Screening and bioinformatics analysis of key autophagy-related genes in alcoholic hepatitis / 器官移植

Objective To screen key autophagy-related genes in alcoholic hepatitis (AH) and investigate potential biomarkers and therapeutic targets for AH. Methods Two AH gene chips in Gene Expression Omnibus (GEO) and autophagy-related data sets obtained from MSigDB and GeneCards databases were used, and the key genes were verified and obtained by weighted gene co-expression network analysis (WGCNA). The screened key genes were subject to gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), protein-protein interaction (PPI) and immune infiltration analyses. Messenger RNA (mRNA)- microRNA (miRNA) network was constructed to analyze the expression differences of key autophagy-related genes during different stages of AH, which were further validated by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) in the liver tissues of AH patients and mice. Results Eleven autophagy-related genes were screened in AH (EEF1A2, CFTR, SOX4, TREM2, CTHRC1, HSPB8, TUBB3, PRKAA2, RNASE1, MTCL1 and HGF), all of which were up-regulated. In the liver tissues of AH patients and mice, the relative expression levels of SOX4, TREM2, HSPB8 and PRKAA2 in the AH group were higher than those in the control group. Conclusions SOX4, TREM2, HSPB8 and PRKAA2 may be potential biomarkers and therapeutic targets for AH.

Studies on the enzymatic specificity of diguanylate cyclases domains and the production of new second messengers / Estudos na especificidade enzimática de diguanilato ciclases e na produção de novos segundos mensageiros

In the first chapter, studies on substrate recognition and enzymatic activity of GGDEF domains are presented. Many proteins containing GGDEF domains are diguanylate cyclases (DGCs, EC 2.7.7.65), enzymes that catalyze the conversion of 2 GTP molecules into the second messenger c-di-GMP in prokaryotes. This molecule is primarily implicated in the transition between motile and sessile lifestyles, as well several other phenotypes. Redundancy and diversity of GGDEF domain sequences in many bacterial genomes raises the possibility that other enzymatic functions may yet be discovered. To test this hypothesis, i) the effect of point mutations on the structure and enzymatic activity of GGDEF domains is analyzed, ii) the enzymatic specificity of wild-type GGDEF domains from different proteins is also tested, and iii) when non-canonical products are detected, enzymatic models are studied to understand its preferential production. The principal results obtained from these studies are as follows. Seven mutants of the DGC PleD (a GGDEF containing-protein from Caulobacter crescentus) were constructed and the crystallographic structure of two of them was solved, showing that they are unlikely to bind the guanine moiety in its active site. Additionally, five mutants of XAC0610, another DGC from Xanthomonas citr, were constructed and their substrate specificities were evaluated. None of those mutants were able to use ATP as a substrate. Finally, seven different GGDEF domain-containing DGCs from different sources were expressed and purified and their enzymatic specificities were tested with several nucleotide triphosphates. One enzyme, GSU1658 from Geobacter sulfurreducens was particularly promiscuous and shown to produce c-di-GMP, c-di-AMP, c-di-IMP, c-di-2´dGMP, cGAMP, c-GIMP, and c-AIMP. Interestingly, XAC0610 was able to recognize 2´dGTP as substrate. Analysis of enzyme kinetics of XAC0610 in presence of 2´dGTP and/or GTP showed the preferential formation of the hybrid linear