Role of Reactive Oxygen Species in Triptolide-induced Apoptosis of Renal Tubular Cells and Renal Injury in Rats

This study investigated the role of reactive oxygen species(ROS) in the pathogenesis of triptolide-induced renal injury in vivo.Rats were randomly divided into 4 groups(n=5 in each):triptolide group in which the rats were intraperitoneally injected with triptolide solution at a dose of 1 mg/kg of body weight on day 8;control group in which the rats received a single intraperitoneal injection of 0.9% physiological saline on day 8;vitamin C group in which the rats were pretreated with vitamin C by gavage at a dose of 250 mg/kg of body weight per day for 7 days before the same treatment as the control group on day 8;triptolide+vitamin C group in which the rats were first subjected to an oral administration of vitamin C at a dose of 250 mg/kg of body weight per day for 7 days,and then to the same treatment as the triptolide group on day 8.All the rats were sacrificed on day 10.Blood samples were collected for detection of plasma creatinine(Pcr) and plasma urea nitrogen(PUN) concentrations.Both kidneys were removed.The histological changes were measured by haematoxylin-eosin(HE) staining.The production of ROS was determined by detecting the fluorescent intensity of the oxida-tion-sensitive probe rhodamine 123 in renal tissue.Renal malondialdehyde(MDA) content was meas-ured to evaluate lipid peroxidation level in renal tissue.TUNEL staining was performed to assess apop-tosis of renal tubular cells.Renal expression of apoptosis-related proteins Bcl-2,Bax,Bid,Bad,Fas and FasL,as well as corresponding encoding genes were assessed by Western Blotting and real-time PCR.The results showed that triptolide treatment promoted the generation of a great amount of ROS,up-regulated the expression of Bax,Bid,Bad,Fas and FasL at both protein and mRNA levels,as well as the ratio of Bax to Bcl-2,and caused the apoptosis of renal tubular cells and renal injury.However,pretreatment with an antioxidant,vitamin C,significantly reduced the generation of ROS and effectively inhibited the triptolide-induced apoptosis of renal tubular cells and renal injury.It was concluded that ROS plays a critical role in triptolide-induced apoptosis of renal tubular cells and renal injury.The protective administration of vitamin C may help alleviate triptolide-induced renal injury and nephrotoxicity.

Bioluminescent Assay of Microbial ATP in Postmortem Tissues for the Estimation of Postmortem Interval

To study the relationship between changes of microbial ATP in four kinds of murine tis-sues and the postmortem interval (PMI), healthy SD rats were sacrificed and their muscles, livers, spleens and kidneys were sampled at different postmortem intervals. The concentration of microbial ATP was detected using bioluminescent assay and the data was statistically analyzed. The concentra-tion of microbial ATP in muscle increased with PMI time. The peak appeared at the 7th day after death, and at the 10th day, microbial ATP in muscle tissue increased again. In internal organs, the peaks of microbial ATP were observed at the 8th day after death and the level decreased during 8–10 d. The differences in microbial ATP concentration in liver, spleen and kidney were not statistically significant. During day 0 to day 9 after death, the correlation was best between PMI and microbial ATP in muscle. With PMI as the independent variable, the cubic polynomial regression equation was Y=0.02X3–0.166X2–0.666X+13.412 (R2=0.989, P<0.01). In internal organs, the best correlation was found between PMI and microbial ATP during day 0 to day 10. With PMI as the independent variable, the cubic polynomial regression equation was Y=0.016X3–0.127X2–0.809X+13.324 (R2=0.986, P<0.01). There existed high correlations between PMI and microbial ATP concentration in rat tissues. Since only a small amount of tissue was needed for the detection and the sample was not affected by self-decomposition, the method may extend the time range of PMI estimation.

