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Background: The 95-95-95 UNAIDS global strategy was adapted to end the AIDS epidemic by 2030. The target is based on the premise that early detection of HIV-infected persons and linking them to treatment regardless of their CD4 counts will lead to sustained viral suppression. HIV testing strategies to increase uptake of testing in Western and Central Africa remain inadequate. Hence, a high proportion of people living with HIV in this region do not know their status. This report describes the implementation of a community-based health screening (also known as “Know Your Status” -KYS), as part of basic science research, in a way that contributed to achieving public health goals. Methods: A community-based health screening was conducted in 7 communities within the Eastern region of Ghana between November 2017 to April 2018, to recruit and match HIV seronegative persons to HIV seropositive persons in a case-control HIV gut microbiota study. Health assessments included blood pressure, body mass index, blood sugar, Hepatitis B virus, syphilis, and HIV testing for those who consented. HIV seronegative participants who consented were consecutively enrolled in an ongoing HIV gut microbiota case-control study. Descriptive statistics (percentages) were used to analyze data. Results: Out of 738 people screened during the exercise, 700 consented to HIV testing and 23 (3%) were HIV positive. Hepatitis B virus infection was detected in 4% (33/738) and Syphilis in 2 % (17/738). Co-infection of HIV and HBV was detected in 4 persons. The HIV prevalence of 3% found in these communities is higher than both the national prevalence of 1.7% and the Eastern Regional prevalence of 2.7 in 2018. Conclusion: Community-based health screening, such as undertaken in our study could be critical for identifying infected persons from the community and linking them to care. This will greatly contribute to achieving the first two 95s and working towards ending AIDS by 2030.

Accurate monitoring of epidemics is a key strategy for controlling human immunodeficiency virus type-1 (HIV-1) infection. To delineate the characteristics of newly diagnosed cases of HIV-1 infection, we assessed the proportion of recent HIV-1 infections using a recent infection-testing algorithm (RITA). In 2015, 248 cases were newly diagnosed with HIV infection at the Regional Hospital Koforidua, Ghana. Of these, 234 cases (94.4%) were infected with HIV-1 only, four (1.6%) were infected with HIV-2 only, and 10 (4.0%) were co-infected with HIV-1 and HIV-2. All HIV-1 single-seropositive samples were used in the HIV-1 LAg avidity assay for RITA. Our analysis revealed that 18 cases (7.7%) were recently infected, indicating that early diagnosis was not achieved in Ghana. This is the first report to assess the proportion of recent infections in Ghana using a biomarker approach. The accumulation of these data will contribute to the accurate estimation of HIV-1 incidence and prevalence in Ghana.

Polymorphisms in human leukocyte antigen (HLA) class I loci are known to have a great impact on disease progression in HIV-1 infection. Prevailing HIV-1 subtypes and HLA genotype distribution are different all over the world, and the HIV-1 and host HLA interaction could be specific to individual areas. Data on the HIV-1 and HLA interaction have been accumulated in HIV-1 subtype B- and C-predominant populations but not fully obtained in West Africa where HIV-1 subtype CRF02_AG is predominant. In the present study, to obtain accurate HLA typing data for analysis of HLA association with disease progression in HIV-1 infection in West African populations, HLA class I (HLA-A, -B, and -C) four-digit allele typing was performed in treatment-naïve HIV-1 infected individuals in Ghana (n = 324) by a super high-resolution single-molecule sequence-based typing (SS-SBT) using next-generation sequencing. Comparison of the SS-SBT-based data with those obtained by a conventional sequencing-based typing (SBT) revealed incorrect assignment of several alleles by SBT. Indeed, HLA-A*23:17, HLA-B*07:06, HLA-C*07:18, and HLA-C*18:02 whose allele frequencies were 2.5%, 0.9%, 4.3%, and 3.7%, respectively, were not determined by SBT. Several HLA alleles were associated with clinical markers, viral load and CD4+ T-cell count. Of note, the impact of HLA-B*57:03 and HLA-B*58:01, known as protective alleles against HIV-1 subtype B and C infection, on clinical markers was not observed in our cohort. This study for the first time presents SS-SBT-based four-digit typing data on HLA-A, -B, and -C alleles in Ghana, describing impact of HLA on viral load and CD4 count in HIV-1 infection. Accumulation of these data would facilitate high-resolution HLA genotyping, contributing to our understanding of the HIV-1 and host HLA interaction in Ghana, West Africa.

