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1.
Euro Surveill ; 25(39)2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33006300

RESUMO

We found that a single nucleotide polymorphism (SNP) in the nucleoprotein gene of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from a patient interfered with detection in a widely used commercial assay. Some 0.2% of the isolates in the EpiCoV database contain this SNP. Although SARS-CoV-2 was still detected by the other probe in the assay, this underlines the necessity of targeting two independent essential regions of a pathogen for reliable detection.


Assuntos
Betacoronavirus/genética , Técnicas de Laboratório Clínico , Infecções por Coronavirus/diagnóstico , Nucleoproteínas/genética , Pandemias , Pneumonia Viral/diagnóstico , Mutação Puntual , Polimorfismo de Nucleotídeo Único , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Virais/genética , Sequência de Bases , Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico/métodos , Busca de Comunicante , Infecções por Coronavirus/virologia , Primers do DNA , Erros de Diagnóstico , Reações Falso-Negativas , Feminino , Genes Virais , Humanos , Pessoa de Meia-Idade , Nasofaringe/virologia , Nucleoproteínas/análise , Filogenia , Pneumonia Viral/virologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Romênia , Doença Relacionada a Viagens , Proteínas Virais/análise
2.
Beijing Da Xue Xue Bao Yi Xue Ban ; 52(5): 971-974, 2020 Oct 18.
Artigo em Chinês | MEDLINE | ID: mdl-33047739

RESUMO

The rearrangement of the gene encoding the transcription factor ETS-related gene (ERG) is thought to play a key role in the development of prostate cancer. However, the studies on the ERG mutations have been rarely reported in non-small cell lung carcinoma (NSCLC). Here, we reported genetic features regarding a case of a 68-year-old male patient who presented the primary synchronous multiple tumor lesions in the separated lungs. The patient was hospitalized due to the presence of tumor lesions at the right and left lungs revealed by a chest computerized tomography (CT) scan. After conducting lobectomies at the both lungs, the tumor nodules were all removed, and the histological analysis suggested adenocarcinoma at the both tumor lesions. The patient was diagnosed with synchronous multiple primary lung cancer (SMPLC) based on Martini-Melamed criteria and American College of Chest Physicians practice guidelines. An exome analysis of 315 genes in the two tumor lesions and a non-tumor lesion was conducted by using Illumina Nextseq500 platform from each tumor region to decipher a potential evolutional progress of SMPLC. Single or pair-end reads were first mapped to a human genome reference and filtered based on the mapping quality score. The read depth was ≥ 1 000× and the depth of coverage was 95%. The data revealed a discordant epidermal growth factor receptor (EGFR) from the separate lungs; additionally, a high frequency of point mutation on exon 9 H310P of the ERG gene was detected at the both sites of the tumor lesions. This case showed that a potential role of the molecular features analysis from each tumor lesion might contribute to the understanding of the evolutional development of SMPLC. This study suggests that the same environment may contribute certain gene(s) mutations in the same sites in the early stages of polyclonal tumor origins; meanwhile the extensive studies on these genes may help us understand the evolution and progress of tumor clones.


Assuntos
Adenocarcinoma , Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Neoplasias Primárias Múltiplas , Idoso , Humanos , Neoplasias Pulmonares/genética , Masculino , Neoplasias Primárias Múltiplas/genética , Mutação Puntual , Regulador Transcricional ERG
3.
Proc Natl Acad Sci U S A ; 117(38): 23652-23662, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32868447

RESUMO

The magnitude of the COVID-19 pandemic underscores the urgency for a safe and effective vaccine. Many vaccine candidates focus on the Spike protein, as it is targeted by neutralizing antibodies and plays a key role in viral entry. Here we investigate the diversity seen in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequences and compare it to the sequence on which most vaccine candidates are based. Using 18,514 sequences, we perform phylogenetic, population genetics, and structural bioinformatics analyses. We find limited diversity across SARS-CoV-2 genomes: Only 11 sites show polymorphisms in >5% of sequences; yet two mutations, including the D614G mutation in Spike, have already become consensus. Because SARS-CoV-2 is being transmitted more rapidly than it evolves, the viral population is becoming more homogeneous, with a median of seven nucleotide substitutions between genomes. There is evidence of purifying selection but little evidence of diversifying selection, with substitution rates comparable across structural versus nonstructural genes. Finally, the Wuhan-Hu-1 reference sequence for the Spike protein, which is the basis for different vaccine candidates, matches optimized vaccine inserts, being identical to an ancestral sequence and one mutation away from the consensus. While the rapid spread of the D614G mutation warrants further study, our results indicate that drift and bottleneck events can explain the minimal diversity found among SARS-CoV-2 sequences. These findings suggest that a single vaccine candidate should be efficacious against currently circulating lineages.