product pppGp2´dG. The second chapter present studies on cyanide metabolism in Bacillus with focus on the cyanide dihydratase of Bacillus safensis. Cyanide is widely used in industries due to its high affinity for metals. This same ability confers potent toxicity to this compound. Thus, industries must reduce the cyanide concentration from wastewater before its final disposal. Physical, chemical, and biological methods have been developed to achieve this goal, but knowledge about metabolic pathways and the biology of enzymes involved in cyanide degradation is still scarce. Here, the isolation of a Bacillus safensis strain from mine tailings in Peru is described. Classification of this strain was done through a comparative analysis of 132 core genomes of strains from the Bacillus pumilus group. Sequence analysis determined that a cyanide dihydratase (CynD, EC 3.5.5.1)) encoded in the genome of the isolated strain was likely the enzyme responsible for cyanide degradation. Confirmation of the cyanide degrading activity of CynD from this strain was achieved by cloning, expression and purification of the enzyme and its enzymatic characterization. CynD from this strain was active up to pH 9 and oligomerization patterns analyzed by SEC-MALS and electron microscopy showed that the enzyme forms large helical structures at pH 8 and smaller structures at higher pHs. Finally, we show that CynD expression is strongly induced in the presence of cyanide. The last two years of graduate studies were carried out in the context of the COVID-19 pandemic. Thanks to the large amount of publicly available genomic data, we were able to carry out studies on the worldwide dynamics of the spread of SARS-CoV-2 mutants forms. In the first year of the pandemic, genomic classification of 171,461 genomes showed the presence of five major haplotypes based on nine mutations. The worldwide distribution and the temporal evolution of frequency of these haplotypes was carefully analyzed. All the haplotypes were identified in the six regions analyzed (South America, North America, Europe, Asia, Africa, and Oceania); however, the frequency of each of them was different in each of these regions. As of September 30, 2020, haplotype 3 (or operational taxonomic unit 3, OTU_3) was the most prevalent in four regions (South America, Asia, Africa, and Oceania). OTU_5 was the most prevalent in North America and OTU_2 in Europe. Temporal dynamics of the haplotypes showed that OTU_1 became nearly extinct after 8 months of pandemic (November 2020). Other OTUs are still present in different frequencies all around the world, while currently generating new variants. Based on their temporal dynamics, a classification scheme of 115 SARS-CoV-2 mutations identified from 1,058,020 SARS-COV-2 genomes was also performed. Three types of temporal dynamics of mutations were identified: i) High-Frequency mutations are characterized by a rapid increase in frequency upon its appearance, ii) medium and iii) low-frequency mutations maintain mid or low-frequencies for several months and can be region-specific. Finally, we performed a correlation analysis of the effective reproduction number (Rt) of SARS-CoV-2 harboring the high-frequency mutation N501Y with the level of control measures adopted in specific jurisdictions. We show that Rt is negatively correlated with the level of control measures in eight of the nine countries analyzed. This negative correlation was similar when we analyzed the Rt of SARS-CoV-2 not-harboring N501Y. Thus, the control measures likely diminish the Rt of both SARSCoV-2 wild-type and N501Y

