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The present study was conducted to confirm the presence of Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae associated with a nosocomial outbreak in a Chinese pediatric hospital. From July 2009 to January 2011, 124 nonduplicated K. pneumoniae isolates were collected from specimens from patients of pediatric units in the hospital. Twelve of the 124 isolates possessed the bla(KPC-2) gene and showed 7 different pulsed-field gel electrophoresis (PFGE) patterns. Meanwhile, 16S rRNA methylase, acc(6')-Ib-cr, and several types of ß-lactamases were also produced by the majority of the KPC-producing isolates. Class 1 integron-encoded intI1 integrase gene was subsequently found in all strains, and amplification, sequencing, and comparison of DNA between 5' conserved segment and 3' conserved segment region showed the presence of several known antibiotic resistance gene cassettes of various sizes. The conjugation and plasmid-curing experiments indicated some KPC-2-encoding genes were transmissible. In addition, conjugal cotransfer of multidrug-resistant phenotypes with KPC-positive phenotypes was observed in KPC-producing strains. Restriction endonuclease analysis and DNA hybridization with a KPC-specific probe showed that the bla(KPC-2) gene was carried by plasmid DNA from K. pneumoniae of PFGE pattern B. The overall results indicate that the emergence and outbreak of KPC-producing K. pneumoniae in our pediatric wards occurred in conjunction with plasmids coharboring 16S rRNA methylase and extended-spectrum ß-lactamases.
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Infecção Hospitalar/microbiologia , Genes Bacterianos/genética , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Carbapenêmicos/farmacologia , Criança , Pré-Escolar , China , Farmacorresistência Bacteriana/genética , Eletroforese em Gel de Campo Pulsado , Feminino , Transferência Genética Horizontal , Genótipo , Hospitais Pediátricos , Humanos , Lactente , Klebsiella pneumoniae/classificação , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/isolamento & purificação , Masculino , Metiltransferases/genética , Testes de Sensibilidade Microbiana , Plasmídeos/genética , beta-Lactamases/genéticaRESUMO
In recent years,with the continuous progress of DNA extraction and detection technology,cell-free DNA(cfDNA)has been widely used in the life science field,and its potential application value in forensic identification is becoming more and more obvious.This paper reviews the concept,formation mechanism,and classification of cfDNA,etc.,and describes the latest research progress of cfDNA in personal identification of crime scene touch DNA samples and non-invasive prenatal pater-nity testing(NIPPT).Meanwhile,this paper summarizes the potential application of cfDNA in injury inference,and discusses the advantages and disadvantages of common cfDNA analysis methods and techniques,and its application prospects,to provide a new idea for the wide application of cfDNA in the field of forensic science.
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Objective To explore the context and hotspot changes of forensic mixed stain research through bibliometric approach.Methods The literature of forensic mixed stain included in the core col-lection of Web of Science database from 2011 to 2022 were collected as the study object,and the an-nual publication number,countrie(region),institution,journal,keywords,etc.were bibliometrically and visually analyzed using the R-based Bibliometrix 1.1.6 package and VOSviewer 1.6.18 software.Re-sults A total of 732 articles on forensic mixed stain were included from 2011 to 2022,with the an-nual number of articles published and the annual citation frequency showing a steady increase year by year.Among the 59 countries(regions)with the most published articles,the United States ranked first with 246 articles,followed by China with 153 articles.The literature came from 104 journals,and the total number of articles published in the top 10 journals was 633.FORENSIC SCI INT GENET ranked first with 307 articles.Visual analysis using VOSviewer software showed that keywords could be divided into four research clusters,namely the genetic marker development group(blue),the mixed stain typing analysis theory group(red),the sequencing analysis group(yellow),and the case sample research group(green).It can be divided into four development stages in terms of different time peri-ods:early development(2011-2013),middle development(2014-2016),rapid development(2017-2020)and latest development(2021-2022).Conclusion The number of publications by domestic and foreign scholars in the study of mixed stain in forensic science is showing a relatively stable trend.Machine learning,next generation sequencing and other research have been the hottest topics that have attracted the most attention in recent years,which is expected to further develop the theory of mixed stain typing and sequencing analysis in forensic mixed stain research.
