98,110 results on '"POINT MUTATION"'
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2. Chapter Seven - Mouse models for understanding physiological functions of ADARs
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Zhang, Qinyi and Walkley, Carl R.
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- 2025
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3. Drosophila models used to simulate human ATP1A1 gene mutations that cause Charcot-Marie-Tooth type 2 disease and refractory seizures.
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Yao Yuan, Lingqi Yu, Xudong Zhuang, Dongjing Wen, Jin He, Jingmei Hong, Jiayu Xie, Shengan Ling, Xiaoyue Du, Wenfeng Chen, and Xinrui Wang
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- 2025
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4. The F204S mutation in adrenodoxin oxidoreductase drives salinomycin resistance in Eimeria tenella.
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Sun, Pei, Wang, Chaoyue, Xie, Fujie, Chen, Linlin, Zhang, Yuanyuan, Tang, Xinming, Hu, Dandan, Gao, Yang, Zhang, Ning, Hao, Zhenkai, Yu, Yonglan, Suo, Jingxia, Suo, Xun, and Liu, Xianyong
- Abstract
Salinomycin is a polyether ionophore widely used for the treatment of coccidiosis in poultry. However, the emergence of coccidia strains resistant to salinomycin presents challenges for control efforts, and the mechanisms underlying this resistance in Eimeria remain inadequately understood. In this study, 78 stable salinomycin-resistant strains were generated through experimental evolution approaches. Whole-genome sequencing of salinomycin-resistant Eimeria tenella isolates revealed single nucleotide polymorphisms (SNPs), with 12 candidate genes harboring nonsynonymous mutations identified. To confirm the candidate gene responsible for conferring salinomycin resistance, we leveraged reverse genetic strategies and identified a key amino acid substitution (F204S) in adrenodoxin oxidoreductase (EtADR), which markedly reduced susceptibility to salinomycin. Our results elucidate the complex interactions among salinomycin resistance, parasite fitness, point mutations, and the structure of EtADR, laying the foundation for future studies on drug resistance in Eimeria and contributing to the development of targeted control strategies. [ABSTRACT FROM AUTHOR]
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- 2024
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5. Mutations on the A and C genomes of <italic>Brassica napus</italic> confer herbicide resistance to acetohydroxyacid synthase inhibitors.
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Christey, Mary C., Braun, Robert H., Dumbleton, Andrew, Jacobs, Jeanne M. E., and Conner, Anthony J.
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ACETOLACTATE synthase , *ETHYL methanesulfonate , *GENE amplification , *DNA sequencing , *MUTAGENESIS , *HERBICIDES , *HERBICIDE resistance - Abstract
The point mutations conferring resistance to the sulfonylurea herbicide, chlorsulfuron, are reported for two
Brassica napus lines (19c and 30a) developed by seed mutagenesis using ethyl methanesulfonate. Crosses with the herbicide-susceptible wild-type line generated F2 and backcrossed progeny and confirmed that chlorsulfuron resistance was inherited as a dominant mutation at a single locus in both lines. F2 progeny from crossing 19c and 30a segregated in a 15 resistant: 1 susceptible ratio, establishing that the two herbicide-resistant loci are independently inherited and that the mutations for resistance occur at different loci. PCR amplification and DNA sequencing of regions from the acetohydroxyacid synthase gene revealed two separate point mutations at different nucleotides in the same codon. This results in the substitution of proline-197 for leucine in 19c and proline-197 for serine in 30a. The different amino acid substitutions in the acetohydroxyacid synthase enzyme explain the differing responses of 19c and 30a to sulfonylurea and imidazolinone herbicides. Based on attempts at introgression of chlorsulfuron resistance to a wide range of forage brassicas, we conclude that the 19c mutation resides in theBrassica C genome, whereas the 30a mutation is in theBrassica A genome. [ABSTRACT FROM AUTHOR]- Published
- 2024
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6. A single-base deletion in exon 2 of Hd1 delineates monogenic recessive photoperiod insensitivity in aromatic Joha rice: a novel allele for seasonal adaptability.
