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Background: Maintaining critical care for non-Coronavirus-disease-2019 (non-COVID-19) patients is a key pillar of tackling the impact of the COVID-19 pandemic. This study aimed to reveal the medium-term impacts of the COVID-19 epidemic on case volumes and quality of intensive care for critically ill non-COVID-19 patients., Methods: Administrative data were used to investigate the trends in case volumes of admissions to intensive care units (ICUs) compared with the previous years. Standardized mortality ratios (SMRs) of non-COVID-19 ICU patients were calculated in each wave of the COVID-19 epidemic in Japan., Results: The ratios of new ICU admissions of non-COVID-19 patients to those in the corresponding months before the epidemic: 21% in May 2020, 8% in August 2020, 9% in February 2021, and 14% in May 2021, approximately concurrent with the peaks in COVID-19 infections. The decrease was greatest for new ICU admissions of non-COVID patients receiving invasive mechanical ventilation (IMV) on the first day of ICU admission: 26%, 15%, 19%, and 19% in the first, second, third, and fourth waves, respectively. No statistically significant change in SMR was observed in any wave of the epidemic; SMRs were 0.990 (95% uncertainty interval (UI), 0.962-1.019), 0.979 (95% UI, 0.953-1.006), 0.996 (95% UI, 0.980-1.013), and 0.989 (95% UI, 0.964-1.014), in the first, second, third, and fourth waves of the epidemic, respectively., Conclusions: Compared to the previous years, the number of non-COVID-19 ICU patients continuously decreased over the medium term during the COVID-19 epidemic. The decrease in case volumes was larger in non-COVID-19 ICU patients initially receiving IMV than those undergoing other initial treatments. The standardized in-hospital mortality of non-COVID-19 ICU patients did not change in any waves of the epidemic., Competing Interests: The authors declare that they have no competing interests.

Introduction: The coronavirus disease (COVID-19) pandemic has caused unprecedented challenges for the medical staff worldwide, especially for those in hospitals where COVID-19-positive patients are hospitalized. The announcement of COVID-19 hospital restrictions by the Japanese government has led to several limitations in hospital care, including an increased use of physical restraints, which could affect the care of elderly dementia patients. However, few studies have empirically validated the impact of physical restraint use during the COVID-19 pandemic. We aimed to evaluate the impact of regulatory changes, consequent to the pandemic, on physical restraint use among elderly dementia patients in acute care hospitals., Methods: In this retrospective study, we extracted the data of elderly patients (aged > 64 years) who received dementia care in acute care hospitals between January 6, 2019, and July 4, 2020. We divided patients into two groups depending on whether they were admitted to hospitals that received COVID-19-positive patients. We calculated descriptive statistics to compare the trend in 2-week intervals and conducted an interrupted time-series analysis to validate the changes in the use of physical restraint., Results: In hospitals that received COVID-19-positive patients, the number of patients who were physically restrained per 1,000 hospital admissions increased after the government's announcement, with a maximum incidence of 501.4 per 1,000 hospital admissions between the 73rd and 74th week after the announcement. Additionally, a significant increase in the use of physical restraints for elderly dementia patients was noted (p = 0.004) in hospitals that received COVID-19-positive patients. Elderly dementia patients who required personal care experienced a significant increase in the use of physical restraints during the COVID-19 pandemic., Conclusion: Understanding the causes and mechanisms underlying an increased use of physical restraints for dementia patients can help design more effective care protocols for similar future situations., Competing Interests: The authors have declared that no competing interests exist.

Background: Gastric cancer (GC) patients frequently develop peritoneal metastasis. Recently, it has been reported that peritoneal mesothelial cells (PMCs) activated by GC cells acquire a migratory capacity and promote GC cell invasion. The invasiveness of PMCs reportedly depends on the activity of Tks5, an adaptor protein required for invadopodia formation. However, the relationship between clinicopathologic features and Tks5 expression in PMCs has been poorly documented. In this study, we evaluated the clinicopathologic significance of the Tks5 expression of PMCs in GC patients., Materials and Methods: A total of 110 GC patients who underwent gastrectomy were enrolled in this study. Tks5 expressions in PMCs from the greater omentum, lesser omentum and retroperitoneum were evaluated by immunohistochemistry. We analyzed the correlation between Tks5 expressions in PMCs and the patients' clinicopathologic features., Results: Tks5 expression was found in 71 (64.5%) of the 110 patients, while 39 (35.5%) were Tks5-negative. Tks5 positivity was significantly (p = 0.038) associated with a greater tumor depth (i.e., T3/4 compared with T1/T2). Peritoneal recurrence was found in 12 of 98 cases within 3 years of surgery. The 3-year peritoneal recurrence-free survival (PRFS) rate in Tks5-positive cases was significantly poorer than that in Tks5-negative cases (80.1% vs 97.4%, p = 0.024). Multivariate analysis revealed that Tks5 positivity and lymph node metastasis were independent factors for PRFS., Conclusion: Tks5 is frequently expressed in PMCs in advanced-stage gastric cancer. Tks5 might be a useful predictor for peritoneal recurrence in GC patients., Competing Interests: The authors have declared that no competing interests exist.

