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Introduction The worldwide pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has continued to date. Given that some of the patients with coronavirus disease 2019 (COVID-19) are asymptomatic, antibody tests are useful to determine whether there is a previous infection with SARS-CoV-2. In this study, we measured IgM and IgG antibody titers against SARS-CoV-2 in the serum of asymptomatic healthy subjects in The University of Tokyo, Japan. Methods From June 2020, we recruited participants, who were students, staff, and faculty members of The University of Tokyo in the project named The University of Tokyo COVID-19 Antibody Titer Survey (UT-CATS). Following blood sample collection, participants were required to answer an online questionnaire about their social and health information. We measured IgG and IgM titers against SARS-CoV-2 using iFlash-SARS-CoV-2 IgM and IgG detection kit which applies a chemiluminescent immunoassay (CLIA) for the qualitative detection. Results There were 6,609 volunteers in this study. After setting the cutoff value at 10 AU/mL, 32 (0.48%) were positive for IgG and 16 (0.24%) for IgM. Of six participants with a history of COVID-19, five were positive for IgG, whereas all were negative for IgM. The median titer of IgG was 0.40 AU/mL and 0.39 AU/mL for IgM. Both IgG and IgM titers were affected by gender, age, smoking status, and comorbidities. Conclusions Positive rates of IgG and IgM titers were relatively low in our university. Serum levels of these antibodies were affected by several factors, which might affect the clinical course of COVID-19.

Abetalipoproteinemia (ABL) is a rare autosomal recessive disorder caused by biallelic pathogenic mutations in the MTTP gene. Deficiency of microsomal triglyceride transfer protein (MTTP) abrogates the assembly of apolipoprotein (apo) B-containing lipoprotein in the intestine and liver, resulting in malabsorption of fat and fat-soluble vitamins and severe hypolipidemia. Patients with ABL typically manifest steatorrhea, vomiting, and failure to thrive in infancy. The deficiency of fat-soluble vitamins progressively develops into a variety of symptoms later in life, including hematological (acanthocytosis, anemia, bleeding tendency, etc.), neuromuscular (spinocerebellar ataxia, peripheral neuropathy, myopathy, etc.), and ophthalmological symptoms (e.g., retinitis pigmentosa). If left untreated, the disease can be debilitating and even lethal by the third decade of life due to the development of severe complications, such as blindness, neuromyopathy, and respiratory failure. High dose vitamin supplementation is the mainstay for treatment and may prevent, delay, or alleviate the complications and improve the prognosis, enabling some patients to live to the eighth decade of life. However, it cannot fully prevent or restore impaired function. Novel therapeutic modalities that improve quality of life and prognosis are awaited. The aim of this review is to 1) summarize the pathogenesis, clinical signs and symptoms, diagnosis, and management of ABL, and 2) propose diagnostic criteria that define eligibility to receive financial support from the Japanese government for patients with ABL as a rare and intractable disease. In addition, our diagnostic criteria and the entry criterion of low-density lipoprotein cholesterol (LDL-C) ＜15 mg/dL and apoB ＜15 mg/dL can be useful in universal or opportunistic screening for the disease. Registry research on ABL is currently ongoing to better understand the disease burden and unmet needs of this life-threatening disease with few therapeutic options.

Introduction The usefulness of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody tests in asymptomatic individuals has not been well validated, although they have satisfied sensitivity and specificity in symptomatic patients. In this study, we investigated the significance of IgM and IgG antibody titers against SARS-CoV-2 in the serum of asymptomatic healthy subjects. Methods From June 2020, we recruited 10,039 participants to the project named the University of Tokyo COVID-19 Antibody Titer Survey (UT-CATS), and measured iFlash-SARS-CoV-2 IgM and IgG (YHLO IgM and IgG) titers in the collected serum. For the samples with increased IgM or IgG titers, we performed additional measurements using Elecsys Anti-SARS-CoV-2 Ig (Roche total Ig) and Architect SARS-CoV-2 IgG (Abbott IgG) and investigated the reactivity to N, S1, and receptor binding domain (RBD) proteins. Results After setting the cutoff value at 5 AU/mL, 61 (0.61%) were positive for YHLO IgM and 104 (1.04%) for YHLO IgG. Few samples with elevated YHLO IgM showed reactivity to S1 or RBD proteins, and IgG titers did not increase during the follow-up in any samples. The samples with elevated YHLO IgG consisted of two groups: one reacted to S1 or RBD proteins and the other did not, which was reflected in the results of Roche total Ig. Conclusions In SARS-CoV-2 seroepidemiological studies of asymptomatic participants, sufficient attention should be given to the interpretation of the results of YHLO IgM and IgG, and the combined use of YHLO IgG and Roche total Ig might be more reliable.