Spectrophotometric Determination of Trimethylamine-nitrogen in Cadaver Tissues for the Estimation of Late Postmortem Interval:A Pilot Study

To study the relationship between the late postmortem interval (PMI) and trimethyl-amine-nitrogen (TMA-N) in postmortem tissues of cadaver, TMA-N in muscles, livers and kidneys of rats was measured at different postmortem intervals (PMI) by using a modified spectrophotometric method. The results indicated that the detection sensitivity of TMA-N was 1 mg/L, and there was a good linear correlation between the value of absorbance (A value) and TMA-N at the concentration of 1-10 mg/L (R2=0.9991). Although TMA variation in muscles was different from that in inner organs during the time since death, TMA-N changes in cadaver tissues was positively correlated with PMI. During 2 to 7d since death, the best correlation between PMI and TMA-N concentration was found in muscles. With PMI as an independent variable, the cubic polynomial regression equation was y=-0.457x3 +6.519x2-24.574x+27.207 (R2=0.969). During 3 to 8 days since death, PMI was best correlated with TMA-N concentration in inner organs. With PMI as the independent variable, the cubic polynomial regression equation was y=0.509x3-9.153x2+55.727x-95.819 (R2=0.953). It was concluded that TMA-N in tissues could be used as a new estimator for late PMI. The method used in this study offered advantages such as accuracy, sensitivity, little samples required and wide PMI estimation.

Characterization of Protein in Old Myocardial Infarction by FTIR Micro-spectroscopy

The aim of the present study was to assess whether Fourier transform infrared spectrometry (FTIR) micro-spectroscopy could produce distinct spectral information on protein of old myocardial infarction (OMI) and to set them as molecular markers to diagnose atypical OMI. Paraffin-embedded heart samples were derived from victims dying of OMI. In combination with histological stain, FTIR and infrared micro-spectroscopy, the characteristics of OMI were analyzed morphologically and molecularly. The most relevant bands identified were the amide A, B, Ⅰ and Ⅱ, showing crucial spectral differences between apparent normal region and OMI region, including the peak position blue shift and the increased intensity of OMI, moreover relative increase in α-helix and decrease in β-sheet of protein secondary structures in OMI. Comparing to single spectral band, the I1650/I1550 ratio was increased and rationally used as a molecular marker for diagnosing OMI. These novel preliminary findings supported further exploration of FTIR molecular profiling in clinical or forensic study, and were in accordance with histopathology.

The Exact Estimation of Visual Acuity by VEP Technology:A Report of 726 Cases of Eye Injury

This study explored the accuracy of using visual evoked potentials(VEP)technology for visual acuity estimation.The enrolled 726 patients with post-traumatic unilateral decrease in visual acuity included the injured eyes served as the experimental group,and the healthy eyes as the control group.The least signal visual angle(LSVA),and amplitude and latency of P100 were chosen as test indexes.The results under different experimental conditions were recorded by PRVEP technology.All data collected were processed and analyzed by SPSS software.The results showed that the coincidence between subjective and VEP visual acuity was 96.7% in control group,but there was very significant difference in experimental group.It was concluded that with the regression formulation for the amplitude of P100 and vision under LSVA,visual acuity can be estimated more accurately and impartially.

Determination of the Early Time of Death by Computerized Image Analysis of DNA Degradation: Which Is the Best Quantitative Indicator of DNA Degradation?

This study evaluated the correlation between DNA degradation of the splenic lympho-cytes and the early time of death,examined the early time of death by computerized image analysis technique(CIAT) and identified the best parameter that quantitatively reflects the DNA degradation.The spleen tissues from 34 SD rats were collected,subjected to cell smearing every 2 h within the first 36 h after death,stained by Feulgen-Van's staining,three indices reflecting DNA content in splenic lymphocytes,including integral optical density(IOD),average optical density(AOD),aver-age gray scale(AG) were measured by the image analysis.Our results showed that IOD and AOD decreased and AG increased over time within the first 36 h.A stepwise linear regression analysis showed that only AG was fitted.A correlation between the postmortem interval(PMI) and AG was identified and the corresponding regression equation was obtained.Our study suggests that CIAT is a useful and promising tool for the estimation of early PMI with good objectivity and reproducibility,and AG is a more effective and better quantitative indicator for the estimation of PMI within the first 36 h after death in rats.

