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1.
Sheng Wu Gong Cheng Xue Bao ; 37(11): 3915-3932, 2021 Nov 25.
Artigo em Chinês | MEDLINE | ID: mdl-34841795

RESUMO

Targeted protein degradation (TPD) technology facilitates specific and efficient degradation of disease-related proteins through hijacking the two major protein degradation systems in mammalian cells: ubiquitin-proteasome system and lysosome pathway. Compared with traditional small molecule-inhibitors, TPD-based drugs exhibit the characteristics of a broader target spectrum. Compared with techniques interfere with protein expression on the gene and mRNA level, TPD-based drugs are target-specific, efficaciously rapid, and not constrained by post-translational modification of proteins. In the past 20 years, various TPD-based technologies have been developed. Most excitingly, two TPD-based therapeutic drugs have been approved by FDA for phase Ⅰ clinical trials in 2019. Despite of the early stage characteristics and various obstructions of the TPD technology, it could serve as a powerful tool for the development of novel drugs. This review summarizes the advances of different degradation systems based on TPD technologies and their applications in disease therapy. Moreover, the advantages and challenges of various technologies were discussed systematically, with the aim to provide theoretical guidance for further application of TPD technologies in scientific research and drug development.


Assuntos
Complexo de Endopeptidases do Proteassoma , Proteínas , Animais , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas/genética , Proteínas/metabolismo , Proteólise , Tecnologia
3.
Talanta ; 235: 122797, 2021 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-34517655

RESUMO

As the outbreak of coronavirus disease 2019 (COVID-19), on-site molecular diagnosis is becoming increasingly important. In this study, a freeze-drying method was introduced for PCR reagents to meet the requirements of microfluidic molecular diagnosis. Using this method, PCR components were pre-mixed and freeze-dried as a bead, which could be transferred into microfluidic chips easily. As this bead only required reconstitution in water, operational steps of PCR were simplified, pipetting errors and errors associated with improper handling of wet reagents could also be reduced. In addition, 19 PCR mixes for different targets (including both RNA and DNA) detection were stable when stored at room temperature (18-25 °C) for 1-2 years and may be stored longer as activity monitoring remains ongoing. To shorten the stability testing time, accelerated stability testing at higher temperatures was proposed. The evaluation periods of the freeze-dried PCR mixes were shortened to less than one month when stored at 56 °C and 80 °C. When attempts were further tried to predict the shelf lives for freeze-dried PCR mixes, our findings challenged the classic view of the Q10 method as a prediction model for freeze-dried PCR mixes and confirmed for the first time that this prediction was influenced by different factors at varying degrees. These studies and findings are important for the development of molecular diagnosis at both central laboratories and resource-limited areas.


Assuntos
COVID-19 , Microfluídica , Humanos , Patologia Molecular , Reação em Cadeia da Polimerase , SARS-CoV-2 , Temperatura
4.
Lancet Reg Health West Pac ; 12: 100182, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34527973

RESUMO

Background: Universal screening of congenital cytomegalovirus (cCMV) infection is important for monitoring and intervention during critical stages of speech and language development. This study aimed to explore the optimal detection strategy for cCMV infection screening. Methods: Serum samples from pregnant women and saliva and urine samples from their newborns were collected for the anti-CMV IgG and CMV DNA PCR tests, respectively. The sensitivity, specificity, and predictive values as well as the likelihood ratios of 12 potential screening strategies for cCMV infection, based on tests for saliva, urine, and their combination, were evaluated. Findings: A total of 6729 pregnant women were enrolled, and the seroprevalence was 98.1%. Among 6350 newborns that were followed up, 49 were defined as having cCMV infection. In the screening test, the CMV DNA positivity rate remained similar from day 0 to day 5, increased slowly from day 6 to day 13, and became high in newborns beyond 13 days of birth. In the confirmatory testing, the positive rates increased significantly beyond day 21. For the 49 newborns with cCMV infection, the proportion of agreement between saliva and urine testing was poor. Upon evaluating alternative screening strategies, using saliva and urine screening with saliva and urine confirmation as the reference strategy, saliva screening with saliva and urine confirmation showed good diagnostic accuracy and feasibility, with sensitivity, specificity, positive predictive and negative predictive values of 85.7%, 100.0%, 100.0% and 99.9%, respectively. Interpretation: In populations with high seroprevalence, saliva screening with saliva and urine confirmation might be an alternative strategy for screening cCMV infections. The suggested timeframes for screening and confirmation are within 13 (ideally 5) and 21 (ideally 13) days of birth, respectively. Funding: National Natural Science Foundation of China, National Science and Technology Major Project of China and Merck & Co., Inc., Kenilworth, New Jersey, U.S.A.