Familial hypercholesterolemia (FH) is an inherited disorder with retarded clearance of plasma LDL caused by mutations of the genes involved in the LDL receptor-mediated pathway and most of them exhibit autosomal dominant inheritance. Homozygotes of FH (HoFH) may have plasma LDL-C levels, which are at least twice as high as those of heterozygous FH (HeFH) and therefore four times higher than normal levels. Prevalence of HoFH had been estimated as 1 in 1,000,000 before but more recent genetic analysis surveys predict 1 in 170,000 to 300,000. Since LDL receptor activity is severely impaired, HoFH patients do not or very poorly respond to medications to enhance activity, such as statins, and have a poorer prognosis compared to HeFH. HoFH should therefore be clinically distinguished from HeFH. Thorough family studies and genetic analysis are recommended for their accurate diagnosis. Fatal cardiovascular complications could develop even in the first decade of life for HoFH, so aggressive lipid-lowering therapy should be initiated as early as possible. Direct removal of plasma LDL by lipoprotein apheresis has been the principal measure for these patients. However, this treatment alone may not achieve stable LDL-C target levels and combination with drugs should be considered. The lipid-lowering effects of statins and PCSK9 inhibitors substantially vary depending on the remaining LDL receptor activity of individual patients. On the other hand, the action an MTP inhibitor is independent of LDL receptor activity, and it is effective in most HoFH cases. This review summarizes the key clinical issues of HoFH as well as insurance coverage available under the Japanese public healthcare system.

Introduction: Familial hypercholesterolemia (FH) is caused by genetic defects in low-density lipoprotein (LDL) receptor pathway, resulting in a persistent elevation of plasma LDL-cholesterol (LDL-C) levels as well as an increased risk of cardiovascular diseases. Early diagnosis and treatment are imperative for its management. Currently, FH patients are mainly identified by cascade screening. However, FH is underdiagnosed and undertreated, particularly in young adults, which warrants a systematic screening strategy. Hypothesis: Considering that the prevalence of FH is high (~0.4% in the general population) and that treatment of FH should be initiated earlier at the latest in young adults, we hypothesized that the universal screening of LDL-C in young adults is beneficial for early diagnosis and treatment of FH. Methods: At the University of Tokyo, plasma LDL-C levels have been measured at heath checkups for employees as well as new students. Young adults (18-30 years old) with hypercholesterolemia (LDL-C ≥ 160 mg/dL) identified by universal screening and referred to the University of Tokyo Hospital were eligible (the Young FH study). Those who have been identified by cascade screening were excluded. Mutation in FH-causing genes, as well as clinical and biochemical parameters useful for the clinical diagnosis of FH, were analyzed. The protocol was approved by the human genome, gene analysis research ethics committee of the University of Tokyo. Results: Of the total participants (n = 89, mean age 23.4 ± 3.4 years), 36 (40%) had mutations in LDLR or PCSK9 . The prevalence of mutation carriers was higher in non-obese than in obese participants (47% for body mass index (BMI) < 24 kg/m 2 vs. 26% for BMI ≥ 24). The sensitivity and specificity of clinical diagnosis were 28% and 85%, respectively, by Dutch Lipid Clinic Network Criteria (definite + probable FH), or 22% and 98%, respectively, by Japanese diagnostic criteria of FH. Conclusions: The universal screening of LDL-C effectively identifies FH high-risk patients in Japanese young adults. This screening may be beneficial in countries with low-prevalence of obesity or in non-obese populations. The use of clinical diagnostic criteria of FH for universal screening may lead to underdiagnosis.

Bovine respiratory disease complex (BRDC) is frequently found in cattle worldwide. The etiology of BRDC is complicated by infections with multiple pathogens, making identification of the causal pathogen difficult. Here, we developed a detection system by applying TaqMan real-time PCR (Dembo respiratory-PCR) to screen a broad range of microbes associated with BRDC in a single run. We selected 16 bovine respiratory pathogens (bovine viral diarrhea virus, bovine coronavirus, bovine parainfluenza virus 3, bovine respiratory syncytial virus, influenza D virus, bovine rhinitis A virus, bovine rhinitis B virus, bovine herpesvirus 1, bovine adenovirus 3, bovine adenovirus 7, Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, Trueperella pyogenes, Mycoplasma bovis and Ureaplasma diversum) as detection targets and designed novel specific primer-probe sets for nine of them. The assay performance was assessed using standard curves from synthesized DNA. In addition, the sensitivity of the assay was evaluated by spiking solutions extracted from nasal swabs that were negative by Dembo respiratory-PCR for nucleic acids of pathogens or synthesized DNA. All primer-probe sets showed high sensitivity. In this study, a total of 40 nasal swab samples from cattle on six farms were tested by Dembo respiratory-PCR. Dembo respiratory-PCR can be applied as a screening system with wide detection targets.