Acute gastroenteritis associated with diarrhea is considered a serious disease in Africa and South Asia. In this study, we examined the trends in the causative pathogens of diarrhea and the corresponding gut microbiota in Ghana using microbiome analysis performed on diarrheic stools via 16S rRNA sequencing. In total, 80 patients with diarrhea and 34 healthy adults as controls, from 2017 to 2018, were enrolled in the study. Among the patients with diarrhea, 39 were norovirus-positive and 18 were rotavirus-positive. The analysis of species richness (Chao1) was lower in patients with diarrhea than that in controls. Beta-diversity analysis revealed significant differences between the two groups. Several diarrhea-related pathogens (e.g., Escherichia-Shigella, Klebsiella and Campylobacter) were detected in patients with diarrhea. Furthermore, co-infection with these pathogens and enteroviruses (e.g., norovirus and rotavirus) was observed in several cases. Levels of both Erysipelotrichaceae and Staphylococcaceae family markedly differed between norovirus-positive and -negative diarrheic stools, and the 10 predicted metabolic pathways, including the carbohydrate metabolism pathway, showed significant differences between rotavirus-positive patients with diarrhea and controls. This comparative study of diarrheal pathogens in Ghana revealed specific trends in the gut microbiota signature associated with diarrhea and that pathogen-dependent dysbiosis occurred in viral gastroenteritis.

In human immunodeficiency virus type-1 (HIV-1) infections, cytotoxic T-lymphocyte (CTL) responses targeting human leukocyte antigen (HLA)-restricted viral epitopes exert strong suppressive pressure on viral replication and frequently select for mutations resulting in viral escape from CTL recognition. Numerous data on these HLA-associated mutations in HIV-1 subtypes B and C have been amassed with few reports described in other subtypes. In the present study, we investigated the HLA-associated mutations in HIV-1 subtype CRF02_AG prevailing in Ghana, Western Africa. We determined viral gag sequences in 246 out of 324 HIV-1-infected Ghanaians. Phylogeny analysis revealed that 200 (81.3%) individuals were infected with HIV-1 CRF02_AG. Full gag and vif sequences were obtained from 199 and 138, respectively, out of the 200 individuals infected with CRF02_AG and subjected to determination of HLA-associated mutations. The analysis found HLA-associated HIV-1 CRF02_AG non-synonymous polymorphisms at 19 sites; 13 in gag and six in vif, including those that were newly determined. Generation of this data is an important contribution to our understanding of HIV-1 CRF02_AG and host T cell interaction.

HIV-1 infected individuals under antiretroviral therapy can control viremia but often develop non-AIDS diseases such as cardiovascular and metabolic disorders. Gut microbiome dysbiosis has been indicated to be associated with progression of these diseases. Analyses of gut/fecal microbiome in individual regions are important for our understanding of pathogenesis in HIV-1 infections. However, data on gut/fecal microbiome has not yet been accumulated in West Africa. In the present study, we examined fecal microbiome compositions in HIV-1 infected adults in Ghana, where approximately two-thirds of infected adults are females. In a cross-sectional case-control study, age- and gender-matched HIV-1 infected adults (HIV+; n = 55) and seronegative controls (HIV-; n = 55) were enrolled. Alpha diversity of fecal microbiome in HIV+ was significantly reduced compared to HIV- and associated with CD4 counts. HIV+ showed reduction in varieties of bacteria including Faecalibacterium, the most abundant in seronegative controls, but enrichment of Proteobacteria. Ghanaian HIV+ exhibited enrichment of Dorea and Blautia; bacteria groups whose depletion has been reported in HIV-1 infected individuals in several other cohorts. Furthermore, HIV+ in our cohort exhibited a depletion of Prevotella, a genus whose enrichment has recently been shown in men having sex with men (MSM) regardless of HIV-1 status. The present study revealed the characteristics of dysbiotic fecal microbiome in HIV-1 infected adults in Ghana, a representative of West African populations.

Recent studies have indicated an association between gut microbiome composition and various disorders, including infectious diseases. The composition of the microbiome differs among ethnicities and countries, possibly resulting in diversified interactions between host immunity and the gut microbiome. Characterization of baseline microbiome composition in healthy people is an essential step for better understanding of the biological interactions associated with individual populations. However, data on the gut/fecal microbiome have not been accumulated for individuals in West Africa. In the present study, we examined the fecal microbiome composition in healthy adults in Ghana. Toward this, 16S rRNA gene libraries were prepared using bacterial fractions derived from 55 Ghanaian adults, which were then subjected to next-generation sequencing. The fecal microbiome of the Ghanaian adults was dominated by Firmicutes (Faecalibacterium, Subdoligranulum, and Ruminococcaceae UCG-014), Proteobacteria (Escherichia-Shigella and Klebsiella), and Bacteroidetes (Prevotella 9 and Bacteroides), consistent with previous observations in African cohorts. Further, our analysis revealed differences in microbiome composition and a lower diversity of the fecal microbiome in the Ghanaian cohort compared with those reported in non-African countries. This is the first study to describe substantial fecal microbiome data obtained using high-throughput metagenomic tools on samples derived from a cohort in Ghana. The data may provide a valuable basis for determining the association between the fecal microbiome and progression of various diseases in West African populations.

