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1.
BMC Res Notes ; 14(1): 10, 2021 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-33407800

RESUMO

OBJECTIVE: This study describes the occurrence of a silent mutation in the RNA binding domain of nucleocapsid phosphoprotein (N protein) coding gene from SARS-CoV-2 that may consequence to a missense mutation by onset of another single nucleotide mutation. RESULTS: In the DNA sequence isolated from severe acute respiratory syndrome (SARS-CoV-2) in Iran, a coding sequence for the RNA binding domain of N protein was detected. The comparison of Chinese and Iranian DNA sequences displayed that a thymine (T) was mutated to cytosine (C), so "TTG" from China was changed to "CTG" in Iran. Both DNA sequences from Iran and China have been encoded for leucine. In addition, the second T in "CTG" in the DNA or uracil (U) in "CUG" in the RNA sequences from Iran can be mutated to another C by a missense mutation resulting from thymine DNA glycosylase (TDG) of human and base excision repair mechanism to produce "CCG" encoding for proline, which consequently may increase the affinity of the RNA binding domain of N protein to viral RNA and improve the transcription rate, pathogenicity, evasion from human immunity system, spreading in the human body, and risk of human-to-human transmission rate of SARS-CoV-2.


Assuntos
/genética , RNA Viral/genética , Motivos de Ligação ao RNA/genética , /genética , China , Bases de Dados Genéticas , Humanos , Irã (Geográfico) , Mutação de Sentido Incorreto , Fosfoproteínas/genética , Análise de Sequência de DNA , Mutação Silenciosa
2.
Genome Med ; 13(1): 4, 2021 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-33413610

RESUMO

During COVID-19, diagnostic serological tools and vaccines have been developed. To inform control activities in a post-vaccine surveillance setting, we have developed an online "immuno-analytics" resource that combines epitope, sequence, protein and SARS-CoV-2 mutation analysis. SARS-CoV-2 spike and nucleocapsid proteins are both vaccine and serological diagnostic targets. Using the tool, the nucleocapsid protein appears to be a sub-optimal target for use in serological platforms. Spike D614G (and nsp12 L314P) mutations were most frequent (> 86%), whilst spike A222V/L18F have recently increased. Also, Orf3a proteins may be a suitable target for serology. The tool can accessed from: http://genomics.lshtm.ac.uk/immuno (online); https://github.com/dan-ward-bio/COVID-immunoanalytics (source code).


Assuntos
/genética , /imunologia , /diagnóstico , Simulação por Computador , /imunologia , Epitopos de Linfócito B/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Mutação , Fosfoproteínas/genética , Fosfoproteínas/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , /imunologia
3.
Chemosphere ; 262: 127792, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32805656

RESUMO

Tebuconazole is a triazole fungicide, used in agriculture to treat phytopathogenic fungi, and as a biocide, has been reported to be related to reproductive and developmental toxicity. The purpose of this study was to investigate the effect of tebuconazole exposure on rat fetal Leydig cells and fetal testis during pregnancy. Pregnant Sprague-Dawley rats were randomly divided into 4 groups, daily gavaged with corn oil (as a control), 25, 50, and 100 mg/kg body weight tebuconazole for 10 days (from the 12th day of pregnancy). Tebuconazole increased fetal serum testosterone and progesterone levels at a dose of 100 mg/kg. Exposure to 100 mg/kg tebuconazole significantly caused an increase in the number of fetal Leydig cells per testis without inducing cell aggregation. Tebuconazole up-regulated the expression of Star, Cyp11a1, Hsd17b3, and Fshr and their proteins. Further investigation found that tebuconazole caused increased phosphorylation of AKT1, ERK1/2, and mTOR, the level of BCL2, as well as the decrease of Beclin1, LC3B, and BAX, which may contribute to the fetal Leydig cell autophagy and proliferation. In conclusion, in utero exposure of tebuconazole causes the proliferation of fetal Leydig cells.


Assuntos
Fungicidas Industriais/toxicidade , Células Intersticiais do Testículo/efeitos dos fármacos , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Reprodução/efeitos dos fármacos , Triazóis/toxicidade , Animais , Feminino , Células Intersticiais do Testículo/metabolismo , Masculino , Fosfoproteínas/genética , Fosforilação , Gravidez , Efeitos Tardios da Exposição Pré-Natal/patologia , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Testículo/efeitos dos fármacos , Testículo/embriologia , Testículo/patologia , Testosterona/sangue , Regulação para Cima
4.
Diagn Microbiol Infect Dis ; 99(1): 115197, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32977117

