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p53 is a tumor suppressor gene involved mainly in the regulation of the G1/S cell cycle phase, DNA repair, and senescence. Although p53 is frequently altered in human cancer, the consequences of its depletion in human embryonic stem cells (hESCs) are unknown. We generated NERCe003-A-3, a p53 knockout hESC line, from the normal NERCe003-A hESC line by using CRISPR/Cas9 editing. This cell line maintained a normal 46, XY karyotype. Further analysis suggested that the cells expressed pluripotency-related markers and had the capacity to differentiate in vitro into derivatives of all three germ layers.

Oxidative stress is significant in numerous types of cancer. Tobacco smoke, an important risk factor for oral squamous cell carcinoma (OSCC), is able to generate reactive oxygen species (ROS) and cause oxidative DNA damage. Superoxide dismutase (SOD) is an endogenous antioxidant enzyme that is critical in limiting the oxidative burden effectively. The purpose of this study was to investigate the effects of the mitochondrial SOD2 and Cu/Zn enzyme SOD1 gene polymorphisms on the susceptibility to and clinicopathological characteristics of OSCC, as well as the synergistic effect between these gene polymorphisms and the well-known risk factor of tobacco consumption. Patients with clinically diagnosed OSCC (n=362) and healthy normal individuals (n=358) were investigated for four single nucleotide polymorphisms (SNPs; rs4880, rs5746136, rs1804450 and rs11556620) by polymerase chain reaction-restriction fragment length polymorphism and DNA sequencing methods. Following adjustment for other confounders, no significant difference was observed in the rs5746136 SOD2 SNPs between the patients and controls. However, the incidence of the CT genotype of SOD2 SNP rs4880 was higher in the patients than in normal subjects in the additive model [CT vs. TT; P=0.045; adjusted odds ratio (AOR)=1.484; 95% confidence interval (CI), 1.009-2.182] and in the dominant model (CT/CC vs. TT; P=0.022; AOR=1.559; 95% CI, 1.067-2.278). For those who smoked, the incidence of the CT genotype of rs4880 increased markedly in the patients compared with the controls in the additive model (CT vs. TT; P=0.003; AOR=2.325; 95% CI, 1.330-4.064) and in the dominant model (CT/CC vs. TT; P=0.001; AOR=2.448; 95% CI, 1.417-4.230). For SOD1, polymorphisms at rs1804450 and rs11556620 were not present in any of the OSCC or control subjects. The results suggest that SOD2 rs4880 may be involved in the tumorigenesis of OSCC and may be useful as a genetic susceptibility marker for OSCC.

The field of microbial forensics focuses on the attribution of numerous microbes used by perpetrators inbiocrime and bioattack, including many foodborne pathogens such as Escherichia coli O157:H7 caused serious zoonotic disease. Recent source tracing analysis with the 16S rRNA gene, even genome-wide variations of a pathogen, is increasing as sequencing technologies advance. By using whole genome sequence analysis, more SNPs have been used to create a very finely resolved phylogenetic tree for discriminating close evolutionary relationship among strains. In our study, 1401bp of 16S rRNA gene in Enterohemorrhagic Escherichia coli (EHEC) O157:H7 strains were sequenced. The result showed that nucleotide transition and nucleotide transversion happened at 14 positions of sequence in the research strains, and variation of seven positions was located at one of nine separate hypervariable regions (V7). Another SNP within the constant region of 16S rRNA gene was also observed as the previous studies. Through comparing the available sequences obtained from the NCBI database, SNP was the only 16S rRNA polymorphism detected among the closely related EHEC O157:H7 stains. Our investigation demonstrates that unique SNPs in 16S rRNA gene have potential discriminatory power as other species-/strain-specific informative markers for attribution purpose.

Nonbinary single-nucleotide polymorphisms (SNPs) are potential forensic genetic markers because their discrimination power is greater than that of normal binary SNPs, and that they can detect highly degraded samples. We previously developed a nonbinary SNP multiplex typing assay. In this study, we selected additional 20 nonbinary SNPs from the NCBI SNP database and verified them through pyrosequencing. These 20 nonbinary SNPs were analyzed using the fluorescent-labeled SNaPshot multiplex SNP typing method. The allele frequencies and genetic parameters of these 20 nonbinary SNPs were determined among 314 unrelated individuals from Han populations from China. The total power of discrimination was 0.9999999999994, and the cumulative probability of exclusion was 0.9986. Moreover, the result of the combination of this 20 nonbinary SNP assay with the 20 nonbinary SNP assay we previously developed demonstrated that the cumulative probability of exclusion of the 40 nonbinary SNPs was 0.999991 and that no significant linkage disequilibrium was observed in all 40 nonbinary SNPs. Thus, we concluded that this new system consisting of new 20 nonbinary SNPs could provide highly informative polymorphic data which would be further used in forensic application and would serve as a potentially valuable supplement to forensic DNA analysis.

