Magnetic bead-based separation of sperm cells from semen-vaginal fluid mixed stains using an anti-ACRBP antibody.

Forensic DNA analysis of semen-vaginal fluid mixed stains is essential and necessary in sexual assault cases. Here, we used a magnetic bead conjugated acrosin binding protein (ACRBP) antibody to separate and enrich sperm cells from mixed stains. Previously, western blotting indicated that ACRBP was specifically expressed in sperm cells, but not in female blood and epithelial cells, while immunofluorescence data showed ACRBP was localized to the acrosome in sperm cells. In our study, sperm were separated from mixed samples at three sperm cell/female buccal epithelial cell ratios (103:103; 103:104; and 103:105) using a magnetic bead conjugated ACRBP antibody. Subsequently, 23 autosomal short tandem repeat (STR) loci were amplified using the Huaxia™ Platinum PCR Amplification System and genotyped using capillary electrophoresis. The genotyping success rate for STR loci was 90% when the sperm to female buccal epithelial cell ratio was > 1:100 in mixed samples. Our results suggest that the magnetic bead conjugated ACRBP antibody is effective for isolating sperm cells in sexual assault cases.

Individual Identification in Facial Appearance Biometrics Based on Macroscopical Comparison.

Individual identification is one of the research hotspots in the practice of forensic science, and the judgment is usually built on the comparison of the unique biological characteristics of the individual, such as fingerprints, iris and DNA. With the dramatic increase in the number of cases related to video image investigations, there is an increasing need for the technology to identify individuals based on the macroscopic comparison of facial appearance biometrics. At present, with the introduction of computer three-dimensional (3D) modeling and 3D superimposition comparison technology, considerable progress has been made in individual identification methods based on macroscopic comparison of facial appearance biometrics. This paper reviews individual facial appearance biometric methods based on macroscopical comparison, comprehensively analyzes the advantages and limitations of different methods, and puts forward recommendations and prospects for subsequent research.

Association between MTHFR (677C>T and 1298A>C) polymorphisms and psychiatric disorder: A meta-analysis.

Recent studies showed that genetic polymorphism of 5,10-methylenetetrahydrofolate reductase (MTHFR) is related to attention-deficit hyperactivity disorder (ADHD), bipolar disorder (BD) and schizophrenia (SCZ). However, no consistent conclusion has been determined. This meta-analysis aims to interrogate the relationship between MTHFR gene polymorphisms (677C>T and 1298A>C) and the occurrence of ADHD, BD and SCZ. We retrieved case-control studies that met the inclusion criteria from the PubMed database. Associations between MTHFR polymorphisms (677C>T and 1298A>C) and ADHD, BD and SCZ were measured by means of odds ratios (ORs) using a random effects model and 95% confidence intervals (CIs). Additionally, sensitivity analysis and publication bias were performed. After inclusion criteria were met, a total of five studies with ADHD including 434 cases and 670 controls, 18 studies with BD including 4167 cases and 5901 controls and 44 studies with SCZ including 16,098 cases and 19913 controls were finally included in our meta-analysis. Overall, our meta-analytical results provided evidence that the MTHFR 677C>T was associated with occurrence of BD and SCZ, while the 1298A>C polymorphism was related to ADHD and BD, and additionally the sensitivity analysis indicated these results were stable and reliable. This may provide useful information for relevant studies on the etiology of psychiatric disorders.

Sperm Cell Capture Based on ABH Antigen Differences to Separate Two Men in Mixed Seminal Stains.

Personal identification of two individuals in mixed semen samples in forensic DNA testing in general usually involves analysis using autosomal and Y chromosome short tandem repeats (STRs). Results may exclude unrelated donors but cannot identify individuals. In this study, sperm cell capture based on ABH antigen differences was used to obtain the cells with the single ABO blood type. Immunohistochemical staining using labeled anti-A, anti-B, and anti-H antibodies and the laser microdissection system can be used to enrich sperm with different ABO types in mixed seminal stains from two individuals. Then, PCR amplification and capillary electrophoresis were performed to genotype the STR loci. To some extent, after sperm cell capture based on ABH antigen differences, autosomal STR typing using enriched single blood group cells can be utilized to partially identify different individuals in a mixed seminal stain sample from two individuals.

