Study on the underlying molecular mechanism of benzene-induced nervous system damage in mice based on tandem mass tag (TMT) proteomics.

Benzene is known to be a common toxic industrial chemical, and prolonged benzene exposure may cause nervous system damage. At present, there were few studies on benzene-induced neurological damage. This research aimed to identify the protein biomarkers to explore the mechanism of nervous system damage caused by benzene. We established a benzene poisoning model of C57 mice by gavage of benzene-peanut oil suspension and identified differentially expressed proteins (DEPs) in brain tissue using tandem mass tag (TMT) proteomics. The results showed a significant weight loss and decrease in leukocyte and neutrophil counts in benzene poisoning mice compared to the control group. We also observed local cerebral oedema and small vessel occlusion in the cerebral white matter of benzene poisoning mice. TMT proteomic results showed that a total 6,985 proteins were quantified, with a fold change (FC) > 1.2 (or < 1/1.2) and P value <0.05 were considered as DEPs. Compared with the control group, we identified 43 DEPs, comprising 14 upregulated and 29 downregulated proteins. Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis results showed that the candidate proteins were mainly involved in cholesterol metabolism, complement and coagulation cascades, african trypanosomiasis, PPAR signaling pathway, and vitamin digestion and absorption. Three proteins, 2-hydroxyacylsphingosine 1-beta-galactosyltransferase (UGT8), Apolipoprotein A-I (APOA1) and Complement C3 (C3) were validated using immunoblotting and immunohistochemical. In conclusion, our study preliminarily investigated the mechanism of benzene toxicity to the nervous system by analyzing DEPs changes in the brain.

Bioinformatics analysis of sequential gene expression profiling after skin and skeletal muscle wound in mice.

It is of great value to use bioinformatics methods to screen the core differentially expressed genes (DEGs) at different times after mouse skin and skeletal muscle wound, and to explore the relationship between them and the wound age. To this end, we downloaded the gene expression profiles of GSE140517 and GSE23006 from the NCBI-GEO gene database, used GEO2R online tools and Venn diagrams to screen out DEGs at different times and common-DEGs. The Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) channel analysis were carried out through the DAVID website respectively. Use STRING tool to build a Protein-protein Interaction (PPI) network, and use Cytoscape software to screen out core DEGs. The results showed that 13, 53, 43 and 13 core DEGs were screened out in the 6 h, 12 h, 24 h and common-DEGs group after wound. There were 7 core DEGs (Cxcl2, Cxcl3, Il1b, Ptgs2, Cxcl1, Timp1, Ccl3) in both the different time point and the common DEGs group. Meanwhile, there are 1 core DEGs (Ccl4) specifically expressed in the 6 h, 29 specifically expressed core DEGs (Isg20, Rtp4, Fcgr1, Ifi44, Trim30a, etc.) in the 12 h, and 18 specifically expressed core DEGs (Ccr7, Myd88, Igsf6, Ccr2, Gpsm3, etc.) in the 24 h, there are 6 core DEGs (Ccl4, Ccl7, Saa3, Cxcl5, Ccl2, Lcn2) specifically expressed in the common-DEGs group. The results of GO and KEGG analysis showed that the deterioration and exudation of the inflammatory response were the main cause at 6 h after wound. In addition to inflammation at 12 h and 24 h, the systemic immune response against viral and bacterial infections also gradually increased. In summary, the core DEGs selected in this study have combined characteristics, consistent with the healing function at the corresponding time point, and they are also has specificity and correlation with wound age. Therefore, by detecting the changes in the expression of co-expressed core DEGs at different times after wound, as well as detecting specific expressed DEGs at a specific time point or a specific period of time, it is very promising to provide help for the wound age estimation. However, limited by the GSE140517 gene expression profile in the database, only the difference in gene expression at different times within 24 h after wound was explored, and the research on the late wound age still needs to be further in-depth.

New contributions to the relationship between sequential changes of ATP-related metabolites and post-mortem interval in rats.