Prostaglandin E2 (PGE2) is known to have important roles in labor, but the detailed mechanism underlying the spontaneous human labor remains unknown. Here, we examined the involvement of prostaglandin biosynthetic enzymes and transporter in the accumulation of PGE2 in amniotic fluid in human labor. PGE2 and its metabolites were abundant in amniotic fluid in deliveries at term in labor (TLB), but not at term not in labor (TNL). In fetal-membrane Transwell assays, levels of PGE2 production in both maternal and fetal compartments were significantly higher in the TLB group than the TNL group. In fetal-membrane, the mRNA level of PTGES3, which encodes cytosolic prostaglandin E synthase (cPGES), was significantly higher in TLB than in TNL, but the mRNA levels of the other PGE2-synthase genes were not affected by labor. Moreover, the mRNA level of PTGS2, which encodes cyclooxygenase-2 (COX-2) in the amnion was significantly higher in TLB than in TNL. Western blot analyses revealed that the levels of COX-1 and COX-2 were comparable between the two groups, however, the level of cPGES was relatively higher in TLB than in TNL. COXs, cPGES, and prostaglandin transporter (SLCO2A1) proteins were all expressed in both chorionic trophoblasts and amniotic epithelium. These findings suggest that COXs, cPGES and SLCO2A1 contribute to PGE2 production from fetal-membrane in labor., Competing Interests: The authors have declared that no competing interests exist.

Intra-operative autologous blood donation is a blood conservation technique with limited evidence. We evaluated the association between intra-operative autologous blood donation and decrease in peri-operative transfusion in cardiovascular surgery based on evidence from a Japanese administrative database. We extracted the data of patients who had undergone cardiovascular surgery from the Diagnosis Procedure Combination database in Japan (2016-2019). Based on the surgery type, we examined the association of intra-operative autologous blood donation with the transfusion rate and amount of blood used in cardiac and aortic surgeries using multilevel propensity score matching. We enrolled 32,433 and 4,267 patients who underwent cardiac and aortic surgeries and received 5.0% and 6.7% intra-operative autologous blood donation with mean volumes of 557.68 mL and 616.96 mL, respectively. The red blood cell transfusion rates of the control and intra-operative autologous blood donation groups were 60.6% and 38.4%, respectively, in the cardiac surgery cohort (p < .001) and 91.4%, and 83.8%, respectively, in the aortic surgery cohort (p = .037). The transfusion amounts for the control and intra-operative autologous blood donation groups were 5.9 and 3.5 units of red blood cells, respectively, for cardiac surgery patients (p < .001) and 11.9 and 7.9 units, respectively, for aortic surgery patients (p < .001). Intra-operative autologous blood donation could reduce the transfusion rate or amount of red blood cells and fresh frozen plasma for patients undergoing index cardiovascular surgery and could be an effective blood transfusion strategy in cardiovascular surgery for Japanese patients., Competing Interests: The authors have declared that no competing interests exist.

Background: In response to the coronavirus diseases 2019 (COVID-19) pandemic, the Japanese government declared a state of emergency on April 7, 2020. Six days earlier, the Japan Surgical Society had recommended postponing elective surgical procedures. Along with the growing public fear of COVID-19, hospital visits in Japan decreased., Methods: Using claims data from the Quality Indicator/Improvement Project (QIP) database, this study aimed to clarify the impact of the first wave of the pandemic, considered to be from March to May 2020, on case volume and claimed hospital charges in acute care hospitals during this period. To make year-over-year comparisons, we considered cases from July 2018 to June 2020., Results: A total of 2,739,878 inpatient and 53,479,658 outpatient cases from 195 hospitals were included. In the year-over-year comparisons, total claimed hospital charges decreased in April, May, June 2020 by 7%, 14%, and 5%, respectively, compared to the same months in 2019. Our results also showed that per-case hospital charges increased during this period, possibly to compensate for the reduced case volumes. Regression results indicated that the hospital charges in April and May 2020 decreased by 6.3% for hospitals without COVID-19 patients. For hospitals with COVID-19 patients, there was an additional decrease in proportion with the length of hospital stay of COVID-19 patients including suspected cases. The mean additional decrease per COVID-19 patient was estimated to 5.5 million JPY., Conclusion: It is suggested that the hospitals treating COVID-19 patients were negatively incentivized., Competing Interests: The authors have declared that no competing interests exist.

