Transcriptome-Wide Identification and Functional Analysis of PgSQE08-01 Gene in Ginsenoside Biosynthesis in Panax ginseng C.A.Mey.

Panax ginseng C.A.Mey.is an important plant species used in traditional Chinese medicine,whose primary active ingredient is a ginsenoside.Ginsenoside biosynthesis is not only regulated by transcription factors but also controlled by a variety of structural genes.Nonetheless,the molecular mechanism underlying ginsenoside biosynthesis has always been a topic in the discussion of ginseng secondary metabolites.Squalene epoxidase(SQE)is a key enzyme in the mevalonic acid pathway,which affects the biosynthesis of secondary metabolites such as terpenoid.Using ginseng transcriptome,expression,and ginsenoside content databases,this study employed bioinformatic methods to systematically analyze the genes encoding SQE in ginseng.We first selected six PgSQE candidates that were closely involved in ginsenoside biosynthesis and then identified PgSQE08-01 to be highly associated with ginsenoside biosynthesis.Next,we constructed the overexpression vector pCAMBIA3301-PgSQE08-01 and the RNAi vector pART27-PgSQE08-01 and transformed ginseng adventitious roots using Agrobacterium rhizogenes,to obtain positive hairy-root clones.Thereafter,quantitative reverse transcriptionpolymerase chain reaction and high-performance liquid chromatography were used to determine the expression of relevant genes and ginsenoside content,respectively.Then,we focused on the function of PgSQE08-01 gene,which was noted to be involved in ginsenoside biosynthesis.Thus,these findings not only provided a molecular basis for the identification of important functional genes in ginseng but also enriched genetic resources for the biosynthesis of ginsenosides using synthetic biology.

基于CRISPR/Cas9技术构建IDO1基因敲除的THP-1细胞株及其表型研究

目的 采用CRISPR/Cas9技术构建吲哚胺-2,3-双加氧酶1(IDO1)基因敲除的THP-1细胞株,为研究IDO1在巨噬细胞中的作用提供细胞模型。方法 设计靶向IDO1基因的3条向导RNA(guide RNA,gRNA),分别构建IDO1-gRNA重组质粒,酶切及测序鉴定后,包装成Lenti-IDO1-gRNA慢病毒。利用Cas9慢病毒感染THP-1细胞获得稳定表达Cas9蛋白的细胞株,再经Lenti-IDO1-gRNA慢病毒感染敲除IDO1基因,有限稀释法获得单克隆细胞株。T7E1酶切检测gRNA打靶效率,PCR产物测序和Western blot鉴定IDO1基因敲除效果。CCK8法检测IDO1基因敲除对THP-1细胞增殖活性的影响,流式细胞术检测对巨噬细胞标志物CD11b、CD68和CD14表达的影响,中性红法检测巨噬细胞吞噬功能。结果 成功构建了3种IDO1-gRNA重组质粒,3条gRNA均能有效编辑IDO1基因,以gRNA2编辑效率最高。经PCR产物测序和Western blot验证获得了3株THP-1 IDO1-KO单克隆细胞株,并发现IDO1基因敲除可抑制THP-1细胞增殖,下调THP-1巨噬细胞CD11b、CD68表达,上调CD14表达,增强THP-1巨噬细胞吞噬功能。结论 成功构建IDO1基因敲除的THP-1细胞株;IDO1对THP-1细胞增殖活性、分化调节以及THP-1巨噬细胞吞噬功能调节有重要作用,为后续进一步探讨IDO1基因在巨噬细胞中的功能及机制研究奠定基础。

