Distinct spectrum of microRNA expression in forensically relevant body fluids and probabilistic discriminant approach.

MicroRNA is attracting worldwide attention as a new marker for the identification of forensically relevant body fluids. A probabilistic discriminant model was constructed to identify venous blood, saliva, semen, and vaginal secretion, based on microRNA expression assessed via RT-qPCR. We quantified 15 candidate microRNAs in four types of body fluids by RT-qPCR and found that miR-144-3p, miR-451a-5p, miR-888-5p, miR-891a-5p, miR-203a-3p, miR-223-3p and miR-1260b were helpful to discriminate body fluids. Using the relative expression of seven candidate microRNAs in each body fluid, we implemented a partial least squares-discriminant analysis (PLS-DA) as a probabilistic discriminant model and distinguished four types of body fluids. Of 14 testing samples, 13 samples were correctly identified with >90% posterior probability. We also investigated the effects of microRNA expression in skin, semen infertility, and vaginal secretion during different menstrual phases. Semen infertility and menstrual phases did not affect our body fluid identification system. Therefore, the selected microRNAs were effective in identifying the four types of body fluids, indicating that probabilistic evaluation may be practical in forensic casework.

Prevention of Cell Growth by Suppression of Villin Expression in Lithocholic Acid-Stimulated HepG2 Cells.

Cholestasis is a condition wherein bile flow is interrupted and lithocholic acid is known to play a key role in causing severe liver injury. In this study, we performed in-depth analysis of the morphological changes in bile canaliculi and the biological role of villin in cholestasis using lithocholic acid-stimulated HepG2 human hepatocarcinoma cells. We confirmed disruption of the bile canaliculi in liver sections from a liver allograft patient with cholestasis. Lithocholic acid caused strong cytotoxicity in HepG2 cells, which was associated with abnormal morphology. Lithocholic acid reduced villin expression, which recovered in the presence of nuclear receptor agonists. Furthermore, villin mRNA expression increased following small interfering RNA (siRNA)-mediated knockdown of the nuclear farnesoid X receptor and pregnane X receptor. Villin knockdown using siRNA caused cell growth arrest in HepG2 cells. The effect of villin-knockdown on whole-genome expression in HepG2 cells was analyzed by DNA microarray. Our data suggest that lithocholic acid caused cell growth arrest by suppressing villin expression via farnesoid X receptor and pregnane X receptor in HepG2 cells.

Optimal small-molecular reference RNA for RT-qPCR-based body fluid identification.

MicroRNA (miRNA) -based body fluid identification (BFID) plays a prominent role in a forensic practice, and the selected reference RNA is indispensable for a robust normalization in BFID performed using reverse transcription-quantitative PCR. In this study, we first examined sample quality using RNA integrity number, then evaluated the consistency of expression of candidate reference RNAs in 4 forensically relevant body fluids using NormFinder and BestKeeper, and lastly used each rank and index output from these tools for selecting the optimal reference RNA and the combination of the multiple RNAs using the RankAggreg package of R. We found that RNA integrity number was small in our samples, despite the use of pristine body fluids; 5S-rRNA was the optimal reference RNA for the identification of forensically relevant body fluids; and the combination of 5S-rRNA and miR-92a-3p and/or miR-484 enhanced the normalization quality. Our findings enable us to perform stringent normalization of the expression of body fluid-specific RNAs, and thus, can contribute to the development of small RNA-based BFID systems.

Forensic age prediction for dead or living samples by use of methylation-sensitive high resolution melting.