Purpose. There have been hardly any reports on the human immunodeficiency virus type 1 (HIV-1) drug-resistance profile from northern Ghana since antiretroviral therapy (ART) was introduced over a decade ago. This study investigated prevailing HIV-1 subtypes and examined the occurrence of drug resistance in ART-experienced patients in Tamale, the capital of the Northern Region of Ghana. Methodology. A cross-sectional study was carried out on HIV-infected adult patients receiving first-line ART. HIV viral load (VL) and CD4+ T-cell counts were measured. The pol gene sequences were analysed for genotypic resistance by an in-house HIV-1 drug-resistance test; the prevailing HIV-1 subtypes were analysed in detail. Results/Key findings. A total of 33 subjects were studied. Participants comprised 11 males (33.3 %) and 22 (66.7 %) females, with a median age of 34.5 years [interquartile range (IQR) 30.0–40.3]. The median duration on ART was 12 months (IQR 8.0–24). Of the 24 subjects successfully genotyped, 10 (41.7 %) viruses possessed at least one mutation conferring resistance to nucleoside or non-nucleoside reverse-transcriptase inhibitors (NRTIs/NNRTIs). Two-class drug resistance to NRTI and NNRTI was mostly detected (25 %, 6/24). The most frequent mutations were lamivudine-resistance M184V and efavirenz/nevirapine-resistance K103N. HIV-1 subtype CRF02_AG was predominant (79.2 %). Other HIV-1 subtypes detected were G (8.3 %), A3 (4.2 %) and importantly two (8.3 %) unique HIV-1 recombinant forms with CRF02_AG/A3 mosaic. Conclusion. HIV-1 shows high genetic diversity and on-going viral genetic recombination in the study region. Nearly 42 % of the patients studied harboured a drug-resistant virus. The study underscores the need for continued surveillance of HIV-1 subtype diversity; and of drug-resistance patterns to guide selection of second-line regimens in northern Ghana.

In human immunodeficiency virus type-1 (HIV-1) infections, cytotoxic T-lymphocyte (CTL) responses targeting human leukocyte antigen (HLA)-restricted viral epitopes exert strong suppressive pressure on viral replication and frequently select for mutations resulting in viral escape from CTL recognition. Numerous data on these HLA-associated mutations in HIV-1 subtypes B and C have been amassed with few reports described in other subtypes. In the present study, we investigated the HLA-associated mutations in HIV-1 subtype CRF02_AG prevailing in Ghana, Western Africa. We determined viral gag sequences in 246 out of 324 HIV-1-infected Ghanaians. Phylogeny analysis revealed that 200 (81.3%) individuals were infected with HIV-1 CRF02_AG. Full gag and vif sequences were obtained from 199 and 138, respectively, out of the 200 individuals infected with CRF02_AG and subjected to determination of HLA-associated mutations. The analysis found HLA-associated HIV-1 CRF02_AG non-synonymous polymorphisms at 19 sites; 13 in gag and six in vif, including those that were newly determined. Generation of this data is an important contribution to our understanding of HIV-1 CRF02_AG and host T cell interaction.

BackgroundAn outbreak of novel coronavirus (SARS-CoV-2)-associated respiratory infectious diseases (COVID-19) emerged in 2019 and has spread rapidly in humans around the world. The demonstration of in vitro infectiousness of respiratory specimens is an informative surrogate for SARS-CoV-2 transmission from patients with COVID-19; accordingly, viral isolation assays in cell culture are an important aspect of laboratory diagnostics for COVID-19.MethodsWe developed a simple and rapid protocol for isolating SARS-CoV-2 from respiratory specimens using VeroE6/TMPRSS2 cells, a cell line that is highly susceptible to the virus. We also investigated a correlation between isolation of SARS-CoV-2 and viral load detected by real-time RT-PCR (rRT-PCR) using N2 primer/probe set that has been developed for testing of COVID-19 in Japan.ResultsThe SARS-CoV-2 isolation protocol did not require blind passage of inoculated cells and yielded the results of viral isolation within 7 days after inoculation. Specimens with cycle threshold (Ct) values of 35 were virus isolation-positive, indicating that low viral loads (high Ct values) in upper respiratory specimens do not always indicate no risk of containing transmissible virus.ConclusionIn combination with rRT-PCR, the SARS-CoV-2 isolation protocol provides a means for assessing the potential risk of transmissible virus in upper respiratory specimens.