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Reddy, Bodeddula Jayasankar, Baradkar, Shreekant M., Manogna, Tamma V. S. S., Bordoloi, Dibosh, Bihani, Subhash C., Barua, Nagendra Sarma, Baruah, Akhil Ranjan, Das, Bikram Kishore, Mondal, Suvendu, and Sarma, Debojit
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RECESSIVE genes ,FRAMESHIFT mutation ,GENETIC mutation ,RICE ,MOLECULAR cloning ,RICE breeding - Abstract
Background: Assam's aromatic Joha rice is a unique rice class famous for its aroma, taste, and nutritional benefits, which fetch high market prices in domestic and international markets. Joha landraces are inherently poor yielders due to their strong aroma and predominantly photoperiod sensitivity. Hybridization involving non-aromatic HYVs improves yield with concomitant loss of quality. In this context, mutation breeding, a sustainable approach where genetic mutations are induced to create desirable traits, often provides useful allelic variation in specific morpho-agronomic traits. The present study delves into the genetic characterization of a photoperiod-insensitive mutant. As part of our mutation breeding programme, this mutant was isolated from a gamma ray-induced M
2 population of a Joha rice landrace, Kon Joha. Results: The mutant was unique, and a single recessive gene conditions the induced photoperiod insensitivity. Mutant gene tagging involved 402 SSR and InDel markers, and later polymorphic markers were used for bulk segregant analysis (BSA) in the F2 population of 'mutant × Kalijeera (distant parent)'. BSA revealed an association between the SSR marker RM527 and this mutant trait. This marker is present on chromosome 6 of the rice genome. Using chromosome 6-specific SSR markers in polymorphic screening and BSA revealed another associated marker, RM19725, for the mutant trait. The genomic interval between RM527 and RM19725 harbors a photoperiod-insensitive gene, Hd1, on chromosome 6. Cloning and sequencing of Hd1 genomic fragments from the parents and mutants revealed a single-base deletion in exon 2, leading to a frameshift mutation in the Hd1 protein. This mutation in exon 2 leads to severe structural abnormalities in the CCT domain of the Hd1 protein that is critical for the interaction of the repressing complex with conserved response elements in the florigen gene under long-day conditions, thereby causing photoperiod insensitivity. Conclusions: The mutant's pleasant aroma and other quality characteristics, comparable to those of the parent cultivar, hold significant promise. They expand its potential use in a structured breeding programme aimed at developing high-value aromatic Joha rice. This rice, resilient to winter- and summer-growing environments and with broad seasonal adaptability, could revolutionize the rice market. The practical value of our research is underscored by this exciting possibility. [ABSTRACT FROM AUTHOR]- Published
- 2024
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7. A case of adrenomyeloneuropathy caused by a novel point mutation in the ABCD1 gene and functional verification.
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Xiaoxue Shi, Xuelin Qi, Jinhua Zheng, Jianjun Ma, and Dongsheng Li
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ADRENOLEUKODYSTROPHY ,PHENOTYPES ,GENE expression ,GENETIC mutation ,DRUG target ,PROTEIN expression - Abstract
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]
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- 2024
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8. The F204S mutation in adrenodoxin oxidoreductase drives salinomycin resistance in Eimeria tenella
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Pei Sun, Chaoyue Wang, Fujie Xie, Linlin Chen, Yuanyuan Zhang, Xinming Tang, Dandan Hu, Yang Gao, Ning Zhang, Zhenkai Hao, Yonglan Yu, Jingxia Suo, Xun Suo, and Xianyong Liu
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Eimeria tenella ,salinomycin ,drug resistance ,point mutation ,Veterinary medicine ,SF600-1100 - Abstract
Abstract Salinomycin is a polyether ionophore widely used for the treatment of coccidiosis in poultry. However, the emergence of coccidia strains resistant to salinomycin presents challenges for control efforts, and the mechanisms underlying this resistance in Eimeria remain inadequately understood. In this study, 78 stable salinomycin-resistant strains were generated through experimental evolution approaches. Whole-genome sequencing of salinomycin-resistant Eimeria tenella isolates revealed single nucleotide polymorphisms (SNPs), with 12 candidate genes harboring nonsynonymous mutations identified. To confirm the candidate gene responsible for conferring salinomycin resistance, we leveraged reverse genetic strategies and identified a key amino acid substitution (F204S) in adrenodoxin oxidoreductase (EtADR), which markedly reduced susceptibility to salinomycin. Our results elucidate the complex interactions among salinomycin resistance, parasite fitness, point mutations, and the structure of EtADR, laying the foundation for future studies on drug resistance in Eimeria and contributing to the development of targeted control strategies.
