Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 5.172
Filtrar
1.
Pestic Biochem Physiol ; 158: 77-87, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31378364

RESUMO

Pyrethroid-resistance in onion thrips, Thrips tabaci, has been reported in many countries including Japan. Identifying factors of the resistance is important to correctly monitoring the resistance in field populations. To identify pyrethroid-resistance related genes in T. tabaci in Japan, we performed RNA-Seq analysis of seven T. tabaci strains including two pyrethroid-resistant and five pyrethroid-susceptible strains. We identified a pair of single point mutations, T929I and K1774N, introducing two amino acid mutations, in the voltage-gated sodium channel gene, a pyrethroid target gene, in the two resistant strains. The K1774N is a newly identified mutation located in the fourth repeat domain of the sodium channel. Genotyping analysis of field-collected populations showed that most of the T. tabaci individuals in resistant populations carried the mutation pair, indicating that the mutation pair is closely associated with pyrethroid-resistance in Japan. Another resistance-related mutation, M918L, was also identified in part of the resistant populations. Most of the individuals with the mutation pair were arrhenotokous while all individuals with the M918L single mutation were thelytokous. The result of differentially expressed gene analysis revealed a small number of up-regulated detoxification genes in each resistant strain which might be involved in resistance to pyrethroid. However, no up-regulated detoxification genes common to the two resistant strains were detected. Our results indicate that the mutation pair in the sodium channel gene is the most important target for monitoring pyrethroid-resistance in T. tabaci, and that pyrethroid-resistant arrhenotokous individuals with the mutation pair are likely to be widely distributed in Japan.


Assuntos
Piretrinas/farmacologia , Tisanópteros/efeitos dos fármacos , Tisanópteros/metabolismo , Canais de Sódio Disparados por Voltagem/metabolismo , Animais , Resistência a Inseticidas/genética , Inseticidas/farmacologia , Japão , Mutação/genética , Mutação Puntual/genética , Tisanópteros/genética , Canais de Sódio Disparados por Voltagem/genética
2.
Pestic Biochem Physiol ; 159: 17-21, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31400779

RESUMO

Capsella bursa-pastoris is a serious broadleaf weed in winter wheat fields in China. It has evolved high levels of resistance to acetolactate synthase (ALS) inhibiting herbicides and has caused substantial losses of wheat yield in recent years. We monitored the herbicide resistance of Capsella bursa-pastoris collected from 18 regions of Shandong Province in 2009, 2013 and 2017, respectively. Compared with the 2009 populations, the number of populations resistant to florasulam had increased in 2013 and 2017. Resistance to tribenuron-methyl increased in 2013, but decreased in 2017. The 2009 and 2013 populations developed resistance only to tribenuron-methyl, but some 2017 populations developed cross-resistance to imazethapyr and florasulam as well. Mutations in ALS (Pro-197-Thr/Ser/His/Arg/Leu/Gln) were identified in the 2009 and 2013 populations; however, two ALS mutations (Pro197 and/or Trp574) were identified in 2017 plants. Meanwhile, plants containing both point mutations (Pro197 + Trp574) were identified in the 2017 populations. This study demonstrated that target site gene mutations were the main reason for Capsella bursa-pastoris resistance to ALS-inhibiting herbicides. Although target-site mutation is the reason for resistance to ALS-inhibiting herbicides in Capsella bursa-pastoris, the resistance patterns and mutations identified have changed over time.


Assuntos
Acetolactato Sintase/genética , Capsella/efeitos dos fármacos , Resistência a Herbicidas/genética , Herbicidas/farmacologia , Proteínas de Plantas/genética , Sulfonatos de Arila/farmacologia , Capsella/enzimologia , Capsella/genética , Mutação/genética , Ácidos Nicotínicos/farmacologia , Mutação Puntual/genética , Pirimidinas/farmacologia , Sulfonamidas/farmacologia
3.
Gene ; 714: 143990, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31326550

