Binding interaction of laccases from Bacillus Subtilis after industrial dyes exposure: Molecular docking and molecular dynamics simulation studies

Dyes are becoming more widely used around the world wide, but there is no effective bioremediation approach for removing them completely from the environment. Several dyes are mentioned to be degraded through bacteria; however, it's still unknown how the particular enzymes act throughout the dye degradation. The behavior and function of these enzymes in the biodegradation of azo dyes (Textile dyes) had been investigated experimentally by the numbers of the researchers, however, the molecular mechanisms remain unclear. Therefore, the interaction mechanisms of textile dye (methyl orange) with laccase from B. subtilis were explored through molecular docking and molecular dynamics simulations, the three selected dyes (methyl orange, malachite green, and acid blue 62) that interact positively with laccase on the basis of their maximum binding energy, molecular docking results indicate that one of the three dyes is more stable as a target for degradation through Bacillus subtilis laccase. Therefore, subsequent research focused solely on one substrate: methyl orange. Molecular Dynamics simulation study was applied after the molecular docking to determine the interaction between laccases and methyl orange dyes. The trajectory was proved with root mean square deviation and root mean square fluctuation analysis. According to the molecular dynamics simulation results, laccase-methyl orange complexes remain stable during the catalytic reaction. So, this study demonstrates how laccase is involved in methyl orange bioremediation.

Network pharmacological mechanism and experimental validation of Yinjiushu in the treatment of non-alcoholic fatty liver disease based on the homology of medicine and food / 国际中医中药杂志

Objective:To the molecular mechanism of Yinjiushu in the treatment of non-alcoholic fatty liver disease (NAFLD) by network pharmacology based on the theory of homology of medicine and food; To conduct experimental verification.Methods:The active components and targets of the Yinjiushu were screened through the TCMSP platform. Cytoscape 3.7.2 was used to construct the "Chinese materia medica-component-target" network of Yinjiushu. The potential targets of NAFLD were obtained by using TTD, GeneCards database and DisGeNET database, and the intersection targets of Yinjiushu and NAFLD were obtained by mapping targets with Venn diagram. The high confidence interaction relationship of intersection targets was obtained in STRING database, and the core targets of Yinjiushu in treating NAFLD were screened out. GO function and KEGG pathway enrichment of common targets were analyzed by David database, and the above results were further verified by animal experiments. The rats were divided into blank group, model group, Western medicine group and Yinjiushu high-, medium- and low-dosage groups according to random number table method, with 8 rats in each group. Except the blank group, rats in other groups were fed with high-fat diet to prepare NAFLD model. Each group was given corresponding drugs for intervention. The rats were weighed regularly. The serum contents of GPT, GOT, TC, TG, IL-6, TNF-α, MPO of rats were detected by ELISA. The liver index was calculated. The degree of fatty degeneration of hepatocytes was observed by HE. The expressions of CAT, NOS3, SOD, PI3K, p-Akt, Akt protein were detected by Western blot.Results:A total of 8 418 NAFLD-related targets, 118 kinds of active components from Yinjiushu, and 137 targets acting on NAFLD were screened. The core targets included IL-6, TNF, VEGFA, TP53, JUN, CAT, NOS3, SOD, etc. 20 related signaling pathways were screened from KEGG enrichment pathway, among which PI3K/Akt pathway, calcium ion pathway, cAMP pathway and TNF pathway may play key roles in the treatment. Yinjiushu was closely related to inflammatory reaction, oxidative stress, angiogenesis, autophagy, cell proliferation, differentiation, metabolism, apoptosis, etc., or it could treat NAFLD by promoting cell apoptosis, inhibiting cell proliferation, inhibiting cell migration, etc. The animal experiment proved that Yinjiushu could reduce the body weight, wet liver weight and liver-body ratio of NAFLD rats, reduce some liver function and blood lipid indexes (GPT, GOT, TG, TC), down-regulate serum IL-6, TNF-α and MPO, up-regulate the expression of CAT, NOS3 and SOD in hepatocytes, and activate the expression of PI3K/Akt key protein.Conclusion:Yinjiushu can play a role in treating NAFLD by inhibiting the release of inflammatory mediators, improving lipid metabolism disorder of hepatocytes, repairing oxidative stress injury and promoting the recovery of liver function.

