Screening and identification of autophagy-related biomarkers for oral squamous cell carcinoma (OSCC) via integrated bioinformatics analysis.

Increasing evidences have showed that autophagy played a significant role in oral squamous cell carcinoma (OSCC). Purpose of our study was to explore the prognostic value of autophagy-related genes (ATGs) and screen autophagy-related biomarkers for OSCC. RNA-seq and clinical data were downloaded from The Cancer Genome Atlas (TCGA) database following extracting ATG expression profiles. Then, differentially expressed analysis was performed in R software and a risk score model according to ATGs was established. Moreover, comprehensive bioinformatics analyses were used to screen autophagy-related biomarkers which were later verified in OSCC tissues and cell lines. A total of 232 ATGs were extracted, and 37 genes were differentially expressed in OSCC. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis demonstrated that these genes were mainly located in autophagosome membrane and associated with autophagy. Furthermore, the risk score on basis of ATGs was identified as potential independent prognostic biomarker. Moreover, ATG12 and BID were identified as potential autophagy-related biomarkers of OSCC. This study successfully constructed a risk model, and the risk score could predict the prognosis of OSCC patients accurately. Moreover, ATG12 and BID were identified as two potential independent prognostic autophagy-related biomarkers and might provide new OSCC therapeutic targets.

[Pathological changes in rats with acute Dysosma versipellis poisoning].

OBJECTIVE: To observe the pathological changes of major organs in rats with acute Dysosma versipellis poisoning and investigate the toxic mechanism and the injuries of target tissues and organs. METHODS: Forty Sprague-Dawley (SD) rats were randomly divided into three experimental groups, which were given the gavage with 0.5, 1.0 and 2.0 LDo doses of Dysosma versipellis decoction, and one control group, which was given the gavage with 1.0 LD0 dose of normal saline. The rats were sacrificed 14 days after Dysosma versipellis poisoning and samples including brain, heart, liver, lung, and kidney were taken. After pathological process, the pathological changes of the major organs and tissues were observed by light microscope and electron microscope. The experimental data were statistical analyzed by chi2 test. RESULTS: The observations of light microscopy: loose cytoplasm of neurons with loss of most Nissl bodies; swelling of myocardial cells with disappearance of intercalated disk and striations; hepatocellular edema with ballooning degeneration; and swelling epithelial cells of renal proximal convoluted tubule with red light coloring protein-like substances in the tube. The observations of electron microscopy: the structures of cell membrane and nuclear membrane of neurons were destroyed; cytoplasm of neurons, obvious edema; and most organelles, destroyed and disappeared. The mortalities of rats after acute poisoning of the four groups increased with doses (P < 0.05). CONCLUSION: Acute Dysosma versipellis poisoning can cause multi-organ pathological changes. There is a positive correlation between the toxic effect and the dosage. The target tissues and organs are brain (neurons), heart, liver and kidney.

[Expression of fas protein of myocardium in dilated cardiomyopathy].

OBJECTIVE: To investigate Fas protein expression of the myocardium in dilated cardiomyopathy (DCM) and its relationship with occurrence of sudden death caused by DCM. METHODS: Nine autopsy cases of sudden death caused by DCM along with the heart samples were chosen from the archives in the Department of Forensic Medicine, Tongji Medical College, HUST from 1997 to 2007. Other 11 cases which died of violence and other diseases were selected as the control group. Expressions of myocardial Fas protein in the samples were quantitatively detected by immunohistochemistry and computerized imaging analysis. RESULTS: Myocardial Fas protein expression increased significantly in the DCM group. Positive color showed brown-yellow granulated or striped distribution in the longitudinal section of myocardial within the cell membrane and cytoplasm, and showed circular brown granules in the cross section of the cell membrane, while these changes were not observed in the control group though there was focal weak staining noted. Statistical significance was observed between the experimental and control groups (P = 0.002), but no statistical significance was found for the average optical density value between these two groups (P = 0.675). CONCLUSION: The expression of Fas protein increased obviously in the DCM group. Such alteration in expression quantity and distribution of myocardial Fas protein may be related to arrhythmia and heart failure in the patients with DCM.

[Comparative analysis of 607 autopsy cases of poisoning death].