Age prediction with epigenetic information is now edging closer to practical use in forensic community. Many age-related CpG (AR-CpG) sites have proven useful in predicting age in pyrosequencing or DNA chip analyses. In this study, a wide range methylation status in the ELOVL2 and FHL2 promoter regions were detected with methylation-sensitive high resolution melting (MS-HRM) in a labor-, time-, and cost-effective manner. Non-linear-distributions of methylation status and chronological age were newly fitted to the logistic curve. Notably, these distributions were revealed to be similar in 22 living blood samples and 52 dead blood samples. Therefore, the difference of methylation status between living and dead samples suggested to be ignorable by MS-HRM. Additionally, the information from ELOVL2 and FHL2 were integrated into a logistic curve fitting model to develop a final predictive model through the multivariate linear regression of logit-linked methylation rates and chronological age with adjusted R(2)=0.83. Mean absolute deviation (MAD) was 7.44 for 74 training set and 7.71 for 30 additional independent test set, indicating that the final predicting model is accurate. This suggests that our MS-HRM-based method has great potential in predicting actual forensic age.

Abnormal Localization of STK17A in Bile Canaliculi in Liver Allografts: An Early Sign of Chronic Rejection.

The biological significance of STK17A, a serine/threonine kinase, in the liver is not known. We analyzed STK17A expression in HepG2 cells and human liver tissue. Accordingly, we investigated whether STK17A could help in identifying earlier changes during the evolution of chronic rejection (CR) after liver transplantation. RT-PCR and immunofluorescence were used to analyze STK17A expression in HepG2 cells. Antibody microarray was performed using human liver samples from CR and healthy donors. Immunohistochemistry was used to verify the clinical utility of STK17A on sequential biopsies for the subsequent development of CR. A novel short isoform of STK17A was found in HepG2 cells. STK17A was localized in the nuclei and bile canaliculi in HepG2 cells and human livers. Microarray of STK17A revealed its decrease in failed liver allografts by CR. During the evolution of CR, the staining pattern of bile canalicular STK17A gradually changed from diffuse linear to focal intermittent. The focal intermittent staining pattern was observed before the definite diagnosis of CR. In conclusion, the present study was the first to find localization of STK17A in normal bile canaliculi. Abnormal expression and localization of STK17A were associated with CR of liver allografts since the early stage of the rejection process.

Development of a novel miniplex DNA identification system for the Japanese population.

To aid DNA identification using 36 short tandem repeat (STR) loci for kinship analysis, likelihood ratio (LR) distributions were estimated using the allele frequency data evaluated for the Japanese population in our previous study. The results revealed that the LR tended to be higher when kinship analysis was performed using the 36 STR loci than when the analysis was performed using Identifiler®, the most commonly used commercial DNA typing kit in Japan, even when a sibship case was analyzed. However, a typing kit targeting 36 STR loci is not suitable for the analysis of damaged DNA. In this study, we developed a novel miniplex DNA identification system targeting 7 STR loci (D3S1744, D5S818, D8S1179, D10S2325, Penta D, Penta E, and vWA) that was optimized for use in combination with MiniFiler™ for the Japanese population. The combined matching probability of the MiniFiler plus miniplex system was 4.8×10(-19), which is lower than that of Identifiler (4.3×10(-17)). All expected alleles were detected successfully on DNA isolated from HepG2 human hepatocarcinoma cells by the miniplex system, but no significant signal was observed from a DNA sample isolated from COS-7 African green monkey kidney cells.

Accelerated telomere reduction and hepatocyte senescence in tolerated human liver allografts.

BACKGROUND: In living donor liver transplantation, the biological organ age of the donated allograft is unknown in young patients who receive grafts from older donors. Few studies have focused on the effects of aging on allografts in the state of tolerance. The purpose of this study was to assess the biological organ age of liver grafts. METHODS: In 20 tolerated allografts over a 10-year post-transplant follow-up period, the relative telomere lengths were measured by multiplex quantitative polymerase chain reaction, and hepatocyte nuclear size and cell cycle phase markers were determined by immunohistochemistry. The results were compared with the same measurements that had been obtained prior to transplantation in the recipients' pre-implantation donor livers. Tolerance was defined strictly as a condition in which the allograft functioned normally and showed normal histology without any histological signs of rejection, fibrosis or inflammation in the absence of immunosuppression. RESULTS: First, telomere length correlated with chronological donor age (n=41). Accelerated telomere reduction was seen in tolerated grafts compared with the predicted telomere length of each allograft calculated from the regression line of donor livers. Tolerated grafts were associated with higher hepatocyte p21 expression and greater nuclear area than in the donor livers prior to transplantation. CONCLUSIONS: These findings suggest that allografts age more rapidly than in the normal population, and that grafts may reach the limit of proliferative capacity even in the state of tolerance.