小白链霉菌中CRISPR-Cas9基因敲除系统的构建与优化

小白链霉菌(Streptomyces albulus)是工业上生产ε-聚赖氨酸的主要菌株。为提高S.albulus的ε-聚赖氨酸合成能力,基于诱变和抗性筛选的传统育种方法被普遍应用。然而,由于传统育种存在“疲劳效应”,当前S.albulus的育种陷入了瓶颈。为利用代谢工程方法进一步提高S.albulus的ε-聚赖氨酸合成能力,亟需建立基于CRISPR-Cas9的基因编辑方法。为此,该文以S.albulus GS114为研究对象,以ε-聚赖氨酸合成酶基因pls为靶基因,构建了CRISPR-Cas9基因敲除系统,成功敲除了pls,编辑效率为100%。为进一步提高获得的敲除株数量,对S.albulus GS114结合转移条件进行了系统优化,获得的最优结合转移条件为:供受体比例1∶1,镁离子浓度30 mmol/L,55℃热激孢子10 min,培养20 h后覆盖抗生素。在最优转移条件下,结合转移效率达到2.5×10^(-8),较优化前提高了78%。该研究结果一方面为后续S.albulus的代谢工程改造提供了重要工具,另一方面为其他工业链霉菌构建CRISPR-Cas系统提供了重要参考。

Creutzfeldt-Jakob disease presenting as Korsakoff syndrome caused by E196A mutation in PRNP gene:A case report

BACKGROUND Prion diseases are a group of degenerative nerve diseases that are caused by infectious prion proteins or gene mutations.In humans,prion diseases result from mutations in the prion protein gene(PRNP).Only a limited number of cases involving a specific PRNP mutation at codon 196(E196A)have been reported.The coexistence of Korsakoff syndrome in patients with Creutzfeldt-Jakob disease(CJD)caused by E196A mutation has not been documented in the existing literature.CASE SUMMARY A 61-year-old Chinese man initially presented with Korsakoff syndrome,followed by rapid-onset dementia,visual hallucinations,akinetic mutism,myoclonus,and hyperthermia.The patient had no significant personal or familial medical history.Magnetic resonance imaging of the brain revealed extensive hyperintense signals in the cortex,while positron emission tomography/computed tomography showed a diffuse reduction in cerebral cortex metabolism.Routine biochemical and microorganism testing of the cerebrospinal fluid(CSF)yielded normal results.Tests for thyroid function,human immunodeficiency virus,syphilis,vitamin B1 and B12 levels,and autoimmune rheumatic disorders were normal.Blood and CSF tests for autoimmune encephalitis and autoantibody-associated paraneoplastic syndrome yielded negative results.A test for 14-3-3 protein in the CSF yielded negative results.Whole-genome sequencing revealed a diseasecausing mutation in PRNP.The patient succumbed to the illness 11 months after the initial symptom onset.CONCLUSION Korsakoff syndrome,typically associated with alcohol intoxication,also manifests in CJD patients.Individuals with CJD along with PRNP E196A mutation may present with Korsakoff syndrome.

基于label-free定量蛋白质组学方法筛选沉默CHAF1B基因后心肌细胞差异表达蛋白及调控网络分析

目的分析沉默染色质装配因子1亚基B(chromatin assembly factor 1 subunit B,CHAF1B)基因后心肌细胞中差异表达蛋白,预测CHAF1B基因调控网络,为寻找促进心肌细胞修复的潜在治疗靶点提供参考。方法采用转染和蛋白质印迹法筛选沉默CHAF1B基因的有效小干扰RNA(small interfering RNA,siRNA)。应用有效siRNA沉默人源心肌AC16细胞CHAF1B基因后,采用细胞活力检测方法检测细胞活力;提取总蛋白质进行定量、还原、烷基化和胰蛋白酶裂解成肽段,利用高效液相串联质谱法鉴定肽段;搜索UniProt蛋白库筛选差异表达的蛋白质进行基因本体(Gene Ontology,GO)富集分析、京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)通路富集和蛋白质互作网络(protein-protein interaction networks,PPI)分析。结果siRNA有效沉默CHAF1B基因后,心肌细胞存活明显受到抑制;label-free定量蛋白质组学方法鉴定结果显示,共有69个差异表达蛋白质,其中50个表达显著上调(差异倍数≥2,P<0.05),19个表达显著下调(差异倍数≤0.5,P<0.05)。GO分析显示,差异表达蛋白质主要参与大分子复合亚基体、细胞组分生物合成和组装等生物学过程,分布在细胞质和囊泡等区域,发挥蛋白质结合等分子功能。KEGG通路富集和PPI分析显示,差异表达蛋白质参与的信号通路包括蛋白酶体、氨酰tRNA生物合成、胞吞、嘧啶代谢和氨基酸生物合成等10条信号途径;表达显著上调的蛋白质如蛋白酶体亚单位A2和B7、26 S蛋白酶体调节亚单位6B和10B参与蛋白酶体途径,丝氨酸、甘氨酸、谷氨酰胺和赖氨酸tRNA合成酶介导氨酰tRNA生物合成;表达显著下调的蛋白质包括骨架相关蛋白2/3复合体亚单位3和热休克70蛋白1样参与胞吞作用,核糖核苷-二磷酸还原酶大亚基介导嘧啶代谢等通路。实时荧光定量聚合酶链式反应结果证实,转染CHAF1B siRNA后心肌细胞中合成骨架相关蛋白2/3复合体亚单位3的基因ARPC3和氨酰tRNA生物合成关键基因QARS1的mRNA水平均显著降低。结论CHAF1B为心肌细胞存活的关键蛋白质,参与调控心肌细胞的胞吞和氨基酸生物合成等多种生物学过程,参考其调控网络可帮助寻找促进心肌细胞修复的干预环节。

