Short tandem repeat typing of cells transferred via micromanipulation with whole-genome amplification.

This study attempted to determine the minimum number of cells required to conduct DNA analyses effectively. Oral mucosal cells obtained from eight persons were suspended and individually collected by using micromanipulation technique. DNA was extracted and amplified by whole-genome amplification (WGA). Nuclear DNA was extracted to evaluate the feasibility of autosomal short tandem repeat (STR) polymorphism and Y-chromosomal STR polymorphism analyses. Tests were conducted with 20 and 30 cells, to determine the minimum number of cells required for each DNA analysis. Tests with 20 cells were repeated 5 times, to examine reproducibility. When five or 10 cells were used, loci could not be identified for most alleles. Furthermore, DNA polymorphism analyses of a single cell transferred directly to a polymerase chain reaction solution were unsuccessful. The present findings suggest that, in forensic identification, 20 or more cells are required in order to obtain clear results from autosomal and Y-chromosomal STR polymorphism analyses. Furthermore, the feasibility of sample preservation and reexamination was also confirmed by DNA amplification with WGA.

Social contribution of forensic odontology in Japan.

Half a century has passed since the department for education and research on forensic odontology was established at dentistry-related universities in Japan in 1964. In order to meet the demands of society, the number of universities with a department of forensic odontology increased up until around 2005. In 2007, the Japanese Society of Forensic Dental Science was established, and then a series of reforms such as establishment of the Study Council on Death Cause Investigation in both the National Police Agency and the Cabinet Office of the Japanese government, cabinet decision of enactment and enforcement of new laws on death cause investigation, publication of an article on the Model Core Curriculum of Dental Education, publication of the results of a fact-finding survey on education and research on forensic odontology conducted by the Ministry of Education, Culture, Sports, Science and Technology, inclusion of questions about forensic odontology in the National Board Dental Examination, and compilation of a database on dental findings by the Ministry of Health, Labor and Welfare, proceeded in succession. We introduced the half century of forensic odontology in Japan in chronological order.

DNA analysis of root canal-filled teeth.

Teeth are markedly useful as samples for DNA analysis; however, intact teeth are not always available. This study examined the possibility of identifying autosomal and Y-chromosome short tandem repeat (STR) types in samples from 34 teeth (15 intact and 19 root canal filled) that had been preserved for 10-33years after dental extraction. The aim was to explore the feasibility of individual identification by DNA analysis of samples obtained from highly decomposed and skeletonized corpses. Only one out of 24 autosomal STR loci was not identified in two of the 15 intact teeth, whereas all 23 loci of the Y chromosome STR were detected. One or two autosomal STR loci remained unidentified in eight of the 19 root-filled teeth, and four or five of the 23 Y STR loci were undetected in three cases. However, the types were identified in about 20 loci in all samples, and the composition of the root canal filling material did not appear to interfere with the PCR. This study demonstrates that the storage period of the teeth had no influence on our results indicating that root canal filled teeth can be used for DNA analysis.

Real-time PCR assay for the detection of picoplankton DNA distribution in the tissues of drowned rabbits.

The detection of plankton DNA is one of the important methods for the diagnosis of drowning from postmortem tissues. This study investigated the quantities of picoplankton (Cyanobacteria) DNA in the lung, liver, kidney tissues and blood of drowned and non-drowned rabbits, and the sensitivity of detection of picoplankton DNA by polymerase chain reaction (PCR) detect for the diagnosis of death from drowning. For this purpose, the DNA of the 16S ribosomal RNA gene of picoplankton was quantitatively assayed from the tissues of drowned and non-drowned rabbits immersed in water after death. Each of the liver, kidney and lung tissues and blood were obtained from drowned and non-drowned rabbits. Picoplankton DNA in the tissues was extracted using the DNeasy® Blood & Tissue kit to determine the yield of picoplankton DNA from each tissue. TaqMan real-time PCR was performed for quantitative analysis of picoplankton DNA. Target DNA was detected in the liver, kidney and lung samples obtained from the drowned rabbits, while no picoplankton DNA was detected in the non-drowned rabbit tissues (except in lung samples). The results verified that direct PCR for the detection of picoplankton DNA is useful for the diagnosis of drowning. Although we observed seasonal changes in the quantity of picoplankton in river water, we were able to detect DNA from various organs of drowned bodies during the season when picoplankton were not the most abundant.