Chronic enteropathy associated with SLCO2A1 gene (CEAS) is caused by loss-of-function mutations in SLCO2A1, which encodes a prostaglandin (PG) transporter. In this study, we report a sibling case of CEAS with a novel pathogenic variant of the SLCO2A1 gene. Compound heterozygous variants in SLCO2A1 were identified in an 8-year-old boy and 12-year-old girl, and multiple chronic nonspecific ulcers were observed in the patients using capsule endoscopy. The splice site mutation (c.940 + 1G>A) of the paternal allele was previously reported to be pathogenic, whereas the missense variant (c.1688T>C) of the maternal allele was novel and had not yet been reported. The affected residue (p.Leu563Pro) is located in the 11th transmembrane domain (helix 11) of SLCO2A1. Because SLCO2A1 mediates the uptake and clearance of PGs, the urinary PG metabolites were measured by liquid chromatography coupled to tandem mass spectrometry. The urinary tetranor-prostaglandin E metabolite levels in the patients were significantly higher than those in unaffected individuals. We established cell lines with doxycycline-inducible expression of wild type SLCO2A1 (WT-SLCO2A1) and the L563P mutant. Immunofluorescence staining showed that WT-SLCO2A1 and the L563P mutant were dominantly expressed on the plasma membranes of these cells. Cells expressing WT-SLCO2A1 exhibited time- and dose-dependent uptake of PGE2, while the mutant did not show any uptake activity. Residue L563 is very close to the putative substrate-binding site in SLCO2A1, R561 in helix 11. However, in a molecular model of SLCO2A1, the side chain of L563 projected outside of helix 11, indicating that L563 is likely not directly involved in substrate binding. Instead, the substitution of Pro may twist the helix and impair the transporter function. In summary, we identified a novel pathogenic variant of SLCO2A1 that caused loss-of-function and induced CEAS., Competing Interests: The authors have declared that no competing interests exist.

[This corrects the article DOI: 10.1371/journal.pone.0225958.].

Psychiatric disorders including depression and anxiety comprise a broad range of conditions with different symptoms. We have developed a mouse model of depression/anxiety in mice deficient in the St3gal4 gene. In this study, we performed a comparative analysis of urinary volatile organic compounds (VOCs) in St3gal4-deficient (St3gal4-KO) and wild-type mice using gas chromatography-mass spectrometry, and we screened 18 putative VOCs. Principal component analysis (PCA) based on these VOCs identified a major group of 11 VOCs, from which two groups were clarified by hierarchical clustering analysis. One group including six VOCs (pentanoic acid, 4-methyl-, ethyl ester; 3-heptanone, 6-methyl; benzaldehyde; 5,9-undecadien-2-ol, 6,10-dimethyl; and unknown compounds RI1291 and RI1237) was correlated with the startle response (r = 0.620), which is related to an unconscious defensive response. The other group including two VOCs (beta-farnesene and alpha-farnesene) comprised pheromones which increased in KO mice. Next, male mice underwent a social behavior test with female mice in the estrus stage, showing reduced access of KO male mice to female mice. Comparative analysis of urinary VOCs before and after encounters revealed that the six VOCs were not changed by these encounters. However, in WT mice, the two farnesenes increased after the encounters, reaching the level observed in KO mice, which was not altered following the encounter. Taken together, these results indicated that St3gal4 was involved in modulating urinary VOCs. Moreover, VOC clusters discovered by comparison of St3gal4-KO mice with WT mice were correlated with differential emotional behaviors., Competing Interests: The authors have declared that no competing interests exist.

Background: Peritoneal recurrence is one of the most frequent recurrent diseases in gastric cancer. Although the exposure of cancer cells to the serosal surface is considered a common risk factor for peritoneal recurrence, there are some cases of peritoneal recurrence without infiltration to the serosal surface even after curative surgery. This study sought to clarify the risk factors of peritoneal recurrence in the absence of invasion to the serosal surface., Materials and Methods: Ninety-six patients with gastric cancer who underwent curative surgery were enrolled. In all 96 cases, the depth of tumor invasion was subserosal (T3). The microscopic distance from the tumor invasion front to the serosa (DIFS) was measured using tissue slides by H&E staining and pan-cytokeratin staining. E-cadherin expression was evaluated by immunohistochemical staining., Results: Among the 96 patients, 16 developed peritoneal recurrence after curative surgery. The DIFS of the tumors with peritoneal recurrence (156±220 μm) was significantly shorter (p = 0.011) than that without peritoneal recurrence (360±478 μm). Peritoneal recurrence was significantly correlated with DIFS ≤234 μm (p = 0.023), but not with E-cadherin expression. The prognosis of DIFS ≤234 μm was significantly poorer than that of DIFS >234 μm (log rank, p = 0.007). A multivariate analysis of the patients' five-year overall survival revealed that DIFS ≤234 μm and lymph node metastasis were significantly correlated with survival (p = 0.005, p = 0.032, respectively)., Conclusion: The measurement of the DIFS might be useful for the prediction of peritoneal recurrence in T3-gastric cancer patients after curative surgery., Competing Interests: The authors have declared that no competing interests exist.