RESUMO

BACKGROUND: Progressive cardiac conduction defect (PCCD), also known as Lenegre-Lev disease, is one of the most common heart conduction abnormalities. Previous studies have screened for known mutation sites that cause heart block in a 68-person family with a history of PCCD, revealed no mutations. OBJECTIVE: To screen pathogenic genes of the PCCD family and to study the function of the gene mutations related to heart block diseases. METHODS: Whole exome sequencing (WES) was performed on two PCCD patients and one non-PCCD family member to find the related pathogenic gene. After family co-segregation and preliminary functional analysis, we identified the mutant gene CLCA2. To study the function of this gene, we constructed mutant-gene mice using CRISPR-Cas9 technology, and electrocardiogram monitoring was performed after genotype verification. RESULTS: The CLCA2 c.G1725T mutation was identified and co-segregated with the phenotype. The analysis showed that the CLCA2 c.G1725T mutation is harmful and mainly affects protein glycosylation. Immunofluorescence staining revealed that CLCA2 was highly expressed in the sinoatrial node (SAN) tissues. Electrocardiogram monitoring of the mice revealed that CLCA2 point mutations induced mild conduction block and ectopic pacemakers. CONCLUSION: Our findings indicate that a novel heterozygous missense mutation c.G1725T of the CLCA2 gene may be associated with heart block disease and the mutation in this gene may lead to sinus node lesions and conduction blocking.


Assuntos
Canais de Cloreto/genética , Bloqueio Cardíaco/genética , Mutação de Sentido Incorreto/genética , Sequência de Aminoácidos , Animais , Eletrocardiografia/métodos , Feminino , Heterozigoto , Humanos , Masculino , Camundongos , Linhagem , Fenótipo , Mutação Puntual/genética , Nó Sinoatrial/patologia
4.
Pestic Biochem Physiol ; 157: 80-87, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31153480

RESUMO

The European red mite Panonychus ulmi (Koch) is a major pest of apple trees worldwide and causes significant damage to apple orchards in Iran. Pyrethroid insecticides/acaricides, such as fenpropathrin and fenvalerate, are widely used to control P. ulmi, but their long-term use may lead to low efficacy. Earlier studies investigating pyrethroid resistance in closely related mites such as Tetranychus urticae revealed that pyrethroid resistance was associated with point mutations in the voltage-gated sodium channel gene (vgsc). The aim of this study was to investigate the biochemical and molecular mechanisms of fenpropathrin and fenvalerate resistance in Iranian populations of P. ulmi. Pyrethroid toxicity bioassays were carried out on different P. ulmi field populations. Marand (resistance ratio, RR = 149), Maraqeh (RR = 90) and Mianeh2 (RR = 71) populations exhibited high levels of resistance to fenpropathrin, compared to a susceptible field population (Shahin Dej). Resistance was also observed for fenvalerate with resistance ratio's ranging from 2- to 20-fold. Synergism experiments and enzyme activity assays predicted a minor role for classical detoxification enzymes. In contrast, two amino acid substitutions in the VGSC, L1024V and F1538I, that were previously shown to confer pyrethroid resistance, were detected in all three resistant P. ulmi populations and point towards target-site insensitivity as the most likely resistance mechanism. Furthermore, sequencing after cloning of vgsc fragments from single haploid males revealed the presence of multiple copies of vgsc in a highly resistant strain. The link between resistance mutations and vgsc copy number variation should be the subject of future study, as this might be used to develop molecular markers for monitoring pyrethroid resistance of P. ulmi in the field.


Assuntos
Mutação Puntual/genética , Piretrinas/farmacologia , Canais de Sódio Disparados por Voltagem/genética , Animais , Variações do Número de Cópias de DNA/genética , Duplicação Gênica/efeitos dos fármacos , Duplicação Gênica/genética , Resistência a Inseticidas/genética , Irã (Geográfico) , Ácaros , Canais de Sódio Disparados por Voltagem/metabolismo
5.
Nat Commun ; 10(1): 2569, 2019 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-31189880

RESUMO

Synonymous mutations have been viewed as silent mutations, since they only affect the DNA and mRNA, but not the amino acid sequence of the resulting protein. Nonetheless, recent studies suggest their significant impact on splicing, RNA stability, RNA folding, translation or co-translational protein folding. Hence, we compile 659194 synonymous mutations found in human cancer and characterize their properties. We provide the user-friendly, comprehensive resource for synonymous mutations in cancer, SynMICdb ( http://SynMICdb.dkfz.de ), which also contains orthogonal information about gene annotation, recurrence, mutation loads, cancer association, conservation, alternative events, impact on mRNA structure and a SynMICdb score. Notably, synonymous and missense mutations are depleted at the 5'-end of the coding sequence as well as at the ends of internal exons independent of mutational signatures. For patient-derived synonymous mutations in the oncogene KRAS, we indicate that single point mutations can have a relevant impact on expression as well as on mRNA secondary structure.


