Bloodstain examination and DNA typing from hand-washed bloodstains on clothes.

We investigated whether bloodstain examination and DNA typing can be performed on washed bloodstains on clothes. Blood was dropped onto T-shirts made from 100% cotton or 100% polyester. After drying, the T-shirts were hand-washed with handwashing soap, dishwashing detergent, laundry detergent, soap, or just water until the bloodstains could not be seen. After drying the T-shirts, DNA and RNA were extracted simultaneously from the bloodstained areas using commercial kits. RNA was reverse-transcribed to DNA, and then the detection of the mRNAs for HBB, ACTB, and 18S rRNA was examined. DNA was quantified via real-time PCR, and then STR typing was performed with a commercial kit. The luminol and leucomalachite green tests were used as preliminary bloodstain tests, and an immuno-chromatography kit was used to identify human bloodstains. DNA could be extracted from all washed bloodstains, but more DNA was extracted from cotton T-shirts than from polyester T-shirts. STR typing was successful for all bloodstains without issues such as PCR inhibition. In the human bloodstain identification test using mRNA, almost all bloodstains produced a Ct value for HBB and all bloodstains produced a Ct value for 18S rRNA, whereas few bloodstains produced a Ct value for ACTB. All bloodstains reacted positively to luminol, but some were negative for leucomalachite green. Most of the bloodstains did not react positively in the human bloodstain identification test using the immuno-chromatography kit. The results suggest that human bloodstain identification and DNA typing can still be performed after clothes with bloodstains are washed.

Endoscopic full-thickness resection of gastric gastrointestinal stromal tumor: a Japanese case series.

BACKGROUND: Gastrointestinal stromal tumors (GISTs) are potentially malignant and are indicated for resection. The standard treatment for resectable GISTs is surgery, although endoscopic resection has been reported outside Japan. This study retrospectively analyzed the results of endoscopic resection of GISTs in Japan. METHOD: We identified patients with GISTs treated only by endoscopic resection in our institute between January 2016 and December 2018, and analyzed their clinical and pathological characteristics. RESULTS: During the study period, 8 GISTs were resected only by endoscopy: 7 were located in the upper third of the stomach and 1 in the middle. All were intraluminal growth type. Median (range) tumor diameter was 20 (10-35) mm. All tumors were resected en bloc with a median (range) operation time of 67.5 (50-166) min. Complete perforation occurred in 5 cases, but the serosa remained in 2 and the outer layer of the muscularis propria remained in 1. The defect was endoscopically closed with clip-and-endoloop purse-string suturing (n=3), simple endoclipping (n=2), or over-the-scope clipping (n=2), and 1 did not require closure because the outer longitudinal muscle was preserved. Oral feeding was commenced on postoperative day (POD) 3 (median; range 2-4), and the patient was discharged on POD 6 (median; range 4-11). No serious adverse event developed after the procedures. CONCLUSION: Endoscopic resection for selected cases of small intraluminal GISTs is feasible, making it a viable alternative treatment option to laparoscopic surgery.

Time to initiation or duration of S-1 adjuvant chemotherapy; which really impacts on survival in stage II and III gastric cancer?

BACKGROUND: Surgical resection with S-1 adjuvant chemotherapy (AC) is the standard of care for stage II-III gastric cancer (GC). However, it is unclear if time to initiation and duration of S-1 AC impact on survival. METHODS: A multi-institutional GC database identified 498 patients who were treated with S-1 AC after D2 or more extended radical surgery for stage II-III gastric cancer. Patients were divided into four groups according to the interval between surgery and initiation of AC and the duration of AC as follows: group A (n = 226), who received AC earlier (≤6 weeks) and for longer (≥6 months) after surgery; group B (n = 160), who received AC later (>6 weeks) and for longer after surgery; group C (n = 46), who received AC earlier but for a shorter period (<6 months) after surgery; and group D (n = 66), who received AC later and for a shorter period after surgery. Prognostic factors for overall survival (OS) were investigated using multivariate analysis. RESULTS: The 5-year OS was 69.5%. Pathological stage II disease (hazard ratio (HR), 0.334; 95% confidence interval (CI), 0.215-0.499), with an OS of 85.8% versus 60.5% for stage III disease, as well as a longer duration (≥6 months) of S-1 (HR, 0.498; 95% CI, 0.355-0.706), with an OS of 74.3% versus 53.0% for a shorter duration (<6 months) of S-1, were identified as significant prognostic factors for long-term survival. Time to initiation was not associated with OS. CONCLUSIONS: A duration of S-1 AC of ≥6 months, but not time to initiation within 6 weeks, impacts on OS in stage II-III gastric cancer.