- Published
- 2024
- Full Text
- View/download PDF
9. Identification of de-novo CREBBP gene variants in patients with Rubinstein–Taybi syndrome.
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Ji, Qinghong, Ma, Weihong, Xin, Gang, Xin, Qian, Duan, Shuhong, Ding, Mingxia, Dong, Lihua, Li, Zhiqiang, and Hong, Fanzhen
- Abstract
Rubinstein–Taybi syndrome (RSTS) is an autosomal dominant genetic disease characterized by growth retardation, psychomotor retardation, and distinctive facial features. It is primarily caused by mutations in CREBBP or EP300. In this study, we aimed to describe the clinical manifestations and genetic analyses of two cases with RSTS. Clinical analysis was performed on two cases with RSTS. Molecular diagnoses were made via whole exome sequencing, and potential pathogenic variants were filtered and selected. PCR followed by Sanger sequencing was used to verify candidate variants in the family members. Case 1 involved a 7-year-old boy (patient 1) who exhibited delayed language development, growth retardation, and intellectual disability. We did not find any other characteristics of RSTS, such as thumb or hallux abnormalities. Case 2 involved a fetus who had severe congenital heart disease, low conus medullaris, and a large gallbladder. Whole exome and Sanger sequencing results revealed that a missense mutation c.5120G>A (p. Cys1707Tyr) was present in patient 1 and that the fetus carried a heterozygous nonsense mutation c.1984C>T (p. Gln662Ter). In conclusion, whole exome sequencing combined with Sanger sequencing revealed that c.5120G>A (p. Cys1707Tyr) and c.1984C>T (p. Gln662Ter) are two new mutation sites that cause RSTS. This study expands the clinical phenotypes and is helpful in identifying gene-phenotype correlations in RSTS. [ABSTRACT FROM AUTHOR]
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- 2025
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10. Molecular Mutations in Histiocytosis: A Comprehensive Survey of Genetic Alterations.
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Pai, Padmini, Nirmal, Arnav, Mathias, Lian, Jain, Siya, Shetty, Manasa Gangadhar, and Sundara, Babitha Kampa
- Abstract
Histiocytosis represents a group of uncommon disorders characterized by the abnormal accumulation of specialized immune cells, such as macrophages, dendritic cells, or monocyte-derived cells, in various tissues and organs. Over 100 distinct subtypes have been documented, each displaying a broad spectrum of clinical presentations and histological characteristics. Till today, histiocytosis has been addressed through a combination of chemotherapy, radiotherapy, and surgery, with varying responses from individual patients. Due to its atypical symptoms, it has been prone to misdiagnosis. Advances in our understanding of the cellular and molecular aspects of these conditions are paving the way for improved diagnostic methods and targeted therapies. Researchers have extensively investigated various mutations in patient samples. However, no paper has yet provided a comprehensive summary of the collective analysis of mutations and pathways. Hence, this paper consolidates research efforts that specifically concentrate on gene mutations identified in patient samples of different subtypes of histiocytosis. These insights are essential for developing targeted therapies and improving diagnosis. Further, it provides potential insights to enhance the development of more effective therapeutic approaches for rare diseases. Parts of the figure were drawn by using pictures from Servier Medical Art. Servier Medical Art by Servier is licensed under a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0/). [ABSTRACT FROM AUTHOR]
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- 2025
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11. Substrate specificity controlled by the exit site of human P4-ATPases, revealed by de novo point mutations in neurological disorders.
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Calianese, David C., Tomoyasu Noji, Sullivan, Jennifer A., Schoch, Kelly, Shashi, Vandana, McNiven, Vanda, Ramos, Luiza Lorena Pires, Jordanova, Albena, Kárteszi, Judit, Hiroshi Ishikita, and Shigekazu Nagata
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GENE expression , *MOLECULAR dynamics , *CELL membranes , *BIOCHEMICAL substrates , *LYSIS - Abstract
The maintenance of lipid asymmetry on the plasma membrane is regulated by flippases, such as ATP8A2, ATP11A, and ATP11C, which translocate phosphatidylserine and phosphatidylethanolamine from the outer leaflet to the inner leaflet. We previously identified a patient-derived point mutation (Q84E) in ATP11A at the phospholipid entry site, which acquired the ability to flip phosphatidylcholine (PtdCho). This mutation led to elevated levels of sphingomyelin (SM) in the outer leaflet of the plasma membrane. We herein present two de novo ATP11A dominant mutations (E114G and S399L) in heterozygous patients exhibiting neurological and developmental disorders. These mutations, situated near the predicted phospholipid exit site, similarly confer the ability for ATP11A to recognize PtdCho as a substrate, resulting in its internalization into cells. Cells expressing these mutants had increased SM levels on their surface, attributed to the up-regulated expression of the sphingomyelin synthase-1 gene, rendering them more susceptible to SM phosphodiesterase-mediated cell lysis. Corresponding mutations in ATP11C and ATP8A2, paralogs of ATP11A, exerted similar effects on PtdCho-flipping activity and increased SM levels on the cell surface. Molecular dynamics simulations, based on the ATP11C structure, suggest that the E114G and S399L mutations enhance ATP11C's affinity toward PtdCho. These findings underscore the importance of the well-conserved exit and entry sites in determining phospholipid substrate specificity and indicate that aberrant flipping of PtdCho contributes to neurological disorders. [ABSTRACT FROM AUTHOR]
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- 2024
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12. Development of a High-Resolution Melting Method for the Detection of Clarithromycin-Resistant Helicobacter pylori in the Gastric Microbiome.