Assuntos
Betacoronavirus/genética , Genoma Viral , Vacinas Virais/genética , Betacoronavirus/imunologia , Infecções por Coronavirus/genética , Infecções por Coronavirus/prevenção & controle , Variação Genética , Humanos , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Mutação Puntual , Seleção Genética
4.
PLoS One ; 15(8): e0237300, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32785274

RESUMO

The outbreak of COVID-19 across the world has posed unprecedented and global challenges on multiple fronts. Most of the vaccine and drug development has focused on the spike proteins and viral RNA-polymerases and main protease for viral replication. Using the bioinformatics and structural modelling approach, we modelled the structure of the envelope (E)-protein of novel SARS-CoV-2. The E-protein of this virus shares sequence similarity with that of SARS- CoV-1, and is highly conserved in the N-terminus regions. Incidentally, compared to spike proteins, E proteins demonstrate lower disparity and mutability among the isolated sequences. Using homology modelling, we found that the most favorable structure could function as a gated ion channel conducting H+ ions. Combining pocket estimation and docking with water, we determined that GLU 8 and ASN 15 in the N-terminal region were in close proximity to form H-bonds which was further validated by insertion of the E protein in an ERGIC-mimic membrane. Additionally, two distinct "core" structures were visible, the hydrophobic core and the central core, which may regulate the opening/closing of the channel. We propose this as a mechanism of viral ion channeling activity which plays a critical role in viral infection and pathogenesis. In addition, it provides a structural basis and additional avenues for vaccine development and generating therapeutic interventions against the virus.


Assuntos
Betacoronavirus/química , Infecções por Coronavirus/prevenção & controle , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Betacoronavirus/isolamento & purificação , Simulação por Computador , Infecções por Coronavirus/virologia , Humanos , Hidrogênio , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Pneumonia Viral/virologia , Mutação Puntual , Conformação Proteica , Homologia Estrutural de Proteína , Vacinas Atenuadas , Vacinas de Produtos Inativados , Proteínas do Envelope Viral/imunologia , Vacinas Virais , Água/química
5.
Nat Commun ; 11(1): 4147, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32811837

RESUMO

Mutated receptor tyrosine kinases (MT-RTKs) such as internal tandem duplication of FMS-like tyrosine kinase 3 (FLT3 ITD) and a point mutation KIT D816V are driver mutations for acute myeloid leukemia (AML). Clathrin assembly lymphoid myeloid leukemia protein (CALM) regulates intracellular transport of RTKs, however, the precise role for MT-RTKs remains elusive. We here show that CALM knock down leads to severely impaired FLT3 ITD- or KIT D814V-dependent cell growth compared to marginal influence on wild-type FLT3- or KIT-mediated cell growth. An antipsychotic drug chlorpromazine (CPZ) suppresses the growth of primary AML samples, and human CD34+CD38- AML cells including AML initiating cells with MT-RTKs in vitro and in vivo. Mechanistically, CPZ reduces CALM protein at post transcriptional level and perturbs the intracellular localization of MT-RTKs, thereby blocking their signaling. Our study presents a therapeutic strategy for AML with MT-RTKs by altering the intracellular localization of MT-RTKs using CPZ.


Assuntos
Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Clorpromazina/farmacologia , Leucemia Mieloide Aguda/genética , Proteínas Monoméricas de Montagem de Clatrina/metabolismo , Proteínas Proto-Oncogênicas c-kit/genética , Tirosina Quinase 3 Semelhante a fms/genética , Tirosina Quinase 3 Semelhante a fms/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Proliferação de Células/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Feminino , Células HL-60 , Humanos , Leucemia Mieloide Aguda/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Proteínas Monoméricas de Montagem de Clatrina/genética , Mutação Puntual , Transdução de Sinais/efeitos dos fármacos , Sequências de Repetição em Tandem/genética , Transplante Heterólogo , Adulto Jovem
6.
PLoS Genet ; 16(8): e1008941, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32760060

RESUMO

Apolipoprotein B-containing lipoproteins (B-lps) are essential for the transport of hydrophobic dietary and endogenous lipids through the circulation in vertebrates. Zebrafish embryos produce large numbers of B-lps in the yolk syncytial layer (YSL) to move lipids from yolk to growing tissues. Disruptions in B-lp production perturb yolk morphology, readily allowing for visual identification of mutants with altered B-lp metabolism. Here we report the discovery of a missense mutation in microsomal triglyceride transfer protein (Mtp), a protein that is essential for B-lp production. This mutation of a conserved glycine residue to valine (zebrafish G863V, human G865V) reduces B-lp production and results in yolk opacity due to aberrant accumulation of cytoplasmic lipid droplets in the YSL. However, this phenotype is milder than that of the previously reported L475P stalactite (stl) mutation. MTP transfers lipids, including triglycerides and phospholipids, to apolipoprotein B in the ER for B-lp assembly. In vitro lipid transfer assays reveal that while both MTP mutations eliminate triglyceride transfer activity, the G863V mutant protein unexpectedly retains ~80% of phospholipid transfer activity. This residual phospholipid transfer activity of the G863V mttp mutant protein is sufficient to support the secretion of small B-lps, which prevents intestinal fat malabsorption and growth defects observed in the mttpstl/stl mutant zebrafish. Modeling based on the recent crystal structure of the heterodimeric human MTP complex suggests the G865V mutation may block triglyceride entry into the lipid-binding cavity. Together, these data argue that selective inhibition of MTP triglyceride transfer activity may be a feasible therapeutic approach to treat dyslipidemia and provide structural insight for drug design. These data also highlight the power of yolk transport studies to identify proteins critical for B-lp biology.