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Talent is one of the basic and strategic supports for building a modern socialist country in all aspects. Since the 1980s, the establishment of forensic medicine major and the cultivation of innovative talents in forensic medicine have become hot topics in higher education in forensic medicine. Over the past 43 years, the forensic medicine team of Shanxi Medical University has adhered to the joint education of public security and colleges, and made collaborative innovation, forming a training mode of "One Combination, Two Highlights, Three Combinations, Four in One" for innovative talents in forensic medicine. It has carried out "5+3/X" integrated reform, and formed a relatively complete talent training innovation mode and management system in teaching, scientific research, identification, major, discipline, team, platform and cultural construction. It has made a historic contribution to China's higher forensic education, accumulated valuable experience for the construction of first-class major and first-class discipline of forensic medicine, and provided strong support for the construction of the national new forensic talent training system. The popularization of this training mode is conducive to the rapid and sustainable development of forensic science, and provides more excellent forensic talents for national building, regional social development and the discipline construction of forensic science.
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Humanos , Medicina Legal/educação , AptidãoRESUMO
Objective To study the heteroplasmy of the whole mitochondrial genome genotyping result of hair shaft samples using HID Ion GeneStudioTM S5 Sequencing System. Methods The buccal swabs and blood of 8 unrelated individuals, and hair shaft samples from different parts of the same individual were collected. Amplification of whole mitochondrial genome was performed using Precision ID mtDNA Whole Genome Panel. Analysis and detection of whole mitochondrial genome were carried out using the HID Ion GeneStudioTM S5 Sequencing System. Results The mitochondrial DNA sequences in temporal hair shaft samples from 2 individuals showed heteroplasmy, while whole mitochondrial genome genotyping results of buccal swabs, blood, and hair samples from the other 6 unrelated individuals were consistent. A total of 119 base variations were observed from the 8 unrelated individuals. The numbers of variable sites of the individuals were 29, 40, 38, 35, 13, 36, 40 and 35, respectively. Conclusion Sequence polymorphism can be fully understood using HID Ion GeneStudioTM S5 Sequencing system.
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Humanos , DNA Mitocondrial/genética , Genoma Mitocondrial/genética , Heteroplasmia , Sequenciamento de Nucleotídeos em Larga Escala , Análise de Sequência de DNARESUMO
Objective To preliminarily discuss the feasibility of geolocation inference of forensic individual origin by soil metagenomic analysis. Methods The 33 soil samples from Heilongjiang, Qinghai and Tibet were collected, total bacterial DNA in the samples were extracted, and universal primers were used to amplify the V3 and V4 hypervariable region of bacterial 16S rDNA. The region was sequenced by high-throughput sequencing (HTS) with the MiSeq sequencer. Bioinformatics analysis such as species composition and sample comparison was performed on sequencing data. The richness index and diversity index were calculated based on operational taxonomic unit (OTU) results. Results A total of 2 720 149 sequences were generated by sequencing. Those sequences were clustered into 114 848 OTUs. The Chao1 indexes of soil microorganisms in Heilongjiang, Qinghai, and Tibet were 797.45, 745.11 and 535.98, respectively, and Shannon indexes were 6.46, 6.36 and 6.25, respectively. The number of bacterial species and the community diversity in the soil from high to low were Heilongjiang > Qinghai > Tibet. The composition of soil bacteria in three provinces at various classification levels were obtained, the dominant genuses in Heilongjiang were Chthoniobacteraceae DA101 and an unannotated genus of Thermogemmatisporaceae; the dominant genuses in Qinghai were an unannotated genus of Cytophagaceae and an unannotated genus of Nocardioidaceae; the dominant genuses in Tibet were an unannotated genus of Comamonadaceae and Verrucomicrobiaceae Luteolibacter. The results of principal co-ordinates analysis demonstrated that, according to the weighted UniFrac analysis, the three principle components represented 56.36% of the total variable, and according to the unweighted UniFrac analysis, the three principle components represented 34.81% of the total variable. The samples from the same province could be clustered together, and the species and content of soil microorganisms from different provinces were significantly different. Conclusion Based on the metagenomic analysis method, soil samples from different regions can be effectively distinguished, which has potential application value in geolocation inference of forensic individual origin in the future.
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Bactérias/genética , Sequenciamento de Nucleotídeos em Larga Escala , RNA Ribossômico 16S/genética , Solo , Microbiologia do SoloRESUMO
Single-cell sequencing is a technique that analyzes DNA and RNA sequences on the cellular level with next generation sequencing. The ultra high resolution of single-cell sequencing provides new perspectives and opens new frontiers for our understanding of many areas of life sciences, including forensic genome. This paper summarizes the recent advancements in single-cell sequencing and the prospect of its forensic application.