Sorbin and SH3 domain-containing protein 2 is released from infarcted heart in the very early phase: proteomic analysis of cardiac tissues from patients.

BACKGROUND: Few proteomic studies have examined human cardiac tissue following acute lethal infarction. Here, we applied a novel proteomic approach to formalin-fixed, paraffin-embedded human tissue and aimed to reveal the molecular changes in the very early phase of acute myocardial infarction. METHODS AND RESULTS: Heart tissue samples were collected from 5 patients who died within 7 hours of myocardial infarction and from 5 age- and sex-matched control cases. Infarcted and control myocardia were histopathologically diagnosed and captured using laser microdissection. Proteins were extracted using an originally established method and analyzed using liquid chromatography-tandem mass spectrometry. The label-free quantification demonstrated that the levels of 21 proteins differed significantly between patients and controls. In addition to known biomarkers, the sarcoplasmic protein sorbin and SH3 domain-containing protein 2 (SORBS2) was greatly reduced in infarcted myocardia. Immunohistochemical analysis of cardiac tissues confirmed the decrease, and Western blot analysis showed a significant increase in serum sorbin and SH3 domain-containing protein 2 in acute myocardial infarction patients (n=10) compared with control cases (n=11). CONCLUSIONS: Our advanced comprehensive analysis using patient tissues and serums indicated that sarcoplasmic sorbin and SH3 domain-containing protein 2 is released from damaged cardiac tissue into the bloodstream upon lethal acute myocardial infarction. The proteomic strategy presented here is based on precise microscopic findings and is quite useful for candidate biomarker discovery using human tissue samples stored in depositories.

Allele frequencies of 37 short tandem repeat loci in a Japanese population.

Allele frequencies for 37 STR autosomal loci (D1S1656, D2S1338, D2S1360, D2S441, D3S1358, D3S1744, D4S2366, D5S2500, D5S818, D6S474, D7S1517, D7S820, D8S1132, D8S1179, D10S1248, D10S2325, D12S391, D13S317, D16S539, D18S51, D19S433, D21S11, D21S2055, D22S1045, CSF1PO, F13A01, F13B, FESFPS, FGA, LPL, Penta C, Penta D, Penta E, SE33, TH01, TPOX, vWA) were obtained from a sample of 175 unrelated individuals in a Japanese population.

Dialysis purification of integrase-DNA complexes provides high-resolution atomic force microscopy images: dimeric recombinant HIV-1 integrase binding and specific looping on DNA.

It remains difficult to obtain high-resolution atomic force microscopy images of HIV-1 integrase bound to DNA in a dimeric or tetrameric fashion. We therefore constructed specific target DNAs to assess HIV-1 integrase binding and purified the complex by dialysis prior to analysis. Our resulting atomic force microscopy analyses indicated precise size of binding human immunodeficiency virus type 1 (HIV-1) recombinant integrase in a tetrameric manner, inducing formation of a loop-like or figure-eight-like secondary structure in the target DNA. Our findings regarding the target DNA secondary structure provide new insights into the intermediate states of retroviral integration.

Frequent hepatocyte chimerism in long-term human liver allografts independent of graft outcome.

