RESUMO
Evolution of SARS-CoV-2 requires the reassessment of current vaccine measures. Here, we characterized BA.2.86 and XBB-derived variant FLip by investigating their neutralization alongside D614G, BA.1, BA.2, BA.4/5, XBB.1.5, and EG.5.1 by sera from 3-dose-vaccinated and bivalent-vaccinated healthcare workers, XBB.1.5-wave-infected first responders, and monoclonal antibody (mAb) S309. We assessed the biology of the variant spikes by measuring viral infectivity and membrane fusogenicity. BA.2.86 is less immune evasive compared to FLip and other XBB variants, consistent with antigenic distances. Importantly, distinct from XBB variants, mAb S309 was unable to neutralize BA.2.86, likely due to a D339H mutation based on modeling. BA.2.86 had relatively high fusogenicity and infectivity in CaLu-3 cells but low fusion and infectivity in 293T-ACE2 cells compared to some XBB variants, suggesting a potentially different conformational stability of BA.2.86 spike. Overall, our study underscores the importance of SARS-CoV-2 variant surveillance and the need for updated COVID-19 vaccines.
Assuntos
Vacinas contra COVID-19 , COVID-19 , Evasão da Resposta Imune , SARS-CoV-2 , Humanos , Anticorpos Monoclonais , Anticorpos Neutralizantes , Anticorpos Antivirais , COVID-19/imunologia , SARS-CoV-2/classificação , SARS-CoV-2/fisiologiaRESUMO
Spatial omics is a rapidly evolving approach for exploring tissue microenvironment and cellular networks by integrating spatial knowledge with transcript or protein expression information. However, there is a lack of databases for users to access and analyze spatial omics data. To address this limitation, we developed Aquila, a comprehensive platform for managing and analyzing spatial omics data. Aquila contains 107 datasets from 30 diseases, including 6500+ regions of interest, and 15.7 million cells. The database covers studies from spatial transcriptome and proteome analyses, 2D and 3D experiments, and different technologies. Aquila provides visualization of spatial omics data in multiple formats such as spatial cell distribution, spatial expression and co-localization of markers. Aquila also lets users perform many basic and advanced spatial analyses on any dataset. In addition, users can submit their own spatial omics data for visualization and analysis in a safe and secure environment. Finally, Aquila can be installed as an individual app on a desktop and offers the RESTful API service for power users to access the database. Overall, Aquila provides a detailed insight into transcript and protein expression in tissues from a spatial perspective. Aquila is available at https://aquila.cheunglab.org.
Assuntos
Bases de Dados Genéticas , Genômica , Animais , Bases de Dados Factuais , Proteoma/genética , Proteômica , Transcriptoma/genéticaRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible coronavirus responsible for the global COVID-19 pandemic. Herein, we provide evidence that SARS-CoV-2 spreads through cell-cell contact in cultures, mediated by the spike glycoprotein. SARS-CoV-2 spike is more efficient in facilitating cell-to-cell transmission than is SARS-CoV spike, which reflects, in part, their differential cell-cell fusion activity. Interestingly, treatment of cocultured cells with endosomal entry inhibitors impairs cell-to-cell transmission, implicating endosomal membrane fusion as an underlying mechanism. Compared with cell-free infection, cell-to-cell transmission of SARS-CoV-2 is refractory to inhibition by neutralizing antibody or convalescent sera of COVID-19 patients. While angiotensin-converting enzyme 2 enhances cell-to-cell transmission, we find that it is not absolutely required. Notably, despite differences in cell-free infectivity, the authentic variants of concern (VOCs) B.1.1.7 (alpha) and B.1.351 (beta) have similar cell-to-cell transmission capability. Moreover, B.1.351 is more resistant to neutralization by vaccinee sera in cell-free infection, whereas B.1.1.7 is more resistant to inhibition by vaccinee sera in cell-to-cell transmission. Overall, our study reveals critical features of SARS-CoV-2 spike-mediated cell-to-cell transmission, with important implications for a better understanding of SARS-CoV-2 spread and pathogenesis.