Prevalence and distribution of anomalies of permanent dentition in 9584 Japanese high school students.

This study investigated the prevalence and distribution of anomalies of permanent dentition in the current Japanese population by examining an unbiased sample. We conducted a survey of dental anomalies by mass dental screening at eight high schools in 2012. Participants were all students with permanent dentition. Dental anomalies were classified as hypodontia, supernumerary teeth, peg-shaped teeth, fused teeth, and talon cusps. Students with one or more dental anomalies on oral examination were given a differential diagnosis by three specialists. The final sample comprised 9584 participants (5062 boys, 4522 girls). Hypodontia was present in 372 students (3.88 %) with no significant sex difference (191 boys, 181 girls). Frequent sites were the right or left mandibular second premolar, right or left maxillary second premolar, and right or left maxillary lateral incisor. Supernumerary teeth were observed in three boys (0.06 %) and one girl (0.02 %). Peg-shaped teeth were observed in 74 students (0.77 %; 27 boys, 47 girls), differing significantly between sexes; they were most prevalent among maxillary lateral incisors. Of affected students, 18 students (0.19 %) also had hypodontia (3 boys, 15 girls). Fused teeth were present in two boys (0.04 %) and three girls (0.07 %) (gemination in one boy and fusion in the remaining four students). Sites were limited to maxillary and mandibular central and lateral incisors. Talon cusps were observed in two boys (0.04 %) and four girls (0.09 %). The present survey of a large unbiased sample can be considered to reflect the prevalence and distribution of anomalies of permanent dentition in the current Japanese population.

Analysis of human mitochondrial DNA polymorphisms in the Japanese population.

The highly polymorphic nature and high amplification efficiency of mitochondrial DNA (mtDNA) is valuable for the analysis of biological evidence in forensic casework, such as the identification of individuals and assignment of race/ethnicity. To be useful, a mtDNA polymorphism database for the Japanese population requires an understanding of the range of haplotype variation and phylogenies of mtDNA sequences. To extend current knowledge on the haplotypes in the Japanese population, this study defines new lineages and provides more detail about some of those previously described. We compared the hypervariable regions (HVRs) of 270 healthy, unrelated Japanese individuals and demonstrated 192 haplotypes. Combining HVR1 and HVR2, the genetic diversity was 0.9935, thus providing a high level of identification capability. Haplogroup status was defined for 160 individuals using HVR1, HVR2, and particular coding region polymorphisms; these individuals belonged to 94 haplotypes, four of which were new lineages. The complete mtDNA sequence was also determined from seven individuals.

ABO blood group genotyping by quenching probe method.

We developed a multiplex ABO genotyping method with quenching probes (Q-probe). In this method, it is possible to discriminate the mutations, not only frequently used positions 261 and 796 but also position 703 in a single PCR. Each probe was designed to have cytosine residue at 5' or 3' end and labeled with three different fluorescence dyes, enabling the triplex detections of these polymorphisms. All polymorphisms were successfully detected by using fluorescence labeled Q-probe in a specifically amplified PCR product. Each Q-probe showed unique dissociation patterns depending on the polymorphism types. All of the results obtained with Q-probe were compared with standard serotyping and TaqMan PCR method and resulted in complete match with each other. Consequently, these results indicated that multiplex ABO genotyping method is quite accurate and convenient method for the determination of ABO genotype.

A case of personal identification due to detection of rare DNA types from seminal stain.

Following a rape incident in an apartment in Japan, we were requested to perform a DNA analysis on a body fluid stain left on a bath towel to determine whether it could be attributed to the suspect. The acid phosphatase and prostatic-specific antigen tests confirmed it to be a seminal stain. Based on the DNA analysis by autosomal and Y-chromosome short tandem repeat (STR) systems, no inconsistency was found with the profile of the suspect with African ancestry. In this case, allele 21 of DYS390 at the Y-STR locus was examined, as it is reported to have a distinctly lower frequency in the Japanese population. Furthermore, the haplotype combinations of Y-STR at the DYS389I, DYS389II and DYS390 loci are powerful for personal identification, as these have not yet been found in the Japanese population.

Genetic studies of eight X-STRs in a Japanese population.

