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Aim: Whole-exome sequencing (WES) studies have revealed that germline de novo variants (gDNVs) contribute to the genetic etiology of schizophrenia. However, the contribution of mosaic DNVs (mDNVs) to the risk of schizophrenia remains to be elucidated. In the present study, we systematically investigated the gDNVs and mDMVs that contribute to the genetic etiology of schizophrenia in a Japanese population., Methods: We performed deep WES (depth: 460×) of 73 affected offspring and WES (depth: 116×) of 134 parents from 67 families with schizophrenia. Prioritized rare nonsynonymous gDNV and mDNV candidates were validated using Sanger sequencing and ultra-deep targeted amplicon sequencing (depth: 71,375×), respectively. Subsequently, we performed a Gene Ontology analysis of the gDNVs and mDNVs to obtain biological insights. Lastly, we selected DNVs in known risk genes for psychiatric and neurodevelopmental disorders., Results: We identified 62 gDNVs and 98 mDNVs. The Gene Ontology analysis of mDNVs implicated actin filament and actin cytoskeleton as candidate biological pathways. There were eight DNVs in known risk genes: splice region gDNVs in AKAP11 and CUL1; a frameshift gDNV in SHANK1; a missense gDNV in SRCAP; missense mDNVs in CTNNB1, GRIN2A, and TSC2; and a nonsense mDNV in ZFHX4., Conclusion: Our results suggest the potential contributions of rare nonsynonymous gDNVs and mDNVs to the genetic etiology of schizophrenia. This is the first report of the mDNVs in schizophrenia trios, demonstrating their potential relevance to schizophrenia pathology., (© 2024 The Author(s). Psychiatry and Clinical Neurosciences © 2024 Japanese Society of Psychiatry and Neurology.)

Background: The organized functioning of the anisotropic myocardial layers-including the inner longitudinal, middle circular, and outer longitudinal layers-is essential for stable systemic circulation. However, the proteomic profile of each myocardial layer has not been studied yet. Here, we aimed to elucidate the layer-specific proteomic profile of human cardiac tissue using microscopic sampling., Methods: Normal hearts were obtained from five autopsy cases, and cardiomyocytes were microdissected separately from the three myocardial layers of the left ventricle. Histological analysis and shotgun proteomic profiling were performed, followed by immunohistochemical analysis., Results: Histologically, no significant changes were observed among the three layers regarding cardiomyocyte diameter and myocardial fibrosis. Totally 1220 proteins-comprising 9404 peptides-were identified from 15 samples, of which the expression levels of 92 proteins were significantly altered among the layers. Gene ontology enrichment analysis revealed that the proteins specifically elevated in the inner and outer layers mostly belonged to the actin filament-binding protein group. In particular, MYH1 was highly expressed in cardiomyocytes in the outer layer, and CTNNA3 was highly expressed at the intercalated disc in the inner layer., Conclusions: This is the first report on layer-specific proteomic profiling of human normal hearts. Anisotropic profiles of actin filament-binding proteins in myocardial layers may contribute to the anisotropic contractile and conductive abilities of the heart. Knowledge of the layer-specific proteome profiles of a human heart in the normal state can aid in further research on cardiac pathology, such as the prognosis and treatment of focal myocardial infarction., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationship that could have appeared to influence the work reported in this study., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Trauma has been suspected as a factor leading to development of angiosarcoma, a malignant soft-tissue tumor. We conducted a forensic autopsy to investigate a putative relation between a motor vehicle collision and the driver's later death from angiosarcoma. A vehicle operated by a man in his 60 s collided with an oncoming vehicle at a curve. The victim noticed no injury at the scene. However, 45 days later, he was transferred to an emergency room with dyspnea and bloody sputum. After diagnosis of angiosarcoma, he died of respiratory failure 132 days later. The bereaved family speculated about a relation between the collision and angiosarcoma onset. At autopsy, tumor cells of the scalp had metastasized to the lung, pleura, liver, and spleen. Histopathological examinations revealed characteristic features of angiosarcoma with positive immune-staining for CD31, CD34, and factor VIII. When a person dies some time after a collision, it is designated as a delayed death. In such cases, the relevance of trauma to the person's death is often an issue of concern. Because the interval between trauma and angiosarcoma development was short, only 45 days, the angiosarcoma might be coincidental. Therefore, we rejected the relation. Forensic experts sometimes need to investigate such inquiries., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

