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Adrenoleukodystrophy is a rare neurogenetic disease, and adrenomyeloneuropathy is the most common phenotype in adults. The clinical data of a patient with adrenoleukodystrophy and spinal-peripheral neuropathy caused by a novel point mutation in exon 4 of the ABCD1 gene (c.1256T > G (p.Val419Gly)) were retrospectively analyzed. Furthermore, we constructed wild-type and mutant vectors of the ABCD1 (NM0000334) gene to validate the effect of this mutation on the expression of the ABCD1 gene and protein and to explore the mechanism of X-linked adrenoleukodystrophy occurrence and development to identify therapeutic targets for clinical treatment. [ABSTRACT FROM AUTHOR]

Background: The issue of Helicobacter pylori (H. pylori) resistance to clarithromycin (CLR) has consistently posed challenges for clinical treatment. Hence, a rapid susceptibility testing (AST) method urgently needs to be developed. Methods: In the present study, 35 isolates of H. pylori were isolated from 203 gastritis patients of the Guangzhou cohort, and the antimicrobial resistance phenotypes were associated with their genomes to analyze the relevant mutations. Based on these mutations, a rapid detection system utilizing high-resolution melting (HRM) curve analysis was designed and verified by the Shenzhen cohort, which consisted of 38 H. pylori strains. Results: Genomic analysis identified the mutation of the 2143 allele from A to G (A2143G) of 23S rRNA as the most relevant mutation with CLR resistance (p < 0.01). In the HRM system, the wild-type H. pylori showed a melting temperature (Tm) of 79.28 ± 0.01 °C, while the mutant type exhibited a Tm of 79.96 ± 0.01 °C. These differences enabled a rapid distinction between two types of H. pylori (p < 0.01). Verification examinations showed that this system could detect target DNA as low as 0.005 ng/μL in samples without being affected by other gastric microorganisms. The method also showed a good performance in the Shenzhen validation cohort, with 81.58% accuracy, and 100% specificity. Conclusions: We have developed an HRM system that can accurately and quickly detect CLR resistance in H. pylori. This method can be directly used for the detection of gastric microbiota samples and provides a new benchmark for the simple detection of H. pylori resistance. [ABSTRACT FROM AUTHOR]

Chimeric mouse models have recently been developed to study human microglia in vivo. However, widespread engraftment of donor microglia within the adult brain has been challenging. Here, we present a protocol to introduce the G795A point mutation using CRISPR-Cas9 into the CSF1R locus of human pluripotent stem cells. We also describe an optimized microglial differentiation technique for transplantation into newborn or adult recipients. We then detail pharmacological paradigms to achieve widespread and near-complete engraftment of human microglia. For complete details on the use and execution of this protocol, please refer to Chadarevian et al. (2023).1.

Abstract 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.

Abstract 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.

SUMMARY: 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. [ABSTRACT FROM AUTHOR]

This research cloned and expressed the sugar transporter gene KM_SUT5 from Kluyveromyces marxianus GX-UN120, which displayed remarkable sugar transportation capabilities, including pentose sugars. To investigate the impact of point mutations on xylose transport capacity, we selected four sites, predicted the suitable amino acid sites by molecular docking, and altered their codons to construct the corresponding mutants, Q74D, Y195K, S460H, and Q464F, respectively. Furthermore, we conducted site-directed truncation on six sites of KM_SUT5p. The molecular modification resulted in significant changes in mutant growth and the D-xylose transport rate. Specifically, the S460H mutant exhibited a higher growth rate and demonstrated excellent performance across 20 g L−1 xylose, achieving the highest xylose accumulation under xylose conditions (49.94 μmol h−1 gDCW-1, DCW mean dry cell weight). Notably, mutant delA554-, in which the transporter protein SUT5 is truncated at position delA554-, significantly increased growth rates in both D-xylose and D-glucose substrates. These findings offer valuable insights into potential modifications of other sugar transporters and contribute to a deeper understanding of the C-terminal function of sugar transporters. [ABSTRACT FROM AUTHOR]

BACKGROUND: Sclerotium rolfsii is a destructive soil‐borne fungal pathogen which is distributed worldwide. In previous study, the succinate dehydrogenase inhibitor (SDHI) fungicide benzovindiflupyr has been identified for its great antifungal activity against Sclerotium rolfsii. This study is aimed to investigate the resistance risk and mechanism of benzovindiflupyr in Sclerotium rolfsii. RESULTS: Eight stable benzovindiflupyr‐resistant isolates were generated by fungicide adaptation. Although the obtained eight resistant isolates have a stronger pathogenicity than the parental sensitive isolate, they have a fitness penalty in the mycelial growth and sclerotia formation compared to the parental isolate. A positive cross‐resistance existed in the resistant isolates between benzovindiflupyr and thifluzamide, carboxin, boscalid and isopyrazam. Three‐point mutations, including SdhBN180D, SdhCQ68E and SdhDH103Y, were identified in the benzovindiflupyr‐resistant isolates. However, molecular docking analysis indicated that only SdhDH103Y could influence the sensitivity of Sclerotium rolfsii to benzovindiflupyr. After mycelial co‐incubation of resistant isolates and the sensitive isolate, resistance genes may be transmitted to the sensitive isolate. The in vivo efficacy of benzovindiflupyr and thifluzamide against benzovindiflupyr‐resistant isolates was a little lower than that against the sensitive isolate but with no significant difference. CONCLUSION: The results suggested a low to medium resistance risk of Sclerotium rolfsii to benzovindiflupyr. However, once resistance occurs, it is possible to spread in the population of Sclerotium rolfsii. This study is helpful to understanding the risk and mechanism of resistance to benzovindiflupyr in multinucleate pathogens such as Sclerotium rolfsii. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

