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1.
Methods Mol Biol ; 2147: 175-183, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32840820

RESUMO

Bioprinting has emerged as a promising method for precise spatiotemporal patterning of biological materials such as living cells, genetic materials, and proteins, which are sensitive to any other fabrication techniques. Bioprinting allows the generation of tissue constructs and models that closely mimic the anatomical and physiological attributes of a chosen tissue. In vitro toxicology assays can greatly benefit from bioprinting as drugs can be screened with higher efficiencies in a significantly reduced period. This protocol describes a method for fabricating bioprinted cartilage constructs which can be used for in vitro toxicology studies employing a scalable "tissue strand" bioprinting modality.

2.
BMC Pediatr ; 20(1): 483, 2020 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-33076884

RESUMO

BACKGROUND: Fetus in fetu (FIF) is a rare congenital anomaly. The preoperative diagnosis of FIF and differentiating it from teratoma and other abdominal tumors can be challenging for radiologists. Clarification of the blood supply and the relationship with the surrounding vessels is especially helpful for successful surgery; however, multimode ultrasound (US) performed for FIF has rarely been explored. Here, we first report a "humanoid" FIF case diagnosed by multimode US examinations, with the use of contrast-enhanced ultrasound (CEUS) for clarifying the blood supply features. CASE PRESENTATION: A 25-day-old preterm male infant was referred to our hospital for surgery. The US and computed tomography (CT) examinations led to a diagnosis of teratoma at the local hospital. The laboratory workup at our hospital revealed an elevation of total bilirubin, direct bilirubin, indirect bilirubin, alpha-fetoprotein, and neuron-specific enolase levels. A precise diagnosis and differentiation from teratoma, hepatoblastoma, neuroblastoma and other abdominal tumors were needed. In addition, the blood supply and the relationship with the surrounding vessels needed clarification prior to surgery. Multimode US examinations were performed and the features of a "humanoid" FIF as well as the blood supply for the abdominal lesion of the infant were suggested by grayscale US, color Doppler flow imaging (CDFI), and CEUS. Furthermore, CDFI and CEUS revealed an aorta-like structure and umbilical cord-like blood vessels in the "humanoid" FIF, and the CEUS helped with marking the surface of the infant's abdominal wall. To the best of our knowledge, this is the first case report of CEUS in FIF, and the blood supply was clearly demonstrated in the FIF. The intraoperative findings confirmed our multimode US findings and revealed a "humanoid" FIF. The infant quickly recovered after the operation and had no positive findings at the 2-year follow-up visit. CONCLUSIONS: Multimode US was helpful in diagnosing the rare FIF without radiation exposure. Specifically, CEUS clearly demonstrated the limb branch vessel-like structures, the abdominal aorta-like structure and the blood supply, which was useful for the FIF diagnosis and for avoiding damage to important vessels during the operation.

3.
Nat Metab ; 2(10): 1135-1148, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-33067605

RESUMO

Circulating proteins are vital in human health and disease and are frequently used as biomarkers for clinical decision-making or as targets for pharmacological intervention. Here, we map and replicate protein quantitative trait loci (pQTL) for 90 cardiovascular proteins in over 30,000 individuals, resulting in 451 pQTLs for 85 proteins. For each protein, we further perform pathway mapping to obtain trans-pQTL gene and regulatory designations. We substantiate these regulatory findings with orthogonal evidence for trans-pQTLs using mouse knockdown experiments (ABCA1 and TRIB1) and clinical trial results (chemokine receptors CCR2 and CCR5), with consistent regulation. Finally, we evaluate known drug targets, and suggest new target candidates or repositioning opportunities using Mendelian randomization. This identifies 11 proteins with causal evidence of involvement in human disease that have not previously been targeted, including EGF, IL-16, PAPPA, SPON1, F3, ADM, CASP-8, CHI3L1, CXCL16, GDF15 and MMP-12. Taken together, these findings demonstrate the utility of large-scale mapping of the genetics of the proteome and provide a resource for future precision studies of circulating proteins in human health.