[Eleven Patients with Gastric Cancer Who Received Chemotherapy after Stent Placement for Gastric Outlet Obstruction].

Endoscopic placement of self-expandable metallic stents is reportedly effective for gastric outlet obstructions due to advanced gastric cancer, and is less invasive than gastrojejunostomy. For patients who have good performance status, we administer chemotherapy after stent placement, although the safety and feasibility of this chemotherapy have not yet been discussed in full. Between 2011 and 2015, 15 patients at our institution underwent endoscopic gastroduodenal stent placement for gastric outlet obstruction due to gastric cancer. Eleven of these patients were administered chemotherapy after stent placement. In our case series, we did not observe any specific adverse event caused by stent placement plus chemotherapy. Adverse events after chemotherapy included anemia of CTCAE Grade 3 in 7 patients. Stent-in-stent placement was needed in 2 patients. Neither stent migration nor perforation was observed. Therefore, chemotherapy after stent placement for gastric outlet obstruction due to gastric cancer was considered safe and feasible. Stent placement is useful not only as palliative care for patients with terminal-stage disease, but also as one of the multimodal therapeutic strategies for gastric cancer.

Identification of canine saliva using mRNA-based assay.

A dog saliva analysis in addition to a bite-mark analysis may be important for evidence when a crime involves a dog bite. In this study, the utility of detecting canine saliva-specific mRNAs to identify canine saliva was evaluated. Canine saliva swabs (n = 20), urine swabs (n = 20), body surface swabs (n = 20), whole blood samples (n = 10), human saliva (n = 20), human skin surface swabs (n = 20), and human whole blood (n = 20) were tested. The saliva-specific genes encoding statherin (STATH), carbonic anhydrase VI (CA-VI), and dog allergens (Canf1 and Canf2) were analyzed as candidate genes. Moreover, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as confirmation of canine mRNA extraction. STATH, CA-VI, Canf1, Canf2, and GAPDH mRNAs were detected in 19/20, 1/20, 11/20, 4/20, and 20/20 saliva samples, respectively. The STATH, CA-VI, Canf1, Canf2, and GAPDH mRNAs did not exhibit cross-reactivity with samples of human origin. This mRNA-based assay was also able to detect canine saliva in mock forensic samples. The results of this study indicated that the detection of STATH mRNA is useful for the identification of canine saliva, and GAPDH is a suitable marker for canine mRNA extraction.

[A Case of Hepatocellular Carcinoma with Portal Vein Tumor Thrombosis Treated Successfully with Multidisciplinary Therapy Including Preoperative Stereotactic Body Radiotherapy(SBRT)].

A 61-year-old man untreated with hepatitis C virus presented to our hospital.He was diagnosed advanced hepatocellular carcinoma(HCC)with portal vein tumor thrombosis(PVTT).The hazy tumor was located in the left lobe, and the tumor thrombus extended into the left portal vein.The patient received stereotactic body radiotherapy(SBRT, 48 Gy/4 Fr)for PVTT. The extended left lobectomy with thrombectomy was performed 12 days after SBRT.Resected specimen was diagnosed histopathologically as a poorly differentiated HCC, vp1, and no viable tumor cells in the tumor thrombosis.The patient's postoperative course was uneventful, and hepatic arterial infusion chemotherapy was started 1 month after the operation.He remains free of recurrence 5 years after the hepatectomy.Multidisciplinary therapy including preoperative SBRT was feasible and might be a treatment option for HCC with PVTT.

[Surgical Resection of Peritoneal Recurrence of Hepatocellular Carcinoma with Endoscopic Fluorescence Imaging System].

A 66-year-old man presented with recurrences in the peritoneum 3 years after heavy particle beam therapy with hepatocellular carcinoma(HCC)and underwent laparoscopic surgery.Five months after surgery, a new peritoneal dissemination found in the vicinity of the colon hepatic flexure area by CT examination, and laparoscopic resection was planned again.Indocyanine green(ICG)0.5 mg/kg was administered on the day before surgery.ICG imaging by the PINPOINT®system revealed 2 small ICG accumulation sites in the diaphragm, as well as the main lesion, and each lesion was excised laparoscopically.All lesions were diagnosed as peritoneal dissemination of HCC, and the postoperative course was uneventful.Although new dissemination nodules were appeared 6 months after surgery, he underwent laparoscopic surgery again and survives.In PINPOINT fluorescence mode, high-definition white-light image and fluorescence image was combined, and it was easy to determine the cut line but also to visualize the small lesion difficult to identify in the visible light mode.It was suggested that the PINPOINT®system might be useful in cases of HCC peritoneal dissemination.