OBJECTIVE: To provide references for forensic expertise by investigating the kinds of toxicant, routes of exposure and manners of poisoning deaths, etc. METHODS: Six hundred and seven autopsy cases of poisoning deaths from 1957 to 2008 in Department of Forensic Medicine, Tongji Medical College (Tongji Forensic Science Identification Center of Hubei), were comparatively reviewed. RESULTS: In 218 cases from 1999 to 2008, more than 50% of decedents were male in the ages of 30-49. The toxicants are usually taken orally and the most common manner of death was accidental. The common substances involved in poisoning death were rodenticide, poisoning gas and insecticide. Compared to the data of 1983-1998 and 1957-1982, the common toxic agents had changed significantly. The number of cases involving insecticide and cyanide poisoning decreased in recent years, and the number of cases of rodenticide, poisoning gas, alcohols poisoning displayed an increase tendency, especially for drugs abuse. CONCLUSION: Poisoning deaths of pesticides remain a major public health problem for a long time and the awareness of prevention need to be raised, especially for the prevention of deaths from multiple poisons.

Pan-Cancer Analyses of the Tumor Microenvironment Reveal That Ubiquitin-Conjugating Enzyme E2C Might Be a Potential Immunotherapy Target.

Increasing evidence indicated that the tumor microenvironment (TME) played a crucial role in cancer initiation and progression. Ubiquitin-conjugating enzyme E2C (UBE2C) was differentially expressed in many cancer types. However, the immunological and prognostic roles of UBE2C were unclear. Differentially expressed genes (DEGs) of 29 cancer types were downloaded from GEPIA2 and 4 cancer types failed to download owing to no DEGs. Furthermore, the gene expression profiles, mutation data, and survival data of 33 cancer types were obtained from UCSC Xena. Clinical stage relevance, tumor mutational burden (TMB), TME relevance analysis, and gene set enrichment analysis (GSEA) of DEGs in 33 cancer types were performed. And DEGs were identified in oral squamous cell carcinoma (OSCC) by biological experiments. Previous studies indicated that UBE2C was related to the prognosis of many cancers. In our study, the higher UBE2C expression level meant a terminal clinical stage in 8 cancer types and the expression level of UBE2C was related to TMB in 20 cancer types. In addition, both immune relevance analysis and GSEA showed that UBE2C might participate in immune response in many cancers. Furthermore, the UBE2C mRNA level and protein level were all identified as upregulated in OSCC cell lines and tissues. UBE2C was differentially expressed in many cancer types and related to the pathogenesis and TME of many cancers, which might be a potential diagnostic and therapeutic biomarker.

Bioinformatics Analyses Indicate That Cathepsin G (CTSG) is a Potential Immune-Related Biomarker in Oral Squamous Cell Carcinoma (OSCC).

PURPOSE: Plenty of studies showed that the immune system was associated with cancer initiation and progression. This study aimed to explore the prognostic biomarkers from immune-related genes (IRGs) in oral squamous cell carcinoma (OSCC). MATERIALS AND METHODS: RNA-seq data were downloaded from The Cancer Genome Atlas (TCGA) and IRGs and transcription factors (TFs) were extracted. Then, the co-expression network between IRGs and TFs was constructed using the "WGCNA" package in R software. Furthermore, a gene expression signature according to IRGs was constructed to predict OSCC prognosis and its accuracy was validated by survival analysis. Subsequently, correlation analyses between risk-score and immune cells level and clinical parameters were performed. Finally, immune-related biomarkers were selected and further investigated using gain-of-function assays in vitro. RESULTS: A total of 32 normal cases and 317 OSCC cases were selected in our study. Differentially-expressed analysis indicated that there were 381 differentially-expressed IRGs and 62 TFs in OSCC. Among them, 25 TFs and 21 IRGs were enrolled in the co-expression network. Furthermore, we found that gene expression signature on the basis of 10 IRGs could predict the prognosis accurately and a high-risk score based on gene expression signature meant a high T classification, terminal clinical stage, and low immune cells level in OSCC. Finally, cathepsin G (CTSG) was identified as a potential immune-related biomarker and therapeutic target in OSCC. CONCLUSION: In conclusion, IRGs were directly involved in the development and progression of OSCC. Furthermore, CTSG was identified as a potential independent biomarker and might be an immunotherapeutic target in OSCC treatment.

[Effects of triptolide on hypothalamic-pituitary-adrenal axis of rats].