To investigate the bacterial succession on rat carcasses and to evaluate the use of bacterial succession for postmortem interval (PMI) estimation.Adult female SD rat remains were placed in carton boxes. The bacterial colonization of circumocular skin, mouth and vagina was collected to be identified using culture-dependent biochemical methods. The changes in community composition were regularly documented.The bacterial succession in three habitats showed that Staphylococcus and Neisseria were predominated in early PMI, especially Staphylococcus aureus and Neisseria lactamica in 6 hours after death. Lactobacillus casei developed on the 3-4 days regularly, and kept stable at a certain level in late PMI.The involvement of normal and putrefactive bacteria in three body habitats of rat remains can be used for PMI estimation.

Decomposition is a complex process involving the interaction of both biotic and abiotic factors. Microbes play a critical role in the process of carrion decomposition. In this study, we analysed bacterial communities from live rats and rat remains decomposed under natural conditions, or excluding sarcosaphagous insect interference, in China using Illumina MiSeq sequencing of 16S rRNA gene amplicons. A total of 1,394,842 high-quality sequences and 1,938 singleton operational taxonomic units were obtained. Bacterial communities showed notable variation in relative abundance and became more similar to each other across body sites during the decomposition process. As decomposition progressed, Proteobacteria (mostly Gammaproteobacteria) became the predominant phylum in both the buccal cavity and rectum, while Firmicutes and Bacteroidetes in the mouth and rectum, respectively, gradually decreased. In particular, the arrival and oviposition of sarcosaphagous insects had no obvious influence on bacterial taxa composition, but accelerated the loss of biomass. In contrast to the rectum, the microbial community structure in the buccal cavity of live rats differed considerably from that of rats immediately after death. Although this research indicates that bacterial communities can be used as a “microbial clock” for the estimation of post-mortem interval, further work is required to better understand this concept.

To evaluate the association of HSD17B1 and HSD17B2 gene polymorphisms with uterine leiomyoma in Chinese women.121 Chinese women with clinically diagnosed uterine leiomyoma and 217 healthy normal Chinese women were investigated to compare three single nucleotide polymorphisms (SNPs) (rs605059 and rs676387 of HSD17B1 gene and rs8191246 of HSD17B2 gene) by polymerase chain reaction-restriction fragment length polymorphism and DNA sequencing method.All the SNPs were polymorphisms in Chinese women. Frequencies of rs605059 AA genotype and A allele were significantly increased in patients with uterine leiomyoma compared to healthy controls (GG vs. AA, OR 0.40, 95 % CI 0.20-0.82; G vs. A, OR 0.68, 95 % CI 0.50-0.94).The results suggest that the genotype of HSD17B1 rs605059 may play a role in the tumourgenesis of uterine leiomyoma.

SNPs, abundant in human genome with lower mutation rate, are attractive to genetic application like forensic, anthropological and evolutionary studies. Universal SNPs showing little allelic frequency variation among populations while remaining highly informative for human identification were obtained from previous studies. However, genotyping tools target only dozens of markers simultaneously, limiting their applications. Here, 124 SNPs were simultaneous tested using Ampliseq technology with Ion Torrent PGM platform. Concordance study was performed with 2 reference samples of 9947A and 9948 between NGS and Sanger sequencing. Full concordance were obtained except genotype of rs576261 with 9947A. Parameter of FMAR (%) was introduced for NGS data analysis for the first time, evaluating allelic performance, sensitivity testing and mixture testing. FMAR values for accurate heterozygotes should be range from 50% to 60%, for homozygotes or Y-SNP should be above 90%. SNPs of rs7520386, rs4530059, rs214955, rs1523537, rs2342747, rs576261 and rs12997453 were recognized as poorly performing loci, either with allelic imbalance or with lower coverage. Sensitivity testing demonstrated that with DNA range from 10 ng-0.5 ng, all correct genotypes were obtained. For mixture testing, a clear linear correlation (R2 = 0.9429) between the excepted FMAR and observed FMAR values of mixtures was observed.