Exercise-induced modulation of miR-149-5p and MMP9 in LPS-triggered diabetic myoblast ER stress: licorice glycoside E as a potential therapeutic target

Background:This study explores the relationship between endoplasmic reticulum(ER)stress and diabetes,particularly focusing on the impact of physical exercise on ER stress mechanisms and identifying potential therapeutic drugs and targets for diabetes-related sepsis.The research also incorporates traditional physical therapy perspectives,emphasizing the genomic insights gained from exercise therapy in disease management and prevention.Methods:Gene analysis was conducted on the GSE168796 and GSE94717 datasets to identify ER stress-related genes.Gene interactions and immune cell correlations were mapped using GeneCard and STRING databases.A screening of 2,456 compounds from the TCMSP database was performed to identify potential therapeutic agents,with a focus on their docking potential.Techniques such as luciferase reporter gene assay and RNA interference were used to examine the interactions between microRNA-149-5p and MMP9.Results:The study identified 2,006 differentially expressed genes and 616 miRNAs.Key genes like MMP9,TNF-α,and IL1B were linked to an immunosuppressive state.Licorice glycoside E demonstrated high affinity for MMP9,suggesting its potential effectiveness in treating diabetes.The constructed miRNA network highlighted the regulatory roles of MMP9,IL1B,IFNG,and TNF-α.Experimental evidence confirmed the binding of microRNA-149-5p to MMP9,impacting apoptosis in diabetic cells.Conclusion:The findings highlight the regulatory role of microRNA-149-5p in managing MMP9,a crucial gene in diabetes pathophysiology.Licorice glycoside E emerges as a promising treatment option for diabetes,especially targeting MMP9 affected by ER stress.The study also underscores the significance of physical exercise in modulating ER stress pathways in diabetes management,bridging traditional physical therapy and modern scientific understanding.Our study has limitations.It focuses on the microRNA-149-5p-MMP9 network in sepsis,using cell-based methods without animal or clinical trials.Despite strong in vitro findings,in vivo studies are needed to confirm licorice glycoside E’s therapeutic potential and understand the microRNA-149-5p-MMP9 dynamics in real conditions.

Differences of aroma development and metabolic pathway gene expression between Kyoho and 87-1 grapes