Assuntos
Bases de Dados de Ácidos Nucleicos , Regulação Neoplásica da Expressão Gênica/genética , Neoplasias/genética , Mutação Silenciosa/genética , Conjuntos de Dados como Assunto , Humanos , Mutação de Sentido Incorreto/genética , Mutação Puntual/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Dobramento de RNA/genética , Processamento de RNA/genética , RNA Mensageiro/química , RNA Mensageiro/genética
6.
Biosens Bioelectron ; 139: 111326, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31129389

RESUMO

In view of their critical function in metastasis, characterization of single circulating tumor cells (CTCs) can provide important clinical information to monitor tumor progression and guide personal therapy. Single-cell genetic analysis methods based on microfluidics have some inherent shortcomings such as complicated operation, low throughput, and expensive equipment requirements. To overcome these barriers, we developed a simple and open micro-well array containing 26,208 units for either nuclear acids or single-cell genetic analysis. Through modification of the polydimethylsiloxane surface and optimization of chip packaging, we addressed protein adsorption and solution evaporation for PCR amplification on a chip. In the detection of epidermal growth factor receptor (EGFR) exon gene 21, this micro-well array demonstrated good linear correlation at a DNA concentration from 1 × 101 to 1 × 105 copies/µL (R2 = 0.9877). We then successfully integrated cell capture, lysis, PCR amplification, and signal read-out on the micro-well array, enabling the rapid and simple genetic analysis of single cells. This device was used to detect duplex EGFR mutation genes of lung cancer cell lines (H1975 and A549 cells) and normal leukocytes, demonstrating the ability to perform high-throughput, massively parallel duplex gene analysis at the single-cell level. Different types of point mutations (EGFR-L858R mutation or EGFR-T790M mutation) were detected in single H1975 cells, further validating the significance of single-cell level gene detection. In addition, this method showed a good performance in the heterogeneity detection of individual CTCs from lung cancer patients, required for micro-invasive cancer monitoring and treatment selection.


Assuntos
Técnicas Biossensoriais , Genes erbB-1/genética , Neoplasias Pulmonares/diagnóstico , Análise de Célula Única/métodos , Humanos , Neoplasias Pulmonares/genética , Células Neoplásicas Circulantes/química , Mutação Puntual/genética
7.
BMJ Case Rep ; 12(3)2019 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-30936345

RESUMO

Leber's hereditary optic neuropathy (LHON) is a mitochondrially inherited disorder characterised by bilateral, painless visual loss which leads to severe optic atrophy. It can be associated with other conditions including multiple sclerosis (MS), movement disorders, epilepsy and cardiac arrhythmias. The association of LHON with an MS-like illness is often referred to as Harding's disease (or Harding's syndrome). We report two siblings, who both harbour the 11 778 mitochondrial DNA (mtDNA) mutation, but who manifest markedly different clinical phenotypes; a male with classical LHON and a female with an MS-like illness. LHON affects males four to five times more often than females. By contrast, Harding's disease is seen predominantly in females, in a pattern comparable to that seen in MS. The pathogenic basis behind the variation in penetrance and phenotype between genders and individual family members remains unclear.


Assuntos
Esclerose Múltipla/fisiopatologia , Atrofia Óptica Hereditária de Leber/fisiopatologia , Mutação Puntual/genética , Transtornos da Visão/etiologia , DNA Mitocondrial/genética , Família , Feminino , Transtornos Neurológicos da Marcha/genética , Transtornos Neurológicos da Marcha/fisiopatologia , Aconselhamento Genético , Humanos , Imunoterapia , Pessoa de Meia-Idade , Esclerose Múltipla/complicações , Esclerose Múltipla/genética , Atrofia Óptica Hereditária de Leber/diagnóstico , Atrofia Óptica Hereditária de Leber/genética , Encaminhamento e Consulta , Distúrbios da Fala/genética , Distúrbios da Fala/fisiopatologia , Resultado do Tratamento , Transtornos da Visão/genética , Transtornos da Visão/fisiopatologia
8.
BMJ Case Rep ; 12(3)2019 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-30936349

RESUMO

Mutations in the nuclear POLG1 gene compromise the integrity of mitochondrial DNA and show great allelic and clinical heterogeneity. Among adult POLG1-associated mitochondrial disease, the main clinical feature is chronic progressive external ophthalmoplegia. Other related clinical manifestations are sensory or cerebellar ataxia, peripheral neuropathy, myopathy or extrapyramidal symptoms. We report the case of a 72-year-old man who presented with a late onset sensory neuronopathy, chronic progressive external ophthalmoplegia, gait ataxia and parkinsonism. Genetic studies showed a compound heterozygosity of known pathogenic mutations in the POLG1 gene (variant T252I/P587 L in cis configuration in allele 1 and variant R807C in allele 2). Late life presentation highlights that mitochondrial disorders should be considered regardless of age of onset of symptoms.