Assuntos
Proteínas de Transporte/genética , Lipídeos/genética , Lipoproteínas/genética , Triglicerídeos/genética , Animais , Fígado Gorduroso/genética , Fígado Gorduroso/patologia , Trato Gastrointestinal/metabolismo , Humanos , Imunoprecipitação , Gotículas Lipídicas/metabolismo , Lipoproteínas/metabolismo , Mutação de Sentido Incorreto/genética , Mutação Puntual/genética , Transporte Proteico/genética , Triglicerídeos/metabolismo , Peixe-Zebra/genética
7.
J Chin Med Assoc ; 83(8): 725-732, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32773643

RESUMO

BACKGROUND: The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused severe pneumonia at December 2019. Since then, it has been wildly spread from Wuhan, China, to Asia, European, and United States to become the pandemic worldwide. Now coronavirus disease 2019 were globally diagnosed over 3 084 740 cases with mortality of 212 561 toll. Current reports variants are found in SARS-CoV-2, majoring in functional ribonucleic acid (RNA) to transcribe into structural proteins as transmembrane spike (S) glycoprotein and the nucleocapsid (N) protein holds the virus RNA genome; the envelope (E) and membrane (M) alone with spike protein form viral envelope. The nonstructural RNA genome includes ORF1ab, ORF3, ORF6, 7a, 8, and ORF10 with highly conserved information for genome synthesis and replication in ORF1ab. METHODS: We apply genomic alignment analysis to observe SARS-CoV-2 sequences from GenBank (http://www.ncbi.nim.nih.gov/genebank/): MN 908947 (China, C1); MN985325 (United States: WA, UW); MN996527 (China, C2); MT007544 (Australia: Victoria, A1); MT027064 (United States: CA, UC); MT039890 (South Korea, K1); MT066175 (Taiwan, T1); MT066176 (Taiwan, T2); LC528232 (Japan, J1); and LC528233 (Japan, J2) and Global Initiative on Sharing All Influenza Data database (https://www.gisaid.org). We adopt Multiple Sequence Alignments web from Clustalw (https://www.genome.jp/tools-bin/clustalw) and Geneious web (https://www.geneious.com. RESULTS: We analyze database by genome alignment search for nonstructural ORFs and structural E, M, N, and S proteins. Mutations in ORF1ab, ORF3, and ORF6 are observed; specific variants in spike region are detected. CONCLUSION: We perform genomic analysis and comparative multiple sequence of SARS-CoV-2. Large scaling sequence alignments trace to localize and catch different mutant strains in United possibly to transmit severe deadly threat to humans. Studies about the biological symptom of SARS-CoV-2 in clinic animal and humans will be applied and manipulated to find mechanisms and shield the light for understanding the origin of pandemic crisis.


Assuntos
Betacoronavirus/genética , Genoma Viral , Fases de Leitura Aberta , Glicoproteína da Espícula de Coronavírus/fisiologia , Humanos , Filogenia , Mutação Puntual , Glicoproteína da Espícula de Coronavírus/genética
8.
Ann Hematol ; 99(10): 2393-2404, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32803312

RESUMO

The prognostic impacts of BCR-ABL1 fusion gene mutations in Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph + ALL) remain unknown. Using data from a nationwide Japanese registry, we have evaluated the prognostic impact of BCR-ABL1 mutations prior to the first allogeneic hematopoietic cell transplantation (HCT). The cohort included 289 patients with a median of 48 years of age (range: 16-70). Point mutations were detected in 110 patients. Of these, 90 (82%) harbored T315I mutations, while 20 had other mutations. With a median follow-up period of 29 months (range: 1-125), outcomes after 2 years were worse with mutations than without (overall survival [OS]: 34% vs 68%, p < 0.001; relapse rate [RR]: 48% vs 18%, p < 0.001), particularly with the presence of the T315I mutation (OS: 29% vs 68%, p < 0.001; RR: 54% vs 18%, p < 0.001). OS was significantly worse in the T315I group even among the cohort with hematological (p < 0.001) or molecular complete remission (p = 0.025) as compared to the no mutation group. Multivariate analysis determined the prognostic impact of the T315I mutation (OS: hazard ratio [HR] = 2.19, 95% confidence interval [CI]: 1.5-3.3, p < 0.001; RR: HR = 2.51, 95% CI: 1.5-4.2, p < 0.001). This study is the first to report on the prognostic significance of BCR-ABL1 mutations in Ph + ALL.