Assuntos
COVID-19/imunologia , COVID-19/transmissão , SARS-CoV-2/imunologia , Internalização do Vírus , Enzima de Conversão de Angiotensina 2 , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais , COVID-19/terapia , Fusão Celular , Chlorocebus aethiops , Células HEK293 , Humanos , Imunização Passiva , Glicoproteína da Espícula de Coronavírus/imunologia , Células Vero , Soroterapia para COVID-19RESUMO
The evolution of SARS-CoV-2 paired with immune imprinting by prototype messenger RNA (mRNA) vaccine has challenged the current vaccination efficacy against newly emerged Omicron subvariants. In our study, we investigated a cohort of macaques infected by SIV and vaccinated with two doses of bivalent Pfizer mRNA vaccine containing wildtype and BA.5 spikes. Using a pseudotyped lentivirus neutralization assay, we determined neutralizing antibody (nAb) titers against new XBB variants, i.e., XBB.1.5, XBB.1.16, and XBB.2.3, alongside D614G and BA.4/5. We found that compared to humans vaccinated with three doses of monovalent mRNA vaccine plus a bivalent booster, the monkeys vaccinated with two doses of bivalent mRNA vaccines exhibited relatively increased titers against XBB subvariants. Of note, SIV-positive dam macaques had reduced nAb titers relative to SIV-negative dams. Additionally, SIV positive dams that received antiretroviral therapy had lower nAb titers than untreated dams. Our study underscores the importance of reformulating the COVID-19 vaccine to better protect against newly emerged XBB subvariants as well as the need for further investigation of vaccine efficacy in individuals living with HIV-1.
Assuntos
COVID-19 , Vacinas de mRNA , Humanos , Animais , Macaca mulatta , Vacinas Combinadas , SARS-CoV-2/genética , Vacinas contra COVID-19 , COVID-19/prevenção & controle , Vacinação , Anticorpos Neutralizantes , RNA Mensageiro , Anticorpos AntiviraisRESUMO
Androgen receptor (AR) is a master transcription factor that drives prostate cancer (PCa) development and progression. Alterations in the expression or activity of AR coregulators significantly impact the outcome of the disease. Using a proteomics approach, we identified the tripartite motif-containing 33 (TRIM33) as a novel transcriptional coactivator of AR. We demonstrate that TRIM33 facilitates AR chromatin binding to directly regulate a transcription program that promotes PCa progression. TRIM33 further stabilizes AR by protecting it from Skp2-mediated ubiquitination and proteasomal degradation. We also show that TRIM33 is essential for PCa tumor growth by avoiding cell-cycle arrest and apoptosis, and TRIM33 knockdown sensitizes PCa cells to AR antagonists. In clinical analyses, we find TRIM33 upregulated in multiple PCa patient cohorts. Finally, we uncover an AR-TRIM33-coactivated gene signature highly expressed in PCa tumors and predict disease recurrence. Overall, our results reveal that TRIM33 is an oncogenic AR coactivator in PCa and a potential therapeutic target for PCa treatment.
Assuntos
Neoplasias da Próstata , Receptores Androgênicos , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Recidiva Local de Neoplasia/genética , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Receptores Androgênicos/uso terapêutico , Proteínas Quinases Associadas a Fase S/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The aggregation and limited activity of nanoscale zero-valent iron (NZVI) in aqueous media hinder its practical application. In this study, a cost-effective, environmentally friendly, robust, and efficient synthesis method for NZVI-based composite was developed. NZVI@Chitin-modified ZSM-5 (NZVI@C-ZSM) composite was facilely and greenly synthesized by loading NZVI into alkali-modified ZSM-5 molecular sieves after modifying with chitin as a surfactant and binder. NZVI@C-ZSM exhibited remarkable efficacy in TC removal, achieving a removal efficiency of 97.72% within 60 min. Compared with pristine NZVI, NZVI@C-ZSM demonstrated twice the removal efficiency, indicating that NZVI@C-ZSM effectively improved the dispersion and stability of NZVI. This enhancement provided more reactive sites for generating reactive oxygen species (ROS), significantly boosting catalytic activity and durability while reducing the potential risk of secondary pollution. An improved two-parameter pseudo-first-order kinetic model was used to effectively characterize the reaction kinetics. The mechanism for TC removal primarily involved an adsorption process and chemical oxidation-reduction reactions induced by hydroxyl radicals (â¢OH) and superoxide radicals (â¢O2-). Three potential degradation pathways for TC were suggested. Furthermore, NZVI@C-ZSM exhibited good resistance to interference, suggesting its broad potential for practical applications in complex environmental conditions. This study offers a viable material and method for addressing the issue of antibiotic-contaminated water, with potential applications in water resource management.