DNA methylation modifications are known to influence epigenetic phenomena and have been a focus of forensic science research for some time. Degraded DNA after bisulfite treatment is widely used in DNA methylation analysis. In this study, we analyzed methylation levels at 12 CpG sites of four selected genomic regions by pyrosequencing after bisulfite treatment. DNA was extracted from buccal swab samples collected from 102 Japanese individuals who were 21-77 years old. We also developed a simple method to quantify the degradation levels of bisulfite-converted DNA by real-time PCR, and evaluated the effect of DNA degradation on age estimation. We found that the methylation levels and chronological ages were highly correlated in the four selected regions, and the mean absolute deviation (MAD) between chronological and estimated ages was low at 3.88 years. These results indicated that pyrosequencing analysis at the 12 CpGs was useful for age estimation in the Japanese population. To develop a sensitive quantification method, we analyzed the amplification efficiency of short and long fragments from 10 regions by real-time PCR. The amplification efficiency was highest for CCDC102B, and the degradation levels of bisulfite-converted DNA for the 102 samples were categorized as moderately or heavily degraded. For the younger age groups (20-49 years), the MADs were lower for moderately degraded DNA than they were for heavily degraded DNA. This finding indicates that degradation levels affected the accuracy of age estimation in most of the samples; the exception was the samples from the 50-77 years age group., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Coronavirus disease 2019 (COVID-19) is an emerging respiratory infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). COVID-19 has been reported to lead to acute cardiac injury, but previous research indicated that the mechanism is different from that of other viruses and remains poorly understood. Herein, we describe a case of COVID-19-associated sudden death, in a healthy 47-year-old man after developing diffuse cardiac necrosis. Two days before death, the patient developed general malaise without respiratory symptoms. The patient's fatigue worsened with time, and he ultimately developed cardiac arrest in an ambulance; however, resuscitation was unsuccessful. Antigen testing performed at the hospital revealed that the patient was positive for SARS-CoV-2 virus. At autopsy, contraction band necrosis was observed insularly in all areas of the myocardium. CD42b immunohistochemical staining indicated platelet aggregation in the microvessels around the cardiac necrosis area, suggesting COVID-19 can be fatal for healthy people by microcirculatory disturbance due to diffuse cardiac injury arising from platelet activiation. This unique mechanism can be a novel therapeutic target of COVID-19-related cardiac injury., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

Homicide by electrocution is rare in forensics, and the identification of the cause of death can be quite difficult when the electric device is removed from the scene. We present an instance where the police were unsure of homicide in the initial investigation. The offender used hand-made electrode plates for electrocution, which produced unique electric marks different from those produced by common electric devices such as electric wires. To the best of our knowledge, this is the first report of homicide by electrocution with electrode plates. We believe that the macroscopic and microscopic findings in this instance are quite valuable for forensic practitioners., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Cardiac hypertrophy, which develops in middle-aged and older individuals as a consequence of hypertension and obesity, is an established risk factor for sudden cardiac death (SCD). However, it is sometimes difficult to differentiate SCD with acquired cardiac hypertrophy (SCH) from compensated cardiac hypertrophy (CCH), at autopsy. We aimed to elucidate the proteomic alteration in SCH, which can be a guideline for future postmortem diagnosis., Methods: Cardiac tissues were sampled at autopsy. SCH group consisted of ischemic heart failure, hypertensive heart failure, and aortic stenosis. CCH group included cases of non-cardiac death with cardiac hypertrophy. The control group comprised cases of non-cardiac death without cardiac hypertrophy. All patients were aged > 40 years, and hypertrophic cardiomyopathy was not included in this study. We performed histological examination and shotgun proteomic analysis, followed by quantitative polymerase chain reaction analysis., Results: Significant obesity and myocardial hypertrophy, and mild myocardial fibrosis were comparable in SCH and CCH cases compared to control cases. The proteomic profile of SCH cases was distinguishable from those of CCH and control cases, and many sarcomere proteins were increased in SCH cases. Especially, the protein and mRNA levels of MYH7 and MYL3 were significantly increased in SCH cases., Conclusion: This is the first report of cardiac proteomic analysis in SCH and CCH cases. The stepwise upregulation of sarcomere proteins may increase the risk for SCD in acquired cardiac hypertrophy before cardiac fibrosis progresses significantly. These findings can possibly aid in the postmortem diagnosis of SCH in middle-aged and older individuals., (© 2023. The Author(s).)