Quality regulatory issues and suggestions for the Lonicerae Japonicae Flos with property of drug and food homologous substance / 国际中医中药杂志

In the context of the national strategy of promoting the research and development of food and drug homologous substances, it is necessary to study the regulatory model of food and drug homologous substances as food. Medicinal and food homologous substances are regulated as "Chinese medicinal materials" or "Chinese decoction pieces" in the drug category, and as "food and drug homologous substances", "agricultural products" or "traditional Chinese medicine healthy food" in the food category. Drug supervision focuses on quality, while food supervision focuses on safety, and it is worth exploring whether there is a blind spot in the supervision of food and drug substances completely based on the food concept. In this paper, Lonicerae Japonicae Flos, as drug and food homologous substance, is selected as the research object, and its quality regulation status is investigated by reviewing the relevant laws and regulations and standards on food, analyzing the quality information and test results of the samples, and analyzing the necessity of quality regulation when serving as food, in order to provide a basis and reference for the way food substances are regulated.

Perilla frutescens: A traditional medicine and food homologous plant / 中草药·英文版

Perilla frutescens, an annual herb of the Labiatae family, has been cultivated in China for more than 2000 years. P. frutescens is the one of the first medicinal and edible plant published by the Ministry of Health. Its leaves, stems and seeds can be used as medicine and edible food. Because of the abundant nutrients and bioactive components in this plant, P. frutescens has been studied extensively in medicine, food, health care and chemical fields with great prospects for development. This paper reviews the cultivation history, chemical compositions and pharmacological activities of P. frutescens, which provides a reference for the development and utilization of P. frutescens resources.

Phyllanthi Fructus: A modal medicinal and food homologous item in quality evaluation / 中草药·英文版

Phyllanthi Fructus is a highly unique medicine and food homologous item, which exhibits distinctive flavor, notable nutritional value, and abundant pharmacological activity. It has enormous potential in the creation of health products and pharmaceuticals. However, due to the unique laws of quality formation and transfer of Phyllanthi Fructus, its appearance, shape, chemical compositions, nutrients, and sensory flavors are frequently greatly influenced by botanical resources, the processing and storage conditions. As a result, the current quality evaluation model is difficult to meet the needs of Phyllanthi Fructus as a medicine and food homologous item in the development of diversified products. This paper constructs the hierarchical utilization mode of Phyllanthi Fructus based on its unique quality formation and transmission laws, explores the quality evaluation model for food-oriented use and medicinal-oriented use, respectively, and systematically describes the quality evaluation idea under diversified application scenarios. This paper aims to serve as a reference for the construction of a quality evaluation model suitable for the medicine and food homologous item of Phyllanthi Fructus.

Value of drug resistance and homology of Acinetobacter baumannii in tracing the source of nosocomial infection in Jianyang, Sichuan / 中国热带医学

@#Abstract: Objective　To analyze the drug sensitivity and the carrying of carbapenem resistant gene of Acinetobacter baumannii isolated from clinical patients and clinical objects, and analyze the homology of strains to provide support for the control of nosocomial infection. Methods　A total of 38 strains of Acinetobacter baumannii isolated from patients and clinical objects surface were collected from January 2019 to August 2020. The antimicrobial susceptibility was tested by the minimum inhibitory concentration method. In addition, the resistance related genes were detected by polymerase chain reaction method, and homology analysis was performed by enterobacterial repetitive Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR). Results All 34 strains of Acinetobacter baumannii isolated from Clinical patients and 4 strains isolated from clinical objects carried blaOXA-51 and imp resistance genes, neither of them carried blaVIM gene. 32 Acinetobacter baumannii carrying blaOXA-23 gene, 28 strains carrying blaTEM gene, 7 strains carrying blaOXA-58 gene. After cluster analysis, 38 Acinetobacter baumannii isolates were classified into 7 genotypes (expressed A, B, C, D, E, F, G), and cluster E and cluster G were the main clusters, containing 12 strains (12/38, 31.6%) and 18 strains (18/38, 47.4%), respectively, as the main prevalent clonal strains. Conclusions　Acinetobacter baumannii isolated from different sources have the significant differences in drug resistance and carry different resistance genes. There is no direct correlation between patients and environmental isolates of Acinetobacter baumannii belonging to different clonal strains. Also, there aren’t significant correlation between clinical patients infected with Acinetobacter baumannii.