The CRISPR system, as an effective genome editing technology, has been extensively utilized for the construction of disease models in human pluripotent stem cells. Establishment of a gene mutant or knockout stem cell line typically relies on Cas nuclease-generated double-stranded DNA breaks and exogenous templates, which can produce uncontrollable editing byproducts and toxicity. The recently developed adenine base editors (ABE) have greatly facilitated related research by introducing A/T > G/C mutations in the coding regions or splitting sites (AG-GT) of genes, enabling mutant gene knock-in or knock-out without introducing DNA breaks. In this study, we edit the AG bases in exons anterior to achieve gene knockout via the ABE8e-SpRY, which recognizes most expanded protospacer adjacent motif to target the genome. Except for gene-knockout, ABE8e-SpRY can also efficiently establish disease-related A/T-to-G/C variation cell lines by targeting coding sequences. The method we generated is simple and time-saving, and it only takes two weeks to obtain the desired cell line. This protocol provides operating instructions step-by-step for constructing knockout and point mutation cell lines. [ABSTRACT FROM AUTHOR]

β-Carotene is an attractive compound and that its biotechnological production can be achieved by using engineered Saccharomyces cerevisiae. In a previous study, we developed a technique for the efficient establishment of diverse mutants through the introduction of point and structural mutations into the yeast genome. In this study, we aimed to improve β-carotene production by applying this mutagenesis technique to S. cerevisiae strain that had been genetically engineered for β-carotene production. Point and structural mutations were introduced into β-carotene-producing engineered yeast. The resulting mutants showed higher β-carotene production capacity than the parental strain. The top-performing mutant, HP100_74, produced 37.6 mg/L of β-carotene, a value 1.9 times higher than that of the parental strain (20.1 mg/L). Gene expression analysis confirmed an increased expression of multiple genes in the glycolysis, mevalonate, and β-carotene synthesis pathways. In contrast, expression of ERG9, which functions in the ergosterol pathway competing with β-carotene production, was decreased in the mutant strain. The introduction of point and structural mutations represents a simple yet effective method for achieving mutagenesis in yeasts. This technique is expected to be widely applied in the future to produce chemicals via metabolic engineering of S. cerevisiae. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Porcine reproductive and respiratory syndrome (PRRS) is a globally prevalent contagious disease caused by the positive-strand RNA PRRS virus (PRRSV), resulting in substantial economic losses in the swine industry. Modifying the CD163 SRCR5 domain, either through deletion or substitution, can eff1ectively confer resistance to PRRSV infection in pigs. However, large fragment modifications in pigs inevitably raise concerns about potential adverse effects on growth performance. Reducing the impact of genetic modifications on normal physiological functions is a promising direction for developing PRRSV-resistant pigs. In the current study, we identified a specific functional amino acid in CD163 that influences PRRSV proliferation. Viral infection experiments conducted on Marc145 and PK-15CD163 cells illustrated that the mE535G or corresponding pE529G mutations markedly inhibited highly pathogenic PRRSV (HP-PRRSV) proliferation by preventing viral binding and entry. Furthermore, individual viral challenge tests revealed that pigs with the E529G mutation had viral loads two orders of magnitude lower than wild-type (WT) pigs, confirming effective resistance to HP-PRRSV. Examination of the physiological indicators and scavenger function of CD163 verified no significant differences between the WT and E529G pigs. These findings suggest that E529G pigs can be used for breeding PRRSV-resistant pigs, providing novel insights into controlling future PRRSV outbreaks. [ABSTRACT FROM AUTHOR]

Acetate esters comprise a wide range of products including fragrances and industrial solvents. Biosynthesis of esters offers a promising alternative to chemical synthesis because such routes use renewable carbohydrate resources and minimize the generation of waste. One biochemical method for ester formation relies on the ATF1 gene from Saccharomyces cerevisiae, which encodes alcohol-O-acyltransferase (AAT) which converts acetyl-CoA and an exogenously supplied alcohol into the ester. In this study, the formation of several acetate esters via AAT was examined in Escherichia coli chromosomally expressing citrate synthase variants, which create a metabolic bottleneck at acetyl-CoA. In shake flask cultures, variant strains generated more acetate esters than the strains expressing the wild-type citrate synthase. In a controlled bioreactor, E. coli GltA[A267T] generated 3.9 g propyl acetate in 13 h, corresponding to a yield of 0.155 g propyl acetate/g glucose, which is 18% greater than that obtained by the wild-type GltA control. These results demonstrate the ability of citrate synthase variants to redistribute carbon from central metabolism into acetyl-CoA-derived biochemicals. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Objectives To determine the frequencies and clonal distributions of putative genetic determinants of resistance to antimicrobials applied for treatment of Clostridioides difficile infection (CDI), as documented in the genomic record. Methods We scanned 26 557 C. difficile genome sequences publicly available from the EnteroBase platform for plasmids, point mutations and gene truncations previously reported to reduce susceptibility to vancomycin, fidaxomicin or metronidazole, respectively. We measured the antimicrobial susceptibility of 143 selected C. difficile isolates. Results The frequency of mutations causing reduced susceptibility to vancomycin and metronidazole, respectively, increased strongly after 2000, peaking at up to 52% of all sequenced C. difficile genomes. However, both mutations declined sharply more recently, reflecting major changes in CDI epidemiology. We detected mutations associated with fidaxomicin resistance in several major genotypes, but found no evidence of international spread of resistant clones. The pCD-METRO plasmid, conferring metronidazole resistance, was detected in a single previously unreported C. difficile isolate, recovered from a hospital patient in Germany in 2008. The pX18-498 plasmid, putatively associated with decreased vancomycin susceptibility, was confined to related, recent isolates from the USA. Phenotype measurements confirmed that most of those genetic features were useful predictors of antibiotic susceptibility, even though ranges of MICs typically overlapped among isolates with and without specific mutations. Conclusions Genomic data suggested that resistance to therapeutic antimicrobial drugs is rare in C. difficile. Public antimicrobial resistance marker databases were not equipped to detect most of the genetic determinants relevant to antibiotic therapy of CDI. [ABSTRACT FROM AUTHOR]