RESUMO

Automated assays for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies in coronavirus disease 2019 (COVID-19) diagnostics have recently come available. We compared the performance of the Elecsys® Anti-SARS-CoV-2 and LIAISON® SARS-CoV-2 S1/S2 IgG tests. The seroconversion panel comprised of 120 samples from 13 hospitalized COVID-19 patients. For the sensitivity and specificity testing, samples from COVID-19 outpatients >15 days after positive nucleic acid amplification test (NAAT) result (n = 35) and serum control samples collected before the COVID-19 era (n = 161) were included in the material. Samples for the detection of possible cross-reactions were also tested. Based on our results, the SARS-CoV-2 antibodies can be quite reliably detected 2 weeks after NAAT positivity and 3 weeks after the symptom onset with both tests. However, since some COVID-19 patients were positive only with Elecsys®, the antibodies should be screened against N-antigen (Elecsys®) and reactive samples confirmed with S antigen (LIAISON®), but both results should be reported. In some COVID-19 patients, the serology can remain negative.


Assuntos
Anticorpos Antivirais/sangue , /imunologia , /imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Adulto , Idoso , /diagnóstico , Reações Cruzadas , Feminino , Humanos , Cinética , Masculino , Pessoa de Meia-Idade , Fosfoproteínas/imunologia , /isolamento & purificação , Sensibilidade e Especificidade , Soroconversão , Adulto Jovem
5.
Talanta ; 224: 121726, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33379001

RESUMO

The outbreak of COVID-19 caused by a novel Coronavirus (termed SARS-CoV-2) has spread to over 210 countries around the world. Currently, reverse transcription quantitative qPCR (RT-qPCR) is used as the gold standard for diagnosis of SARS-CoV-2. However, the sensitivity of RT-qPCR assays of pharyngeal swab samples are reported to vary from 30% to 60%. More accurate and sensitive methods are urgently needed to support the quality assurance of the RT-qPCR or as an alternative diagnostic approach. A reverse transcription digital PCR (RT-dPCR) method was established and evaluated. To explore the feasibility of RT-dPCR in diagnostic of SARS-CoV-2, a total of 196 clinical pharyngeal swab samples from 103 suspected patients, 77 close contacts and 16 supposed convalescents were analyzed by RT-qPCR and then measured by the proposed RT-dPCR. For the 103 fever suspected patients, 19 (19/25) negative and 42 (42/49) equivocal tested by RT-qPCR were positive according to RT-dPCR. The sensitivity of SARS-CoV-2 detection was significantly improved from 28.2% by RT-qPCR to 87.4% by RT-dPCR. For 29 close contacts (confirmed by additional sample and clinical follow up), 16 (16/17) equivocal and 1 negative tested by RT-qPCR were positive according to RT-dPCR, which is implying that the RT-qPCR is missing a lot of asymptomatic patients. The overall sensitivity, specificity and diagnostic accuracy of RT-dPCR were 91%, 100% and 93%, respectively. RT-dPCR is highly accurate method and suitable for detection of pharyngeal swab samples from COVID-19 suspected patients and patients under isolation and observation who may not be exhibiting clinical symptoms.


Assuntos
/métodos , RNA Viral/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , /genética , /genética , Humanos , Faringe/virologia , Fosfoproteínas/genética , Poliproteínas/genética , Proteínas Virais/genética
6.
Zhonghua Nan Ke Xue ; 26(3): 258-264, 2020 Mar.
Artigo em Chinês | MEDLINE | ID: mdl-33346967

RESUMO

Objective: To investigate the effects of Xiongcan Yishen Prescription (XYP) on the expressions of cholesterol transport proteins, steroidogenic enzymes and steroidogenic factor-1 (SF-1) in the Leydig cells of the rats with late-onset hypogonadism (LOH). METHODS: Twenty-five 18-month-old male SD rats were randomly divided into five groups of equal number, LOH model control, testosterone propionate (TP) and low-, medium- and high-dose XYP, and another 5 two-month-old male SD rats included as normal controls. After modeling, the animals in the TP group were treated by intramuscular injection of TP at 5.21 mg/kg qd alt, those in the low-, medium- and high-dose XYP groups intragastrically with XYP at 10.4, 20.8 and 41.6 g/kg qd alt respectively, and those in the LOH model and normal control groups with saline, all for 28 successive days. Then, all the rats were sacrificed for determination of the expressions of the cholesterol transport proteins StAR and TSPO, steroidogenic enzymes CYP11A1, HSD3B7 and HSD17B4, and SF-1 in the Leydig cells by Western blot. RESULTS: The expressions of StAR, TSPO, CYP11A1, HSD3B7, HSD17B4 and SF-1 in the Leydig cells were significantly decreased in the LOH model controls compared with those in the normal controls (P< 0.05), but remarkably increased in the low-, medium- and high-dose XYP groups in comparison with those in the LOH model control group (P< 0.05). CONCLUSIONS: Xiongcan Yishen Prescription can up-regulate the expressions of the cholesterol transport proteins StAR and TSPO, steroidogenic enzymes CYP11A1, HSD3B7 and HSD17B4, and SF-1 in the rat Leydig cells, which might be one of the possible mechanisms of the prescription in the treatment of LOH.