Assuntos
Proteínas de Fusão bcr-abl/genética , Transplante de Células-Tronco Hematopoéticas , Mutação de Sentido Incorreto , Cromossomo Filadélfia , Mutação Puntual , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Adolescente , Adulto , Idoso , Aloenxertos , Antineoplásicos/uso terapêutico , Terapia Combinada , Intervalo Livre de Doença , Feminino , Seguimentos , Humanos , Japão/epidemiologia , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidade , Prognóstico , Inibidores de Proteínas Quinases/uso terapêutico , Recidiva , Sistema de Registros , Estudos Retrospectivos , Resultado do Tratamento , Adulto Jovem
9.
Bioresour Technol ; 317: 124024, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32836036

RESUMO

To enhance the biomass conversion efficiency, the R17L mutant of the lytic polysaccharide monooxygenase (LPMO) MtC1LPMO with improved catalytic efficiency was constructed via rational point mutation based on the HotSpot Wizard 3.0 and dezyme web servers. Compared with the wild-type (WT) MtC1LPMO, R17L exhibited a 1.8-fold increase of specific activity and 1.92-fold increase of catalytic efficiency (kcat/Km). The degree of increase of the reducing sugar yield from microcrystalline cellulose and three plant biomass materials during synergistic hydrolysis using cellulase in combination with R17L was about 2 times higher than with the WT. Molecular dynamics simulations revealed that the R17L mutation reduced the stability of the region R18-I36, which then weakened the direct interactions between region N24-V31 and the substrate cellohexaose. Consequently, the deflection time of the cellohexaose conformation in R17L was prolonged compared to the WT, which enhanced its catalytic efficiency.


Assuntos
Simulação de Dinâmica Molecular , Mutação Puntual , Biomassa , Lignina
10.
Nat Commun ; 11(1): 4319, 2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32859923

RESUMO

Disrupted energy metabolism drives cell dysfunction and disease, but approaches to increase or preserve ATP are lacking. To generate a comprehensive metabolic map of genes and pathways that regulate cellular ATP-the ATPome-we conducted a genome-wide CRISPR interference/activation screen integrated with an ATP biosensor. We show that ATP level is modulated by distinct mechanisms that promote energy production or inhibit consumption. In our system HK2 is the greatest ATP consumer, indicating energy failure may not be a general deficiency in producing ATP, but rather failure to recoup the ATP cost of glycolysis and diversion of glucose metabolites to the pentose phosphate pathway. We identify systems-level reciprocal inhibition between the HIF1 pathway and mitochondria; glycolysis-promoting enzymes inhibit respiration even when there is no glycolytic ATP production, and vice versa. Consequently, suppressing alternative metabolism modes paradoxically increases energy levels under substrate restriction. This work reveals mechanisms of metabolic control, and identifies therapeutic targets to correct energy failure.


Assuntos
Trifosfato de Adenosina/metabolismo , Redes e Vias Metabólicas/genética , Redes e Vias Metabólicas/fisiologia , Trifosfato de Adenosina/genética , Sistemas CRISPR-Cas , Linhagem Celular , Metabolismo Energético , Feminino , Fibroblastos , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Glucose/metabolismo , Glicólise/fisiologia , Hexoquinase/genética , Hexoquinase/metabolismo , Humanos , Células K562 , Metabolômica , Mitocôndrias/metabolismo , Via de Pentose Fosfato , Mutação Puntual
11.
Proc Natl Acad Sci U S A ; 117(35): 21299-21307, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32817557

RESUMO

Densely O-glycosylated mucin domains are found in a broad range of cell surface and secreted proteins, where they play key physiological roles. In addition, alterations in mucin expression and glycosylation are common in a variety of human diseases, such as cancer, cystic fibrosis, and inflammatory bowel diseases. These correlations have been challenging to uncover and establish because tools that specifically probe mucin domains are lacking. Here, we present a panel of bacterial proteases that cleave mucin domains via distinct peptide- and glycan-based motifs, generating a diverse enzymatic toolkit for mucin-selective proteolysis. By mutating catalytic residues of two such enzymes, we engineered mucin-selective binding agents with retained glycoform preferences. StcEE447D is a pan-mucin stain derived from enterohemorrhagic Escherichia coli that is tolerant to a wide range of glycoforms. BT4244E575A derived from Bacteroides thetaiotaomicron is selective for truncated, asialylated core 1 structures commonly associated with malignant and premalignant tissues. We demonstrated that these catalytically inactive point mutants enable robust detection and visualization of mucin-domain glycoproteins by flow cytometry, Western blot, and immunohistochemistry. Application of our enzymatic toolkit to ascites fluid and tissue slices from patients with ovarian cancer facilitated characterization of patients based on differences in mucin cleavage and expression patterns.