OBJECTIVE: To observe the effects of triptolide on the hypothalamic-pituitary-adrenal axis (HPAA) of rats in light of morphological and functional changes. METHODS: Thirty Sprague-Dawley (SD) male rats were randomized into 3 groups and given 2% propylene glycol, mixture of propylene glycol and prednisone acetate or compounds of propylene glycol and triptolide by gavage, respectively, for consecutive 7 weeks. Determination in the 3 groups was conducted concerning the contents of blood plasma cortisol (COR), adrenocorticotropic hormone (ACTH) and corticotropin-releasing hormone (CRH) besides measurement of the rats' body weight, coefficient of the adrenal gland and observation of the histopathological changes in fascicular zone of adrenal cortex. Immunohistochemical staining technique was used to detect the expression of ACTH in pituitary in the 3 groups. RESULTS: (1) The content of COR in the groups of triptolide and prednisone acetate appeared lower and serum ACTH showed no significant difference, but CRH in the group of triptolide was augmented as compared with the control group (P < 0.05). (2) The rats' weight in the groups of triptolide and prednisone acetate was declined, and yet, the coefficient of the adrenal gland remained no significant change in comparison with the controls. HE staining and electron microscopy examination revealed thinned and constricted zona fasciculata in adrenal gland in the rats of triptolide and prednisone acetate, with hypofunction. ACTH expression in the group of triptolide was higher than that of the control group (P < 0.05). CONCLUSION: Morphologically and functionally, the findings suggest that long-term use of triptolide may result in atrophied cortex and hypofunction of the adrenal gland, leading to augmented production and secretion of CRH and ACTH from respective hypothalamic and pituitary.

Fibroblast Activation Protein (FAP) Overexpression Induces Epithelial-Mesenchymal Transition (EMT) in Oral Squamous Cell Carcinoma by Down-Regulating Dipeptidyl Peptidase 9 (DPP9).

PURPOSE: Fibroblast activation protein (FAP) acts as a tumor promoter via epithelial-mesenchymal transition (EMT) in human oral squamous cell carcinoma (OSCC). The present study was designed to investigate the FAP targeting proteins and explore the precise mechanism by which FAP promotes EMT in OSCC. PATIENTS AND METHODS: Proteins interacting with FAP were found and filtered by immunoprecipitation-mass spectrometry (IP-MS). Both DPP9 protein and mRNA were examined in 90 paired OSCC samples and matched normal tissue. DPP9 knockdown was conducted to determine its function in OSCC in vitro and in vivo. RESULTS: Dipeptidyl peptidase 9 (DPP9) was identified as interacting with FAP intracellularly by IP-MS. The levels of both DPP9 protein and mRNA were down-regulated in OSCC tissue. Lower DPP9 expression was correlated with unfavorable survival rates of OSCC patients. DPP9 knockdown accelerates the proliferation of OSCC cells in vitro and in vivo. Overexpression of FAP leads to a reduction in DPP9 expression. Likewise, DPP9 overexpression reverses the proliferation, migration, invasion and EMT induced by FAP during OSCC. CONCLUSION: Our study finds that FAP promotes EMT of OSCC by down-regulating DPP9 in a non-enzymatic manner. FAP-DPP9 pathway could be a potential therapeutic target of OSCC.

NF-κB-mediated lncRNA AC007271.3 promotes carcinogenesis of oral squamous cell carcinoma by regulating miR-125b-2-3p/Slug.

Oral squamous cell carcinoma (OSCC) is the most common oral cancer. The molecular mechanisms of this disease are not fully understood. Our previous studies confirmed that dysregulated function of long non-coding RNA (lncRNA) AC007271.3 was associated with a poor prognosis and overexpression of AC007271.3 promoted cell proliferation, migration, invasion, and inhibited cell apoptosis in vitro, and promoted tumor growth in vivo. However, the underlying mechanisms of AC007271.3 dysregulation remained obscure. In this study, our investigation showed that AC007271.3 functioned as competing endogenous RNA by binding to miR-125b-2-3p and by destabilizing primary miR-125b-2, resulted in the upregulating expression of Slug, which is a direct target of miR-125b-2-3p. Slug also inhibited the expression of E-cadherin but N-cadherin, vimentin, and ß-catenin had no obvious change. The expression of AC007271.3 was promoted by the canonical nuclear factor-κB (NF-κB) pathway. Taken together, these results suggested that the classical NF-κB pathway-activated AC007271.3 regulates EMT by miR-125b-2-3p/Slug/E-cadherin axis to promote the development of OSCC, implicating it as a novel potential target for therapeutic intervention in this disease.