4.
Adv Healthc Mater ; : e2001657, 2020 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-33073548

RESUMO

The heterogeneous and anisotropic articular cartilage is generally studied as a layered structure of "zones" with unique composition and architecture, which is difficult to recapitulate using current approaches. A novel hybrid bioprinting strategy is presented here to generate zonally stratified cartilage. Scaffold-free tissue strands (TSs) are made of human adipose-derived stem cells (ADSCs) or predifferentiated ADSCs. Cartilage TSs with predifferentiated ADSCs exhibit improved mechanical properties and upregulated expression of cartilage-specific markers at both transcription and protein levels as compared to TSs with ADSCs being differentiated in the form of strands and TSs of nontransfected ADSCs. Using the novel hybrid approach integrating new aspiration-assisted and extrusion-based bioprinting techniques, the bioprinting of zonally stratified cartilage with vertically aligned TSs at the bottom zone and horizontally aligned TSs at the superficial zone is demonstrated, in which collagen fibers are aligned with designated orientation in each zone imitating the anatomical regions and matrix orientation of native articular cartilage. In addition, mechanical testing study reveals a compression modulus of ≈1.1 MPa, which is similar to that of human articular cartilage. The prominent findings highlight the potential of this novel bioprinting approach for building biologically, mechanically, and histologically relevant cartilage for tissue engineering purposes.

5.
J Med Virol ; 2020 Oct 07.
Artigo em Inglês | MEDLINE | ID: covidwho-833874

RESUMO

The recent coronavirus disease (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has been spreading rapidly throughout the continents. The insights in how this viral disease affects general population is thus urgently needed. Diabetes mellitus is one of the leading threats for morbidity and mortality globally. Infection of coronavirus in diabetic patients may trigger acute hyperglycemia due to increased secretion of hyperglycemic hormones, extensive application of glucocorticoids to patients with severe symptoms and the potential pathogenicity of coronavirus in pancreas expressing angiotensin-converting enzyme 2 (ACE2) This article is protected by copyright. All rights reserved.

6.
J Med Virol ; 2020 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-33026669

RESUMO

The recent coronavirus disease (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has been spreading rapidly throughout the continents. The insights in how this viral disease affects general population is thus urgently needed. Diabetes mellitus is one of the leading threats for morbidity and mortality globally. Infection of coronavirus in diabetic patients may trigger acute hyperglycemia due to increased secretion of hyperglycemic hormones, extensive application of glucocorticoids to patients with severe symptoms and the potential pathogenicity of coronavirus in pancreas expressing angiotensin-converting enzyme 2 (ACE2) This article is protected by copyright. All rights reserved.

7.
Virol J ; 17(1): 157, 2020 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-33081802

RESUMO

BACKGROUND: Street rabies virus (RABV) usually infects hosts at peripheral sites and migrates from motor or sensory nerves to the central nervous system. Several studies have found that inflammation is mild in a mouse model of street RABV infection. However, the pathogenetic mechanisms of street RABV in naturally infected dogs or humans are not well understood. METHODS: Brain tissues collected from 3 dogs and 3 humans were used; these tissue samples were collected under the natural condition of rabies-induced death. The inflammatory response and pathway activation in the brain tissue samples of dogs and humans were evaluated by HE, IHC, ARY006, WB and ELISA. The clinical isolate street RABV strains CGS-17 and CXZ-15 from 30 six-week-old ICR mice were used to construct the mouse infection model presented here. RESULTS: Neuronal degeneration and increased lymphocyte infiltration in the cerebral cortex, especially marked activation of microglia, formation of glial nodules, and neuronophagy, were observed in the dogs and humans infected with the street RABV strains. The various levels of proinflammatory chemokines, particularly CXCL1, CXCL12, CCL2, and CCL5, were increased significantly in the context of infection with street RABV strains in dogs and humans in relation to healthy controls, and the levels of MAPK and NF-κB phosphorylation were also increased in dogs and humans with natural infection. We also found that the degrees of pathological change, inflammatory response, MAPK and NF-κB signaling pathway activation were obviously increased during natural infection in dogs and humans compared with artificial model infection in mice. CONCLUSION: The data obtained here provide direct evidence for the RABV-induced activation of the inflammatory response in a dog infection model, which is a relatively accurate reflection of the pathogenic mechanism of human street RABV infection. These observations provide insight into the precise roles of underlying mechanisms in fatal natural RABV infection.