Resistance to the quinone outside inhibitor (QoI) fungicides in the tea gray blight-causing fungus Pestalotiopsis longiseta is a serious problem in Japanese tea cultivation. We conducted a population genetic analysis of QoI-resistant P. longiseta isolates on the Makinohara Plateau, Shizuoka Prefecture, Japan’s largest tea-growing area, to elucidate the disease’s epidemiology and the spread of QoI resistance. Inter simple sequence repeat (ISSR) analysis of 1,083 isolates from 395 fields collected from 2009 to 2012 detected 42 ISSR types, designated as PL01 to PL42. A total of 18, seven, and 38 ISSR types were detected in highly resistant, moderately resistant, and sensitive isolates, respectively. No distinct phylogenetic relationship corresponding to QoI sensitivity or sampling location was observed. No annual changes in the population genetic structure of highly resistant isolates were observed during the study period. A different ISSR type was predominant among QoI-resistant isolates in each region. Analysis of molecular variance revealed significant genetic differentiation in populations of highly resistant isolates among regions (FCT = 0.213) and farmers (FCT = 0.071). Consequently, we speculate that QoI-resistant P. longiseta strains occurred in a number of clonal lineages and spread by both natural and artificial transmission, such as rain splash and plucking machines, throughout each region on the Makinohara Plateau.

Numbers of HLA-associated polymorphisms have been reported on HIV-1 subtypes B and C, but few on other subtypes. Here, we analyzed HLA-associated gag and nef polymorphisms in HIV-1 subtype A/E prevalent in Vietnam. We determined HLA-A, B and C genotypes in 179 HIV-1-infected Vietnamese by next generation sequencing and analyzed proviral genome sequences in 144 of them, showing that 142 of the 144 were subtype A/E. Analysis revealed HLA-associated subtype A/E gag and nef polymorphisms at nineteen residues including those newly determined. Accumulation of these data would contribute to our understanding of HIV-1 subtype A/E and host immune interaction.

Antiretroviral therapy (ART) and drug resistance studies worldwide have focused almost exclusively on human immunodeficiency virus type 1 (HIV-1). As a result, there is limited information on ART and drug resistance in HIV-2 patients. In Ghana, the HIV epidemic is characterized by the domination of HIV-1, with cocirculating HIV-2. We, therefore, sought to determine viral load and drug resistance mutations in HIV-2 patients to inform the clinical management of such individuals in Ghana. We used purposive sampling to collect blood from 16 consented patients, confirmed as HIV-2 or HIV-1/2 dual infections by serology. A 2-step real-time RT-PCR assay was used to determine plasma HIV-2 RNA viral loads. For drug resistance testing, nucleic acids were extracted from plasma and peripheral blood mononuclear cells. The reverse transcriptase and protease genes of HIV-2 were amplified, sequenced and analyzed for drug resistance mutations and HIV-2 group. HIV-2 viral load was detected in 9 of 16 patients. Six of these had quantifiable viral loads (range: 2.62–5.45 log IU/mL) while 3 had viral loads below the limit of quantification. Sequences were generated from 7 out of 16 samples. Five of these were classified as HIV-2 group B and 2 as HIV-2 group A. HIV-2 drug resistance mutations (M184V, K65R, Y115F) were identified in 1 patient. This study is the first to report HIV-2 viral load and drug resistance mutations in HIV-2 strains from Ghana. The results indicate the need for continuous monitoring of drug resistance among HIV-2- infected patients to improve their clinical management.

Sensitivity of the novolac positive resist THMR iP-3300 to violet and blue light emitting diodes with central wavelengths of 404-470 nm was investigated to utilize for developing a new numbering exposure system using a matrix array of such light emitting diodes. In addition, the spectral transmittance of the resist was measured, and the relationship between transmittance and sensitivity was discussed. It was clarified that the sensitivity greatly differed between exposures to violet and blue light rays as much as more than 30 times, and the resist was sensitized only by 3 s exposure to the violet light at minimum. Therefore, reasonable exposure time performance will be obtained if the relevant reduction projection lens is applied.

Differential speed rolling (DSR) was applied to the AZ31 magnesium alloy, intended to modify the texture and thus to enhance the room temperature ductility. Especially, effect of DSR temperature on resulting room temperature tensile properties and texture was investigated at a fixed rolling speed ratio of 1.1. The strength was not affected by the rolling temperature: the yield strength and the ultimate tensile strength for all materials processed by DSR were approximately 240 and 290 MPa, respectively. On the other hand, elongation-to-failure increased from 13.6 to 18.5% with decreasing rolling temperature from 573 to 473 K. In addition, the material processed by DSR exhibited approximately 1.5 times larger ductility than that by conventional symmetric rolling at a rolling temperature of 473 K. The DSR at 523 and 473 K resulted in the basal plane orientation at the position inclined by ∼5 and 8° from the normal direction toward the rolling direction, respectively. It was suggested that the higher ductility of the material processed at lower temperatures is attributed to the slight change in the basal plane orientation and/or reduction in grain size.