M6A-related bioinformatics analysis reveals that HNRNPC facilitates progression of OSCC via EMT.

Increasing evidence suggests that N6-methyladenosine(m6A) has a vital role in cancer progression. Therefore, we aimed to explore the prognostic relevance of m6A-related genes in oral squamous cell carcinoma (OSCC). First, Expression profiles were downloaded from The Cancer Genome Atlas (TCGA) and m6A-related genes were extracted afterwards. Then, cluster analysis and principal component analysis (PCA) were used to analyze m6A-related genes. And differentially-expressed analysis was performed in R software. Furthermore, a risk model was constructed, and crucial m6A genes were selected to explore its biological effects in OSCC cells. Total of 13 m6A-related genes were extracted and 8 differentially-expressed genes were identified. Subsequently, m6A-based clustering showed 2 subtypes with different clinical outcome. In addition, a risk model was successfully established. Of 13 m6A-related genes, only heterogeneous nuclear ribonucleoprotein C (HNRNPC) might be an independent biomarker and mean unfavorable overall survival in OSCC by univariate and multivariate cox regression analysis. Functional studies revealed that overexpression of HNRNPC promoted carcinogenesis of OSCC via epithelial- mesenchymal transition (EMT). In total, a risk model of m6A-related genes in OSCC was established. Subsequently, HNRNPC was proved to promote OSCC carcinogenesis and be an independent biomarker prognostic biomarker of OSCC, suggesting that it might be a new biomarker and therapeutic target of OSCC.

Contraction band necrosis in two ecstasy abusers: a latent lethal lesion associated with ecstasy.

Cardiotoxicity associated with ecstasy has been reported in recent years but is very rare. We described 2 lethal cases associated with Ecstasy use. One whole tablet of ecstasy in one case and a half-tablet ecstasy in another case led to lethal effect, but death was not simply attributed to a lethal intoxication. Severe myocardial contraction band necrosis was observed in these 2 cases as revealed by the histopathologic examination. Focal interstitial infiltration of lymphocyte and monocyte around myocardial lesions in case one was also found. The results suggest that contraction band necrosis could be induced by ecstasy and is a potential lethal lesion in some cases.

[Pathological study on autopsy died of Tripterygium intoxication--report of 4 cases].

The Tripterygium preparation, a Chinese herbal medicine, has been widely used to treat various autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus. Its significant clinical effects have received a great praise and attention by the public health in China, but its toxicity also definitely exists, with the therapeutic dosage approaching the minimal toxic dosage. In order to provide reference for the safe use of Tripterygium preparation in clinical practice, the pathological changes of 4 autopsy cases by Tripterygium poisoning were reported in this paper. In them, 2 cases died of acute cardiogenic shock caused by myocardial damage, showing hydropic degeneration of the myocardial cells, even with obvious contraction band necrosis in the papillary muscles; the other 2 died of severe acute renal failure due to severe acute toxic nephrosis; cerebral edema and gastrointestinal inflammatory changes were found in all cases. The authors suggested that careful dosage control is the key step to prevent Tripterygium intoxication during the medical treatments; directly using the crude Tripterygium in clinics should be prohibited; and the Tripterygium preparation used should be produced by the pharmaceutical companies regulated by the government.

Identification of Candidate Biomarkers and Analysis of Prognostic Values in Oral Squamous Cell Carcinoma.