BACKGROUND: Bacterial leaf blight caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most serious diseases of rice, and there is a lack of bactericides for controlling this disease. We previously found parthenolide (PTL) is a potential lead for developing bactericides against Xoo, and subunit F of respiratory chain complex I (NuoF) is an important target protein of PTL. However, the binding modes of PTL with NuoF need further elucidation. RESULTS: In this study, we obtained the crystal structure of Xoo NuoEF (complex of subunit E and F of respiratory chain complex I) with a resolution of 2.36 Å, which is the first report on the protein structure of NuoEF in plant‐pathogenic bacteria. The possible binding sites of PTL with NuoF (Cys105 and Cys187) were predicted with molecular docking and mutated into alanine using a base mismatch method. The mutated proteins were expressed in Escherichia coli and purified with affinity chromatography. The binding abilities of PTL with mutated proteins were investigated via pull‐down assay and BIAcore analysis, which revealed that double mutation of Cys105 and Cys187 in NuoF severely affected the binding ability of PTL with NuoF. In addition, the binding modes were further simulated with combined quantum mechanical/molecular mechanical calculations, and the results indicated that PTL may have a stronger binding with Cys105 than Cys187. CONCLUSION: NuoEF protein structure of Xoo was resolved, and Cys105 and Cys187 in NuoF are important binding sites of PTL. This study further clarified the action mechanism of PTL against Xoo, and will promote the innovation of bactericides targeting Xoo complex I. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

FAM86A is a class I lysine methyltransferase (KMT) that generates trimethylation on the eukaryotic translation elongation factor 2 (EEF2) at Lys525. Publicly available data from The Cancer Dependency Map project indicate high dependence of hundreds of human cancer cell lines on FAM86A expression. This classifies FAM86A among numerous other KMTs as potential targets for future anticancer therapies. However, selective inhibition of KMTs by small molecules can be challenging due to high conservation within the S-adenosyl methionine (SAM) cofactor binding domain among KMT subfamilies. Therefore, understanding the unique interactions within each KMT-substrate pair can facilitate developing highly specific inhibitors. The FAM86A gene encodes an N-terminal FAM86 domain of unknown function in addition to its C-terminal methyltransferase domain. Here, we used a combination of X-ray crystallography, the AlphaFold algorithms, and experimental biochemistry to identify an essential role of the FAM86 domain in mediating EEF2 methylation by FAM86A. To facilitate our studies, we also generated a selective EEF2K525 methyl antibody. Overall, this is the first report of a biological function for the FAM86 structural domain in any species and an example of a noncatalytic domain participating in protein lysine methylation. The interaction between the FAM86 domain and EEF2 provides a new strategy for developing a specific FAM86A small molecule inhibitor, and our results provide an example in which modeling a protein-protein interaction with AlphaFold expedites experimental biology.

Single-nucleotide variants (SNVs) are the most common type variation of sequence alterations at a specific location in the genome, thus involving significant clinical and biological information. The assay of SNVs has engaged great awareness, because many genome-wide association studies demonstrated that SNVs are highly associated with serious human diseases. Moreover, the investigation of SNV expression levels in single cells are capable of visualizing genetic information and revealing the complexity and heterogeneity of single-nucleotide mutation-related diseases. Thus, developing SNV assay approaches in vitro , particularly in single cells, is becoming increasingly in demand. In this review, we summarized recent progress in the enzyme-free and enzyme-mediated strategies enabling SNV assay transition from sensing interface to the test tube and single cells, which will potentially delve deeper into the knowledge of SNV functions and disease associations, as well as discovering new pathways to diagnose and treat diseases based on individual genetic profiles. The leap of SNV assay achievements will motivate observation and measurement genetic variations in single cells, even within living organisms, delve into the knowledge of SNV functions and disease associations, as well as open up entirely new avenues in the diagnosis and treatment of diseases based on individual genetic profiles. [ABSTRACT FROM AUTHOR]

Manganese superoxide dismutase (MnSOD) is an antioxidant that exists in mitochondria and can effectively remove superoxide anions in mitochondria. In a dark, high-pressure, and low-temperature deep-sea environment, MnSOD is essential for the survival of sea cucumbers. Six MnSODs were identified from the transcriptomes of deep and shallow-sea sea cucumbers. To explore their environmental adaptation mechanism, we conducted environmental selection pressure analysis through the branching site model of PAML software. We obtained night positive selection sites, and two of them were significant (97F→H, 134K→V): 97F→H located in a highly conservative characteristic sequence, and its polarity change might have a great impact on the function of MnSOD; 134K→V had a change in piezophilic ability, which might help MnSOD adapt to the environment of high hydrostatic pressure in the deepsea. To further study the effect of these two positive selection sites on MnSOD, we predicted the point mutations of F97H and K134V on shallow-sea sea cucumber by using MAESTROweb and PyMOL. Results show that 97F→H, 134K→V might improve MnSOD's efficiency of scavenging superoxide anion and its ability to resist high hydrostatic pressure by moderately reducing its stability. The above results indicated that MnSODs of deep-sea sea cucumber adapted to deep-sea environments through their amino acid changes in polarity, piezophilic behavior, and local stability. This study revealed the correlation between MnSOD and extreme environment, and will help improve our understanding of the organism's adaptation mechanisms in deep sea. [ABSTRACT FROM AUTHOR]

Rotavirus A (RVA) is the leading cause of diarrhea requiring hospitalization in children and causes over 100,000 annual deaths in Sub-Saharan Africa. In order to generate next-generation vaccines against African RVA genotypes, a reverse genetics system based on a simian rotavirus strain was utilized here to exchange the antigenic capsid proteins VP4, VP7 and VP6 with those of African human rotavirus field strains. One VP4/VP7/VP6 (genotypes G9-P[6]-I2) triple-reassortant was successfully rescued, but it replicated poorly in the first cell culture passages. However, the viral titer was enhanced upon further passaging. Whole genome sequencing of the passaged virus revealed a single point mutation (A797G), resulting in an amino acid exchange (E263G) in VP4. After introducing this mutation into the VP4-encoding plasmid, a VP4 mono-reassortant as well as the VP4/VP7/VP6 triple-reassortant replicated to high titers already in the first cell culture passage. However, the introduction of the same mutation into the VP4 of other human RVA strains did not improve the rescue of those reassortants, indicating strain specificity. The results show that specific point mutations in VP4 can substantially improve the rescue and replication of recombinant RVA reassortants in cell culture, which may be useful for the development of novel vaccine strains. [ABSTRACT FROM AUTHOR]