Background: Most platelets are present in peripheral blood, but some are stored in the spleen. Because the tissue environments of peripheral blood vessels and the spleen are quite distinct, the properties of platelets present in each may also differ. However, no studies have addressed this difference. We previously reported that hypothermia activates splenic platelets, but not peripheral blood platelets, whose biological significance remains unknown. In this study, we focused on platelet-derived microvesicles (PDMVs) and analyzed their biological significance connected to intrasplenic platelet activation during hypothermia., Methods: C57Bl/6 mice were placed in an environment of -20 °C, and their rectal temperature was decreased to 15 °C to model hypothermia. Platelets and skeletal muscle tissue were collected and analyzed for their interactions., Results: Transcriptomic changes between splenic and peripheral platelets were greater in hypothermic mice than in normal mice. Electron microscopy and real-time RT-PCR analysis revealed that platelets activated in the spleen by hypothermia internalized transcripts, encoding tissue repairing proteins, into PDMVs and released them into the plasma. Plasma microvesicles from hypothermic mice promoted wound healing in the mouse myoblast cell line C2C12. Skeletal muscles in hypothermic mice were damaged but recovered within 24 h after rewarming. However, splenectomy delayed recovery from skeletal muscle injury after the mice were rewarmed., Conclusions: These results indicate that PDMVs released from activated platelets in the spleen play an important role in the repair of skeletal muscle damaged by hypothermia., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Age prediction based on methylation analysis has been reported in many populations, with 10 ng or more of DNA usually required for each determination. In this study, we designed thermostable locked nucleic acid (LNA) primers by replacing a small number of DNA bases in standard DNA primers with LNAs. We evaluated these primer sets by single-base extension analysis using 10, 5, or 2 ng of DNA that would be less than template DNA used in standard methylation testing, and determined sensitivity and accuracy. We analyzed EDARADD, SST, and KLF14 genes, targeting one CpG site in each gene. Melting temperature values of most LNA primers were 4°C higher than those of DNA primers. The intensities of signals from the EDARADD and SST genes were significantly improved by the LNA primers, by 3.3 times and 1.4 times, respectively, compared with the DNA primers using 2 ng of DNA. Coefficient of variation (CV) analysis was used to assess the accuracy of the determined methylation levels. CVs were increased using small amounts of DNA, but lower CVs were detected using LNA primers. We also showed high accuracy of age prediction for 51 individuals using LNA primers. The lowest mean absolute deviation was obtained using 10 ng of DNA and was 3.88 years with the LNA primers. Thermostable PCR primers were simply designed, and the LNAs improved the sensitivity and accuracy of methylation analysis for 10 ng or less of DNA., (© 2022 American Academy of Forensic Sciences.)