Objectives: Oral squamous cell carcinoma (OSCC) is the most common oral cancer with a poor prognosis owing to limited understanding of the disease mechanisms. The aim of this study was to explore and identify the potential biomarkers in OSCC by integrated bioinformatics analysis. Materials and Methods: Expression profiles of long non-coding RNAs (lncRNAs), microRNAs (miRNAs), and messenger RNAs (mRNAs) were downloaded from The Cancer Genome Atlas (TCGA) and differentially expressed RNAs (DERNAs) were subsequently identified in OSCC by bioinformatics analysis. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were used to analyze DERNAs. Then, the competing endogenous RNA (ceRNA) network was constructed in Cytoscape and the protein -protein interaction (PPI) network was established in the STRING database. We established a risk model to predict the overall survival of OSCC on the basis of DElncRNAs with Kaplan-Meier analysis and combined with logrank p test. Furthermore, we identified potential biomarkers by combining univariate Cox regression with overall survival rate, which were then validated in Gene Expression Omnibus (GEO), OSCC cell lines and OSCC specimens. Results: A total of 1,919 DEmRNAs, 286 DElncRNAs and 111 DEmiRNAs were found to be dysregulated in OSCC. A ceRNA network included 46 DElncRNAs,7 DEmiRNAs and 10 DEmRNAs, and the PPI network included 712 DEmRNAs including 31 hub genes. Moreover, a 7 lncRNAs risk model was established and four genes (CMA1, GNA14, HCG22, HOTTIP) were identified as biomarkers on overall survival in patients with OSCC. Conclusions: This study successfully constructed a ceRNA network and a PPI network which play a crucial role in OSCC. A risk model was established to predict the prognosis, and four DERNAs are revealed with overall survival in patients with OSCC, suggesting that they may be potential biomarkers in tumor diagnosis and treatment.

LncRNA AC007271.3 promotes cell proliferation, invasion, migration and inhibits cell apoptosis of OSCC via the Wnt/ß-catenin signaling pathway.

AIMS: Long noncoding RNA (lncRNA) AC007271.3 has been identified to be dysregulated in oral squamous cell carcinoma (OSCC) in our previous study. However, the precise role of AC007271.3 in OSCC remains unclear. In this study, we investigated the potential functions and the underlying mechanisms of AC007271.3 in OSCC. MATERIALS AND METHODS: The expression levels of AC007271.3 in OSCC tissues and cell lines were examined using RT-qPCR. The relationship between AC007271.3 level and clinicopathological characteristics was analyzed, and its association with patient prognosis was assessed by the Kaplan-Meier method. The biological function of AC007271.3 and its role in the development of OSCC through Wnt/ß-catenin signaling pathway were studied. KEY FINDINGS: We identified that AC007271.3 was up-regulated and positively correlated with advanced clinical stage, lymph node metastasis, poor histological differentiation and unfavorable prognosis. We explored the expression, function, and molecular mechanism of AC007271.3 in OSCC cells. Overexpression of AC007271.3 remarkably promoted cell proliferation in vitro and in vivo, induced cell migration, invasion and inhibited apoptosis in vitro, while knockdown of AC007271.3 attenuated cell proliferation, migration, invasion and induced apoptosis. Mechanistically, AC007271.3 overexpression substantially increased the expression of ß-catenin and the downstream target molecules CyclinD1, c-myc and Bcl-2, while silencing of AC007271.3 has the opposite effect. Rescued experiments showed that the ability to promote cell proliferation, migration, invasion and inhibiting apoptosis could be reversed when treated with the Wnt/ß-catenin pathway inhibitor. SIGNIFICANCE: Our data indicated that AC007271.3 could promote cell proliferation, invasion and inhibit cell apoptosis of OSCC via the Wnt/ß-catenin signaling pathway, which might provide a novel therapeutic approach for OSCC.

[Sudden infant death syndrome (SIDS) and its forensic investigation].

During the past two decades tremendous efforts have been made by the medical community, especially in the fields of forensic medicine and pediatrics, to better understand the etiology, epidemiology and pathophysiology of SIDS. There have been many SIDS reports from developed countries, but few from developing Asian countries. Despite a recent significant decrease in the incidence of SIDS in many developed countries, SIDS continues to be the most common cause of post-neonatal infant death in these countries. This article analyzes the SIDS data (1990-2006) from the Office of the Chief Medical Examiner for the State of Maryland, USA, along with review of the literature with regard to the history, epidemiological and pathophysiological characteristics of SIDS, as well as the recent advances in SIDS research. The changing trends in the diagnosis of SIDS and current challenges to its forensic investigation are also discussed.

Aconitine alters connexin43 phosphorylation status and [Ca2+] oscillation patterns in cultured ventricular myocytes of neonatal rats.