Lead (Pb) is one of the most effective alloying elements to improve the machinability of steels. However the movement of eliminating Pb contents is recently increasing because of reducing environmental impact. Therefore new types of free cutting steels without Pb are strongly required. Ti4C2S2 is one of the promissing candidates of free cutting elements. However the mechanism of improvement in machinability by Ti4C2S2 has not been confirmed yet. In this study we investgated the effect of Ti4C2S2 in 13 % Cr stainless steels. Ti4C2S2 had notch effect like other sulfides such as MnS. Thereby chip breakability was improved. Ti4C2S2 also had tool-protecting effect by forming stable adhesion. On the other hand Ti4C2S2 was not shown remarkable lubricant effect as Pb. The improvement of machinability in 13 mass%Cr ferritic stainless steels by including Ti4C2S2 attribute to the chip breakability and restraint of the tool-wear by forming stable adhesion.

The microstructures and mechanical properties of fullerene powder dispersed AZ91 magnesium alloy composites were examined. The AZ91 with 0, 1 and 5 mass% fullerene additions were produced by extruding a mixture of AZ91 machined chips and fullerene powder. The matrix grains of the extruded materials were equiaxed, and the size was � 5 mm irrespective of the fullerene content. The agglomerated fullerene powder was aligned parallel to the extrusion direction in the composites. The addition of 1 mass% fullerene had little effect on the elastic moduli, tensile strength and ductility, whereas the addition of 5 mass% fullerene slightly decreased them. A comparison between the tensile properties of the present composites and those of AZ91 alloys processed by various methods was also made. The damping capacity increased steeply by the fullerene addition. The effectiveness of the fullerene addition for the enhancement of the damping capacity of metallic alloys was verified. A good combination of damping capacity, elastic moduli, strength and ductility was attained in the present composites.

Cerebrospinal fluid (CSF) induced neurite retraction of differentiated PC12 cells; the action was observed in 15 min (a rapid response) and the activity further increased until 6 h (a long-acting response) during exposure of CSF to the cells. The CSF action was sensitive to monoglyceride lipase and diminished by homologous desensitization with lysophosphatidic acid (LPA) and by pretreatment with an LPA receptor antagonist Ki16425. Although fresh CSF contains LPA to some extent, the LPA content in the medium was increased during culture of PC12 cells with CSF. The rapid response was mimicked by exogenous LPA, and a long-acting response was duplicated by a recombinant autotaxin, lysophospholipase D (lyso-PLD). Although the lyso-PLD substrate lysophosphatidylcholine (LPC) was not detected in CSF, lyso-PLD activity and an approximately 120-kDa autotaxin protein were detected in CSF. On the other hand, LPC but not lyso-PLD activity was detected in the conditioned medium of a PC12 cell culture without CSF. Among neural cells examined, leptomeningeal cells expressed the highest lyso-PLD activity and autotaxin protein. These results suggest that leptomeningeal cells may work as one of the sources for autotaxin, which may play a critical role in LPA production and thereby regulate axonal and neurite morphological change.

特発性大腸穿孔の中でも稀な横行結腸穿孔の1例を経験した.症例は壊疽性膿皮症で加療中の65歳,女性.平成15年10月22日に出現し経過観察していた腹痛が28日夕方に突然増強し当科紹介となった.腹部所見とCT検査にて,穿孔性腹膜炎と診断し緊急手術となった.横行結腸中央に約3cmの穿孔を認めたため,横行結腸部分切除,腹腔内洗浄ドレナージ,および人工肛門造設を行った.病理組織像では,穿孔部に粘膜と筋層の急峻な断裂像を認めるのみで,潰瘍,腫瘍,憩室などの所見はなかった.特発性横行結腸穿孔は,比較的稀であるが,穿孔性腹膜炎の鑑別疾患の一つとして念頭に置く必要がある.