Background: Upon the emergence of the Eris variant in our country, we aimed to develop an RT-qPCR kit to detect the SARS-CoV-2 Eris variant. Methods: By studying the genome sequences uploaded to GISAID, target regions were designed by focusing on the mutation regions of EG.5 and EG.5.1, which are the main lineage of the Eris variant. When developing the kit, the hydrolysis probe-based detection (e.g., TaqMan®) method was chosen. Target sequences specific to the SARSCoV-2 EG.5 variant were then specifically amplified, with amplification monitored in real time using fluorescent labeled probes. In the study, 470 samples were used, 109 of which were positive for SARS-CoV-2 RNA, from various Hospitals. Results: Of the 109 samples that were positive for SARS-CoV-2 RNA, 67 (61%) were also detected positive for Eris variant RNA. Conclusions: It was determined that the developed kit detected the Eris variant and the rate was 61%. [ABSTRACT FROM AUTHOR]

Simple Summary: One of the most destructive pests of cruciferous plants that quickly develops resistance to most pesticide groups is Plutella xylostella. Investigating the resistant strain of P. xylostella revealed the molecular mechanisms underlying resistance to chlorpyrifos, concentrating specifically on the ace1 gene. The sequencing results revealed amino acid substitutions in ace1 of the resistant strain. The structures of the wild-type and mutant ace1 strains were compared via molecular dynamics (MDs) simulations and docking investigations. The results showed that the mutant ace1 has different substrate entry points and structural modifications that affect the enzyme inhibitor affinity. Significant differences in ace1 gene expression between the mutant and wild-type strains were revealed by real-time quantitative PCR, which raises the possibility of a relationship between ace1 mutations and changes in mRNA transcription levels. Plutella xylostella, a destructive crucifer pest, can rapidly develop resistance to most classes of pesticides. This study investigated the molecular resistance mechanisms to chlorpyrifos, an organophosphate pesticide. Two P. xylostella genes, ace1 and ace2, were described. The nucleotide sequence results revealed no variation in ace2, while the resistant strain (Kar-R) had four amino acid alterations in ace1, two of which (A298S and G324A) were previously shown to confer organophosphate resistance in P. xylostella. In the present study, the 3D model structures of both the wild-type (Gu-S) and mutant (Kar-R) of P. xylostella ace1 strains were studied through molecular dynamics (MDs) simulations and molecular docking. Molecular dynamics simulations of RMSD revealed less structural deviation in the ace1 mutant than in its wild-type counterpart. Higher flexibility in the 425–440 amino acid region in the mutant active site (Glu422 and Acyl pocket) increased the active site's entropy, reducing the enzyme's affinity for the inhibitors. Gene expression analysis revealed that the relative transcription levels of ace1 were significantly different in the Kar-R strain compared with the Gu-S strain. This study enhances the understanding of the mechanisms governing ace1′s resistance to insecticide and provides essential insights for new insecticides as well as valuable insights into environmentally conscious pest management techniques. [ABSTRACT FROM AUTHOR]

Ethyl methanesulphonate (EMS), is a widely used chemical mutagen that causes high‐frequency germline null mutation by inserting an alkyl group into the nucleotide guanine in eukaryotic cells. The effect of EMS on the dynamics of the aneuploid genome, increased cellular instability, and carcinogenicity in relation to benign and malignant tumors are reported, but the molecular level understanding of morphological changes of higher‐order chromatin structure has poorly been understood. This is due to a lack of sufficient resolution in conventional microscopic techniques to see small structures below the diffraction limit. Here, using super‐resolution radial fluctuation, a largely fragmented, decompaction, and less dense heterochromatin structure upon EMS treatment to HEK 293A cells without any change in nuclear DNA domains is observed. This result suggests an early stage of carcinogenicity happened due to the point mutation. In addition, the distinct structural changes with an elongated morphology of lysosomes are also observed. On the other hand, fragmented and increased heterogeneous populations with an increased cytoplasmic occupancy of mitochondria are observed. [ABSTRACT FROM AUTHOR]

Background: The recently identified PROS1 mutation Protein S Erlangen c.1904T>C, resulting in amino acid exchange F635S, is associated with severe quantitative protein S (PS) deficiency and clinical thrombosis. It was hypothesized that this deficiency is due to a secretion defect [1]. This report aims to further elucidate the potential secretion defect of PS Erlangen. Methods: Coding sequences (CDS) of wild type (WT) PROS1 (encoding PS) and mutated PROS1c.1904T>C (encoding PSF635S) were cloned in front of the CDS of green fluorescent protein (GFP), and the respective plasmids were introduced into HEK293T cells. PROS1-GFP and PROS1c.1904T>C-GFP expressing HEK293T cell lines were analyzed by confocal laser scanning microscopy and western blot for cellular proteins and proteins secreted to the growth medium. Results: Western blot analysis revealed a significantly reduced secretion of PSF635S compared to WT PS. This observation was confirmed by the detection of mutant PSF635S-GFP fusion exclusively in the endoplasmic reticulum (ER), while PS-GFP passed through the entire secretory pathway, as indicated by the localization within both the ER and Golgi apparatus. Conclusions: The Protein S Erlangen mutation results in type I PS deficiency caused by a secretion defect. [ABSTRACT FROM AUTHOR]

The RET receptor tyrosine kinase plays a pivotal role in cell survival, proliferation, and differentiation, and its abnormal activation leads to cancers through receptor fusions or point mutations. Mutations that disrupt the disulfide network in the extracellular domain (ECD) of RET drive multiple endocrine neoplasia type 2A (MEN2A), a hereditary syndrome associated with the development of thyroid cancers. However, structural details of how specific mutations affect RET are unclear. Here, we present the first structural insights into the ECD of the RET(C634R) mutant, the most common mutation in MEN2A. Using electron microscopy, we demonstrate that the C634R mutation causes ligand-independent dimerization of the RET ECD, revealing an unusual tail-to-tail conformation that is distinct from the ligand-induced signaling dimer of WT RET. Additionally, we show that the RETC634R ECD dimer can form complexes with at least two of the canonical RET ligands and that these complexes form very different structures than WT RET ECD upon ligand binding. In conclusion, this structural analysis of cysteine-mutant RET ECD suggests a potential key mechanism of cancer induction in MEN2A, both in the absence and presence of its native ligands, and may offer new targets for therapeutic intervention.