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Kuang, Zupeng, Huang, Huishu, Chen, Ling, Shang, Yanyan, Huang, Shixuan, Liu, Jun, Chen, Jianhui, Xie, Xinqiang, Chen, Moutong, Wu, Lei, Gao, He, Zhao, Hui, Li, Ying, and Wu, Qingping
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HELICOBACTER pylori ,MICROBIAL sensitivity tests ,MELTING points ,GENOMICS ,DRUG resistance in microorganisms - Abstract
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]
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- 2024
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13. 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.
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Li, Xuecheng, Zhang, Yongli, Wen, Xin, and Pan, Junmin
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GENOME editing , *GENE expression , *CHLAMYDOMONAS reinhardtii , *GREEN algae , *DRUG resistance in bacteria - Abstract
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]
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- 2024
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14. Molecular Modification Enhances Xylose Uptake by the Sugar Transporter KM_SUT5 of Kluyveromyces marxianus.
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Luo, Xiuyuan, Tao, Xi, Ran, Guangyao, Deng, Yuanzhen, Wang, Huanyuan, Tan, Liyan, and Pang, Zongwen
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KLUYVEROMYCES marxianus , *CARRIER proteins , *MOLECULAR docking , *XYLOSE , *BIOCHEMICAL substrates - Abstract
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]
- Published
- 2024
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15. Resistance risk assessment for benzovindiflupyr in Sclerotium rolfsii and transmission of resistance genes among population.
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Cui, Kaidi, Jiang, Chaofan, Sun, Longjiang, Wang, Mengke, He, Leiming, and Zhou, Lin
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SCLEROTIUM rolfsii ,SUCCINATE dehydrogenase ,RISK assessment ,MOLECULAR docking ,GENES - Abstract
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]
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- 2024
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16. CRISPR generation of CSF1R-G795A human microglia for robust microglia replacement in a chimeric mouse model
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Chadarevian, Jean Paul, Davtyan, Hayk, Lombroso, Sonia I, Bennett, F Chris, and Blurton-Jones, Mathew
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Medical Biotechnology ,Biomedical and Clinical Sciences ,Transplantation ,Stem Cell Research - Embryonic - Human ,Neurosciences ,Stem Cell Research - Induced Pluripotent Stem Cell ,Stem Cell Research ,5.2 Cellular and gene therapies ,Adult ,Animals ,Mice ,Infant ,Newborn ,Humans ,Microglia ,Brain ,Disease Models ,Animal ,Pluripotent Stem Cells ,Point Mutation ,CRISPR ,Cell Culture ,Cell Differentiation ,Immunology ,Model Organisms ,Neuroscience ,Single Cell ,Stem Cells - Abstract
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.
- Published
- 2023
17. The FAM86 domain of FAM86A confers substrate specificity to promote EEF2-Lys525 methylation.
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Francis, Joel, Shao, Zengyu, Narkhede, Pradnya, Trinh, Annie, Lu, Jiuwei, Song, Jikui, and Gozani, Or
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EEF2 ,EEF2KMT ,FAM86A ,alphafold ,lysine methylation ,mRNA translation ,methyltransferase ,protein synthesis ,ribosome ,translation elongation ,Humans ,Lysine ,Methylation ,Methyltransferases ,Peptide Elongation Factor 2 ,S-Adenosylmethionine ,Substrate Specificity ,Protein Domains ,Protein Structure ,Tertiary ,Models ,Molecular ,Crystallography ,X-Ray ,Point Mutation - Abstract
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.
- Published
- 2023
18. Salmonella enteritidis acquires phage resistance through a point mutation in rfbD but loses some of its environmental adaptability
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Yukun Zeng, Ping Li, Shenglong Liu, Mangmang Shen, Yuqing Liu, and Xin Zhou
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Salmonella enteritidis ,rfbD gene ,point mutation ,phage resistance ,environmental adaptability ,Veterinary medicine ,SF600-1100 - Abstract
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.
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- 2024
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19. Citrate synthase variants improve yield of acetyl-CoA derived 3-hydroxybutyrate in Escherichia coli
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Hemshikha Rajpurohit and Mark A. Eiteman
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Batch ,Repeated batch ,Fermentation ,Point mutation ,3-hydroxybutyrate ,Microbiology ,QR1-502 - Abstract
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.
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- 2024
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20. Crispr-Based Editing of Human Pluripotent Stem Cells for Disease Modeling.
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Chang, Yun, Lan, Feng, Zhang, Yongshuai, and Ma, Shuhong
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PLURIPOTENT stem cells , *DOUBLE-strand DNA breaks , *HUMAN stem cells , *GENE knockout , *STEM cells , *GENOME editing - Abstract
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]
- Published
- 2024
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21. Induction of point and structural mutations in engineered yeast Saccharomyces cerevisiae improve carotenoid production.