On the Distribution of Acidic Proline-rich Proteins Phenotypes in Chinese Han Population in Liaoning Area

The distribution of salivary acidic proline-rich proteins pheno-types was investigated by using the ultra-thin PAGIEF technique in 258 ChineseHan populations in Liaoning area. The gene frequencies were as follows: Pr^10.8101, Pr^20.1899; Db^+0.0416, Db^-0.9584; Pa^+0.1717, Pa^-0.8283; PIF^+0.6647, PIF^-0.3353. The observed numbers of the phenotypes are in good a-greement with the expected numbers under the Hardy-Weinberg equilibrium.The gene frequencies among the Chinese and other populations are compared.

Modified Method for Showing Gc Subtypes and the Discovery of Two New Variants of Gc Subtypes from Chinese

An improved method for showing Gc band by sulfosalicylic acidafter IEF is reported. This method provides us an effective mean to identify theGc subtypes and its variants within 4 h without using anti-serum for immunofix-ation. And new variants Gc1c3 and Gc2c7 are discovered in Chinese people, andtheir frequencies are 0. 0008 and 0. 0004 respectively.

Typing of 24 mtDNA SNPs in a Chinese Population Using SNaPshot Minisequencing

Three SNaPshot multiplex assays were developed to test 23 coding region single nucleotide polymorphisms(SNPs) and one control region SNP outside hypervariable regions(HVR)Ⅰand Ⅱ,which was aimed at increasing the discrimination power of the mitochondrial DNA(mtDNA) typing in forensic casework,and confirming haplogroup assignments of mtDNA profiles in both human population studies and medical research.The selected SNPs targeted the East Asian phylogeny.These multiplex assays were validated by comparing with the sequencing analysis of samples chosen randomly.The mtDNA variations of 100 unrelated individuals from the Wuhan population in China were examined and classified into 31 haplotypes,and the haplotype diversity was estimated to be 0.952.The multiplex SNaPshot method is rapid and robust,and suitable for large-scale screening studies of mtDNA variability.

Correlations between Event-related Potentials with Pictures Recognition and WMS-RC Scores in Patients with Memory Disorder Caused by Severe Traumatic Brain Injury

This study explored the possibility of using event-related potentials (ERP) for the measurement of picture-recognition memory and examined its correlation with the Chinese Wechsler Memory Scale-revised (WMS-RC) in patients with memory disorder caused by severe traumatic brain injury (sTBI). The subjects included 20 sTBI patients with memory disorder and 22 healthy individuals. Memory function was measured by using WMS-RC. Behavioral and ERP responses were recorded on-line during performance on a battery of picture recognition and the responses were analyzed off-line for recognition memory effects. Mean memory quotient (MQ) of patients with sTBI was significantly lower than that of the control group. Mean reaction time (RT) was significantly longer and the mean correctness rate (CR) of picture recognition was significantly lower in sTBI group than that of the controls. In controls, the main components of average ERP of picture recognition includes two positive-going waves, designated as P170 and P500, that appear 170 ms and 500 ms after stimulation when the subject could later successfully recall and recognize the pictures. P500 amplitude of target stimulus was significantly higher than that of non-target stimulus. Compared to controls, P500 responses of sTBI group were significantly delayed in latency (P<0.001) and lower in amplitude (P<0.001). P500 latency showed significant negative correlation with MQ and the scores of "addition", "visual recognition", "picture recall", "visual reproduction" and "tactile memory" in WMS-RC. ERP of picture recognition provides a neurophysiological approach to directly assess memory impairment, and P500 may serve as a helpful index for memory disorder caused by sTBI in forensic practice.

Violent offences of methamphetamine users and dilemmas of forensic psychiatric assessment

Methamphetamine(MA),an extremely addictive synthetic stimulant,has quickly spread to become the most frequently used illicit drug in China.People with a history of chronic and heavy MA use have a high possibility of exhibiting schizophrenia-like psychotic symptoms,mainly delusions of reference,auditory hallucinations and cognitive deficits.These emerging findings suggest MA use increases aggression and violence and that there is a correlation between MA use and violence.However,it is unclear how to assess the capacity of criminal responsibility in“MA-induced”psychosis and how to set clear boundaries between schizophrenia and MA-induced psychosis when only limited and inconsistent evidence is available.Furthermore,a final persuasive differential diagnostic method based on improved understanding of schizophrenia and MA-induced psychotic disorders has yet to be developed.This paper will evaluate the epidemiology,social harm,and forensic psychiatric assessment of MA users,propose a future direction for the differential diagnosis between MA-induced psychotic disorders and schizophrenia,and put forward some practical solutions to assess the capacity of criminal responsibility of defendants with drug-induced psychotic disorder.