The appearance of Meadow saffron (Colchicum autumnale), which contains colchicine, closely resembles Alpine leek (Allium victorialis), a popular edible wild vegetable in Northern Japan. This often results in the accidental ingestion of Meadow saffron and acute colchicine poisoning deaths. Here, we report on a case of acute colchicine poisoning death caused by the accidental ingestion of Meadow saffron. A man in his 70 s had been given wild vegetables from his neighborhood, which were then cooked and eaten by himself and his wife. Several hours later, they suffered from abdominal pain, vomiting, and diarrhea. They immediately went to the hospital and received routine treatment. While his wife made a full recovery, he died at home two days after consumption of the vegetables. A forensic autopsy was conducted five days after ingestion of the Meadow saffron and a lethal concentration (21.5 ng/mL) of colchicine in the peripheral blood sample was detected by liquid chromatography-tandem mass spectrometry. Distribution of colchicine in body fluids, tissues and gastrointestinal contents was also investigated. Some of the plants he had eaten were identified as Alpine leek or Meadow saffron by genetic analysis of his stomach contents. Histopathological examination showed apoptotic cells and cell cycle arrest at the metaphase in the intestinal crypts and testis. In addition, we detected high concentrations of endotoxins and tumor necrosis factor-α in his blood, indicating that intestinal mucosal injury induced by colchicine poisoning had allowed endotoxins to invade the body, causing death by endotoxin shock., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Frostbite occurs when the skin is exposed to localized low temperatures. The main causes of frostbite are thought to be direct cell injury due to freezing of cells and tissue ischemia due to abnormal blood circulation. However, the molecular mechanism of frostbite has not been elucidated. This study aims to explain the molecular dynamics of frostbite using a mouse frostbite model and keratinocyte cell culture. Comprehensive gene expression analysis performed on mouse skin samples revealed that β-catenin signaling is activated by frostbite. Immunohistochemistry showed nuclear translocation of β-catenin in the skin of frostbite model mice that was not observed in mice subjected to a mechanical skin damage model induced by tape stripping. Tissue hypoxia, as detected by pimonidazole staining, coexisted with nuclear expression of β-catenin. In keratinocyte cell cultures, nuclear translocation of β-catenin was induced by hypoxia, but not by low temperature. Hypoxia induced epithelial-mesenchymal transition - an important biological event in the healing process of skin - and in vitro wound-healing activity, both of which were suppressed by β-catenin inhibition. Our results suggest that during frostbite, impaired blood flow causes hypoxia, which in turn activates β-catenin that promotes keratinocyte motility and tissue repair., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Hepatocellular carcinoma (HCC) activates platelets through the action of adjacent sinusoidal cells. Activated platelets bind to tumor-associated endothelial cells and release growth factors that promote tumor progression. We hypothesized that platelets encapsulated with tumor inhibitors would function as drug carriers for tumor therapy. We propose a therapeutic strategy for HCC using autologous platelets encapsulating multiple tyrosine kinase inhibitors in a rat chemically induced HCC model. Sorafenib or lenvatinib was encapsulated in platelets isolated from tumor-bearing rats in vitro. The rats were divided into groups that received repeated intravenous injections (twice a week for 10 weeks) of the following materials: placebo, sorafenib (SOR), lenvatinib (LEN), autologous platelets, autologous platelets encapsulating sorafenib (SOR-PLT) and autologous platelets encapsulating lenvatinib (LEN-PLT). The therapeutic effect was then analyzed by ultrasonography (US) and histopathological analysis. Histopathological and US analysis demonstrated extensive tumor necrosis in the tumor tissue of SOR-PLT or LEN-PLT, but not in other experimental groups. By liquid chromatography-mass spectrometry, more abundant sorafenib was detected in tumor tissues after SOR-PLT administration than in surrounding normal tissues, but no such difference in sorafenib level was observed with SOR administration. Therefore, the use of autologous platelets encapsulating drugs might be a novel therapeutic strategy for HCC., (© 2021 UICC.)

Background: Several microorganisms inhabit the mammalian gastrointestinal tract and are associated with the pathogenesis of various diseases, including cancer. Recent studies have indicated that several probiotics produce antitumor molecules and inhibit host tumor progression. We demonstrated that heptelidic acid (HA), a sesquiterpene lactone derived from the probiotic Aspergillus oryzae, exerts antitumor effects against pancreatic cancer in vitro and in vivo. In this study, the antitumor effects of HA against extraintestinal melanoma were assessed in vitro and in vivo., Results: Sulforhodamine B (SRB) assay revealed that the growth of B16F10 cells was significantly inhibited by HA in a concentration-dependent manner. The enzymatic activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) decreased in proportion with the growth inhibition effect of HA. Moreover, oral HA administration significantly suppressed the growth of transplanted B16F10 tumors without any significant changes in biochemical test values. Moreover, GAPDH activity in the transplanted tumor tissues in the HA group significantly decreased compared with that in the PBS group., Conclusion: This study suggests that orally administered HA was absorbed in the gastrointestinal tract, reached the cancer cells transplanted in the skin, and inhibited GAPDH activity, thereby inhibiting the growth of extraintestinal melanoma cells. Thus, this study proposes a novel system for extraintestinal tumor regulation via gut bacteria-derived bioactive mediators., (© 2022. The Author(s).)