Preliminary Construction of Evaluation Index System for Traditional Chinese Medicine Medicated Diet based on Delphi Method and Analytic Hierarchy Process / 中医杂志

ObjectiveTo build an evaluation index system for traditional Chinese medicine （TCM） medicated diet， promoting the scientific and standardized development of medicated diet in TCM. MethodsThe framework of the evaluation index system was constructed through literature review and Delphi expert consultation method. The analytic hierarchy process was used to construct a hierarchical structure model. Pairwise comparisons between the indicators were conducted using the Saaty 1-9 scale method， and the weight of each indicator was calculated using Yaahp 10.3. ResultsThe response rates for the two rounds of expert consultation were 93.33% and 100%， respectively. The Kendall's W coefficients for the first-level and second-level indicators in the second round were 0.270 and 0.281， respectively （both P＜0.001）. Finally， an evaluation index system for TCM medicated diet therapy was constructed， consisting of 6 primary indicators and 27 secondary indicators. The weightings of the primary indicators were as follows： sensory appearance （0.1843）， health value （0.3569）， ingredient compatibility （0.1271）， packaging （0.0370）， production and preparation （0.1005）， and reliability （0.1940）. ConclusionA comprehensive and universally applicable evaluation index system for TCM medicated diet has been developed， taking into conside-rations of color， taste， appearance， efficacy， preparation， quality and others. This system can provide valuable reference for the evaluation of the value of medicated diet as well as its development.

Epidemiological investigation and genomic characterization of the first case of fever with thrombocytopenia syndrome in Puyang / 中华微生物学和免疫学杂志

Objective:To report the first case of sever fever with thrombocytopenia syndrome caused by severe fever with thrombocytopenia syndrome virus (SFTSV) in Puyang city, and to study the epidemiological and molecular characteristics of S, M, L fragments of the SFTSV isolate.Methods:The epidemiological characteristics of this case was analyzed with epidemiological methods. SFTSV was isolated from the patient′s serum sample. Nucleic acid of SFTSV was extracted and detected by fluorescent RT-PCR. A multiplex PCR method was constructed to amplify the nucleic acid sequence of the virus. whole-genome sequencing was performed on the next-generation sequencing platform. MEGA11 and DNAStar was used for homology analysis and a phylogenetic tree was constructed.Results:Epidemiological investigation showed that the patient and his close contacts had no history of travel or tick bite within 14 d, but had a history of fieldwork. The patient′s serum sample was positive for SFTSV nucleic acid. Genetic analysis showed that the S, M, L gene fragments of the first SFTSV isolate in Puyang belonged to genotype E. This isolate shared 94.8%-99.6%, 94.0%-99.8% and 95.7%-99.7% nucleotide sequence homology with the representative strains acquired from GeneBank in S, M, L gene fragments, respectively.Conclusions:This case was the first case of SFTSV-caused severe fever with thrombocytopenia syndrome in Puyang. The SFTSV isolate shared a close homology with domestic isolates, but its genotype was significantly different from the SFTSV strains isolated in Henan in recent years, indicating that it might an imported case from other places in Henan Province or Hubei Province. Disease monitoring and professional training for medical personnel should be strengthened and more attention should be paid to the evolution and mutation of SFTSV.