Microchimerism after liver transplantation is considered to promote graft tolerance or tissue repair, but its significance is controversial. By using multiplex polymerase chain reaction (PCR) of short tandem repeat (STR) loci after laser capture microdissection of hepatocyte nuclei, we compared the proportions of recipient-derived hepatocytes in long-term stable liver allografts and late dysfunctional allografts caused by chronic rejection or idiopathic post-transplantation hepatitis. Through fluorescence in situ hybridization (FISH), we also analyzed the presence of recipient-derived Y-positive hepatocytes in the biopsies of livers transplanted from female donors to male recipients. The study population comprised 24 pediatric liver transplant recipients who survived with the initial graft, whose 10-year protocol biopsy records were available, and who had normal liver function (stable graft, SG; n=13) or a late dysfunctional graft (LDG; n=11) with similar follow-up periods (mean 10.8years in the SG group and 11.2years in the LDG group). STR analysis revealed that hepatocyte chimerism occurred in 7 of 13 (54%) SGs and 5 of 11 (45%) LDGs (p=0.68). The proportion of hepatocyte chimerism was low, with a mean of 3% seen in 2 of 3 female-to-male transplanted livers (one each of SG and LDG). In conclusion, hepatocyte chimerism was a constant event. The extent of engraftment of recipient-derived hepatocytes does not seem to correlate with the degree of hepatic injury in long-term liver allografts.

Bile canalicular abnormalities in the early phase of a mouse model of sclerosing cholangitis.

BACKGROUND: The bile canaliculus is the smallest and first biliary channel and is formed by two or three adjacent hepatocytes. Previous studies of chronic cholangiopathies such as primary sclerosing cholangitis have focused on the bile ductules. However, little is known about the pathological alterations in bile canaliculi in the early phase of cholangiopathies. AIM: To characterize the bile canalicular morphology in the early phase of sclerosing cholangitis we used 3,5-diethoxycarbonyl-1,4-dihydrocollidine-induced mouse model of sclerosing cholangitis. METHODS: Mice were fed a diet with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (0.1%). Serum biochemical, histological, immunohistochemical, and electron microscopic analyses were performed 1, 2, 4, and 7 days after feeding. RESULTS: All experimental groups showed significantly increased serum aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase levels. From day 1, bile canalicular abnormalities such as dilatation and meandering and loss of microvilli were observed. After bile canalicular abnormalities had appeared, substantial infiltration of inflammatory cells was observed amongst the necrotic cells and periductal region. After these inflammatory changes, cholangiocytes proliferated in the portal area and formed ductular reactions. Finally, periductal fibrosis appeared. CONCLUSION: This study provides novel evidence of the occurrence of bile canalicular abnormalities during the early phase of sclerosing cholangitis.

Mixture interpretation: Experimental and simulated reevaluation of qualitative analysis.

We present here analytical data using the 15 STR typing (Identifiler) kit regarding heterozygote balance in experimental DNA samples including one or two persons. Surprisingly, the allelic imbalance was observed even in samples consisting of only one person but adequate DNA for the standard protocol. The variance of heterozygote balance was more expanded in two-person mixtures than in one-person samples. Therefore, it is not suitable to use allelic peak heights/areas for estimating the genotypes of the contributors such as the quantitative analysis. We also reevaluated the effectiveness of qualitative analysis by simulation, i.e. consideration of the probability of all possible genotype combinations from the typing results of a mixed DNA sample. As demonstrated, the qualitative analysis using 15 STR loci is still extremely effective even in a mixture from two or three individuals.

Novel in situ pretreatment method for significantly enhancing the signal in MALDI-TOF MS of formalin-fixed paraffin-embedded tissue sections.

The application of matrix-assisted laser desorption/ionization (MALDI)-based mass spectrometry (MS) to the proteomic analysis of formalin-fixed paraffin-embedded (FFPE) tissue presents significant technical challenges. In situ enzymatic digestion is frequently used to unlock formalin-fixed tissues for analysis, but the results are often unsatisfactory. Here, we report a new, simplified in situ pretreatment method for preparing tissue sections for MS that involves heating with vapor containing acetonitrile in a small airtight pressurized space. The utility of the novel method is shown using FFPE tissue of human colon carcinoma. The number and intensity of MALDI peaks obtained from analysis of pretreated tissue was significantly higher than control tissue not subjected to pretreatment. A prominent peak (m/z 850) apparently specific to cancerous tissue was identified as a fragment of histone H2A in FFPE tissue pretreated using our method. This highly sensitive treatment may enable MALDI-MS analysis of archived pathological FFPE samples, thus leading to the identification of new biomarkers.