Assuntos
Antiparkinsonianos/uso terapêutico , Polimerase do DNA Mitocondrial/genética , Levodopa/uso terapêutico , Doenças Mitocondriais/diagnóstico , Oftalmoplegia Externa Progressiva Crônica/diagnóstico , Transtornos Parkinsonianos/fisiopatologia , Idade de Início , Idoso , Blefaroptose , Progressão da Doença , Humanos , Masculino , Doenças Mitocondriais/tratamento farmacológico , Doenças Mitocondriais/genética , Doenças Mitocondriais/fisiopatologia , Oftalmoplegia Externa Progressiva Crônica/tratamento farmacológico , Transtornos Parkinsonianos/tratamento farmacológico , Transtornos Parkinsonianos/etiologia , Transtornos Parkinsonianos/genética , Mutação Puntual/genética , Resultado do Tratamento
9.
J Med Case Rep ; 13(1): 63, 2019 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-30837005

RESUMO

BACKGROUND: Mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes, the most common maternally inherited mitochondrial disease, can present with a wide range of neurological manifestations including both central and peripheral nervous system involvement. The most frequent genetic mutation reported in mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes syndrome is A3243G in MT-TL1 gene. Stroke-like episodes, dementia, epilepsy, lactic acidemia, myopathy, recurrent headaches, hearing impairment, diabetes, and short stature constitute the known presentations in this syndrome. Among the abnormal involuntary movements in mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes syndrome, myoclonus is the commonest. Other movement disorders, including chorea, are rarely reported in this disorder. CASE PRESENTATION: A 14-year-old South Asian boy from rural Bengal (India), born of a second degree consanguineous marriage, with normal birth and development history, presented with abnormal brief jerky movements involving his trunk and limbs, with recurrent falls for 10 months. We present here a case of heteroplasmic mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes syndrome with A3251G mutation, in which the clinical picture was dominated by a host of involuntary abnormal movements including chorea-ballism, myoclonus, and oromandibular dystonia in a backdrop of cognitive decline, seizure, and stroke-like episode. A final diagnosis was established by muscle biopsy and genetic study. Haloperidol was administered to control the involuntary movements along with introduction of co-enzyme Q, besides symptomatic management for his focal seizures. Six months into follow-up his seizures and abnormal movements were controlled significantly with slight improvement of cognitive abilities. CONCLUSION: The dominance of hyperkinetic movements in the clinical scenario and the finding of a point mutation A3251G in MT-TL1 gene make this a rare presentation.


Assuntos
Antidiscinéticos/uso terapêutico , Coreia/diagnóstico , DNA Mitocondrial/genética , Haloperidol/uso terapêutico , Síndrome MELAS/diagnóstico , Mutação Puntual/genética , Adolescente , Coreia/genética , Coreia/fisiopatologia , Testes Genéticos , Humanos , Síndrome MELAS/tratamento farmacológico , Síndrome MELAS/genética , Síndrome MELAS/fisiopatologia , Masculino , Micronutrientes/uso terapêutico , Resultado do Tratamento , Ubiquinona/uso terapêutico
10.
Methods Mol Biol ; 1961: 29-44, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30912038

RESUMO

Current genome editing tools enable targeted mutagenesis of selected DNA sequences in many species. However, the efficiency and the type of introduced mutations by the genome editing method are largely dependent on the target site. As a consequence, the outcome of the editing operation is difficult to predict. Therefore, a quick assay to quantify the frequency of mutations is vital for a proper assessment of genome editing actions. We developed two methods that are rapid, cost-effective, and readily applicable: (1) TIDE, which can accurately identify and quantify insertions and deletions (indels) that arise after introduction of double strand breaks (DSBs); (2) TIDER, which is suited for template-mediated editing events including point mutations. Both methods only require a set of PCR reactions and standard Sanger sequencing runs. The sequence traces are analyzed by the TIDE or TIDER algorithm (available at https://tide.nki.nl or https://deskgen.com ). The routine is easy, fast, and provides much more detailed information than current enzyme-based assays. TIDE and TIDER accelerate testing and designing of DSB-based genome editing strategies.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Edição de Genes/métodos , Quebras de DNA de Cadeia Dupla , Mutação INDEL/genética , Mutagênese , Mutação/genética , Mutação Puntual/genética
11.
Mol Cell ; 74(2): 320-329.e6, 2019 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-30853402