Assuntos
Mucinas/análise , Polissacarídeo-Liase/metabolismo , Adenocarcinoma/química , Motivos de Aminoácidos , Western Blotting , Feminino , Citometria de Fluxo , Humanos , Neoplasias Ovarianas/química , Mutação Puntual , Polissacarídeo-Liase/química , Polissacarídeo-Liase/genética
12.
Nat Commun ; 11(1): 3382, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32636381

RESUMO

The Stimulator of Interferon Genes (STING) pathway initiates potent immune responses upon recognition of DNA. To initiate signaling, serine 365 (S365) in the C-terminal tail (CTT) of STING is phosphorylated, leading to induction of type I interferons (IFNs). Additionally, evolutionary conserved responses such as autophagy also occur downstream of STING, but their relative importance during in vivo infections remains unclear. Here we report that mice harboring a serine 365-to-alanine (S365A) mutation in STING are unexpectedly resistant to Herpes Simplex Virus (HSV)-1, despite lacking STING-induced type I IFN responses. By contrast, resistance to HSV-1 is abolished in mice lacking the STING CTT, suggesting that the STING CTT initiates protective responses against HSV-1, independently of type I IFNs. Interestingly, we find that STING-induced autophagy is a CTT- and TBK1-dependent but IRF3-independent process that is conserved in the STING S365A mice. Thus, interferon-independent functions of STING mediate STING-dependent antiviral responses in vivo.


Assuntos
Herpes Simples/imunologia , Fator Regulador 3 de Interferon/imunologia , Interferon Tipo I/imunologia , Proteínas de Membrana/genética , Animais , Autofagia , Feminino , Herpesvirus Humano 1 , Evasão da Resposta Imune , Macrófagos/imunologia , Masculino , Proteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Mutantes , Mutação Puntual , Transdução de Sinais
13.
PLoS One ; 15(7): e0235641, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32614888

RESUMO

We sequenced 25 isolates of phenotypically multidrug-resistant Salmonella Indiana (n = 11), Typhimurium (n = 8), and Enteritidis (n = 6) using both MinION long-read [SQK-LSK109 and flow cell (R9.4.1)] and MiSeq short-read (Nextera XT and MiSeq Reagent Kit v2) sequencing technologies to determine the advantages of each approach in terms of the characteristics of genome structure, antimicrobial resistance (AMR), virulence potential, whole-genome phylogeny, and pan-genome. The MinION reads were base-called in real-time using MinKnow 3.4.8 integrated with Guppy 3.0.7. The long-read-only assembly, Illumina-only assembly, and hybrid assembly pipelines of Unicycler 0.4.8 were used to generate the MinION, MiSeq, and hybrid assemblies, respectively. The MinION assemblies were highly contiguous compared to the MiSeq assemblies but lacked accuracy, a deficiency that was mitigated by adding the MiSeq short reads through the Unicycler hybrid assembly which corrected erroneous single nucleotide polymorphisms (SNPs). The MinION assemblies provided similar predictions of AMR and virulence potential compared to the MiSeq and hybrid assemblies, although they produced more total false negatives of AMR genotypes, primarily due to failure in identifying tetracycline resistance genes in 11 of the 19 MinION assemblies of tetracycline-resistant isolates. The MinION assemblies displayed a large genetic distance from their corresponding MiSeq and hybrid assemblies on the whole-genome phylogenetic tree, indicating that the lower read accuracy of MinION sequencing caused incorrect clustering. The pan-genome of the MinION assemblies contained significantly more accessory genes and less core genes compared to the MiSeq and hybrid assemblies, suggesting that although these assemblies were more contiguous, their sequencing errors reduced accurate genome annotations. Our research demonstrates that MinION sequencing by itself provides an efficient assessment of the genome structure, antimicrobial resistance, and virulence potential of Salmonella; however, it is not sufficient for whole-genome phylogenetic and pan-genome analyses. MinION in combination with MiSeq facilitated the most accurate genomic analyses.