8.
Bioorg Med Chem ; 28(20): 115686, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-33069071

RESUMO

Enzyme assemblies such as type II polyketide synthases (PKSs) produce a wide array of bioactive secondary metabolites. While the molecules produced by type II PKSs have found remarkable clinical success, the biosynthetic prowess of these enzymes has been stymied by 1) the inability to reconstitute the bioactivity of the minimal PKS enzymes in vitro and 2) limited exploration of type II PKSs from diverse phyla. To begin filling this unmet need, we expressed, purified, and characterized the ketosynthase chain length factor (KS-CLF) and acyl carrier protein (ACP) from Ktedonobacter racemifer (Kr). Using E. coli as a heterologous host, we obtained soluble proteins in titers signifying improvements over previous KS-CLF heterologous expression efforts. Characterization of these enzymes reveals that KrACP has self-malonylating activity. Sedimentation velocity analytical ultracentrifugation (SV-AUC) analysis of holo-KrACP and KrKS-CLF indicates that these enzymes do not interact in vitro, suggesting that the acylated state of these proteins might play an important role in facilitating biosynthetically relevant interactions. These results lay important groundwork for optimizing the interaction between KrKS-CLF and KrACP and exploring the biosynthetic potential of other non-actinomycete type II PKSs.

9.
Anal Methods ; 12(37): 4562-4571, 2020 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-33001064

RESUMO

Xuebijing (XBJ) is a compound Chinese medicine that contains Paeoniae Radix Rubra, ChuanXiong Rhizoma, Salvia Miltiorrhiza Radix et Rhizoma, Carthami Flos, and Angelicae Sinensis Radix. It is widely used in China to treat sepsis. Previous studies have demonstrated that XBJ can decrease mortality in patients with moderate paraquat poisoning. However, the mechanism by which it exerts this effect is not completely clear. In this study, an ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS/MS)-based metabolomics approach was used to perform a metabolic profiling analysis. Principal component analysis (PCA), random forest (RF), and partial least squares discriminant analysis (PLS-DA) were used to identify metabolites to clarify the mechanism of XBJ's activity. XBJ clearly alleviated lung injury in a Sprague Dawley (SD) rat model of paraquat (PQ) poisoning. Seven metabolites related to four pathways, including those involved in sphingolipid and phospholipid metabolism, amino acid metabolism, unsaturated fatty acid metabolism, and pantothenic acid and CoA biosynthesis, were present at different levels in PQ-poisoned rats treated with XBJ compared with untreated rats. XBJ can ameliorate the effects of PQ poisoning in SD rats. Using a metabolomics approach enabled us to gain new insight into the mechanism underlying this effect.