Screening Test for Shed Skin Cells by Measuring the Ratio of Human DNA to Staphylococcus epidermidis DNA.

A novel screening method for shed skin cells by detecting Staphylococcus epidermidis (S. epidermidis), which is a resident bacterium on skin, was developed. Staphylococcus epidermidis was detected using real-time PCR. Staphylococcus epidermidis was detected in all 20 human skin surface samples. Although not present in blood and urine samples, S. epidermidis was detected in 6 of 20 saliva samples, and 5 of 18 semen samples. The ratio of human DNA to S. epidermidisDNA was significantly smaller in human skin surface samples than in saliva and semen samples in which S. epidermidis was detected. Therefore, although skin cells could not be identified by detecting only S. epidermidis, they could be distinguished by measuring the S. epidermidis to human DNA ratio. This method could be applied to casework touch samples, which suggests that it is useful for screening whether skin cells and human DNA are present on potential evidentiary touch samples.

Expression of recombinant organophosphorus hydrolase in the original producer of the enzyme, Sphingobium fuliginis ATCC 27551.

The plasmid encoding His-tagged organophosphorus hydrolase (OPH) cloned from Sphingobium fuliginis was modified to be transferred back to this bacterium. The replication function of S. amiense plasmid was inserted at downstream of OPH gene, and S. fuliginis was transformed with this plasmid. The transformant produced larger amount of active OPH with His-tag than E. coli.

Comparison of surgical techniques for stoma closure: A retrospective study of purse-string skin closure versus conventional skin closure following ileostomy and colostomy reversal.

The aim of this study was to compare the incidence of postoperative complications, including superficial incisional surgical site infection (SSI) following purse-string skin closure (PS) and conventional skin closure with a drainage tube (CD) following stoma closure. A total of 55 consecutive patients who underwent loop colostomy and loop ileostomy closures in our hospital between October, 2011 and September, 2014 were retrospectively assessed. The patients were divided into two groups, namely the PS group (26 patients) and the CD group (29 patients). There were no significant differences in the characteristics of the patients between the two groups. The baseline and operative characteristics also did not differ significantly between the two groups. However the incidence of superficial incisional SSI was lower in the PS group compared to that in the CD group (0 vs. 13.8%, respectively; P=0.049). The overall incidence of complications did not differ significantly between the two groups (P=0.313). The duration of postoperative hospital stay in the PS group was shorter compared to that in the CD group. In conclusion, the results of this study suggest that PS may an effective technique to reduce the incidence of superficial incisional SSI. This technique appears to be superior to the conventional technique, allowing for better cosmesis.

A novel method for sex determination by detecting the number of X chromosomes.

A novel method for sex determination, based on the detection of the number of X chromosomes, was established. Current methods, based on the detection of the Y chromosome, can directly identify an unknown sample as male, but female gender is determined indirectly, by not detecting the Y chromosome. Thus, a direct determination of female gender is important because the quality (e.g., fragmentation and amelogenin-Y null allele) of the Y chromosome DNA may lead to a false result. Thus, we developed a novel sex determination method by analyzing the number of X chromosomes using a copy number variation (CNV) detection technique (the comparative Ct method). In this study, we designed a primer set using the amelogenin-X gene without the CNV region as the target to determine the X chromosome copy number, to exclude the influence of the CNV region from the comparative Ct value. The number of X chromosomes was determined statistically using the CopyCaller software with real-time PCR. All DNA samples from participants (20 males, 20 females) were evaluated correctly using this method with 1-ng template DNA. A minimum of 0.2-ng template DNA was found to be necessary for accurate sex determination with this method. When using ultraviolet-irradiated template DNA, as mock forensic samples, the sex of the samples could not be determined by short tandem repeat (STR) analysis but was correctly determined using our method. Thus, we successfully developed a method of sex determination based on the number of X chromosomes. Our novel method will be useful in forensic practice for sex determination.

Identification of feces by detection of Bacteroides genes.