Objective— APOA5 variants are strongly associated with hypertriglyceridemia, as well as increased risks of cardiovascular disease and acute pancreatitis. Hypertriglyceridemia in apo AV dysfunction often aggravates by environmental factors such as high-carbohydrate diets or aging. To date, the molecular mechanisms by which these environmental factors induce hypertriglyceridemia are poorly defined, leaving the high-risk hypertriglyceridemia condition undertreated. Previously, we reported that LXR (liver X receptor)-SREBP (sterol regulatory element-binding protein)-1c pathway regulates large-VLDL (very low-density lipoprotein) production induced by LXR agonist. However, the pathophysiological relevance of the finding remains unknown. Approach and Results— Here, we reconstitute the environment-induced hypertriglyceridemia phenotype of human APOA5 deficiency in Apoa5 −/− mice and delineate the role of SREBP-1c in vivo by generating Apoa5 −/− ;Srebp-1c −/− mice. The Apoa5 −/− mice, which showed moderate hypertriglyceridemia on a chow diet, developed severe hypertriglyceridemia on high-carbohydrate feeding or aging as seen in patients with human apo AV deficiency. These responses were nearly completely abolished in the Apoa5 −/− ;Srebp-1c −/− mice. Further mechanistic studies revealed that in response to these environmental factors, SREBP-1c was activated to increase triglyceride synthesis and to permit the incorporation of triglyceride into abnormally large-VLDL particles, which require apo AV for efficient clearance. Conclusions— Severe hypertriglyceridemia develops only when genetic factors (apo AV deficiency) and environmental effects (SREBP-1c activation) coexist. We demonstrate that the regulated production of large-sized VLDL particles via SREBP-1c determines plasma triglyceride levels in apo AV deficiency. Our findings explain the long-standing enigma of the late-onset hypertriglyceridemia phenotype of apo AV deficiency and suggest a new approach to treat hypertriglyceridemia by targeting genes that mediate environmental effects.

During surveillance for bovine diarrhea of unknown causes in Japanese black cattle in Kagoshima Prefecture, Japan, we found two types of novel kobu-like viruses in fecal samples of calves. Sequence analyses revealed that they had L protein and 2A protein with H-box/NC sequence motif, which are present in kobuviruses. Phylogenetic analysis revealed that they were related to kobuviruses; however, they clustered apart from other kobuviruses. In the prevalence study of two types of novel kobu-like viruses, 16.9% and 10.4% prevalence of these viruses were observed in the feces of diarrheal calves in this area.

Bats are natural hosts of many zoonotic viruses. Monitoring bat viruses is important to detect novel bat-borne infectious diseases. In this study, next generation sequencing techniques and conventional PCR were used to analyze intestine, lung, and blood clot samples collected from wild bats captured at three locations in Davao region, in the Philippines in 2012. Different viral genes belonging to the Retroviridae and Herpesviridae families were identified using next generation sequencing. The existence of herpesvirus in the samples was confirmed by PCR using herpesvirus consensus primers. The nucleotide sequences of the resulting PCR amplicons were 166-bp. Further phylogenetic analysis identified that the virus from which this nucleotide sequence was obtained belonged to the Gammaherpesvirinae subfamily. PCR using primers specific to the nucleotide sequence obtained revealed that the infection rate among the captured bats was 30 %. In this study, we present the partial genome of a novel gammaherpesvirus detected from wild bats. Our observations also indicate that this herpesvirus may be widely distributed in bat populations in Davao region.

Japanese eel endothelial cells-infecting virus (JEECV) has spread in eel farms and caused serious economic loss. In this study, we examined the prevalence of JEECV infection in 100 wild Japanese eel (Anguilla japonica) elvers caught from Yamaguchi prefecture, Japan, using quantitative PCR and conventional PCR. Total genomic DNA was obtained from the cranial quarter of the body in 70 of 100 eels and from the gill in the remaining. Of 30 gill samples, 20 were analyzed after pooling with other samples, and the remaining 10 were analyzed separately. A single positive result for JEECV was detected following analysis of the 10 separately analyzed samples. This result constitutes the first report of JEECV infection in wild A. japonica elvers.

Aim: Myriad biological effects of leptin may lead to broad therapeutic applications for various metabolic diseases, including diabetes and its complications; however, in contrast to its anorexic effect, the molecular mechanisms underlying adipopenic and glucose-lowering effects of leptin have not been fully understood. Here we aim to clarify the role of hormone-sensitive lipase (HSL) in leptin's action. Methods: Wild-type (WT) and HSL-deficient (HSLKO) mice were made hyperleptinemic by two commonly-used methods: adenovirus-mediated overexpression of leptin and continuous subcutaneous infusion of leptin by osmotic pumps. The amount of food intake, body weights, organ weights, and parameters of glucose and lipid metabolism were measured. Results: Hyperleptinemia equally suppressed the food intake in WT and HSLKO mice. On the other hand, leptin-mediated fat loss and glucose-lowering were significantly blunted in the absence of HSL when leptin was overexpressed by recombinant adenovirus carrying leptin. By osmotic pumps, the fat-losing and glucose-lowering effects of leptin were milder due to lower levels of hyperleptinemia; although the difference between WT and HSLKO mice did not reach statistical significance, HSLKO mice had a tendency to retain more fat than WT mice in the face of hyperleptinemia. Conclusions: We clarify for the first time the role of HSL in leptin's effect using a genetic model: leptin-promoted fat loss and glucose-lowering are at least in part mediated via HSL-mediated lipolysis. Further studies to define the pathophysiological role of adipocyte lipases in leptin action may lead to a new therapeutic approach to circumvent leptin resistance.