We studied eight X-STRs (DXS7132, DXS7423, DXS8378, DXS10074, DXS10101, DXS10134, DXS10135, HPRTB) polymorphism in 494 unrelated Japanese individuals (313 males, 181 females) using Mentype Argus X-8 PCR Amplification Kit. PD of the eight X-STRs ranged from 0.558 (male) to 0.987 (female). Allele frequencies, number of alleles, and PIC were 0.001-0.587, 6-20, and 0.470-0.913, respectively.

The suppression of proinflammatory cytokines improves heart function from non-heart-beating donors following transplantation in a canine model.

We evaluated the effectiveness of a suppressant of the production of proinflammatory cytokines such as interleukin (IL)-1 and tumor necrosis factor (TNF)-alpha on a canine heart transplantation model with non-heart-beating donors (NHBDs).Adult mongrel dogs were divided into 3 groups of 5: a control group; FR-1 in which donors were given FR167653, a potent suppressant of IL-1beta and TNF-alpha production; and FR-2 in which both donors and recipients were given FR167653. After measuring the baseline hemodynamic parameters, including cardiac output (CO), left ventricular pressure (LVP), and maximum and minimum rates of increase in LVP (+/- LVdp/dt), FR167653 was administered continuously for 30 minutes before ischemia in the FR-1 and FR-2 groups. Cardiac arrest was obtained by rapid exsanguination from the abdominal aorta and inferior vena cava. The organ was left in the cadaver for 30 minutes. The coronary vascular beds were washed out with 4 degrees C Celsior solution, and then the donor heart was preserved in 4 degrees C Celsior solution for 4 hours. The donor heart was transplanted orthotopically with cardiopulmonary bypass (CPB). FR167653 was administered intravenously from 15 minutes before aortic-cross clamping until the end of the experiment in the FR-2 group. The recipient was weaned from CPB 1 hour after reperfusion. We compared the hemodynamic parameters at 3 hours after reperfusion with the preoperative values in donor animals with the right atrial pressure at 10 mmHg and a 5 microg/kg/min dopamine infusion. Histopathological analysis was also performed.There were no significant differences in the recovery rates of the hemodynamic parameters between the control and FR-1 groups and between the FR-1 and FR-2 groups. However, the recovery rates of CO and -LVdp/dt in the FR-2 group were significantly (P < 0.05) higher than those in the control group. Histopathological analysis showed that myofilaments were better preserved in the FR-2 group compared with the control group.The administration of a suppressant of proinflammatory cytokines before both ischemia and reperfusion effectively preserves donor heart function after transplantation with NHBDs.

Rapid and simple sex determination method from dental pulp by loop-mediated isothermal amplification.

Sex determination from dental pulp DNA was examined by loop-mediated isothermal amplification (LAMP) method. Amelogenin locus was analyzed for sex determination. A set of four specially designed primers was prepared based on database from Gene Bank, and loop primers were designed to shorten the analysis time. Analysis was performed using 32 dental pulp DNA samples removal from permanent teeth stored at room temperature for 1-25 years after extraction. The X allele was detected in approximately 32min with real-time turbidimeter and the Y allele was detected in approximately 34min. Analysis time was reduced to half when using loop primers. Visual detection was also possible as the amplified product showed white turbidity. Sex determination by LAMP method was rapid and simple, and it should prove useful in unknown bodies of mass disasters.

Polymorphism of the vWA locus located in the center of the three vWA STRs in the Japanese population.

The structural polymorphism of the vWA locus (vWA-T) located between the two polymorphic vWA loci (vWA-K and -P) was analyzed in 100 Japanese individuals using DNA samples isolated from dental pulp. The polymorphism of this locus was based on the difference in the number of tcta repeat. New interallele 11.1 was found in two samples. All together 9 alleles and 19 genotypes were observed. In addition, one mutant allele contained tcga in the common tcta repeat structure. The value of PD was calculated to be 0.900. Inheritance of the polymorphism was confirmed in a family including 23 individuals and 6 matings.

Combined liver and inferior vena cava resection for adrenocortical carcinoma.