Assuntos
Colesterol/metabolismo , Medicamentos de Ervas Chinesas/uso terapêutico , Hidroxiesteroide Desidrogenases/metabolismo , Hipogonadismo , Células Intersticiais do Testículo/efeitos dos fármacos , Animais , Transporte Biológico , Proteínas de Transporte , Hipogonadismo/tratamento farmacológico , Células Intersticiais do Testículo/metabolismo , Masculino , Fosfoproteínas/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Esteroides/metabolismo , Testosterona
7.
Sci Rep ; 10(1): 22387, 2020 12 28.
Artigo em Inglês | MEDLINE | ID: mdl-33372181

RESUMO

In the genome of SARS-CoV-2, the 5'-terminus encodes a polyprotein, which is further cleaved into 15 non-structural proteins whereas the 3' terminus encodes four structural proteins and eight accessory proteins. Among these 27 proteins, the present study aimed to discover likely antigenic proteins and epitopes to be used for the development of a vaccine or serodiagnostic assay using an in silico approach. For this purpose, after the full genome analysis of SARS-CoV-2 Wuhan isolate and variant proteins that are detected frequently, surface proteins including spike, envelope, and membrane proteins as well as proteins with signal peptide were determined as probable vaccine candidates whereas the remaining were considered as possible antigens to be used during the development of serodiagnostic assays. According to results obtained, among 27 proteins, 26 of them were predicted as probable antigen. In 26 proteins, spike protein was selected as the best vaccine candidate because of having a signal peptide, negative GRAVY value, one transmembrane helix, moderate aliphatic index, a big molecular weight, a long-estimated half-life, beta wrap motifs as well as having stable, soluble and non-allergic features. In addition, orf7a, orf8, and nsp-10 proteins with signal peptide were considered as potential vaccine candidates. Nucleocapsid protein and a highly antigenic GGDGKMKD epitope were identified as ideal antigens to be used in the development of serodiagnostic assays. Moreover, considering MHC-I alleles, highly antigenic KLNDLCFTNV and ITLCFTLKRK epitopes can be used to develop an epitope-based peptide vaccine.


Assuntos
/imunologia , /genética , Glicoproteína da Espícula de Coronavírus/imunologia , /diagnóstico , Simulação por Computador , /imunologia , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Genoma Viral/genética , Humanos , Simulação de Acoplamento Molecular , Fosfoproteínas/genética , Fosfoproteínas/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/imunologia , Proteínas Virais/genética , Proteínas Virais/imunologia
8.
BMC Infect Dis ; 20(1): 818, 2020 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-33167900

RESUMO

BACKGROUND: To explore the kinetic changes in virology, specific antibody response and imaging during the clinical course of COVID-19. METHODS: This observational study enrolled 20 patients with COVID-19, who were hospitalized between January 20-April 6, 2020, in the two COVID-19 designated hospitals of Zhoushan, Zhejiang and Rushan, Shandong, China, The laboratory findings, imaging, serum response to viral infection, and viral RNA level in the throat and stool samples were assessed from onset to recovery phase in patients with COVID-19. RESULTS: SARS-COV-2 RNA was positive as early as day four. It remained positive until day 55 post-onset in the sputum-throat swabs and became negative in most cases (55%) within 14 days after onset. Lymphocytopenia occurred in 40% (8/20) of patients during the peak infection period and returned to normal at week five. The most severe inflammation in the lungs appeared in week 2 or 3 after onset, and this was completely absorbed between week 6 and 8 in 85.7% of patients. All patients had detectable antibodies to the receptor binding domain (RBD), and 95% of these patients had IgG to viral N proteins. The antibody titer peaked at week four. Anti-S IgM was positive in 7 of 20 patients after week three. CONCLUSIONS: All COVID-19 patients in this study were self-limiting and recovered well though it may take as long as 6-8 weeks. Our findings on the kinetic changes in imaging, serum response to viral infection and viral RNA level may help understand pathogenesis and define clinical course of COVID-19.


Assuntos
Anticorpos Antivirais/sangue , Betacoronavirus/imunologia , Técnicas de Laboratório Clínico , Infecções por Coronavirus/diagnóstico por imagem , Infecções por Coronavirus/imunologia , Pulmão/diagnóstico por imagem , Pneumonia Viral/diagnóstico por imagem , Pneumonia Viral/imunologia , Adolescente , Adulto , Idoso , Betacoronavirus/genética , Criança , China/epidemiologia , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/virologia , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Masculino , Pessoa de Meia-Idade , Proteínas do Nucleocapsídeo/imunologia , Pandemias , Fosfoproteínas , Pneumonia Viral/epidemiologia , Pneumonia Viral/virologia , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Escarro/virologia , Tomografia Computadorizada por Raios X , Adulto Jovem
9.
Biomedica ; 40(Supl. 2): 166-172, 2020 10 30.
Artigo em Inglês, Espanhol | MEDLINE | ID: mdl-33152200

RESUMO

Introduction: The 2019 coronavirus pandemic (COVID-19) has caused around 25 million cases worldwide. Asymptomatic patients have been described as potential sources of transmission. However, there are difficulties to detect them and to establish their role in the dynamics of virus transmission, which hinders the implementation of prevention strategies. Objective: To describe the behavior of asymptomatic SARS-CoV-2 virus infection in a cohort of workers at the El Dorado "Luis Carlos Galán Sarmiento" International Airport in Bogotá, Colombia. Materials and methods: A prospective cohort of 212 workers from the El Dorado airport was designed. The follow-up began in June, 2020. A survey was used to characterize health and work conditions. Every 21 day, a nasopharyngeal swab was taken to identify the presence of SARS-CoV-2 using RT-PCR. We analyzed the behavior of the cycle threshold (ORF1ab and N genes) according to the day of follow-up. Results: In the first three follow-ups of the cohort, we found an incidence of SARS-CoV-2 infection of 16.51%. The proportion of positive contacts was 14.08%. The median threshold for cycle threshold was 33.53. Conclusion: We characterized the asymptomatic SARS-CoV-2 infection in a cohort of workers. The identification of asymptomatic infected persons continues to be a challenge for epidemiological surveillance systems.


Assuntos
Aeroportos , Infecções Assintomáticas , Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico , Infecções por Coronavirus/diagnóstico , Nasofaringe/virologia , Pandemias , Pneumonia Viral/diagnóstico , Adulto , Infecções Assintomáticas/epidemiologia , Betacoronavirus/genética , Betacoronavirus/fisiologia , Colômbia , Busca de Comunicante , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/virologia , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas do Nucleocapsídeo/genética , Fosfoproteínas , Pneumonia Viral/epidemiologia , Pneumonia Viral/virologia , Poliproteínas , Estudos Prospectivos , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Inquéritos e Questionários , Proteínas Virais/genética , Replicação Viral/genética , Local de Trabalho
10.
Immunity ; 53(5): 925-933.e4, 2020 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-33129373

RESUMO

We conducted a serological study to define correlates of immunity against SARS-CoV-2. Compared to those with mild coronavirus disease 2019 (COVID-19) cases, individuals with severe disease exhibited elevated virus-neutralizing titers and antibodies against the nucleocapsid (N) and the receptor binding domain (RBD) of the spike protein. Age and sex played lesser roles. All cases, including asymptomatic individuals, seroconverted by 2 weeks after PCR confirmation. Spike RBD and S2 and neutralizing antibodies remained detectable through 5-7 months after onset, whereas α-N titers diminished. Testing 5,882 members of the local community revealed only 1 sample with seroreactivity to both RBD and S2 that lacked neutralizing antibodies. This fidelity could not be achieved with either RBD or S2 alone. Thus, inclusion of multiple independent assays improved the accuracy of antibody tests in low-seroprevalence communities and revealed differences in antibody kinetics depending on the antigen. We conclude that neutralizing antibodies are stably produced for at least 5-7 months after SARS-CoV-2 infection.


Assuntos
Betacoronavirus/imunologia , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/imunologia , Imunidade Humoral , Pneumonia Viral/epidemiologia , Pneumonia Viral/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Arizona/epidemiologia , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/sangue , Infecções por Coronavirus/diagnóstico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas do Nucleocapsídeo/imunologia , Pandemias , Fosfoproteínas , Pneumonia Viral/sangue , Pneumonia Viral/diagnóstico , Prevalência , Domínios e Motivos de Interação entre Proteínas , Estudos Soroepidemiológicos , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Adulto Jovem
11.
Nat Commun ; 11(1): 5387, 2020 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-33144593

RESUMO

The Human Silencing Hub (HUSH) complex is necessary for epigenetic repression of LINE-1 elements. We show that HUSH-depletion in human cell lines and primary fibroblasts leads to induction of interferon-stimulated genes (ISGs) through JAK/STAT signaling. This effect is mainly attributed to MDA5 and RIG-I sensing of double-stranded RNAs (dsRNAs). This coincides with upregulation of primate-conserved LINE-1s, as well as increased expression of full-length hominid-specific LINE-1s that produce bidirectional RNAs, which may form dsRNA. Notably, LTRs nearby ISGs are derepressed likely rendering these genes more responsive to interferon. LINE-1 shRNAs can abrogate the HUSH-dependent response, while overexpression of an engineered LINE-1 construct activates interferon signaling. Finally, we show that the HUSH component, MPP8 is frequently downregulated in diverse cancers and that its depletion leads to DNA damage. These results suggest that LINE-1s may drive physiological or autoinflammatory responses through dsRNA sensing and gene-regulatory roles and are controlled by the HUSH complex.


Assuntos
Epigênese Genética/fisiologia , Regulação Neoplásica da Expressão Gênica , Inativação Gênica/fisiologia , Interferon Tipo I/metabolismo , Elementos Nucleotídeos Longos e Dispersos/fisiologia , Proteína DEAD-box 58/genética , Proteína DEAD-box 58/metabolismo , Dano ao DNA , Regulação para Baixo , Técnicas de Inativação de Genes , Células HEK293 , Células HeLa , Humanos , Inflamação , Helicase IFIH1 Induzida por Interferon/metabolismo , Elementos Nucleotídeos Longos e Dispersos/genética , Fosfoproteínas/metabolismo , RNA de Cadeia Dupla , Análise de Sequência de RNA , Transdução de Sinais
12.
Molecules ; 25(21)2020 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-33147821

RESUMO

With an increasing fatality rate, severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) has emerged as a promising threat to human health worldwide. Recently, the World Health Organization (WHO) has announced the infectious disease caused by SARS-CoV-2, which is known as coronavirus disease-2019 (COVID-2019), as a global pandemic. Additionally, the positive cases are still following an upward trend worldwide and as a corollary, there is a need for a potential vaccine to impede the progression of the disease. Lately, it has been documented that the nucleocapsid (N) protein of SARS-CoV-2 is responsible for viral replication and interferes with host immune responses. We comparatively analyzed the sequences of N protein of SARS-CoV-2 for the identification of core attributes and analyzed the ancestry through phylogenetic analysis. Subsequently, we predicted the most immunogenic epitope for the T-cell and B-cell. Importantly, our investigation mainly focused on major histocompatibility complex (MHC) class I potential peptides and NTASWFTAL interacted with most human leukocyte antigen (HLA) that are encoded by MHC class I molecules. Further, molecular docking analysis unveiled that NTASWFTAL possessed a greater affinity towards HLA and also available in a greater range of the population. Our study provides a consolidated base for vaccine design and we hope that this computational analysis will pave the way for designing novel vaccine candidates.


Assuntos
Betacoronavirus/imunologia , Proteínas do Nucleocapsídeo/imunologia , Sequência de Aminoácidos , Linfócitos T CD8-Positivos/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Hipersensibilidade a Drogas/imunologia , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Antígenos de Histocompatibilidade Classe I , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Proteínas do Nucleocapsídeo/química , Fragmentos de Peptídeos/imunologia , Fosfoproteínas , Estrutura Secundária de Proteína , Vacinas Virais/imunologia
13.
Zhong Nan Da Xue Xue Bao Yi Xue Ban ; 45(9): 1053-1060, 2020.
Artigo em Inglês, Chinês | MEDLINE | ID: mdl-33051418

RESUMO

OBJECTIVES: Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide, especially in Asia and Africa. However, the underlying mechanism is still unclear. Consequently, it is important to explore its key genes and prognosis-related genes via bioinformatics. This study aimed to explore the molecular mechanism of HCC by using bioinformatics analysis for HCC gene chip data. METHODS: Microarray data of HCC genes were downloaded from public GEO database and screened for differentially expressed genes (DEGs) by GEO2R analysis. Then DAVID online tool was used for GO annotation and KEGG pathway enrichment analysis. STRING-DB online database and Cytoscape software were used for protein interaction network analysis.GEPIA and Ualcan were applied to evaluate prognosis and promoter methylation level. RESULTS: A total of 87 DEGs of HCC were screened, of which 15 genes were up-regulated and 72 genes were down-regulated. GO annotation indicated that most of the genes were involved in oxidation reduction,cellular amino acid derivative metabolic process, carboxylic acid catabolic process, and response to wounding. KEGG pathways were enriched in linoleic acid metabolism, retinol metabolism, complement and coagulation cascades,steroid hormone biosynthesis, drug metabolism, and other pathways. Two key modules and key genes AURKA and SPP2 were obtained by protein interaction network analysis. Prognostic analysis showed that the 2 genes were significantly correlated with the total survival time of patients with HCC. There was no significant difference in the methylation level of AURKA promoter between the primary tumor group and the normal group (P=0.296) and the methylation level of SPP2 promoter was significantly lower in the primary tumor group than that in the normal group (P<0.001). CONCLUSIONS: HCC-relevant AURKA and SPP2 are obtained via bioinformatics analysis, which are closely related to the prognosis of patients with HCC. Gene promoter methylation is not the main factor for AURKA and SPP2 expression levels.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Carcinoma Hepatocelular/genética , Biologia Computacional , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Neoplasias Hepáticas/genética , Análise de Sequência com Séries de Oligonucleotídeos , Fosfoproteínas
14.
Sci Rep ; 10(1): 18289, 2020 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-33106569

RESUMO

The World Health Organization characterized COVID-19 as a pandemic in March 2020, the second pandemic of the twenty-first century. Expanding virus populations, such as that of SARS-CoV-2, accumulate a number of narrowly shared polymorphisms, imposing a confounding effect on traditional clustering methods. In this context, approaches that reduce the complexity of the sequence space occupied by the SARS-CoV-2 population are necessary for robust clustering. Here, we propose subdividing the global SARS-CoV-2 population into six well-defined subtypes and 10 poorly represented genotypes named tentative subtypes by focusing on the widely shared polymorphisms in nonstructural (nsp3, nsp4, nsp6, nsp12, nsp13 and nsp14) cistrons and structural (spike and nucleocapsid) and accessory (ORF8) genes. The six subtypes and the additional genotypes showed amino acid replacements that might have phenotypic implications. Notably, three mutations (one of them in the Spike protein) were responsible for the geographical segregation of subtypes. We hypothesize that the virus subtypes detected in this study are records of the early stages of SARS-CoV-2 diversification that were randomly sampled to compose the virus populations around the world. The genetic structure determined for the SARS-CoV-2 population provides substantial guidelines for maximizing the effectiveness of trials for testing candidate vaccines or drugs.


Assuntos
Betacoronavirus/genética , Polimorfismo Genético , Betacoronavirus/classificação , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Genótipo , Humanos , Proteínas do Nucleocapsídeo/genética , Pandemias , Fosfoproteínas , Filogenia , Pneumonia Viral/epidemiologia , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Glicoproteína da Espícula de Coronavírus/genética , Proteínas não Estruturais Virais/genética , Proteínas Virais/genética
15.
Eur J Endocrinol ; 183(5): 497-504, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33107440

RESUMO

Background: Hypophosphataemic rickets (HR) comprise a clinically and genetically heterogeneous group of conditions, defined by renal-tubular phosphate wasting and consecutive loss of bone mineralisation. X-linked hypophosphataemia (XLH) is the most common form, caused by inactivating dominant mutations in PHEX, a gene encompassing 22 exons located at Xp22.1. XLH is treatable by anti-Fibroblast Growth Factor 23 antibody, while for other forms of HR such as therapy may not be indicated. Therefore, a genetic differentiation of HR is recommended. Objective: To develop and validate a next-generation sequencing panel for HR with special focus on PHEX. Design and methods: We designed an AmpliSeq gene panel for the IonTorrent PGM next-generation platform for PHEX and ten other HR-related genes. For validation of PHEX sequencing 50 DNA-samples from XLH-patients, in whom 42 different mutations in PHEX and 1 structural variation have been proven before, were blinded, anonymised and investigated with the NGS panel. In addition, we analyzed one known homozygous DMP1 mutation and two samples of HR-patients, where no pathogenic PHEX mutation had been detected by conventional sequencing. Results: The panel detected all 42 pathogenic missense/nonsense/splice-site/indel PHEX-mutations and in one the known homozygous DMP1 mutation. In the remaining two patients, we revealed a somatic mosaicism of a PHEX mutation in one; as well as two variations in DMP1 and a very rare compound heterozygous variation in ENPP1 in the second patient. Conclusions: This developed NGS panel is a reliable tool with high sensitivity and specificity for the diagnosis of XLH and related forms of HR.


Assuntos
Raquitismo Hipofosfatêmico Familiar/diagnóstico , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Nefropatias/diagnóstico , Endopeptidase Neutra Reguladora de Fosfato PHEX/análise , Distúrbios do Metabolismo do Fósforo/diagnóstico , Proteínas da Matriz Extracelular/análise , Raquitismo Hipofosfatêmico Familiar/genética , Feminino , Doenças Genéticas Ligadas ao Cromossomo X , Humanos , Nefropatias/genética , Masculino , Mutação , Fosfoproteínas/análise , Distúrbios do Metabolismo do Fósforo/genética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Análise de Sequência de DNA
16.
Nat Commun ; 11(1): 4093, 2020 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-33097703

RESUMO

A major challenge in genetic association studies is that most associated variants fall in the non-coding part of the human genome. We searched for variants associated with bone mineral density (BMD) after enriching the discovery cohort for loss-of-function (LoF) mutations by sequencing a subset of the Nord-Trøndelag Health Study, followed by imputation in the remaining sample (N = 19,705), and identified ten known BMD loci. However, one previously unreported variant, LoF mutation in MEPE, p.(Lys70IlefsTer26, minor allele frequency [MAF] = 0.8%), was associated with decreased ultradistal forearm BMD (P-value = 2.1 × 10-18), and increased osteoporosis (P-value = 4.2 × 10-5) and fracture risk (P-value = 1.6 × 10-5). The MEPE LoF association with BMD and fractures was further evaluated in 279,435 UK (MAF = 0.05%, heel bone estimated BMD P-value = 1.2 × 10-16, any fracture P-value = 0.05) and 375,984 Icelandic samples (MAF = 0.03%, arm BMD P-value = 0.12, forearm fracture P-value = 0.005). Screening for the MEPE LoF mutations before adulthood could potentially prevent osteoporosis and fractures due to the lifelong effect on BMD observed in the study. A key implication for precision medicine is that high-impact functional variants missing from the publicly available cosmopolitan panels could be clinically more relevant than polygenic risk scores.


Assuntos
Densidade Óssea/genética , Proteínas da Matriz Extracelular/genética , Fraturas Ósseas/genética , Estudos de Associação Genética , Predisposição Genética para Doença/genética , Glicoproteínas/genética , Fosfoproteínas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Biologia Computacional , Feminino , Frequência do Gene , Testes Genéticos , Genoma Humano , Humanos , Islândia , Masculino , Pessoa de Meia-Idade , Osteoporose/genética
17.
Immunity ; 53(5): 1095-1107.e3, 2020 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-33128877

RESUMO

Developing effective strategies to prevent or treat coronavirus disease 2019 (COVID-19) requires understanding the natural immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We used an unbiased, genome-wide screening technology to determine the precise peptide sequences in SARS-CoV-2 that are recognized by the memory CD8+ T cells of COVID-19 patients. In total, we identified 3-8 epitopes for each of the 6 most prevalent human leukocyte antigen (HLA) types. These epitopes were broadly shared across patients and located in regions of the virus that are not subject to mutational variation. Notably, only 3 of the 29 shared epitopes were located in the spike protein, whereas most epitopes were located in ORF1ab or the nucleocapsid protein. We also found that CD8+ T cells generally do not cross-react with epitopes in the four seasonal coronaviruses that cause the common cold. Overall, these findings can inform development of next-generation vaccines that better recapitulate natural CD8+ T cell immunity to SARS-CoV-2.


Assuntos
Betacoronavirus/imunologia , Linfócitos T CD8-Positivos/imunologia , Infecções por Coronavirus/imunologia , Pneumonia Viral/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Adulto , Idoso , Betacoronavirus/isolamento & purificação , Convalescença , Coronavirus/imunologia , Infecções por Coronavirus/diagnóstico , Mapeamento de Epitopos , Epitopos de Linfócito T , Feminino , Humanos , Epitopos Imunodominantes , Memória Imunológica , Masculino , Pessoa de Meia-Idade , Proteínas do Nucleocapsídeo/imunologia , Pandemias , Fosfoproteínas , Pneumonia Viral/diagnóstico , Poliproteínas , Proteínas Virais/imunologia , Adulto Jovem
18.
Sci Rep ; 10(1): 16219, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-33004978

RESUMO

COVID-19 pandemic has resulted in 16,114,449 cases with 646,641 deaths from the 217 countries, or territories as on July 27th 2020. Due to multifaceted issues and challenges in the implementation of the safety and preventive measures, inconsistent coordination between societies-governments and most importantly lack of specific vaccine to SARS-CoV-2, the spread of the virus that initially emerged at Wuhan is still uprising after taking a heavy toll on human life. In the present study, we mapped immunogenic epitopes present on the four structural proteins of SARS-CoV-2 and we designed a multi-epitope peptide based vaccine that, demonstrated a high immunogenic response with a vast application on world's human population. On codon optimization and in-silico cloning, we found that candidate vaccine showed high expression in E. coli and immune simulation resulted in inducing a high level of both B-cell and T-cell mediated immunity. The results predicted that exposure of vaccine by administrating three injections significantly subsidized the antigen growth in the system. The proposed candidate vaccine found promising by yielding desired results and hence, should be validated by practical experimentations for its functioning and efficacy to neutralize SARS-CoV-2.


Assuntos
Epitopos/imunologia , Simulação de Acoplamento Molecular , Vacinas de Subunidades/imunologia , Vacinas Virais/imunologia , Complexo Antígeno-Anticorpo/química , Complexo Antígeno-Anticorpo/imunologia , Antígenos Virais/imunologia , Linfócitos B/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Epitopos/química , Antígenos HLA/química , Antígenos HLA/imunologia , Humanos , Imunogenicidade da Vacina , Proteínas do Nucleocapsídeo/química , Proteínas do Nucleocapsídeo/imunologia , Fosfoproteínas , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Linfócitos T/imunologia , Receptores Toll-Like/imunologia , Vacinas de Subunidades/química , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/imunologia , Vacinas Virais/química
19.
Nat Commun ; 11(1): 4940, 2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-33009411

RESUMO

The HUSH complex represses retroviruses, transposons and genes to maintain the integrity of vertebrate genomes. HUSH regulates deposition of the epigenetic mark H3K9me3, but how its three core subunits - TASOR, MPP8 and Periphilin - contribute to assembly and targeting of the complex remains unknown. Here, we define the biochemical basis of HUSH assembly and find that its modular architecture resembles the yeast RNA-induced transcriptional silencing complex. TASOR, the central HUSH subunit, associates with RNA processing components. TASOR is required for H3K9me3 deposition over LINE-1 repeats and repetitive exons in transcribed genes. In the context of previous studies, this suggests that an RNA intermediate is important for HUSH activity. We dissect the TASOR and MPP8 domains necessary for transgene repression. Structure-function analyses reveal TASOR bears a catalytically-inactive PARP domain necessary for targeted H3K9me3 deposition. We conclude that TASOR is a multifunctional pseudo-PARP that directs HUSH assembly and epigenetic regulation of repetitive genomic targets.


Assuntos
Elementos de DNA Transponíveis/genética , Epigênese Genética , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Sequência de Aminoácidos , Antígenos de Neoplasias/metabolismo , Sítios de Ligação , Éxons/genética , Genoma , Células HEK293 , Células HeLa , Histonas/metabolismo , Humanos , Lisina/metabolismo , Espectroscopia de Ressonância Magnética , Metilação , NAD/metabolismo , Proteínas Nucleares/química , Fosfoproteínas/metabolismo , Ligação Proteica , Domínios Proteicos , RNA/metabolismo , Processamento Pós-Transcricional do RNA , Transcrição Genética
20.
Sci Rep ; 10(1): 16561, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-33024213

RESUMO

As the Coronavirus Disease 2019 (COVID-19), which is caused by the novel SARS-CoV-2, continues to spread rapidly around the world, there is a need for well validated serological assays that allow the detection of viral specific antibody responses in COVID-19 patients or recovered individuals. In this study, we established and used multiple indirect Enzyme Linked Immunosorbent Assay (ELISA)-based serological assays to study the antibody response in COVID-19 patients. In order to validate the assays we determined the cut off values, sensitivity and specificity of the assays using sera collected from pre-pandemic healthy controls, COVID-19 patients at different time points after disease-onset, and seropositive sera to other human coronaviruses (CoVs). The developed SARS-CoV-2 S1 subunit of the spike glycoprotein and nucleocapsid (N)-based ELISAs not only showed high specificity and sensitivity but also did not show any cross-reactivity with other CoVs. We also show that all RT-PCR confirmed COVID-19 patients tested in our study developed both virus specific IgM and IgG antibodies as early as week one after disease onset. Our data also suggest that the inclusion of both S1 and N in serological testing would capture as many potential SARS-CoV-2 positive cases as possible than using any of them alone. This is specifically important for tracing contacts and cases and conducting large-scale epidemiological studies to understand the true extent of virus spread in populations.


Assuntos
Anticorpos Antivirais/sangue , Betacoronavirus/imunologia , Infecções por Coronavirus/diagnóstico , Proteínas do Nucleocapsídeo/imunologia , Pneumonia Viral/diagnóstico , Soroconversão , Testes Sorológicos/métodos , Glicoproteína da Espícula de Coronavírus/imunologia , Adulto , Idoso , Betacoronavirus/genética , Estudos de Coortes , Infecções por Coronavirus/virologia , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Masculino , Pessoa de Meia-Idade , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Pandemias , Fosfoproteínas , Pneumonia Viral/virologia , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade , Adulto Jovem
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