O presente trabalho está dividido em três capítulos sobre linhas de pesquisa diferentes desenvolvidas pelo autor durante o período de doutorado No primeiro capítulo, são apresentados estudos relacionados ao reconhecimento estrutural de substratos e análise enzimática de domínios GGDEF com atividade diguanilato ciclase (EC 2.7.7.65). As proteínas contendo domínios GGDEF estão relacionados à produção enzimática do segundo mensageiro c-di-GMP, a partir de duas moléculas de GTP, em procariotos. Esta molécula está principalmente envolvida na transição entre os estilos de vida móveis e sésseis, bem como vários outros fenótipos. Redundância e diversidade de sequências de domínio GGDEF aumentam a possibilidade de que outras funções enzimáticas ainda possam ser descobertas. Para testar esta hipótese, i) o efeito de mutações pontuais na estrutura e atividade enzimática dos domínios GGDEF é analisado, ii) a especificidade enzimática de domínios GGDEF de enzimas diferentes também é testada e iii) quando produtos não canônicos são detectados, modelos enzimáticos são estudados para entender sua produção preferencial. Como resultados mais importantes, sete mutantes do PleD (uma proteína contendo GGDEF) foram construídos e a estrutura cristalográfica de dois delas foi resolvida, mostrando que é improvável que eles liguem à porção guanina em seu sítio ativo. Além disso, cinco mutantes da proteína XAC0610 de Xanthomonas citri foram construídos e sua capacidade de usar ATP ou GTP como substrato foi avaliada. Nenhum desses mutantes foi capaz de usar ATP como substrato. Finalmente, sete outras proteínas contendo GGDEF foram purificadas e sua especificidade enzimática foi avaliada com vários trifosfatos de nucleotídeos. Uma enzima promíscua chamada GSU1658 mostrou produzir c-di-GMP, c-di-AMP, c-di-IMP, c-di-2´dGMP, c-GAMP, cGIMP e c-AIMP. Curiosamente, o XAC0610 foi capaz de reconhecer 2´dGTP como substrato. A análise da cinética enzimática de XAC0610 na presença de 2´dGTP e GTP mostrou a formação preferencial do produto linear híbrido pppGp2´dG. O segundo capítulo aborda estudos sobre o metabolismo do cianeto em Bacillus com foco na cianeto dihidratase de Bacillus safensis. O cianeto é amplamente utilizado nas indústrias devido à sua alta afinidade com os metais. Esta mesma capacidade confere toxicidade potente a este composto. Assim, as indústrias têm que reduzir a concentração de cianeto das águas residuais antes de sua disposição final. Métodos físicos, químicos e biológicos têm sido desenvolvidos para atingir esse objetivo, mas o conhecimento sobre as vias metabólicas e a biologia das enzimas envolvidas na degradação do cianeto ainda é escasso. Aqui, é descrito o isolamento de uma cepa de Bacillus safensis de rejeitos de minas no Peru. A classificação desta cepa foi feita através de uma análise comparativa de 132 core genomes de cepas do grupo de Bacillus pumilus. Em seguida, determinamos que uma cianeto dihidratase (CynD, EC 3.5.5.1) codificada no genoma da cepa isolada era provavelmente a enzima responsável pela degradação do cianeto. A confirmação da atividade degradante de cianeto de CynD desta cepa foi feita por clonagem, expressão e purificação da enzima e realização de caracterização enzimática. O CynD desta cepa é ativo até pH 9 e os padrões de oligomerização analisados por SEC-MALS mostraram que a enzima forma longas estruturas helicoidais em pH 8 e estruturas menores enquanto o pH aumenta. Finalmente, foi demonstrado que a expressão de CynD é fortemente induzida na presença de cianeto. Os últimos dois anos do doutorado foram realizados no contexto da pandemia COVID- 19. Vários laboratórios se dedicaram a gerar conhecimento para ajudar no combate à pandemia. Nesta situação e graças à grande quantidade de dados genômicos disponíveis publicamente, estudos sobre a dinâmica das mutações do SARS-CoV-2 foram realizados. No primeiro ano da pandemia, a classificação genômica de 171.461 genomas mostrou a presença de cinco haplótipos principais com base em nove mutações. A distribuição mundial e a mudança de frequência desses haplótipos foram analisadas cuidadosamente. Todos os haplótipos foram identificados nas seis regiões analisadas (América do Sul, América do Norte, Europa, Ásia, África e Oceania); no entanto, a frequência de cada um deles foi diferente em cada uma dessas regiões. Em 30 de setembro de 2020, o haplótipo 3 (ou unidade taxonômica operacional 3, OTU_3) era o mais prevalente em quatro regiões (América do Sul, Ásia, África e Oceania). OTU_5 foi o mais prevalente na América do Norte e OTU_2 na Europa. A dinâmica temporal dos haplótipos mostrou que OTU_1 parece perto da extinção após 8 meses de pandemia (novembro de 2020). Outros OTUs ainda estão presentes em diferentes frequências em todo o mundo, mesmo atualmente gerando novas variantes. Com base em sua dinâmica temporal, um esquema de classificação de 115 mutações SARS-CoV-2 identificadas a partir de 1.058.020 genomas SARS-COV-2 também foi feito. Três tipos de dinâmica temporal de mutações foram identificados: i) Mutações de alta frequência, ii) mutações de média frequência e iii) mutações de baixa frequência. Finalmente, foi analisada a correlação do número de reprodução efetiva (Rt) do SARS-CoV-2 que contém a mutação de alta frequência N501Y com o nível de medidas de controle, mostrando que seu Rt está negativamente correlacionado com o nível de medidas de controle em oito dos nove países analisados. Esta correlação negativa foi semelhante quando foi analisado o Rt de SARS-CoV-2 sem a mutação N501Y. Assim, as medidas de controle provavelmente diminuirão o Rt de SARS-CoV-2 tipo selvagem e N501Y