In forensic science, the identification of feces is very important in a variety of crime investigations. However, no sensitive and simple fecal identification method using molecular biological techniques has been reported. Here, we focused on the fecal bacteria, Bacteroides uniformis, Bacteroides vulgatus and Bacteroides thetaiotaomicron, and developed a novel fecal identification method by detection of the gene sequences specific to these bacteria in various body (feces, blood, saliva, semen, urine, vaginal fluids and skin surfaces) and forensic (anal adhesions) specimens. Bacterial gene detection was performed by real-time PCR using a minor groove binding probe to amplify the RNA polymerase ß-subunit gene of B. uniformis and B. vulgatus, and the α-1-6 mannanase gene of B. thetaiotaomicron. At least one of these bacteria was detected in the feces of 20 donors; the proportions of B. uniformis, B. vulgatus and B. thetaiotaomicron were 95, 85 and 60%, respectively. Bacteroides vulgatus was also detected in one of six vaginal fluid samples, but B. thetaiotaomicron and B. uniformis were not detected in body samples other than feces. Further, we applied this method to forensic specimens from 18 donors. Eighteen anal adhesions also contained at least one of three bacteria; B. uniformis, B. vulgatus and B. thetaiotaomicron were detected in 89, 78 and 56%, respectively, of the specimens. Thus, these bacteria were present at a high frequency in the fecal and forensic specimens, while either B. uniformis or B. vulgatus was detected in all samples. Therefore, B. uniformis and B. vulgatus represent more appropriate target species than B. thetaiotaomicron for the identification of fecal material. If B. vulgatus and/or B. uniformis are detected, it is likely that the sample contains feces. Taken together, our results suggest that the use of molecular biological techniques will aid the detection of feces in forensic practice, although it is possible that the samples contained both feces and vaginal fluid.

Chromic behaviors of hexagonal columnar liquid crystalline platinum complexes with catecholato, 2-thiophenolato, and benzenedithiolato.

Liquid crystalline (LC) platinum(II) complexes with 1,2-thiophenolato and 1,2-benzendithiolato have been newly synthesized and investigated by spectroscopy together with the catecholato analogue. The variations in coordinating atoms (O or S or O/S mixed) lead to significant modulation in electrochemical properties in solution and absorption and emission properties of the complexes both in solution media and in the condensed phases. The asymmetric, polar mesogens/chromophores consisting of Pt(II), redox-active ligands, and alkyl-substituted bipyridine commonly play important roles not only in stabilizing the columnar LC phases, but also in fluctuations of the ground state energies. A key finding of the present work is the chromic properties of LC complexes induced by the interplay of self-association of the mesogens/chromophores and their fluctuating properties.

A simple identification method of saliva by detecting Streptococcus salivarius using loop-mediated isothermal amplification.

We previously reported that detection of Streptococcus salivarius is feasible for proving the presence of saliva in a forensic sample. Here, a simple and rapid method for the detection of S. salivarius in forensic samples was developed that uses loop-mediated isothermal amplification (LAMP). The LAMP primer set was designed using S. salivarius-specific sequences of glucosyltransferase K. To simplify the procedure, the sample was prepared by boiling and mutanolysin treatment only, and the entire analytical process was completed within 2.5 h. The cut-off value was set at 0.1 absorbance units, measured at 660 nm, upon termination of the reaction. S. salivarius was identified in all saliva samples, but was not detected in other body fluids or on the skin surface. Using this method, S. salivarius was successfully detected in various mock forensic samples. We therefore suggest that this approach is useful for the identification of saliva in forensic practice.

A novel method for the identification of saliva by detecting oral streptococci using PCR.

We have used DNA amplification methods to detect common oral bacterial strains to test for the presence of saliva in forensic samples. Streptococcus salivarius and Streptococcus mutans were detected in various forms of saliva samples, whereas these streptococci were not detected in semen, urine, vaginal fluid, or on skin surfaces. Therefore, we demonstrated that these streptococci are promising new marker for the forensic identification of saliva. Our data indicated that S. salivarius is more reliable than S. mutans as an indicator of saliva presence, because the detection rates for S. salivarius and S. mutans by this method were 100% and 90%, respectively. Furthermore, S. salivarius was detected in all saliva stain samples, whereas S. mutans was only identified in 60% of the stains. Finally, using this method we were able to successfully detect S. salivarius and S. mutans in mock forensic samples. We therefore suggested that this method is useful for the identification of saliva in forensic science.