10.
J Formos Med Assoc ; 2020 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-32883568

RESUMO

BACKGROUND/PURPOSE: Gastric parietal cell antibody (GPCA), thyroglobulin antibody (TGA), and thyroid microsomal antibody (TMA) are organ-specific autoantibodies. This study mainly assessed the frequencies of serum GPCA, TGA, and TMA positivities in burning mouth syndrome (BMS) patients. METHODS: Serum GPCA, TGA, and TMA levels were measured in 884 BMS patients and in 442 age- and sex-matched healthy control subjects. RESULTS: We found that 12.3%, 21.6%, and 22.7% of 884 BMS patients and 1.8%, 2.3%, and 2.9% of 442 healthy control subjects had the serum GPCA, TGA, and TMA positivities, respectively. BMS patients had significantly higher frequencies of GPCA, TGA, and TMA positivities than healthy control subjects (all P-values < 0.001). We also found that 20 (2.3%), 130 (14.7%), and 181 (20.5%) BMS patients and 3 (0.7%), 8 (1.8%), and 6 (1.4%) healthy control subjects had the presence of three (GPCA + TGA + TMA), two (GPCA + TGA, GPCA + TMA, or TGA + TMA), or one (GPCA only, TGA only, or TMA only) organ-specific autoantibody in their sera, respectively. Of 255 TGA/TMA-positive BMS patients whose serum thyroid-stimulating hormone (TSH) levels were measured, 87.8%, 5.1%, and 7.1% of these TGA/TMA-positive BMS patients had normal, lower, and higher serum TSH levels, respectively. CONCLUSION: Approximately 37.5% of 884 BMS patients have serum GPCA/TGA/TMA positivity. Moreover, 12.3%, 21.6%, and 22.7% of 884 BMS patients have the serum GPCA, TGA, and TMA positivities, respectively. Only 5.1% and 7.1% of TGA/TMA-positive BMS patients have hyperthyroidism and hypothyroidism, respectively. It needs further studies to know whether GPCA-positive BMS patients may finally become as having autoimmune atrophic gastritis.

11.
J Immunol Res ; 2020: 2141508, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32908938

RESUMO

Berberine (BBR), a natural compound extracted from a Chinese herb, has been shown to effectively attenuate insulin resistance (IR) and inflammation in the clinic. However, its ameliorative mechanism against IR is not well defined. This study is aimed at investigating the effect of BBR and protein phosphatase, Mg2+/Mn2+-dependent 1B (PPM1B) on IR. Biochemical measurements and liver histopathology were detected using the biochemical analyzer and HE staining in ZDF rats, respectively. Microarray analysis of liver tissues was performed, and differentially expressed gene (DEG) levels were examined by quantitative real-time PCR (qPCR) and Western blot. Additionally, the effect of BBR was also explored in HepG2-IR cells. The glucose oxidase method and the fluorescent glucose analog were used to detect glucose consumption and uptake, respectively. The PKA inhibitor H89, ELISA, qPCR, Western blot, and immunofluorescence staining were employed to estimate the expression levels of related signaling pathways. To evaluate the roles of PPM1B, HepG2-IR cells were stably infected with lentivirus targeting PPM1B. The administration of BBR drastically decreased the body weight, urine volume, blood glucose, blood urea nitrogen (BUN), CHOL, hepatic index levels, and pathologic changes and improved ALB levels in ZDF rats with PPM1B upregulation. Furthermore, BBR effectively improves glucose consumption, uptake, and inflammation in HepG2-IR cells. The knockdown of PPM1B expression aggravated the inflammatory response and glycometabolism disorder in HepG2-IR cells. Mechanistically, a reversal in the expression of cAMP, PKA, PPM1B, PPARγ, LRP1, GLUT4, NF-κB p65, JNK, pIKKß Ser181, IKKß, IRS-1 Ser307, IRS-1, IRS-2 Ser731, IRS-2, PI3K p85, and AKT Ser473 contributes to ameliorate IR in HepG2-IR cells with BBR treatment. Altogether, these results suggest that BBR might regulate IR progression through the regulation of the cAMP, PKA, PPM1B, PPARγ, LRP1, GLUT4, NF-κB p65, JNK, pIKKß Ser181, IKKß, IRS-1 Ser307, IRS-1, IRS-2 Ser731, IRS-2, PI3K p85, and AKT Ser473 expression in the liver.