Introduction: Thrombospondin-4 [1] is an extracellular glycoprotein involved in wound healing and tissue remodeling. Although THBS4 is reportedly frequently expressed in solid tumors, there are few reports of the clinicopathological features of carcinomas with THBS4 expression. We evaluated the clinicopathologic significance of THBS4 expression in gastric carcinoma (GC)., Materials and Methods: We retrospectively analyzed the cases of 584 GC patients. The expression of THBS4 in each tumor was evaluated by immunohistochemistry. We then divided the patients into the THBS4-high (n = 223, 38.2%) group and THBS4-low (n = 361, 61.8%) group. THBS4 expression in cancer-associated fibroblasts (CAFs), normal-associated fibroblasts (NFs) and gastric cancer cell lines was examined by western blotting., Results: THBS4 is expressed on stromal cells with αSMA or Podoplanin expression in the GC microenvironment, but not expressed on cancer cells with cytokeratin expression. The western blot analysis results showed that CAFs (but not NFs and cancer cells) expressed THBS4. Compared to the THBS4-low expression status, the THBS4-high expression status was correlated with higher αSMA expression, higher invasion depth, lymph-node metastasis, lymphatic invasion, peritoneal cytology, peritoneal metastasis, larger tumor size, microscopic diffuse type, and the macroscopic diffuse infiltrating type. The THBS4-high group's 5-year overall survival rate was significantly poorer than that of the THBS4-low group. A multivariate analysis revealed that THBS4 expression was an independent prognostic factor., Conclusion: THBS4 is expressed on CAFs in the gastric cancer microenvironment. THBS4 from CAFs is associated with the metastasis of cancer cells, and is a useful prognostic indicator for gastric cancer patients., Competing Interests: The authors have declared that no competing interests exist.

Background: It has been reported that CD63, an exosome marker, is expressed in solid cancer tissues. However, its significance in patients with gastric cancer has not been clarified. Exosomes derived from cancer cells and stromal cells might play an important role in the intracellular communications involved in the development of carcinoma. This study aimed to clarify the relationship between CD63 expression in cancer cells and stromal cells and clinical-pathologic factors., Methods: A total of 595 gastric cancer patients were enrolled in this study. CD63 expression in cancer cells and stromal cells was analyzed by immunohistochemistry. The correlations between CD63 expression and several clinicopathological factors were investigated., Results: CD63 expression was mainly observed on the cell membranes of cancer cells, and in the cytoplasm of stromal cells. Of 595 patients, 247 cases had CD63-positive cancer cells, and 107 cases had CD63-positive stromal cells. Cases with CD63-positive cancer cells were significantly correlated with scirrhous-type gastric cancer, tumor depth, lymph node metastasis, lymphatic invasion, and tumor size. Cases with CD63-positive stromal cells were significantly correlated with age (≥65), tumor depth (T3-4), lymphatic invasion, and tumor size (≥ 5 cm). The 5-year survival rate was significantly lower (p<0.001) in patients with CD63-positive than CD63-negative tumors. Multivariate analysis showed that CD63 expression in cancer cells was a significant independent prognostic factor in patients with gastric cancer., Conclusion: CD63 might be a prognostic marker for patients with gastric cancer. CD63-positive exosomes might be associated with the interaction between stromal cells and cancer cells., Competing Interests: The authors have declared that no competing interests exist.

We previously demonstrated that patients with multiple sclerosis (MS) of high serum Sema4A levels are resistant to IFN-β therapy. To further elucidate the role of serum Sema4A as a biomarker for therapeutic stratification in MS patients, it is important to clarify the efficacy of other disease-modifying drugs (DMD) in those with high serum Sema4A levels. Thus, in this study we investigated whether fingolimod has beneficial effects on MS patients with high Sema4A levels. We retrospectively analyzed annualized relapse rate (ARR) and Expanded Disability Status Scale (EDSS) change in 56 relapsing-remitting multiple sclerosis (RRMS) patients who had been treated with fingolimod, including those who switched from IFN-β therapy. The levels of Sema4A in the sera were measured by sandwich ELISA. The implications of Sema4A on the efficacy of fingolimod were investigated by administering recombinant Sema4A-Fc and fingolimod to mice with experimental autoimmune encephalomyelitis (EAE). Retrospective analysis of MS cohort (17 high Sema4A and 39 low Sema4A) demonstrated the effectiveness of fingolimod in those with high serum Sema4A levels, showing reduction of ARR (from 1.21 to 0.12) and EDSS progression (from 0.50 to 0.04). Consistent with this observation, improvement in the disease severity of EAE mice receiving recombinant Sema4A-Fc was also observed after fingolimod treatment. These data suggest that fingolimod could serve as a candidate DMD for managing the disease activity of MS patients with high Sema4A levels.