Background: Accidental hypothermia (AH) sometimes leads to coagulation disorder, especially in severe AH. We previously demonstrated that intrasplenic platelet activation caused aberrant hemostasis and thrombus formation after rewarming in a murine AH model. However, no study has focused on the appropriate management of platelets causing coagulation activation after rewarming of AH. We investigated whether or not recombinant soluble thrombomodulin (rTM) can suppress thrombosis formation after rewarming using a rat AH model., Methods: Wistar rats were exposed to an ambient temperature of -20 °C under general anesthesia until their rectal temperature decreased to 26 °C. The Hypo group rats (n = 5) were immediately euthanized, while the Hypo/Re group (n = 5) and rTM group rats (n = 5), which were administered rTM (1 mg/kg) via the tail vein, were rewarmed until the rectal temperature returned to 34 °C and then euthanized 6 h later. Tissue and blood samples were collected from all rats for histopathological and coagulation analyses at euthanasia., Results: There was no significant change in the D-dimer level in the Hypo group rats, while the D-dimer level was significantly elevated at 6 h after rewarming in the Hypo/Re group rats (P = 0.015), and histopathology detected both fibrin and platelets in the renal glomerulus. However, the rTM group rats did not show any elevation of the D-dimer levels at 6 h after rewarming, and no fibrin was noted on histopathology., Conclusions: rTM may be useful as an appropriate anticoagulant in cases of aberrant hemostasis after rewarming of AH., Competing Interests: Declaration of competing interest ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:, (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: We previously showed that Lactobacillus brevis-derived polyphosphate (poly P) exerts a curative effect on intestinal inflammation. However, whether or not poly P improves the inflammation and injury of distant organs remains unclear., Aims: We aimed to investigate the change in the intestinal microbiome and to evaluate the protective effect of poly P on injuries in a cerulein-induced acute pancreatitis (AP) mouse., Methods: Poly P was orally administered to BALB/C mice every day for 24 days, and then mice were intraperitoneally injected with cerulein. Before cerulein injection, stool samples were collected and analyzed by 16S rRNA gene sequencing. Mice were sacrificed at 24 h after the last cerulein injection; subsequently, the serum, pancreas, and colon were collected., Results: The microbial profile differed markedly between poly P and control group. Notably, the levels of beneficial bacteria, including Alistipes and Candidatus_Saccharimonas, were significantly increased, while those of the virulent bacteria Desulfovibrio were decreased in the poly P group. The elevations of the serum amylase and lipase levels by cerulein treatment were suppressed by the pre-administration of poly P for 24 days, but not for 7 days. The numbers of cells MPO-positive by immunohistology were decreased and the levels of MCP-1 significantly reduced in the AP + Poly P group. An immunofluorescence analysis showed that the ZO-1 and occludin in the colon was strongly augmented in the epithelial cell membrane layer in the AP + Poly P group., Conclusions: Poly P attenuates AP through both modification of the intestinal microbiome and enhancement of the intestinal barrier integrity., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

Intake of probiotics or fermented food produced by some probiotic bacteria is believed to exert anti-tumor functions in various cancers, including pancreatic cancer, because several studies have demonstrated the anti-tumor effects of probiotic bacteria in vitro and in vivo in animal carcinogenesis models. However, the mechanisms underlying the anticancer effects of probiotics on pancreatic cancer have not been clarified. In this study, we assessed the anti-tumor effects of probiotic bacteria against pancreatic cancer cells. Among the known probiotic bacteria, Aspergillus oryzae exhibited a strong pancreatic tumor suppression effect. The culture supernatant of A. oryzae was separated by HPLC. Heptelidic acid was identified as an anti-tumor molecule derived from A. oryzae by LC-MS and NMR analysis. The anti-tumor effect of heptelidic acid was exhibited in vitro and in vivo in a xenograft model of pancreatic cancer cells. The anti-tumor effect of heptelidic acid was exerted by the p38 MAPK signaling pathway. Heptelidic acid traverses the intestinal mucosa and exerts anti-tumor effects on pancreatic cancer cells. This is a novel anti-tumor mechanism induced by beneficial bacteria against pancreatic cancer in which bacterial molecules pass through the intestinal tract, reach the extra-intestinal organs, and then induce apoptosis via an inducible signaling pathway.

Inflammatory bowel disease (IBD), such as ulcerative colitis (UC) and Crohn's disease (CD), is an intractable intestinal inflammation associated with the disruption of the intestinal mucosa. We previously demonstrated that Lactobacillus brevis -derived long-chain polyphosphate (poly P) improved the intestinal barrier function by the upregulation of cell adhesion and relieved intestinal inflammation, thereby exerting a curing effect on colitis in vitro , in vivo , and in an investigator-initiated clinical study of UC. However, how poly P improves mucosal defects induced by intestinal inflammation has not been elucidated. In this study, we detected the accumulation of platelets in inflamed tissues induced by poly P in a dextran sulfate sodium- (DSS-) induced colitis mouse model. A light transmission aggregometry analysis and scanning electron microscopy showed that poly P promoted the platelet aggregation. An SRB assay and ki-67 staining showed that the supernatant of poly P-treated platelet-rich plasma (PRP) increased intestinal epithelial cell growth. A wound healing assay showed that the supernatant of poly P-treated PRP, but not poly P itself, accelerated wound healing. A Western blotting analysis indicated that mitogen-activated protein kinase activation was induced by the supernatant of poly P-treated human PRP in the epithelial cells and its wound healing effect was significantly decreased by the inhibition of ERK signaling. These data suggested that platelet-derived mediators induced by poly P improved intestinal inflammation through the promotion of epithelial cell growth by the activation of the ERK signaling pathway. The mechanism is a novel host-microbe interaction through mammalian platelet-derived mediators induced by bacterial molecules., Competing Interests: Dr. Fujiya obtained sodium polyphosphate used for the production of calcium polyphosphate in this study from Kamui Pharma Inc., (Copyright © 2021 Shotaro Isozaki et al.)