12.
J Formos Med Assoc ; 2020 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-32888843

RESUMO

BACKGROUND/PURPOSE: Our previous study found the serum gastric parietal cell antibody (GPCA) positivity in 12.3% of burning mouth syndrome (BMS) patients. This study assessed whether GPCA-positive BMS (GPCA+BMS) patients had significantly higher frequencies of macrocytosis, anemia, hematinic deficiencies, and hyperhomocysteinemia than healthy control subjects or GPCA-negative BMS (GPCA-BMS) patients. METHODS: The mean corpuscular volume, blood hemoglobin (Hb), and serum iron, vitamin B12, folic acid, homocysteine, and GPCA levels were measured and compared between any two of three groups of 109 GPCA+BMS patients, 775 GPCA-BMS patients, and 442 healthy control subjects. RESULTS: We found that 109 GPCA+BMS patients had significantly higher frequencies of macrocytosis, blood Hb and serum iron and vitamin B12 deficiencies, and hyperhomocysteinemia than 442 healthy control subjects (all P-values < 0.001) and significantly higher frequencies of macrocytosis, blood Hb and serum vitamin B12 deficiencies, and hyperhomocysteinemia than 775 GPCA-BMS patients (all P-values < 0.01). Moreover, 775 GPCA-BMS patients had significantly higher frequencies of macrocytosis, blood Hb and serum iron, vitamin B12, and folic acid deficiencies, and hyperhomocysteinemia than 442 healthy control subjects (all P-values < 0.005). Pernicious anemia (45.5%) and normocytic anemia (24.2%) were the two most common types of anemia in 33 anemic GPCA+BMS patients. Moreover, normocytic anemia (61.3%), thalassemia trait-induced anemia (15.5%), and iron deficiency anemia (14.1%) were the three most common types of anemia in 142 anemic GPCA-BMS patients. CONCLUSION: GPCA+BMS patients have significantly higher frequencies of macrocytosis, blood Hb and serum vitamin B12 deficiencies, and hyperhomocysteinemia than healthy control subjects or GPCA-BMS patients.

16.
Eur J Clin Invest ; : e13409, 2020 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-32916764

RESUMO

BACKGROUND: Accurate classification of coronary artery abnormalities (CAAs) is essential for clinical decision-making and long-term management in Kawasaki disease (KD) patients. To date, there are several echocardiographic criteria of CAA assessment. MATERIALS AND METHODS: The Japanese Ministry of Health (JMH) criteria and the Z-score criteria from 2004 American Heart Association guidelines were adopted and their detective efficacies for CAAs were compared in 251 Chinese patients with KD Z scores were calculated by 6 published methods. RESULTS: According to the JMH criteria, 19 (7.57%) KD patients were classified as CAAs during the acute KD episode. However, the detective number of CAAs was highest and had a 0.68-fold increase by the Dallaire et al method with a Z-score cut point of ≥2.5 as compared with the JMH criteria; in contrast, more than 78.95% of patients with CAAs identified by the JMH criteria had a coronary artery Z score ≥2.5. All 6 different Z-score methods had satisfactory accuracies with a range from 93.23% to 97.61% in screening CAAs. For the 19 patients with CAAs identified by the JMH criteria, their Z scores presented the widest variation calculated by the McCrindle et al method. CONCLUSIONS: The JMH criteria underestimate the prevalence of CAAs as compared with the Z-score criteria. Quantitative assessment of coronary artery luminal dimensions, normalized as Z scores adjusted for body surface, should be recommended. The larger coronary artery luminal dimensions vary, the more heterogeneous Z scores calculated by different methods have.