Oxidative stress and mitochondrial dysfunction are important determinants of neurodegeneration in secondary progressive multiple sclerosis (SPMS). We previously showed that febuxostat, a xanthine oxidase inhibitor, ameliorated both relapsing-remitting and secondary progressive experimental autoimmune encephalomyelitis (EAE) by preventing neurodegeneration in mice. In this study, we investigated how febuxostat protects neuron in secondary progressive EAE. A DNA microarray analysis revealed that febuxostat treatment increased the CNS expression of several mitochondria-related genes in EAE mice, most notably including GOT2, which encodes glutamate oxaloacetate transaminase 2 (GOT2). GOT2 is a mitochondrial enzyme that oxidizes glutamate to produce α-ketoglutarate for the Krebs cycle, eventually leading to the production of adenosine triphosphate (ATP). Whereas GOT2 expression was decreased in the spinal cord during the chronic progressive phase of EAE, febuxostat-treated EAE mice showed increased GOT2 expression. Moreover, febuxostat treatment of Neuro2a cells in vitro ameliorated ATP exhaustion induced by rotenone application. The ability of febuxostat to preserve ATP production in the presence of rotenone was significantly reduced by GOT2 siRNA. GOT2-mediated ATP synthesis may be a pivotal mechanism underlying the protective effect of febuxostat against neurodegeneration in EAE. Accordingly, febuxostat may also have clinical utility as a disease-modifying drug in SPMS.

Leukotriene B4 (LTB4) receptor 1 (BLT1) is a G protein-coupled receptor expressed in various leukocyte subsets; however, the precise expression of mouse BLT1 (mBLT1) has not been reported because a mBLT1 monoclonal antibody (mAb) has not been available. In this study, we present the successful establishment of a hybridoma cell line (clone 7A8) that produces a high-affinity mAb for mBLT1 by direct immunization of BLT1-deficient mice with mBLT1-overexpressing cells. The specificity of clone 7A8 was confirmed using mBLT1-overexpressing cells and mouse peripheral blood leukocytes that endogenously express BLT1. Clone 7A8 did not cross-react with human BLT1 or other G protein-coupled receptors, including human chemokine (C-X-C motif) receptor 4. The 7A8 mAb binds to the second extracellular loop of mBLT1 and did not affect LTB4 binding or intracellular calcium mobilization by LTB4. The 7A8 mAb positively stained Gr-1-positive granulocytes, CD11b-positive granulocytes/monocytes, F4/80-positive monocytes, CCR2-high and CCR2-low monocyte subsets in the peripheral blood and a CD4-positive T cell subset, Th1 cells differentiated in vitro from naïve CD4-positive T cells. This mAb was able to detect Gr-1-positive granulocytes and monocytes in the spleens of naïve mice by immunohistochemistry. Finally, intraperitoneal administration of 7A8 mAb depleted granulocytes and monocytes in the peripheral blood. We have therefore succeeded in generating a high-affinity anti-mBLT1 mAb that is useful for analyzing mBLT1 expression in vitro and in vivo.

Background: The intestine is one of the first affected organs in Parkinson's disease (PD). PD subjects show abnormal staining for Escherichia coli and α-synuclein in the colon., Methods: We recruited 52 PD patients and 36 healthy cohabitants. We measured serum markers and quantified the numbers of 19 fecal bacterial groups/genera/species by quantitative RT-PCR of 16S or 23S rRNA. Although the six most predominant bacterial groups/genera/species covered on average 71.3% of total intestinal bacteria, our analysis was not comprehensive compared to metagenome analysis or 16S rRNA amplicon sequencing., Results: In PD, the number of Lactobacillus was higher, while the sum of analyzed bacteria, Clostridium coccoides group, and Bacteroides fragilis group were lower than controls. Additionally, the sum of putative hydrogen-producing bacteria was lower in PD. A linear regression model to predict disease durations demonstrated that C. coccoides group and Lactobacillus gasseri subgroup had the largest negative and positive coefficients, respectively. As a linear regression model to predict stool frequencies showed that these bacteria were not associated with constipation, changes in these bacteria were unlikely to represent worsening of constipation in the course of progression of PD. In PD, the serum lipopolysaccharide (LPS)-binding protein levels were lower than controls, while the levels of serum diamine oxidase, a marker for intestinal mucosal integrity, remained unchanged in PD., Conclusions: The permeability to LPS is likely to be increased without compromising the integrity of intestinal mucosa in PD. The increased intestinal permeability in PD may make the patients susceptible to intestinal dysbiosis. Conversely, intestinal dysbiosis may lead to the increased intestinal permeability. One or both of the two mechanisms may be operational in development and progression of PD.