Assuntos
Farmacorresistência Bacteriana Múltipla/genética , Genoma Bacteriano , Salmonella enterica/genética , Sequenciamento Completo do Genoma/métodos , Antibacterianos/farmacologia , Genótipo , Testes de Sensibilidade Microbiana , Fenótipo , Filogenia , Plasmídeos/genética , Plasmídeos/metabolismo , Mutação Puntual , Salmonella enterica/classificação , Salmonella enterica/efeitos dos fármacos , Salmonella enterica/patogenicidade , Salmonella enteritidis/classificação , Salmonella enteritidis/efeitos dos fármacos , Salmonella enteritidis/genética , Salmonella enteritidis/patogenicidade , Salmonella typhimurium/classificação , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/genética , Salmonella typhimurium/patogenicidade , Virulência
14.
Arterioscler Thromb Vasc Biol ; 40(9): 2143-2158, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32640903

RESUMO

OBJECTIVE: ERα (estrogen receptor alpha) exerts nuclear genomic actions and also rapid membrane-initiated steroid signaling. The mutation of the cysteine 451 into alanine in vivo has recently revealed the key role of this ERα palmitoylation site on some vasculoprotective actions of 17ß-estradiol (E2) and fertility. Here, we studied the in vivo role of the arginine 260 of ERα which has also been described to be involved in its E2-induced rapid signaling with PI-3K (phosphoinositide 3-kinase) as well as G protein in cultured cell lines. Approach and Results: We generated a mouse model harboring a point mutation of the murine counterpart of this arginine into alanine (R264A-ERα). In contrast to the C451A-ERα, the R264A-ERα females are fertile with standard hormonal serum levels and normal control of hypothalamus-pituitary ovarian axis. Although R264A-ERα protein abundance was normal, the well-described membrane ERα-dependent actions of estradiol, such as the rapid dilation of mesenteric arteries and the acceleration of endothelial repair of carotid, were abrogated in R264A-ERα mice. In striking contrast, E2-regulated gene expression was highly preserved in the uterus and the aorta, revealing intact nuclear/genomic actions in response to E2. Consistently, 2 recognized nuclear ERα-dependent actions of E2, namely atheroma prevention and flow-mediated arterial remodeling were totally preserved. CONCLUSIONS: These data underline the exquisite role of arginine 264 of ERα for endothelial membrane-initiated steroid signaling effects of E2 but not for nuclear/genomic actions. This provides the first model of fertile mouse with no overt endocrine abnormalities with specific loss-of-function of rapid ERα signaling in vascular functions.


Assuntos
Lesões das Artérias Carótidas/tratamento farmacológico , Endotélio Vascular/efeitos dos fármacos , Estradiol/farmacologia , Receptor alfa de Estrogênio/agonistas , Terapia de Reposição de Estrogênios , Estrogênios/farmacologia , Fertilidade/efeitos dos fármacos , Artérias Mesentéricas/efeitos dos fármacos , Mutação Puntual , Animais , Aterosclerose/metabolismo , Aterosclerose/patologia , Aterosclerose/prevenção & controle , Lesões das Artérias Carótidas/metabolismo , Lesões das Artérias Carótidas/patologia , Lesões das Artérias Carótidas/fisiopatologia , Proliferação de Células/efeitos dos fármacos , Endotélio Vascular/lesões , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiopatologia , Ativação Enzimática , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Ciclo Estral/efeitos dos fármacos , Feminino , Masculino , Artérias Mesentéricas/metabolismo , Artérias Mesentéricas/fisiopatologia , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo III/metabolismo , Ovariectomia , Reepitelização/efeitos dos fármacos , Transdução de Sinais , Fatores de Tempo , Útero/efeitos dos fármacos , Útero/metabolismo , Remodelação Vascular/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos
15.
Nucleic Acids Res ; 48(14): 7856-7863, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32619224

RESUMO

The Fragile X-related disorders (FXDs) are Repeat Expansion Diseases, genetic disorders that result from the expansion of a disease-specific microsatellite. In those Repeat Expansion Disease models where it has been examined, expansion is dependent on functional mismatch repair (MMR) factors, including MutLγ, a heterodimer of MLH1/MLH3, one of the three MutL complexes found in mammals and a minor player in MMR. In contrast, MutLα, a much more abundant MutL complex that is the major contributor to MMR, is either not required for expansion or plays a limited role in expansion in many model systems. How MutLγ acts to generate expansions is unclear given its normal role in protecting against microsatellite instability and while MLH3 does have an associated endonuclease activity, whether that contributes to repeat expansion is uncertain. We show here, using a gene-editing approach, that a point mutation that eliminates the endonuclease activity of MLH3 eliminates expansions in an FXD mouse embryonic stem cell model. This restricts the number of possible models for repeat expansion and supports the idea that MutLγ may be a useful druggable target to reduce somatic expansion in those disorders where it contributes to disease pathology.