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Yamada, Ryosuke, Ando, Kazuya, Sakaguchi, Rumi, Matsumoto, Takuya, and Ogino, Hiroyasu
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SACCHAROMYCES cerevisiae , *ERGOSTEROL , *CAROTENES , *GENE expression , *GENETIC mutation , *YEAST , *MUTAGENESIS - Abstract
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]
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- 2024
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22. Genetically modified pigs with CD163 point mutation are resistant to HP-PRRSV infection.
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Ying Liu, Lin Yang, Hong-Yong Xiang, Ming Niu, Jia-Cheng Deng, Xue-Yuan Li, Wen-Jie Hao, Hong-Sheng OuYang, Tong-Yu Liu, Xiao-Chun Tang, Da-Xin Pang, and Hong-Ming Yuan
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PORCINE reproductive & respiratory syndrome ,SWINE ,COMMUNICABLE diseases ,AFRICAN swine fever ,CIRCOVIRUS diseases - Abstract
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-15
CD163 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]- Published
- 2024
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23. Production of Esters in Escherichia coli Using Citrate Synthase Variants.
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Shipmon, Jacoby C., Rathinasabapathi, Pasupathi, Broich II, Michael L., Hemansi, and Eiteman, Mark A.
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CITRATE synthase ,ESCHERICHIA coli ,CHEMICAL synthesis ,RENEWABLE natural resources ,ESTERS - Abstract
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]
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- 2024
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24. Investigation of genotype-phenotype and familial features of Turkish dystrophinopathy patients.
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Ozkalayci, Hande, Bora, Elcin, Cankaya, Tufan, Kocabey, Mehmet, Zubari, Nadide Cemre, Yis, Uluc, Giray Bozkaya, Ozlem, Turan, Serkan, Pekcanlar Akay, Aynur, Caglayan, Ahmet Okay, and Ulgenalp, Ayfer
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TURKS ,BECKER muscular dystrophy ,DUCHENNE muscular dystrophy ,NONSENSE mutation ,DYSTROPHIN genes - Abstract
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]
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- 2024
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25. Citrate synthase variants improve yield of acetyl-CoA derived 3-hydroxybutyrate in Escherichia coli.
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Rajpurohit, Hemshikha and Eiteman, Mark A.
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ACETYLCOENZYME A ,CITRATE synthase ,PYRUVATES ,ESCHERICHIA coli ,KREBS cycle ,3-Hydroxybutyric acid ,MICROBIAL products - 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. [ABSTRACT FROM AUTHOR]- Published
- 2024
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26. Pygo-F773W Mutation Reveals Novel Functions beyond Wnt Signaling in Drosophila.
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Li, Youfeng, Jiang, Zhigang, Xu, Yue, Yan, Jing, Wu, Qiong, Huang, Sirui, Wang, Lingxiao, Xie, Yulian, Wu, Xiushan, Wang, Yuequn, Li, Yongqing, Fan, Xiongwei, Li, Fang, and Yuan, Wuzhou
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DROSOPHILA , *DROSOPHILA melanogaster , *CELLULAR signal transduction , *WNT signal transduction , *CLUSTER analysis (Statistics) , *HUMAN locomotion , *POST-translational modification , *HISTONES - Abstract
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]
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- 2024
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27. Genetic determinants of resistance to antimicrobial therapeutics are rare in publicly available Clostridioides difficile genome sequences.
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Kolte, Baban and Nübel, Ulrich
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CLOSTRIDIOIDES difficile , *DRUG resistance in microorganisms , *PLASMID genetics , *ANTI-infective agents , *PHENOTYPES , *CILIARY motility disorders - Abstract
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]
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- 2024
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28. Structural insight into subunit F of respiratory chain complex I from Xanthomonas oryzae pv. oryzae inhibition by parthenolide.
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Li, Lei, Zhou, Qian, Li, Linwei, Ran, Tingting, Wang, Weiwu, Liu, Chenyang, Chen, Jin, Sun, Tiemin, Chen, Yu, Feng, Xu, Zhang, Feng, and Xu, Shu
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XANTHOMONAS oryzae ,RICE diseases & pests ,BINDING sites ,PROTEIN structure ,MOLECULAR docking ,XANTHOMONAS ,BACTERICIDES - Abstract
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]
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- 2024
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29. Recent advances in enzyme-free and enzyme-mediated single-nucleotide variation assay in vitro.