Eukaryotic positive-strand RNA [(+)RNA] viruses are intracellular obligate parasites replicate using the membrane-bound replicase complexes that contain multiple viral and host components. To replicate, (+)RNA viruses exploit host resources and modify host metabolism and membrane organization. Phospholipase D (PLD) is a phosphatidylcholine- and phosphatidylethanolamine-hydrolyzing enzyme that catalyzes the production of phosphatidic acid (PA), a lipid second messenger that modulates diverse intracellular signaling in various organisms. PA is normally present in small amounts (less than 1% of total phospholipids), but rapidly and transiently accumulates in lipid bilayers in response to different environmental cues such as biotic and abiotic stresses in plants. However, the precise functions of PLD and PA remain unknown. Here, we report the roles of PLD and PA in genomic RNA replication of a plant (+)RNA virus, Red clover necrotic mosaic virus (RCNMV). We found that RCNMV RNA replication complexes formed in Nicotiana benthamiana contained PLDα and PLDβ. Gene-silencing and pharmacological inhibition approaches showed that PLDs and PLDs-derived PA are required for viral RNA replication. Consistent with this, exogenous application of PA enhanced viral RNA replication in plant cells and plant-derived cell-free extracts. We also found that a viral auxiliary replication protein bound to PA in vitro, and that the amount of PA increased in RCNMV-infected plant leaves. Together, our findings suggest that RCNMV hijacks host PA-producing enzymes to replicate.

Cryopreservation of mature oocytes and embryos has provided numerous benefits in reproductive medicine. Although successful cryopreservation of germinal-vesicle stage (GV) oocytes holds promise for further advances in reproductive biology and clinical embryology fields, reports regarding cryopreservation of immature oocytes are limited. Oocyte survival and maturation rates have improved since vitrification is being performed at the GV stage, but the subsequent developmental competence of GV oocytes is still low. The purpose of this study was to evaluate the effects of supplementation of the maturation medium with cyclic adenosine monophosphate (cAMP) modulators on the developmental competence of vitrified-warmed GV bovine oocytes. GV oocytes were vitrified-warmed and cultured to allow for oocyte maturation, and then parthenogenetically activated or fertilized in vitro. Our results indicate that addition of a cAMP modulator forskolin (FSK) or 3-isobutyl-1-methylxanthine (IBMX) to the maturation medium significantly improved the developmental competence of vitrified-warmed GV oocytes. We also demonstrated that vitrification of GV oocytes led to a decline in cAMP levels and maturation-promoting factor (MPF) activity in the oocytes during the initial and final phases of maturation, respectively. Nevertheless, the addition of FSK or IBMX to the maturation medium significantly elevated cAMP levels and MPF activity during IVM. Taken together, our results suggest that the cryopreservation-associated meiotic and developmental abnormalities observed in GV oocytes may be ameliorated by an artificial increase in cAMP levels during maturation culture after warming.

The leukotriene B4 (LTB4) receptor 1 (BLT1) is a high affinity receptor for LTB4, a chemotactic and inflammatory eicosanoid. The LTB4 receptor 2 (BLT2) was originally identified as a low affinity receptor for LTB4, and, more recently, as a high affinity receptor for 12-hydroxyheptadecatrienoic acid (12-HHT). The zebrafish BLT receptors have not been previously identified and the in vivo functions of these receptors have been unknown. In this paper, we describe one zebrafish BLT1-like receptor, Blt1, and two zebrafish BLT2-like receptors, Blt2a and Blt2b. Cells expressing Blt1 exhibited LTB4-induced intracellular [Ca2+] increases, inhibition of cAMP production, ligand-dependent [35S]GTPγS binding, and transforming growth factor-α (TGFα) shedding activity in a dose-dependent manner, similar to human BLT1. Cells expressing Blt2a and Blt2b exhibited 12-HHT- and LTB4-induced intracellular [Ca2+] increases, inhibition of cAMP production, [35S]GTPγS binding, and TGFα shedding activity, with a dose-dependency similar to human BLT2. Reverse transcription (RT)-PCR analysis and whole-mount in situ hybridization revealed that blt1, blt2a, blt2b, zebrafish LTA4 hydrolase (lta4h), and zebrafish 5-lipoxiganase (5lo) are expressed in zebrafish embryos. Knockdown of blt1 by morpholino antisense oligonucleotides resulted in delayed epiboly at gastrulation. Consistently, knockdown of lta4h, an enzyme mediating LTB4 production, induced a phenotype similar to knockdown of blt1. These results suggest that the LTB4-BLT1 axis is involved in epiboly in zebrafish development.