We investigated the expression of 50 cytochrome P450 (CYP) genes in the post-ischemic reperfused rat liver using a DNA microarray. Thirteen CYPs showed absent expression in all experiments and 2 CYPs were induced by pentobarbital, which was used as an anesthetic. Eight of the remaining 35 CYPs showed significantly decreased expression following ischemia-reperfusion. Monitoring CYP expression may be a powerful approach for elucidation of pathways regulating drug metabolism that may be involved in postischemic reperfusion liver injury. Keywords:: cytochrome P450, postischemic liver reperfusion, gene expression

Lettuce big-vein virus (LBVV) is the type species of the genus Varicosavirus and is a two-segmented negative-sense single-stranded RNA virus. The larger LBVV genome segment (RNA1) consists of 6797 nt and encodes an L polymerase that resembles that of rhabdoviruses. Here, the nucleotide sequence of the second LBVV genome segment (RNA2) is reported. LBVV RNA2 consisted of 6081 nt and contained antisense information for five major ORFs: ORF1 (nt 210–1403 on the viral RNA), ORF2 (nt 1493–2494), ORF3 (nt 2617–3489), ORF4 (nt 3843–4337) and ORF5 (nt 4530–5636), which had coding capacities of 44, 36, 32, 19 and 41 kDa, respectively. The gene at the 3′ end of the viral RNA encoded a coat protein, while the other four genes encoded proteins of unknown functions. The 3′-terminal 11 nt of LBVV RNA2 were identical to those of LBVV RNA1, and the 5′-terminal regions of LBVV RNA1 and RNA2 contained a long common nucleotide stretch of about 100 nt. Northern blot analysis using probes specific to the individual ORFs revealed that LBVV transcribes monocistronic RNAs. Analysis of the terminal sequences, and primer extension and RNase H digestion analysis of LBVV mRNAs, suggested that LBVV utilizes a transcription termination/initiation strategy comparable with that of rhabdoviruses.

It has been suggested that lipoproteins in the central nervous system are involved in the regulation of several neural functions independent of cholesterol metabolism as well as those related to lipid metabolism. We recently demonstrated that lipoproteins are carriers for sphingosine 1-phosphate (S1P). This raised the possibility that S1P mediates the neural cell functions induced by lipoproteins. In the current study, we examined the effects of plasma high-density lipoprotein (HDL) on astroglial cell functions, focusing especially on the role of the lipoprotein-associated S1P. In rat type I astrocytes or C6 glioma cells, similar to S1P, HDL stimulated DNA synthesis and mRNA expression of fibroblast growth factor-2, a potent neurotrophic factor, which was associated with the activation of extracellular signal-regulated kinase (ERK) in a pertussis toxin-sensitive manner. The data from fractionation studies of HDL indicated that S1P may be a major component for the activation of ERK. In C6 glioma cells, HDL also induced phospholipase C-dependent intracellular Ca2+ mobilization. Desensitization of the C6 glioma cells with S1P abolished these HDL-induced actions. Furthermore, overexpression of S1P receptors in C6 glioma cells led to a significant enhancement of HDL-induced ERK activation and Ca2+ mobilization. Thus, at least some HDL-induced actions may be mediated by cell-surface S1P receptors in astroglial cells. These results imply that S1P might partially mediate lipoprotein-induced cholesterol metabolism-independent neural cell functions in the central nervous system.

The complete nucleotide sequence of RNA1 from Lettuce big-vein virus (LBVV), the type member of the genus Varicosavirus , was determined. LBVV RNA1 consists of 6797 nucleotides and contains one large ORF that encodes a large (L) protein of 2040 amino acids with a predicted M r of 232,092. Northern blot hybridization analysis indicated that the LBVV RNA1 is a negative-sense RNA. Database searches showed that the amino acid sequence of L protein is homologous to those of L polymerases of nonsegmented negative-strand RNA viruses. A cluster dendrogram derived from alignments of the LBVV L protein and the L polymerases indicated that the L protein is most closely related to the L polymerases of plant rhabdoviruses. Transcription termination/polyadenylation signal-like poly(U) tracts that resemble those in rhabdovirus and paramyxovirus RNAs were present upstream and downstream of the coding region. Although LBVV is related to rhabdoviruses, a key distinguishing feature is that the genome of LBVV is segmented. The results reemphasize the need to reconsider the taxonomic position of varicosaviruses.

GeneChip (Affymetrix, Inc., USA) employs a specific method for spotting DNA probes on chips, which is different from any other DNA chips, and can complete the whole process from sample preparation to data construction and analysis. The GeneChip system can be applied to both gene expression analysis and genomic mutation analysis, which would play an important role in human genome analysis in the future. Techniques for data construction ("wet" experimental techniques), which are the major components in the GeneChip system, are generally established as routine work in the first screening process in most laboratories worldwide. The most important point would be how we exchange experimental data produced by researchers and gene/genome information available both on the public and the commercial bases so that we reduce useful information on gene expression. Recently, the center of the research has been shifting to computing technology for data processing ("bioinformatics"). This article separately deals with gene expression analysis and genomic analysis, with emphasis on bioinformatics. We describe the data on gene expression screening, the gene targeting process, the analysis of genomic DNA mutations using the P53 probe array, and the HuSNP mapping assays, by presenting our experimental examples.