Genetic polymorphisms of 20 autosomal STR loci in the Han population of Zhangzhou City, Southeastern China.

China harbors 56 ethnic groups and Han is the largest population. It is informative and useful to explore the available population genetic characteristics of Chinese Han population from Fujian Province, Southeast China. In our study, we explored the genetic characteristics of 20 autosomal Short tandem repeat (STR) loci in 1555 unrelated Chinese Han individuals from Zhangzhou City, Southeastern China using the SureID® 21G Human STR Identification Kit. Moreover, phylogenetic analysis was performed between the Zhangzhou Han population and other relevant populations based on the shared autosomal STR genotyping. The neighbor-joining tree and multidimensional scaling analysis were analyzed based on the Nei's standard genetic distance. We found 262 alleles among 1555 unrelated individuals and the corresponding allele frequencies ranged from 0.5521 to 0.0003. The combined power of discrimination and exclusion of the 20 autosomal STR loci were 0.99999999999999999999999943 and 0.999999996166537, respectively. Population comparison revealed that the Zhangzhou Han population were lining up together with the southern Han populations in China while showed significant differences from other China populations. Our results found that the 20 autosomal STR loci in Zhangzhou Han population are meaningful for forensic medicine and human genetic. The genetics characteristic of Zhangzhou Han population is similar with the southern Han population in China.

No association between the Ser9Gly polymorphism of the dopamine receptor D3 gene and schizophrenia: a meta-analysis of family-based association studies.

BACKGROUND: Previous studies found that Ser9Gly (rs6280) might be involved in the occurrence of schizophrenia. However, no consist conclusion has yet been achieved. Compared to the case-control study, the family-based study took into account stratification bias. Thus, we conducted a meta-analysis of family-based studies to measure a pooled effect size of the association between Ser9Gly and the risk of schizophrenia. METHODS: The relevant family-based studies were screened using the electronic databases by the inclusion criteria. Odds ratios (ORs) and 95% confidence intervals (CIs) were used to measure the correction between Ser9Gly polymorphism and schizophrenia susceptibility. Subgroup analysis was performed by stratification of ethnicity (i.e., East Asian, Caucasian, and other populations). Additionally, publication bias was evaluated by the funnel plot. RESULTS: After literature searching, a total of 13 family-based association studies were included, which contained 11 transmission disequilibrium test (TDT) studies with 1219 informative meiosis and 5 haplotype-based haplotype relative risk (HRR) studies. No statistical significance of the heterogeneity was detected in TDT and HRR studies. Thus, the pooled effect size was calculated under the fixed effect model. The results found that the association was significantly protective in East Asian in TDT studies (204 informative meiosis, OR = 0.744, 95% CI = 0.564-0.980, Z-value = - 2.104, p = 0.035). CONCLUSIONS: The meta-analysis based on the family study found a protective association of Ser9Gly in East Asian. In future, large sample molecular epidemiology studies are needed to validate our findings.

Population genetics of 24 Y-STR loci in Chinese Han population from Jilin Province, Northeast China.

BACKGROUND: It is meaningful to expand the available population information on forensic medicine and to investigate the genetic characteristics of Han population from Jilin Province, Northeast China. METHODS: In this study, we investigated the genetic characteristics of 24 Y-chromosomal short tandem repeat (STR) loci in 1,088 unrelated Chinese Han male individuals from Jilin Province, using DNATyperTM Y24 amplification kit. Additionally, we performed the population comparison between the Jilin population and the other nine reported populations based on the Y-STR genotyping haplotypes. RESULTS: A total of 1,067 different haplotypes were found from 1,088 unrelated individuals, of which 1,046 were unique and 21 were shared by two individuals. The gene diversity values of 22 loci ranged from 0.3870 (DYS391) to 0.9668 (DYS385ab). The random match probability was 0.0010 with the discrimination capacity of 0.9807. Population comparison showed that there were minor differences compared to Beijing Chinese Han, China Manchu, Gansu Chinese Han, and Jiangsu Chinese Han, but major differences with respect to the populations of Guangdong Chinese Han, Yunnan Chinese Han, China Hui, China Korean, and China Tibetan. CONCLUSION: Our results showed that the 24 Y-STR loci in Jilin Han population are valuable for forensic application and human genetics.