Genetic variation characteristics of HA gene of influenza A virus (H3N2) in Guizhou province from 2017 to 2019 / 中国基层医药

Objective:To analyze the genetic variation characteristics of the HA gene of influenza A virus (H3N2) in Guizhou province from 2017 to 2019. Methods:Twenty strains of influenza A virus (H3N2) were randomly selected from 10 network laboratories in Guizhou province for RNA extraction. Reverse transcriptase-polymerase chain reaction and sequencing were performed. The products were analyzed using bioinformatics software.Results:The nucleotide homology of the HA gene of the 20 strains was 97.7%-100%, which was highly homologous to the vaccine strains A/Hong-Kong/4801/2014 recommended by WHO in 2017 and A/Singapore-INFIMH/16-0019/2016 recommended by WHO in 2018, but they were significantly different from the vaccine strain A/Kansas/14/2017 recommended by WHO in 2019. Genetic analysis showed that the 20 strains were divided into two branches, and the strains that were prevalent in 2019 were located in different branches, with marked genetic differences. Key site analysis showed mutations in antigenic determinants A, B, C, and E and mutations in the anterior and posterior walls of receptor binding sites. Key site analysis also showed that there was an increase in the number of glycosylation sites compared with the vaccine strains prevalent in the same year. Genetic distance, antigen sites, and glycosylation sites were slightly different between virus strains prevalent in 2017-2018 and virus strains prevalent in 2019. Conclusion:The HA gene of the influenza A virus subtype H3N2 in Guizhou province from 2017 to 2019 showed heterogeneity and gene mutation, especially in 2019. Therefore, close monitoring of the genetic evolution of the influenza A virus subtype H3N2 is necessary.

In Silico Modeling and Docking Analysis of CTX-M-5, Cefotaxime-Hydrolyzing ?-Lactamase from Human-Associated Salmonella Typhimurium

Objective: To computationally model the CTX-M-5 ?-lactamase and establish its structure, which is exclusively present in human-associated Salmonella. Methods: The CTX-M-5 aminoacid sequence (Uniprot ID:O65975) of Salmonella enterica subsp. enterica serovar typhimurium was retrieved from UniProt database and subjected to homology modeling using MODELLER 9v7. The homology models were duly validated using RAMPAGE tool by generating Ramachandran plots, ERRAT graphs, and ProSA score. DoGSiteScorer server and ConSurf server were used to detect the cavities, pockets, and clefts to identify conserved amino acid sites in the predicted model. Subsequently, the modeled structure was docked using CLC Drug Discovery Workbench against proven drugs and known inhibitors. Results: Obtained high-quality homology model with 91.7% of the residues in favorable regions in Ramachandran plot and qualified in other quality parameters. Docking studies resulted in a higher dock score for PNK (D-benzylpenicilloic acid) molecule when compared to other reported inhibitors. Conclusion: This in silico study suggests that the compound PNK could be an efficient ligand for CTX-M-5 ?-lactamase and serve as a potent inhibitor of CTX-M-5.

In silico study to predict promiscuous T cell and B cell-epitopes derived from the vaccine candidate antigens of Plasmodium vivax binding to MHC class-II alleles

Malaria is one of the major causes of health and disability globally, even after tremendous efforts to eradicate it. Till date no highly effective vaccine is available for its control. The primary reason for the low efficacy of vaccines is extensive polymorphism in potential vaccine candidate antigen genes and HLA polymorphisms in the human population. This problem can be resolved by developing a vaccine using promiscuous peptides to combine the number of HLA alleles. This study predicted T and B cell epitopes (promiscuous peptides) by targeting PPPK-DHPS and DHFR-TS proteins of Plasmodium vivax, using different in silico tools. Selected peptides were characterized as promiscuous peptides on the basis of their immunogenicity, antigenicity and hydrophobicity. Furthermore, to confirm their immunogenicity, these peptides were utilized for molecular modelling and docking analysis. For determining the requisite affinity with distinct HLA Class-I, and HLA Class-II alleles, only five peptides for DHFR-TS and 3 peptides for PPPK-DHPS were chosen as promiscuous peptides. The D1 peptide has the maximum binding energy with HLA alleles, according to HLA-peptide complex modelling and binding interaction analyses. These findings could lead to the development of epitope-based vaccinations with improved safety and efficacy. These epitopes could be major vaccine targets in P. vivax as they possess a higher number of promiscuous peptides. Also, the B cell epitopes possess maximum affinity towards different alleles as analyzed by docking scores. However, further investigation is warranted in vitro and in vivo.