Background Bovine enterovirus (BEV) belongs to the species Enterovirus E or F, genus Enterovirus and family Picornaviridae. Although numerous studies have identified BEVs in the feces of cattle with diarrhea, the pathogenicity of BEVs remains unclear. Previously, we reported the detection of novel kobu-like virus in calf feces, by metagenomics analysis. In the present study, we identified a novel BEV in diarrheal feces collected for that survey. Complete genome sequences were determined by deep sequencing in feces. Secondary RNA structure analysis of the 5′ untranslated region (UTR), phylogenetic tree construction and pairwise identity analysis were conducted. Results The complete genome sequences of BEV were genetically distant from other EVs and the VP1 coding region contained novel and unique amino acid sequences. We named this strain as BEV AN12/Bos taurus/JPN/2014 (referred to as BEV-AN12). According to genome analysis, the genome length of this virus is 7414 nucleotides excluding the poly (A) tail and its genome consists of a 5′UTR, open reading frame encoding a single polyprotein, and 3′UTR. The results of secondary RNA structure analysis showed that in the 5′UTR, BEV-AN12 had an additional clover leaf structure and small stem loop structure, similarly to other BEVs. In pairwise identity analysis, BEV-AN12 showed high amino acid (aa) identities to Enterovirus F in the polyprotein, P2 and P3 regions (aa identity ≥82.4%). Therefore, BEV-AN12 is closely related to Enterovirus F. However, aa sequences in the capsid protein regions, particularly the VP1 encoding region, showed significantly low aa identity to other viruses in genus Enterovirus (VP1 aa identity ≤58.6%). In addition, BEV-AN12 branched separately from Enterovirus E and F in phylogenetic trees based on the aa sequences of P1 and VP1, although it clustered with Enterovirus F in trees based on sequences in the P2 and P3 genome region. Conclusions We identified novel BEV possessing highly divergent aa sequences in the VP1 coding region in Japan. According to species definition, we proposed naming this strain as “Enterovirus K”, which is a novel species within genus Enterovirus. Further genomic studies are needed to understand the pathogenicity of BEVs. Electronic supplementary material The online version of this article (doi:10.1186/s12866-016-0923-0) contains supplementary material, which is available to authorized users.

There are few reports describing diarrhea of adult cattle caused by group A rotaviruses. Here, we report the identification of a novel bovine group A rotavirus from diarrhea of adult cows. A group A rotavirus was detected from an epizootic outbreak of diarrhea in adult cows with a decrease in milk production in Japan in 2013. The comprehensive genomic analyses from fecal samples by viral metagenomics using a next-generation sequencer revealed that it had an unreported genotype combination G15P[14]. The genome constellation of this strain, namely, RVA/Cow-wt/JPN/Tottori-SG/2013/G15P[14] was G15-P[14]-I2-R2-C2-M2-A3-N2-T6-E2-H3 representing VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5, respectively. Each gene segment of Tottori-SG was most closely related to Japanese bovine group A rotaviruses suggesting that Tottori-SG might have derived from multiple reassortment events from group A rotavirus strains circulating among Japanese cattle. No other diarrhea pathogen of adult cattle was detected by routine diagnosis and metagenomics. Viral metagenomics, using a next-generation sequencer, is useful to characterize group A rotaviruses from fecal samples and offers unbiased comprehensive investigations of pathogen.

Aim Familial apolipoprotein C-II (apoC-II) deficiency is a rare autosomal recessive disorder with marked hypertriglyceridemia resulting from impaired activation of lipoprotein lipase. In most cases of apoC-II deficiency, causative mutations have been found in the protein-coding region of APOC2; however, several atypical cases of apoC-II deficiency were reported to have markedly reduced, but detectable levels of plasma apoC-II protein (hereafter referred to as hypoapoC-II), which resulted from decreased promoter activity or improper splicing of apoC-II mRNA due to homozygous mutations in APOC2. Here we aim to dissect the molecular bases of a new case of hypoapoC-II. Methods We performed detailed biochemical/genetic analyses of our new case of hypoapoC-II, manifesting severe hypertriglyceridemia (plasma triglycerides, 3235 mg·dL(-1)) with markedly reduced levels of plasma apoC-II (0.6 mg·dL(-1)). Results We took advantage of a monocyte/macrophage culture system to prove that transcription of apoC-II mRNA was decreased in the patient's cells, which is compatible with the reported features of hypoapoC-II. Concomitantly, transcriptional activity of the minigene reporter construct of the patient's APOC2 gene was decreased; however, no rare variant was detected in the patient's APOC2 gene. Fifty single nucleotide variants were detected in the patient's APOC2, but all were common variants (allele frequencies ＞35%) that are supposedly not causative. Conclusions A case of apoC-II deficiency was found that is phenotypically identical to hypoapoC-II but with no causative mutations in APOC2, implying that other genes regulate apoC-II levels. The clinical entity of hypoapoC-II is discussed.

We investigated the sensitivity of human rotavirus rapid antigen detection (RAD) kits, RT-PCR and next-generation DNA sequencing (NGS) for detection of bovine group A rotavirus (RVA). The Dipstick ‘Eiken’ Rota (Dipstick) showed the highest sensitivity out of the seven RAD kits against all selected strains in limited dilution analyses, which was consistent with the results for equine rotavirus previously reported. RT-PCR had 100–103-fold higher sensitivity than the Dipstick. NGS using thirteen RT-PCR-negative fecal samples revealed that all samples yielded RVA reads and especially that two of them covered all 11 genome segments. Moreover, mapping reads to reference sequences allowed genotyping. The NGS would be sensitive and useful for analysis of less dependent on specific primers and screening of genotypes.