As a multi-ethnic country, China has some indigenous population groups which vary in culture and social customs, perhaps as a result of geographic isolation and different traditions. However, upon close interactions and intermarriage, admixture of different gene pools among these ethnic groups may occur. In order to gain more insight on the genetic background of X-Chromosome from these ethnic groups, a set of X-markers (18 X-STRs and 16 X-Indels) was genotyped in 5 main ethnic groups of China (HAN, HUI, Uygur, Mongolian, Tibetan). Twenty-three private alleles were detected in HAN, Uygur, Tibetan and Mongolian. Significant differences (p

Recent studies have begun to reveal critical roles of microRNAs (miRNAs) in the pathogenesis of cardiac hypertrophy and dysfunction. In this study, we tested whether a transforming growth factor-β (TGF-β)-regulated miRNA played a pivotal role in the development of cardiac hypertrophy and heart failure (HF). We observed that miR-27b was upregulated in hearts of cardiomyocyte-specific Smad4 knockout mice, which developed cardiac hypertrophy. In vitro experiments showed that the miR-27b expression could be inhibited by TGF-β1 and that its overexpression promoted hypertrophic cell growth, while the miR-27b suppression led to inhibition of the hypertrophic cell growth caused by phenylephrine (PE) treatment. Furthermore, the analysis of transgenic mice with cardiomyocyte-specific overexpression of miR-27b revealed that miR-27b overexpression was sufficient to induce cardiac hypertrophy and dysfunction. We validated the peroxisome proliferator-activated receptor-γ (PPAR-γ) as a direct target of miR-27b in cardiomyocyte. Consistently, the miR-27b transgenic mice displayed significantly lower levels of PPAR-γ than the control mice. Furthermore, in vivo silencing of miR-27b using a specific antagomir in a pressure-overload-induced mouse model of HF increased cardiac PPAR-γ expression, attenuated cardiac hypertrophy and dysfunction. The results of our study demonstrate that TGF-β1-regulated miR-27b is involved in the regulation of cardiac hypertrophy, and validate miR-27b as an efficient therapeutic target for cardiac diseases.

The quantitative determination of H2S in the blood can provide valid evidence for H2S poisoning through occupational exposure. However, known traditional methods for the detection of H2S in blood are time consuming, require complicated pretreatments, and have low sensitivity. In this paper, a new fluorescence sensing assay is proposed for the rapid detection of H2S poisoning in forensic cases based on bovine serum albumin (BSA)-stabilised zinc/copper (Zn/Cu) bi-metal nanoclusters (Zn/Cu@BSA NCs). The as-prepared Zn/Cu@BSA NCs probes have been characterised by UV-vis absorption and fluorescence spectroscopy. The fluorescence of Zn/Cu@BSA NCs can be quenched through specific interactions between HS−/S2− and the Zn2+/Cu2+ bi-metal ions. Under optimised conditions, the fluorescence sensing method was linear in the concentration range of 2.5 nM to 30 mM with 0.69 nM as the limit of detection. Moreover, the practical feasibility of this fluorescence sensing method has also been demonstrated by the analysis of mice blood samples containing different levels of sulfide and human blood samples from forensic cases of H2S poisoning. Compared with gas chromatography/mass spectrometry (GC/MS), this fluorescence sensing method is quite simple, straightforward, and can be accurate for the quantitative determination of H2S poisoning in a few minutes for forensic analysis. Overall, this is the first report of a bi-metal fluorescence sensing assay for detecting H2S poisoning directly in blood. This research may provide a new approach for forensic toxicologists to monitor poisoning by H2S using a fluorescence-sensing method.

In this study, we sequence the complete mitochondrial genome of Muscina stabulans (Diptera: Muscidae), a forensically important entomology, for the first time. The 15,933 bp circular genome contains the 37 genes found in a typical metazoan genome: 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes and 1 non-coding A + T-rich region. All the protein initiation codons are ATN, except for cox1 and ND1 that begin with TCG and TTG. The arrangement of the genes was the same as that found in the other insect. The overall base composition on heavy strand was as follows A: 40.00%, G: 8.80%, C: 13.34%, T: 37.86% and the A + T content 77.86%. The mitochondrial genome of Muscina stabulans presented will be valuable for resolving phylogenetic relationships within the family Muscidae and order Diptera, and could be used to identify favourable genetic markers for species identifications for forensic purposes.