Background and Purpose: The effectiveness of organized stroke care on stroke mortality and morbidity remains uncertain. We examined whether organized stroke care index (OCI), which graded 0-3 based on the presence of rehabilitation, stroke team assessment, and admission to a stroke unit developed by Saposnik (Neurology 2010) influence in-hospital mortality and morbidity of patients with ischemic and hemorrhagic stroke in a nationwide study. Methods: Of the 1369 certified training institutions in Japan, 749 hospitals responded to a questionnaire survey regarding comprehensive stroke care capacities. Among the institutions that responded, data on patients hospitalized between April 1, 2010 and March 31, 2011, because of stroke were obtained from the Japanese Diagnosis Procedure Combination database. In-hospital mortality morbidity was analyzed using hierarchical logistic regression analysis adjusted for age, sex, level of consciousness on admission, and the number of OCI fulfilled in each component and in total.It was supported by Grants-in-Aid from the Ministry of Health, Labour and Welfare of Japan Results: Data from 265 institutions and 53,170 emergency-hospitalized patients were analyzed. Patients fulfilled the criteria for admission to a SCU, stroke team assessment and the presence of rehabilitation were 28.9%, 42.1% and 95.1%, respectively. Mortality adjusted for age, sex, and level of consciousness was significantly correlated with admission to a SCU (OR=0.87, p=0.039), SCU team assessment (OR=0.88,p=0.029), and OCI ( OR=0.93, p=0.031). Modified ranking scale 0 to 2 rate were also associated with significantly SCU admission (p=0.003) .These association holds for ischemic stroke and subarachnoid hemorrhage. Conclusion: A strong association between organized stroke care and lower mortality was apparent. These data suggest that organized stroke care should be provided to stroke patients regardless of stroke subtype.

A sequence of 1425 nt was established that included the complete coat protein (CP) gene of Lettuce big-vein virus (LBVV). The LBVV CP gene encodes a 397 amino acid protein with a predicted M r of 44486. Antisera raised against synthetic peptides corresponding to N-terminal or C-terminal parts of the LBVV CP reacted in Western blot analysis with a protein with an M r of about 48000. RNA extracted from purified particles of LBVV by using proteinase K, SDS and phenol migrated in gels as two single-stranded RNA species of approximately 7·3 kb (ss-1) and 6·6 kb (ss-2). After denaturation by heat and annealing at room temperature, the RNA migrated as four species, ss-1, ss-2 and two additional double-stranded RNAs (ds-1 and ds-2). The Northern blot hybridization analysis using riboprobes from a full-length clone of the LBVV CP gene indicated that ss-2 has a negative-sense nature and contains the LBVV CP gene. Moreover, ds-2 is a double-stranded form of ss-2. Database searches showed that the LBVV CP most resembled the nucleocapsid proteins of rhabdoviruses. These results indicate that it would be appropriate to classify LBVV as a negative-sense single-stranded RNA virus rather than as a double-stranded RNA virus.

Dynamic recrystallization (DRX) behavior was systematically examined in two commercial Mg-Al-Zn alloys in order to clarify the relationship between deformation conditions and the resulting microstructure. The materials were deformed by upset forging at temperatures ranging from 473 to 673 K at an initial strain rate of 3.3 x 10 -2 s -1 . Grain refinement was observed during deformation. It was found that the dynamically recrystallized grain size decreases with an increasing Zener-Hollomon parameter and/or a decreasing initial grain size. A phenomenological constitutive equation was developed in order to provide a guideline for the control of the grain size of hot deformed AZ61 alloy.

An examination of consolidation conditions by hot extrusion of AZ31 magnesium alloy machined chips was conducted to enhance the bonding of individual chips, in order to improve the mechanical properties. Hot extrusions were carried out in the superplastic and non-superplastic region. Microstructural observations revealed that grain refinement was attained by extruding machined chips, and the grain sizes of the chip-extruded materials were smaller than 5 μm. The interfaces of individual chips of extruded materials were not identified when the chips were extruded in the superplastic region. The ultimate tensile strength was about 300 MPa and elongation-to-failure was about 10% for chip-extruded materials that were extruded in the superplastic region. These materials were comparable with the as-received alloy with respect to the room temperature strength, although the ductility was reduced to half. It was confirmed that chip consolidation utilizing superplastic flow is useful to enhance the bonding of individual grains.