RESUMO

Xenophagy, a selective autophagy pathway that protects the cytosol against bacterial invasion, relies on cargo receptors that juxtapose bacteria and phagophore membranes. Whether phagophores are recruited from a constitutive pool or are generated de novo at prospective cargo remains unknown. Phagophore formation in situ would require recruitment of the upstream autophagy machinery to prospective cargo. Here, we show that, essential for anti-bacterial autophagy, the cargo receptor NDP52 forms a trimeric complex with FIP200 and SINTBAD/NAP1, which are subunits of the autophagy-initiating ULK and the TBK1 kinase complex, respectively. FIP200 and SINTBAD/NAP1 are each recruited independently to bacteria via NDP52, as revealed by selective point mutations in their respective binding sites, but only in their combined presence does xenophagy proceed. Such recruitment of the upstream autophagy machinery by NDP52 reveals how detection of cargo-associated "eat me" signals, induction of autophagy, and juxtaposition of cargo and phagophores are integrated in higher eukaryotes.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Autofagia/genética , Proteínas Nucleares/genética , Proteínas Tirosina Quinases/genética , Proteínas Adaptadoras de Transdução de Sinal/química , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Sítios de Ligação/genética , Citoplasma/microbiologia , Citosol/microbiologia , Humanos , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Proteínas Nucleares/química , Mutação Puntual/genética , Ligação Proteica/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/química , Salmonella typhimurium/genética , Salmonella typhimurium/patogenicidade
12.
Zhonghua Er Ke Za Zhi ; 57(3): 206-210, 2019 Mar 02.
Artigo em Chinês | MEDLINE | ID: mdl-30818898

RESUMO

Objective: To explore the clinical characteristics and treatment of a family with inherited generalized epilepsy with febrile seizures plus (GEFS+) caused by STX1B gene mutation and to review the literature. Methods: Clinical data of a child with GEFS+ and his family members who visited Guangzhou Women and Children Medical Center in August 2017 were collected. DNA samples of the proband and his parents, his grandparents were analyzed by the next-generation sequencing and confirmed by Sanger sequencing. A literature search with "STX1B" as the key word was conducted at PubMed, China National Knowledge Infrastructure and Wanfang databases to include recently published studies (up to May 2018). Results: The proband was a 2-year-old boy who was admitted to our hospital because of frequent epileptic seizures in a month, with focal seizures evolving into the generalized bilateral tonic-clonic epileptic attacks after febrile seizure. His seizures were well controlled by levetiracetam. His father presented febrile seizure in early childhood stage and epilepsy in adult stage, which were under control with lamotrigine. The proband's grandmother had a history of febrile seizure. Other family members had no history of convulsion. Ictal electroencephalogram showed low voltage fast activities (8-10 Hz) originating from the central region of the brain. Heterozygous mutation of STX1B gene (c.705C>G, p. Asn235Lys) was detected in the proband, his father and grandmother. The mutation has not been reported previously. Furthermore, no other family members carried the mutation at c. 705 locus of STX1B gene. No article in Chinese was found, and 4 articles in a language other than Chinese provided the complete case data of 9 mutation loci in 33 patients (4 families and 4 sporadic cases). With this research data (3 cases in 1 family) included, there were 36 cases and 10 mutation sites in STX1B gene. Of these mutations, there were 5 missense mutations, 3 nonsense mutations, 1 insert mutation and 1 deletion mutation; and according to the mutation distribution, there were 4 mutation sites in exon 3, 2 in exon 8, 2 in exon 9, and 1 in exon 1.There was incomplete penetrance in the family, so different types of epileptic attacks occurred among different family members. Conclusions: In this study, a de novo mutation of STX1B gene in a family with GEFS+ was defined, which would expand the gene mutation spectrum and provide basis for family genetic counseling. Clinical heterogeneity was found in this family. Seizures caused by STX1B gene mutation were sensitive to antiepileptic drugs.