Characteristics of Mechanical Asphyxia:A Retrospective Epidemiological Study in Two Developed Cities in China

Background:Mechanical asphyxia(MA)is multi-classified.Some epidemiological characteristics are shared and the others different among different MA types.Aims and Objectives:To reveal the characteristics of different types of MA and provide reference data for forensic identification of MA.Materials and Methods:We analyzed 913 cases to reveal the epidemiological characteristics of MA in Dongguan and Guangzhou,China.Results:Seven types of MA were identified and strangulation accounted for up to 89.2%.Those aged 20-29 were the most susceptible to all types,except for traumatic and positional asphyxia.Suicide accounted for 96.6%of hanging cases,and accident was the main manner of death in cases of choking,traumatic,and positional asphyxia.Homicide was the most common in cases of ligature strangulation(LS),manual strangulation(MS),smothering,and asphyxia concomitant with lethal mechanical injury.The homicide rate in LS was positively correlated with the rate of female victims(P<0.001,OR=21.14).In MS,the ratio of female:male was 101:15 in homicide.Compared to native residents,migrant workers were more likely to suffer from LS and MS.Rope was used in 50.8%of hanging cases,while LS tools were evenly distributed.Ligature marks were mostly found above the thyroid cartilage in both hanging and LS cases.The U shaped marks were shown in 51.01%of hanging cases but absent in LS cases.In LS,ligature marks completely encircling the neck,discontinuous marks,as well as≥two marks,were more(P<0.001)than in hanging.In LS and MS cases,abrasion and subcutaneous hemorrhages on the neck were more frequent than in hanging cases(P=0.004 and P<0.001),but the difference in frequency of blister marks was opposite(P<0.001).Conclusion:In summary,this study revealed the epidemiological characteristics of the MA cases in Guangzhou and Dongguan cities.The characteristics of death scene and autopsy findings are important in differentiation of different types of MA,especially between suicide and homicide.

Guide to forensic pathology practice for death cases related to coronavirus disease 2019(COVID-19)(Trial draft)

Autopsy is of great significance for elucidating the pathological changes,pathogenesis and cause of death of Coronavirus Disease 2019(COVID-19)and can provide a theoretical basis for scientific and accurate prevention and control of its outbreak.Based on related laws and regulations,such as the Law of the People's Republic of China on Prevention and Control of Infectious Diseases,clinical manifestations and epidemiological characteristics of COVID-19 and guidelines on the prevention and control of this epidemic,combined with the practical work of forensic pathology examinations,we developed the Guide to Forensic Pathology Practice for Death Cases Related to Coronavirus Disease 2019(COVID-19)(Trial Draft).This guide describes the background investigation of the death cases,autopsy room requirements,personal pre-vention and protections,external examinations,autopsy practices and auxiliary examinations,and thus offers a reference for forensic and pathological examination institutions and staff.

Regulatory variation within 3’UTR of STAT5A correlates with sudden cardiac death in Chinese populations

Definitive diagnosis to sudden cardiac death(SCD)is often challenging since the postmortem examination on SCD victims could hardly demonstrate an adequate cause of death.It is therefore important to uncover the inherited risk component to SCD.Signal transducer and activators of transcription 5 A(STAT5A)is a member of the STAT family and a transcription factor that is activated by many cell ligands and associated with various cardiovascular processes.In this study,we performed a systematic variant screening on the STAT5A to filter potential functional genetic variations.Based on the screening results,an insertion/deletion polymorphism(rs3833144)in 3’UTR of STAT5A was selected as the candidate variant.A total of 159 SCD cases and 668 SCD matched healthy controls was enrolled to perform a case-control study and evaluate the association between rs3833144 and SCD susceptibility in Chinese populations.Logistic regression analysis showed that the deletion allele of rs3833144 had significantly increased the SCD risk(odds ratio(OR)=1.54;95%confidence interval(CI)=1.18-2.01;P=0.000955).Further genotype-expression eQTL analysis showed that samples with deletion allele appeared to lower expression of STAT5A,and in silico prediction suggested the local 3 D structure changes of STAT5A mRNA caused by the variant.On the other hand,the bioinformatic analysis presented that promoters of RARA and PTGES3L-AARSD1 could interact with rs3833144,and eQTL analysis showed the higher expression of both genes in samples with deletion allele.Dual-luciferase activity assays also suggested the significant regulatory role of rs3833144 in gene transcription.Our current data thus suggested a possible involvement of rs3833144 to SCD predisposition in Chinese populations and rs3833144 with potential function roles may become a candidate marker for SCD diagnosis and prevention.