5.
Nat Commun ; 12(1): 5131, 2021 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-34446736

RESUMO

Protein delivery with cell-penetrating peptide is opening up the possibility of using targets inside cells for therapeutic or biological applications; however, cell-penetrating peptide-mediated protein delivery commonly suffers from ineffective endosomal escape and low tolerance in serum, thereby limiting in vivo efficacy. Here, we present an intracellular protein delivery system consisting of four modules in series: cell-penetrating peptide, pH-dependent membrane active peptide, endosome-specific protease sites and a leucine zipper. This system exhibits enhanced delivery efficiency and serum tolerance, depending on proteolytic cleavage-facilitated endosomal escape and leucine zipper-based dimerisation. Intravenous injection of protein phosphatase 1B fused with this system successfully suppresses the tumour necrosis factor-α-induced systemic inflammatory response and acetaminophen-induced acute liver failure in a mouse model. We believe that the strategy of using multifunctional chimaeric peptides is valuable for the development of cell-penetrating peptide-based protein delivery systems, and facilitate the development of biological macromolecular drugs for use against intracellular targets.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Falência Hepática Aguda/tratamento farmacológico , Peptídeos/química , Proteína Fosfatase 1/administração & dosagem , Animais , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/genética , Peptídeos Penetradores de Células/metabolismo , Endossomos/genética , Endossomos/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Falência Hepática Aguda/genética , Falência Hepática Aguda/metabolismo , Camundongos Endogâmicos BALB C , Peptídeos/genética , Peptídeos/metabolismo , Proteína Fosfatase 1/química , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo , Transporte Proteico
6.
Emerg Microbes Infect ; 10(1): 1824-1831, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34392819

RESUMO

Background Congenital human cytomegalovirus (CMV) infection remains largely unrecognized and underemphasized in medical practice. This study aimed to describe the maternal CMV seroprevalence rate in early gestation and congenital CMV infection in a Chinese population. Methods This prospective cohort study was conducted in three hospitals in China from 2015 through 2018. Pregnant women were enrolled in early gestation and followed up in middle and late gestation with serological testing. CMV serostatus was determined by IgG testing in serum during early gestation. Their newborns were screened for cCMV infection by PCR testing in both saliva and urine at two time points. The cCMV prevalence, maternal seroprevalence and associated factors were analyzed. Results In China, the CMV seroprevalence was 98.11% (6602/6729, 95% CI: 97.76%-98.41%), and the cCMV prevalence was 1.32% (84/6350, 95% CI: 1.07%-1.64%). Over 98% of cCMV-positive newborns were from pregnant women who were seropositive in early gestation in China. The prevalence of cCMV infection in newborns from seropositive and seronegative pregnant women was similar (crude prevalence: 1.33% vs 0.82%, P = 1.00; estimated prevalence: 1.27% vs 1.05%, P = 0.32). Pregnant women who were under 25 years old or primiparous had a lower seroprevalence. Newborns from pregnant women under 25 years old or from twin pregnancies had a higher prevalence of cCMV infection. Conclusion in China, the cCMV prevalence was high, and the rates were similar in newborns from pregnant women who were seropositive and seronegative in early gestation. The vast majority of cCMV newborns were from seropositive mothers.Trial registration: ClinicalTrials.gov identifier: NCT02645396..

7.
PeerJ ; 9: e11549, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34221714

RESUMO

Objective: To investigate the association between DNA methylation and the stable warfarin dose through genome-wide DNA methylation analysis and pyrosequencing assay. Method: This study included 161 patients and genome-wide DNA methylation analysis was used to screen potential warfarin dose-associated CpGs through Illumina Infinium HumanMethylation 450 K BeadChip; then, the pyrosequencing assay was used to further validate the association between the stable warfarin dose and alterations in the methylation of the screened CpGs. GenomeStudio Software and R were used to analyze the differentially methylated CpGs. Results: The methylation levels of CpGs surrounding the xenobiotic response element (XRE) within the CYP1A1 promoter, differed significantly between the different dose groups (P < 0.05), and these CpGs presented a positive correlation (r> 0, P < 0.05) with an increase in the stable dose of warfarin. At the VKORC1 promoter, two CpGs methylation levels were significantly different between the differential dose groups (P < 0.05), and one CpG (Chr16: 31106793) presented a significant negative correlation (r <  0, P <  0.05) among different dose (low, medium, and high) groups. Conclusion: This is a novel report of the methylation levels of six CpGs surrounding the XRE within the CYP1A1 promoter and one differential CpG at the VKORC1 promoter associated with stable warfarin dosage; these methylation levels might be applied as molecular signatures for warfarin.