The formation of virus movement protein (MP)-containing punctate structures on the cortical endoplasmic reticulum is required for efficient intercellular movement of Red clover necrotic mosaic virus (RCNMV), a bipartite positive-strand RNA plant virus. We found that these cortical punctate structures constitute a viral replication complex (VRC) in addition to the previously reported aggregate structures that formed adjacent to the nucleus. We identified host proteins that interacted with RCNMV MP in virus-infected Nicotiana benthamiana leaves using a tandem affinity purification method followed by mass spectrometry. One of these host proteins was glyceraldehyde 3-phosphate dehydrogenase-A (NbGAPDH-A), which is a component of the Calvin-Benson cycle in chloroplasts. Virus-induced gene silencing of NbGAPDH-A reduced RCNMV multiplication in the inoculated leaves, but not in the single cells, thereby suggesting that GAPDH-A plays a positive role in cell-to-cell movement of RCNMV. The fusion protein of NbGAPDH-A and green fluorescent protein localized exclusively to the chloroplasts. In the presence of RCNMV RNA1, however, the protein localized to the cortical VRC as well as the chloroplasts. Bimolecular fluorescence complementation assay and GST pulldown assay confirmed in vivo and in vitro interactions, respectively, between the MP and NbGAPDH-A. Furthermore, gene silencing of NbGAPDH-A inhibited MP localization to the cortical VRC. We discuss the possible roles of NbGAPDH-A in the RCNMV movement process.

Background: Activation of glial cells is a cardinal feature in multiple sclerosis (MS) pathology, and acetate has been reported to be selectively uptaken by astrocytes in the CNS. The aim of this study was to investigate the efficacy of PET with (11)C-acetate for MS diagnosis., Materials and Methods: Six patients with relapsing-remitting MS and 6 healthy volunteers (HV) were enrolled. The (11)C-acetate brain uptake on PET was measured in patients with MS and HV. Volume-of-interest analysis of cerebral gray and white matter based on the segmentation technique for co-registered MRI and voxel-based statistical parametric analysis were performed. Correlation between 11C-acetate uptake and the lesion number in T1- and T2- weighted MR images were also assessed., Results: The standardized uptake value (SUV) of 11C-acetate was increased in both white and gray matter in MS patients compared to HV. Voxel-based statistical analysis revealed a significantly increased SUV relative to that in the bilateral thalami (SUVt) in a broad area of white matter, particularly in the subcortical white matter of MS patients. The numbers of T2 lesions and T1 black holes were significantly correlated with SUV of (11)C-acetate in white and gray matter., Conclusions: The 11C-acetate uptake significantly increased in MS patients and correlated to the number of MRI lesions. These preliminary data suggest that (11)C-acetate PET can be a useful clinical examination for MS patients.

Objectives: Oxidative stress plays an important role in the pathogenesis of multiple sclerosis (MS). Though reactive oxygen species (ROS) are produced by various mechanisms, xanthine oxidase (XO) is a major enzyme generating ROS in the context of inflammation. The objectives of this study were to investigate the involvement of XO in the pathogenesis of MS and to develop a potent new therapy for MS based on the inhibition of ROS., Methods: XO were assessed in a model of MS: experimental autoimmune encephalomyelitis (EAE). The contribution of XO-generated ROS to the pathogenesis of EAE was assessed by treating EAE mice with a novel XO inhibitor, febuxostat. The efficacy of febuxostat was also examined in in vitro studies., Results: We showed for the first time that the expression and the activity of XO were increased dramatically within the central nervous system of EAE mice as compared to naïve mice. Furthermore, prophylactic administration of febuxostat, a XO inhibitor, markedly reduced the clinical signs of EAE. Both in vivo and in vitro studies showed infiltrating macrophages and microglia as the major sources of excess XO production, and febuxostat significantly suppressed ROS generation from these cells. Inflammatory cellular infiltration and glial activation in the spinal cord of EAE mice were inhibited by the treatment with febuxostat. Importantly, therapeutic efficacy was observed not only in mice with relapsing-remitting EAE but also in mice with secondary progressive EAE by preventing axonal loss and demyelination., Conclusion: These results highlight the implication of XO in EAE pathogenesis and suggest XO as a target for MS treatment and febuxostat as a promising therapeutic option for MS neuropathology.

Various neuroprotective factors have been shown to help prevention of neuronal cell death, which is responsible for the progression of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). However, most of these therapeutic potentials have been tested by administration of recombinant proteins, transgenic expression or virus vector-mediated gene transfer. Therefore, it remains to be clarified whether any endogenous factors has advantage for neuroprotection in a pathological nervous system. Here we show the role of BAFF-R signaling pathway in the control of neural cell survival. Both B cell-activating factor (BAFF) and its receptor (BAFF-R) are expressed in mouse neurons and BAFF-R deficiency reduces the survival of primary cultured neurons. Although many studies have so far addressed the functional role of BAFF-R on the differentiation of B cells, impaired BAFF-R signaling resulted in accelerated disease progression in an animal model of inherited ALS. We further demonstrate that BAFF-R deficient bone marrow cells or genetic depletion of B cells does not affect the disease progression, indicating that BAFF-mediated signals on neurons, not on B cells, support neural cell survival. These findings suggest opportunities to improve therapeutic outcome for patients with neurodegenerative diseases by synthesized BAFF treatment.