Assuntos
Quitina , Ferro , Oxirredução , Tetraciclina , Poluentes Químicos da Água , Quitina/química , Poluentes Químicos da Água/química , Ferro/química , Tetraciclina/química , Química Verde/métodos , Antibacterianos/química , Zeolitas/químicaRESUMO
Although long noncoding RNAs (lncRNAs) have significant tissue specificity, their expression and variability in single cells remain unclear. Here, we developed ColorCells (http://rna.sysu.edu.cn/colorcells/), a resource for comparative analysis of lncRNAs expression, classification and functions in single-cell RNA-Seq data. ColorCells was applied to 167 913 publicly available scRNA-Seq datasets from six species, and identified a batch of cell-specific lncRNAs. These lncRNAs show surprising levels of expression variability between different cell clusters, and has the comparable cell classification ability as known marker genes. Cell-specific lncRNAs have been identified and further validated by in vitro experiments. We found that lncRNAs are typically co-expressed with the mRNAs in the same cell cluster, which can be used to uncover lncRNAs' functions. Our study emphasizes the need to uncover lncRNAs in all cell types and shows the power of lncRNAs as novel marker genes at single cell resolution.
Assuntos
Bases de Dados de Ácidos Nucleicos , Regulação da Expressão Gênica , RNA Longo não Codificante , Análise de Célula Única , Software , Animais , Humanos , Anotação de Sequência Molecular , RNA Longo não Codificante/biossíntese , RNA Longo não Codificante/genéticaRESUMO
PURPOSE: We aimed to study the association between adjusted mtDNA levels in human trophectoderm biopsy samples and the developmental potential of euploid and mosaic blastocysts. METHODS: We analyzed relative mtDNA levels in 2,814 blastocysts obtained from 576 couples undergoing preimplantation genetic testing for aneuploidy from June 2018 to June 2021. All patients underwent in vitro fertilization in a single clinic; the study was blinded-mtDNA content was unknown at the time of single embryo transfer. The fate of the euploid or mosaic embryos transferred was compared with mtDNA levels. RESULTS: Euploid embryos had lower mtDNA than aneuploid and mosaic embryos. Embryos biopsied on Day 5 had higher mtDNA than those biopsied on Day 6. No difference was detected in mtDNA scores between embryos derived from oocytes of different maternal ages. Linear mixed model suggested that blastulation rate was associated with mtDNA score. Moreover, the specific next-generation sequencing platform used have a significant effect on the observed mtDNA content. Euploid embryos with higher mtDNA content presented significantly higher miscarriage rates and lower live birth rates, while no significant difference was observed in the mosaic cohort. CONCLUSION: Our results will aid in improving methods for analyzing the association between mtDNA level and blastocyst viability.
Assuntos
DNA Mitocondrial , Fertilização in vitro , Feminino , Humanos , Aneuploidia , Blastocisto , DNA Mitocondrial/genética , Fertilização in vitro/métodos , Testes Genéticos/métodos , Idade Materna , Estudos Retrospectivos , Diagnóstico Pré-ImplantaçãoRESUMO
Clustered regularly interspaced short palindromic repeats (CRISPR) system provides adaptive immunity against plasmids and phages in prokaryotes. This system inspires the development of a powerful genome engineering tool, the CRISPR/CRISPR-associated nuclease 9 (CRISPR/Cas9) genome editing system. Due to its high efficiency and precision, the CRISPR/Cas9 technique has been employed to explore the functions of cancer-related genes, establish tumor-bearing animal models and probe drug targets, vastly increasing our understanding of cancer genomics. Here, we review current status of CRISPR/Cas9 gene editing technology in oncological research. We first explain the basic principles of CRISPR/Cas9 gene editing and introduce several new CRISPR-based gene editing modes. We next detail the rapid progress of CRISPR screening in revealing tumorigenesis, metastasis, and drug resistance mechanisms. In addition, we introduce CRISPR/Cas9 system delivery vectors and finally demonstrate the potential of CRISPR/Cas9 engineering to enhance the effect of adoptive T cell therapy (ACT) and reduce adverse reactions.