An attempt to use combination therapy with anti-tumor necrosis factor α (TNFα) antibodies and tacrolimus (TAC) has been tried to induce remission in ulcerative colitis (UC). However, the optimal dose of TAC in combination therapy with anti-TNFα antibodies (TAC + anti-TNFα therapy) remains unclear. We examined the efficacy of various doses of TAC + anti-TNFα therapy in a mouse colitis model. Dextran sulfate sodium induced colitis model mice were divided into an anti-TNFα antibody monotherapy group and the groups that received various doses of TAC + anti-TNFα therapy. The nuclear factor expression of activated T-cells, cytoplasmic 1 (NFATc1) in the nuclei and the mRNA expression of inflammatory cytokines were assessed by immunohistochemistry and RT-PCR, respectively. The serum anti-TNFα antibody concentration was measured with an enzyme-linked immunosorbent assay. The colon length and histological severity were significantly improved in the groups that received any dose of TAC + anti-TNFα therapy. The nuclear expression of NFATc1 was inversely proportional to the administered doses of TAC. The expression levels of inflammatory cytokines tended to decrease in proportion to the dose of TAC. The serum concentration of anti-TNFα antibodies in the high-dose TAC + anti-TNFα therapy was significantly higher than those in the other groups. Low-dose TAC exerted its immunosuppressive effect on T-cells, and additionally, high-dose TAC maintained the serum anti-TNFα antibody concentration. When administered in combination with anti-TNFα antibodies, the dose of TAC should be adjusted according to the disease severity.

Background: Hypothermia triggers coagulation, which can lead to the development of a life-threatening condition. We previously reported that hypothermia induces platelet activation in the spleen, resulting in microthrombosis after rewarming. However, the changes in whole blood clotting properties that occur remain unclear. Using thromboelastography, we investigated blood clotting activity and the effects of rewarming in a murine model of hypothermia., Methods: C57Bl/6 mice were exposed to an ambient temperature of -20 °C under general anesthesia until their rectal temperature decreased to 15 °C. One group of mice was kept at 4 °C for 2 h and then euthanized. Another group was rewarmed, kept in normal conditions for 24 h, and then euthanized. Tissue and citrated whole blood samples were obtained from the mice for histopathological analysis, flow cytometry, and thromboelastography., Results: Hypothermia induced the activation of platelets in the spleen; however, rewarming significantly reduced the number of activated platelets in the spleen while their numbers significantly increased in peripheral blood. In hypothermic mice not subjected to rewarming, no increase in activated platelets was observed in peripheral blood. Thromboelastography analysis showed that whole blood samples from the rewarmed mice displayed an enhanced clotting strength., Conclusions: Rewarming from hypothermia enhances whole blood coagulation activity accompanied by an increase in the number of active platelets in peripheral blood. This phenomenon may lead to formation of microthrombi and thrombotic disorders., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