Profile Interpretation of Extremely Long Alleles at DYF387S1 and DYS447 Migrated into Allele Range of Adjacent Loci

Two rare cases of long alleles at Y‑chromosome short tandem repeat(Y‑STR)loci(DYF387S1 and DYS447)were identified when two father-son pairs were analyzed by multiplex amplification.“Null alleles”were observed at DYF387S1 and DYS447,and duplicated alleles were displayed at DYS533 and DYS19.We secondly amplified DYF387S1,DYS533,DYS447,and DYS19 loci by singleplex polymerase chain reaction(PCR)and sequence analysis of the long alleles at DYF387S1 and DYS447 loci.The results showed that alleles from DYF387S1(allele 55)and DYS447(allele 41)were longer than their common sizes in the allelic ladder ranges(33-42 for DYF387S1 and 18-30 for DYS447)and located in the neighboring loci(DYS533 and DYS19,respectively).Therefore,to identify these cases involving this unusual phenomenon,not only re‑amplification using the same kit but also additional amplification(using alternative multiplex kits with different adjoining markers or additional singleplex PCR amplification)should be performed to avoid misinterpreting Y‑STR profiles.

A Case of Maternal Half‑sisters Sharing Alleles at 18 X‑chromosomal Short Tandem Repeat Loci

Analysis of X‑chromosome short tandem repeats(STRs)is very helpful in deficiency paternity testing.Here,we reported a case of kinship analysis that showed a potentially erroneous inclusion of paternal sisters between two women.The two women shared alleles at 18 X‑chromosomal STR loci spanned from 14.76cM(DXS6807)to 184.19cM(DXS7423).When their relatives were not available for testing,biostatistical analysis for the 18 X‑chromosomal STR loci and 24 autosomal STR loci revealed the most possible relationship between the two women was paternal sisters.However,when the father of one woman was available,the other father‑daughter possibility was excluded.In the end,the likelihood ratio of STR marker and mitochondrial DNA(mtDNA)sequences confirmed the two women were maternal sisters.This case emphasizes a cautionary interpretation of X chromosomal marker in deficiency paternity cases with female offspring.Even though large parts of the X‑chromosome haplotypes shared by two females,additional relatives and extended DNA typing(such as mtDNA)may be needed further to ascertain whether they are paternal or maternal sisters.

Post‑traumatic Stress Disorder:Relationship to Traumatic Brain Injury and Approach to Forensic Psychiatry Evaluation

Posttraumatic stress disorder(PTSD)has an important and complex relationship with traumatic brain injury(TBI).The prevalence of comorbid PTSD and TBI is increasing in both military and civilian populations.Moreover,TBI has emerged as an important risk factor for the development and manifestation of PTSD.Meanwhile,PTSD is also a significant mediator of the negative sequelae of TBI.PTSD and TBI,especially mild TBI(mTBI),have overlapping neural substrates and neuroanatomical functional features.Given that comorbid PTSD and TBI remain a challenge for forensic psychiatry evaluation,we review the relationship between the two disorders and discuss special considerations during evaluation of the condition.

Chinese Forensic Psychiatry: History, Development and Challenges

China has a long history of forensic psychiatry,which can be traced even back to Zhou dynasty(11th century B.C.).However,modern forensic psychiatry has not been set up in China until the 1970s.After the interruption of Cultural Revolution of 1966‑1976,there was a period of rapid development.In the past five years,a series of judicial identification system reforms have been taking place.In 2010,China had 225 forensic psychiatry agencies,2,090 forensic psychiatry experts and 40,822 cases.The experts are organized within the separate agencies of psychiatric hospitals,universities,the Institute of Forensic Sciences of the Ministry of Justice and other forensic agencies.Apart from criminal forensic psychiatry,the experts are often asked to evaluate civil competency for the clients involved in civil litigation.Chinese forensic psychiatry has developed rapidly during the past 30 years,and formed the characteristics of its own under the special legal system.However,there is still much work for Chinese forensic psychiatrists to do for the future.

