Objective To investigate the temporal changes of intestinal bacterial communities in rats that died from combined ethanol and carbon monoxide （CO） poisoning using microbiome analysis techniques, and to explore potential correlations between these changes and the postmortem interval （PMI）. Methods Four experimental groups were established： fatal CO poisoning, fatal CO poisoning following ethanol exposure, ethanol exposure, and blank controls. Rectal specimens were collected at 0, 1, 3, 7, and 14 days postmortem. High-throughput sequencing of the 16S rRNA gene was employed to analyze bacterial communities. Bacterial diversity, relative abundance, and community differences were analyzed. A predictive model for PMI estimation using bacterial community succession patterns was constructed using a random forest algorithm. Results The intestinal bacterial community structure of rats changed at both the phylum and genus levels within 14 days postmortem in the experimental groups. The PMI estimation model based on bacterial characteristics demonstrated good predictive performance across different experimental groups, with model fitting accuracies of 0.60, 0.59, 0.61 and 0.60 for each of the four groups. The average prediction error across all groups was approximately 1 day. Conclusion Ethanol exposure, CO poisoning, and the combined effects of ethanol exposure and CO poisoning all affect postmortem bacterial succession in rat cadavers. In forensic practice, it is recommended to adopt a predictive model specifically developed for the corresponding poisoning condition to improve the accuracy of PMI estimation.

Objective To screen biomarkers of acute myocardial ischemia （AMI） using bioinformatics approaches. Methods Datasets related to myocardial ischemia, including GSE415, GSE775, GSE4648, GSE19875 and GSE8322, were downloaded from the Gene Expression Omnibus （GEO） database. The data were divided into an AMI dataset （20 control samples and 5 experimental samples） and the myocardial ischemia dataset （16 control samples and 18 experimental samples）. Weighted gene co-expression network analysis （WGCNA）, least absolute shrinkage and selection operator （LASSO） regression, and receiver operating characteristic （ROC） curve analysis were applied to identify potential biomarkers. A mouse myocardial ischemia model, with myocardial ischemia induced for 15 min and 60 min, was established, and real-time quantitative PCR （qPCR） was utilized to verify whether the potential biomarkers could assist in the identification of AMI. Results The most accurate indicator for identifying AMI was inhibitor of nuclear factor-κB alpha （IκBα, AUC=0.91）. The accurate indicators for identifying myocardial ischemia were B cell translocation gene 2 （BTG2, AUC=0.78） and vesicle transport protein USE1 （AUC=0.77）. The validation results revealed that the expression levels of IκBα differed significantly among the control, myocardial ischemia and AMI groups （P<0.05）. BTG2 expression was the highest in the myocardial ischemia group （P<0.05）, whereas no statistically significant difference in USE1 expression was observed across the three groups （P>0.05）. Conclusion IκBα can distinguish AMI and myocardial ischemia groups, and BTG2 serves as an auxiliary biomarker for the diagnosis of myocardial ischemia.

Objective To investigate the genetic characteristics of homoplasmic and heteroplasmic mitochondrial DNA （mtDNA） variations in four-generation maternal lineages. Methods Buccal swabs were collected from 23 individuals belonging to 4 four-generation maternal lineages. DNA was extracted, and mtDNA whole genome libraries were constructed. Sequencing was performed using the Ion S5 XL sequencing system, followed by sequence alignment and variation analysis. Results Following quality control and analysis of obtained mtDNA whole genome sequencing data, the sequencing coverage reached 99.73%, and the average sequencing depth was 3 458×. A total of 939 genetic variations were observed, including 883 point mutations, 36 insertion/deletion variations, and 20 heteroplasmic variations. No differentiated homoplasmic point mutations were found in the mtDNA sequences of individuals from the same maternal lineage, whereas inconsistent point heteroplasmies （PHPs） were detected. Conclusion Mitochondrial PHP variations can change during inheritance, and there is a risk of using PHP differences as an exclusion criterion for determining whether individuals come from the same maternal lineage. When comparing mtDNA sequences, if the same PHP is observed in two samples with identical haplotypes and its major and minor alleles are consistent, it can be considered as enhanced evidence to determine that the two samples come from the same maternal lineage.