In recent years, it has become a new research direction to post-mortem interval (PMI) estimation from the changes in metabolite content generated during the process of cadaver corruption, and lead to more and more attention has been paid to the biochemical changes of muscles after death. The ATP degradation process has long been accepted as a precise manner to estimate freshness of meat products in food science. ATP-related metabolites may also serve as an indicator for PMI estimation of corpses. The purpose of this study was to test this possibility. Firstly, this study optimized the detection method of ATP-related metabolites in skeletal muscles of rat. Moreover, in animal experiment, ATP-related metabolites (ATP, ADP, AMP, IMP, Ino, Hx) in lower hind limb skeletal muscles of sixty rats were measured by RP-HPLC at different PMI for 168 h at 20 °C ± 1 °C, the change of freshness indicators (K value, K1 value, K0 value, H value, P value, G value, IMP ratio) were carefully analyzed, and investigate the relationship between them and PMI. The overall results showed that IMP, AMP, Hx, Total metabolites, K value, K1 value, K0 value, H value, P value, G value, and IMP ratio in skeletal muscles changed significantly with PMI (R2 = 0.864-0.971, all P < 0.01) while ATP, ADP, Ino, IMP precursors and IMP degradants had minor changes during the same period (R2 = 0.171-0.706). Specifically, significant linear positive correlations between H value of skeletal muscles and PMI were found, and the coefficients of their regression functions were R2 = 0.971. The correlation between AMP and PMI was slightly lower (R2 = 0.864), but the scope of PMI estimation was the widest (168 h). It can be concluded that the determination of ATP-related metabolites in skeletal muscle may be a potential tool in the PMI estimation. However, more researches on its influencing factors are needed to facilitate its final use in practice.

GDF15 is a heart-derived hormone that regulates body growth.

The endocrine system is crucial for maintaining whole-body homeostasis. Little is known regarding endocrine hormones secreted by the heart other than atrial/brain natriuretic peptides discovered over 30 years ago. Here, we identify growth differentiation factor 15 (GDF15) as a heart-derived hormone that regulates body growth. We show that pediatric heart disease induces GDF15 synthesis and secretion by cardiomyocytes. Circulating GDF15 in turn acts on the liver to inhibit growth hormone (GH) signaling and body growth. We demonstrate that blocking cardiomyocyte production of GDF15 normalizes circulating GDF15 level and restores liver GH signaling, establishing GDF15 as a bona fide heart-derived hormone that regulates pediatric body growth. Importantly, plasma GDF15 is further increased in children with concomitant heart disease and failure to thrive (FTT). Together these studies reveal a new endocrine mechanism by which the heart coordinates cardiac function and body growth. Our results also provide a potential mechanism for the well-established clinical observation that children with heart diseases often develop FTT.

Conductometric titration to determine total volatile basic nitrogen (TVB-N) for post-mortem interval (PMI).

Precise measurement of cadaver decomposition rate is the basis to accurate post-mortem interval (PMI) estimation. There are many approaches explored in recent years, however, it is still unsolved completely. Total volatile basic nitrogen (TVB-N), which is an important index to predict meat freshness and shelf life in food science, could serve as an indicator for measuring PMI associated decomposition rate of cadavers. The aim of this work was to establish a practical method to determine TVB-N in cadaver soft tissues (mainly skeletal muscle) for measuring decomposition rate. Determination of TVB-N in the simulation and animal experiments was conducted by steam distillation and conductometric titration using Kjeldahl distillation unit and conductivity meter. In simulation, standard concentrations of ammonium were used as TVB analogies, TVB-N contents were determined and the recovery rates of nitrogen were calculated. In animal experiment, TVB-N in skeletal muscle of forty-two rats was determined at different PMIs for 312 h at 24 °C ± 1 °C. The relationship between PMI and TVB-N was investigated also. The method showed high precision with 99%-100% recovery rates. TVB-N in skeletal muscle changed significantly with PMI especially after 24 h, and the data fit well to y = 3.35 E-5x3-2.17 E-2x2+6.13x-85.82 (adj. R2 = 0.985). ECi (initial electrical conductivity in the samples just before titration) had positive linear relationship to final measured TVB-N values, y = 1.98x+16.16 (adj. R2 = 0.985). The overall results demonstrated that the method is accurate, rapid and flexible, which could be expected as a basic technique for measuring decomposition rate in later PMI-estimation researches. Further studies are needed to validate our findings.

[Research Progress on Forensic Entomotoxicology].

Forensic entomotoxicology is a branch of forensic medicine, which applies entomology, toxicology and other related studies to solve the poisoning cases. It has an obvious advantage in the investigation on poisoning death. Based on the expounding definition and research of entomotoxicology, this paper reviews research progress and application value in some aspects of forensic medicine, such as the effects of drugs/toxins on the growth and development of sarcosaphagous insects and the qualitative and quantitative analysis of the drugs/toxins in the poisoned body tissue.

Developing a multiplex mtSNP assay for forensic application in Han Chinese based on mtDNA phylogeny and hot spot.