8.
Trends Analyt Chem ; 143: 116377, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34188341

RESUMO

PCR has been widely used in different fields including molecular biology, pathogen detection, medical diagnosis, food detection and etc. However, the difficulty of promoting PCR in on-site point-of-care testing reflects on challenges relative to its speed, convenience, complexity, and even cost. With the emerging state-of-art of microfluidics, rapid PCR can be achieved with more flexible ways in micro-reactors. PCR plays a critical role in the detection of SARS-CoV-2. Under this special background of COVID-19 pandemic, this review focuses on the latest rapid microfluidic PCR. Rapid PCR is concluded in two main features, including the reactor (type, size, material) and the implementation of thermal cycling. Especially, the compromise between speed and sensitivity with microfluidic PCR is explored based on the system ratio of (thermal cycling time)/(reactor size). Representative applications about the detection of pathogens and SARS-CoV-2 viruses based on rapid PCR or other isothermal amplification are discussed as well.

9.
J Med Virol ; 93(8): 5033-5039, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33942328

RESUMO

Congenital cytomegalovirus infection (cCMVi) is an important cause of sensorineural hearing loss in newborns. Detection of human cytomegalovirus (HCMV) DNA in urine has been used to screen for cCMVi in newborns. However, the matrix effect of urine on HCMV DNA detection is unclear. To evaluate the matrix effect of urine on HCMV DNA detection and optimize the sample process strategy to eliminate or minimize the impact of urine on HCMV DNA detection, DNA in spiked samples was extracted using different DNA extraction methods, and urine samples that could inhibit HCMV DNA detection were mixed to evaluate the inhibitory substances, inhibitory mechanism, and elimination of the inhibitory effect. The optimal urine sample process strategy was evaluated using 42 adult female urine samples and 42 newborn urine samples spiked with HCMV. Some urine samples were found to inhibit HCMV DNA detection due to DNA degradation. The addition of ≥5 mM EDTA to the urine before extraction eliminated the inhibitory effect of urine and did not affect the detection results of urine exhibiting no inhibition. Of the 42 adult female and 42 newborn urine samples, four and two samples, respectively, could inhibit HCMV DNA detection. However, the inhibitory effects of these six urine samples were eliminated after the addition of EDTA. The collective results indicate that the addition of EDTA can completely eliminate the impact of inhibitors present in urine on HCMV DNA extraction and improve the detection of HCMV in urine.


Assuntos
Infecções por Citomegalovirus/diagnóstico , Citomegalovirus/isolamento & purificação , DNA Viral/urina , Adulto , Citomegalovirus/genética , Infecções por Citomegalovirus/urina , DNA Viral/metabolismo , Ácido Edético/química , Feminino , Humanos , Recém-Nascido , Urina/química , Urina/virologia
10.
Arch Virol ; 166(8): 2263-2266, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34008106

RESUMO

Enterovirus 71 (EV71) has caused large hand, foot, and mouth disease (HFMD) epidemics among young children, and EV71 infection is the leading cause of severe HFMD cases and deaths. In mainland China, the prevalence and risk factors of non-C4 EV71 strains are still unclear. In this study, we monitored non-C4 strains over a 10-year HFMD epidemiological surveillance period in Xiamen. The 5'UTR and VP1 coding region of EV71 strains were amplified by RT-nested PCR and sequenced. Thirty-two non-C4 EV71 strains were identified during 2009-2018. This study provides important information about the prevalence of EV71 in China that will be applicable for development of vaccines and diagnostic reagents as well as establishment of policies for HFMD prevention and control.