Real advances in biotechnology made it possible to complete human whole genome sequencing within a short duration. Although the genome includes a huge amount of information about biological functions and the interest is now directed to the study using genomic information, the genomic strategy is not clearly understood. The following 4 studies were therefore presented and discussed about the strategy after the completion of the genomic sequence in the 74th Annual Meeting of Japanese Pharmacological Society: 1) Asthma and atopic dermatitis: models for genetic and genomic investigations of complex genetic diseases, by W.C.O. Cookson (University of Oxford, Asthma Genetics Group, Wellcome Trust Centre for Human Genetics); 2) Molecular classification by global gene expression profiling: application on oncogenomic research, by H. Aburatani (Genome Science Division, Research Center for Advanced Science and Technology, University of Tokyo); 3) Functional genomic search of disease-related genes using microarrays with normalized rat cDNA library, by G. Tsujimoto, et al. (Department of Molecular, Cell Pharmacology, National Children's Medical Research Center: and 4) Acute ischemic change of mRNA expression in the hippocampus by GeneChip array analysis: a starting point for post-genome strategy, by S. Asai, et al.

Using a dialysis electrode, previous studies showed a clear biphasic release of glutamate during anoxia and ischemia. In this study, we examined two hypotheses: (1) glutamate is of vesicular origin and its release is thus Ca2+- and ATP-dependent in the first phase, while in the second phase glutamate is derived primarily from the metabolic pool, and (2) reversed glutamate uptake, due to electrogenic stoichiometry, produces the second phase during anoxic insult in the rat brain. A dialysis electrode continuously perfused with glutamate oxidase and ferrocene-conjugated bovine serum albumin (BSA) optimized the time resolution of monitoring, allowing quantitative oxygen-independent, real-time measurement of the extracellular glutamate concentration ([Glu]e) during anoxia. [Glu]e dynamics were analyzed during anoxia by combining the dialysis electrode with focal microinjection of substances inducing glutamate release. Following anoxia in the rat brain, a sharp and rapid [Glu]e elevation took place (first phase). The [Glu]e elevation then shifted, continuing a gently sloping rise throughout the anoxic period (second phase). This first phase disappeared with intracranial administration of either Co2+ or omega-conotoxin. The second phase rise increased with focal microinjection of KCl (300 mM, 1 microL) and decreased with NaCl (300 mM, 1 microL), ultimately reaching a plateau in both cases. Preloading with a novel glutamate transporter inhibitor (tPDC) decreased both the first and second phases of [Glu]e elevation. This dialysis electrode system provides data supporting in vivo evidence that the peak of the first phase of [Glu]e elevation is derived from the "neurotransmitter pool," while the second phase is derived from the neuronal and glial "metabolic pool," which is, at least, partly related to a "reversed uptake" mechanism in the anoxic rat brain.

Newly discovered TT virus (TTV) is widely distributed in human populations. To understand more about the relationship between TTV and its hosts, we tested 400 sera from various nonhuman primates for the presence of TTV DNA by PCR assay. We collected serum samples from 24 different species of nonhuman primates. TTV DNA was determined by PCR with primers designed from the 5′-end region of the TTV genome. Nucleotide sequencing and phylogenetic analysis of viral genomes were also performed. TTV DNA was detected in 87 of 98 (89%) chimpanzees and 3 of 21 (14%) crab-eating macaques. Nucleotide sequences of the PCR products obtained from both animals were 80 to 100% identical between two species. In contrast, the sequences differed from TTV isolates in humans by 24 to 33% at the nucleotide level and 36 to 50% at the amino acid level. Phylogenetic analysis demonstrated that all TTV isolates obtained from simians were distinct from the human TTV isolates. Furthermore, TTV in simians, but not in humans, was classified into three different genotypes. Our results indicate that TTV in simians represents a group different from, but closely related to, TTV in humans. From these results, we tentatively named this TTV simian TTV (s-TTV). The existence of the s-TTV will be important in determining the origin, nature, and transmission of human TTV and may provide useful animal models for studies of the infection and pathogenesis of this new DNA virus.

DIF-1 is a putative morphogen that induces stalk cell formation in the cellular slime mold Dictyostelium discoideum. We have previously discovered that DIF-1 suppresses cell growth and induces cell differentiation in vitro in some tumor cells, and also that relatively low concentrations of DIF-1 promote retinoic acid-induced cell differentiation in the human myeloid leukemia HL-60 cells. In this study, to verify cell biological and therapeutic potential of DIF-1, we have examined whether and how DIF-1 affects normal mammalian cells in vitro, using rat leptomeningeal (RLM) cells and rat gastric mucosal RGM-1 cells. In growing phase of both cells, DIF-1 at 5–40 μM suppressed cell growth in a dose-dependent manner. High concentrations (15–40 μM) of DIF-1 were toxic to the growing cells so that the cells showed unusual morphology, but many of them were still alive even at Day 3–4. Withdrawal of DIF-1 allowed the cells to grow. In confluent phase of the cells, DIF-1 at more than 15 μM promoted medium ...