Aconitine, a highly poisonous type of alkaloid, has a widespread effect in stimulating the membranes of cardiomyocyte. However, other effects of aconitine on cardiomyocyte are unknown. In this study, we investigated whether aconitine also affects the phosphorylation status of connexin43 (Cx43) and intracellular [Ca(2+)] oscillation patterns in cultured ventricular myocytes of neonatal rats. As determined by Western blot analysis, a decreased percentage (47.68+/-2.29%) of phosphorylated Cx43 (P-Cx43) and a concomitant increased percentage (52.32+/-2.29%) of nonphosphorylated Cx43 (NP-Cx43) were found in aconitine-treated cultures, compared to the controls (82.77+/-2.04% for P-Cx43 and 17.23+/-2.04% for NP-Cx43). Quantitative immunofluorescent microscopy revealed similar changes in phosphorylation status occurring in Cx43 containing gap junctions in the cultures under the same treatment conditions. Real-time laser scanning microscopy indicated that intracellular [Ca(2+)] oscillations were relatively stable in control cultures, with occasional calcium sparks; after being treated with aconitine, high frequency [Ca(2+)] oscillations emerged, whereas typical calcium sparks disappeared. Furthermore, Western blot analysis revealed that, after aconitine treatment, the amount of phosphorylated PKCalpha decreased significantly. These observations suggest that aconitine not only induces dephosphorylation of Cx43 and PKCalpha, but also alters intracellular [Ca(2+)] oscillation patterns in cultured cardiomyocytes.

[Analysis and consider of technical identification for 32 cases medical tangle in medical association].

OBJECTIVE: To analyze cause of medical accidents and actuality of technical identification in medical tangle. METHODS: 32 cases (17 death, 15 survive) of medical tangle by technical identification (according to sex, age, mostly diseases, sequel) and identified results (whether or not mistake, cause and effect connection, duty degree) have been studied. RESULTS: 13 cases of 32 medical accidents have been determined.19 cases have been attributed to no medical accidents. Causes of medical accidents were most due to negligence of sense of duty. CONCLUSION: The incidence rate of medical accidents can be decreased by strengthen colligated stuff of medical affairs personnal. We suggest that our state bring out more perfect legislation of autopsy in order to gain positive effect of technical identification in medical tangle. The medical mistake among grade of medical accidents should be added so that justice of identification could be improved.

[Influence of triptolide on neuronal apoptosis in rat with cerebral injury after focal ischemia reperfusion].

OBJECTIVE: To study effect of triptolide (TL) on neuronal apoptosis in cerebral tissue of rat after ischemia-reperfusion. METHOD: Triptolide at dose 0.2 or 0.4 mg x kg(-1) was intraperitoneally injected once a day for 4 d. The focal cerebral ischemia-reperfusion model was established with thread embolism in middle artery before triptolide injection on the fourth day. Neurological deficit score of rats was evaluated; and immunohistochemical techniques were used to count positive cells of express of MPO and TUNEL in cerebraltissue. RESULT: Compared with the control group, the deficit of neural function was significantly improved, and the number of infiltrate of neutrophil and neuronal apoptosis in cerebral tissue was remarkably reduced in two TL-treated groups. CONCLUSION: The results suggested that TL can inhibit infiltration of neutrophil and decrease the degree of neuronal apoptosis in cerebral tissue.

[Study of toxicology of strychnos].

Because of its officinal value, strychnos is widely used by clinic and individual. Since toxic dose and therapeutic dose are very close, strychnos poisoning cases are frequently reported. In this paper the chemical component, toxic dose, mechanisms of toxicity, poisoning symptom and pathological changes after strychnos poisoning are reviewed.

[Reasons why the quality of medico-legal autopsy in the medical tangle varies from two areas].

OBJECTIVE: To study reasons that the quality of medico-legal autopsy in the medical tangle varies from different area. METHODS: Collecting the cases of medical tangle in two medico-legal agencies, then counting percent of classes on the ten key-points, analyzing the data of the cases by chi-square test and t-test. RESULTS: It is indicated that the applied methods and standards of the two agencies are different. There are more different in seven keypoint of medicolegal autopsy by chi-square test. CONCLUSION: Six key-points are found to be more important to medico-legal appraiser, standardization of forensic autopsy, standardization of picking up specimen from the body, diagnosis standardization of the cause of death, consultation system and standardization of writing documents on medico-legal autopsy.