The estimation of postmortem interval (PMI) is one of the most difficult tasks in forensic practice, especially in putrefied bodies. After death, organisms are decomposed by a variety of enzymes and microorganisms. To investigate the succession of bacterial community in the decomposition process, rat remains were placed outside to decompose under natural conditions in Changsha city, China. Bacterial communities from two regions (buccal cavity and rectum) were sampled when experiment animals were alive, soon after they died and at various time span after death. Bacterial samples were analyzed by high throughput metagenomic sequencing of 16S rRNA gene conducted on an Illumina MiSeq platform. Our data showed that several bacteria genera were potentially useful for estimating the PMI, such as Streptococcus , Ignatzschineria , Acinetobacter , Aggregatibacter , Prevotella and Proteus . There were significant bacterial community structure differences in taxon richness and relative abundance patterns through the decomposition process and across different body sites. As decomposition progressed, a negative linear relationship for taxon richness was found along with a shift from aerobic bacteria to anaerobic bacteria. We first reported the bacterial biodiversity in the decomposition process in Chinese terrestrial scenarios and climatic conditions. Bacteria have a remarkable potential for estimate the PMI and next generation sequencing is a novel method to support the application of bacteria in forensic science.

Bacterial genotyping can be used for crime scene investigations and contribute to the attribution of biological attacks for microbial forensics. PyroMark ID Pyrosequencer as an accurate detection platform for single nucleotide polymorphisms (SNPs) has been applied to identify and resolve microorganisms involved in closely Escherichia coli O157:H7 (E. coli O157:H7). To explore more applications and improve the efficiency for pyrosequencing in this field, we developed a method integrated multiplex pyrosequencing with a universal primer. Two multiplex pyrosequencing assays with a universal biotinylated primer were designed to analyze five SNPs located in four gene of E. coli O157:H7 strain. The accuracy of the established assays was validated by genotyping reference strain E. coli O157:H7 EDL933 and E. coli K-12. We also demonstrated that two multiplex pyrosequencing assays were specific and sensitive for genotyping closely related E. coli O157 strains. Reproducibility of results and multiplexing capability were evaluated by a comparison of this method with the monoplex pyrosequencing. Furthermore, these two multiplex pyrosequencing assays have been successfully applied to detect 11 E. coli O157 strains isolated from 1504 Chinese livestock samples. This method reduces costs and time consumption in the process of pyrosequencing analysis, and potentially serves as a rapid tool and reliable candidate strategy for the microbial identification and other genotyping application.

Unequivocal identification of insect specimens is an essential requirement in forensic entomology. With the development of molecular identification, spate of discussions about the feature of the DNA fragments have been raised. Relying solely on single DNA fragment for delimiting closely related species is supposed to be dangerous. Aiming at obtaining more reliable markers that might be universally used, we explore the utility of 700-bp COI fragment and 678-bp period gene fragment in the identification of Sarcophagidae (Diptera). Thirty-six sarcophagid fly specimens were collected from 19 locations in 11 Chinese provinces. Phylogenetic analysis of the sequenced segments showed that all sarcophagid specimens were properly assigned into nine species with relatively strong supporting values, which indicated the possibility of separation congeneric species with COI and period gene fragments. The difference between intraspecific threshold and interspecific divergence confirmed that the combination of nuclear and mitochondrial genes for species identification is much more accurate. The results of this research will be instrumental for implementation of the Chinese Sarcophagidae database.

Tri-allelic single nucleotide polymorphisms (SNPs) are potential forensic markers for DNA analysis. Currently, only a limited number of tri-allelic SNP loci have been proved to be fit for forensic application. In this study, we aimed to develop an effective method to select and genotype tri-allelic SNPs based on both Pyrosequencing (PSQ) and the SNaPshot methods. 50 candidate SNPs were chosen from NCBI's dbSNP database and were analyzed by PSQ. The results revealed that 20 SNPs were tri-allelic and were located on 16 autosomal chromosomes. Then 20 SNP loci were combined in one multiplex polymerase chain reaction to develop a single base extension (SBE)-based SNP-typing assay. A total of 100 unrelated Chinese individuals were genotyped by this assay and allele frequencies were estimated. The total discrimination power was 0.999999999975 and the cumulative probability of exclusion was 0.9937. These data demonstrated that the strategy is a rapid and effective method for seeking and typing tri-allelic SNPs. In addition, the 20 tri-allelic SNP multiplex typing assay may be used to supplement paternity testing and human identification.