Assuntos
Epilepsia Generalizada/genética , Mutação Puntual/genética , Convulsões Febris/genética , Sintaxina 1/genética , Criança , Pré-Escolar , China , Epilepsia Generalizada/diagnóstico , Feminino , Humanos , Masculino , Mutação , Convulsões Febris/diagnóstico
13.
Mem Inst Oswaldo Cruz ; 114: e180425, 2019 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-30726345

RESUMO

BACKGROUND AND OBJECTIVE: Brazil is responsible for a large number of Plasmodium vivax cases in America. Given the emergence of P. vivax parasites resistant to chloroquine and the effectiveness of antifolates in vivax malaria treatment together with a correlation between mutations in P. vivax dhfr and dhps genes and SP treatment failure, the point mutations in these genes were investigated. METHODS: Blood samples from 54 patients experiencing vivax malaria symptomatic episodes in the Amazonian Region were investigated. Genomic DNA was extracted using a DNA extraction kit (QIAGENTM). Nested polymerase chain reaction (PCR) amplification was carried out followed by Sanger sequencing to detect single nucleotide polymorphisms (SNPs). FINDINGS: All tested isolates showed non-synonymous mutations in pvdhfr gene: 117N (54/54, 100%) and 58R (25/54, 46%). Double mutant allele 58R/117N (FRTNI, 28%) was the most frequent followed by triple mutant alleles (58R/117N/173L, FRTNL, 11%; 58R/61M/117N, FRMNI, 5% 117N/173L, FSTNL, 4%) and quadruple mutant allele (58R/61M/117N/173L, FRMNL, 2%). A single mutation was observed at codon C383G in pvdhps gene (SGKAV, 48%). CONCLUSION: No evidence of molecular signatures associated with P. vivax resistance to SP was observed in the Brazilian samples.


Assuntos
Antimaláricos/farmacologia , Resistência a Medicamentos/genética , Malária Vivax/parasitologia , Plasmodium vivax/genética , Mutação Puntual/genética , Proteínas de Protozoários/genética , Pirimetamina/farmacologia , Sulfadoxina/farmacologia , Alelos , Brasil , DNA de Protozoário/genética , Combinação de Medicamentos , Doenças Endêmicas , Humanos , Plasmodium vivax/efeitos dos fármacos , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único
14.
Biosens Bioelectron ; 130: 236-244, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30769288

RESUMO

Here we present a gold nanorod-based platform for the sequence-specific detection of circulating tumor DNA (ctDNA) point mutations without the need for amplification or fluorescence labeling. Peptide nucleic acid probes complimentary to the G12V mutation in the KRAS gene were conjugated to gold nanorods, and the localized surface plasmon resonance absorbance through the sample was measured after exposure to synthetic ctDNA at various concentrations. Each step of the reaction was thoroughly controlled, starting from reagent concentrations and including conjugation, sonication, and incubation time. The platform was evaluated in both buffer and spiked healthy patient serum, demonstrating a linear working range below 125 nanograms of ctDNA per milliliter solution, and an effective limit of detection of 2 nanograms of ctDNA per milliliter. A clear distinction between mutant and wild type synthetic ctDNA was also found using this platform. In order to improve upon the selectivity of the sensor, a DNA hybridization simulation was performed to understand how the addition of mutations to the peptide nucleic acid probe could enhance the selectivity for capture of mutant over wild type sequences. The top candidate from the simulations, which had an additional mutation two base pairs away from the mutation of interest, had a significant impact on the selectivity between mutant and wild type capture. This paper provides a framework for sequence-specific capture of ctDNA, and a method of improving selectivity for desired point mutations through careful probe design.


Assuntos
Técnicas Biossensoriais , DNA Tumoral Circulante/genética , Neoplasias/sangue , Ácidos Nucleicos Peptídicos/química , DNA Tumoral Circulante/sangue , Ouro/química , Humanos , Nanopartículas Metálicas/química , Nanotubos/química , Neoplasias/patologia , Hibridização de Ácido Nucleico , Ácidos Nucleicos Peptídicos/genética , Mutação Puntual/genética , Ressonância de Plasmônio de Superfície
15.
PLoS Comput Biol ; 15(2): e1006799, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30794536

RESUMO

Mutation signatures in cancer genomes reflect endogenous and exogenous mutational processes, offering insights into tumour etiology, features for prognostic and biologic stratification and vulnerabilities to be exploited therapeutically. We present a novel machine learning formalism for improved signature inference, based on multi-modal correlated topic models (MMCTM) which can at once infer signatures from both single nucleotide and structural variation counts derived from cancer genome sequencing data. We exemplify the utility of our approach on two hormone driven, DNA repair deficient cancers: breast and ovary (n = 755 samples total). We show how introducing correlated structure both within and between modes of mutation can increase accuracy of signature discovery, particularly in the context of sparse data. Our study emphasizes the importance of integrating multiple mutation modes for signature discovery and patient stratification, and provides a statistical modeling framework to incorporate additional features of interest for future studies.