Modelamiento in silica de la liasa organomercurial (MerB) de Pseudamanas fluarescens / In silico modeling of Pseudomonas fluoresceins organomercurial lyase (MerB) / Modelagem in silica da liase organomercurial (MerB) de Pseudamanas fluarescens

Resumen El modelamiento ¡n silíco ha sido de gran contribución en los procesos proteómicos, desarrollando estructuras de las secuencias proteicas ya existentes, que por motivos de altos costos y las diferentes tecnologías necesarias para el desarrollo de estas metodologías, se encuentran deficientes en el número de modelamientos de proteínas disponibles. Entre aquellas secuencias con carencia de estructura proteica se encuentra la proteína liasa organomercurial (MerB) de Pseudomonas /luorescens, importante en la resistencia al mercurio. En el presente artículo se analizó tanto estructural como funcionalmente la proteína MerB en Pseudomonas jluorescens, utilizando la herramienta de la química estructural "modelamiento por homología" mediante plataformas bioinformáticas, con el fin de obtener un modelo que represente la estructura 3D más precisa y que capturen las mejores variantes estructurales entre todas las posibles conformaciones de las proteínas en la familia. En este trabajo, se desarrolló un método comparativo de la secuencia estudiada con las reportadas en las bases de datos para las proteínas MerB del género Pseudomonas. Se propone un modelo tridimensional para la enzima (MerB) en P. jluorescens, mediante el modelamiento por homología, se muestra la caracterización en la estructura secundaria, terciaria, la caracterización del dominio catalítico y los motivos estructurales presentes.

Abstract In silico modeling has made a great contribution to proteomic processes, developing structures of the already existing protein sequences, which for reasons of high costs and the different technologies necessary for the development of these methodologies, are deficient in the number of models of available proteins. Among those sequences lacking protein structure is the organomercurial lyase (MerB) protein from Pseudomonas fluoresceins, important in mercury resistance. In this article, the MerB protein in Pseudomonas fluorescens was analyzed both structurally and functionally, using the structural chemistry tool "homology modeling" using bioinformatic platforms, in order to obtain a model that represents the most accurate 3D structure and that captures the best structural variants among all the possible conformations of the proteins in the family. In this work, a comparative method of the sequence studied with those reported in the databases for MerB proteins of the genus Pseudomonas was developed. A three-dimensional model for the enzyme (MerB) in P. fluorescens is proposed, through homology modeling, the characterization at the secondary and tertiary structure level, the characterization of the catalytic domain and the structural motifs present is shown.

Resumo A modelagem in silico tem dado um grande contributo para os processos proteómicos, desenvolvendo estruturas de sequências de proteínas já existentes, as quais, pelos elevados custos e pelas diferentes tecnologias necessárias ao desenvolvimento destas metodologias, são deficientes no número de modelos de proteínas disponíveis. Entre as sequências sem estrutura protéica está a proteína organomercurial liase (MerB) de Pseudomonas fluorescens, importante na resistência ao mercúrio. Neste artigo, a proteína MerB em Pseudomonas fluorescens foi analisada estrutural e funcionalmente, usando a ferramenta de química estrutural "modelagem de homologia" usando plataformas de bioinformática, a fim de obter um modelo que represente a estrutura 3D mais precisa e que capture as melhores variantes estruturais. entre todas as conformações possíveis das proteínas da família. Neste trabalho, foi desenvolvido um método comparativo da sequência estudada com aqueles relatados em bancos de dados para proteínas MerB do gênero Pseudomonas. Um modelo tridimensional para a enzima (MerB) em P. fluorescens é proposto, através de modelagem por homologia, a caracterização em nível de estrutura secundária e terciária, a caracterização do domínio catalítico e os motivos estruturais presentes são mostradas.