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 (SRP14P124A) among the faster-migrating variants, while the slower-migrating variants lack this mutation. Subsequent ectopic expression of wild-type SRP14P124 or SRP14WT and SRP14P124A 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 SRP14WT and SRP14P124A yields similar outcomes in terms of SRP RNA stability, cell morphology, and cell growth, indicating that SRP14P124A 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.

Certain amino acids changes in the human Na+/K+-ATPase pump, ATPase Na+/K+ transporting subunit alpha 1 (ATP1A1), cause Charcot-Marie-Tooth disease type 2 (CMT2) disease and refractory seizures. To develop in vivo models to study the role of Na+/K+-ATPase in these diseases, we modified the Drosophila gene homolog, Atpα, to mimic the human ATP1A1 gene mutations that cause CMT2. Mutations located within the helical linker region of human ATP1A1 (I592T, A597T, P600T, and D601F) were simultaneously introduced into endogenous Drosophila Atpα by CRISPR/Cas9-mediated genome editing, generating the AtpαTTTF model. In addition, the same strategy was used to generate the corresponding single point mutations in flies (AtpαI571T, AtpαA576T, AtpαP579T, and AtpαD580F). Moreover, a deletion mutation (Atpαmut) that causes premature termination of translation was generated as a positive control. Of these alleles, we found two that could be maintained as homozygotes (AtpαI571T and AtpαP579T). Three alleles (AtpαA576T, AtpαP579 and AtpαD580F) can form heterozygotes with the Atpαmut allele. We found that the Atpα allele carrying these CMT2-associated mutations showed differential phenotypes in Drosophila. Flies heterozygous for AtpαTTTF mutations have motor performance defects, a reduced lifespan, seizures, and an abnormal neuronal morphology. These Drosophila models will provide a new platform for studying the function and regulation of the sodium-potassium pump.

Abstract Background Since 2014, dengue epidemics have occurred almost annually in Nouakchott, the capital city of Mauritania, coinciding with the recent establishment of Aedes aegypti, the primary vector of dengue, in the city. Anopheles arabiensis, the primary vector of malaria, is also abundant not only in Nouakchott but also in most areas of the country. Resistance to insecticides has been studied in An. arabiensis but not in Ae. aegypti in Mauritania. The objective of the present study was to establish the baseline data on the frequencies of knockdown resistance (kdr) mutations in the voltage-gated sodium channel (vgsc) gene in Ae. aegypti collected in Nouakchott to improve vector control. Methods Resting Ae. aegypti mosquitoes were collected in 2017 and 2018 in Teyarett and Dar Naim districts in Nouakchott using a battery-powered aspirator. Polymerase chain reaction (PCR) and DNA sequencing were performed to detect the presence of five kdr mutations known to be associated with pyrethroid resistance: L982W, S989P, I1011M/G, V1016G/I, and F1534C. Results A total of 100 female Ae. aegypti mosquitoes were identified among collected resting culicid fauna, of which 60% (60/100) were unfed, 12% (12/100) freshly blood-fed, and 28% (28/100) gravid. Among the mutations investigated in this study, 989P, 1016G, and 1534C were found to be widespread, with the frequencies of 0.43, 0.44, and 0.55, respectively. Mutations were not found in codons 982 and 1011. No other mutations were detected within the fragments analyzed in this study. Genotype distribution did not deviate from Hardy–Weinberg equilibrium. The most frequent co-occurring point mutation patterns among Ae. aegypti mosquitoes were the heterozygous individuals 989SP/1016VG/1534FC detected in 45.1% of mosquitoes. In addition, homozygous mutant 1534CC co-occurred simultaneously with homozygous wild type 989SS and 1016VV in 30.5% of mosquito specimens. Inversely, homozygous wild-type 1534FF co-occurred simultaneously with homozygous mutant 989PP and 1016GG in 19.5% of the mosquitoes. Conclusions To our knowledge, this is the first study reporting the presence of three point mutations in the vgsc gene of Ae. aegypti in Mauritania. The findings of the present study are alarming because they predict a high level of resistance to pyrethroid insecticides which are commonly used in vector control in the country. Therefore, further studies are urgently needed, in particular phenotypic characterization of insecticide resistance using the standardized test. Graphical Abstract

Anthracnose, caused by Colletotrichum truncatum and C. gloeosporioides, is amongst the most serious diseases of soybean in China. Picoxystrobin, a quinone outside inhibitor fungicide, is commonly used for the control of anthracnose. Its resistance risk and mechanism in C. truncatum and C. gloeosporioides are unclear. In this study, the sensitivities of 128 C. truncatum and 121 C. gloeosporioides isolates to picoxystrobin were investigated, and unimodal distributions were observed with average EC50 values of 0.7740 and 1.1561 μg mL−1, respectively. Eleven picoxystrobin-resistant mutants of C. truncatum and six mutants of C. gloeosporioides were acquired, with EC50 values varying from 5.40–152.96 and 13.53–28.30 μg mL−1, respectively. Compared to the parental isolates, mutants showed similar or higher relative fitness in conidial production and germination, and pathogenicity. Collectively, the resistance risk of C. truncatum and C. gloeosporioides to picoxystrobin is moderate to high. There was positive cross-resistance between picoxystrobin and pyraclostrobin, but not between picoxystrobin and fluazinam, difenoconazole, or propiconazole. The G143S mutation in Cyt b protein was detected in seven high-resistant mutants of C. truncatum (RF>100), and G137R occurred in four moderate-resistant mutants (RF