Assuntos
Proteínas do Capsídeo/genética , Enterovirus Humano A/classificação , Doença de Mão, Pé e Boca/epidemiologia , Regiões 5' não Traduzidas , Criança , China/epidemiologia , Enterovirus Humano A/genética , Enterovirus Humano A/isolamento & purificação , Doença de Mão, Pé e Boca/virologia , Humanos , Masculino , Filogenia , Prevalência , Reação em Cadeia da Polimerase Via Transcriptase Reversa
11.
Anal Chim Acta ; 1167: 338599, 2021 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-34049623

RESUMO

Convenient and accurate nucleic acid quantification (NAQ) is crucial to clinical diagnosis, forensic medicine, veterinary medicine and food analysis. However, traditional NAQ relies on the preparation of a laborious, time-consuming and expensive calibration curve, which would also propagate pipette errors through serially dilutions. Besides, traditional NAQ is run in different tubes, which introduces bias from random tube-to-tube variations and is unable to detect inhibitors from biological samples. To solve these problems, a single-tube quantitative PCR (stqPCR) technique is proposed which enables accurate quantification without the need for a calibration curve. In this method, an internal quantitative standard DNA (IQS-DNA) for quantification was screened out by co-amplification with the target DNA. Then the difference between the quantification cycle value (ΔCq) of the IQS-DNA and the target DNA was used for NAQ. The method permitted high accuracy quantification with reliable data for concentrations in plasmid, serum standard, and clinical samples being confirmed (R2 values of 0.9951, 0.9889, and 0.9727, slope values of 1.011, 1.028, and 0.9327, and intercept values of -0.06037, -0.1486, and 0.3325, respectively). Accurate NAQ could also be achieved by stqPCR even though inhibitors were present in a sample; however, in the case of using a commercial assay kit, satisfactory performance was only attained after the same sample was diluted some 32-fold. Moreover, integration of the present method into a microfluidic system could achieve super-fast NAQ in less than 30 min and achieve super-fast "sample in, quantitative answer out" testing in less than 40 min. Thus, the stqPCR method present here would promote the development of NAQ in the laboratory and on site.


Assuntos
DNA , Análise de Alimentos , Calibragem , Reação em Cadeia da Polimerase em Tempo Real
12.
Signal Transduct Target Ther ; 6(1): 136, 2021 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-33790236

RESUMO

Epidemiological studies of the COVID-19 patients have suggested the male bias in outcomes of lung illness. To experimentally demonstrate the epidemiological results, we performed animal studies to infect male and female Syrian hamsters with SARS-CoV-2. Remarkably, high viral titer in nasal washings was detectable in male hamsters who presented symptoms of weight loss, weakness, piloerection, hunched back and abdominal respiration, as well as severe pneumonia, pulmonary edema, consolidation, and fibrosis. In contrast with the males, the female hamsters showed much lower shedding viral titers, moderate symptoms, and relatively mild lung pathogenesis. The obvious differences in the susceptibility to SARS-CoV-2 and severity of lung pathogenesis between male and female hamsters provided experimental evidence that SARS-CoV-2 infection and the severity of COVID-19 are associated with gender.


Assuntos
COVID-19 , SARS-CoV-2/metabolismo , Caracteres Sexuais , Animais , COVID-19/metabolismo , COVID-19/patologia , Modelos Animais de Doenças , Suscetibilidade a Doenças , Feminino , Masculino , Mesocricetus
13.
Small Methods ; 5(2): 2001031, 2021 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-33614907

RESUMO

The ongoing corona virus disease 2019 (COVID-19) pandemic, caused by SARS-CoV-2 infection, has resulted in hundreds of thousands of deaths. Cellular entry of SARS-CoV-2, which is mediated by the viral spike protein and ACE2 receptor, is an essential target for the development of vaccines, therapeutic antibodies, and drugs. Using a mammalian cell expression system, a genetically engineered sensor of fluorescent protein (Gamillus)-fused SARS-CoV-2 spike trimer (STG) to probe the viral entry process is developed. In ACE2-expressing cells, it is found that the STG probe has excellent performance in the live-cell visualization of receptor binding, cellular uptake, and intracellular trafficking of SARS-CoV-2 under virus-free conditions. The new system allows quantitative analyses of the inhibition potentials and detailed influence of COVID-19-convalescent human plasmas, neutralizing antibodies and compounds, providing a versatile tool for high-throughput screening and phenotypic characterization of SARS-CoV-2 entry inhibitors. This approach may also be adapted to develop a viral entry visualization system for other viruses.