Methamphetamine body packer: acute poisoning death due to massive leaking of methamphetamine.

We encountered three methamphetamine (MA) body packers presenting simultaneously, one of whom died. Three Nigerian men (39, 35, and 37 years old) who attempted to smuggle were found to contain 35 (498 g), 21 (292 g), and 5 packages (73 g) of methamphetamine hydrochloride (MA-HCl) in their stomachs, respectively. Packages were wrapped with plastic film and Scotch tape. The 39-year-old man died with acute poisoning from c. 20 g of MA-HCl that had leaked from the packages into the stomach. His plasma MA concentration was 8.6 microg/mL when he was hospitalized (17 h before his death). Autopsy findings showed extreme pulmonary congestion and edema as well as moderate hepatic edema and several petechiae. Quantitative analysis was performed by gas chromatography/mass spectrometry. Extremely high concentrations of MA and its metabolite amphetamine (AP) were found in cardiac blood (63.5 microg/mL and 1.2 microg/mL), urine (4,518 microg/mL and 72.4 microg/mL), gastric contents (8,490 microg/mL and 16.9 microg/mL), and in all other autopsy samples. These high concentrations confirmed that the cause of death was acute MA poisoning. Furthermore, impurity-profiling analysis of the seized MA revealed that the MA smuggled by the three suspects originated from the same batch.

Preparation of latex reagents combined with IgM and its F(ab')2 fragment from commercial ABO blood grouping reagent.

To achieve a rapid assay for ABO blood grouping using a latex reagent, two latex reagents were produced, one of which combined with mouse monoclonal immunoglobulin M (IgM) isolated from commercial ABO blood grouping reagent, and the other of which combined with its F(ab')2 fragment prepared by cold pepsin digestion. The latex reagent adsorbing the F(ab')2 fragment was able to detect the 1000-fold diluted saliva and provided much better sensitivity than that of IgM. This suggests that the difference in sensitivity between the two latex reagents is responsible for adsorption orientation of the antigen site on the latex particles. The new assay successfully completed the ABO blood grouping of cigarette ends within 30 min.

New safe method for preparation of sarin-exposed human erythrocytes acetylcholinesterase using non-toxic and stable sarin analogue isopropyl p-nitrophenyl methylphosphonate and its application to evaluation of nerve agent antidotes.

INTRODUCTION: A non-toxic and stable sarin analogue, isopropyl p-nitrophenyl methylphosphonate (INMP), was synthesized for safe preparation of sarin-exposed acetylcholinesterase (AChE). RESULTS AND DISCUSSION: This agent was stable for years, able to be handled in an ordinary laboratory without special care, and its 50% inhibitory concentration (IC50) on 0.04 U/ml human erythrocytes AChE was 15 nM. This reagent was thought to be especially useful since it enables experiments that require sarin-inhibited AChE, such as the development of antidotes for sarin, in a usual laboratory. To demonstrate the usefulness of this method, 40 known and novel pyridinealdoxime methiodide (PAM)-type oxime antidotes were synthesized, and their reactivation activities to INMP-exposed AChE and structure-activities correlation were studied. CONCLUSION: Among the antidotes tested in this experiment except for 2-PAM, the compound found to have the highest reactivation activity, was the novel hydrophobic 2-PAM-type compound, 2-[(hydroxyimino)methyl]-1-[4-(tert-butyl)benzyl] pyridinium bromide.

Individual comparisons of the levels of (E)-3-methyl-2-hexenoic acid, an axillary odor-related compound, in Japanese.

The (E)-3-methyl-2-hexenoic acid (E3M2H), an axillary odor-related compound, is known to occur in Caucasians. The aims of this study were to clarify whether E3M2H contributes to axillary odor in Asians and to quantify and compare individual levels of E3M2H. Quantitative determination of E3M2H was performed by means of gas chromatography-mass spectrometry of sweat extracted from the axillary areas of T-shirts worn for 24 h by Japanese subjects. The amount of E3M2H was 15.9-34.6 nmol/ml in six of 30 subjects. Our method succeeded in quantitative analysis of E3M2H from axillary sweat collected individually; we also showed that E3M2H could be detected in Asians. This is the first report in which the amount of E3M2H in axillary sweat was quantified on an individual basis and compared to reveal individual differences. The results of this study indicate that E3M2H might contribute to axillary malodor in Asians as well as Caucasians.