Pituitary abscess presenting a very rapid progression: report of a fatal case.

Pituitary abscess is a rare disease presenting with nonspecific clinical symptoms, and diagnosis is often difficult. This disease is potentially life-threatening, but most cases have a chronic and indolent course. We report a case of a 60-year-old man with a pituitary abscess associated with pituitary adenoma who died 5 days after the onset of clinical symptoms without a definitive diagnosis. Postmortem computed tomography and autopsy findings revealed a sellar mass with cystic change and extension toward the optic chiasm. Histopathology of the lesion demonstrated an abscess with suppurative meningitis and encephalitis. The disturbance of the cardiac autonomic nervous system because of hypothalamus involvement was suggested as the cause of rapid progression and death. This case provides useful information for clinicians to avoid a lethal outcome.

Telomere shortening and karyotypic alterations in hepatocytes in long-term transplanted human liver allografts.

The long-term fate of aged liver allografts in young recipients who received grafts from older donors is unknown. We evaluated graft aging by analyzing hepatocytic telomere length and karyotypic changes. Seventeen pediatric individuals who underwent living-donor liver transplantation for congenital biliary diseases were selected. At a median of 10.4 years post-transplant, ten had tolerated grafts with weaned off immunosuppressants, and seven had idiopathic post-transplantation hepatitis. Fluorescence in situ hybridization was used to evaluate the telomere signal intensity (TI) and karyotypic changes. First, we measured predictive age-dependent TI decline with regression analysis of donor livers. The mean TI at the earliest (within a year) and latest biopsies was significantly lower than the predicted TI of the studied allografts. With univariate analysis, a higher abnormal karyotype ratio in the donor liver was correlated with development of idiopathic post-transplantation hepatitis. With multivariate analysis that included clinical parameters, a greater TI decline at the earliest biopsy was correlated with the development of idiopathic post-transplantation hepatitis. In conclusion, graft aging as measured by TI decline and donor abnormal karyotype ratio was associated with idiopathic post-transplantation hepatitis of long-term transplanted liver allografts.

Subdural hemorrhage: A unique case involving secondary vitamin K deficiency bleeding due to biliary atresia.

Extrahepatic biliary atresia (EHBA) is a rare disease characterized by progressive and obliterative cholangiopathy in infants and is one of the major causes of secondary vitamin K deficiency bleeding (VKDB) due to cholestasis-induced fat malabsorption. Breast feeding increases the tendency of bleeding in EHBA patients because breast milk contains low amounts of vitamin K. A 2-month-old female infant unexpectedly died, with symptoms of vomiting and jaundice prior to death. She had been born by uncomplicated vaginal delivery and exhibited normal growth and development with breastfeeding. There was no history of trauma. She received vitamin K prophylaxis orally. In an emergency hospital, a CT scan showed a right intracranial hematoma and mass effect with midline shift to the left. In the postmortem examination, severe atresia was observed in the whole extrahepatic bile duct. Histologically, cholestasis, periductal fibrosis, and distorted bile ductules were noted. The gallbladder was not identified. A subdural hematoma and cerebellar tonsillar herniation were found; however, no traumatic injury in any part of the body was observed. Together, these findings suggest that the subdural hemorrhage was caused by secondary vitamin K deficiency resulting from a combination of cholestasis-induced fat malabsorption and breastfeeding. Subdural hemorrhage by secondary VKDB sometimes occurs even when vitamin K prophylaxis is continued. This case demonstrated that intrinsic factors, such as secondary VKDB (e.g., EHBA, neonatal hepatitis, chronic diarrhea), should also be considered in infant autopsy cases presenting with subdural hemorrhage.

Transmesenteric hernia due to double-loop formation in the small intestine: a fatal case involving a toddler.