Assuntos
Biologia Computacional/métodos , Neoplasias/genética , Análise de Sequência de DNA/métodos , Variação Genética/genética , Genoma , Humanos , Aprendizado de Máquina , Modelos Estatísticos , Mutação , Mutação Puntual/genética , Prognóstico , Transcriptoma/genética
16.
BJOG ; 126(11): 1380-1389, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30801962

RESUMO

OBJECTIVE: Mitochondrial disease is a disorder of energy metabolism that affects 1 in 4300 adults in the UK. Pregnancy is associated with physiological demands that have implications for energy metabolism. We were interested to know how pregnancy was affected in women with mitochondrial disease, particularly those with the most common pathogenic mutation m.3243A>G. DESIGN: Retrospective case-comparison study. POPULATION/SETTING: Sixty-seven women with genetically confirmed mitochondrial disease from the UK Mitochondrial Diseases Cohort and 69 unaffected women participated. METHODS: Participants answered questionnaires regarding each of their pregnancies. Patients were divided into two groups according to genetic mutation, with those harbouring m.3243A>G comprising a single group. MAIN OUTCOME MEASURES: Pregnancy-related complications, mode of delivery, gestational age and birthweight of newborns. RESULTS: Of 139 live births in the comparison group, 62 were in the m.3243A>G group and 87 were in the 'all other mutations' group. Pregnancies of women with the m.3243A>G mutation had significantly more gestational diabetes (odds ratio [OR] = 8.2, 95% CI 1.3-50.1), breathing difficulties (OR = 7.8, 95% CI 1.0-59.1) and hypertension (OR = 8.2, 95% CI 3.1-21.5) than the comparison group. Only half of the pregnancies in the m.3243A>G group had normal vaginal delivery, with emergency caesarean section accounting for 24.2% of deliveries. Babies were born significantly earlier to mothers harbouring m.3243A>G with 53.3% of them preterm (<37 weeks). These babies were also more likely to require resuscitation and admission. CONCLUSION: Women who carried the m.3243A>G mutation appeared to be at higher risk of complications during pregnancies, caesarean section and preterm delivery than the unaffected women or those with other forms of mitochondrial disease. TWEETABLE ABSTRACT: Pregnant women with mitochondrial disease - m.3243A>G mutation - are at greatly increased risk of complications and preterm delivery.


Assuntos
Doenças Mitocondriais/genética , Mutação Puntual/genética , Complicações na Gravidez/genética , Adolescente , Adulto , Estudos de Casos e Controles , DNA Mitocondrial/genética , Feminino , Humanos , Recém-Nascido , Pessoa de Meia-Idade , Doenças Mitocondriais/epidemiologia , Gravidez , Complicações na Gravidez/epidemiologia , Resultado da Gravidez , Estudos Retrospectivos , Reino Unido/epidemiologia , Adulto Jovem
17.
Proc Natl Acad Sci U S A ; 116(8): 3294-3299, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30718391

RESUMO

The parathyroid hormone (PTH) and its related peptide (PTHrP) activate PTH receptor (PTHR) signaling, but only the PTH sustains GS-mediated adenosine 3',5'-cyclic monophosphate (cAMP) production after PTHR internalization into early endosomes. The mechanism of this unexpected behavior for a G-protein-coupled receptor is not fully understood. Here, we show that extracellular Ca2+ acts as a positive allosteric modulator of PTHR signaling that regulates sustained cAMP production. Equilibrium and kinetic studies of ligand-binding and receptor activation reveal that Ca2+ prolongs the residence time of ligands on the receptor, thus, increasing both the duration of the receptor activation and the cAMP signaling. We further find that Ca2+ allostery in the PTHR is strongly affected by the point mutation recently identified in the PTH (PTHR25C) as a new cause of hypocalcemia in humans. Using high-resolution and mass accuracy mass spectrometry approaches, we identified acidic clusters in the receptor's first extracellular loop as key determinants for Ca2+ allosterism and endosomal cAMP signaling. These findings coupled to defective Ca2+ allostery and cAMP signaling in the PTHR by hypocalcemia-causing PTHR25C suggest that Ca2+ allostery in PTHR signaling may be involved in primary signaling processes regulating calcium homeostasis.