Background: The issue of Helicobacter pylori (H. pylori) resistance to clarithromycin (CLR) has consistently posed challenges for clinical treatment. Hence, a rapid susceptibility testing (AST) method urgently needs to be developed. Methods: In the present study, 35 isolates of H. pylori were isolated from 203 gastritis patients of the Guangzhou cohort, and the antimicrobial resistance phenotypes were associated with their genomes to analyze the relevant mutations. Based on these mutations, a rapid detection system utilizing high-resolution melting (HRM) curve analysis was designed and verified by the Shenzhen cohort, which consisted of 38 H. pylori strains. Results: Genomic analysis identified the mutation of the 2143 allele from A to G (A2143G) of 23S rRNA as the most relevant mutation with CLR resistance (p < 0.01). In the HRM system, the wild-type H. pylori showed a melting temperature (Tm) of 79.28 ± 0.01 °C, while the mutant type exhibited a Tm of 79.96 ± 0.01 °C. These differences enabled a rapid distinction between two types of H. pylori (p < 0.01). Verification examinations showed that this system could detect target DNA as low as 0.005 ng/μL in samples without being affected by other gastric microorganisms. The method also showed a good performance in the Shenzhen validation cohort, with 81.58% accuracy, and 100% specificity. Conclusions: We have developed an HRM system that can accurately and quickly detect CLR resistance in H. pylori. This method can be directly used for the detection of gastric microbiota samples and provides a new benchmark for the simple detection of H. pylori resistance.

Single-point mutation diseases in which substitution of one nucleotide with another in a gene occurs include familial Alzheimer's disease (fAD), familial Parkinson's disease (fPD), and familial Creutzfeldt-Jacob disease (fCJD) as well as Huntington's disease (HD), sickle cell anemia, and hemophilia. Inevitability of occurrence of these diseases is certain. However, the time of appearance of symptoms could be influenced by the diet, environment, and possibly other genetic factors. There are no effective approaches to delay the onset or progression of symptoms of these diseases. The fact that increased oxidative stress and inflammation significantly contribute to the initiation and progression of these point mutation diseases shows that antioxidants could be useful. The major objectives are (a) to present evidence that increased oxidative stress and chronic inflammation are associated with selected single-point mutation diseases, such as fAD, fPD, and fCJD, HD, sickle cell anemia, and hemophilia; (b) to describe limited studies on the role of individual antioxidants in experimental models of some of these diseases; and (c) to discuss a rationale for utilizing a comprehensive mixture of micronutrients, which may delay the development and progression of symptoms of above diseases by simultaneously reducing oxidative and inflammatory damages.Key teaching pointsSelected single-point mutation diseases and their pattern of inheritanceCharacteristics of each selected single-point mutation diseaseEvidence for increased oxidative stress and inflammation in each diseasePotential reasons for failure of single antioxidants in human studiesRationale for using a comprehensive mixture of micronutrients in delaying the onset and progression of single-point mutation diseases.

BackgroundPrader-Willi syndrome (PWS) is an imprinting disorder caused by the absence of paternal expressed genes in the Prader-Willi critical region (PWCR) on chromosome 15q11.2-q13. Three molecular mechanisms have been known to cause PWS, including a deletion in the PWCR, uniparental disomy 15 and imprinting defects.ResultsWe report the first case of PWS associated with a single-nucleotide SNRPN variant in a 10-year-old girl presenting with clinical features consistent with PWS, including infantile hypotonia and feeding difficulty, developmental delay with cognitive impairment, excessive eating with central obesity, sleep disturbances, skin picking and related behaviour issues. Whole-exome sequencing revealed a de novo mosaic nonsense variant of the SNRPN gene (c.73C>T, p.R25X) in 10% of DNA isolated from buccal cells and 19% of DNA from patient-derived lymphoblast cells. DNA methylation study did not detect an abnormal methylation pattern in the SNRPN locus. Parental origin studies showed a paternal source of an intronic single-nucleotide polymorphism within the locus in proximity to the SNRPN variant.ConclusionsThis is the first report that provides evidence of a de novo point mutation of paternal origin in SNRPN as a new disease-causing mechanism for PWS. This finding suggests that gene sequencing should be considered as part of the diagnostic workup in patients with clinical suspicion of PWS.

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. [ABSTRACT FROM AUTHOR]

Oncogenic Ras proteins are known to present multiple conformational states, as reported by the great variety of crystallographic structures. The GTP-bound states are grouped into two main states: the "inactive" state 1 and the "active" state 2. Recent reports on H-Ras have shown that state 2 exhibits two substates, directly related to the orientation of Tyr32: toward the GTP-bound pocket and outwards. In this paper, we show that N-Ras exhibits another substate of state 2, related to a third orientation of Tyr32, toward Ala18 and parallel to the GTP-bound pocket. We also show that this substate is highly sampled in the G12V mutation of N-Ras and barely present in its wild-type form, and that the G12V mutation prohibits the sampling of the GTPase-activating protein (GAP) binding substate, rendering this mutation oncogenic. Furthermore, using molecular dynamics simulations, we explore the importance of the membrane on N-Ras' conformational state dynamics and its strong influence on Ras protein stability. Moreover, the membrane has a significant influence on the conformational (sub)states sampling of Ras. This, in turn, is of crucial importance in the activation/deactivation cycle of Ras, due to the binding of guanine nucleotide exchange factor proteins (GEFs)/GTPase-activating proteins (GAPs). [ABSTRACT FROM AUTHOR]