We designed one multiplex assay with a reduced number of SNPs from whole mitochondrial genome as a screening approach for forensic purposes and developed a multiplex SNaPshot assay with 26 mitochondrial SNPs (mtSNPs). This assay included 16 target mtSNPs that defined the main haplogroups in Chinese population and ten hot-spot mtSNPs found by pyrosequencing. To validate our multiplex mtSNP assay, we not only analyzed a Chinese Han population sample, but also sequenced the complete control region of same set of individuals. Fifty-one haplotypes were observed in 204 individuals using our multiplex mtSNP assay and the haplotype diversity was estimated to be 0.9626. Our multiplex mtSNP assay could also distinguish some individuals sharing the same control region sequences. The same mtSNP profiles were obtained from the bloodstain, hair shaft, and salivary swab from same individuals. A good profile could be obtained with 50 pg of DNA. It was evident that our multiplex mtSNP assay not only improved the discrimination power, but also allowed allocating mitochondrial DNA profiles to particular haplogroups not clearly defined with the control region alone. We highlight the importance of the balance of target mtSNPs for haplogroup assignment and hot-spot mtSNPs for increasing discrimination power.

Effects of ketamine on the development of forensically important blowfly Chrysomya megacephala (F.) (Diptera: Calliphoridae) and its forensic relevance.

This study investigated effects of ketamine on the development of Chrysomya Megacephala (Diptera: Calliphoridae) at three different temperatures. Larvae of the C. Megacephala were exposed to different concentrations of drugs and temperatures. The larval lengths, weights, and developmental durations of each stage were observed. This study demonstrated that ketamine, low temperature, and their synergistic action significantly suppressed the development of C. Megacephala (p < 0.001). The time that the larvae in all the treatments achieved the maximum length/weight was significantly delayed (p < 0.05), and that resulted in prolonged duration of larval and prepupal stages especially at low temperature. However, no linear correlations were discovered between ketamine concentration and growth rate of larval length/weight.

[One-step methylation variable position analysis technology in single-tube].

OBJECTIVE: To develop the single-tube one-step methylation variable position (MVP) analysis technology-single-tube post-digestion PCR-melting curve analysis (PDP-MCA). METHODS: Based on differentially methylated region (DMR) reported previously as the model, a set of primers with different melting temperatures of products in the two sides of MVP were designed. By using the FastDigest methylation-sensitive restriction enzyme (MSRE), DNA digestion, multiplex amplification, MCA detection and MCA profiles were performed in a single reaction tube. Same samples (peripheral venous blood, semen, and vaginal fluid, 5 samples each type) were tested by single-tube one step MVP and traditional MSRE-PCR MCA technology. To verify the feasibility of this method, the results were compared with that of the traditional technology. The MCA/HRM profiles of different samples were analyzed and compared. RESULTS: When the melting temperature of the fragments had a differential of 2 degrees C, the MCA melting peaks separated well, and MCA detection after multiplex amplification was successful. The single-tube PDP-MCA assay was developed, which integrated multiple reactions (digestion, amplification and detection) into one tube. By this method, the sample-specific profiles and data were analyzed in 2 h, which is similar to that of the traditional method. The rapid classifications of the samples were also realized. CONCLUSION: Multiplex MVPs can be analyzed in a single closed-tube. The single-tube PDP-MCA technology is a simple, fast, and automatable method. It can be used for detection of DNA methylation variations.

[Effect of ketamine on the development of Chrysomya megacephala (Diptera : Calliphoridae)].

OBJECTIVE: To research the pattern of larvae and pupae development when exposed to ketamine. METHODS: The larvae of Chrysomya megacephala were reared in artificial diet containing ketamine with concentration of 0, 25, 50 and 100 mg/kg respectively at 32 degrees C, 28 degrees C and 24 degrees C in environmental chamber with a 12-h photoperiod and 75% humidity. 10 samples were collected from each group every 12 h from the 16th h after hatching to pupation. The max body length, weight of the larvae, growth rate of body length, weight and developmental duration of each stage were observed. RESULTS: The average length and weight in the treatment groups were significantly less than the control before achieving the maximum (P < 0.05), and the growth rate of 1/2LD50 group at 24 degrees C was most retarded. No dose dependence were observed among the ketamine fed groups. The effect of ketamine dose, temperature and their interaction on larval length and weight was statistically significant (P < 0.01). The effect of ketamine dose, temperature and their interaction account for, respectively, 20.9%, 60.2% and 18.9% of the total effect on growth of larval length, and they account for 8.3%, 85.6% and 6.1% of the total effect on growth of larval weight. The duration of larval stage in treatment groups was significantly delayed in comparison to the control at different temperatures (P < 0.05), and the duration of prepupal stage in treatment groups was significantly delayed (P < 0.05). However, the duration of pupal stage in treatment groups at 24 degrees C was significantly shorter than the control (P < 0.05). CONCLUSION: The time achieving maximum length and weight was significantly delayed, which results in an increased development duration of larval and prepupal stages, indicating that ketamine inhibits the growth of the larvae of C. megacephala.