Signatures of positive selection on the hepatic lipase gene in human populations

The hepatic lipase plays a central role in the lipid metabolism, catalyzing the hydrolysis of phospholipids, monoglycerides, diglycerides, and triglycerides, and acyl-CoA. It is also implied in the conversion of very low-density lipoprotein and intermediate density lipoprotein to low density lipoproteins. As a consequence, the gene encoding the hepatic lipase (LIPC) is associated with several diseases derived from the imbalance of lipids that are in general derived from the interaction between life styles and genetic architecture. Therefore, it is interesting to understand more about the characteristics of the microevolutionary processes affecting genes that, like LIPC, have a role in nutrition and lipid metabolism in human populations. We explored the selection signatures on LIPC in 26 populations, detecting three regions under recent positive selection

Genomic organization of Polycomb Response Elements and its functional implication in Drosophila and other insects

The epigenetic memory is an essential aspect of multicellular organisms to maintain several cell types and their geneexpression pattern. This complex process uses a number of protein factors and specific DNA elements within thedevelopmental cues to achieve this. The protein factors involved in the process are the Polycomb group (PcG)members, and, accordingly, the DNA sequences that interact with these proteins are called Polycomb ResponseElements (PREs). Since the PcG proteins are highly conserved among higher eukaryotes, including insects, andfunction at thousands of sites in the genomes, it is expected that PREs may also be present across the genome. However,the studies on PREs in insect species, other thanDrosophila, is currently lacking.We took a bioinformatics approach todevelop an inclusive PRE prediction tool, ‘PRE Mapper’, to address this need. By applying this tool on the Drosophilamelanogaster genome, we predicted[20,000 PREs. When compared with the available PRE prediction methods, thistool shows far better performance by correctly identifying the in vivo binding sites of PcG proteins, identified bygenome-scale ChIP experiments. Further analysis of the predicted PREs shows their cohabitation with chromatindomain boundary elements at several places in the Drosophila genome, possibly defining a composite epigeneticmodule. We analysed 10 insect genomes in this context and find several conserved features in PREs across the insectspecies with some variations in their occurrence frequency. These analyses leading to the identification of PRE in insectgenomes contribute to our understanding of epigenetic mechanisms in these organisms.

Bioinformatic Analysis of Related Genes and Therapeutic Drugs of Asthma / 中国实验方剂学杂志

Objective::Bioinformatic analysis was used to compare the gene expression profile between asthma patients and healthy people, and the gene characteristics of asthma were preliminarily identified and the potential mechanism and drugs were revealed. Method::The GSE74986 gene expression profile was downloaded from the gene expression omnibus (GEO) and the differentially expressed genes (DEGs) were analyzed by GEO2R. Then the gene heat map of DEGs was made by Morpheus, and their gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) analysis were performed by DAVID 6.8. Moreover, the protein-protein interaction (PPI) network and hub genes were constructed by String 10.5. Finally, the significant modules were analyzed by MCODE in Cytoscape 3.6.1, small molecule drugs related to asthma were screened through Coremine Medical. Result::A total of 510 DEGs were screened, including 29 up-regulated genes and 481 down-regulated genes. DEGs were mainly involved in these biological processes and pathways, including chromatin silencing, transcriptional regulation of RNA polymerase Ⅱ promoter, protein transport, messenger RNA (mRNA) processing, RNA splicing, ubiquitin-mediated proteolysis, protein processing in the endoplasmic reticulum, RNA transport, and myeloid differentiation factor (MyD)-dependent Toll-like receptor signaling pathway, platelet activation, nucleotide binding oligomerization domain (NOD)-like receptor signaling pathway and so on. A total of 9 hub genes were obtained, including T-complex protein 1 subunit theta (CCT8), T-complex protein 1 subunit alpha (TCP1), 26S protease regulatory subunit S10B (PSMC6), heat shock protein 90 alpha (HSP90A)A1, cell cycle protein C (CCNC), HSP90AB1, 26S proteasome non-ATPase regulatory subunit 6 (PSMD6), ubiquitin-specific protease 14 (USP14) and eukaryotic translation initiation factor 4E (EIF4E). Two important modules were obtained. The genes in two modules mainly involved these biological process, such as splice, ubiquitin-mediated proteolysis, protein modification, RNA modification and so on. Some potential molecular drugs for the treatment of asthma, such as anisomycin and genistein, have been developed. Conclusion::DEGs and hub genes can contribute to understanding the molecular mechanism of asthma and providing potential therapeutic targets and drugs for the diagnosis and treatment of asthma.

Comparison of Treponema pallidum genomes for the prediction of resistance genes

Syphilis is a sexually transmitted infection caused by Treponema pallidum, which is highly prevalent in several countries,including Brazil. The use of bioinformatics’ tools for the identification of resistance genes is an important practice for thestudy of microorganisms, such as T. pallidum. In this study, the complete genomes of 43 strains of T. pallidum, isolatedfrom different countries, were analyzed. A total of 41,514 sequences were obtained, and compared against prokaryoteresistance gene databases using BLASTn, BLASTx and RGI for gene alignment and prediction. From the alignments, itwas possible to identify antibiotic resistance genes for each strain. The genes identified in each comparison were groupedaccording to the antibiotic category in which they show resistance to. The antibiotic-resistant genes related to drugs used totreat syphilis were grouped separately. The in silico tools used have shown to be effective in identifying resistance genes ingenomes of T. pallidum strains. Due to the lack of research and accurate information regarding the antibiotic resistancegenes in T. pallidum, this study serves as a basis for studies in molecular biology whose aim is the identification of thesegenes, besides being a reference to help in the control and treatment of this infection.

SeqSQC: A Bioconductor Package for Evaluating the Sample Quality of Next-generation Sequencing Data / 基因组蛋白质组与生物信息学报·英文版

As next-generation sequencing (NGS) technology has become widely used to identify genetic causal variants for various diseases and traits, a number of packages for checking NGS data quality have sprung up in public domains. In addition to the quality of sequencing data, sample quality issues, such as gender mismatch, abnormal inbreeding coefficient, cryptic relatedness, and population outliers, can also have fundamental impact on downstream analysis. However, there is a lack of tools specialized in identifying problematic samples from NGS data, often due to the limitation of sample size and variant counts. We developed SeqSQC, a Bioconductor package, to automate and accelerate sample cleaning in NGS data of any scale. SeqSQC is designed for efficient data storage and access, and equipped with interactive plots for intuitive data visualization to expedite the identification of problematic samples. SeqSQC is available at http://bioconductor.org/packages/SeqSQC.

Toxicology of Asari Radix et Rhizoma Based on Network Analysis / 中国实验方剂学杂志

Objective:To analyze the known mechanism of toxicology and predict the unknown toxicity in Asari Radix et Rhizoma sinensis by establishing the network relationship of compound, protein, gene and toxicant reaction. Method:After comparing the Asari Radix et Rhizoma candidate compounds obtained from the traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP) database and the toxicological information obtained from the Comparative Toxicogenomics Database(CTD) database, we screened out 13 toxic components from Asari Radix et Rhizoma. And use the Pharm Mapper Server website to find the detailed information of target proteins of the 13 components. The network structure of these 13 chemical components and their corresponding target proteins were drawn by using Cytospace software, and several target proteins with the highest degree of association were found. ClueGO+CluePedia plug-in of Cytospace software was applied in gene ontology(GO) enrichment analysis of genes and kyoto encyclopedia of genes and genomes(KEGG) pathway enrichment analysis, so as to determine the pathways through which toxic substances in Asari Radix et Rhizoma might be harmful to human body. Result:The toxic substances in Asari Radix et Rhizoma may induce tumor and cancer formation through p53 signaling pathway, interleukin(IL)-17 signaling pathway, nuclear factor(NF)-kappa B signaling pathway, tumor necrosis factor(TNF)-signaling pathway. Asari Radix et Rhizoma could inhibit the central nervous system by regulating apoptosis pathways and neurons, and may also cause other autoimmune diseases by IL-17, TNF-α pathway and apoptosis regulation. Conclusion:This study preliminarily explores related mechanisms of toxicity of Asari Radix et Rhizoma,this method can be used to predict toxicity and explain toxicity mechanism of traditional Chinese medicine.

Identification of prognosis-related genes in hepatocellular carcinoma based on bioinformatical analysis / 中国肿瘤生物治疗杂志

@# Objective: To identify the differentially expressed genes (DEGs) between hepatocellular carcinoma (HCC) tissues and normal liver tissues by bioinformatic methods, and to explore the intrinsic mechanism of these candidate genes involving in the occurrence and development of HCC from transcriptome level as well as the clinical significance of their associations with the prognosis of HCC patients. Methods: Gene expression profiles of GSE45267, GSE64041, GSE84402 and TCGA were downloaded from GEO (Gene Expression Omnibus) and TCGA(The Cancer GenomeAtlas), respectively. R software and Bioconductor packages were used to identify the DEGs between HCC tissues and para-cancer tissues, and then Gene Ontology (GO) Enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, Protein-Protein Interaction (PPI) network analysis and survival analysis were performed. Results: Forty-six up-regulated genes and 154 down-regulated genes were screened out,and GO enrichment analysis showed that these DEGs were mainly related to cell division, proliferation, cycle regulation, oxidation-reduction process and certain metabolic pathways. KEGG pathway analysis revealed that DEGs were mainly involved in tryptophan metabolism, retinol metabolism and other metabolic pathways as well as p53 pathway. Over-expression of a panel of up-regulated genes (CCNA2, CDK1, DLGAP5, KIF20A, KPNA2 and MELK) was shown to be significantly negatively correlated with the prognosis of HCC patients in the TCGA dataset (all P<0.01). Conclusion: A set of up-regulated hub genes that are negatively correlated with prognosis will provide potential guiding value for the clinical research on the diagnosis and treatment of HCC.

Draft genomes and reference transcriptomes extend the coding potential of the fish pathogen Piscirickettsia salmonis

Background: Draft and complete genome sequences from bacteria are key tools to understand genetic determinants involved in pathogenesis in several disease models. Piscirickettsia salmonis is a Gram-negative bacterium responsible for the Salmon Rickettsial Syndrome (SRS), a bacterial disease that threatens the sustainability of the Chilean salmon industry. In previous reports, complete and draft genome sequences have been generated and annotated. However, the lack of transcriptome data underestimates the genetic potential, does not provide information about transcriptional units and contributes to disseminate annotation errors. Results: Here we present the draft genome and transcriptome sequences of four P. salmonis strains. We have identified the transcriptional architecture of previously characterized virulence factors and trait-specific genes associated to cation uptake, metal efflux, antibiotic resistance, secretion systems and other virulence factors. Conclusions: This data has provided a refined genome annotation and also new insights on the transcriptional structures and coding potential of this fish pathogen.

Whole genome sequence of Mycobacterium kansasii isolates of the genotype 1 from Brazilian patients with pulmonary disease demonstrates considerable heterogeneity

Mycobacterium kansasii is an opportunistic pathogen and one of the most commonly encountered species in individuals with lung disease. We here report the complete genome sequence of 12 clinical isolates of M. kansasii from patients with pulmonary disease in Brazil.

Approaches for in silico finishing of microbial genome sequences

Abstract The introduction of next-generation sequencing (NGS) had a significant effect on the availability of genomic information, leading to an increase in the number of sequenced genomes from a large spectrum of organisms. Unfortunately, due to the limitations implied by the short-read sequencing platforms, most of these newly sequenced genomes remained as "drafts", incomplete representations of the whole genetic content. The previous genome sequencing studies indicated that finishing a genome sequenced by NGS, even bacteria, may require additional sequencing to fill the gaps, making the entire process very expensive. As such, several in silico approaches have been developed to optimize the genome assemblies and facilitate the finishing process. The present review aims to explore some free (open source, in many cases) tools that are available to facilitate genome finishing.

Evaluation of MC1R high-throughput nucleotide sequencing data generated by the 1000 Genomes Project

Abstract The advent of next-generation sequencing allows simultaneous processing of several genomic regions/individuals, increasing the availability and accuracy of whole-genome data. However, these new approaches may present some errors and bias due to alignment, genotype calling, and imputation methods. Despite these flaws, data obtained by next-generation sequencing can be valuable for population and evolutionary studies of specific genes, such as genes related to how pigmentation evolved among populations, one of the main topics in human evolutionary biology. Melanocortin-1 receptor (MC1R) is one of the most studied genes involved in pigmentation variation. As MC1R has already been suggested to affect melanogenesis and increase risk of developing melanoma, it constitutes one of the best models to understand how natural selection acts on pigmentation. Here we employed a locally developed pipeline to obtain genotype and haplotype data for MC1R from the raw sequencing data provided by the 1000 Genomes FTP site. We also compared such genotype data to Phase 3 VCF to evaluate its quality and discover any polymorphic sites that may have been overlooked. In conclusion, either the VCF file or one of the presently described pipelines could be used to obtain reliable and accurate genotype calling from the 1000 Genomes Phase 3 data.

Advances in CRISPR-Cas-mediated genome editing system in plants / 生物工程学报

Targeted genome editing technology is an important tool to study the function of genes and to modify organisms at the genetic level. Recently, CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins) system has emerged as an efficient tool for specific genome editing in animals and plants. CRISPR-Cas system uses CRISPR-associated endonuclease and a guide RNA to generate double-strand breaks at the target DNA site, subsequently leading to genetic modifications. CRISPR-Cas system has received widespread attention for manipulating the genomes with simple, easy and high specificity. This review summarizes recent advances of diverse applications of the CRISPR-Cas toolkit in plant research and crop breeding, including expanding the range of genome editing, precise editing of a target base, and efficient DNA-free genome editing technology. This review also discusses the potential challenges and application prospect in the future, and provides a useful reference for researchers who are interested in this field.

Analysis on structure and phylogeny of chloroplast genomes in Araliaceae species / 中草药

Objective: To obtain the structural characteristics and phylogentic relationships of the chloroplast genome in Araliaceae species. Methods: we used 20 chloroplast genomes which have sequenced as materials, they were from 20 species belonging to 10 genus of Araliaceae. Analysis the differences of genomes and the dilation or shrink of four boundaries for IR, we used MEGA 4.0 to build the phylogenetic tree with Angelica gigas of sibling species as the outgroup and analysis their phylogentic relationships. Results: There was a small difference among the chloroplast genomes size, and the largest difference is 1 909 bp. All of species had existed gene replacements, cemA replaced ycf10, and number of genes existed some differences, they were mainly caused by tRNA. The four boundaries of IR was relatively conservative, only Panax vietnamensi, Panax notoginseng and Schefflera delavayi were special, their boundary genes were in IR. All nodes of the phylogenetic tree of Araliaceae which was based on Angelica gigas were of high supports, and the tree had good resolution to reflect the genetic relationships among Araliaceae. Conclusion: Chloroplast genomes have a lot of information, it can be used to analysis phylogeny among the species which are affinity or faster evolution.

Differential expression of circular RNA in patients with chronic HBV infection of different stages and related bioinformatic analysis / 中华临床感染病杂志

Objective To investigate the differential expression of circular RNA ( circRNA ) in patients with chronic HBV infection of different stages.Methods Seven patients with chronic HBV infection admitted in Taizhou People's Hospital from October 2014 to October 2015 were enrolled, including 4 with chronic hepatitis B ( CHB ) and 3 chronic HBV carriers;3 healthy subjects served as controls. Peripheral blood mononuclear cells (PBMCs) were separated,and the expression of circRNA molecules in PBMCs were detected by new generation of circRNA microarray and validated by fluorescent quantitative PCR.The interaction sites between circRNA and miRNA were predicted with Arraystar miRNA target prediction software.Target genes regulated by the circRNA related to miRNA were analyzed by Gene oncology (Go) and Kyoto encyclopedia of genes and genomes (KEGG) analysis.SPSS 17.0 software was used for statistical analysis.Results Compared with the healthy controls , 137 circRNA molecules of differential expression were found in patients with chronic hepatitis B , of which 89 were up-regulated and 48 were down-regulated; while 444 circRNA molecules of differential expression , of which 130 were up-regulated (>5 fold in 34 ) and 314 down-regulated , were found in chronic HBV carriers.Compared with chronic HBV carriers , 1041 circRNA molecules of differential expression were found in CHB patients , including 663 up-regulated and 378 down-regulated (>5 fold in 54).There were many miRNA responsive elements which complementary with seed regions on miRNA in different circRNA molecules.Target gene analysis demonstrated that 533 target genes regulated by hsa_circ_0038646 were related to miRNAs , 249 target genes found in hsa_circ_0087354 were related to microRNAs.GO analysis showed that function of target genes regulated by hsa_circ_0038646 related to miRNA mainly enriched in activin binding.Function of target genes regulated by hsa_circ_0087354 related to miRNA mainly enriched in armadillo repeat domain binding.KEGG analysis showed that hsa_circ_0038646 molecules related to miRNA mainly involved in T cell receptor , estrogen receptor signaling pathway and so on.Hsa_circ_0087354 molecules related to miRNA mainly involved in adherens junction , MAPK signaling pathway and so on. Conclusion There are differential expressions of circRNA in patients at different clinical stages of chronic HBV infection , which might be involved in immune regulation of chronic HBV infection through the regulation of multiple target genes and signaling pathways.

Effects of Single Nucleotide Polymorphism Marker Density on Haplotype Block Partition

Many researchers have found that one of the most important characteristics of the structure of linkage disequilibrium is that the human genome can be divided into non-overlapping block partitions in which only a small number of haplotypes are observed. The location and distribution of haplotype blocks can be seen as a population property influenced by population genetic events such as selection, mutation, recombination and population structure. In this study, we investigate the effects of the density of markers relative to the full set of all polymorphisms in the region on the results of haplotype partitioning for five popular haplotype block partition methods: three methods in Haploview (confidence interval, four gamete test, and solid spine), MIG++ implemented in PLINK 1.9 and S-MIG++. We used several experimental datasets obtained by sampling subsets of single nucleotide polymorphism (SNP) markers of chromosome 22 region in the 1000 Genomes Project data and also the HapMap phase 3 data to compare the results of haplotype block partitions by five methods. With decreasing sampling ratio down to 20% of the original SNP markers, the total number of haplotype blocks decreases and the length of haplotype blocks increases for all algorithms. When we examined the marker-independence of the haplotype block locations constructed from the datasets of different density, the results using below 50% of the entire SNP markers were very different from the results using the entire SNP markers. We conclude that the haplotype block construction results should be used and interpreted carefully depending on the selection of markers and the purpose of the study.

What stories can the Frankia genomes start to tell us.

Among the Actinobacteria, the genus Frankia is well known for its facultative lifestyle as a plant symbiont of dicotyledonous plants and as a free-living soil dweller. Frankia sp. strains are generally classified into one of four major phylogenetic groups that have distinctive plant host ranges. Our understanding of these bacteria has been greatly facilitated by the availability of the first three complete genome sequences, which suggested a correlation between genome size and plant host range. Since that first report, eight more Frankia genomes have been sequenced. Representatives from all four lineages have been sequenced to provide vital baseline information for genomic approaches toward understanding these novel bacteria. An overview of the Frankia genomes will be presented to stimulate discussion on the potential of these organisms and a greater understanding of their physiology and evolution.

Effect of salt stress on the physiology of Frankia sp strain CcI6.

Actinorhizal plants are able to overcome saline soils and reclaim land. Frankia sp strain CcI6 was isolated from nodules of Casuarina cunninghamiana found in Egypt. Phylogenetic analysis of Frankia sp. strain CcI6 revealed that the strain is closely related to Frankia sp. strain CcI3. The strain displays an elevated level of NaCl tolerance. Vesicle production and nitrogenase activity were also influenced by NaCl.

Análise da expressão de RNAs não codificadores longos em adenocarcinoma de pâncreas / Expression analysis of long noncoding RNAs in pancreatic adecarcinoma

RNAs não codificadores longos (lncRNAs) compõem uma fração significativa do transcriptoma. Alterações na expressão de lncRNAs já foram observadas em vários cânceres humanos, mas ainda não foram exploradas no adenocarcinoma pancreático ductal (PDAC), uma doença devastadora e agressiva para a qual faltam métodos para diagnóstico precoce e tratamentos efetivos. Utilizando uma plataforma de microarranjo de cDNA com sondas para 984 lncRNAs e 2371 mRNAs, o presente estudo identificou conjuntos de lncRNAs expressos em 38 amostras clínicas pancreáticas. O enriquecimento de (i) elementos regulatórios associados às regiões promotoras (H3K4me3); (ii) possíveis inícios de transcrição (CAGE-tags); (iii) presença de elementos conservados sugere que ao menos uma fração desses RNAs seja originada a partir de unidades transcricionais independentes, reguladas e possivelmente funcionais. Foram identificadas assinaturas de expressão gênica compostas por mRNA e lncRNAs associadas ao tumor primário e à metástase pancreática. A assinatura gIenica associada à metástase apresentou enriquecimento RNAs intrônicos de loci gênicos associados à via MAPK quinase. O aumento de expressão dos transcritos intrônicos dos loci PPP3CB, MAP3K14 e DAPK1 foi confirmado por qPCR em metástases. Em conjunto, este trabalho aponta para a importância de lncRNAs intrônicos no PDAC e para a necessidade de estudos mais aprofundados para uma melhor compreensão do papel dessa classe de transcritos na biologia da doença

Long noncoding RNAs (lncRNAs) compose a significant fraction of transcriptome. Altered expression of lncRNAs has been observed in diverse human cancers, but has not being investigated in pancreatic ductal adenocarcinoma (PDAC), a devastating and aggressive disease that lack early diagnosis methods and effective treatments. Using a cDNA microarray platform with probes interrogating 984 lncRNAs and 2371 mRNA, the present study identified subsets of lncRNAs expressed in 38 pancreatic clinical samples. Enrichment of (i) regulatory elements associated to promoter region (H3K4me3); (ii) putative transcription start site (CAGEtags) and (iii) conserved elements, suggest that at least a fraction of these RNAs could be independent transcriptional unit, regulated, an possibly functional. Gene expression signatures comprised of mRNAs and lncRNAs and associated to primary or metastatic tumors were found. A gene signature associated to metastasis was enriched in intronic ncRNAs mapping to gene loci associated to the MAPK pathway. Over expression of intronic RNAs from PPP3CB, MAP3K14 and DAPK1 was confirmed by qPCR in metastatic samples. Taken together, this study points to the importance of intronic lncRNAs in PDAC and for the need to study this class of ncRNAs in greater detail to better understand its role in the biology of PDAC