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Xiong, Erhu, Liu, Pengfei, Deng, Ruijie, Zhang, Kaixiang, Yang, Ronghua, and Li, Jinghong
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GENETIC profile , *GENOME-wide association studies , *GENE expression , *GENETIC variation , *THERAPEUTICS - Abstract
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]
- Published
- 2024
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30. Analysis of environmental selection pressure of superoxide dismutase in deep-sea sea cucumber.
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Li, Yanan, Chen, Zongfu, Zhang, Haibin, Liu, Ruoyu, Chen, Shuichun, and Lin, Li
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SUPEROXIDE dismutase , *SEA cucumbers , *ANTIOXIDANTS , *TRANSCRIPTOMES , *HYDROSTATIC pressure , *POINT mutation (Biology) - Abstract
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]
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- 2024
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31. Unexpected structures formed by the kinase RET C634R mutant extracellular domain suggest potential oncogenic mechanisms in MEN2A.
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Liu, Yixin, De Castro Ribeiro, Orquidea, Haapanen, Outi, Craven, Gregory, Sharma, Vivek, Muench, Stephen, and Goldman, Adrian
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GDF15 ,GDNF ,GFRAL ,GFRα1 ,MEN2A ,RET ,RET C634R mutant ,membrane protein ,protein-protein interaction ,Humans ,Carcinogenesis ,Ligands ,Multiple Endocrine Neoplasia Type 2a ,Point Mutation ,Protein Domains ,Protein Multimerization ,Proto-Oncogene Proteins c-ret ,Cysteine ,Arginine - Abstract
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.
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- 2022
32. VP4 Mutation Boosts Replication of Recombinant Human/Simian Rotavirus in Cell Culture.
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Valusenko-Mehrkens, Roman, Schilling-Loeffler, Katja, Johne, Reimar, and Falkenhagen, Alexander
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ROTAVIRUSES , *CHILD death , *CELL culture , *REVERSE genetics , *WHOLE genome sequencing , *VACCINE development , *BASE pairs - Abstract
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]
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- 2024
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33. Development of a Molecular Diagnostic Kit for the Sars-Cov-2 Eris Variant.
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Tatliesme, Burcin and Irvem, Arzu
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DIAGNOSTIC reagents & test kits ,SARS-CoV-2 ,FLUORESCENT probes ,RNA - Abstract
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]
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- 2024
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34. Resistance Mechanism of Plutella xylostella (L.) Associated with Amino Acid Substitutions in Acetylcholinesterase-1: Insights from Homology Modeling, Docking and Molecular Dynamic Simulation.
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Zolfaghari, Maryam, Xiao, Yong, Safiul Azam, Fardous Mohammad, Yin, Fei, Peng, Zheng-Ke, and Li, Zhen-Yu
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DIAMONDBACK moth , *PESTICIDE resistance , *AMINO acids , *MOLECULAR docking , *DYNAMIC simulation , *INSECTICIDES , *CHOLINESTERASE reactivators - Abstract
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]
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- 2024
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35. Super‐Resolution Microscopy Unveils Synergistic Structural Changes of Organelles Upon Point Mutation.
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Anjum, Farhan, Kaushik, Kush, Salam, Abdul, Yadav, Aditya, and Nandi, Chayan Kanti
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ORGANELLES ,MUTAGENS ,GENETIC mutation ,ETHYL methanesulfonate ,NUCLEAR DNA ,LYSOSOMES - Abstract
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]
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- 2024
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36. The Protein S Erlangen Mutation PROS1c.1904T>C (F635S) Suppresses Secretion.
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Reißig, Julian, Cunningham, Sarah, Wandersee, Alexandra, Brox, Regine, Achenbach, Susanne, Strobel, Julian, Hackstein, Holger, and Schneider, Sabine
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PROTEIN S ,GOLGI apparatus ,GREEN fluorescent protein ,SECRETION ,WESTERN immunoblotting ,PROTEIN S deficiency - Abstract
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 PROS1
c.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]- Published
- 2024
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37. Transfer learning to leverage larger datasets for improved prediction of protein stability changes.
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Dieckhaus, Henry, Brocidiacono, Michael, Randolph, Nicholas Z., and Kuhlman, Brian
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PROTEIN stability , *AMINO acid sequence , *DEEP learning , *PROTEIN engineering , *FORECASTING - Abstract
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]
- Published
- 2024
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38. GTP-Bound N-Ras Conformational States and Substates Are Modulated by Membrane and Point Mutation.
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Farcas, Alexandra and Janosi, Lorant
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RAS oncogenes , *RAS proteins , *GTPASE-activating protein , *GUANINE nucleotide exchange factors , *GUANOSINE triphosphate , *ONCOGENIC proteins - Abstract
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]
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- 2024
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39. Naturally occurring PAE206K point mutation in 2009 H1N1 pandemic influenza viruses impairs viral replication at high temperatures.