14.
Sheng Wu Gong Cheng Xue Bao ; 37(1): 30-39, 2021 Jan 25.
Artigo em Chinês | MEDLINE | ID: mdl-33501787

RESUMO

With the advantages of low immunogenicity and long half-life, human monoclonal antibody has become an indispensable biological agent in vivo. Immortalization of human B cells is a potential and effective method to obtain natural human antibody library, which can provide a rich source for the preparation of human monoclonal antibodies. As there are urgent problems to be solved in each platform, the preparation of antibodies based on human B cell immortalization is still limited to the laboratory research stage. At present, there is a lack of a systematic review to clarify the advantages and disadvantages of the existing human B cell immortalization antibody preparation platform and its feasibility analysis. This paper reviews the research on the preparation of human monoclonal antibody based on human B cells immortalization, and describes an in vitro cell culture method, in which hCD40L vesicles are used instead of feeder cells, in order to provide references for the further development of human monoclonal antibody preparation technology.


Assuntos
Anticorpos Monoclonais , Linfócitos B , Técnicas de Cultura de Células , Humanos
16.
Emerg Microbes Infect ; 10(1): 37-50, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33296295

RESUMO

Hepatitis B e antigen (HBeAg) is a widely used marker both for chronic hepatitis B (CHB) clinical management and HBV-related basic research. However, due to its high amino acid sequence homology to hepatitis B core antigen (HBcAg), most of available anti-HBe antibodies are cross-reactive with HBcAg resulting in high interference against accurate measurement of the status and level of HBeAg. In the study, we generated several monoclonal antibodies (mAbs) targeting various epitopes on HBeAg and HBcAg. Among these mAbs, a novel mAb 16D9, which recognizes the SKLCLG (aa -10 to -5) motif on the N-terminal residues of HBeAg that is absent on HBcAg, exhibited excellent detection sensitivity and specificity in pairing with another 14A7 mAb targeting the HBeAg C-terminus (STLPETTVVRRRGR, aa141 to 154). Based on these two mAbs, we developed a novel chemiluminescent HBeAg immunoassay (NTR-HBeAg) which could detect HBeAg derived from various HBV genotypes. In contrast to widely used commercial assays, the NTR-HBeAg completely eliminated the cross-reactivity with secreted HBcAg from precore mutant (G1896A) virus in either cell culture or patient sera. The improved specificity of the NTR-HBeAg assay enables its applicability in cccDNA-targeting drug screening in cell culture systems and also provides an accurate tool for clinical HBeAg detection.


Assuntos
Anticorpos Anti-Hepatite B/análise , Antígenos E da Hepatite B/química , Vírus da Hepatite B/genética , Hepatite B Crônica/imunologia , Motivos de Aminoácidos , Anticorpos Monoclonais/análise , Técnicas de Cultura de Células , Linhagem Celular , Epitopos/imunologia , Genótipo , Células Hep G2 , Antígenos do Núcleo do Vírus da Hepatite B/química , Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Antígenos E da Hepatite B/imunologia , Vírus da Hepatite B/imunologia , Hepatite B Crônica/sangue , Humanos , Medições Luminescentes
17.
PeerJ ; 8: e9995, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33083118

RESUMO

Background: We used bioinformatic analysis and quantitative reverse transcription polymerase chain reaction (RT-qPCR) assays to investigate the association between plasma microRNAs (miRNAs) and stable warfarin dosage in a Chinese Han population. Methods: Bioinformatics analysis was used to screen out potential warfarin dose-associated miRNAs. Three plasma miRNAs were validated in 99 samples by RT-qPCR. Kruskal-Wallis test and multivariate logistic regression were used to compare differences in plasma miRNAs expression levels between three warfarin dosage groups. Results: There were significant between-group differences among the three dose groups for hsa-miR-133b expression (p = 0.005), but we observed an "n-shaped" dose-dependent curve rather than a linear relationship. Expression levels of hsa-miR-24-3p (p = 0.475) and hsa-miR-1276 (p = 0.558) were not significantly different in the multivariate logistic regression. Conclusion: miRNAs have received extensive attention as ideal biomarkers and possible therapeutic targets for various diseases. However, they are not yet widely used in precision medicine. Our results indicate that hsa-miR-133b may be a possible reference factor for the warfarin dosage algorithm. These findings emphasize the importance of a comprehensive evaluation of complex relationships in warfarin dose prediction models and provide new avenues for future pharmacogenomics studies.