We report a unique case of transmesenteric hernia resulting in death, which went undiagnosed during a recent hospital visit. The victim was a 2.5-year-old girl who - with the exception of chronic constipation - had no medical history. One night she complained of abdominal pains and was taken to a pediatric hospital where doctors performed an abdominal X-ray and echography. No significant findings suggesting bowel obstruction (e.g. air-fluid levels or dilation of the bowel) were obtained on examinations and bloody feces were not observed in this particular episode. As her abdominal pain gradually attenuated, the doctor allowed her to return home. A few hours later, she lost consciousness and expired despite resuscitation efforts attempted at an emergency hospital. A subsequent autopsy revealed that the small bowel had herniated through a defect in the mesentery resulting in two consecutive and inversely forming loops, in which each loop protruded on either side of the mesentery. This rare morphological anatomy seems to have progressed in a two-step process. The girl's mild abdominal pain was likely induced by herniation and formation of the first intestinal loop, followed by severe shock occurring when the subsequent intestinal segment invaginated into the same defect forming the second loop on the opposite side of the mesentery. This case illustrates the difficulty of diagnosing transmesenteric hernia due to the presentation of unspecific symptoms; especially in infants and toddlers. Furthermore, this report demonstrates the value of a complete autopsy in cases of sudden and unexpected deaths involving children.

Murine leukemia retrovirus integration induces the formation of transcription factor complexes on palindromic sequences in the signal transducer and activator of transcription factor 5a gene during the development of pre-B lymphomagenesis.

Murine leukemia retrovirus (MLV) vectors are highly effective tools for introducing a foreign gene into a target host genome. However, it remains unclear how integrated retroviral promoter activity is influenced by the upstream or downstream sequences and how the host cell phenotype is influenced by the integrated promoter activity. Herein, we analyzed a set of pre-B lymphoma clones in which the MLV genome was integrated into the signal transducer and activator of transcription factor 5a (Stat5a) gene. Among the clones, the lymphoma clones with a provirus integrating into the middle position of the palindromic target sequences showed significantly higher transcription of the Stat5a gene; and p300 and other transcriptional factors formed complexes, with binding to the proviral-host junctional DNA segment. By using a luciferase assay, the upstream and downstream sequences of the provirus contributed to the up-regulation of proviral promoter activity. In concomitance with the higher Stat5a transcription, the immunoglobulin gene recombination was arrested. Antiapoptotic activity was significantly higher, with an increase in Bcl-xL, one of the targets of STAT5A, when IL-7 was supplied. Thus, a minute difference between MLV integration sites can lead to large differences in the host phenotype through the formation of transcription factor complexes on the proviral-host junctional DNA segment, suggesting that caution is necessary in monitoring integration sites when working with MLV vectors.

Dual retrovirus integration tagging: identification of new signaling molecules Fiz1 and Hipk2 that are involved in the IL-7 signaling pathway in B lymphoblastic lymphomas.

IL-7R, FLT3, and CD43 are surface antigens expressed during the transition from pro-B to pre-B cells in BM. To understand interactions between their signaling pathways, we analyzed spontaneous mouse B-LBLs with dual MLV integration into Stat5a and Fiz1 or Stat5a and Hipk2. MLV integration resulted in up-regulation of these genes in lymphoma cells compared with normal pro-B cells from the BM. In lymphomas with both integrations into Stat5a and Fiz1, increases in phosphorylated STAT5A and expression of c-Myc, a target gene of STAT5A, were observed following stimulation of the FLT3. Clones with the dual integrations grew faster in IL-7 and FLT3L-supplemented medium than clones with Stat5a integration alone. On the other hand, in lymphomas with integrations into Stat5a and Hipk2, increases in phosphorylated STAT5A and expression of c-Myc were observed following cross-linking of CD43. In conclusion, FLT3 and CD43 signaling pathways involve STAT5A via Fiz1 and Hipk2 in B-LBLs. Identification of the dual MLV integration sites in B-LBLs, therefore, will provide an excellent tool for identification of the signaling pathways in B-LBLs.