Objective To establish a qualitative and quantitative analytical method for the determination of 21 etomidate analogues and etomidate acid in hair and to investigate its application value in actual forensic cases. Methods Hair samples were extracted following cryogenic wet grinding. The supernatant was filtered through a 0.22 μm organic membrane filter. The Kinetex^® Biphenyl （100 mm×2.1 mm, 1.7 μm） column was used, and gradient elution was performed with methanol and 5 mmol/L ammonium acetate within 15 minutes. The data acquisition was acquired by UPLC-MS/MS with electrospray ionization in positive ion mode, followed by method verification. Results The 21 etomidate analogues and etomidate acid exhibited good chromatographic separation in hair samples. In particular, all eight compounds within three distinct groups of isomers, including propoxate and isopropoxate; butomidate, sec-butomidate, iso-butomidate and tert-butomidate; 1-（methoxycarbonyl）-2-methylpropan-2-yl 1-（1-phenylethyl）-1H-imidazole-5-carboxylate and 1-（methoxycarbonyl）-2-methylpropanyl 1-（1-phenylethyl）-1H-imidazole-5- carboxylate, were effectively separated. The method showed a good linear relationship in the quantitative linear range （r>0.999）， with the limits of detection （LOD） ranging from 0.005 to 0.020 ng/mg and the lower limit of quantification （LLOQ） of 0.05 ng/mg. The selectivity， accuracy and precision of the established method met the verification standards. The matrix effects and extraction recoveries showed good reproducibility at different concentration levels， and the quantitative results for processed hair samples remained stable after storage at 4 ℃ for 36 hours. Practical application results showed that etomidate, metomidate, isopropoxate, sec-butomidate, iso-butomidate, TF-etomidate, and ABP-700, and the metabolite etomidate acid could be detected in the hair of abusers, although the content of etomidate acid was relatively low. Conclusion The method established in this study is simple to perform and provides accurate quantification. It enables the simultaneous determination of etomidate acid, 13 scheduled etomidate analogues, and 8 unscheduled potential derivatives in hair. The analytical scope encompasses alkyl, alkoxy, alkoxycarbonyl, halogen, and sulfhydryl substitutions distributed across the ester side chain, benzene ring, and imidazole ring. It can provide technical support for combating the abuse of etomidate analogue substances.

Objective To investigate a strategy for the qualitative analysis of synthetic cannabinoids with unknown structures in the absence of reference standards. Methods Column chromatography was used to purify the suspicious sample and obtain a high-purity prepared sample. The prepared sample was analyzed and its structure was confirmed by comprehensive use of gas chromatography-mass spectrometry （GC-MS）， ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry （UPLC-QTOF-MS）， and nuclear magnetic resonance spectroscopy （NMR）. The differences between collision-induced dissociation （CID） and electron-activated dissociation （EAD） fragmentation modes of UPLC-QTOF-MS were compared, and the structure was finally confirmed. Results A 129.9 mg high-purity prepared sample was obtained through column chromatography. Through comprehensive analysis of GC-MS, UPLC-QTOF-MS and NMR, it was confirmed that the synthetic cannabinoid with an unknown structure was N-（adamantan-1-yl）-2-[1-（4-fluorobenzyl）-1H-indol-3-yl]acetamide, abbreviated as AFUBIATA. Comparison of the CID and EAD fragmentation modes revealed that the EAD provided richer fragment ion information, which facilitated the structural elucidation of the unknown substance. Conclusion Column chromatography purification yields high-purity samples, while the integration of GC-MS, UPLC-QTOF-MS, and NMR data enables the structural elucidation of synthetic cannabinoids with unknown structures.

Objective To investigate the risk factors associated with mental disability caused by traumatic subdural hydroma （TSH）. Methods A retrospective analysis was conducted on 162 cases of isolated TSH that underwent forensic evaluation at Anhui Wanyi Forensic Appraisal Center and Anhui Huatai Forensic Appraisal Institute from 2007 to 2023. Mental disability was assessed according to the Classification of the Degree of Disability Caused by Human Injury. The cases were grouped according to sex, age, educational level, extent of fluid collection, location of fluid collection, and duration of fluid collection. Differences in mental disability scores among groups were compared using the t test or one-way analysis of variance （ANOVA）. Spearman correlation analysis was performed to evaluate the associations between mental disability scores and the relevant variables, and multiple linear regression analysis was used to identify influencing factors associated with the mental disability scores. Receiver operating characteristic （ROC） curve analysis was performed to evaluate the predictive performance of the influencing factors. Results （1） The TSH disability scores were negatively correlated with age, extent of fluid collection, and duration of fluid collection. （2） Multiple linear regression analysis showed that the duration of fluid collection had the strongest association with TSH mental disability scores, followed by the extent of fluid collection. （3） For a mental disability score ≤10, ROC curve analysis identified an extent of fluid collection involving >2 sites and a duration >51 days as the optimal cutoff values. For a mental disability score ≤9, the corresponding cutoff values were >2 sites and >78 days, respectively. For a mental disability score ≤8, the corresponding cutoff values were >2 sites and >97 days, respectively. Conclusion The risk factors associated with adverse outcomes following isolated TSH are the duration and extent of fluid collection. The duration of fluid collection has a stronger association with mental disability and better predictive performance than the extent of fluid collection. TSH with a duration of >51 days and involving >2 sites is more likely to be associated with mental disability.