Assuntos
Síndrome do Cromossomo X Frágil/genética , Proteínas MutL/genética , Expansão das Repetições de Trinucleotídeos , Alelos , Animais , Linhagem Celular , Modelos Animais de Doenças , Masculino , Camundongos , Mutação Puntual , Domínios Proteicos/genética , Células-Tronco
16.
Nat Commun ; 11(1): 3790, 2020 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-32728032

RESUMO

Menthol in mints elicits coolness sensation by selectively activating TRPM8 channel. Although structures of TRPM8 were determined in the apo and liganded states, the menthol-bounded state is unresolved. To understand how menthol activates the channel, we docked menthol to the channel and systematically validated our menthol binding models with thermodynamic mutant cycle analysis. We observed that menthol uses its hydroxyl group as a hand to specifically grab with R842, and its isopropyl group as legs to stand on I846 and L843. By imaging with fluorescent unnatural amino acid, we found that menthol binding induces wide-spread conformational rearrangements within the transmembrane domains. By Φ analysis based on single-channel recordings, we observed a temporal sequence of conformational changes in the S6 bundle crossing and the selectivity filter leading to channel activation. Therefore, our study suggested a 'grab and stand' mechanism of menthol binding and how menthol activates TRPM8 at the atomic level.


Assuntos
Ativação do Canal Iônico/efeitos dos fármacos , Mentol/farmacologia , Canais de Cátion TRPM/agonistas , Sítios de Ligação/genética , Células HEK293 , Humanos , Mentol/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mutagênese , Técnicas de Patch-Clamp , Mutação Puntual , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Canais de Cátion TRPM/química , Canais de Cátion TRPM/genética
17.
Am J Kidney Dis ; 76(3): 384-391, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32660897

RESUMO

RATIONALE & OBJECTIVE: Fibrinogen A α-chain amyloidosis (AFib amyloidosis) is a form of amyloidosis resulting from mutations in the fibrinogen A α-chain gene (FGA), causing progressive kidney disease leading to kidney failure. Treatment may include kidney transplantation (KT) or liver-kidney transplantation (LKT), but it is not clear what factors should guide this decision. The aim of this study was to characterize the natural history and long-term outcomes of this disease, with and without organ transplantation, among patients with AFib amyloidosis and various FGA variants. STUDY DESIGN: Case series. SETTING & PARTICIPANTS: 32 patients with AFib amyloidosis diagnosed by genetic testing in France between 1983 and 2014, with a median follow-up of 93 (range, 4-192) months, were included. RESULTS: Median age at diagnosis was 51.5 (range, 12-77) years. Clinical presentation consisted of proteinuria (93%), hypertension (83%), and kidney failure (68%). Manifestations of kidney disease appeared on average at age 57 (range, 36-77) years in patients with the E526V variant, at age 45 (range, 12-59) years in those with the R554L variant (P<0.001), and at age 24.5 (range, 12-31) years in those with frameshift variants (P<0.001). KT was performed in 15 patients and LKT was performed in 4. In KT patients with the E526V variant, recurrence of AFib amyloidosis in the kidney graft was less common than with a non-E526V (R554L or frameshift) variant (22% vs 83%; P=0.03) and led to graft loss less frequently (33% vs 100%). Amyloid recurrence was not observed in patients after LKT. LIMITATIONS: Analyses were based on clinically available historical data. Small number of patients with non-E526V and frameshift variants. CONCLUSIONS: Our study suggests phenotypic variability in the natural history of AFib amyloidosis, depending on the FGA mutation type. KT appears to be a viable option for patients with the most common E526V variant, whereas LKT may be a preferred option for patients with frameshift variants.


Assuntos
Amiloidose Familiar/cirurgia , Fibrinogênio/genética , Transplante de Rim , Transplante de Fígado , Adolescente , Adulto , Idoso , Amiloidose Familiar/genética , Amiloidose Familiar/patologia , Criança , Terapia Combinada , Progressão da Doença , Feminino , Seguimentos , Mutação da Fase de Leitura , França/epidemiologia , Estudos de Associação Genética , Humanos , Rim/patologia , Falência Renal Crônica/epidemiologia , Falência Renal Crônica/etiologia , Falência Renal Crônica/cirurgia , Falência Renal Crônica/terapia , Transplante de Rim/estatística & dados numéricos , Transplante de Fígado/estatística & dados numéricos , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Mutação Puntual , Diálise Renal , Resultado do Tratamento , Adulto Jovem
18.
Exp Parasitol ; 217: 107957, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32687847

RESUMO

The ruminant livestock production sector is under threat due to the infections with gastrointestinal nematode parasites and the subsequent development of anthelmintic resistance. One of most common and pathogenic species in small ruminants is Haemonchus contortus. The ability to control the infections with this and other gastrointestinal nematodes relies heavily on the use of anthelmintic drugs. Although resistance to all major classes of anthelmintics has been shown in H. contortus, the precise mechanism of resistance acquisition is only known for benzimidazoles. F200Y (TAC) is a common point mutation in the isotype 1 ß tubulin gene which is associated with an effective increase in the resistance towards benzimidazole drugs. Here, we show the utility of using this mutation as a marker in a droplet digital PCR assay to track how two H. contortus laboratory strains, characterized by different resistance levels, change with respect to this mutation, when subjected to increasing concentrations of thiabendazole. Additionally, we wanted to investigate whether exposure to a discriminating dose of thiabendazole in the egg hatch test resulted in the death of all H. contortus eggs with a susceptible genotype. We found the MHco5 strain to maintain an overall higher frequency of the F200Y mutation (80-100%) over all drug concentrations, whilst a steady, gradual increase from around 30%-60% was observed in the case of the MHco4 strain. This is further supported by the dose-response curves, displaying a much higher tolerance of the MHco5 strain (LD50 = 0.38 µg/ml) in comparison to the MHco4 strain (LD50 = 0.07 µg/ml) to the effects of thiabendazole. All things considered, we show that the F200Y mutation is still a viable and reliable marker for the detection and surveillance of benzimidazole drug resistance in H. contortus in Europe.