von Willebrand factor (vWF) is a large plasma glycoprotein that plays an important role in hemostasis by forming molecular bridges with platelets following vascular injury. Previously, we reported that hypothermia enhanced vWF production in the spleen, which resulted in the activation of the platelet pool in a hypothermia-induced murine model. However, the mechanisms that regulate vWF expression under hypothermic conditions remain unclear. In this study, we focused on vWF expression under hypothermic conditions in splenic endothelial cell culture. Human splenic endothelial cells (HSEC) were incubated at 20 °C for 1 h. Total RNA was extracted from the cells, and cDNA microarray gene expression analysis was performed. Genes that may be associated with vWF expression in low temperature culture conditions were then selected for further analysis. Gene expression analysis showed that low temperature conditions increased the expression of FOS and EGR1. We then hypothesized that these factors upregulate vWF mRNA expression in HSEC. The transcriptional inhibitors of EGR1 significantly inhibited vWF mRNA expression in HSEC cultured at a low temperature. Our analysis revealed that low temperatures enhance the gene expression of EGR1, which transcriptionally increases vWF expression. This acute-phase reaction may play an important role in platelet activation in the spleen during hypothermia., Competing Interests: Declaration of competing interest The authors have no conflict of interest regarding the contents of this article., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Colchicine binds to intracellular tubulin and prevents mitosis. Colchicine is also used as an anti-inflammatory drug. Meanwhile, excess administration of medication or accidental ingestion of colchicine-containing plants can cause acute colchicine poisoning, which initially results in gastrointestinal effects that may be followed by multiorgan dysfunction. However, the mechanism of colchicine poisoning remains unclear, and there are no standard therapeutic strategies., Aims: We focused on intestinal barrier function and attempted to reveal the underlying mechanism of colchicine poisoning using an animal model., Methods: Colchicine was orally administered to C57Bl/6 mice. Then, we performed histopathological analysis, serum endotoxin assays, and intestinal permeability testing. Additionally, the LPS-TLR4 signaling inhibitor TAK-242 was intraperitoneally injected after colchicine administration to analyze the therapeutic effect., Results: We observed villus height reduction and increased numbers of apoptotic cells in the gastrointestinal epithelium of colchicine-treated mice. Both intestinal permeability and serum endotoxin levels were higher in colchicine-treated mice than in control mice. Although colchicine-poisoned mice died within 25 h, those that also received TAK-242 treatment survived for more than 48 h., Conclusion: Colchicine disrupted intestinal barrier function and caused endotoxin shock. Therapeutic inhibition of LPS-TLR4 signaling might be beneficial for treating acute colchicine poisoning.

Background: Hypothermia, either therapeutically induced or accidental (ie, an involuntary decrease in core body temperature to <35°C), results in hemostatic disorders. However, it remains unclear whether hypothermia enhances or inhibits coagulation, especially in severe hypothermia. The present study evaluated the thrombocytic and hemostatic changes in hypothermic mice., Methods: C57Bl/6 mice were placed at an ambient temperature of -20°C under general anesthesia. When the rectal temperature decreased to 15°C, 10 mice were immediately euthanized, while another 10 mice were rewarmed, kept in normal conditions for 24 hours, and then euthanized. These treatments were also performed in 20 splenectomized mice., Results: The hypothermic mice had adhesion of CD62P-positive platelets with high expression of von Willebrand factor (vWF) in their spleens, while the status of the peripheral platelets was unchanged. Furthermore, the plasma levels of platelet factor 4 (PF4) and pro-platelet basic protein (PPBP), which are biomarkers for platelet degranulation, were significantly higher in hypothermic mice than in control mice, indicating that hypothermia activated the platelets in the splenic pool. Thus, we analyzed these biomarkers in asplenic mice. There was no increase in either PF4 or PPBP in splenectomized hypothermic mice. Additionally, the plasma D-dimer elevation and microthrombosis were caused in rewarmed mice, but not in asplenic rewarmed mice., Conclusions: Our results indicate that hypothermia leads to platelet activation in the spleen via the upregulation of vWF, and this activation causes hypercoagulability after rewarming., (© 2019 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.)

Background: Recent reports of some studies have described that the cognitive function of cancer patients often declines by a phenomenon designated as cancer related cognitive impairment (CRCI). For patients' decision-making, detecting CRCI is important. To do so, this study uses language-based CRCI screening to examine participants' language ability., Objective: This study was conducted to ascertain whether a Natural Language Processing (NLP) based system can detect CRCI, or not., Materials and Methods: We obtained materials of two types from cancer patients (n = 116): (1) speech samples on three topics, and (2) cognitive function level test scores from Hasegawa's Dementia Scale - Revised (HDS-R), a test used in Japan for dementia patients. The test is similar to the Mini-Mental State Examination., Results and Discussion: Cancer patients with lower HDS-R scores showed a significantly lower Type Token Ratio (TTR)., Conclusion: This result demonstrates the feasibility of the proposed speech-language-based CRCI screening method.