PURPOSE: Adrenocortical carcinoma (ACC) is a rare malignancy, usually diagnosed at an advanced stage when it has invaded or adhered to adjacent organs. We report our experience of performing combined liver and inferior vena cava (IVC) resection for ACC. METHODS: Six patients with clinical stage III (n = 4) or IV (n = 2) ACC underwent combined resection of the liver and IVC. Two patients underwent extended right hepatectomy, and four underwent segmentectomy. In four patients, the IVC was resected segmentally: it was replaced with expanded polytetrafluoroethylene (ePTFE) in three of these patients, and not reconstructed in one. In two patients, the IVC was partially resected and closed directly. RESULTS: Perioperative mortality was zero, and morbidity was 33.3%, with temporary liver failure in two patients and renal failure in one patient. Recurrence was found within 8.1 months in three (50%) of the six patients. The mean recurrence-free survival period was 20.1 +/- 7.7 months (95% confidence interval [CI]: 5.1-35.4), and the median survival time was 6.1 +/- 9.8 months (95% CI: 00-25.3). The 5-year disease-free survival rate was 16.7%. CONCLUSIONS: Patients with ACC involving both the liver and IVC are candidates for partial hepatectomy and segmental IVC resection. Resection affords the possibility of negative margins, acceptable perioperative morbidity and mortality, and prolonged survival in some patients.

Hypervariable region structure and polymorphism of mtDNA from dental pulp and a family analysis.

Nucleotide sequences of the hypervariable region in the D-loop of mitochondrial DNA (mtDNA) were analyzed using DNA extracted from 140 old dental pulp samples. These sequences were compared with the sequence reported by Anderson et al. Nucleotide substitution in the HV1 region was identified at 77 positions. A C-to-T transition at position 16223 (C16223T) was most frequently detected (77.9%). Fourteen types of C-stretch sequence patterns were detected and the same sequence as Anderson had the highest frequency (57.9%). In the HV2 region, base transitions were identified at 56 positions. A263G was identified in all samples. Seven types of C-stretch were detected, but none had the same sequence as Anderson. In the HV3 region, base transitions were identified at 21 positions. T489C was most frequently identified (64.3%). Five types of C-stretch were detected, and the same sequence as Anderson accounted for 92.9%. The 140 samples were classified into 128 kinds by the sequence patterns of the HV region. Next, using the blood and oral mucosa epithelium from 23 subjects comprising four generations in a family line, the hereditary relationship of mtDNA was examined. All mtDNA types of the first-generation mother were infallibly inherited by the fourth generation.

Inhibition of cyclooxygenase-2 improves cardiac function following long-term preservation.

BACKGROUND: Cyclooxygenase (COX) is an intracellular enzyme that converts arachidonic acid to prostaglandin endoperoxide (PGG(2)). There are two isoforms of COX, namely constitutive COX-1 and inducible COX-2. It has been reported that COX-2 plays an important role in ischemia-reperfusion injury and that COX-2 mRNA and protein expression were up-regulated during cardiac allograft rejection. FK3311 is a suppressor of COX-2 activation. The purpose of this study was to evaluate the effectiveness of inhibiting COX-2 with FK3311 for the minimization of ischemia-reperfusion injury and for the improvement of donor heart function following transplantation in a canine model. MATERIALS AND METHODS: Adult mongrel dogs were used. After the measurement of hemodynamic parameters [cardiac output (CO), left ventricular pressure (LVP), and the maximum rates of increase and decrease in LVP (+/-LVdp/dt)], coronary vascular beds were washed out with a hypothermic (4 degrees C) University of Wisconsin (UW) solution following cardiac arrest in response to cold (4 degrees C) glucose-insulin-potassium solution. The heart was then excised and preserved in hypothermic (4 degrees C) UW solution for 12 h. FK3311 (3 mg/kg) was administered intravenously to five dogs prior to reperfusion, while vehicle was administered intravenously to a control group (n = 5). After 3 h of orthotopic transplantation using cardiopulmonary bypass, the hemodynamic parameters were compared with preoperative values of the donor animals under the condition of 10 mm Hg right atrial pressure and 5 mug/kg/min dopamine support. RESULTS: The recovery rates of CO and +/-LVdP/dt were significantly (P < 0.05) higher in the FK-treated dogs than in the controls (CO: 93 +/- 6 versus 66% +/- 4%; +LVdp/dt: 125 +/- 8 versus 77 +/- 10%; and -LVdp/dt: 81 +/- 7 versus 52 +/- 6%; for FK-treated versus control dogs, respectively). The recovery rate of LVP was higher in the FK-treated dogs than in the controls (90 +/- 5 versus 72 +/- 5%), but this difference was not statistically significant. Immunohistochemical staining revealed that COX-2 expression was reduced significantly in the myocardium of FK-treated dogs compared with controls. CONCLUSION: Hemodynamic parameters following transplantation were improved significantly in dogs treated with FK3311. Therefore, the inhibition of COX-2 improves transplanted cardiac function following long-term preservation.