Chemical constituents and pharmacological activities of medicinal plants from Rosa genus / 中草药·英文版

The genus Rosa (Rosaceae family) includes about 200 species spread in the world, and this genus shows unique advantages in medicine and food. To date, several scholars concentrated on compounds belonging to flavonoids, triterpenes, tannins, polysaccharide, phenolic acids, fatty acids, organic acids, carotenoids, and vitamins. Pharmacological effects such as antineoplastic and anti-cancer properties, anti-inflammatory, antioxidant, liver protection, regulate blood sugar, antimicrobial activity, antiviral activity, as well as nervous system protection and cardiovascular protection were wildly reported. This article reviews the chemical constituents, pharmacological effects, applications and safety evaluations of Rosa plants, which provides a reference for the comprehensive utilization of medicine and food resources and gives a scientific basis for the development of medicinal plants of the genus Rosa.

Construction of recombinant herpes simplex virus 2 expressing enhanced green fluorescent protein using CRISPR/Cas9 / 中华微生物学和免疫学杂志

Objective:To construct a recombinant herpes simplex virus 2 (HSV-2) expressing enhanced green fluorescent protein (EGFP) using clustered, regularly interspaced, short palindromic repeat/CRISPR-associated nuclease 9 (CRISPR/Cas9) technology.Methods:Four strategies for inserting exogenous EGFP gene into HSV-2 genome using CRISPR/Cas9 technology were designed: (1) conventional homology-directed repair: circular two homology arm donor-mediated gene knock-in; (2) linearized single homology arm donor-mediated gene knock-in; (3) homology-independent targeted integration; (4) conventional homology-directed repair-mediated by cell lines stably expressing Cas9 and sgRNA.Results:The recombinant virus HSV-2-EGFP was successfully constructed based on the second, the third and the fourth strategies. The second strategy was the most efficient, followed by the third and the fourth strategies. The purified recombinant virus could stably express green fluorescent protein in seven passages and shared similar growth characteristics in Vero cells to the parental virus.Conclusions:Linearized single homology arm donor could increase the efficiency of gene knock-in, and cell lines stably expressing Cas9 and sgRNA could increase the efficiency of gene knock-in mediated by homology-directed repair.

Intervention of Hei Xiaoyaosan in Alzheimer's Disease Immune Inflammation Based on Liver-kidney Homology / 中国实验方剂学杂志

Alzheimer's disease （AD） is a neurological disease highly related to age， which is the main cause of senile dementia and the most common disease leading to the loss of daily living ability of the elderly. AD brings heavy mental burden and economic pressure to patients， families， and society. Traditional Chinese medicine （TCM） ascribes AD to category of "dementia"， believing that the treatment should start from kidney because kidney deficiency is the root cause. Combined with the physiological and pathological characteristics of liver， this paper proposed that liver-kidney homology was an important idea for the prevention and treatment of AD. The main pathological manifestations of AD were amyloid β-protein （Aβ） deposition and neurofibrillary tangles （NFT）， and the pathogenesis was complex. A growing number of studies showed that immune inflammation played an important role in the pathogenesis of AD. The important target of treating AD was the regulation of neuro-immune inflammation through the nuclear factor kappa B （NF-κB）/NOD-like receptor thermal protein domain associated protein 3 （NLRP3）/Caspase-1/interleukin-1β （IL-1β） signaling pathway. Based on the idea of liver-kidney homology， this paper selected the representative formula Hei Xiaoyaosan to explore its effect on the prevention and treatment of AD and the mechanism from the perspective of regulating NF-κB/NLRP3/Caspase-1/IL-1β signaling pathway and inhibiting neuro-immune inflammation， expecting to further promote the in-depth study on the prevention and treatment of AD， and provide references for the prevention and treatment of AD by TCM.

Mechanism of Zhizi Prescription in Protection of CCl4-induced Acute and Subacute Liver Injury in Mice / 中国实验方剂学杂志

