The Potential of High-throughput Metagenomic Sequencing of Aquatic Bacterial Communities to Estimate the Postmortem Submersion Interval

Human remains can be discovered in freshwater or marine ecosystems, circumstances where insects and other invertebrates have infrequently been used for understanding the time of postmortem submersion. In this study, the identification and succession of epinecrotic bacterial communities on vertebrate remains were described during decomposition in a temperate headwater stream during two seasons (summer and winter). Bacterial communities were characterized with 454 pyrosequencing and analyzed at phyletic and generic taxonomic resolutions. There was a significant increase in genera richness over decomposition during both seasons. Additionally, multivariate statistical modeling revealed significant differences in bacterial communities between seasons at both taxonomic resolutions and siginificant genera differences among sampling days within each season, suggesting a succession of these communities. These data are the first to describe aquatic bacterial succession using high-throughput metagenomic sequencing on vertebrate remains submerged in a freshwater habitat, and provide initial evidence for their potential use in forensic investigations.     

家兔玻璃体液2种酶活性变化与死亡时间的相关性

目的探讨死后家兔玻璃体液胆碱脂酶(CHE)和谷草转氨酶(GOT)活性与死后经过时间(PM I)的相关性。方法应用光度法检测在25~30℃和10~15℃下死后即刻至54h之间玻璃体液CHE和GOT活性。结果死后即刻至54h之间,ChE、GOT活性分别由约850U/L、220U/L逐步降解趋于零。死后6h内,两种酶活性降解存在平台期。统计分析,得出相应的单元和多元回归方程,相关系数或决定系数均在0.896以上,P<0.01。结论两种酶活性的降解规律可作为较为客观的推断PM I的参考指标。     

TUNEL法检测大鼠死后心肌细胞核DNA断裂与死亡时间关系研究

目的用原位脱氧糖核苷酸末端转移酶介导的缺口末端标记法(TUNEL)观察大鼠死后心肌细胞DNA断裂与死亡时间(postmortem interval,PMI)的关系。方法建立SD大鼠死亡模型,在25.1℃,分别于死后6、12、24、36、48、60h提取大鼠心肌组织进行TUNEL检测,显微成像系统采集图像,经计算机图像系统分析及统计学处理。结果死后6~60h,心肌细胞核荧光染色依次由绿色、黄绿色亮度增强、亮黄色到荧光亮度减弱;形态由扁圆形或柱状,大小较均匀,到体积增大、呈毛虫状、多数核碎裂到形态不规整,大部分核形成碎片;心肌组织细胞核的IG,死后6h至36h依次增高,36h达到高峰,随后下降;心肌细胞核DNA的AG死后24h最高。结论死后各时间点,心肌细胞核的荧光颜色和亮度以及细胞核的大小和形态有一定的变化规律。     

87例大脑及脑干挫伤HSP70变化的研究

目的探讨人大脑及脑干挫伤后神经细胞HSP70变化与损伤时间的关系及大脑与脑干HSP70变化的差异。方法对87例闭合性颅脑损伤死亡脑挫伤组织进行病理学及HSP70免疫组织化学染色,结果进行图像分析。结果脑挫伤24h后.HSP70阳性神经细胞最多见,且免疫反应性最强,随后逐渐下降,14d组及30d组HSP70表达与对照组类似。脑挫伤部位可见较多HSP70阳性的淀粉样小体。图像分析显示大脑与脑干HSP70阳性细胞3个参数(A、IOD及AOD)之间呈高度显著正相关或显著正相关,由高至低排列为:A>IOD>AOD。结论神经细胞HSP70免疫组化染色结合图像分析技术作为推断脑挫伤经过时间的参考,大脑与脑干挫伤后神经细胞HSP70变化规律基本一致,但脑挫伤灶周围淀粉样小体形成的意义有待于进一步研究。     

85例成人闭合性脑挫伤组织病理学研究

目的研究大脑及脑干脑挫伤后组织病理学变化及与损伤时间的关系,并比较大脑与脑干损伤之间有无差异。方法对85例闭合性颅脑损伤死亡的脑挫伤组织进行病理学GFAP、HSP70、NF200免疫组织化学染色。结果脑损伤早期出现的胞浆嗜酸性肿胀胶质细胞(ECSG)是脑损伤后一种早期应激性反应,挫伤部位淀粉样小体(CA)的增多及噬神经细胞现象也可在脑挫伤后早期形成,而脑干挫伤ECSG发生率高于大脑。中性粒细胞浸润、泡沫细胞及胶质瘢痕形成有一定时序性。结论ECSG在判断生前脑挫伤及推断脑挫伤时间中有重要的参考价值。在ECSG较多的部位,泡沫细胞及CA也较多,三者之间可能存在联系,并提出CA是脑挫伤后反应性产物之一,可用于推断脑损伤时间。     