Aroma is an important quality trait of grapes and often the focus of consumers,viticulturists and grapevine breeders.Kyoho is a hybrid between Vitis vinifera and Vitis labrusca with a strawberry-like scent,while 87-1 is an early-ripening mutant of Muscat hamburg,belonging to Vitis vinifera,with a rose scent.In this study,we compared their aroma compositions and concentrations during berry development by headspace-SPME combined with gas chromatography-mass spectrometry(GC-MS),and analyzed the expression differences of enzyme-encoding genes in the LOX-HPL,MEP and MVA metabolic pathways by qRT-PCR.Twelve esters were detected in Kyoho during the whole berry development and they were abundant after veraison,but no esters were detected in 87-1 berries.Linalool was the dominant terpene among the 14 terpenes detected in 87-1 berries,while limited amounts of terpenes were detected in Kyoho berries.qRT-PCR analysis indicated that the low expression of VvAAT might explain the low content of ester volatiles in 87-1 berries,and the low expression of coding genes in the MEP pathway,especially VvPNLin Ner1,might be the reason for the low content of volatile terpenes in Kyoho berries.The results from this work will promote our understanding of aroma metabolic mechanisms of grapes,and offer some suggestions for grape aromatic quality improvement.

Transgenic Pigs Carrying a Synthesized Fatty Acid Desaturase Gene Yield High Level of ω-3 PUFAs

Polyunsaturated fatty acids （PUFAs） are essential for normal growth in mammals, especially the ω-3 PUFAs, which play important roles in preventing several life-threatening diseases, such as coronary heart disease and diabetes. In this study, we aimed to investigate whether the sFat-1 gene from Caenorhabditis briggsae could be functionally expressed in transgenic pigs, and whether the transgenic could synthesize high quality ω-3 PUFAs endogenously. In this study, a gene construct consisting of CMV promoter and 1.9 kb cDNA of ω-3 fatty acid desaturase gene （sFat-1） from C. briggsae was injected into the male pronucleus of pig embryos by microinjection. The piglets were screened for the transgene by PCR, Southern blot and reverse transcription-PCR analysis. Pigs that give positive results were mated with wild-type pigs to produce the next generation and the transmission of transgene was examined by PCR analysis. Fatty acids compositions of various tissues in the transgenic pigs were then analyzed by gas chromatograph. A total of 878 embryos were transferred into 42 recipients, among which 29 successfully got pregnant and gave birth to a total of 162 piglets, and 8 of them were identified to be transgenic. Fatty acid compositions in the transgenic pigs were altered, and the levels of ω-6：ω-3 ratios were decreased from 14.53 in the control to 2.62 in Fat-1 transgenic pigs. A number of primary sFat-1-transgenic pigs were bred in this study, which lays the foundation for cultivation of new varieties of transgenic pigs.

Use of gene-editing technology to introduce targeted modifications in pigs

Pigs are an important resource in agriculture and serve as a model for human diseases. Due to their physiological and anatomical similarities with humans, pigs can recapitulate symptoms of human diseases, making them a useful model in biomedicine. However, in the past pig models have not been widely used partially because of the difficulty in genetic modification. The lack of true embryonic stem cells in pigs forced researchers to utilize genetic modification in somatic cells and somatic cell nuclear transfer(SCNT) to generate genetically engineered(GE) pigs carrying site-specific modifications. Although possible, this approach is extremely inefficient and GE pigs born through this method often presented developmental defects associated with the cloning process. Advancement in the gene-editing systems such as Zinc-Finger Nucleases(ZFNs), Transcription activator-like effector nucleases(TALENs), and the Clustered regularly interspaced short palindromic repeat(CRISPR)/CRISPR-associated 9(Cas9) system have dramatically increased the efficiency of producing GE pigs. These gene-editing systems, specifically engineered endonucleases, are based on inducing double-stranded breaks(DSBs) at a specific location, and then site-specific modifications can be introduced through one of the two DNA repair pathways: non-homologous end joining(NHEJ) or homology direct repair(HDR).Random insertions or deletions(indels) can be introduced through NHEJ and specific nucleotide sequences can be introduced through HDR, if donor DNA is provided. Use of these engineered endonucleases provides a higher success in genetic modifications, multiallelic modification of the genome, and an opportunity to introduce site-specific modifications during embryogenesis, thus bypassing the need of SCNT in GE pig production. This review will provide a historical prospective of GE pig production and examples of how the gene-editing system, led by engineered endonucleases, have improved GE pig production. We wil also present some of our current progress related to the optimal use of CRISPR/Cas9 system during embryogenesis.

The effect of microgene pSVPoMcat to modify Schwann cell on GAP -43 expression after spinal cord injury in adult rats

Objective To study the effect of microgene pSVP oMcat implanted to modify schwann cell on growth associated protein -43(GAP -43)expression after spinal cord injury in adult rats.Method Hemisected of the T8segment of the sp inal cord was performed for all the experiment rats.The rats were randomly divided into three groups as follows:Group Awith microgene pSVPoMca t implanted to genetically modify SC;Group B with SC implanted ;Group C with hemisection of the spinal cord o nly.The changes of expression of GAP-43in spinal cord were observed by immunochemistry with antibodies against GAP -43.Simultaneous,the combined behavioral scores(CBS)was measured.Result There were not any different (P >0.05)among the three groups in first week a nd 12week.There were significant di ffeence(P <0.05)among three groups in 2nd,8th,and more dxpression of GAP -43at the 2nd week in gr oup A.The neurofunctional recovery was best in group A.Conclusion The microgene pSVPoMcat implanted t o modify schwann cell can promote the expression of GAP -43in spinal cord a nd func-tional recovery in adults rats after SCI.

Identification of the keratin-associated protein 13-3 (KAP13-3) gene in sheep

Keratin-associated proteins (KAPs) are a major structural component of hair and wool fibres, and play a critical role in determining the properties of the fibre. To date, forty functional high sulphur KAP genes from fourteen families have been identified in humans, but only six functional high sulphur KAP genes have been identified in sheep. This led us to search for the ovine KAP13-3 gene, a gene encoding a high sulphur KAP. In this study, the notional KAP13- 3 gene (KRTAP13-3) was amplified using primers designed based on a reported bovine KRTAP13-3 se- quence. PCR-single stranded conformational polymorphism (PCR-SSCP) analysis was used to screen amplicons derived from the gene in one hundred and forty seven New Zealand Romney crossbred sheep. Five unique banding patterns were revealed. Either one PCR-SSCP pattern (homozygous) or a combination of two patterns (heterozygous) was observed for each sheep. Sequencing of PCR amplicons representtative of different SSCP patterns revealed five different DNA sequences. The sequences derived from the amplicons showed a low homology to other known ovine KRTAPs, but had a high homology with previous reported KRTAP13-n sequences from human and cattle, with the closest homology being with bovine KRTAP13-3, suggesting the sequences represent the ovine KRTAP13-3 locus. Among the five allele sequences, four nucleotide substitutions were identified within the coding region. Of these substitutions, three were non-synonymous and would result in amino acid changes (p.Arg79Cys, p.Arg81Gln and p.Tyr130His). This variation in the KAP13-3 gene may affect gene expression, the structure and assembly of the protein, and consequently influence wool traits, if KAP13-3 is of importance to wool fibre structure.

The Sequence Variations of Intron-3 of the α-Amylase Gene in Adzuki Bean

This study describes variation of intron-3 of α-amylase gene from 156 breeds of adzuki beans using SSCP(single-strand conformation polymorphism)analysis. Based on α-amylase gene structure and sequence, A pair of PCR primers, F (CCTACATTCTAACACACCCT) and R (GCATATTGTGCCAGTACAAT) were designed to amplify intron-3 fragments of α-amylase gene. 14 variant types were detected, including 13, 9, 10, 4 variant types in the wild, weed, locally cultivated and modern brought-up adzuki beans respectively, 9, 8, 7 variant types of the wild adzuki beans from Japan, China and Korea respectively, and some other variant types in the local adzuki beans from China and Bhutan. 60% of subjects of cultivated races were found to be EE type in the experiment. In addition, sequence analysis of intron-3 of α-amylase gene from 8 variant types reveals the evolution process of various variant types in adzuki beans.

Cloning and Expression of a Chitinase Gene from Serratia marcescens Strain C8-8

A 1 692 bp long chitinase-encoding ch/A gene was cloned from the genomic DNA of Serrat/a marcescens strain C8-8 by PCR, which was speculated to en- code a 563 aa long polypeptide chain with molecular weight of about 60.9 kD. Homolog analysis showed that the chiA gene sequence cloned from C8-8 shared the highest similarity with cMA sequences from Serrat/a maresscens strains 141 （ DQ 990373.1 ） and 14041 （ DQ 493896. 1 ）, which reached 99%. Domain analysis showed that N-termlnal （23 aa） of the chiA gene cloned from C8-8 harbored typical signal peptide sequence, while C-telminal harbored the other two domains, in- eluding the PKD region （73 aa） and chitinase catalytic region （387 aa）. The PCR fragment was digested with restriction endonucleases and cloned into plasmid pET28a. The recombinant plasmid pET＇28a-ch/A was firstly transformed into Escherichia coli DI-I5 , and then transformed into expression host E. coli DH3 to express ch/A gene. The recombinant strain DH3 chiA could produce transparent hydrolysis circles on the colloidal chitin plate induced by isopropyl-l-thiogalactopyranoside （IFrG）. SDS-PAGE electrophoresis analysis showed that, a protein with relative molecular weight of about 60 kD was expressed by the recombinant strain DH3 chiA, which was consistent with the except molecular weight. After initial purification, biological activity test showed that the recombinant expression product could hydrolyze chitin, which produced transparent hydrolysis circles on the colloidal chitin plates. Results indicated that chiA gene from Serrat/a marcescens strain C8-8 had biological functions and could be utilized as a potential biological control factor.

Protective effects of proanthocyanidins on beta-amyloid peptide (25-35)-induced PC12 cell apoptosis by blocking S-phase and increasing p53 gene expression

BACKGROUND： Current studies related to the effects of proanthocyanidins on Alzheimer＇s disease have focused primarily on the signal transduction pathway of cellular apoptosis. However, the influence of p53 gene expression on cell cycle regulation, with regard to the protective mechanisms of proanthocyanidins, has not been reported. OBJECTIVE： To observe the effect of proanthocyanidins on cell cycle distribution, cellular apoptosis and p53 gene expression in β-amyloid peptide （25-35） （Aβ25-35）-induced PC12 cells cultured in serum-free media, and to investigate the molecular neuroprotective mechanisms of proanthocyanidins with regard to cell cycle regulation. DESIGN, TIME AND SETTING： A parallel, controlled, at the Institute of Biochemistry and Molecular Biology cellular, and molecular study was performed Guangdong Medical College from July 2006 to July 2008. MATERIALS： Proanthocyanidins were provided by Nanjing Xuezi Medical and Chemical Research Center, China; Aβ25-35 was provided by Sigma, USA; PC12 cells were provided by the Institute of Basic Medical Science, Academy of Military Medical Sciences; and rabbit anti-p53 polyclonal antibody was provided by Santa Cruz Biotechnology, USA. METHODS： PC12 cells were cultured in serum-free media for 24 hours. Cells from the model group were treated with 25 μmol/L Aβ25-35 for 24 hours. Cells in the drug protection group were pre-treated with 30 mg/L proanthocyanidins for 1 hour and then treated with 25 μmol/LAβ2^-35 for 24 hours. The control group was not treated. MAIN OUTCOME MEASURES： Flow cytometry was used to detect cell cycle distribution and rate of apoptosis; reverse-transcriptase polymerase chain reaction was used to detect p53 mRNA expression; and Western blot was used to detect p53 protein expression. RESULTS： After treating with 25 μmol/LAβ25-35 for 24 hours, the rate of apoptosis and the percentage of cells in S phase were significantly increased （P 〈 0.01 ）, and p53 mRNA and protein expressions were decreased. Pretreatment with proanthocyanidins for 1 hour blocked the increase in apoptosis and the percentage of cells in S phase in Aβ25-35-induced PC12 cells （P 〈 0.01 ） and increased p53 mRNA and protein expressions. CONCLUSION： Proanthocyanidins blocked apoptosis and S-phase arrest in Aβ25-35-induced PC12 cells cultured in serum-free media. The protective mechanism could be related to increased p53 mRNA and protein expressions.

Study of Swine Leukocyte Antigen Class I-3 (SLA-3) Gene for Inbreeding Wuzhishan Pig

To elucidate the structure of SLA-3 alleles on inbred line of Wuzhishan pig （WZSP） population, we examined the partial exon 1, completed exon 2, and partial exon 3 of SLA-3 loci using the reverse transcription-polymerase chain reaction （RT- PCR） and the sequencing-based method in 32 WZSPs. According to pedigree and amplification results, PCR products of 8 WZSPs were selected to clone and sequence. Nine different nucleotide sequences were obtained. After comparing the DNA and protein sequences of the WZSPs SLA-3 alleles with the published GenBank SLA sequences, it was found that the SLA-3 alleles in WZSPs were all novel, but there were very few variations among them. Comparision of SLA-3 and HLA-A protein sequences indicated that there was more sequence homology. Meanwhile, the construction of a phylogenetic tree using the nucleotide sequences of 23 SLA-3 alleles and 1 HLA-A allele represented that the WZSP population owns its unique genetics resource. In this study, the alleles of SLA-3 on WZSP group were successfully detected and analyzed, which provided the firm basis on the genotype of SLA-3 for breeding specific haplotypes WZSPs.

Antimicrobial Resistance,Virulence Profile,and Molecular Characterization of Listeria monocytogenes Isolated from Ready-to-eat Food in China,2013-2014

We aimed to investigate the potential pathogenic profile and antibiotic resistance of Listeria monocytogenes isolated from ready-to-eat food in China.Antimicrobial resistance was determined by broth microdilution following the Clinical and Laboratory Standards Institute protocol Molecular serotyping,virulence,and resistance genes were identified using PCR.Multi-locus

Cloning and Bioinformatics Analysis of Rosa rugosa β-1,3-Glucanase Gene (RrGlu)

In order to reveal which role the callose played in R. rugosa pollination incompatibility, the full-length cDNA sequence of β-1,3-glucanase gene was cloned for the first time from the stylus of Rosa rugosa “Tanghong” with RT-PCR and RACE methods and named as RrGlu. The full-length cDNA is 1380 bp with an open reading frame of 1041 bp, encoding 346 amino acids. The derived protein has a molecular weight of 37.85 kD, a calculated pI of 9.12, a pfam00332 conserved domain at position 36 - 345, and belongs to glycosyl hydrolase family 17. The derived protein is a hydrophilic protein secreted into the vacuole. There is a signal peptide cleavage site at position 34 - 35, a transmembrane domain at position 13 - 32, six Ser phosphorylation sites, three Thr phosphorylation sites, three Tyr phosphorylation sites, one N-glycosylation site, and five O-glycosylation sites. There are 31.50% α-helixes, 30.92% random coil, 25.14% extended peptide chain, and 12.43% β-corner structure. This protein and the Glu protein from eight other species, including Prunus persica, share a sequence homology of greater than 72%;all of the proteins contain a pfam00332 conserved domain and a β-1,3-glucanase active center sequence (LIVM)-X-(LIVMFYW)3-(STAG)-E-(ST)-G-W-P-(ST)-X-G. Furthermore, their phylogenetic relationships are consistent with their traditional classifications. These results were meaningful to reveal the molecular mechanism of R. rugosa pollination incompatibility and improve the theory and techniques of breeding ornamental R. rugosa.

Genetic variation of TGF-BR2 as a protective genotype for the development of colorectal cancer in men

BACKGROUND The role of transforming growth factor beta(TGF-β)signaling,including both the cytokine and their receptors,in the etiology of colorectal cancer(CRC)has been of particular interest lately.AIM To investigate the association between promoter polymorphism in TGF-β receptor 2 TGF-BR2G^([-875])A with a CRC risk in a cohort of Bulgarian patients using a casecontrol gene association study approach,as well as the protein levels of TGF-β1 in the peripheral blood.METHODS A cohort of 184 CRC patients and 307 sex and age-matched healthy subjects were recruited in the study.A genotyping of the TGF-BR2G^([-875])A(rs3087465)polymorphism was performed by primer-introduced restriction analysespolymerase chain reaction approaches.RESULTS The frequency of TGF-BR2G^([-875])A genotype was decreased in male patients with CRC than in healthy men(31.3%vs 44.8%;P=0.058).Among males,the TGF-BR2G[-509]G genotype was related to a significantly increased risk of CRC development(OR=1.820,95%CI:0.985-3.362,P=0.055)than the GA+AA genotype.Also,TGF-BR2^([-875])*A-allele itself was rarer in men with CRC than healthy men(19.1%vs 26.9%,P=0.086)and was associated with a protective effect(OR=0.644;95%CI:0.389-1.066;P=0.086).Regarding the genotypes,we found that TGF-β1 serum levels were higher in GG genotype in healthy persons above 50 years than the CRC patients[36.3 ng/mL interquartile range(IQR)19.9-56.5 vs 22.4 ng/mL IQR 14.8-29.7,P=0.014].We found significant differences between higher levels of TGF-β1 serum levels in healthy controls above 50 years(GG genotype)and CRC patients(GG genotype)at the early stage(36.3 ng/mL IQR 19.9-56.5 vs 22.8 ng/mL IQR 14.6-28.6,P=0.037)and advanced CRC(36.3 ng/mL IQR 19.9-56.5 vs 21.6 ng/mL IQR 15.9-33.9,P=0.039).CONCLUSION In summary,our results demonstrated that TGF-BR2 AG and AA genotypes were associated with a reduced risk of CRC,as well as circulating levels of TGF-βcould prevent CRC development in a gender-specific manner.Notably,male carriers of TGF-BR2-875A allele genotypes had a lower risk of CRC development and progression,suggesting that TGF-BR2-875A/G polymorphism significantly affects the protective biological factors that also impact the risk of colon and rectal carcinogenesis.

miR-148a-3p调控血管生成的体内外表型鉴定

目的明确miR-148a-3p调控血管生成的表型。方法提取miR-148a基因敲除小鼠的原代组织,结合免疫组织化学染色,探究miR-148a敲除对血管生成的影响;通过miRNA mimic转染,在人脐静脉血管内皮细胞(hUVECs)中过表达miR-148a-3p,结合Matrigel基质胶管腔形成实验、细胞迁移实验、荧光定量PCR实验等,从敲除和过表达两方面明晰miR-148a-3p对血管生成的作用。结果与同窝对照相比,miR-148a基因敲除小鼠的骨骼组织存在H型血管分布异常。离体实验得到了相似的趋势,miR-148a敲除胎鼠的跖骨具有明显更强的血管形成能力。通过miR-148a-3p mimic过表达血管内皮细胞中的miR-148a-3p表达水平,发现miR-148a-3p过表达不影响hUVECs的增殖能力,但可通过抑制其迁移能力干扰血管生成,影响伪足形成、管腔形成等过程。结论miR-148a-3p可抑制体内外血管生成,包括细胞出芽、迁移、管腔形成等生物学过程。

Association of UCP3,APN,and TNF-α Gene Polymorphisms with Type 2 Diabetes in a Population of Northern Chinese Han Patients

We observed the polymorphism distribution and coaction of uncoupling protein 3（UCP3）-55C/T,adiponectin（APN）＋45T/G and tumor necrosis factor（TNF）-α-308G/A on the onset and development of T2DM in a Northern Chinese Han population of 213[100 type 2 diabete（T2DM） patients and 113 health control subjects] by polymerase chain reaction-restriction fragment length polymorphisum（PCR-RFLP） method.Results demonstrate the polymorphism of UCP3-55C/T,APN＋45T/G,and TNF-α-308G/A related to T2DM onset and developement.And the individuals carrying UCP3-55T,APN＋45G and TNF-α-308A allele had higher T2DM risk.Those results are the first report to evaluate the association of the coaction of UCP3,APN,TNF-α genes polymorphism on T2DM risk and the susceptibility of T2DM in the Northern Chinese Han population.