Japanese eel endothelial cell-infecting virus (JEECV) causes viral endothelial cell necrosis of eel (VECNE), resulting in severe economic losses in eel aquaculture in Japan. Here, we report the complete genome sequences of two new JEECV strains isolated from farmed Japanese eels.

Japanese eel endothelial cells-infecting virus (JEECV) has spread in eel farms and caused serious economic loss. In this study, we examined the prevalence of JEECV infection in 100 wild Japanese eel (Anguilla japonica) elvers caught from Yamaguchi prefecture, Japan, using quantitative PCR and conventional PCR. Total genomic DNA was obtained from the cranial quarter of the body in 70 of 100 eels and from the gill in the remaining. Of 30 gill samples, 20 were analyzed after pooling with other samples, and the remaining 10 were analyzed separately. A single positive result for JEECV was detected following analysis of the 10 separately analyzed samples. This result constitutes the first report of JEECV infection in wild A. japonica elvers.

Highlights • Novel viruses were identified in feces from cattle by metagenomics approach. • These viruses had a standard picornavirus genome organization. • They are related to unclassified Chinese picornaviruses from bat, cat and dog. • These viruses were detected in 23% (20/87) feces from cattle in Hokkaido in Japan., We identified novel viruses in feces from cattle with diarrhea collected in 2009 in Hokkaido Prefecture, Japan, by using a metagenomics approach and determined the (near) complete sequences of the virus. Sequence analyses revealed that they had a standard picornavirus genome organization, i.e. 5′ untranslated region (UTR) - L- P1 (VP4- VP3- VP2- VP1) - P2 (2A- 2B- 2C) - P3 (3A- 3B- 3C-3D) - 3′UTR- poly(A). They are closely related to other unclassified Chinese picornaviruses; bat picornaviruses group 1–3, feline picornavirus, and canine picornavirus, sharing 45.4–51.4% (P1), 38.0–44.9% (P2), and 49.6–53.3% (P3) amino acid identities, respectively. The phylogenetic analyses and detailed genome characterization showed that they, together with the unclassified Chinese picornaviruses, grouped as a cluster for the P1, 2C, 3CD and VP1 coding regions. These viruses had conserved features (e.g. predicted protein cleavage sites, presence of a leader protein, 2A, 2C, 3C, and 3D functional domains), suggesting they have a common ancestor. Reverse-transcription-PCR assays, using specific primers designed from the 5′UTR sequence of these viruses, showed that 23.0% (20/87) of fecal samples from cattle with diarrhea were positive, indicating the prevalence of these picornavirus in the Japanese cattle population in Hokkaido Prefecture. However, further studies are needed to investigate the pathogenic potential and etiological role of these viruses in cattle.

A viral metagenomics approach was used to investigate fecal samples of Japanese calves with and without diarrhea. Of the different viral pathogens detected, read counts gave nearly complete astrovirus-related RNA sequences in 15 of the 146 fecal samples collected in three distinct areas (Hokkaido, Ishikawa, and Kagoshima Prefectures) between 2009 and 2015. Due to the lack of genetic information about bovine astroviruses (BoAstVs) in Japan, these sequences were analyzed in this study. Nine of the 15 Japanese BoAstVs were closely related to Chinese BoAstVs and clustered into a lineage (tentatively named lineage 1) in all phylogenetic trees. Three of 15 strains were phylogenetically separate from lineage 1, showing low sequence identities, and clustered instead with an American strain isolated from cattle with respiratory disease (tentatively named lineage 2). Interestingly, two of 15 strains clustered with lineage 1 in the open reading frame (ORF)1a and ORF1b regions, while they clustered with lineage 2 in the ORF2 region. Remarkably, one of 15 strains exhibited low amino acid sequence similarity to other BoAstVs and was clustered separately with porcine astrovirus type 5 in all trees, and ovine astrovirus in the ORF2 region, suggesting past interspecies transmission.

Molecular mechanisms underlying lipolysis, as defined by mobilization of fatty acids from adipose tissue, are not fully understood. A database search for enzymes with α/β hydrolase folds, the GXSXG motif for serine esterase and the His-Gly dipeptide motif, has provided a previously unannotated gene that is induced during 3T3-L1 adipocytic differentiation. Because of its remarkable structural resemblance to triacylglycerol hydrolase (TGH) with 70.4% identity, we have tentatively designated this enzyme as TGH-2 and the original TGH as TGH-1. TGH-2 is also similar to TGH-1 in terms of tissue distribution, subcellular localization, substrate specificity, and regulation. Both enzymes are predominantly expressed in liver, adipose tissue, and kidney. In adipocytes, they are localized in microsome and fatcake. Both enzymes hydrolyzed p-nitophenyl butyrate, triolein, and monoolein but not diolein, cholesteryl oleate, or phospholipids; hydrolysis of short-chain fatty acid ester was 30,000-fold more efficient than that of long-chain fatty acid triacylglycerol. Fasting increased the expression of both genes in white adipose tissue, whereas refeeding suppressed their expression. RNA silencing of TGH-2 reduced isoproterenol-stimulated glycerol release by 10% in 3T3-L1 adipocytes, while its overexpression increased the glycerol release by 20%. Thus, TGH-2 may make a contribution to adipocyte lipolysis during period of increased energy demand.