The emergence of influenza virus A pandemic H1N1 in April 2009 marked the first pandemic of the 21st century. In this study, we observed significant differences in the polymerase activities of two clinical 2009 H1N1 influenza A virus isolates from Chinese and Japanese patients. Sequence comparison of the three main protein subunits (PB2, PB1, and PA) of the viral RNA-dependent RNA polymerase complex and subsequent mutational analysis revealed that a single amino acid substitution (E206K) was responsible for the observed impaired replication phenotype. Further in vitro experiments showed that presence of PAE206K decreased the replication of influenza A/ WSN/33 virus in mammalian cells and a reduction in the virus's pathogenicity in vivo. Mechanistic studies revealed that PAE206K is a temperature-sensitive mutant associated with the inability to transport PB1-PA complex to the nucleus at high temperature (39.5 °C). Hence, this naturally occurring variant in the PA protein represents an ideal candidate mutation for the development of live attenuated influenza vaccines. [ABSTRACT FROM AUTHOR]

Introduction: In Colombia, thyroid cancer ranks among the highest incidences, yet our population lacks studies on its molecular profile. This study aims to characterize clinical, histopathologic and molecular data in a Colombian cohort with papillary thyroid carcinoma (PTC). Methods: A retrospective review of clinical history, clinicopathologic characteristics, treatment and 5–10-year follow-up for all patients was done. DNA and RNA were extracted from formalin-fixed paraffin-embedded (FFPE) tissue using the Quick-DNA & RNA FFPE Min iPrep kit (Zymo Research). Next-generation sequencing (NGS) analysis was performed with SOPHiA Solid Tumor Solutions kit (SOPHiA GENETICS). Tumor mutation genomic analysis used SOPHiA DDM™ platform, with descriptive analysis reporting frequencies, means and associations via chi-square analysis. Results: Among 231 sequenced patients, mean age at diagnosis was 46 (± 12.35) years, with higher frequency in women (81.82%). Two cases were reclassified as non-invasive follicular thyroid neoplasm (NIFT-P); an NRAS mutation was found in one of them. Predominant histologic subtype was classic PTC (57.64%) followed by tall cell (28.82%). Of the 229 sequenced carcinomas, mutations were identified in 186 cases, including BRAF, IDH1, RAS and PIK3CA. Notable copy number variations (CNVs) were PDGFRA, CDK4 and KIT, with RET being the most frequent gene fusion, including CCDC6-RET in two classic subtype cases. Conclusion: This is the first study in Colombia (TIROSEC) to our knowledge that integrates molecular and histopathologic profiles enriching our local comprehension and knowledge of PTC. The identification of target mutations such as BRAF, RET and NTRK fusions holds the potential to guide targeted therapies for tumor recurrence and predict aggressive behavior. [ABSTRACT FROM AUTHOR]

Background: Since 2014, dengue epidemics have occurred almost annually in Nouakchott, the capital city of Mauritania, coinciding with the recent establishment of Aedes aegypti, the primary vector of dengue, in the city. Anopheles arabiensis, the primary vector of malaria, is also abundant not only in Nouakchott but also in most areas of the country. Resistance to insecticides has been studied in An. arabiensis but not in Ae. aegypti in Mauritania. The objective of the present study was to establish the baseline data on the frequencies of knockdown resistance (kdr) mutations in the voltage-gated sodium channel (vgsc) gene in Ae. aegypti collected in Nouakchott to improve vector control. Methods: Resting Ae. aegypti mosquitoes were collected in 2017 and 2018 in Teyarett and Dar Naim districts in Nouakchott using a battery-powered aspirator. Polymerase chain reaction (PCR) and DNA sequencing were performed to detect the presence of five kdr mutations known to be associated with pyrethroid resistance: L982W, S989P, I1011M/G, V1016G/I, and F1534C. Results: A total of 100 female Ae. aegypti mosquitoes were identified among collected resting culicid fauna, of which 60% (60/100) were unfed, 12% (12/100) freshly blood-fed, and 28% (28/100) gravid. Among the mutations investigated in this study, 989P, 1016G, and 1534C were found to be widespread, with the frequencies of 0.43, 0.44, and 0.55, respectively. Mutations were not found in codons 982 and 1011. No other mutations were detected within the fragments analyzed in this study. Genotype distribution did not deviate from Hardy–Weinberg equilibrium. The most frequent co-occurring point mutation patterns among Ae. aegypti mosquitoes were the heterozygous individuals 989SP/1016VG/1534FC detected in 45.1% of mosquitoes. In addition, homozygous mutant 1534CC co-occurred simultaneously with homozygous wild type 989SS and 1016VV in 30.5% of mosquito specimens. Inversely, homozygous wild-type 1534FF co-occurred simultaneously with homozygous mutant 989PP and 1016GG in 19.5% of the mosquitoes. Conclusions: To our knowledge, this is the first study reporting the presence of three point mutations in the vgsc gene of Ae. aegypti in Mauritania. The findings of the present study are alarming because they predict a high level of resistance to pyrethroid insecticides which are commonly used in vector control in the country. Therefore, further studies are urgently needed, in particular phenotypic characterization of insecticide resistance using the standardized test. [ABSTRACT FROM AUTHOR]

SignificanceUnderstanding and treating neurological disorders are global priorities. Some of these diseases are engendered by mutations that cause defects in the cellular synthesis of transfer RNAs (tRNAs), which function as adapter molecules that translate messenger RNAs into proteins. During tRNA biogenesis, ribonuclease P catalyzes removal of the transcribed sequence upstream of the mature tRNA. Here, we focus on a cytoplasmic tRNAArgUCU that is expressed specifically in neurons and, when harboring a particular point mutation, contributes to neurodegeneration in mice. Our results suggest that this mutation favors stable alternative structures that are not cleaved by mouse ribonuclease P and motivate a paradigm that may help to understand the molecular basis for disease-associated mutations in other tRNAs.

Abstract 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.

Naturally occurring point mutations in the HBG promoter switch hemoglobin synthesis from defective adult beta-globin to fetal gamma-globin in sickle cell patients with hereditary persistence of fetal hemoglobin (HPFH) and ameliorate the clinical severity. Inspired by this natural phenomenon, we tiled the highly homologous HBG proximal promoters using adenine and cytosine base editors that avoid the generation of large deletions and identified novel regulatory regions including a cluster at the -123 region. Base editing at -123 and -124 bp of HBG promoter induced fetal hemoglobin (HbF) to a higher level than disruption of well-known BCL11A binding site in erythroblasts derived from human CD34+ hematopoietic stem and progenitor cells (HSPC). We further demonstrated in vitro that the introduction of -123T > C and -124T > C HPFH-like mutations drives gamma-globin expression by creating a de novo binding site for KLF1. Overall, our findings shed light on so far unknown regulatory elements within the HBG promoter and identified additional targets for therapeutic upregulation of fetal hemoglobin.