Assuntos
Edição de Genes , Neoplasias , Animais , Sistemas CRISPR-Cas , Edição de Genes/métodos , Genômica , Humanos , Neoplasias/genética , Neoplasias/terapia , OncogenesRESUMO
BACKGROUND & AIMS: Despite remarkable advances in treatment, most patients with hepatocellular carcinoma (HCC) respond poorly to anti-programmed cell death 1 (anti-PD1) therapy. A deeper insight into the tolerance mechanism of HCC against this therapy is urgently needed. METHODS: We performed next-generation sequencing, multiplex immunofluorescence, and dual-color immunohistochemistry and constructed an orthotopic HCC xenograft tumor model to identify the key gene associated with anti-PD1 tolerance. A spontaneously tumorigenic transgenic mouse model, an in vitro coculture system, mass cytometry, and multiplex immunofluorescence were used to explore the biological function of zinc finger protein 64 (ZFP64) on tumor progression and immune escape. Molecular and biochemical strategies like RNA-sequencing, chromatin immunoprecipitation-sequencing and mass spectrometry were used to gain insight into the underlying mechanisms of ZFP64. RESULTS: We showed that ZFP64 is frequently upregulated in tumor tissues from patients with anti-PD1-resistant HCC. Elevated ZFP64 drives anti-PD1 resistance by shifting macrophage polarization toward an alternative activation phenotype (M2) and fostering an inhibitory tumor microenvironment. Mechanistically, we primarily demonstrated that protein kinase C alpha (PKCα) directly phosphorylates ZFP64 at S226, leading to its nuclear translocation and the transcriptional activation of macrophage colony-stimulating factor (CSF1). HCC-derived CSF1 transforms macrophages to the M2 phenotype to drive immune escape and anti-PD1 tolerance. Notably, Gö6976, a protein kinase inhibitor, and lenvatinib, a multi-kinase inhibitor, reset the tumor microenvironment and restore sensitivity to anti-PD1 by blocking the PKCα/ZFP64/CSF1 axis. CONCLUSIONS: We propose that the PKCα/ZFP64/CSF1 axis is critical for triggering immune evasion and anti-PD1 tolerance. Inhibiting this axis with Gö6976 or lenvatinib overcomes anti-PD1 resistance in HCC. LAY SUMMARY: Despite remarkable treatment progress, most patients with hepatocellular carcinoma respond poorly to anti-PD1 therapy (a type of immunotherapy). A deeper insight into the tolerance mechanisms to this therapy is urgently needed. Herein, we unravel a previously unexplored mechanism linking tumor progression, macrophage polarization, and anti-PD1 resistance, and offer an attractive novel target for anti-PD1 combination therapy, which may benefit patients with hepatocellular carcinoma.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Animais , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Fatores Estimuladores de Colônias , Proteínas de Ligação a DNA , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Camundongos , Proteína Quinase C-alfa/genética , Inibidores de Proteínas Quinases , Fatores de Transcrição , Microambiente TumoralRESUMO
The T cell Ig and mucin domain (TIM) proteins inhibit release of HIV-1 and other enveloped viruses by interacting with cell- and virion-associated phosphatidylserine (PS). Here, we show that the Nef proteins of HIV-1 and other lentiviruses antagonize TIM-mediated restriction. TIM-1 more potently inhibits the release of Nef-deficient relative to Nef-expressing HIV-1, and ectopic expression of Nef relieves restriction. HIV-1 Nef does not down-regulate the overall level of TIM-1 expression, but promotes its internalization from the plasma membrane and sequesters its expression in intracellular compartments. Notably, Nef mutants defective in modulating membrane protein endocytic trafficking are incapable of antagonizing TIM-mediated inhibition of HIV-1 release. Intriguingly, depletion of SERINC3 or SERINC5 proteins in human peripheral blood mononuclear cells (PBMCs) attenuates TIM-1 restriction of HIV-1 release, in particular that of Nef-deficient viruses. In contrast, coexpression of SERINC3 or SERINC5 increases the expression of TIM-1 on the plasma membrane and potentiates TIM-mediated inhibition of HIV-1 production. Pulse-chase metabolic labeling reveals that the half-life of TIM-1 is extended by SERINC5 from <2 to â¼6 hours, suggesting that SERINC5 stabilizes the expression of TIM-1. Consistent with a role for SERINC protein in potentiating TIM-1 restriction, we find that MLV glycoGag and EIAV S2 proteins, which, like Nef, antagonize SERINC-mediated diminishment of HIV-1 infectivity, also effectively counteract TIM-mediated inhibition of HIV-1 release. Collectively, our work reveals a role of Nef in antagonizing TIM-1 and highlights the complex interplay between Nef and HIV-1 restriction by TIMs and SERINCs.