Sterol regulatory element-binding proteins (SREBPs) are transcription factors that are predominately involved in the regulation of lipogenic and cholesterogenic enzyme gene expression. To identify unknown proteins that interact with SREBP, we screened nuclear extract proteins with 35S-labeled SREBP-1 bait in Far Western blotting analysis. Using this approach, high mobility group protein-B1 (HMGB1), a chromosomal protein, was identified as a novel SREBP interacting protein. In vitro glutathione S-transferase pull-down and in vivo coimmunoprecipitation studies confirmed an interaction between HMGB1 and both SREBP-1 and -2. The protein-protein interaction was mediated through the helix-loop-helix domain of SREBP-1, residues 309-344, and the A box of HMGB1. Furthermore, an electrophoretic mobility shift assay demonstrated that HMGB1 enhances SREBPs binding to their cognate DNA sequences. Moreover, luciferase reporter analyses, including RNA interference technique showed that HMGB1 potentiates the transcriptional activities of SREBP in cultured cells. These findings raise the intriguing possibility that HMGB1 is potentially involved in the regulation of lipogenic and cholesterogenic gene transcription.

目的: 下顎前歯部舌側歯槽面においては, 部分床義歯の義歯床および大連結子をいかに設計するかが問題となる.本実験では同部の血流動態に着目し, 安静時の状態および義歯を設置した状態を想定した加圧時の状態を, レーザードップラー血流計を用いて測定した.方法: 測定には特製のスプリントを使用した.測定点は左右犬歯間の舌側歯槽面に設定した18点で, 同部の無圧状態の安静時血流量と加圧時血流量とを測定した.加圧に関しては歯槽面を上下的に3分割し, 3部位すべてを加圧する全加圧, 上部だけ加圧する上部加圧, 下方2部位を加圧する下方加圧, 下部だけを加圧する下部加圧の4条件とした.また, 同時に測定点の粘膜厚径を, 超音波粘膜厚径測定器を用いて測定した.結果: 舌側歯槽面の安静時血流量は, 歯種別では差は認められなかったが, 高低別では差がみられ, 口腔底部は歯肉縁部の約1.8倍の血流量であり, 底部になるほど血流量は増加した.加圧時血流量は, 測定部位間で下部はほかの加圧部位と比較して血流量が多かった.加圧条件では, 下部加圧と全加圧で差が認められ, 下部加圧ではすべての部位で血流量が増加し, 全加圧では下部のみ増加した.舌側歯槽面の平均粘膜厚径は0.83mmであった.結論: 下顎前歯部歯槽面は血流の観点からみると, 安静時は近遠心的には差がなく, 高低別では下部ほど血流量が多く, 加圧時には上部のほうが下部に比べて影響を受けやすい.

目的: 義歯床下粘膜の健康状態の指標を得ることを最終的な目的とし, 義歯に機能力が加わった場合, 義歯床下粘膜の血流量の実態を確認するために, 義歯装着時の血流量を計測, 検討した.方法: 上顎無歯顎者10名と, 同年代の上顎有歯顎者5名を被験者として, 口蓋粘膜の29点における血流量を2台のレーザードップラー血流計を用いて測定した. 各測定点を解剖学的形状に基づいて, 無歯顎者においては, 顎堤頂臼歯部, 顎堤頂前歯部, 口蓋正中部, 口蓋前方部, 口蓋側方斜面部の5部位に分けて, それぞれの血流量を比較検討した.結果: 1. 安静時血流量の日差に関しては, 無歯顎者, 有歯顎者ともに, 良好な再現性がみられた. 2. 同年代の無歯顎者と有歯顎者の安静時血流量に有意な差は認められなかった. 3. 無歯顎者の安静時血流量は, 顎堤頂臼歯部において最も少なかった. 4. 無歯顎者の血流量は, 測定点すべてにおいて加圧により減少する傾向がみられた. 5. 粘膜加圧時の血流量変化率は, 口蓋部に比較して顎堤頂部でより著明であった.結論: 義歯床下粘膜の血流動態は, 各部の解剖学的特徴に対応し, また義歯機能時を想定した加圧時の力学的反応として, 合理的な変化を示すことが確認されたことから, 今後, より詳細な検討を加えることで, 各種臨床術式の判定や粘膜部の健康状態を評価する指標の1つとして利用できる可能性が示唆された.

During group A rotavirus (RVA) surveillance of pig farms in Japan, we detected three RVA strains (G4P[6], G5P[7], and G9P[23] genotypes), which showed super-short RNA patterns by polyacrylamide gel electrophoresis, in samples from a healthy eight-day-old pig and two pigs of seven and eight days old with diarrhea from three farms. Reverse transcription PCR and sequencing revealed that the full-length NSP5 gene of these strains contained 952 or 945 nucleotides, which is consistent with their super-short electropherotypes. Due to a lack of whole genome data on Japanese porcine RVAs, we performed whole genomic analyses of the three strains. The genomic segments of these RVA strains showed typical porcine RVA constellations, except for H2 NSP5 genotype, (G4,5,9-P[6,7,23]-I5-R1-C1-M1-A8-N1-T1-E1-H2 representing VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5 genes). In phylogenetic analyses, these porcine RVA strains clustered with porcine and porcine-like human RVA strains and showed a typical porcine RVA backbone, except for the NSP5 gene; however, intra-genotype reassortment events among porcine and porcine-like human RVA strains were observed. The NSP5 gene segments of these strains were clustered within the H2b genotype with super-short human RVA strains. The H2 genotype has to date only been identified in human and lapine RVA strains. Thus, to our knowledge, this report presents the first case of H2 NSP5 genotype showing a super-short RNA pattern in porcine RVA. These data suggest the possibility of interspecies transmission between pigs and humans and imply that super-short porcine RVA strains possessing H2 genotype are circulating among both asymptomatic and diarrheic porcine populations in Japan.