[A recurrent endocrine cell cancer of the stomach showing almost complete remission after chemotherapy for 1 year].

We report a recurrent case of gastric endocrine cell cancer that showed a remarkable response to systemic chemotherapy. A 70-year-old male who underwent gastroscopy at our hospital showed a 0-IIa-like lesion, but no abnormal CT findings. He was diagnosed with gastric cancer, and underwent a proximal gastrectomy. The resected specimen showed endocrine cell cancer. The tumor was Grimelius-positive histologically and chromogranin A-and NSE-positive immunohistochemically. About 2 years after surgery, liver, lymph node, and bone metastases were detected. Systemic chemotherapy with TS-1 and CDDP was started, and the lesions progressed. Then, by approximately 1 year after CDDP and CPT-11 treatments, the recurrent lesions had diminished remarkably and were no longer seen on CT or FDG-PET.

Effects of a free radical scavenger, MCI-186, on ischemia-reperfusion injury during extended liver resection in dogs.

BACKGROUND/AIMS: In liver surgery, total clamping of the portal triad (Pringle's procedure) is commonly used, and this sometimes causes liver failure. This study evaluated the effects of a free radical scavenger, MCI-186, on ischemia-reperfusion injury during liver resection in dogs. METHODOLOGY: The experimental animals were divided into two groups. In the MCI group (n = 6), MCI-186 (6mg/kg/h) was administered twice, through a catheter placed in the right hepatic vein: the first time was from 0.5 hours before the onset of ischemia until ischemia by partial inflow occlusion, and the second was from 0.5-hours before reperfusion until reperfusion. In the control group (n = 6), vehicle (physiological saline) was administered in the same manner. RESULTS: The serum AST, ALT, and LDH levels were significantly (P < 0.05) lower in the MCI group than in the control group. Hepatic tissue blood flow 0.5 hours after reperfusion was significantly (P < 0.05) higher in the MCI group than in the control group. Histological tissue damage was mild, and tissue MDA levels were significantly (P < 0.05) lower in the MCI group than in the control group. CONCLUSIONS: MCI-186 ameliorates the ischemia-reperfusion injury caused by Pringle's procedure during extended liver resection.

Long-term preservation using a new apparatus combined with suppression of pro-inflammatory cytokines improves donor heart function after transplantation in a canine model.

OBJECTIVE: We developed a new apparatus for long-term heart preservation that combines simple immersion with coronary perfusion. In a previous study, we reported that suppression of pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNF-alpha) and interleukin-1beta (IL-1beta), improved results after transplantation. In this study, we evaluated whether long-term preservation using our apparatus for continuous coronary perfusion, combined with suppression of pro-inflammatory cytokines, improves donor heart function after transplantation in a canine model. METHODS: We used adult mongrel dogs in this study. Coronary vascular beds were washed with University of Wisconsin (UW) solution after arresting hearts with glucose-insulin-potassium solution. The heart was then excised and preserved for 12 hours with a combination of immersion and coronary perfusion using a preservation apparatus. Adult mongrel dogs were divided into 2 groups: the coronary perfusion (CP) group (n = 7) and the FR167653 (FR-CP) group (n = 6). In the CP group, we used a 4 degrees C UW solution for immersion and coronary perfusion. In the FR-CP group, we used a 4 degrees C UW solution supplemented with 20 mg/liter of the anti-inflammatory agent FR167653 for immersion and coronary perfusion. At 2 and at 3 hours after orthotopic transplantation, we compared hemodynamic parameters with pre-operative values in donor animals, with right atrial pressure at 10 mm Hg and with 5 microg/kg/min dopamine infusion. We compared serum concentrations of TNF-alpha from the coronary sinus and compared electron microscopic studies between the 2 groups. RESULTS: Three hours after transplantation, cardiac output (CO), left ventricular pressure (LVP), and -LVdp/dt were significantly greater (p < 0.05) in the FR-CP group than in the CP group (CO, 178% +/- 65% vs 93% +/- 40%; LVP, 115% +/- 22% vs 73% +/-26%; -LVdp/dt, 168% +/- 13% vs 61% +/- 17%, respectively). Electron microscopic studies showed that glycogen was well preserved in the FR-CP group compared with the CP group. Serum concentrations of TNF-alpha were decreased significantly in the FR-CP group compared with the CP group at 3 hours after reperfusion (161 +/- 54 pg/dl vs 642 +/- 636 pg/dl, respectively). CONCLUSION: Hemodynamics after transplantation were significantly better in the FR-CP group than in the CP group. The combined preservation method of continuous perfusion and immersion using our apparatus in conjunction with suppression of pro-inflammatory cytokines improves donor heart function after transplantation.