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Humanos , DNA , Genética Forense , Sequenciamento de Nucleotídeos em Larga Escala , Análise de Sequência de DNA/métodosRESUMO
A study was designed to characterize three carbapenemase-producing Klebsiella pneumoniae isolated from pediatric patients in China. Molecular characterization was done using polymerase chain reaction and sequencing for blaVIM, blaNDM, blaIMP, blaKPC, blaCTX-Ms, blaOXAs, blaTEMs, and blaSHV; plasmid-mediated quinolone resistance determinants; aminoglycoside resistance determinants; multilocus sequencing typing; plasmid replicon typing; addiction; and virulence factors. Kp32 belonged to the newly described sequence type 1137, were positive for aac(6')-Ib-suzhou, qnrA1, qnrB4, qnrS1, aac(6')-Ib, rmtB, armA, blaSHV-12, blaCTX-M-15, blaKPC-2, and blaIMP-4; contained IncA/C plasmids that tested positive for K1 capsular antigens, the ccdAB (coupled cell division locus) addiction system and the wabG, ureA, rmpA, magA, allS, fimH, and the aerobactin virulence factors. However, the others belonged to clone ST11, and were positive for aac(6')-Ib-cr, qnrB4, blaCTX-M-14, blaSHV-11, aac(6')-Ib, rmtB, and blaKPC-2; contained IncFIA plasmids that tested positive for K2 capsular antigens, the vagCD addiction system and the uge, wabG, ureA, kfuBC, rpmA, and fimH virulence factors. ST1137 had more virulence factors than the comparative strains ST11. The blaKPC-2 gene was located on the IncFIA and IncA/C replicon groups of plasmids. An analysis of the genetic environment of blaKPC-2 gene has demonstrated that the blaKPC-2 gene was always associated with one of the Tn4401 isoforms (a or b). Our study suggested that K. pneumoniae carbapenemases being found in virulent K. pneumoniae should be emphasized, as this will eventually become a global health threat.
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Proteínas de Bactérias/genética , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/patogenicidade , Fatores de Virulência/genética , beta-Lactamases/genética , Antibacterianos/uso terapêutico , Pré-Escolar , China , Conjugação Genética , Elementos de DNA Transponíveis , Farmacorresistência Bacteriana Múltipla/genética , Humanos , Infecções por Klebsiella/tratamento farmacológico , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/isolamento & purificação , Masculino , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , Plasmídeos/química , VirulênciaRESUMO
The aim of this study was to investigate, for the first time, the combinations of carbapenem resistance mechanisms in clinical isolates of extended-spectrum beta-lactamase (ESBL)-producing Pseudomonas aeruginosa in a Chinese hospital. Pulsed-field gel electrophoresis revealed the presence of eight clonal types among the 15 ESBL producers. Multilocus sequence typing of two isolates harboured blaIMP-1 identified the clonal strain as ST325. All these genes were found either alone or simultaneously in the strains in the following five different arrangements:
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Regulação Bacteriana da Expressão Gênica , Genes MDR , Porinas/genética , Pseudomonas aeruginosa/genética , Resistência beta-Lactâmica/genética , beta-Lactamases/genética , Antibacterianos/uso terapêutico , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Carbapenêmicos/uso terapêutico , China , Células Clonais , Transferência Genética Horizontal , Hospitais Universitários , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Tipagem de Sequências Multilocus , Plasmídeos , Porinas/metabolismo , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/isolamento & purificação , beta-Lactamases/metabolismoRESUMO
Four closely related KPC-producing Klebsiella pneumoniae strains, which were isolated from the patients with neonatal sepsis, harbored bla(CTX-M-14), bla(TEM-1), bla(CTX-M-15), bla(SHV-11),bla(SHV-12), class 1 integron, qnrS1, acc(6')-Ib-cr, and rmtB genes. Multilocus sequence typing experiments showed that all isolates but Kp122 were proven to share the same sequence type (ST), ST11. These isolates have not yet been previously reported in a university-affiliated children's hospital or in the city of Wenzhou.