Goat diarrheal feces were subjected to metagenome analysis by the next-generation sequencing. Nucleotide sequences with homology to enteroviruses were obtained. Primers for RT-PCR were designed based on the nucleotide sequence of these sequences at the 5'-untranslated region, and we determined 563 bp nucleotide sequences that showed homology to bovine-like and ovine enteroviruses (77-87 %). We named the virus detected in this study goat enterovirus G1 (GEV-G1). In the phylogenetic analysis, GEV-G1 belonged to a cluster containing ovine enteroviruses. To our knowledge, this is the first report on nucleotide sequences of an enterovirus infecting Japanese goats.

Recently, Avian bornavirus (ABV) was detected in proventricular dilatation disease (PDD) affected-birds and feather picking diseases affected-birds. However, the pathogenicity of ABV has not been thoroughly investigated. In this study, we surveyed ABV in pet birds in Japan. We found four ABV-infected birds among 93 pet birds using RT-PCR, and genotypes of the ABV were determined as ABV-2 and -4. Two of the birds positive for ABV-4 showed proventricular dilatation typically found in PDD, and chronic stomach disturbance, whereas two of the birds positive for ABV-2 showed unexplained behavioral problems that are tapping, autophagia, and cloaca prolapse.

Overexpression of sterol regulatory element-binding protein-1c (SREBP-1c) in β cells causes impaired insulin secretion and β cell dysfunction associated with diminished pancreatic duodenal homeodomain transcription factor-1 (PDX-1) expression in vitro and in vivo. To identify the molecular mechanism responsible for this effect, the mouse Pdx-1 gene promoter (2.7 kb) was analyzed in β cell and non-β cell lines. Despite no apparent sterol regulatory element-binding protein-binding sites, the Pdx-1 promoter was suppressed by SREBP-1c in β cells in a dose-dependent manner. PDX-1 activated its own promoter. The E-box (−104/−99 bp) in the proximal region, occupied by ubiquitously expressed upstream stimulatory factors (USFs), was crucial for the PDX-1-positive autoregulatory loop through direct PDX-1·USF binding. This positive feedback activation was a prerequisite for SREBP-1c suppression of the promoter in non-β cells. SREBP-1c and PDX-1 directly interact through basic helix-loop-helix and homeobox domains, respectively. This robust SREBP-1c·PDX-1 complex interferes with PDX-1·USF formation and inhibits the recruitment of PDX-1 coactivators. SREBP-1c also inhibits PDX-1 binding to the previously described PDX-1-binding site (−2721/−2646 bp) in the distal enhancer region of the Pdx-1 promoter. Endogenous up-regulation of SREBP-1c in INS-1 cells through the activation of liver X receptor and retinoid X receptor by 9-cis-retinoic acid and 22-hydroxycholesterol inhibited PDX-1 mRNA and protein expression. Conversely, SREBP-1c RNAi restored Pdx-1 mRNA and protein levels. Through these multiple mechanisms, SREBP-1c, when induced in a lipotoxic state, repressed PDX-1 expression contributing to the inhibition of insulin expression and β cell dysfunction.

Conventionally, fabricating a facial prosthesis requires complicated steps and sophisticated skills. Particularly, the facial impression can be uncomfortable for the patient and can cause compression because of the weight of the material. The new approach presented in this report could simplify the fabrication of facial prostheses using a noncontact three-dimensional digitizer and binder multinozzle inkjet printer, without computed tomography or making a conventional impression. Treatment time was reduced, and the patient expressed satisfaction after 6 months follow-up.

Traditional brewing of Fukuyama pot vinegar is a process that has been continued in Fukuyama, Kagoshima, Japan, for almost 200 years. The entire process proceeds from raw materials, including steamed rice, rice koji (steamed rice grown with a fungus, Aspergillus oryzae) and water, to produce vinegar in roughly capped large pots laid in the open air. No special fermentative manipulation is required, except for scattering dried rice koji (called furi-koji) on the surface of the mash to form a cap-like mat on the surface at the start of brewing. As the biochemical mechanism of the natural transition of the fermentative processes during brewing has not been fully explained, we conducted a microbiological and biochemical study on the transition. First, a distinct biochemical change was observed in the brewing of spring preparation; that is, a sharp decline in pH from 6.5 to 3.5 within the first 5 days of brewing was observed due to lactic acid fermentation. Alcoholic fermentation also proceeded with a sharp increase to 4.5% ethanol within the first 5 days under the acidic conditions, suggesting that saccharification and both fermentations proceed in parallel. Acidic conditions and ethanol accumulation restricted the growth of most microorganisms in the mash, and in turn provided a favorable growth condition for acetic acid bacteria which are acid resistant and "ethanol-philic." Acetic acid was detected from day 16 and gradually increased in concentration, reaching a maximum of 7% at day 70 that was maintained thereafter. Empirically furi-koji naturally sinks into the mash after around day 40 by an unknown mechanism, allowing acetic acid bacteria to easily form pellicles on the mash surface and promoting efficient acetic acid fermentation. Dominant microbial species involved in the three fermentations were identified by denaturing gradient gel electrophoresis analysis using PCR-amplified defined-regions of small rDNA from microorganisms in the brewing mash or colony direct PCR applied to isolated microorganisms from the mash.