Assuntos
Anti-Helmínticos/farmacologia , Haemonchus/genética , Taxa de Mutação , Tiabendazol/farmacologia , Tubulina (Proteína)/genética , Animais , DNA de Helmintos/isolamento & purificação , Relação Dose-Resposta a Droga , Resistência a Medicamentos/genética , Frequência do Gene , Marcadores Genéticos , Genótipo , Hemoncose/parasitologia , Hemoncose/veterinária , Haemonchus/classificação , Haemonchus/efeitos dos fármacos , Dose Letal Mediana , Óvulo/efeitos dos fármacos , Fenótipo , Mutação Puntual , Reação em Cadeia da Polimerase , Ovinos , Doenças dos Ovinos/parasitologia
19.
BMC Infect Dis ; 20(1): 518, 2020 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-32677920

RESUMO

BACKGROUND: Campylobacter jejuni is a leading cause of bacterial diarrhea worldwide, and increasing rates of fluoroquinolone (FQ) resistance in C. jejuni are a major public health concern. The rapid detection and tracking of FQ resistance are critical needs in developing countries, as these antimicrobials are widely used against C. jejuni infections. Detection of point mutations at T86I in the gyrA gene by real-time polymerase chain reaction (RT-PCR) is a rapid detection tool that may improve FQ resistance tracking. METHODS: C. jejuni isolates obtained from children with diarrhea in Peru were tested by RT-PCR to detect point mutations at T86I in gyrA. Further confirmation was performed by sequencing of the gyrA gene. RESULTS: We detected point mutations at T86I in the gyrA gene in 100% (141/141) of C. jejuni clinical isolates that were previously confirmed as ciprofloxacin-resistant by E-test. No mutations were detected at T86I in gyrA in any ciprofloxacin-sensitive isolates. CONCLUSIONS: Detection of T86I mutations in C. jejuni is a rapid, sensitive, and specific method to identify fluoroquinolone resistance in Peru. This detection approach could be broadly employed in epidemiologic surveillance, therefore reducing time and cost in regions with limited resources.


Assuntos
Infecções por Campylobacter/diagnóstico , Campylobacter jejuni/genética , DNA Girase/genética , Farmacorresistência Bacteriana/genética , Fluoroquinolonas/uso terapêutico , Mutação Puntual , Reação em Cadeia da Polimerase em Tempo Real/métodos , Substituição de Aminoácidos , Infecções por Campylobacter/tratamento farmacológico , Infecções por Campylobacter/microbiologia , Campylobacter jejuni/isolamento & purificação , Criança , Ciprofloxacino/uso terapêutico , Análise Mutacional de DNA/métodos , Diarreia/diagnóstico , Diarreia/tratamento farmacológico , Diarreia/microbiologia , Humanos , Isoleucina/genética , Testes de Sensibilidade Microbiana , Peru , Treonina/genética
20.
BMC Evol Biol ; 20(1): 89, 2020 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-32689942

RESUMO

BACKGROUND: Tumors are widely recognized to progress through clonal evolution by sequentially acquiring selectively advantageous genetic alterations that significantly contribute to tumorigenesis and thus are termned drivers. Some cancer drivers, such as TP53 point mutation or EGFR copy number gain, provide exceptional fitness gains, which, in time, can be sufficient to trigger the onset of cancer with little or no contribution from additional genetic alterations. These key alterations are called superdrivers. RESULTS: In this study, we employ a Wright-Fisher model to study the interplay between drivers and superdrivers in tumor progression. We demonstrate that the resulting evolutionary dynamics follow global clonal expansions of superdrivers with periodic clonal expansions of drivers. We find that the waiting time to the accumulation of a set of superdrivers and drivers in the tumor cell population can be approximated by the sum of the individual waiting times. CONCLUSIONS: Our results suggest that superdriver dynamics dominate over driver dynamics in tumorigenesis. Furthermore, our model allows studying the interplay between superdriver and driver mutations both empirically and theoretically.


Assuntos
Evolução Clonal/genética , Mutação/genética , Evolução Biológica , Progressão da Doença , Humanos , Neoplasias/genética , Mutação Puntual , Fatores de Tempo
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