Botulinum neurotoxins (BoNTs), produced by the spore-forming bacterium Clostridium botulinum, cause botulism, a rare but fatal illness affecting humans and animals. Despite causing a life-threatening disease, BoNT is a multipurpose therapeutic. Nevertheless, as the most potent natural toxin, BoNT is classified as a Select Agent in the US, placing C. botulinum research under stringent governmental regulations. The extreme toxicity of BoNT, its impact on public safety, and its diverse therapeutic applications urge to devise safe solutions to expand C. botulinum research. Accordingly, we exploited CRISPR/Cas9-mediated genome editing to introduce inactivating point mutations into chromosomal bont/e gene of C. botulinum Beluga E. The resulting Beluga Ei strain displays unchanged physiology and produces inactive BoNT (BoNT/Ei) recognized in serological assays, but lacking biological activity detectable ex- and in vivo. Neither native single-chain, nor trypsinized di-chain form of BoNT/Ei show in vivo toxicity, even if isolated from Beluga Ei sub-cultured for 25 generations. Beluga Ei strain constitutes a safe alternative for the BoNT research necessary for public health risk management, the development of food preservation strategies, understanding toxinogenesis, and for structural BoNT studies. The example of Beluga Ei generation serves as template for future development of C. botulinum producing different inactive BoNT serotypes.

In eukaryotic cells, stressors reprogram the cellular proteome by activating the integrated stress response (ISR). In its canonical form, stress-sensing kinases phosphorylate the eukaryotic translation initiation factor eIF2 (eIF2-P), which ultimately leads to reduced levels of ternary complex required for initiation of mRNA translation. Previously we showed that translational control is primarily exerted through a conformational switch in eIF2's nucleotide exchange factor, eIF2B, which shifts from its active A-State conformation to its inhibited I-State conformation upon eIF2-P binding, resulting in reduced nucleotide exchange on eIF2 (Schoof et al. 2021). Here, we show functionally and structurally how a single histidine to aspartate point mutation in eIF2B's β subunit (H160D) mimics the effects of eIF2-P binding by promoting an I-State like conformation, resulting in eIF2-P independent activation of the ISR. These findings corroborate our previously proposed A/I-State model of allosteric ISR regulation.

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. [ABSTRACT FROM AUTHOR]

This research cloned and expressed the sugar transporter gene KM_SUT5 from Kluyveromyces marxianus GX-UN120, which displayed remarkable sugar transportation capabilities, including pentose sugars. To investigate the impact of point mutations on xylose transport capacity, we selected four sites, predicted the suitable amino acid sites by molecular docking, and altered their codons to construct the corresponding mutants, Q74D, Y195K, S460H, and Q464F, respectively. Furthermore, we conducted site-directed truncation on six sites of KM_SUT5p. The molecular modification resulted in significant changes in mutant growth and the D-xylose transport rate. Specifically, the S460H mutant exhibited a higher growth rate and demonstrated excellent performance across 20 g L−1 xylose, achieving the highest xylose accumulation under xylose conditions (49.94 μmol h−1 gDCW-1, DCW mean dry cell weight). Notably, mutant delA554-, in which the transporter protein SUT5 is truncated at position delA554-, significantly increased growth rates in both D-xylose and D-glucose substrates. These findings offer valuable insights into potential modifications of other sugar transporters and contribute to a deeper understanding of the C-terminal function of sugar transporters.

Acetate esters comprise a wide range of products including fragrances and industrial solvents. Biosynthesis of esters offers a promising alternative to chemical synthesis because such routes use renewable carbohydrate resources and minimize the generation of waste. One biochemical method for ester formation relies on the ATF1 gene from Saccharomyces cerevisiae, which encodes alcohol-O-acyltransferase (AAT) which converts acetyl-CoA and an exogenously supplied alcohol into the ester. In this study, the formation of several acetate esters via AAT was examined in Escherichia coli chromosomally expressing citrate synthase variants, which create a metabolic bottleneck at acetyl-CoA. In shake flask cultures, variant strains generated more acetate esters than the strains expressing the wild-type citrate synthase. In a controlled bioreactor, E. coli GltA[A267T] generated 3.9 g propyl acetate in 13 h, corresponding to a yield of 0.155 g propyl acetate/g glucose, which is 18% greater than that obtained by the wild-type GltA control. These results demonstrate the ability of citrate synthase variants to redistribute carbon from central metabolism into acetyl-CoA-derived biochemicals.

Molecular switch proteins whose cycling between states is controlled by opposing regulators1,2 are central to biological signal transduction. As switch proteins function within highly connected interaction networks3, the fundamental question arises of how functional specificity is achieved when different processes share common regulators. Here we show that functional specificity of the small GTPase switch protein Gsp1 in Saccharomyces cerevisiae (the homologue of the human protein RAN)4 is linked to differential sensitivity of biological processes to different kinetics of the Gsp1 (RAN) switch cycle. We make 55 targeted point mutations to individual protein interaction interfaces of Gsp1 (RAN) and show through quantitative genetic5 and physical interaction mapping that Gsp1 (RAN) interface perturbations have widespread cellular consequences. Contrary to expectation, the cellular effects of the interface mutations group by their biophysical effects on kinetic parameters of the GTPase switch cycle and not by the targeted interfaces. Instead, we show that interface mutations allosterically tune the GTPase cycle kinetics. These results suggest a model in which protein partner binding, or post-translational modifications at distal sites, could act as allosteric regulators of GTPase switching. Similar mechanisms may underlie regulation by other GTPases, and other biological switches. Furthermore, our integrative platform to determine the quantitative consequences of molecular perturbations may help to explain the effects of disease mutations that target central molecular switches.