Dihydromyricetin promotes apoptosis, suppresses proliferation and tumor necrosis factor-α-mediated nuclear factor kappa-B activation in nasopharyngeal carcinoma CNE-2 cells

OBJECTIVE: To investigate the efficacy of dihydromyricetin(DMY) on nasopharyngeal carcinoma(NPC) cell proliferation, apoptosis and to reveal the underlying mechanism in vitro experiments.METHODS: The CNE-2 cell line was treated with different concentrations of DMY and the effects of DMY on cell viability and proliferation were evaluated using cell counting kit-8(CCK-8) assay and plate colony formation assay. Cellular apoptosis was detected by flow cytometry following Annexin V fluorescein isothiocyanate/propidine iodide staining.Nuclei morphology was observed under a fluorescence microscope following Hoechst 333258 staining. The expression of phosphorylated inhibitor of nuclear factor kappa-B kinase subunit beta(p-IKKβ), phosphorylated inhibitor of nuclear factor kappa-B kinase subunit alpha(p-IKKα), inhibitor of nuclear factor kappa-B alpha(IκB-α), nuclear factor kappa-B(NF-κB)/p65 was examined by Western blot analysis and the nuclear translocation of NF-κB/p65 was observed using a confocal laser scanning microscopy.RESULTS: DMY inhibited the proliferative capability and colony formation of NPC CNE-2 cells. Meanwhile, DMY induced apoptosis of CNE-2 cells in a dose and time-dependent manner via upregulating B-cell lymphoma-2 associated X, but downregulating B-cell lymphoma-2 and pro-caspase-3. Importantly, we found that DMY suppressed tumor necrosis factor alpha(TNF-α)-mediated NF-κB activation via inhibiting p-IKKβ, p-IKKα and blocking NF-κB subunit p65.CONCLUSION: Our experiments demonstrated that DMY had significant antiproliferative and apoptosisinducing effects on CNE-2 cells. Additionally, DMY promoted inactivation of p-IKKβ, p-IKKα,and blocked the nuclear translocation of NF-κB subunit p65. These results suggest that DMY may be an important therapeutic approach for NPC.

Ketamine Alleviates Fear Generalization Through GluN2B-BDNF Signaling in Mice

Fear memories are critical for survival.Nevertheless,over-generalization of these memories,depicted by a failure to distinguish threats from safe stimuli,is typical in stress-related disorders.Previous studies have supported a protective role of ketamine against stress-induced depressive behavior.However,the effect of ketamine on fear generalization remains unclear.In this study,we investigated the effects of ketamine on fear generalization in a fear-generalized mouse model.The mice were given a single sub-anesthetic dose of ketamine(30 mg/kg,i.p.)1 h before,1 week before,immediately after,or 22 h after fear conditioning.The behavioral measure of fear(indicated by freezing level)and synaptic protein expression in the basolateral amygdala(BLA)and inferior-limbic pre-frontal cortex(IL-PFC)of mice were examined.We found that only ketamine administered 22 h after fear conditioning significantly decreased the fear generalization,and the effect was dose-dependent and lasted for at least 2 weeks.The fear-generalized mice showed a lower level of brainderived neurotrophic factor(BDNF)and a higher level of GluN2B protein in the BLA and IL-PFC,and this was reversed by a single administration of ketamine.Moreover,the GluN2B antagonist ifenprodil decreased the fear generalization when infused into the IL-PFC,but had no effect when infused into the BLA.Infusion of ANA-12(an antagonist of the BDNF receptor TrkB)into the BLA or ILPFC blocked the effect of ketamine on fear generalization.These findings support the conclusion that a single dose of ketamine administered 22 h after fear conditioning alleviates the fear memory generalization in mice and the GluN2B-related BDNF signaling pathway plays an important role in the alleviation of fear generalization.