The association between MLH1 -93 G>A polymorphism of DNA mismatch repair and cancer susceptibility: a meta-analysis.

DNA mismatch repair, known as a fundamentally biological pathway, plays key roles in maintaining genomic stability, eliminating mismatch bases and preventing both mutagenesis in the short term and cancerogenesis in the long term. Polymorphisms of MLH1 in individuals may have an effect on the DNA repair capacity and therefore on cancer risk. Recently, emerging studies have been done to evaluate the association between MLH1 -93 G/A polymorphism and cancer risk in diverse populations. However, the results remain conflicting rather than conclusive. In this meta-analysis, we assessed reported studies of association between the MLH1 -93 G/A polymorphism and cancer risk including 13 691 cancer cases and 14 068 controls from 17 published studies. A borderline significant association between the MLH1 -93 G/A polymorphism and cancer risk was observed in overall analysis [heterozygote: odds ratio (OR) = 1.15; 95% confidence interval (CI) 1.05-1.26; homozygote: OR = 1.21; 95% CI, 1.04-1.40; dominant model: OR = 1.13; 95% CI 1.01-1.26; recessive model: OR = 1.21; 95% CI 1.07-1.35, respectively]. In subgroup analysis by ethnicity, significantly increased risks were found in Asian population and mixed population but not in Caucasian population. After stratified analysis according to the quality of literature, increased cancer risks were observed in the studies of lower quality but not in the studies of higher quality. Similarly, elevated cancer risks were observed in hospital-based studies but not in population-based studies. These findings showed no persuasive evidence that MLH1 -93 G/A polymorphism was associated with an increased risk of cancer. On the conservative standpoint, well-designed population-based studies with larger sample size in different ethnic groups should be performed to further confirm these results.

False homozygosities at CSF1PO loci revealed by discrepancies between two kits in Chinese population.

During the course of paternity test, three samples in two cases were apparently homozygous at the CSF1PO locus using AmpFlSTRs Identifiler PCR Amplification kits, but using the PowerPlexs 16 kit, the three individuals were found to be heterozygous. This puzzling problem was solved by using multiple analytical approaches, including the use of different primer pairs and the characterization of the mutation causing the ''null allele.'' Dropout was caused by a single mutation event in the presumptive binding site of the forward primer. While the frequency of these silent alleles remains low (0.5% in our study), it is suggested that appropriate measures should be taken for database comparisons and that allelic dropout should be further investigated by sequence analysis and be reported to the forensic community.

No association between estrogen receptor beta polymorphisms and uterine leiomyoma.

Numerous candidate genes have been proposed as susceptibility factors for the development of uterine leiomyoma. Interaction of estrogen receptor alpha (ESR1) and estrogen receptor beta (ESR2) plays a pivotal role in tunica muscularis uteri cell proliferation, differentiation, and tumorigenesis of myometrium. To our knowledge, no study has examined the relationship between the ESR2 and uterine leiomyoma. The aim of the study was to evaluate the association of ESR2 polymorphisms with uterine leiomyoma in Chinese women. We investigated two common ESR2 polymorphisms, rs1256049 (G1082A) and rs928554 (Cx + 56 A --> G), to find their association with uterine leiomyoma risk by polymerase chain reaction-restriction fragment length polymorphism and DNA sequencing method. A total of 150 Chinese women with clinically diagnosed uterine leiomyoma and 150 healthy, normal Chinese women were included in the study. The results suggest that there were no significant differences in the genotype and allele frequencies of ESR2 polymorphisms between uterine leiomyoma patients and controls in Chinese women (p > 0.05). Further studies are still needed to explore the complicated interaction between environmental factors and ESR2 polymorphisms in the risk of uterine leiomyoma, particularly in ethnically different populations.

XRCC1 codon 280 and ERCC2 codon 751 polymorphisms and risk of esophageal squamous cell carcinoma in a Chinese population.