18.
Clin Chim Acta ; 511: 154-159, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33058836

RESUMO

BACKGROUND: Respiratory syncytial virus (RSV) infection is a global public-health problem. Timely diagnostics are needed for high-risk patients. Several methods have been used for RSV detection but not suitable for on-site detection due to the requirement of specialized laboratories and expensive equipment. METHODS: We developed a convenient, rapid and low-cost method of nucleic acids (NA) extraction based on cellulose paper, which could extract NA from nasopharyngeal swabs (NPSs) within 1 min. This extraction method was integrated with fluorescence convection polymerase chain reaction (CPCR), which easily affordable and easy-to-use NA detection of the RSV in 33 min. RESULTS: The developed cellulose-based NA purification combine with CPCR (CP-CPCR) reliably detected as little as 0.01 TCID50/mL of RSV cultures. CP-CPCR performance was tested further using NPSs: it showed sensitivity of 100% and a specificity of 100% compared with traditional extraction and amplification methods. CONCLUSIONS: Our evaluation confirmed high specificity, sensitivity and efficient of the CP-CPCR, which can be used widely for RSV testing in resource-limited settings.


Assuntos
Infecções por Vírus Respiratório Sincicial , Vírus Sincicial Respiratório Humano , Celulose , Humanos , Nasofaringe , Testes Imediatos , Reação em Cadeia da Polimerase , RNA Viral , Infecções por Vírus Respiratório Sincicial/diagnóstico , Vírus Sincicial Respiratório Humano/genética , Sensibilidade e Especificidade
19.
Int J Mol Sci ; 21(18)2020 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-32899704

RESUMO

Precise gene editing is-or will soon be-in clinical use for several diseases, and more applications are under development. The programmable nuclease Cas9, directed by a single-guide RNA (sgRNA), can introduce double-strand breaks (DSBs) in target sites of genomic DNA, which constitutes the initial step of gene editing using this novel technology. In mammals, two pathways dominate the repair of the DSBs-nonhomologous end joining (NHEJ) and homology-directed repair (HDR)-and the outcome of gene editing mainly depends on the choice between these two repair pathways. Although HDR is attractive for its high fidelity, the choice of repair pathway is biased in a biological context. Mammalian cells preferentially employ NHEJ over HDR through several mechanisms: NHEJ is active throughout the cell cycle, whereas HDR is restricted to S/G2 phases; NHEJ is faster than HDR; and NHEJ suppresses the HDR process. This suggests that definitive control of outcome of the programmed DNA lesioning could be achieved through manipulating the choice of cellular repair pathway. In this review, we summarize the DSB repair pathways, the mechanisms involved in choice selection based on DNA resection, and make progress in the research investigating strategies that favor Cas9-mediated HDR based on the manipulation of repair pathway choice to increase the frequency of HDR in mammalian cells. The remaining problems in improving HDR efficiency are also discussed. This review should facilitate the development of CRISPR/Cas9 technology to achieve more precise gene editing.


Assuntos
Sistemas CRISPR-Cas/genética , Reparo do DNA por Junção de Extremidades/genética , Edição de Genes/métodos , Animais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , DNA/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades/fisiologia , Reparo do DNA/genética , Reparo do DNA/fisiologia , Endonucleases/metabolismo , Edição de Genes/tendências , Humanos , RNA Guia/genética , Reparo de DNA por Recombinação/genética
20.
Emerg Microbes Infect ; 9(1): 2105-2113, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32893735

RESUMO

The global pandemic of coronavirus disease 2019 (COVID-19) is a disaster for human society. A convenient and reliable neutralization assay is very important for the development of vaccines and novel drugs. In this study, a G protein-deficient vesicular stomatitis virus (VSVdG) bearing a truncated spike protein (S with C-terminal 18 amino acid truncation) was compared to that bearing the full-length spike protein of SARS-CoV-2 and showed much higher efficiency. A neutralization assay was established based on VSV-SARS-CoV-2-Sdel18 pseudovirus and hACE2-overexpressing BHK21 cells (BHK21-hACE2 cells). The experimental results can be obtained by automatically counting the number of EGFP-positive cells at 12 h after infection, making the assay convenient and high-throughput. The serum neutralizing titer measured by the VSV-SARS-CoV-2-Sdel18 pseudovirus assay has a good correlation with that measured by the wild type SARS-CoV-2 assay. Seven neutralizing monoclonal antibodies targeting the receptor binding domain (RBD) of the SARS-CoV-2 S protein were obtained. This efficient and reliable pseudovirus assay model could facilitate the development of new drugs and vaccines.


Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Betacoronavirus/imunologia , Infecções por Coronavirus/diagnóstico , Testes de Neutralização/métodos , Pneumonia Viral/diagnóstico , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19 , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Pandemias , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/genética , Células Vero , Vírus da Estomatite Vesicular Indiana/genética , Vírus da Estomatite Vesicular Indiana/imunologia
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