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ObjectiveTo explore the cytotoxicity of four wild mushrooms involved in a case of Yunnan sudden unexplained death （YNSUD）, to provide the experimental basis for prevention and treatment of YNSUD.MethodsFour kinds of wild mushrooms that were eaten by family members in this YNSUD incident were collected and identified by expert identification and gene sequencing. Raw extracts from four wild mushrooms were extracted by ultrasonic extraction to intervene HEK293 cells, and the mushrooms with obvious cytotoxicity were screened by Cell Counting Kit-8 （CCK-8）. The selected wild mushrooms were prepared into three kinds of extracts, which were raw, boiled, and boiled followed by enzymolysis. HEK293 cells were intervened with these three extracts at different concentrations. The cytotoxicity was detected by CCK-8 combined with lactate dehydrogenase （LDH） Assay Kit, and the morphological changes of HEK293 cells were observed under an inverted phase contrast microscope.ResultsSpecies identification indicated that the four wild mushrooms were Butyriboletus roseoflavus, Boletus edulis, Russula virescens and Amanita manginiana. Cytotoxicity was found only in Amanita manginiana. The raw extracts showed cytotoxicity at the mass concentration of 0.1 mg/mL, while the boiled extracts and the boiled followed by enzymolysis extracts showed obvious cytotoxicity at the mass concentration of 0.4 mg/mL and 0.7 mg/mL, respectively. In addition to the obvious decrease in the number of HEK293 cells, the number of synapses increased and the refraction of HEK293 cells was poor after the intervention of Amanita manginiana extracts.ConclusionThe extracts of Amanita manginiana involved in this YNSUD case has obvious cytotoxicity, and some of its toxicity can be reduced by boiled and enzymolysis, but cannot be completely detoxicated. Therefore, the consumption of Amanita manginiana is potentially dangerous, and it may be one of the causes of the YNSUD.

ObjectiveTo explore the possible mechanism of Yunaconitine poisoning by studying the changes of urine metabolic profile in rats chronically poisoned by Yunaconitine via non-targeted metabolomics.MethodsA rat model of Yunaconitine poisoning was established, and a metabolomics method based on UPLC-QTOF-MS technology was used to obtain the urine metabolic profile. Principal component analysis （PCA）, orthogonal projections to latent structures-discriminant analysis （OPLS-DA）, variable importance in projection （VIP） value greater than 1, fold change （FC） value greater than 3 or less than 0.33 and P value less than 0.05 were used to screen potential biomarkers related to the toxicity of Yunaconitine. The metabolic pathway analysis was performed through the MetaboAnalyst website and pathological changes of related tissues were observed.ResultsSixteen potential biomarkers including L-isoleucine were screened, which mainly involved six metabolic pathways including the biosynthesis and degradation of valine, leucine and isoleucine, pentose and glucuronate interconversions, and propanoate metabolism, alanine, aspartate and glutamate metabolism, tyrosine metabolism. Pathological studies showed that rat toxic change in nervous system, liver and cardiac caused by Yunaconitine.ConclusionYunaconitine may cause neurotoxicity, hepatotoxicity and cardiotoxicity by affecting amino acid and glucose metabolism.

Microhaplotypes have been reported for applications in different aspects of human forensics. Next generation sequencing (NGS) possesses several advantages in microhaplotype genotyping. Large amounts of NGS-based microhaplotype data has been accumulated, and bioinformatic software designed for microhaplotype NGS data analysis is in urgent need. In this work, we introduced MHTyper, a Linux/Unix pipeline designed for efficient microhaplotype allele-calling and sequencing depth statistics. We developed an NGS-based 30-plex microhaplotype panel and used MHTyper to analyse the data produced. The allele-calling accuracy of MHTyper was evaluated by manually comparing the MHTyper-reported genotypes with those visualized, along with the well-accepted bioinformatic software Integrative Genomics Viewer. No discordance was observed in all 150 pairs for comparison. These data supported that MHTyper could provide an efficient tool for NGS-based forensic microhaplotype data analysis. [ABSTRACT FROM AUTHOR]

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To investigate the feasibility of DNA analysis from free margin of the nail, genomic DNA was extracted from the free margin of nail clipping of 10 volunteers using the proteinase K/SDS -based organic method, the Chelex-100 method, or a combined method. Target DNA was simultaneously amplified using a fluorescent multiplex AmpFlSTR Identifier kit. The PCR products were analyzed on the ABI PRISM 3130 Genetic Analyzer. The results showed that, compared with profiles achieved by genotyping of blood samples from each volunteer as reference, 100% concordance was achieved using the combined method. The STR genotype profiles obtained through the organic method were acceptable, despite preferential amplification at some loci. In contrast, no readable profiles could be determined when DNA was extracted by the Chelex-100 method, and there were a large number of alleles missing. Our data suggest that free margin of nail can be used for nuclear DNA analysis, but the type of DNA isolation method used is critical. The traditional organic extraction method works reasonably well for free margin nail DNA isolation, and combination of organic extraction and the Chelex-100 method works best.

To investigate the genetic polymorphism of FIBRA,DHFRP2 and ACTBP2 in Yunnan Han population as well as their application in forensic science, EDTA-blood specimens were collected from 200 healthy individuals. The DNA were extracted either by the Chloro form, phenol method or by the Chelex-100 method. The PCR products were analyzed by PAG vertical electrophoresis,following by silver staining. All gene frequencies, discrimination power (DP), exclusion of paternity probability (EPP), heterozygosity (H),polymorphisms information content (PIC),matching probability (PM) as well as the Hardy-Weinberg test were calculated. The obtained data are beneficial in the understanding of population genetics of the three STR loci in Yunnan Han population and the results suggest that these loci are valuable genetic markers for paternity testing and personal identification in forensic science practice.