and GQ-3 did not experience fitness costs. Positive cross-resistance was observed only between pyrisoxazole and the DMIs tebuconazole and prochloraz but not between pyrisoxazole and the non-DMIs iprodione, procymidone, pyrimethanil, fludioxonil, fluazinam, and fluopyram. Sequence alignment of the CYP51 gene indicated that three point mutations were observed in the highly resistant mutant, namely, V24I in XD-5, G461S in GQ-3, and R464K in DG-4. When exposed to pyrisoxazole, the induced expression levels of the ABC transporter AtrD and MFS transporter Mfs1 increased in the resistant isolates compared with those in the sensitive isolates, whereas the expression level of the CYP51 gene did not change significantly. Molecular docking suggested that the G461S and R464K mutations both led to a decrease in the binding energy between CYP51 and pyrisoxazole, whereas no change was found with the V24I mutation. Thus, two point mutations in the CYP51 protein combined with induced expression of the Mfs1 and AtrD genes appeared to mediate the pyrisoxazole resistance of the highly resistant mutants DG-4 and GQ-3, while the overexpression of the Mfs1 and AtrD genes was responsible for the highly resistant mutant XD-5.

Revealing the Mechanism of NLRP3 Inflammatory Pathway Activation through K+ Efflux Induced by PLO via Signal Point Mutations.

Trueperella pyogenes is an important opportunistic pathogenic bacterium widely distributed in the environment. Pyolysin (PLO) is a primary virulence factor of T. pyogenes and capable of lysing many different cells. PLO is a member of the cholesterol-dependent cytolysin (CDC) family of which the primary structure only presents a low level of homology with other members from 31% to 45%. By deeply studying PLO, we can understand the overall pathogenic mechanism of CDC family proteins. This study established a mouse muscle tissue model infected with recombinant PLO (rPLO) and its single-point mutations, rPLO N139K and rPLO F240A, and explored its mechanism of causing inflammatory damage. The inflammatory injury abilities of rPLO N139K and rPLO F240A are significantly reduced compared to rPLO. This study elaborated on the inflammatory mechanism of PLO by examining its unit point mutations in detail. Our data also provide a theoretical basis and practical significance for future research on toxins and bacteria.

Pygo-F773W Mutation Reveals Novel Functions beyond Wnt Signaling in Drosophila.

Pygopus (Pygo) has been identified as a specific nuclear co-activator of the canonical Wingless (Wg)/Wnt signaling pathway in Drosophila melanogaster. Pygo proteins consist of two conserved domains: an N-terminal homologous domain (NHD) and a C-terminal plant homologous domain (PHD). The PHD's ability to bind to di- and trimethylated lysine 4 of histone H3 (H3K4me2/3) appears to be independent of Wnt signaling. There is ongoing debate regarding the significance of Pygo's histone-binding capacity. Drosophila Pygo orthologs have a tryptophan (W) > phenylalanine (F) substitution in their histone pocket-divider compared to vertebrates, leading to reduced histone affinity. In this research, we utilized CRISPR/Cas9 technology to introduce the Pygo-F773W point mutation in Drosophila, successfully establishing a viable homozygous Pygo mutant line for the first time. Adult mutant flies displayed noticeable abnormalities in reproduction, locomotion, heart function, and lifespan. RNA-seq and cluster analysis indicated that the mutation primarily affected pathways related to immunity, metabolism, and posttranslational modification in adult flies rather than the Wnt signaling pathway. Additionally, a reduction in H3K9 acetylation levels during the embryonic stage was observed in the mutant strains. These findings support the notion that Pygo plays a wider role in chromatin remodeling, with its involvement in Wnt signaling representing only a specific aspect of its chromatin-related functions.