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Mengmeng Cao, Qiannan Jia, Jinghua Li, Lili Zhao, Li zhu, Yufan Zhang, Shan Li, and Tao Deng
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H1N1 influenza ,INFLUENZA A virus ,POLYMERASES ,VIRAL replication - Abstract
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 PA
E206K 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]- Published
- 2024
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40. TIROSEC: Molecular, Clinical and Histopathological Profile of Papillary Thyroid Carcinoma in a Colombian Cohort.
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Cruz-Romero, Sergio D., González, Sebastián, Juez, José Y., Becerra, David S., Baldión, Ana M., Hakim, José A., González-Devia, Deyanira, Perdomo, Sandra, and Rodríguez-Urrego, Paula A.
- Abstract
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]
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- 2024
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41. Can a Micronutrient Mixture Delay the Onset and Progression of Symptoms of Single-Point Mutation Diseases?
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Prasad, Kedar N and Bondy, Stephen C
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Biomedical and Clinical Sciences ,Nutrition and Dietetics ,Prevention ,Hematology ,Nutrition ,Sickle Cell Disease ,Brain Disorders ,Genetics ,Neurodegenerative ,Rare Diseases ,Neurosciences ,2.1 Biological and endogenous factors ,Aetiology ,Anemia ,Sickle Cell ,Antioxidants ,Hemophilia A ,Humans ,Huntington Disease ,Inflammation ,Micronutrients ,Point Mutation ,Trace Elements ,Single-point mutation diseases ,pattern of inheritance ,oxidative stress ,chronic inflammation ,micronutrients ,Nutrition & Dietetics ,Nutrition and dietetics - Abstract
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.
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- 2022
42. Mosaic de novo SNRPN gene variant associated with Prader-Willi syndrome
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Huang, Yue, Grand, Katheryn, Kimonis, Virginia, Butler, Merlin G, Jain, Suparna, Huang, Alden Yen-Wen, Martinez-Agosto, Julian A, Nelson, Stanley F, and Sanchez-Lara, Pedro A
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Biological Sciences ,Bioinformatics and Computational Biology ,Biomedical and Clinical Sciences ,Genetics ,Rare Diseases ,Congenital Structural Anomalies ,Obesity ,Clinical Research ,Human Genome ,Pediatric ,Brain Disorders ,Intellectual and Developmental Disabilities (IDD) ,2.1 Biological and endogenous factors ,Aetiology ,Child ,Female ,Humans ,Chromosomes ,Human ,Pair 15 ,DNA ,DNA Methylation ,Genomic Imprinting ,Mouth Mucosa ,Prader-Willi Syndrome ,snRNP Core Proteins ,Polymorphism ,Single Nucleotide ,imprinting ,point mutation ,Medical and Health Sciences ,Genetics & Heredity ,Clinical sciences - Abstract
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.
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- 2022
43. The P124A mutation of SRP14 alters its migration on SDS-PAGE without impacting its function
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Liu Yaofu and Zhou Jinqiu
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SRP14 ,proline mutation ,point mutation ,alanine-rich domain ,migration ,Biochemistry ,QD415-436 ,Genetics ,QH426-470 - Abstract
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.
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- 2024
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44. Resistance risk and molecular mechanism associated with resistance to picoxystrobin in Colletotrichum truncatum and Colletotrichum gloeosporioides
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Niu-niu SHI, Jin-pan LIAN, De-zhu QIU, Fu-ru CHEN, and Yi-xin DU
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Colletotrichum truncatum ,Colletotrichum gloeosporioides ,picoxystrobin ,point mutation ,Cyt b ,molecular docking ,Agriculture (General) ,S1-972 - 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
- Published
- 2023
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45. First report of kdr mutations in the voltage-gated sodium channel gene in the arbovirus vector, Aedes aegypti, from Nouakchott, Mauritania
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Mohamed Aly Ould Lemrabott, Sébastien Briolant, Nicolas Gomez, Leonardo Basco, and Ali Ould Mohamed Salem Boukhary
- Subjects
Aedes aegypti ,Mauritania ,Voltage-gated sodium channel ,kdr ,Point mutation ,Pyrethroids ,Infectious and parasitic diseases ,RC109-216 - Abstract
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
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- 2023
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46. Drosophila models used to simulate human ATP1A1 gene mutations that cause Charcot-Marie-Tooth type 2 disease and refractory seizures
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Yao Yuan, Lingqi Yu, Xudong Zhuang, Dongjing Wen, Jin He, Jingmei Hong, Jiayu Xie, Shengan Ling, Xiaoyue Du, Wenfeng Chen, and Xinrui Wang
- Subjects
atp1a1 ,atpα ,bang-sensitive paralysis ,charcot-marie-tooth disease type 2 ,crispr/cas9 ,homology-directed repair ,na+/k+-atpase ,point mutation ,seizures ,sodium pump ,Neurology. Diseases of the nervous system ,RC346-429 - Abstract
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.