Mitochondrial DNA control region sequences may differentiate Yanbian Koreans in China from other Asian populations.

BACKGROUND: Sequence polymorphisms of mitochondrial DNA (mtDNA) are valuable in forensic medicine and anthropological genetics. AIM: This study investigated the mtDNA control region sequences in 295 unrelated individuals living in the Yanbian Korean Autonomous Prefecture in the People's Republic of China. SUBJECTS AND METHODS: DNA was extracted from blood stained filter papers. Hypervariable regions of the mtDNA control region (HVI and HVII) were amplified and sequenced. The resulting sequences were aligned and compared with the revised Cambridge reference sequence (rCRS). RESULTS: A total of 182 variations were confirmed. Population comparison showed the significant difference between Yanbian Korean and other included populations. CONCLUSION: These results provide useful data for human genetic studies and forensic examinations and demonstrate that the Yanbian Korean population is an endogamous Northeast Asian group.

[Relationship between corneal thickness and postmortem interval in rabbit].

OBJECTIVE: To explore the relationship between corneal thickness and postmortem interval (PMI) in rabbit. METHODS: The rabbit model was established by air embolism. The rabbit cornea was sampled at 6-hour-interval from 0 to 72 h postmortem. After routine HE staining, the whole cornea image was collected by the optical microscope. Three markers were observed including corneal epithelial thickness (x1), corneal stromal thickness (x2) and whole corneal thickness (x3) using Motic Images Plus 2.0 image analysis software and the data were statistically analyzed to establish the regression function with PMI (y). RESULTS: Within 72 h postmortem, rabbit corneal stromal thickness and whole corneal thickness increased at 12h postmortem and reached the peak at 54h postmortem. The two markers showed positive correlation with PMI. The regression functions of the two markers were y = -0.070 2 x2(2) +11.398 x2 + 1634 (R2 = 0.712 2, P < 0.05) and y = -0.074 9 x3(2) +12.036 x3 + 1819.4 (R = 0.675 0, P < 0.05), respectively. CONCLUSION: The two markers of corneal stromal thickness and the whole corneal thickness showed the strong linear correlation with PMI. The correlation of the corneal stromal thickness is better than the whole corneal thickness. The two markers can be used to estimate PMI.

Cannabinoid receptor type 2 is time-dependently expressed during skin wound healing in mice.

Dynamic localization of CB2R and quantitative analysis of CB2R mRNA during skin wound healing in mice were performed. Co-localization of CB2R with F4/80 or α-SMA was detected by double-color immunofluorescence microscopy. A total of 110 male mice were divided into control, injury, and postmortem groups. Sixty-five mice were sacrificed, followed by sampling at 0.5 h-21 days post-injury. Five mice without incision were used as control. The other 40 mice that received incised wound were sacrificed at 5 days after injury. The samples were collected at 0 h-3 days postmortem. In the uninjured controls, CB2R immunoreactivity was detected in the epidermis, hair follicles, sebaceous glands, dermomuscular layer, and vascular smooth muscle. In the incision groups, polymorphonulcear cells, macrophages, and myofibroblasts showed positive staining for CB2R. Morphometrically, the average ratios of CB2R-positive cells were more than 50 % at 5 days post-wounding, whereas it was <50 % at the other posttraumatic intervals. The average ratios of CB2R-positive macrophages maximized at 3 days post-wounding, and the average ratios of CB2R-positive myofibroblasts peaked at 5 days post-wounding. The relative quantity of CB2R mRNA expression maximized at posttraumatic 5 days in comparison with control as detected by real-time PCR, with an average ratio of >4.10, which was also confirmed by Western blotting. There was no significant change for CB2R protein within 6 h postmortem and for mRNA within 3 h postmortem as compared with the control group. In conclusion, dynamic distribution and expression of CB2R suggest that CB2R is involved in modulating macrophages and myofibroblasts in response to inflammatory event and repair process in mouse skin wound healing, and CB2R is available as a marker for wound age determination.