Assuntos
Infecções por HIV/metabolismo , Receptor Celular 1 do Vírus da Hepatite A/fisiologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana/fisiologia , Membrana Celular/metabolismo , Regulação para Baixo , Células HEK293 , Soropositividade para HIV , HIV-1/metabolismo , HIV-1/patogenicidade , Receptor Celular 1 do Vírus da Hepatite A/antagonistas & inibidores , Receptor Celular 1 do Vírus da Hepatite A/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Leucócitos Mononucleares/metabolismo , Glicoproteínas de Membrana , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Transporte Proteico , Receptores de Superfície Celular/metabolismo , Vírion/metabolismo , Replicação Viral/efeitos dos fármacos , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismoAssuntos
Vacinas contra COVID-19 , COVID-19 , Imunização Secundária , SARS-CoV-2 , COVID-19/etiologia , COVID-19/prevenção & controle , Vacinas contra COVID-19/imunologia , Vacinas contra COVID-19/uso terapêutico , Humanos , RNA Mensageiro , SARS-CoV-2/genética , SARS-CoV-2/imunologia , Eficácia de Vacinas , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/uso terapêutico , Vacinas de mRNA/imunologia , Vacinas de mRNA/uso terapêuticoRESUMO
Hepatic lipase is an important gene in lipid metabolism, which is crucial in the growth of fish. In this study, the cDNA sequence of genetically improved farmed tilapia (GIFT) HL gene was cloned by aimed rapid amplification of cDNA ends (RACE) method. Then, the characteristics of HL were analyzed with bioinformatics methods, and the expression of HL was assessed by the quantitative real-time PCR. To study the regulation of HL expression, GIFT were fed with diets containing different contents of lipid (40, 80, and 120 g kg-1) and choline (500, 750, and 1000 mg kg-1) and fed with different frequencies (2 or 3 times/day) and amounts (20, 40, 60, 80, and g kg-1 of body weight). Our results revealed that the GIFT HL gene has a full length of 1872 bp, encoding 493 amino acids. Consistent with the study in other species, GIFT HL was specially expressed in the liver. The HL gene of GIFT shared identity of 60.9-96.6% with other species. The expression of HL in 120 g kg-1 dietary lipid and 1000 mg kg-1 dietary choline group was the highest in all groups (P < 0.01). The expression of HL was increased gradually with 3 times/day frequency. All these results revealed the cDNA sequence of GIFT HL, and the expression of HL was affected by dietary choline and lipid levels, feeding frequency, and amount. This would guide the aquaculture of GIFT in the future.
Assuntos
Colina/farmacologia , Ciclídeos/genética , Gorduras na Dieta/farmacologia , Proteínas de Peixes/genética , Lipase/genética , Ração Animal , Animais , Animais Geneticamente Modificados , Clonagem Molecular , Dieta/veterinária , Proteínas de Peixes/química , Lipase/química , Fígado/enzimologiaRESUMO
Ebola virus (EBOV) is a highly pathogenic filovirus that causes hemorrhagic fever in humans and animals. Currently, how EBOV fuses its envelope membrane within an endosomal membrane to cause infection is poorly understood. We successfully measure cell-cell fusion mediated by the EBOV fusion protein, GP, assayed by the transfer of both cytoplasmic and membrane dyes. A small molecule fusion inhibitor, a neutralizing antibody, as well as mutations in EBOV GP known to reduce viral infection, all greatly reduce fusion. By monitoring redistribution of small aqueous dyes between cells and by electrical capacitance measurements, we discovered that EBOV GP-mediated fusion pores do not readily enlarge-a marked difference from the behavior of other viral fusion proteins. EBOV GP must be cleaved by late endosome-resident cathepsins B or L in order to become fusion-competent. Cleavage of cell surface-expressed GP appears to occur in endosomes, as evidenced by the fusion block imposed by cathepsin inhibitors, agents that raise endosomal pH, or an inhibitor of anterograde trafficking. Treating effector cells with a recombinant soluble cathepsin B or thermolysin, which cleaves GP into an active form, increases the extent of fusion, suggesting that a fraction of surface-expressed GP is not cleaved. Whereas the rate of fusion is increased by a brief exposure to acidic pH, fusion does occur at neutral pH. Importantly, the extent of fusion is independent of external pH in experiments in which cathepsin activity is blocked and EBOV GP is cleaved by thermolysin. These results imply that low pH promotes fusion through the well-known pH-dependent activity of cathepsins; fusion induced by cleaved EBOV GP is a process that is fundamentally independent of pH. The cell-cell fusion system has revealed some previously unappreciated features of EBOV entry, which could not be readily elucidated in the context of endosomal entry.