Background: To improve the quality of life of colorectal cancer patients, it is important to establish new screening methods for early diagnosis of colorectal cancer., Methodology/principal Findings: We performed serum metabolome analysis using gas-chromatography/mass-spectrometry (GC/MS). First, the accuracy of our GC/MS-based serum metabolomic analytical method was evaluated by calculating the RSD% values of serum levels of various metabolites. Second, the intra-day (morning, daytime, and night) and inter-day (among 3 days) variances of serum metabolite levels were examined. Then, serum metabolite levels were compared between colorectal cancer patients (N = 60; N = 12 for each stage from 0 to 4) and age- and sex-matched healthy volunteers (N = 60) as a training set. The metabolites whose levels displayed significant changes were subjected to multiple logistic regression analysis using the stepwise variable selection method, and a colorectal cancer prediction model was established. The prediction model was composed of 2-hydroxybutyrate, aspartic acid, kynurenine, and cystamine, and its AUC, sensitivity, specificity, and accuracy were 0.9097, 85.0%, 85.0%, and 85.0%, respectively, according to the training set data. In contrast, the sensitivity, specificity, and accuracy of CEA were 35.0%, 96.7%, and 65.8%, respectively, and those of CA19-9 were 16.7%, 100%, and 58.3%, respectively. The validity of the prediction model was confirmed using colorectal cancer patients (N = 59) and healthy volunteers (N = 63) as a validation set. At the validation set, the sensitivity, specificity, and accuracy of the prediction model were 83.1%, 81.0%, and 82.0%, respectively, and these values were almost the same as those obtained with the training set. In addition, the model displayed high sensitivity for detecting stage 0-2 colorectal cancer (82.8%)., Conclusions/significance: Our prediction model established via GC/MS-based serum metabolomic analysis is valuable for early detection of colorectal cancer and has the potential to become a novel screening test for colorectal cancer.

Background: Certain intestinal microflora are thought to regulate the systemic immune response. Lactic acid bacteria are one of the most studied bacteria in terms of their beneficial effects on health and autoimmune diseases; one of which is Multiple sclerosis (MS) which affects the central nervous system. We investigated whether the lactic acid bacterium Pediococcus acidilactici, which comprises human commensal bacteria, has beneficial effects on experimental autoimmune encephalomyelitis (EAE), an animal model of MS., Methodology/principal Findings: P. acidilactici R037 was orally administered to EAE mice to investigate the effects of R037. R037 treatment suppressed clinical EAE severity as prophylaxis and therapy. The antigen-specific production of inflammatory cytokines was inhibited in R037-treated mice. A significant increase in the number of CD4(+) Interleukin (IL)-10-producing cells was observed in the mesenteric lymph nodes (MLNs) and spleens isolated from R037-treated naive mice, while no increase was observed in the number of these cells in the lamina propria. Because only a slight increase in the CD4(+)Foxp3(+) cells was observed in MLNs, R037 may primarily induce Foxp3(-) IL10-producing T regulatory type 1 (Tr1) cells in MLNs, which contribute to the beneficial effect of R037 on EAE., Conclusions/significance: An orally administered single strain of P. acidilactici R037 ameliorates EAE by inducing IL10-producing Tr1 cells. Our findings indicate the therapeutic potential of the oral administration of R037 for treating multiple sclerosis.

Background: Neurons and glial cells can be efficiently induced from mouse embryonic stem (ES) cells in a conditioned medium collected from rat primary-cultured astrocytes (P-ACM). However, the use of rodent primary cells for clinical applications may be hampered by limited supply and risk of contamination with xeno-proteins., Methodology/principal Findings: We have developed an alternative method for unimpeded production of human neurons under xeno-free conditions. Initially, neural stem cells in sphere-like clusters were induced from human ES (hES) cells after being cultured in P-ACM under free-floating conditions. The resultant neural stem cells could circumferentially proliferate under subsequent adhesive culture, and selectively differentiate into neurons or astrocytes by changing the medium to P-ACM or G5, respectively. These hES cell-derived neurons and astrocytes could procure functions similar to those of primary cells. Interestingly, a conditioned medium obtained from the hES cell-derived astrocytes (ES-ACM) could successfully be used to substitute P-ACM for induction of neurons. Neurons made by this method could survive in mice brain after xeno-transplantation., Conclusion/significance: By inducing astrocytes from hES cells in a chemically defined medium, we could produce human neurons without the use of P-ACM. This self-serving method provides an unlimited source of human neural cells and may facilitate clinical applications of hES cells for neurological diseases.