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- 2023
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47. Structural basis for impaired 5′ processing of a mutant tRNA associated with defects in neuronal homeostasis
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Lai, Lien B, Lai, Stella M, Szymanski, Eric S, Kapur, Mridu, Choi, Edric K, Al-Hashimi, Hashim M, Ackerman, Susan L, and Gopalan, Venkat
- Subjects
Biological Sciences ,Biomedical and Clinical Sciences ,Genetics ,Neurosciences ,2.1 Biological and endogenous factors ,Aetiology ,Neurological ,Animals ,Base Pairing ,Cerebral Cortex ,Homeostasis ,Magnesium ,Mice ,Models ,Molecular ,Neurons ,Nucleic Acid Conformation ,Point Mutation ,Protein Processing ,Post-Translational ,RNA ,Transfer ,Ribonuclease P ,Substrate Specificity ,tRNA processing ,neurodegeneration ,conformational toggling ,tRNA-Arg-TCT-4-1 - Abstract
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.
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- 2022
48. Development of a High-Resolution Melting Method for the Detection of Clarithromycin-Resistant Helicobacter pylori in the Gastric Microbiome
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Zupeng Kuang, Huishu Huang, Ling Chen, Yanyan Shang, Shixuan Huang, Jun Liu, Jianhui Chen, Xinqiang Xie, Moutong Chen, Lei Wu, He Gao, Hui Zhao, Ying Li, and Qingping Wu
- Subjects
Helicobacter pylori ,clarithromycin ,antibiotic susceptibility testing ,point mutation ,high-resolution melting ,Therapeutics. Pharmacology ,RM1-950 - Abstract
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.
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- 2024
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49. A case of exacerbated encephalopathy with stroke-like episodes and lactic acidosis triggered by metformin in a patient with MELAS.
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Shin, Hui Jin, Na, Ji-Hoon, and Lee, Young-Mock
- Subjects
- *
MELAS syndrome , *LACTIC acidosis , *METFORMIN , *MITOCHONDRIAL DNA , *BRAIN diseases , *GENETIC disorders - Abstract
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]
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- 2024
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50. Identification of novel HPFH-like mutations by CRISPR base editing that elevate the expression of fetal hemoglobin
- Author
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Ravi, Nithin Sam, Wienert, Beeke, Wyman, Stacia K, Bell, Henry William, George, Anila, Mahalingam, Gokulnath, Vu, Jonathan T, Prasad, Kirti, Bandlamudi, Bhanu Prasad, Devaraju, Nivedhitha, Rajendiran, Vignesh, Syedbasha, Nazar, Pai, Aswin Anand, Nakamura, Yukio, Kurita, Ryo, Narayanasamy, Muthuraman, Balasubramanian, Poonkuzhali, Thangavel, Saravanabhavan, Marepally, Srujan, Velayudhan, Shaji R, Srivastava, Alok, DeWitt, Mark A, Crossley, Merlin, Corn, Jacob E, and Mohankumar, Kumarasamypet M
- Subjects
Biomedical and Clinical Sciences ,Cardiovascular Medicine and Haematology ,Hematology ,Rare Diseases ,Sickle Cell Disease ,Stem Cell Research ,Stem Cell Research - Nonembryonic - Human ,Genetics ,Aetiology ,2.1 Biological and endogenous factors ,Adenine ,Anemia ,Sickle Cell ,CRISPR-Cas Systems ,Cell Line ,Clustered Regularly Interspaced Short Palindromic Repeats ,Cytosine ,Fetal Hemoglobin ,Gene Editing ,Hematopoietic Stem Cells ,Humans ,Point Mutation ,Promoter Regions ,Genetic ,beta-Globins ,beta-Thalassemia ,gamma-Globins ,base editing ,globin regulation ,HPFH mutations ,beta-hemoglobinopathies ,fetal hemoglobin ,CRISPR ,Cas9 ,Base editing ,Beta hemoglobinopathies ,Fetal hemoglobin ,Globin regulation ,HBGpromoter ,Sickle cell disease ,Beta-thalassemia ,Large deletions ,CD34+HSPCs ,CRISPR/Cas9 ,beta hemoglobinopathies ,beta-thalassemia ,cd34+ hspcs ,chromosomes ,crispr/cas9 ,gene expression ,genetics ,genomics ,hbgpromoter ,hpfh mutations ,large deletions ,sickle cell disease ,Biochemistry and Cell Biology ,Biological sciences ,Biomedical and clinical sciences ,Health sciences - Abstract
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.
- Published
- 2022
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