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Carbapenêmicos/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Klebsiella pneumoniae/efeitos dos fármacos , Antibacterianos/farmacologia , China , DNA Bacteriano/isolamento & purificação , Transferência Genética Horizontal , Genes Bacterianos , Hospitais Universitários , Humanos , Lactente , Integrons , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/isolamento & purificação , Tipagem de Sequências Multilocus , Reação em Cadeia da Polimerase , RNA Ribossômico 16S/isolamento & purificação , Sepse/microbiologia , Sepse/patologia , Análise de Sequência de DNARESUMO
Y chromosome is a male-specific paternal inherited chromosome. The STR markers on Y chromosome have been widely used in forensic practices. This article summarizes the characteristics of Y-STR and some factors are considered of selecting appropriate Y-STR markers for Chinese population. The prospects of existing and potential forensic applications of Y-STR profiles are discussed including familial excluding, familial searching, crowd source deducing, mixture sample testing, and kinship identifying. The research, development, verification of Y-STR kit, Y-STR mutation rate, and search software are explored and some suggestions are given.
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Feminino , Humanos , Masculino , Povo Asiático/genética , Cromossomos Humanos Y , DNA/genética , Impressões Digitais de DNA , Bases de Dados de Ácidos Nucleicos , Medicina Legal/métodos , Genética Populacional , Genótipo , Repetições de Microssatélites , Mutação , Reação em Cadeia da Polimerase , Kit de Reagentes para Diagnóstico , SoftwareRESUMO
Epigenetics is the study of heritable changes in gene expression other than changes in the underlying DNA sequence. Such changes include DNA methylation, histone modification, chromatin remodeling, genomic imprinting, X chromosome inactivation and non-coding RNA regulation. Recent progresses on epigenetics open new possibilities in tackling these challenging problems in forensic science, including identification of fetal paternity testing in embryonic period, determination of the necessary allele in paternity testing, discrimination of identical twins, origination analysis of micro tissue, verification of forged DNA. This review focuses on epigenetics concept and its latest application in the field of paternity testing, age estimation, discrimination between the twins, identification of tissue of origin, and estimation of postmortem interval.
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Humanos , Alelos , Metilação de DNA , Epigênese Genética , Epigenômica , Ciências Forenses , Expressão Gênica , Impressão Genômica , Gêmeos MonozigóticosRESUMO
With the development of molecular biology, the evidences of genetics has been used widely in forensic sciences. DNA technology has played an important role in individual identification and paternity testing, RNA technology is showing more and more wide application in prospect. This article reviews the application and progress of RNA in forensic science including estimation of postmortem interval, bloodstain age, wound age, as well as determination of cause of death and the source of body fluids.
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Animais , Humanos , Actinas/metabolismo , Manchas de Sangue , Líquidos Corporais/metabolismo , Causas de Morte , Medicina Legal/métodos , Expressão Gênica , Marcadores Genéticos/genética , MicroRNAs/genética , Reação em Cadeia da Polimerase/métodos , Mudanças Depois da Morte , RNA/genética , Estabilidade de RNA , RNA Mensageiro/metabolismo , RNA Ribossômico 18S/genéticaRESUMO
Objective of this study was to perform bioinformatics analysis of the characteristics of gene expression profiling regulated by amifostine and predict its novel potential biological function to provide a direction for further exploring pharmacological actions of amifostine and study methods. Amifostine was used as a key word to search internet-based free gene expression database including GEO, affymetrix gene chip database, GenBank, SAGE, GeneCard, InterPro, ProtoNet, UniProt and BLOCKS and the sifted amifostine-regulated gene expression profiling data was subjected to validity testing, gene expression difference analysis and functional clustering and gene annotation. The results showed that only one data of gene expression profiling regulated by amifostine was sifted from GEO database (accession: GSE3212). Through validity testing and gene expression difference analysis, significant difference (p < 0.01) was only found in 2.14% of the whole genome (460/192000). Gene annotation analysis showed that 139 out of 460 genes were known genes, in which 77 genes were up-regulated and 62 genes were down-regulated. 13 out of 139 genes were newly expressed following amifostine treatment of K562 cells, however expression of 5 genes was completely inhibited. Functional clustering displayed that 139 genes were divided into 11 categories and their biological function was involved in hematopoietic and immunologic regulation, apoptosis and cell cycle. It is concluded that bioinformatics method can be applied to analysis of gene expression profiling regulated by amifostine. Amifostine has a regulatory effect on human gene expression profiling and this action is mainly presented in biological processes including hematopoiesis, immunologic regulation, apoptosis and cell cycle and so on. The effect of amifostine on human gene expression need to be further testified in experimental condition.