Assuntos
AMP Cíclico/genética , Hipocalcemia/genética , Hormônio Paratireóideo/genética , Receptor Tipo 1 de Hormônio Paratireóideo/genética , Regulação Alostérica/genética , Animais , Células COS , Sinalização do Cálcio/genética , Cercopithecus aethiops , AMP Cíclico/metabolismo , Humanos , Hipocalcemia/metabolismo , Hipocalcemia/patologia , Cinética , Ligantes , Hormônio Paratireóideo/metabolismo , Proteína Relacionada ao Hormônio Paratireóideo/genética , Mutação Puntual/genética , Ligação Proteica/genética , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo
18.
Int J Mol Sci ; 20(1)2019 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-30625989

RESUMO

We present a genome-wide comparative and comprehensive analysis of three different sequencing methods (conventional next generation sequencing (NGS), tag-based single strand sequencing (e.g., SSCS), and Duplex Sequencing for investigating mitochondrial mutations in human breast epithelial cells. Duplex Sequencing produces a single strand consensus sequence (SSCS) and a duplex consensus sequence (DCS) analysis, respectively. Our study validates that although high-frequency mutations are detectable by all the three sequencing methods with the similar accuracy and reproducibility, rare (low-frequency) mutations are not accurately detectable by NGS and SSCS. Even with conservative bioinformatical modification to overcome the high error rate of NGS, the NGS frequency of rare mutations is 7.0 × 10-4. The frequency is reduced to 1.3 × 10-4 with SSCS and is further reduced to 1.0 × 10-5 using DCS. Rare mutation context spectra obtained from NGS significantly vary across independent experiments, and it is not possible to identify a dominant mutation context. In contrast, rare mutation context spectra are consistently similar in all independent DCS experiments. We have systematically identified heat-induced artifactual variants and corrected the artifacts using Duplex Sequencing. Specific sequence contexts were analyzed to examine the effects of neighboring bases on the accumulation of heat-induced artifactual variants. All of these artifacts are stochastically occurring rare mutations. C > A/G > T, a signature of oxidative damage, is the most increased (170-fold) heat-induced artifactual mutation type. Our results strongly support the claim that Duplex Sequencing accurately detects low-frequency mutations and identifies and corrects artifactual mutations introduced by heating during DNA preparation.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Temperatura Alta , Mutação Puntual/genética , Adulto , Artefatos , Linhagem Celular , Sequência Consenso , DNA Mitocondrial/genética , Genoma Mitocondrial , Humanos , Reprodutibilidade dos Testes , Processos Estocásticos , Adulto Jovem
19.
PLoS One ; 14(1): e0206896, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30699117

RESUMO

Toxin synthesis and endospore formation are two of the most critical factors that determine the outcome of infection by Clostridioides difficile. The two major toxins, TcdA and TcdB, are the principal factors causing damage to the host. Spores are the infectious form of C. difficile, permit survival of the bacterium during antibiotic treatment and are the predominant cell form that leads to recurrent infection. Toxin production and sporulation have their own specific mechanisms of regulation, but they share negative regulation by the global regulatory protein CodY. Determining the extent of such regulation and its detailed mechanism is important for understanding the linkage between two apparently independent biological phenomena and raises the possibility of creating new ways of limiting infection. The work described here shows that a codY null mutant of a hypervirulent (ribotype 027) strain is even more virulent than its parent in a mouse model of infection and that the mutant expresses most sporulation genes prematurely during exponential growth phase. Moreover, examining the expression patterns of mutants producing CodY proteins with different levels of residual activity revealed that expression of the toxin genes is dependent on total CodY inactivation, whereas most sporulation genes are turned on when CodY activity is only partially diminished. These results suggest that, in wild-type cells undergoing nutrient limitation, sporulation genes can be turned on before the toxin genes.


Assuntos
Proteínas de Bactérias/metabolismo , Clostridium difficile/metabolismo , Clostridium difficile/patogenicidade , Ribotipagem , Esporos Bacterianos/fisiologia , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Toxinas Bacterianas/toxicidade , Vias Biossintéticas/efeitos dos fármacos , Vias Biossintéticas/genética , Clostridium difficile/genética , Diarreia/microbiologia , Etanolamina/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Genes Bacterianos , Camundongos Endogâmicos C57BL , Família Multigênica , Óperon/genética , Mutação Puntual/genética , Domínios Proteicos , Esporos Bacterianos/genética , Transcrição Genética/efeitos dos fármacos , Virulência/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA