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1.
Mol Cell ; 79(6): 1037-1050.e5, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32882183

RESUMO

DNA double-stranded breaks (DSBs) are dangerous lesions threatening genomic stability. Fidelity of DSB repair is best achieved by recombination with a homologous template sequence. In yeast, transcript RNA was shown to template DSB repair of DNA. However, molecular pathways of RNA-driven repair processes remain obscure. Utilizing assays of RNA-DNA recombination with and without an induced DSB in yeast DNA, we characterize three forms of RNA-mediated genomic modifications: RNA- and cDNA-templated DSB repair (R-TDR and c-TDR) using an RNA transcript or a DNA copy of the RNA transcript for DSB repair, respectively, and a new mechanism of RNA-templated DNA modification (R-TDM) induced by spontaneous or mutagen-induced breaks. While c-TDR requires reverse transcriptase, translesion DNA polymerase ζ (Pol ζ) plays a major role in R-TDR, and it is essential for R-TDM. This study characterizes mechanisms of RNA-DNA recombination, uncovering a role of Pol ζ in transferring genetic information from transcript RNA to DNA.


Assuntos
DNA/genética , RNA/genética , Saccharomyces cerevisiae/genética , Adolescente , Adulto , DNA/ultraestrutura , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Replicação do DNA/genética , DNA Complementar/genética , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/ultraestrutura , Instabilidade Genômica/genética , Humanos , Pessoa de Meia-Idade , RNA/ultraestrutura , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Adulto Jovem
2.
PLoS One ; 15(9): e0238614, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32936826

RESUMO

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and associated with severe respiratory illness emerged in Wuhan, China, in late 2019. The virus has been able to spread promptly across all continents in the world. The current pandemic has posed a great threat to public health concern and safety. Currently, there are no specific treatments or licensed vaccines available for COVID-19. We isolated SARS-CoV-2 from the nasopharyngeal sample of a patient in Turkey with confirmed COVID-19. We determined that the Vero E6 and MA-104 cell lines are suitable for supporting SARS-CoV-2 that supports viral replication, development of cytopathic effect (CPE) and subsequent cell death. Phylogenetic analyses of the whole genome sequences showed that the hCoV-19/Turkey/ERAGEM-001/2020 strain clustered with the strains primarily from Australia, Canada, England, Iran and Kuwait and that the cases in the nearby clusters were reported to have travel history to Iran and to share the common unique nucleotide substitutions.


Assuntos
Betacoronavirus/isolamento & purificação , Pandemias , Cultura de Vírus/métodos , Animais , Austrália , Betacoronavirus/genética , Betacoronavirus/fisiologia , Canadá , Linhagem Celular , Chlorocebus aethiops , Busca de Comunicante , Infecções por Coronavirus , Efeito Citopatogênico Viral , DNA Complementar/genética , DNA Viral/genética , Inglaterra , Genoma Viral , Células HeLa , Humanos , Irã (Geográfico) , Kuweit , Macaca mulatta , Nasofaringe/virologia , Filogenia , Pneumonia Viral , Análise de Sequência de DNA , Viagem , Turquia/epidemiologia , Células Vero , Replicação Viral
3.
mBio ; 11(5)2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32978313

RESUMO

Infectious coronavirus (CoV) disease 2019 (COVID-19) emerged in the city of Wuhan (China) in December 2019, causing a pandemic that has dramatically impacted public health and socioeconomic activities worldwide. A previously unknown coronavirus, severe acute respiratory syndrome CoV-2 (SARS-CoV-2), has been identified as the causative agent of COVID-19. To date, there are no U.S. Food and Drug Administration (FDA)-approved vaccines or therapeutics available for the prevention or treatment of SARS-CoV-2 infection and/or associated COVID-19 disease, which has triggered a large influx of scientific efforts to develop countermeasures to control SARS-CoV-2 spread. To contribute to these efforts, we have developed an infectious cDNA clone of the SARS-CoV-2 USA-WA1/2020 strain based on the use of a bacterial artificial chromosome (BAC). Recombinant SARS-CoV-2 (rSARS-CoV-2) was readily rescued by transfection of the BAC into Vero E6 cells. Importantly, BAC-derived rSARS-CoV-2 exhibited growth properties and plaque sizes in cultured cells comparable to those of the natural SARS-CoV-2 isolate. Likewise, rSARS-CoV-2 showed levels of replication similar to those of the natural isolate in nasal turbinates and lungs of infected golden Syrian hamsters. This is, to our knowledge, the first BAC-based reverse genetics system for the generation of infectious rSARS-CoV-2 that displays features in vivo similar to those of a natural viral isolate. This SARS-CoV-2 BAC-based reverse genetics will facilitate studies addressing several important questions in the biology of SARS-CoV-2, as well as the identification of antivirals and development of vaccines for the treatment of SARS-CoV-2 infection and associated COVID-19 disease.IMPORTANCE The pandemic coronavirus (CoV) disease 2019 (COVID-19) caused by severe acute respiratory syndrome CoV-2 (SARS-CoV-2) is a major threat to global human health. To date, there are no approved prophylactics or therapeutics available for COVID-19. Reverse genetics is a powerful approach to understand factors involved in viral pathogenesis, antiviral screening, and vaccine development. In this study, we describe the feasibility of generating recombinant SARS-CoV-2 (rSARS-CoV-2) by transfection of a single bacterial artificial chromosome (BAC). Importantly, rSARS-CoV-2 possesses the same phenotype as the natural isolate in vitro and in vivo This is the first description of a BAC-based reverse genetics system for SARS-CoV-2 and the first time that an rSARS-CoV-2 isolate has been shown to be phenotypically identical to a natural isolate in a validated animal model of SARS-CoV-2 infection. The BAC-based reverse genetics approach will facilitate the study of SARS-CoV-2 and the development of prophylactics and therapeutics for the treatment of COVID-19.


Assuntos
Betacoronavirus/genética , Cromossomos Artificiais Bacterianos/genética , Animais , Betacoronavirus/patogenicidade , Betacoronavirus/fisiologia , Chlorocebus aethiops , Infecções por Coronavirus/virologia , Cricetinae , DNA Complementar/genética , Genoma Viral/genética , Pandemias , Pneumonia Viral/virologia , RNA Viral/genética , Genética Reversa , Células Vero , Replicação Viral
4.
PLoS One ; 15(8): e0235102, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32857764

RESUMO

Inosine is ubiquitous and essential in many biological processes, including RNA-editing. In addition, oxidative stress on RNA has been a topic of increasing interest due, in part, to its potential role in the development/progression of disease. In this work we probed the ability of three reverse transcriptases (RTs) to catalyze the synthesis of cDNA in the presence of RNA templates containing inosine (I), 8-oxo-7,8-dihydroinosine (8oxo-I), guanosine (G), or 8-oxo-7,8-dihydroguanosine (8-oxoG), and explored the impact that these purine derivatives have as a function of position. To this end, we used 29-mers of RNA (as template) containing the modifications at position-18 and reverse transcribed DNA using 17-mers, 18-mers, or 19-mers (as primers). Generally reactivity of the viral RTs, AMV / HIV / MMLV, towards cDNA synthesis was similar for templates containing G or I as well as for those with 8-oxoG or 8-oxoI. Notable differences are: 1) the use of 18-mers of DNA (to explore cDNA synthesis past the lesion/modification) led to inhibition of DNA elongation in cases where a G:dA wobble pair was present, while the presence of I, 8-oxoI, or 8-oxoG led to full synthesis of the corresponding cDNA, with the latter two displaying a more efficient process; 2) HIV RT is more sensitive to modified base pairs in the vicinity of cDNA synthesis; and 3) the presence of a modification two positions away from transcription initiation has an adverse impact on the overall process. Steady-state kinetics were established using AMV RT to determine substrate specificities towards canonical dNTPs (N = G, C, T, A). Overall we found evidence that RNA templates containing inosine are likely to incorporate dC > dT > > dA, where reactivity in the presence of dA was found to be pH dependent (process abolished at pH 7.3); and that the absence of the C2-exocyclic amine, as displayed with templates containing 8-oxoI, leads to increased selectivity towards incorporation of dA over dC. The data will be useful in assessing the impact that the presence of inosine and/or oxidatively generated lesions have on viral processes and adds to previous reports where I codes exclusively like G. Similar results were obtained upon comparison of AMV and MMLV RTs.


Assuntos
Vírus da Mieloblastose Aviária/enzimologia , Transcriptase Reversa do HIV/metabolismo , Vírus da Leucemia Murina de Moloney/enzimologia , DNA Polimerase Dirigida por RNA/metabolismo , Animais , Sequência de Bases , DNA Complementar/biossíntese , DNA Complementar/química , DNA Complementar/genética , Guanosina/análogos & derivados , Guanosina/química , Guanosina/metabolismo , Humanos , Técnicas In Vitro , Inosina/análogos & derivados , Inosina/química , Inosina/metabolismo , Cinética , Camundongos , RNA Viral/química , RNA Viral/genética , RNA Viral/metabolismo , Moldes Genéticos
5.
Appl Microbiol Biotechnol ; 104(19): 8427-8437, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32813067

RESUMO

Infectious bronchitis virus (IBV) is a member of genus gamma-coronavirus in the family Coronaviridae, causing serious economic losses to the poultry industry. Reverse genetics is a common technique to study the biological characteristics of viruses. So far, there is no BAC reverse genetic system available for rescue of IBV infectious clone. In the present study, a new strategy for the construction of IBV infectious cDNA clone was established. The full-length genomic cDNA of IBV vaccine strain H120 was constructed in pBAC vector from four IBV fragment subcloning vectors by homologous recombination, which contained the CMV promoter at the 5' end and the hepatitis D virus ribozyme (HDVR) sequence and bovine growth hormone polyadenylation (BGH) sequence after the polyA tail at the 3' end of the full-length cDNA. Subsequently, using the same technique, another plasmid pBAC-H120/SCS1 was also constructed, in which S1 gene from IBV H120 strain was replaced with that of a virulent SC021202 strain. Recombinant virus rH120 and rH120/SCS1 were rescued by transfecting the plasmids into BHK cells and passaged in embryonated chicken eggs. Finally, the pathogenicity of both the recombinant virus strains rH120 and rH120/SCS1 was evaluated in SPF chickens. The results showed that the chimeric rH120/SCS1 strain was not pathogenic compared with the wild-type IBV SC021202 strain and the chickens inoculated with rH120/SCS1 could resist challenge infection by IBV SC021202. Taken together, our results indicate that BAC reverse genetic system could be used to rescue IBV in vitro and IBV S1 protein alone might not be the key factor for IBV pathogenicity. KEY POINTS: • BAC vector was used to construct IBV full-length cDNA by homologous recombination. • Based on four subcloning vectors, a recombinant chimeric IBV H120/SCS1 was constructed and rescued. • Pathogenicity of H120/SCS1 was similar to that of H120, but different to that of SC021202.


Assuntos
Vírus da Bronquite Infecciosa/genética , Vírus da Bronquite Infecciosa/patogenicidade , Proteínas Virais/genética , Animais , Embrião de Galinha , Galinhas , Infecções por Coronavirus/veterinária , DNA Complementar , Recombinação Homóloga , Doenças das Aves Domésticas/virologia , Proteínas Recombinantes/genética , Vacinas Virais/genética , Vacinas Virais/imunologia , Virulência/genética
6.
Med Sci (Paris) ; 36(8-9): 797-802, 2020.
Artigo em Francês | MEDLINE | ID: mdl-32755538

RESUMO

SARS-CoV-2 (severe acute respiratory syndrome-coronavirus-2, which emerged in China at the end of 2019, is responsible for a global health crisis resulting in the confinement of more than 3 billion people worldwide and the sharp decline of the world economy. In this context, a race against the clock is launched in order to develop a treatment to stop the pandemic as soon as possible. A study published in Nature by the Volker Thiel team reports the development of reverse genetics for SARS-CoV-2 allowing them to recreate the virus in just a few weeks. The perspectives of this work are very interesting since it will allow the genetic manipulation of the virus and thus the development of precious tools which will be useful to fight the infection. Even though this approach represents a technological leap that will improve our knowledge of the virus, it also carries the germ of possible misuse and the creation of the virus for malicious purposes. The advantages and disadvantages of recreating SARS-CoV-2 in this pandemic period are discussed in this mini-synthesis.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/virologia , Organismos Geneticamente Modificados , Pandemias , Pneumonia Viral/virologia , Genética Reversa/métodos , Betacoronavirus/patogenicidade , Derramamento de Material Biológico , Cromossomos Artificiais de Levedura , Clonagem Molecular/métodos , Coronaviridae/classificação , Coronaviridae/genética , Coronaviridae/patogenicidade , Infecções por Coronavirus/prevenção & controle , DNA Complementar/genética , Especificidade de Hospedeiro , Humanos , Organismos Geneticamente Modificados/genética , Organismos Geneticamente Modificados/patogenicidade , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , RNA Viral/genética , Recombinação Genética , Risco , Vacinas Virais
7.
J Med Microbiol ; 69(8): 1114-1123, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32783802

RESUMO

Introduction. Coronavirus disease 2019 (COVID-19) is an infectious disease caused by Severe Acute Respiratory Corona Virus-2 (SARS-CoV-2). The disease was first identified in December 2019 in Wuhan, the capital of China's Hubei province, and has since spread globally, resulting in the ongoing 2019-2020 corona virus pandemic. SARS-CoV-2 is closely related to the original SARS-CoV. It is thought to have a zoonotic origin. The virus is primarily spread between people during close contact, often via small droplets produced by coughing, sneezing or talking. People may also become infected by touching a contaminated surface and then touching their face. COVID-19 patients currently remain the primary source of infection. An epidemiological survey indicated that the general population is susceptible to SARS-CoV-2. The spectrum of this disease ranges from mild to life-threatening. Fever is the most common symptom, although older people and those with comorbidities may experience fever later in the disease. Other common symptoms include cough, loss of appetite, fatigue, shortness of breath, sputum production, and muscle and joint pains. Symptoms such as nausea, vomiting and diarrhea have been observed in varying percentages. Some cases might progress promptly to acute respiratory distress syndrome (ARDS) and/or multiple organ function failure. Asymptomatic carriers and those in the incubation period may also be infectious.Aim. To determine the epidemiological and clinical characteristics of patients presenting with COVID-19 at the screening clinic of a tertiary care hospital in Peshawar, Pakistan.Methodology. In this descriptive study, we analysed data of patients presenting to a newly established Covid-19 screening clinic in Rehman Medical Institute. Anyone who reported with new onset fever and/or cough was tested for SARS-CoV-2 in the screening clinic. We documented and analysed demographic, epidemiological and clinical characteristics, which included age, sex, travel history, clinical features, comorbidities and laboratory data of patients confirmed by real-time reverse-transcription (RT)-PCR at Rehman Medical Institute, Peshawar, Pakistan from 15 March till 21 April 2020. Paired specimens of throat swabs and nasal swabs were obtained from 845 patients, ribonucleic acid (RNA) was extracted and tested for SARS-CoV-2 by the RT-PCR assay.Results. A total of 845 specimens were taken as described above. The positive rate for SARS-CoV-2 was about 14.3%. Male and older population had a significantly higher positive rate. Of the 121 patients infected with SARS-CoV-2, the mean age was 43.19 years (sd, 17.57) and the infections were more frequent among male gender accounting for 85 (70.25 %) patients. Common symptoms included fever (88 patients, 72 %), cough (72 patients, 59.5 %) and shortness of breath (69 patients, 57 %). Twenty-two (18 %) patients had recent travel history outside Pakistan in the previous 14 days, the majority of whom had returned back from Saudi Arabia.Conclusion. In this single-centre, prospective, descriptive study, fever, cough and shortness of breath were the most common symptoms. Old age (>50 years), chronic underlying comorbidities and travel history may be risk factors. Therefore, we concluded that viral nucleic acid amplification tests (NAAT) played an important role in identifying SARS-CoV-2 infection in a screening clinic, which helped with isolation and cohorting of these patients.


Assuntos
Betacoronavirus , Infecções por Coronavirus/epidemiologia , Surtos de Doenças , Pneumonia Viral/epidemiologia , Adolescente , Adulto , Distribuição por Idade , Idoso , Idoso de 80 Anos ou mais , Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Criança , Pré-Escolar , Comorbidade , Infecções por Coronavirus/diagnóstico , DNA Complementar/genética , Feminino , Humanos , Lactente , Masculino , Programas de Rastreamento , Pessoa de Meia-Idade , Pandemias , Pneumonia Viral/diagnóstico , Estudos Prospectivos , RNA Viral/genética , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Risco , Distribuição por Sexo , Viagem , Adulto Jovem
8.
PLoS One ; 15(8): e0236477, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32756607

RESUMO

Antibodies function by binding to antigens. Antibodies must be cloned and expressed to determine their binding characteristics, but current methods for high-throughput antibody sequencing yield antibody DNA pooled from many cells and do not readily permit cloning of antibodies from single B cells. We present a strategy for retrieving and cloning antibody DNA from single cells within a pooled library of cells. Our strategy, called selective PCR for antibody retrieval (SPAR), takes advantage of the unique sequence barcodes attached to individual cDNA molecules during sample preparation to enable specific amplification by PCR of antibody heavy- and light-chain cDNA originating from a single cell. We show through computational analysis that most human antibodies sequenced using typical high-throughput methods can be retrieved using SPAR, and experimentally demonstrate retrieval of full-length antibody variable region cDNA from three cells within pools of ~5,000 cells. SPAR enables rapid low-cost cloning and expression of native human antibodies from pooled single-cell sequence libraries for functional characterization.


Assuntos
Anticorpos/genética , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Reação em Cadeia da Polimerase/métodos , Sequência de Aminoácidos/genética , Técnicas de Visualização da Superfície Celular , Clonagem Molecular , DNA Complementar/genética , Biblioteca Gênica , Vetores Genéticos/genética , Humanos , Análise de Célula Única
9.
Exp Parasitol ; 217: 107963, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32781092

RESUMO

This study analyzed the large-subunit (60S) ribosomal protein L12 of Eimeria tenella (Et60s-RPL12). A full-length cDNA was cloned, and the recombinant protein was expressed in E. coli BL21 and inoculated in rabbits to produce the polyclonal antibody. Quantitative real-time polymerase chain reaction and western blotting were used to analyze the transcription levels of Et60s-RPL12 and translation levels in different developmental stages of E. tenella. The results showed that the mRNA transcription level of Et60s-RPL12 was highest in second-generation merozoites, whereas the translation level was highest in unsporulated oocysts. Indirect immunofluorescence showed that Et60s-RPL12 was localized to the anterior region and surface of sporozoites, except for the two refractile bodies. As the invasion of DF-1 cells progressed, fluorescence intensity was increased, and Et60s-RPL12 was localized to the parasitophorous vacuole membrane (PVM). The secretion assay results using staurosporine indicated that this protein was secreted, but not from micronemes. The role of Et60s-RPL12 in invasion was evaluated in vitro. The results of the invasion assay showed that polyclonal antibody inhibited host cell invasion by the parasite, which reached about 12%. However, the rate of invasion was not correlated with the concentration of IgG.


Assuntos
Eimeria tenella/genética , Proteínas Ribossômicas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Ceco/parasitologia , Linhagem Celular , Embrião de Galinha , Galinhas , Biologia Computacional , DNA Complementar/genética , DNA Complementar/metabolismo , Eimeria tenella/química , Eletroforese em Gel de Poliacrilamida , Fezes/parasitologia , Fibroblastos , Técnica Indireta de Fluorescência para Anticorpo , Biossíntese de Proteínas , Coelhos , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Ribossômicas/química , Organismos Livres de Patógenos Específicos , Transcrição Genética
10.
Viruses ; 12(8)2020 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-32824272

RESUMO

Genome sequencing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is increasingly important to monitor the transmission and adaptive evolution of the virus. The accessibility of high-throughput methods and polymerase chain reaction (PCR) has facilitated a growing ecosystem of protocols. Two differing protocols are tiling multiplex PCR and bait capture enrichment. Each method has advantages and disadvantages but a direct comparison with different viral RNA concentrations has not been performed to assess the performance of these approaches. Here we compare Liverpool amplification, ARTIC amplification, and bait capture using clinical diagnostics samples. All libraries were sequenced using an Illumina MiniSeq with data analyzed using a standardized bioinformatics workflow (SARS-CoV-2 Illumina GeNome Assembly Line; SIGNAL). One sample showed poor SARS-CoV-2 genome coverage and consensus, reflective of low viral RNA concentration. In contrast, the second sample had a higher viral RNA concentration, which yielded good genome coverage and consensus. ARTIC amplification showed the highest depth of coverage results for both samples, suggesting this protocol is effective for low concentrations. Liverpool amplification provided a more even read coverage of the SARS-CoV-2 genome, but at a lower depth of coverage. Bait capture enrichment of SARS-CoV-2 cDNA provided results on par with amplification. While only two clinical samples were examined in this comparative analysis, both the Liverpool and ARTIC amplification methods showed differing efficacy for high and low concentration samples. In addition, amplification-free bait capture enriched sequencing of cDNA is a viable method for generating a SARS-CoV-2 genome sequence and for identification of amplification artifacts.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , RNA Viral/genética , Sequência de Bases , Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , DNA Complementar/genética , Genoma Viral , Humanos , Epidemiologia Molecular , Reação em Cadeia da Polimerase Multiplex/métodos , Pandemias , Sequenciamento Completo do Genoma/métodos
11.
Methods Mol Biol ; 2203: 167-184, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32833212

RESUMO

The Escherichia coli and vaccinia virus-based reverse genetics systems have been widely applied for the manipulation and engineering of coronavirus genomes. These systems, however, present several limitations and are sometimes difficult to establish in a timely manner for (re-)emerging viruses. In this chapter, we present a new universal reverse genetics platform for the assembly and engineering of infectious full-length cDNAs using yeast-based transformation-associated recombination cloning. This novel assembly method not only results in stable coronavirus infectious full-length cDNAs cloned in the yeast Saccharomyces cerevisiae but also fosters and accelerates the manipulation of their genomes. Such a platform is widely applicable for the scientific community, as it requires no specific equipment and can be performed in a standard laboratory setting. The protocol described can be easily adapted to virtually all known or emerging coronaviruses, such as Middle East respiratory syndrome coronavirus (MERS-CoV).


Assuntos
Coronavirus/genética , DNA Complementar/genética , Genômica/métodos , Saccharomyces cerevisiae/genética , Animais , Linhagem Celular , Coronavirus/patogenicidade , Recombinação Homóloga , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Coronavírus da Síndrome Respiratória do Oriente Médio/patogenicidade
12.
Mol Biol Rep ; 47(9): 7283-7289, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32772343

RESUMO

It is well established that Escherichia coli represents a powerful tool for the over-expression of human proteins for structure/function studies. In many cases, such as for membrane transporters, the bacterial toxicity or the aggregation of the target protein hamper the expression limiting the application of this tool. The aim of this study was finding the appropriate conditions for the expression of reluctant proteins that is the human neutral amino acid transporters ASCT2 and B0AT1, that have great relevance to human health in cancer therapy and in COVID-19 research, respectively. The cDNAs coding for the proteins of interest were cloned in the pCOLD I vector and different E. coli strains (BL21 codon plus RIL, and RosettaGami2) were cultured in absence or in presence of glucose (0.5-1%), at low temperature (15 °C), and low inducer concentrations (10-100 µM). Cell growth and protein production were monitored by optical density measurements and western blotting assay, respectively. Even though in different conditions, the expression of both amino acid transporters was obtained.Reducing the growth rate of specific E. coli strains by lowering the temperature and the IPTG concentration, together with the addition of glucose, two reluctant human neutral amino acid transporters have been expressed in E. coli. The results have a potentially great interest in drug discovery since ASCT2 is an acknowledged target of anticancer therapy, and B0AT1 together with ACE2 is part of a receptor for the SARS-Cov-2 RBD proteins.


Assuntos
Sistema ASC de Transporte de Aminoácidos/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Betacoronavirus/fisiologia , Infecções por Coronavirus/virologia , Escherichia coli/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Pneumonia Viral/virologia , Sistema ASC de Transporte de Aminoácidos/genética , Sistemas de Transporte de Aminoácidos Neutros/genética , Temperatura Baixa , DNA Complementar/genética , Descoberta de Drogas , Escherichia coli/genética , Expressão Gênica , Humanos , Antígenos de Histocompatibilidade Menor/genética , Pandemias , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo
14.
PLoS One ; 15(7): e0236366, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32702062

RESUMO

Deep sequencing of T-cell receptor (TCR) genes is powerful at profiling immune repertoire. To prepare a TCR sequencing library, multiplex polymerase chain reaction (mPCR) is widely applied and is highly efficient. That is, most mPCR products contain the region critical for antigen recognition, which also indicates regular V(D)J recombination. Multiplex PCR, however, may suffer from primer bias. A promising alternative is 5'-RACE, which avoids primer bias by applying only one primer pair. In 5'-RACE data, however, non-regular V(D)J recombination (e.g., TCR sequences without a V gene segment) has been observed and the frequency varies (30-80%) between studies. This suggests that the cause of or how to reduce non-regular TCR sequences is not yet well known by the science community. Although it is possible to speculate the cause by comparing the 5'-RACE protocols, careful experimental confirmation is needed and such a systematic study is still not available. Here, we examined the 5'-RACE protocol of a commercial kit and demonstrated how a modification increased the fraction of regular TCR-ß sequences to >85%. We also found a strong linear correlation between the fraction of short DNA fragments and the percentage of non-regular TCR-ß sequences, indicating that the presence of short DNA fragments in the library was the main cause of non-regular TCR-ß sequences. Therefore, thorough removal of short DNA fragments from a 5'-RACE library is the key to high data efficiency. We highly recommend conducting a fragment length analysis before sequencing, and the fraction of short DNA fragments can be used to estimate the percentage of non-regular TCR sequences. As deep sequencing of TCR genes is still relatively expensive, good quality control should be valuable.


Assuntos
Sequência de Aminoácidos/genética , DNA/genética , Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T/genética , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Sequência de Bases , Fragmentação do DNA , DNA Complementar/genética , Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T/imunologia , Humanos , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia
15.
J Virol Methods ; 284: 113937, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32659241

RESUMO

Due to the huge demand for SARS-Cov-2 determination,alternatives to the standard qtPCRtestsare potentially useful for increasing the number of samples screened. Our aim was to develop a direct fluorescent PCR capillary-electrophoresis detection of the viral genome. We validated this approach on several SARS-Cov-2 positive and negative samples.We isolated the naso-pharingealRNA from 20 positive and 10 negative samples. The cDNA was synthesised and two fragments of the SARS-Cov-2 were amplified. One of the primers for each pair was 5´-end fluorochrome labelled. The amplifications were subjected to capillary electrophoresis in ABI3130 sequencers to visualize the fluorescent peaks.The two SARS-Cov-2 fragments were successfully amplified in the positive samples, while the negative samples did not render fluorescent peaks. In conclusion, we describe and alternative method to identify the SARS-Cov-2 genome that could be scaled to the analysis of approximately 100 samples in less than 5 h. By combining a standard PCR with capillary electrophoresis our approach would overcome the limits imposed to many labs by the qtPCR and increase the testing capacity.


Assuntos
Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/virologia , Eletroforese Capilar/métodos , Pneumonia Viral/virologia , Reação em Cadeia da Polimerase/métodos , Sequência de Bases , Betacoronavirus/genética , Infecções por Coronavirus/diagnóstico , Primers do DNA/genética , DNA Complementar/genética , Genoma Viral , Humanos , Técnicas de Amplificação de Ácido Nucleico/métodos , Pandemias , Pneumonia Viral/diagnóstico , Sensibilidade e Especificidade
16.
In Vivo ; 34(3 Suppl): 1629-1632, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: covidwho-534630

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a positive-sense single-stranded RNA virus. It is contagious in humans and is the cause of the coronavirus disease 2019 (COVID-19) pandemic. In the current analysis, we searched for SARS-CoV-2 sequences within the human genome. To compare the SARS-CoV-2 genome to the human genome, we used the blast-like alignment tool (BLAT) of the University of California, Santa Cruz Genome Browser. BLAT can align a user sequence of 25 bases or more to the genome. BLAT search results revealed a 117-base pair SARS-CoV-2 sequence in the human genome with 94.6% identity. The sequence was in chromosome 1p within an intronic region of the netrin G1 (NTNG1) gene. The sequence matched a sequence in the SARS-CoV-2 orf1b (open reading frames) gene. The SARS-CoV-2 human sequence lies within non-structural proteins 14 and 15 (NSP14 and NSP15), and is quite close to the viral spike sequence, separated only by NSP16, a 904-base pair sequence. The mechanism for SARS-CoV-2 infection is the binding of the virus spike protein to the membrane-bound form of angiotensin-converting enzyme 2 and internalization of the complex by the host cell. It is probably no accident that a sequence from the SARS-CoV-2 orf1b gene is found in the human NTNG1 gene, implicated in schizophrenia, and that haloperidol, used to treat schizophrenia, may also be a treatment for COVID-19. We suggest, therefore, that it is important to investigate other haloperidol analogs. Among them are benperidol, bromperidol, bromperidol decanoate, droperidol, seperidol hydrochloride, and trifluperidol. These analogs might be valuable in the treatment of COVID-19 and other coronavirus infections.


Assuntos
Betacoronavirus/genética , Cromossomos Humanos Par 1/genética , Genes Virais , Netrina-1/genética , Proteínas Virais/genética , Animais , Antivirais/farmacologia , Antivirais/uso terapêutico , Sequência de Bases , Infecções por Coronavirus/tratamento farmacológico , DNA Complementar/genética , Endorribonucleases/genética , Exorribonucleases/genética , Haloperidol/análogos & derivados , Haloperidol/farmacologia , Haloperidol/uso terapêutico , Humanos , Íntrons/genética , Pan troglodytes/genética , Pandemias , Pneumonia Viral/tratamento farmacológico , RNA Viral/genética , Esquizofrenia/tratamento farmacológico , Esquizofrenia/genética , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Especificidade da Espécie , Proteínas não Estruturais Virais/genética
17.
Viruses ; 12(6)2020 06 03.
Artigo em Inglês | MEDLINE | ID: covidwho-532726

RESUMO

Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory illness in humans; the second-largest and most deadly outbreak to date occurred in Saudi Arabia. The dromedary camel is considered a possible host of the virus and also to act as a reservoir, transmitting the virus to humans. Here, we studied evolutionary relationships for 31 complete genomes of betacoronaviruses, including eight newly sequenced MERS-CoV genomes isolated from dromedary camels in Saudi Arabia. Through bioinformatics tools, we also used available sequences and 3D structure of MERS-CoV spike glycoprotein to predict MERS-CoV epitopes and assess antibody binding affinity. Phylogenetic analysis showed the eight new sequences have close relationships with existing strains detected in camels and humans in Arabian Gulf countries. The 2019-nCov strain appears to have higher homology to both bat coronavirus and SARS-CoV than to MERS-CoV strains. The spike protein tree exhibited clustering of MERS-CoV sequences similar to the complete genome tree, except for one sequence from Qatar (KF961222). B cell epitope analysis determined that the MERS-CoV spike protein has 24 total discontinuous regions from which just six epitopes were selected with score values of >80%. Our results suggest that the virus circulates by way of camels crossing the borders of Arabian Gulf countries. This study contributes to finding more effective vaccines in order to provide long-term protection against MERS-CoV and identifying neutralizing antibodies.


Assuntos
Camelus/virologia , Infecções por Coronavirus/virologia , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Glicoproteína da Espícula de Coronavírus/genética , Sequência de Aminoácidos , Animais , Betacoronavirus/classificação , Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Evolução Biológica , DNA Complementar/química , DNA Viral/química , Epitopos/análise , Epitopos/química , Epitopos/genética , Biblioteca Gênica , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/classificação , Coronavírus da Síndrome Respiratória do Oriente Médio/isolamento & purificação , Filogenia , RNA Viral/análise , RNA Viral/química , RNA Viral/isolamento & purificação , Arábia Saudita
18.
Nucleic Acids Res ; 48(15): e86, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32544240

RESUMO

Specific nucleic acid sequences can be detected in individual cells by in situ hybridization. However, when very few copies of a target sequence are present per cell, its signal is undetectable by flow cytometry. Although various approaches have been developed to increase fluorescence signals for in situ hybridization, flow cytometric detection of specific genomic DNA sequences has not been established. Here, we present a flow cytometry assay for detection of single-copy genomic sequences in human lymphocytes using in situ PCR with universal energy transfer-labelled primers.


Assuntos
DNA/isolamento & purificação , Citometria de Fluxo/métodos , Imagem Individual de Molécula/métodos , DNA/genética , DNA Complementar/química , DNA Complementar/genética , Humanos , Hibridização In Situ/métodos , Linfócitos/química , Reação em Cadeia da Polimerase/métodos
19.
J Vis Exp ; (159)2020 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-32478748

RESUMO

Osteochondral defects in middle-aged patients might be treated with focal metallic implants. First developed for defects in the knee joint, implants are now available for the shoulder, hip, ankle and the first metatarsalphalangeal joint. While providing pain reduction and clinical improvement, progressive degenerative changes of the opposing cartilage are observed in many patients. The mechanisms leading to this damage are not fully understood. This protocol describes a tribological experiment to simulate a metal-on-cartilage pairing and comprehensive analysis of the articular cartilage. Metal implant material is tested against bovine osteochondral cylinders as a model for human articular cartilage. By applying different loads and sliding speeds, physiological loading conditions can be imitated. To provide a comprehensive analysis of the effects on the articular cartilage, histology, metabolic activity and gene expression analysis are described in this protocol. The main advantage of tribological testing is that loading parameters can be adjusted freely to simulate in vivo conditions. Furthermore, different testing solutions might be used to investigate the influence of lubrication or pro-inflammatory agents. By using gene expression analysis for cartilage-specific genes and catabolic genes, early changes in the metabolism of articular chondrocytes in response to mechanical loading might be detected.


Assuntos
Cartilagem Articular/fisiologia , Metais/farmacologia , Próteses e Implantes , Animais , Osso e Ossos/efeitos dos fármacos , Cartilagem Articular/efeitos dos fármacos , Bovinos , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , DNA Complementar/biossíntese , Fricção , Regulação da Expressão Gênica , Fatores de Tempo
20.
Plant Mol Biol ; 103(6): 705-718, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32577984

RESUMO

Fritillariae Bulbus are the most commonly used antitussive and edible herbs in China. Based on UPLC-QTOF-MS and UPLC-QQQ-MS, the validated MRM-based non-targeted quantitative method was applied to determinate the contents of 48 Fritillaria alkaloids (FAs) in three Fritillaria species (F. thunbergii Miq., F. unibracteata and F. ussuriensis). The RNA-Seq results showed that gene transcript levels have different expression patterns in three Fritillaria species. Based on transcriptome data, the full-length cDNA sequences of squalene epoxidase gene were cloned and characterized. Natural evolution of squalene epoxidase genes resulted in four mutations (C236R, M489L, G510A and K517R) in three Fritillaria species. Molecular docking analysis showed that the 236 residue is located inside the pocket and the binding center while other three residues are located on the surface of the protein. Functional verification indicated the mutations of SQE (C236R) could effectively increase the activity of SQE and obtain higher yield of 2,3-oxidosqualene in recombinant yeast. And the mutations of SQE (M489L and G510A), which increased the hydrophobicity of the protein surface, could also enhance the activity of SQE. This study provides major insights into the metabolites differentiation of FAs biosynthesis, and a firm foundation for the quality control and metabolic engineering of Fritillariae bulbus.


Assuntos
Fritillaria/enzimologia , Esqualeno Mono-Oxigenase/metabolismo , Alcaloides/metabolismo , Cromatografia Líquida de Alta Pressão , DNA Complementar/genética , Simulação de Acoplamento Molecular , Mutação/genética , Filogenia , Alinhamento de Sequência , Esqualeno Mono-Oxigenase/genética
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