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Humanos , Amifostina , Farmacologia , Biologia Computacional , Expressão Gênica , Perfilação da Expressão Gênica , Métodos , Análise em Microsséries , Anotação de Sequência MolecularRESUMO
This study was aimed to explore the gene expression profile characteristics of T lymphocytes involved in pathogenesis of severe aplastic anemia (SAA) and to predict putative curative drugs for SAA by using biological principle of similarity contrast of gene expression profiles between drugs and diseases. SAA and T lymphocyte were used as key words to search gene expression datasets related to pathogenesis of SAA in public Gene Expression Omnibus (GEO) of NCBI. After significance test, gene expression profiling involved in pathogenesis of SAA were screened and applied to cluster analysis. And then SAA-related gene expression profiles were thrown into pharmacological gene expression datasets of 3000 candidate drugs for similarity analysis and significantly negative correlation was used as a screening criterion for selecting putative curative drugs of SAA. The results showed that only one gene expression dataset was found out, i.e. GSE3807. Computational bioanalysis identified a total of 515 candidate genes of T lymphocyte involved in pathogenesis of SAA, whose expression level exceeded more than 2-fold. Among them, 202 genes were upregulated and 313 genes were downregulated. Cluster analysis showed that those genes belonged to different pathways, including nucleic acid metabolic process, ubiquitin-dependent protein catabolic process, Golgi apparatus protein transport, protein phosphorylation and immunoglobin/major histocompatibility complex. Similarity analysis of gene expression profiles of SAA and drugs showed that hydroxycamptothecin and metformin might have a potential therapeutic efficacy on SAA. It is concluded that by means of novel bioinformatics method, gene expression profiling combined with similarity analysis between disease-related gene expression and pharmacological gene expression profiles may be a novel way of drug screening for SAA.
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Humanos , Anemia Aplástica , Genética , Biologia Computacional , Avaliação Pré-Clínica de Medicamentos , Perfilação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos , Linfócitos T , MetabolismoRESUMO
<p><b>UNLABELLED</b>The present study was aimed to clone ID4 gene promoter and upstream regulatory region, and to construct a series of recombinant promoter-luciferase reporter for exploring the mechanism of ID4 gene expression regulation.</p><p><b>METHODS AND RESULTS</b>the upstream 5' flanking sequence of 2242 bp from transcriptional start site (TSS) and downstream 5' non-coding region of 212 bp on ID4 gene were searched out and downloaded from human genome databank of NCBI using whole length of ID4 gene cDNA as a probe; On-line promoter analysis softwares, including TESS and Genomax, were employed to analyze the characteristics of ID4 gene promoter and upstream regulatory elements. Then, based on the analytic results, PCR primers were designed and synthesized. Segmental amplification method was adopted to obtain two fragments of 1829 bp and 784 bp. The two fragments were inserted into the plasmid pGEM-T, transformed into TOP10 competent E. coli., and positive recombinants were screened respectively. Subsequently, restriction enzymes KpnI/NheI and KpnI/EcoRI were used to digest the above-mentioned two plasmids pGEM-T and pGL3, and ligation was completed by T4 DNA ligase. After transformation to TOP10 competent E. coli. and screening of positive colonies, the basic recombinant ID4 gene promoter-pGL3 was successfully constructed. KpnI/NheI double digestion and sequencing showed that the target fragment was 2 459 bp and consistent with the corresponding sequence of GenBank; Using the 2459 bp fragment as a template, 5 pairs of primers with identical 3' terminus and different 5' terminus were designed and synthesized for half-nest PCR amplification. 5 fragments with an interval of approximate 400 bp each other, i.e. 2112 bp, 1703 bp, 1290 bp, 784 bp and 496 bp, were produced and inserted into pGEM-T after recovery and purification for transformation to TOP10 competent E. coli. and screening of positive colonies. After that, KpnI/NheI was used to digest the above-mentioned five pGEM-T recombinant plasmids and pGL3 basic vector, and the ligation was completed by T4 DNA ligase. After transformation to TOP10 competent E. coli. and screening for positive colonies, 5 subcloned recombinants of ID4 gene promoter and pGL3 Basic vector cells were constructed. In conclusion, 2.5 kb ID4 gene promoter with upstream expression regulatory sequence was successfully cloned and a series of ID4 promoter subclone-pGL3-Basic recombinant were constructed for further researches on activity, expression regulation and function of ID4 promoter.</p>