Tri-allelic single nucleotide polymorphisms (SNPs) are potential forensic markers for DNA analysis. Currently, only a limited number of tri-allelic SNP loci have been proved to be fit for forensic application. In this study, we aimed to develop an effective method to select and genotype tri-allelic SNPs based on both Pyrosequencing (PSQ) and the SNaPshot methods. 50 candidate SNPs were chosen from NCBI's dbSNP database and were analyzed by PSQ. The results revealed that 20 SNPs were tri-allelic and were located on 16 autosomal chromosomes. Then 20 SNP loci were combined in one multiplex polymerase chain reaction to develop a single base extension (SBE)-based SNP-typing assay. A total of 100 unrelated Chinese individuals were genotyped by this assay and allele frequencies were estimated. The total discrimination power was 0.999999999975 and the cumulative probability of exclusion was 0.9937. These data demonstrated that the strategy is a rapid and effective method for seeking and typing tri-allelic SNPs. In addition, the 20 tri-allelic SNP multiplex typing assay may be used to supplement paternity testing and human identification.

In this research, we investigated the allele frequencies and forensic parameters of 21 non-, CODIS short tandem repeat (STR) loci (D6S474, D12ATA63, D22S1045, D10S1248, D1S1677, D11S4463, D1S1627, D3S4529, D2S441, D6S1017, D4S2408, D19S433, D17S1301, D1GATA113, D18S853, D20S482, D14S1434, D9S1122, D2S1776, D10S1435 and D5S2500) among 523 unrelated, Chinese Mongolians in the city of Tongliao, Horqin district, Inner Mongolia Autonomous Region.

Osteoblasts participate in bone formation, bone mineralization, osteoclast differentiation and many pathological processes. To study the function of genes in osteoblasts using Cre-LoxP system, we generated a mouse line expressing the Cre recombinase under the control of the rat Collagen1alpha1 (Col1alpha1) promoter (Col1alpha1-Cre). Two founders were identified by genomic PCR from 16 offsprings, and the integration efficiency is 12.5%. In order to determine the tissue distribution and the activity of Cre recombinase in the transgenic mice, the Col1alpha1-Cre transgenic mice were bred with the ROSA26 reporter strain and a mouse strain that carries Smad4 conditional alleles (Smad4(Co/Co)). Multiple tissue PCR of Col1alpha1-Cre;Smad4(Co/+)mice revealed the restricted Cre activity in bone tissues containing osteoblasts and tendon. LacZ staining in the Col1alpha1-Cre;ROSA26 double transgenic mice revealed that the Cre recombinase began to express in the osteoblasts of calvaria at E14.5. Cre activity was observed in the osteoblasts and osteocytes of P10 double transgenic mice. All these data indicated that the Col1alpha1-Cre transgenic mice could serve as a valuable tool for osteoblast lineage analysis and conditional gene knockout in osteoblasts.

In microbial forensics arena, the large number of microbes been used as potential bioterrorist agents in a criminal attack, especially many foodborne pathogens such as Escherichia coli O157:H7 caused serious zoonotic disease involved. Further development of high resolution genotyping assays is needed to rapidly and accurately analyze microbiological evidence from a crime for attribution purposes. Some recent approaches support multilocus variable number tandem repeat analysis (MLVA) is a PCR-based subtyping method to discriminate among different strains of a bacterium. In our study, MLVA combined with automated capillary electrophoresis was used to analyze genetic relationships and potential population structure within 31 E. coli O157:H7 isolates from humans and animals in China and standard strains. Alleles of each variable number tandem repeat (VNTR) were validated by sequencing. MLVA resolved 29 distinct genotypes respectively, and were able to largely separate genotypes from humans and all kinds of animals among zoonotic strains. Microvariation events occurred in two VNTR loci. In the light of the advantages of highly discrimination, MLVA can be regarded as powerful tools for detailed tracking of E. coli O157:H7 and could also prove useful in forensic investigations.

Single nucleotide polymorphisms (SNPs) have been proved to be effective forensic marker for supplementary paternity testing and forensic identification due to their low mutation rate and depending on small amplicon size. However, at least a set of 50–60 binary SNP loci can reach discrimination power of 13 STR loci that were routinely used in forensic practice. Meanwhile, binary SNP loci were ineffective to detect mixed samples, which may result in incorrect genotyping. In this study, we aimed to find out the potential forensic markers, tri-allelic SNP loci in Chinese Han population. Pyrosequencing (PSQ) with pooling samples was used to analyze candidate SNP loci and estimate allele frequencies of them. To confirm the existence of three alleles, individual samples were sequenced with PSQ method. Our results revealed that there were 10 tri-allelic SNP loci resided on 10 autosomal chromosomes in Chinese Han population. These data demonstrated that PSQ was an effective method to detect tri-allelic SNPs and estimate allele frequencies of them.