Numerous candidate genes have been proposed as susceptibility factors for the development of esophageal squamous cell carcinoma (ESCC). XRCC1 (X-ray cross-complementing group 1) codon 280 and ERCC2 (excision repair cross complementing group 2) codon 751 polymorphisms were studied in ESCC in a Chinese population. The aim of this study is to investigate the potential association between single-nucleotide polymorphisms (SNP) of XRCC1 codon 280 His and ERCC2 codon 751 Gln polymorphisms and ESCC. Peripheral blood samples of 200 cases and 200 age-and-gender matching controls were collected from a Chinese population and the two polymorphisms were studied by means of polymerase chain reaction (PCR) restriction fragment length polymorphism techniques. Our results showed that XRCC1 codon 280 His allele had no significant difference between ESCC patients and normal controls (P > 0.05), while ERCC2 codon 751Gln allele was associated with a borderline decrease of ESCC (odds ratio [OR] = 0.628, 95% confidence interval [CI]: 0.400-0.986).

Parentally imprinted allele (PIA) typing in the differentially methylated region upstream of the human H19 gene.

The H19 gene is a paternally imprinted gene located on chromosome 11p15.5. In this study, the H19FR1 and H19FR2 haplotype polymorphisms including four and three SNPs, respectively, upstream of the H19 gene according to the GenBank sequence (accession no. AF125183) were investigated. Five haplotypes and nine genotypes were detected for H19FR1 in the Chinese Han population by means of PCR and subsequent denaturing gradient gel electrophoresis (DGGE). The power of discrimination (Dp), polymorphism information content (PIC) and probability of paternity exclusion (PE) were estimated to be 0.803, 0.58 and 0.322, respectively. For the H19FR2, two haplotypes and three genotyes were observed, and the Dp, PIC and PE were 0.626, 0.37 and 0.162, respectively. Sequencing results showed that only two of the four reported SNPs, a7342g and g7547a, were detected in H19FR1 in the Chinese Han population, and two new SNPs, g7351c and a7357g, were found. In the H19FR2 region, only one of the three reported SNPs, a8097g, was detected. Based on the methylation status of the genomic DNA, selective detection of the parental alleles for H19FRs was examined by using two types of enzymes, the methylation-sensitive restriction enzyme (msRE) HpaII or HhaI and McrBC. Genomic DNA digested by either HpaII or HhaI, revealed a single band derived from the paternal allele, as a result of cleavage of unmethylated recognition sites on the maternal allele. On the contrary, the use of McrBC, which can digest a methylated paternal sequence, resulted in exclusively amplifying the maternal allele. This parentally imprinted allele (PIA) typing method could be one of the useful techniques for discriminating the parental origin of alleles.

[Haplotype and heteroplasmy in the HV II region of mtDNA in human blood detected by PCR-DGGE].

OBJECTIVE: To establish a simple and effective technique for detecting haplotype and heteroplasmy of mtDNA, and investigate their frequencies in Chinese Han population. METHODS: The fragments from 29-290 nt of mtDNA HV II from peripheral leukocytes of 200 unrelated Wuhan Han individuals were analyzed by using PCR-DGGE technique. RESULTS: Seventeen haplotypes were found in the range of 29-290 nt, and the haplotype diversity (HD) was 0.8844. The heterogeneity was observed from 4 individuals, and its frequency was 2%. CONCLUSION: PCR-DGGE is a simple, sensitive and effective technique in analyzing polymorphism and heteroplasmy of mtDNA, and can be used in forensic practice.

[Genetic polymorphism of 23 Y chromosome biallelic markers in Wuhan Han population].

To search polymorphic Y chromosome biallelic markers in Chinese Han population, and obtain their population genetic data. Genotyping of 23 biallelic markers on human Y chromosome (M7, M9, M50, M88, M89, M95, M111, M117, M119, M121, M122, M134, M159, M164, M175, M214, LINE1, MSY2, RPS4Y711, SRY465, IMS-JST164520, IMS-JST021354 and IMS-JST003305) were carried out in a sample of 160 unrelated Chinese male individuals living in Wuhan using fragment length discrepant allele specific PCR (FLDAS-PCR) and PAGE technique. In all 23 biallelic markers, genetic polymorphism were identified for 20 loci in Wuhan Han population except for M50, M159 and M164, and the ranges of gene diversity (GD) were 0.0126-0.4855. A total of 35 different haplogroups (Hg1-35) were observed and the haplogroup diversity (HD) was 0.9471. The haplogroups formed by 20 biallelic markers are highly polymorphic, and can be used in forensic science and population evolution studies.