Objective To develop an eight-dye fluorescent multiplex amplification system based on mini-STR and eight-dye fluorescent labeling technique, and to evaluate its applicability in the analysis of degraded samples in forensic medicine. Methods An eight-dye fluorescent labeling technique was used to develop a multiplex amplification system targeting 26 loci, including D22S1045, D13S317, CSF1PO, D2S441, D10S1248, TPOX, D16S539, D5S818, Penta D, TH01, D2S1338, Penta E, rs2032678,Amelogenin, D19S433, D21S11, D6S1043, D8S1179, vWA, D7S820, D1S1656, DYS391, D18S51, D12S391, D3S1358, and FGA. The established amplification system was evaluated and tested for its specificity, sensitivity, accuracy, and the ability to analyze degraded samples. Results An eight-dye fluorescent multiplex amplification system containing 26 loci was successfully developed. All amplicons were less than 310 bp in length. The system demonstrated high specificity, sensitivity, and accurate typing, and performed well in the analysis of degraded samples. Conclusion The mini-STR with the eight-dye fluorescent multiplex amplification system has good potential for the analysis of degraded samples and can be applied to forensic casework.

Bloodstains can provide critical information for criminal investigations and serve as important forensic evidence. The discovery of potential bloodstains at a crime scene is crucial, and can be achieved by chemical, physical, and biological means. Biological search is typically represented by canines. Using canines to search for potential bloodstains at crime scenes is a real-time, rapid, effective and non-destructive detection method, which can meet the needs of various detection scenarios. This paper traces the development history of the canine bloodstain search technique and summarizes the olfactory basis of bloodstain search by canines. From the perspectives of using police dogs to search for trace bloodstains, diluted bloodstains, and cleaned bloodstains, and differentiating human blood from animal blood, this paper reviews the current application of the canine bloodstain search technique in forensic investigations, and looks forward to the development of this technique, with the aim of providing a reference for related research on canine bloodstain searches.

DNA identification technology is widely used in forensic individual identification and kinship analysis. However, the high-temperature environment of fire scenes often causes severe DNA degradation, compromising its evidentiary value. Recent studies have found that certain nucleic acid molecules exhibit significant advantages in high-temperature degraded samples due to their unique biological characteristics. Compared with autosomal STRs, some loci of Y-chromosomal STRs （Y-STRs） have shorter amplicons. During the random breakage of DNA strands induced by high-temperature, the use of Y-STRs can effectively reduce the probability that breakpoints occur within the core repeat regions, which significantly improves the success rate of genotyping. Mitochondrial DNA （mtDNA） maintains a stable detection rate even under high-temperature conditions due to its high copy number and double-stranded closed structure. Although microRNAs （miRNAs） cannot be used for individual identification, their high stability allows them to persist at relatively high levels in high-temperature environments, providing a novel molecular tool for estimating the postmortem interval. This paper systematically reviews the genotyping efficiency of nuclear genomic DNA and mtDNA under high-temperature conditions and the factors affecting their performance. It summarizes the regulatory effect of high-temperature on tissue miRNA expression profiles and integrates the mechanisms and contributing factors of heat-induced nucleic acid damage, with the aim of providing a reference for improving the evidentiary value of nucleic acid evidence recovered from fire scenes.

Since the emergence of indole and indazole-3-carboxamide synthetic cannabinoids, they have caused serious public health and social problems. These substances exhibit considerable structural diversity and undergo extensive and rapid metabolism both in vivo and in vitro. Their parent compounds cannot be detected in biological samples, while the precise mechanisms responsible for their harmful effects are still not fully understood. These characteristics present significant challenges for toxicological screening and forensic toxicology case analysis. Given the similar structural and physicochemical properties of indole and indazole-3-carboxamide synthetic cannabinoids, this review conducts a literature survey of recent research on these substances based on structural considerations, and summarizes their pharmacodynamics, pharmacokinetics, and other relevant research findings, with the aim of providing a reference for forensic identification and the regulation of these substances.

Nitrous oxide （N₂O）, also known as laughing gas, is one of the substances used as an alternative to traditional illicit drugs. Abuse of N₂O can cause cognitive impairment and memory deficits. Inhalation of an excessive amount of N₂O within a short period of time may cause systemic hypoxia and, in severe cases, can result in sudden asphyxial death. Based on relevant domestic and international research, this paper systematically reviews the pharmacological and toxicological effects, toxicological data, symptoms of poisoning, in vivo process and toxicological analysis findings from representative cases involving N₂O with the aim of providing a reference for forensic identification.