Assuntos
Ebolavirus/fisiologia , Doença pelo Vírus Ebola/virologia , Proteínas Virais de Fusão/metabolismo , Internalização do Vírus , Animais , Western Blotting , Células COS , Catepsinas/metabolismo , Chlorocebus aethiops , Citometria de Fluxo , Imunofluorescência , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Imuno-Histoquímica , Técnicas de Patch-ClampRESUMO
Accumulating evidence indicates that T-cell immunoglobulin (Ig) and mucin domain (TIM) proteins play critical roles in viral infections. Herein, we report that the TIM-family proteins strongly inhibit HIV-1 release, resulting in diminished viral production and replication. Expression of TIM-1 causes HIV-1 Gag and mature viral particles to accumulate on the plasma membrane. Mutation of the phosphatidylserine (PS) binding sites of TIM-1 abolishes its ability to block HIV-1 release. TIM-1, but to a much lesser extent PS-binding deficient mutants, induces PS flipping onto the cell surface; TIM-1 is also found to be incorporated into HIV-1 virions. Importantly, TIM-1 inhibits HIV-1 replication in CD4-positive Jurkat cells, despite its capability of up-regulating CD4 and promoting HIV-1 entry. In addition to TIM-1, TIM-3 and TIM-4 also block the release of HIV-1, as well as that of murine leukemia virus (MLV) and Ebola virus (EBOV); knockdown of TIM-3 in differentiated monocyte-derived macrophages (MDMs) enhances HIV-1 production. The inhibitory effects of TIM-family proteins on virus release are extended to other PS receptors, such as Axl and RAGE. Overall, our study uncovers a novel ability of TIM-family proteins to block the release of HIV-1 and other viruses by interaction with virion- and cell-associated PS. Our work provides new insights into a virus-cell interaction that is mediated by TIMs and PS receptors.
Assuntos
Linfócitos T CD4-Positivos/fisiologia , Infecções por HIV/metabolismo , HIV-1/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Receptores Virais/metabolismo , Linfócitos T CD4-Positivos/virologia , Membrana Celular/metabolismo , Membrana Celular/virologia , Técnicas de Silenciamento de Genes , Células HEK293 , Infecções por HIV/virologia , HIV-1/crescimento & desenvolvimento , Células HeLa , Receptor Celular 1 do Vírus da Hepatite A , Receptor Celular 2 do Vírus da Hepatite A , Humanos , Células Jurkat , Glicoproteínas de Membrana/genética , Proteínas de Membrana/genética , Fosfatidilserinas/metabolismo , RNA Interferente Pequeno/genética , Receptores Virais/genética , Vírion/crescimento & desenvolvimento , Vírion/metabolismo , Replicação Viral/fisiologiaRESUMO
To explore the optimum conditions of ß-glucosidase activity in Scrophularia root by using pNPG method. The extraction conditions and reaction conditions (such as extraction liquid type, reaction system, reaction time, temperature, and substrate concentration) were screened by using monofactorial experiment and homogeneous design. Then the changes of ß-glucosidase activity in Scrophularia root were detected at the drying temperature of 40-100 â. The results showed that citric acid phosphate buffer had better extraction effect, and the maximum absorbance produced by enzymatic reaction was present at 50 â environment after reaction for 30 min. Homogeneous design experiment determined that the optimal conditions were as follows: optimal extraction liquid pH 7.0; enzymatic reaction system pH 6.0; substrate concentration 20 mmolâ¢L⻹. The change of enzyme activity was affected by drying temperature and water loss rate. In the drying temperature of 60-100 â, the enzyme activity was reduced rapidly with the increase in water loss rate, while the activity was seen even with 0% of water at 40 and 50 â. This study has laid the theoretical foundation for research of hydrolysis mechanism of iridoid glycosides and optimum drying process.
Assuntos
Dessecação/métodos , Scrophularia/enzimologia , beta-Glucosidase/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Glicosídeos Iridoides/química , Raízes de Plantas/enzimologia , Tecnologia Farmacêutica , TemperaturaRESUMO
To establish a new quantitative method for simultaneous determination of multi-components in Scrophularia root by using high performance liquid chromatography (HPLC) and validate its feasibilitiesï¼Meanwhile,using catalpol as one chemical reference substance to establish the relative correct factors and relative retention values of aucubin,harpagide,acteoside,angoroside C,harpagoside and cinnamic acidï¼Then using the quantitative analysis of multi-components by single-marker (QAMS)model,the six analytes can be quantitatively determined in Scrophularia rootï¼The method was evaluated by comparison of the quantitative results to external standard methodï¼No significant differences were observed between the quantitative results of the two methodsï¼The obtained RCFs were credibleï¼It is feasible and suitable to evaluate the quality of Scrophularia root.