[Time-dependent recruitment and differentiation of fibrocytes in mouse skin wound healing].

OBJECTIVE: To investigate the time-dependent recruitment and differentiation of fibrocytes in skin wound healing. METHODS: Fibrocytes (expressing CD45 and procollagen I ) and myofibroblasts (expressing CD45 and alpha-SMA) were co-localized by immunofluorescent staining. The number of fibrocytes and myofibroblasts was counted at different post-wounding interval. RESULTS: At 3 d after injury, fibrocytes started to recruit at the margin of the wounds. At 5 d after injury, myofibroblasts started to appear in new formed granulation tissue. The number of fibrocytes and myofibroblasts peaked at 7 d post-wounding. CONCLUSION: During skin wound healing, myofibroblasts in granulation tissue originated at least partly from fibrocytic differentiation. The time-dependent recruitment and differentiation of fibrocytes may provide new information for wound age determination.

Nicotinic acetylcholine receptor α7 subunit is time-dependently expressed in distinct cell types during skin wound healing in mice.

Recent studies have shown that nicotinic acetylcholine receptor alpha7 subunit (nAChRα7) plays an important role in regulation of inflammation, angiogenesis and keratinocyte biology, but little is known about its expression after the skin is wounded. A preliminary study on time-dependent expression and distribution of nAChRα7 was performed by immunohistochemistry, Western blotting and RT-PCR during skin wound healing in mice. After a 1-cm-long incision was made in the skin of the central dorsum, mice were killed at intervals ranging from 6 h to 14 days post-injury. In uninjured skin controls, nAChRα7 positive staining was observed in epidermis, hair follicles, sebaceous glands, vessel endothelium and resident dermal fibroblastic cells. In wounded specimens, a small number of polymorphonuclear cells, a large number of mononuclear cells (MNCs) and fibroblastic cells (FBCs) showed positive reaction for nAChRα7 in the wound zones. Simultaneously, nAChRα7 immunoreactivity was evident in endothelial-like cells of regenerated vessels and neoepidermis. By morphometric analysis, an up-regulation of nAChRα7 expression was verified at the inflammatory phase after skin injury and reached a peak at the proliferative phase of wound healing. The expression tendency was further confirmed by Western blotting and RT-PCR assay. By immunofluorescent staining for co-localization, the nAChRα7-positive MNCs and FBCs in skin wounds were identified as macrophages, fibrocytes and myofibroblasts. A number of nAChRα7-positive myofibroblasts were also CD45 positive, indicating that they originated from differentiation of fibrocytes. The results demonstrate that nAChRα7 is time-dependently expressed in distinct cell types, which may be closely involved in inflammatory response and repair process during skin wound healing.

[Expression of cannabinoid receptor I during mice skin incised wound healing course].

OBJECTIVE: To investigate the expression of cannabinoid receptor I (CB1R) during mice skin incised wound healing course and time-dependent changes of CB1R in wound age determination. METHODS: The changes of CBIR expression in skin incised wound were detected by immunohistochemistry and Western blotting. RESULTS: The control group showed a low expression of CB1R detected mainly in epidermis, hair follicles, sebaceous gland and dermomuscular layer. CB1R expression was undetectable in neutrophils in the wound specimens from 6h to 12h post-injury. CB1R positive cells were mostly mononuclear cells (MNCs) and fibroblastic cells (FBCs) from 1 d to 5 d post-injury. CB1R positive cells were mostly FBCs from 7 d to 14d post-injury. The ratio of the CB1R positive cells increased gradually in the wound specimens from 6 h to 3 d post-injury, reached peak level at 5 d, and then decreased gradually from 7d to 14 d post-injury. The positive bands of CB1R were observed in all time points of the wound healing course by Western blotting. The expression peak showed at 5 d post-injury. CONCLUSION: CB1R is activated during the wound healing course. The expression of CB1R is found in mononuclear cells, which could be involved in inflammation reaction. CBIR is observed in fibroblastic cells, which could participate in the wound healing. CB1R may be a potentially useful marker for determination of wound healing age.