Locked nucleic acid (LNA) has been widely used for various genetic analyses, and has many benefits, in terms of the specificity or sensitivity of amplification, because LNA-containing primers/probes form more stable duplexes with template DNA than probes lacking LNA. Here, we developed a new method for discriminating HV1 haplotypes from mitochondrial DNA (mtDNA) mixtures by applying PCR clamping using LNA. PCR clamping is based on the selective inhibition of amplification using LNA-containing probes, which can discriminate single-nucleotide differences. Before designing probes, we selected 171 sequences with single-nucleotide variations from the HV1 region, and evaluated the specificity of LNA-containing probes for them by predicting Tm values. The differences of Tm between mismatched and exactly matched probe-template duplexes depended markedly on the type of LNA nucleotides for discriminating single-nucleotide differences, and the cytosine LNA nucleotide at the site of variations in the probes was most effective to discriminate these differences. For mixture analysis, each probe targeted one or two variations (16209C, 16217C, 16257A/16261T, 16297C/16298C, 16304C, 16362C, or 16362T) that are particularly common in the Japanese population, and seven designed probes completely inhibited the amplification of exactly matched templates. We prepared mixed samples by mixing DNA from two individuals at a ratio of 1:9, 1:4, 1:1, 4:1, or 9:1, and then performed Sanger sequencing analysis after PCR clamping with each probe. Our method distinguished each haplotype at lower ratios from two-person mixtures, and enabled sensitive detection at 12 pg of total DNA including 600 copies of mtDNA. Moreover, we analyzed three-person mixtures with representative sequences, and detected the minor haplotype of one individual present at a rate of 10% by adding two selected probes. The ability to discriminate haplotypes in mixed samples by using LNA-mediated PCR clamping indicates the potential value of mtDNA analysis in criminal investigations., (Copyright © 2019 Elsevier B.V. All rights reserved.)

We analyzed the degradation level of DNA from buccal cells under humid conditions using quantitative PCR analysis. Gauze samples with buccal cells were incubated for up to 12 months under three different conditions (25 °C/dry, 25 °C/humid, or 40 °C/humid). The degradation was evaluated based on two degradation ratios (129:41 and 305:41 bp). DNA degraded slowly under the 25 °C/humid condition, and significant differences in the two degradation ratios were detected between 25 °C/dry and 25 °C/humid conditions after 12 months. Moreover, the degradation rapidly progressed under the 40 °C/humid condition, and the two degradation ratios in this condition were much lower than those from 25 °C/dry and 25 °C/humid conditions after a short incubation period (3 months). To evaluate the effect of DNA repair on low-copy degraded DNA, degenerate oligonucleotide-primed PCR (DOP-PCR) was performed before short tandem repeats (STR) genotyping. As a standard DOP-PCR, we used a 22-base primer with 10 degenerate sequences (5'-CTCGAGNNNNNNNNNNATGTGG-3'), and additionally designed DOP-PCR primers with 2, 4, 6, or 8 locked nucleic acids (LNAs). When slightly degraded DNA (305:41-bp ratio = 0.60) was used, DOP-PCR significantly increased the fluorescent intensity and success rate of genotyping using Identifiler and Globalfiler kits. In particular, the reaction with four LNAs produced the highest value. However, such benefits were not observed in the analysis of moderately degraded DNA (305:41-bp ratio = 0.13). Although the recovery rates of STR profiles by DOP-PCR were dependent on the degradation level of low-copy DNA, the effectiveness of DOP-PCR highlights the potential of LNA for degenerate sequences., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Paraquat (PQ) is a widely used herbicide in the world despite being highly toxic to humans. PQ causes fatal damage to multiple organs, especially the lungs. While oxidative stress is the main toxic mechanism of PQ, there is no established standard therapy for PQ poisoning. In this study, we investigated the cytoprotective effect of 4-phenylbutyrate (4PBA) on PQ toxicity in human lung adenocarcinoma A549 cells. Phosphorylation levels of major survival signaling kinases Akt and ERK, as well as expression levels of antioxidant enzymes catalase and superoxide dismutase 2 (SOD2) were examined. The cytoprotective mechanism of 4PBA against PQ was compared with the antioxidant reagent trolox. We demonstrated that both 4PBA and trolox attenuated PQ toxicity, but their mechanisms were different. 4PBA increased ERK2 phosphorylation levels, which could be inhibited by the PI3K inhibitor LY294002. The cytoprotective effect of 4PBA was also inhibited by LY294002. Catalase expression levels were increased by 4PBA, although this increase was not inhibited by LY294002. 4PBA did not increase SOD2 expression. Trolox did not affect phosphorylation of Akt or ERK, or the expression of antioxidant enzymes. These results suggest that 4PBA attenuated PQ cytotoxicity by ERK2 activation via PI3K. Our study may provide new findings for understanding the molecular mechanism underlying cytoprotection by 4PBA, as well as new therapeutic targets for PQ poisoning., (Copyright © 2018 Elsevier Inc. All rights reserved.)