ObjectiveTo investigate the protective effect of Zhizi prescription （ZZP） on carbon tetrachloride （CCl4）-induced acute and subacute liver injury and its mechanism. MethodAcute and subacute liver injury animal models were induced. C57 mice were randomly divided into a normal group， model group， obeccholic acid group， ZZP high-dose （0.5 g·kg-1） group， and ZZP low-dose （0.25 g·kg-1） group. According to the experiment design， the serum and liver tissue of mice were collected after the last administration. Hematoxylin-eosin （HE） and Sirius staining was used to observe the liver pathological changes. Serum alanine aminotransferase （ALT）， aspartate aminotransferase （AST）， total bilirubin （TBIL）， liver homogenate hydroxyproline （Hyp）， malondialdehyde （MDA）， and superoxide dismutase （SOD） levels were determined by kit. The levels of tumor necrosis factor-α （TNF-α） and interleukin-6 （IL-6） in the liver tissue were determined by enzyme-linked immunosorbent assay （ELISA）. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to detect the mRNA expressions of collagen 1A1 （Col1a1）， collagen 3A1 （Col3a1）， fibronectin （FN）， transforming growth factor β receptor Ⅱ （Tgfbr2） and α-smooth muscle actin （α-SMA） in the liver tissue. ResultIn terms of the acute liver injury， as compared with the normal group， the levels of ALT， AST， TBIL and MDA in the model group were significantly increased （P<0.01）， while the activity of liver SOD was significantly decreased （P<0.01）. Compared with model group， the ZZP high-dose and low-dose groups both significantly reduced the degree of liver cell injury， and protected the acute liver injury induced by CCl4. The ZZP high-dose group had a better effect than the ZZP low-dose group. In terms of the subacute liver injury， the levels of ALT， AST， MDA，TNF-α and IL-6 in the model group were significantly increased （P<0.01）， while the activity of liver SOD was significantly decreased （P<0.01）. As compared with the model group， liver Hyp content in the ZZP high-dose and low-dose groups was significantly decreased （P<0.01）， and the collagen deposition in liver of both groups was significantly reduced. The ZZP high-dose group also significantly down-regulated the mRNA expressions of α-SMA， Col1a1， Col3a1， FN， and Tgfbr2 in the liver of mice （P<0.05， P<0.01）. ConclusionZZP effectively protects the acute and subacute liver injury induced by CCl4， and the protective effect is proportional to its concentration. The mechanism may be related to the increase of the activity of antioxidant enzymes in the liver tissue， the decrease of the level of lipid peroxidation， and the inhibition of inflammatory response， thus reducing collagen deposition and improving early liver fibrosis.

Mechanism of Zhizi Prescription in Protection of CCl4-induced Acute and Subacute Liver Injury in Mice / 中国实验方剂学杂志

ObjectiveTo investigate the protective effect of Zhizi prescription （ZZP） on carbon tetrachloride （CCl4）-induced acute and subacute liver injury and its mechanism. MethodAcute and subacute liver injury animal models were induced. C57 mice were randomly divided into a normal group， model group， obeccholic acid group， ZZP high-dose （0.5 g·kg-1） group， and ZZP low-dose （0.25 g·kg-1） group. According to the experiment design， the serum and liver tissue of mice were collected after the last administration. Hematoxylin-eosin （HE） and Sirius staining was used to observe the liver pathological changes. Serum alanine aminotransferase （ALT）， aspartate aminotransferase （AST）， total bilirubin （TBIL）， liver homogenate hydroxyproline （Hyp）， malondialdehyde （MDA）， and superoxide dismutase （SOD） levels were determined by kit. The levels of tumor necrosis factor-α （TNF-α） and interleukin-6 （IL-6） in the liver tissue were determined by enzyme-linked immunosorbent assay （ELISA）. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to detect the mRNA expressions of collagen 1A1 （Col1a1）， collagen 3A1 （Col3a1）， fibronectin （FN）， transforming growth factor β receptor Ⅱ （Tgfbr2） and α-smooth muscle actin （α-SMA） in the liver tissue. ResultIn terms of the acute liver injury， as compared with the normal group， the levels of ALT， AST， TBIL and MDA in the model group were significantly increased （P<0.01）， while the activity of liver SOD was significantly decreased （P<0.01）. Compared with model group， the ZZP high-dose and low-dose groups both significantly reduced the degree of liver cell injury， and protected the acute liver injury induced by CCl4. The ZZP high-dose group had a better effect than the ZZP low-dose group. In terms of the subacute liver injury， the levels of ALT， AST， MDA，TNF-α and IL-6 in the model group were significantly increased （P<0.01）， while the activity of liver SOD was significantly decreased （P<0.01）. As compared with the model group， liver Hyp content in the ZZP high-dose and low-dose groups was significantly decreased （P<0.01）， and the collagen deposition in liver of both groups was significantly reduced. The ZZP high-dose group also significantly down-regulated the mRNA expressions of α-SMA， Col1a1， Col3a1， FN， and Tgfbr2 in the liver of mice （P<0.05， P<0.01）. ConclusionZZP effectively protects the acute and subacute liver injury induced by CCl4， and the protective effect is proportional to its concentration. The mechanism may be related to the increase of the activity of antioxidant enzymes in the liver tissue， the decrease of the level of lipid peroxidation， and the inhibition of inflammatory response， thus reducing collagen deposition and improving early liver fibrosis.

A high-efficiency and versatile CRISPR/Cas9-mediated HDR-based biallelic editing system / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9), the third-generation genome editing tool, has been favored because of its high efficiency and clear system composition. In this technology, the introduced double-strand breaks (DSBs) are mainly repaired by non-homologous end joining (NHEJ) or homology-directed repair (HDR) pathways. The high-fidelity HDR pathway is used for genome modification, which can introduce artificially controllable insertions, deletions, or substitutions carried by the donor templates. Although high-level knock-out can be easily achieved by NHEJ, accurate HDR-mediated knock-in remains a technical challenge. In most circumstances, although both alleles are broken by endonucleases, only one can be repaired by HDR, and the other one is usually recombined by NHEJ. For gene function studies or disease model establishment, biallelic editing to generate homozygous cell lines and homozygotes is needed to ensure consistent phenotypes. Thus, there is an urgent need for an efficient biallelic editing system. Here, we developed three pairs of integrated selection systems, where each of the two selection cassettes contained one drug-screening gene and one fluorescent marker. Flanked by homologous arms containing the mutated sequences, the selection cassettes were integrated into the target site, mediated by CRISPR/Cas9-induced HDR. Positively targeted cell clones were massively enriched by fluorescent microscopy after screening for drug resistance. We tested this novel method on the amyloid precursor protein (APP) and presenilin 1 (PSEN1) loci and demonstrated up to 82.0% biallelic editing efficiency after optimization. Our results indicate that this strategy can provide a new efficient approach for biallelic editing and lay a foundation for establishment of an easier and more efficient disease model.

Application of homology modeling technique in congenital cardiovascular disease / 国际儿科学杂志

Congenital cardiovascular disease is a kind of congenital disease caused by the interaction of multiple factors such as heredity, environment and pregnancy infection, but its exact etiology is still unclear.With the deepening of research, more and more evidences show that genetic factors play a significant role in its pathogenesis.Many genes are involved in the development of heart embryos, and the mutation of these genes often leads to congenital cardiovascular diseases.Homology modeling is a technique to predict the structural and functional changes of proteins caused by gene mutation through computer algorithm, and its application in the pathogenesis of congenital diseases has become a future trend.The purpose of this paper is to review homology modeling and its application in pathogenesis of congenital cardiovascular diseases briefly.

Homology-based repair induced by CRISPR-Cas nucleases in mammalian embryo genome editing

Recent advances in genome editing, especially CRISPR-Cas nucleases, have revolutionized both laboratory research and clinical therapeutics. CRISPR-Cas nucleases, together with the DNA damage repair pathway in cells, enable both genetic diversification by classical non-homologous end joining (c-NHEJ) and precise genome modification by homology-based repair (HBR). Genome editing in zygotes is a convenient way to edit the germline, paving the way for animal disease model generation, as well as human embryo genome editing therapy for some life-threatening and incurable diseases. HBR efficiency is highly dependent on the DNA donor that is utilized as a repair template. Here, we review recent progress in improving CRISPR-Cas nuclease-induced HBR in mammalian embryos by designing a suitable DNA donor. Moreover, we want to provide a guide for producing animal disease models and correcting genetic mutations through CRISPR-Cas nuclease-induced HBR in mammalian embryos. Finally, we discuss recent developments in precise genome-modification technology based on the CRISPR-Cas system.