[DNA degradation in nucleolus of skeletal muscle, heart, liver, kidney and brain in mice after death].

OBJECTIVE: To study the change of DNA degradation in nucleolus of mice organs and its relationship with the postmortem interval, and to investigate a new accurate method to estimate the postmortem interval. METHODS: Eight parameters of cell nuclei were chosen, including the head DNA level, the tail DNA level, the head radius, the tail length, the tail moment, the Olive moment, the head area and the tail area. The changes of DNA degradation were analyzed in skeletal muscle, myocardium, liver, kidney and brain in mice at different intervals (0-72 h postmortem) by using single-cell gel electrophoresis and fluorescent microscope connected with auto-analysis-image system. RESULTS: The tail DNA level, the tail length, the tail moment, the Olive moment and the tail area showed an increasing tendency. The head DNA level, the head radius and the head area showed a decreasing tendency within 72h postmortem in mice. A quadratic regression equation (P < 0.001) and multiple regression equation of DNA degradation tendency were established (P < 0.000 1). CONCLUSION: The regression equations established can be used as a new method for estimating postmortem interval in forensic practice.

[The time-dependent changes of phospho-JNK expression during the skin incised wound healing in mice].

OBJECTIVE: To investigate the changes of phospho-JNK (p-JNK) during the incised wound healing of the skin in mice and to explore the rule of the time-dependent change of p-JNK in wound age determination. METHODS: The changes of p-JNK expression in incised skin wound were detected by immunohistochemistry and Western blot. RESULTS: There was a minimal baseline staining of p-JNK in control mouse skin. Changes of p-JNK expression were mainly detectable in neutrophils in the wound specimens from 3 hours to 12 hours after injury. Afterwards, the p-JNK positive cells were mostly mononuclear cells and fibroblasts between post-injury day 1 and day 5, whereas the p-JNK positive cells were mostly fibroblasts between post-injury day 7 and day 14. Morphometrically, the ratio of the p-JNK positive cells to the total increased gradually in the wound specimens from 3 hours to day 1, and maximized at day 1 with a slight decrease from post-injury day 3 to day 5. The ratio showed a second peak in the specimens of day 7, and then decreased gradually from post-injury day 10 to day 14. The changes of p-JNK expression were observed throughout the wound healing stages by Western blot as well, with a peak expression occurring between 12 hour and day 3 after injury. CONCLUSION: p-JNK may play a pivotal role in inducing apoptosis of neutrophils, mononuclear cells, and fibroblasts during skin wound healing and meanwhile, p-JNK may be a potentially useful marker for wound age determination.

[Posttraumatic stress disorder (psychiatric injury) after road traffic accidents in forensic medicine: a primary study].

OBJECTIVE: Studying forensic medical problem related with RTA leading PTSD and supplying accumulating evidence for psychiatric compensation in court. METHODS: One hundred and fifty six victims of RTA were recruited who applied to court for a costs order. The victims were examined for psychiatric diagnosis by psychiatrists and for rank of impairment by experts in forensic clinical medicine. The self-report psychopathological status and quality of life were also measured. RESULTS: Eighty one victims of 156 (51.92%) fulfilled the criteria for PTSD (ICD-10). Morbidity difference in male and female were significant; The more serious extent of impairment is, the more PTSD'possibility is; The scores in World Health Organization Quality of Life were lower and in SAS and SDS were higher in PTSD group than in non-PTSD group. Acquirement of awarded costs could obviously prevent PTSD. CONCLUSION: The higher PTSD incidence existed in the RTA victims who applied to court for a costs order, and acquirement of awarded costs could obviously prevent PTSD.