尸体甲状腺球蛋白降解及其与死亡时间的关系

目的探讨尸体甲状腺球蛋白降解程度与死亡时间(PM I)的关系。方法死后21d内尸体的甲状腺组织,经免疫组化(EPOS)法染色,观察甲状腺球蛋白的染色反应;对63例阳性染色的甲状腺组织(PM I≤5d)进行计算机图像分析,并对所得图像参数的数据进行统计学分析。结果PM I≤5d的甲状腺组织,甲状腺腺泡上皮细胞胞浆及甲状腺胶质中均显示了不同程度的免疫反应阳性;PM I超过12d,免疫反应阴性;经图像分析及统计学处理,其积分光密度、分布密度和目标面积与PM I之间的确定系数(R2)分别达0.9794,0.9732及0.9884。结论尸体甲状腺组织的甲状腺球蛋白降解程度随PM I不同而变化,二者呈现较强的相关性。     

RNA指标在死后人体皮肤组织中稳定性研究

目的探讨多种RNA指标的表达水平在死后人体皮肤组织中的稳定性。方法收集死亡时间(PMI)明确的人体皮肤组织样本10例,提取总RNA,运用实时荧光定量PCR(qRT-PCR)技术检测ACTB、GAPDH、18S、miR-203、5S、1et-7a、RPS29、U6共计8个RNA指标的表达水平。geNorm软件评估各RNA指标表达水平的稳定性,挑选出稳定的内参指标并分析其表达水平与相关因素(年龄、性别、死因)的关系。结果人体皮肤组织中5S、U6和miR-203表达水平最为稳定,其平均表达量与年龄、性别、及死因无关(P0.05)。结论 5S、U6和miR-203可以作为qRT-PCR方法研究PMI的理想内参指标。     

大鼠尸体细菌演替规律及其在死亡时间推断中的应用(英文)

目的探讨大鼠尸体细菌演替规律并评估其在死亡时间(PMI)推断中的应用价值。方法选取成年雌性SD大鼠,处死后置于纸箱内自然腐败,采集大鼠眼周皮肤、口腔和阴道三个部位的细菌样本,使用纯培养生化方法鉴定菌种,记录菌群构成变化规律。结果大鼠三个部位细菌演替规律显示,葡萄球菌属和奈瑟菌属为死后初期的优势菌属,尤其是金黄色葡萄球菌与乳糖奈瑟菌在死后6 h内相对含量较高。干酪乳杆菌规律地出现在死后第3或第4天,且在死后晚期维持较恒定水平。结论大鼠死后三个部位的正常细菌及腐败细菌呈现一定的演替规律,可用于PMI推断。     

Developmental Times of Chrysomya megacephala (Fabricius) (Diptera: Calliphoridae) at Constant Temperatures and Applications in Forensic Entomology

The characteristic life stages of infesting blowflies (Calliphoridae) such as Chrysomya megacephala (Fabricius) are powerful evidence for estimating the death time of a corpse, but an established reference of developmental times for local blowfly species is required. We determined the developmental rates of C. megacephala from southwest China at seven constant temperatures (16–34°C). Isomegalen and isomorphen diagrams were constructed based on the larval length and time for each developmental event (first ecdysis, second ecdysis, wandering, pupariation, and eclosion), at each temperature. A thermal summation model was constructed by estimating the developmental threshold temperature D₀ and the thermal summation constant K. The thermal summation model indicated that, for complete development from egg hatching to eclosion, D₀ = 9.07 ± 0.54°C and K = 3991.07 ± 187.26 h °C. This reference can increase the accuracy of estimations of postmortem intervals in China by predicting the growth of C. megacephala.     

Predicting the postmortem interval using human intestinal microbiome data and random forest algorithm

Gradual changes in microbial communities in a human body after death can be used to determine postmortem interval (PMI). In this study, gut microflora samples were collected from the vermiform appendix and the transverse colon of human cadavers with PMIs between 5 and 192 h. The results revealed that the appendix might be an excellent intestinal sampling site and the appendix flora had an inferred succession rule during human body decomposition. Firmicutes, Bacteroidetes, and their respective subclasses showed a predictable succession rule in relative abundance over time. A Random Forest regression model was developed to correlate human gut microbiota with PMI. We believe that our findings have increased the knowledge of the composition and abundance of the gut microbiota in human corpses, and suggest that the use of the human appendix microbial succession may be a potential method for forensic estimation of the time of death.