17.
Nat Commun ; 11(1): 4370, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32873792

RESUMO

BRAF kinase, a critical effector of the ERK signaling pathway, is hyperactivated in many cancers. Oncogenic BRAFV600E signals as an active monomer in the absence of active RAS, however, in many tumors BRAF dimers mediate ERK signaling. FDA-approved RAF inhibitors poorly inhibit BRAF dimers, which leads to tumor resistance. We found that Ponatinib, an FDA-approved drug, is an effective inhibitor of BRAF monomers and dimers. Ponatinib binds the BRAF dimer and stabilizes a distinct αC-helix conformation through interaction with a previously unrevealed allosteric site. Using these structural insights, we developed PHI1, a BRAF inhibitor that fully uncovers the allosteric site. PHI1 exhibits discrete cellular selectivity for BRAF dimers, with enhanced inhibition of the second protomer when the first protomer is occupied, comprising a novel class of dimer selective inhibitors. This work shows that Ponatinib and BRAF dimer selective inhibitors will be useful in treating BRAF-dependent tumors.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Sítio Alostérico/efeitos dos fármacos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linhagem Celular Tumoral , Cristalografia por Raios X , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Imidazóis/farmacologia , Imidazóis/uso terapêutico , Sistema de Sinalização das MAP Quinases/genética , Simulação de Acoplamento Molecular , Mutação , Neoplasias/genética , Neoplasias/patologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/uso terapêutico , Multimerização Proteica/efeitos dos fármacos , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas B-raf/ultraestrutura , Piridazinas/farmacologia , Piridazinas/uso terapêutico , Bibliotecas de Moléculas Pequenas , Relação Estrutura-Atividade
18.
Int J Surg ; 82: 172-178, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32891829

RESUMO

BACKGROUND: This study aimed to describe the epidemiologic and clinical characteristics of coronavirus disease 2019 (COVID-19) in surgical patients and medical staff. METHODS: A single-center case series of 1586 consecutive surgical patients was selected at our hospital from January 13 to March 12, 2020. The epidemiological and clinical characteristics of COVID-19 were analyzed and followed up to May 20, 2020. The transmission of COVID-19 between the surgical patients and medical staff was also recorded. RESULTS: Seventeen (1.07%) surgical patients were diagnosed with COVID-19, with a high incidence in the thoracic department (9.37%), and the median age was 58 years (IQR, 53-73). The median time from hospital admission to COVID-19 diagnosis was 9.0 days (7.0-12.0) and was 6.0 days (4.0-7.0) from the day of surgery to COVID-19 diagnosis. Eleven (64.70%) patients suffered from pulmonary infection before surgery. When COVID-19 was diagnosed, common symptoms were fever (82.35%) and cough (94.12%), and most (82.35%) neutrophil/lymphocyte ratios were high (>3.5). Chest computed tomography (CT) (82.35%) showed bilateral dense shadows. Surgical patients with COVID-19 stayed in the hospital for approximately 35.0 days (25.5-43.0), with a mortality rate of 11.76%. Sixteen medical staff were infected with COVID-19 in the early stage. CONCLUSIONS: In this series of 1586 surgical patients, the COVID-19 infection rate was 1.07%, with an especially high incidence among patients with thoracic diseases. Middle-aged and elderly patients with preoperative pulmonary infection were more susceptible to COVID-19 infection after surgery. Medical staff were infected with COVID-19 and should take protective measures to protect themselves.


Assuntos
Betacoronavirus , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/transmissão , Transmissão de Doença Infecciosa do Paciente para o Profissional , Pneumonia Viral/epidemiologia , Pneumonia Viral/transmissão , Procedimentos Cirúrgicos Operatórios/efeitos adversos , Adulto , Idoso , Infecções por Coronavirus/diagnóstico , Feminino , Febre , Hospitalização , Humanos , Masculino , Pessoa de Meia-Idade , Pandemias , Pneumonia Viral/diagnóstico , Estudos Retrospectivos , Avaliação de Sintomas , Tomografia Computadorizada por Raios X , Adulto Jovem
19.
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 , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Pandemias , Glicoproteína da Espícula de Coronavírus/genética , Células Vero , Vírus da Estomatite Vesicular Indiana/genética , Vírus da Estomatite Vesicular Indiana/imunologia
20.
Emerg Microbes Infect ; 9(1): 2105-2113, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: covidwho-745875

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 , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Pandemias , Glicoproteína da Espícula de Coronavírus/genética , Células Vero , Vírus da Estomatite Vesicular Indiana/genética , Vírus da Estomatite Vesicular Indiana/imunologia
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