The effects of a bradykinin B2 receptor antagonist in extended liver resection with ischemia in dogs.

BACKGROUND/AIMS: In liver surgery, total clamping of the portal triad (Pringle's procedure) is commonly used, and sometimes causes liver failure. This study evaluated the effects of a bradykinin B2 receptor antagonist, FR173657 (FR), on ischemia-reperfusion injury during liver resection in dogs. METHODOLOGY: Experimental animals were divided into two groups. In the FR group (n=6), FR (100 nmol/kg/hr) was administered continuously via the portal vein from 30 min before the onset of ischemia until 2 hr after reperfusion. In the control group (n=6), vehicle was injected in the same manner. The right portal pedicle was clamped for 60 min, while the left portal branch was left patent to avoid portal congestion. Following reperfusion, the non-ischemic lobes were resected, and remnant liver function was evaluated. RESULTS: AST and ALT were significantly (p<0.05) lower in the FR group than in the control group. Hepatic tissue blood flow 30 min after reperfusion was significantly (p<0.05) higher in the FR group than in the control group. Histological tissue damage was mild, and polymorphonuclear neutrophil infiltration was significantly (p<0.05) reduced in the FR group compared with the control group. CONCLUSIONS: A bradykinin B2 receptor antagonist ameliorated the ischemia-reperfusion injury caused by Pringle's procedure during extended liver resection.

Effects of a bradykinin B(2) receptor antagonist on ischemia-reperfusion injury in a canine lung transplantation model.

BACKGROUND: This study investigated the effects of a bradykinin B(2) receptor antagonist, FR173657 (FR), on ischemia-reperfusion (I/R) injury in a canine lung transplantation model. METHODS: Eighteen pairs of weight-matched dogs were randomly divided into 3 groups. Six pairs were assigned to the FR(D+R) group, in which FR (100 nmol/kg/h) was administered to the transplant donor continuously beginning 30 minutes before ischemia until the onset of ischemia, and FR was administered to the transplant recipient beginning 30 minutes before reperfusion and continuing for 2 hours after reperfusion. Another 6 pairs of dogs were assigned to the FR(R) group, in which FR was administered only to the recipient in the same manner as in the FR(D+R) group. The other pairs were assigned to the control group, in which vehicle alone was administered. Orthotopic left lung transplantation was performed after 12-hour cold storage in Euro-Collins solution. Fifteen minutes after reperfusion, the right pulmonary artery and the right stem bronchus were ligated. The animals were measured for 4 hours after reperfusion for left pulmonary vascular resistance (L-PVR), cardiac output (CO), arterial oxygen pressure (PaO(2)) and alveolar-arterial oxygen pressure difference (A-aD(O(2))). Lung specimens were harvested for measurement of the wet-to-dry lung weight ratio (WDR), histopathologic studies and polymorphonuclear neutrophil (PMN) count. RESULTS: Compared with the control group, PaO(2), A-aDO(2), L-PVR and CO were all significantly (p < 0.05) improved and WDR significantly (p < 0.05) lower in both the FR(D+R) and FR(R) groups. Moreover, in the FR-treated groups, histologic tissue edema was mild, and PMN infiltration was significantly (p < 0.05) reduced. CONCLUSIONS: The bradykinin B(2) receptor antagonist, FR173657, ameliorates I/R injury in lung grafts, indicating that protection of lung grafts can be achieved by the administration of FR solely to the transplant recipient.