Unstable lipid-rich plaques in atherosclerosis are characterized by the accumulation of macrophage foam cells loaded with cholesterol ester (CE). Although hormone-sensitive lipase and cholesteryl ester hydrolase (CEH) have been proposed to mediate the hydrolysis of CE in macrophages, circumstantial evidence suggests the presence of other enzymes with neutral cholesterol ester hydrolase (nCEH) activity. Here we show that the murine orthologue of KIAA1363, designated as neutral cholesterol ester hydrolase (NCEH), is a microsomal nCEH with high expression in murine and human macrophages. The effect of various concentrations of NaCl on its nCEH activity resembles that on endogenous nCEH activity of macrophages. RNA silencing of NCEH decreases nCEH activity at least by 50%; conversely, its overexpression inhibits the CE formation in macrophages. Immunohistochemistry reveals that NCEH is expressed in macrophage foam cells in atherosclerotic lesions. These data indicate that NCEH is responsible for a major part of nCEH activity in macrophages and may be a potential therapeutic target for the prevention of atherosclerosis.

Hormone-sensitive lipase (HSL) regulates the hydrolysis of acylglycerol and cholesteryl ester (CE) in var- ious organs, including adipose tissues. However, the hepatic expression level of HSL has been reported to be almost negligible. In the present study, we found that mice lack- ing both leptin and HSL (Lep ob/ob /HSL 2/2 ) showed mas- sive accumulation of CE in the liver compared with Lep ob/ob / HSL 1/1 mice, while triacylglycerol (TG) accumulation was modest. Similarly, feeding with a high-cholesterol diet in- duced hepatic CE accumulation in HSL 2/2 mice. Supporting these observations, we detected significant expression of pro- tein as well as mRNA of HSL in the liver. HSL 2/2 mice showed reduced activity of CE hydrolase, but not of TG li- pase, in the liver compared with wild-type mice. Furthermore, we confirmed the expression of HSL in viable parenchymal cells isolated from wild-type mice. The hepatocytes from HSL 2/2 mice showed reduced activity of CE hydrolase and containedmoreCEthanthosefromHSL 1/1 miceevenwithout the incubation with lipoproteins. Incubation with LDL further augmented the accumulation of CE in the HSL-deficient hepatocytes. From these results, we conclude that HSL is in- volved in the hydrolysis of CE in hepatocyes.—Sekiya, M., J-i. Osuga, N. Yahagi, H. Okazaki, Y. Tamura, M. Igarashi, S. Takase, K. Harada, S. Okazaki, Y. Iizuka, K. Ohashi, H. Yagyu, M. Okazaki, T. Gotoda, R. Nagai, T. Kadowaki, H. Shimano, N. Yamada, and S. Ishibashi. Hormone-sensitive lipase is involved in hepatic cholesteryl ester hydrolysis. J. Lipid Res. 2008. 49: 1829-1838.

PURPOSE The record block for maxillomandibular registration of the denture is absolutely necessary in order to decide the mandibular position. This record block must provide sufficient strength so as not to cause deformation in the oral cavity. It must also fit the alveolar ridge well in order to keep accurate registration. Since most of the conventional materials for this purpose are not sufficient, a new specific light-polymerized resin was experimentally developed for the record base. METHODS The mechanical strength and suitability of the new material were examined in comparison with a conventional autopolymerizing resin. RESULTS 1) The transverse strength was equivalent to that of the autopolymerizing resin. 2) Although the shear bond strength was equivalent to the autopolymerizing resin, the intensity was drastically improved by the application of composite primer. 3) The light-polymerized resin and the autopolymerizing resin did not show any difference in suitability, and it was a clinical value without problem. CONCLUSIONS From these findings, it was confirmed that the new light-polymerized resin had the physical property for clinical application as a material for the record base.

Squalene synthase (SS) is the first committed enzyme for cholesterol biosynthesis, located at a branch point in the mevalonate pathway. To examine the role of SS in the overall cholesterol metabolism, we transiently overexpressed mouse SS in the livers of mice using adenovirus-mediated gene transfer. Overexpression of SS increased de novo cholesterol biosynthesis with increased 3-hydroxy-3-methyglutaryl-CoA (HMG-CoA) reductase activity, in spite of the downregulation of its own mRNA expression. Furthermore, overexpression of SS increased plasma concentrations of LDL, irrespective of the presence of functional LDL receptor (LDLR). Thus, the hypercholesterolemia is primarily caused by increased hepatic production of cholesterol-rich VLDL, as demonstrated by the increases in plasma cholesterol levels after intravenous injection of Triton WR1339. mRNA expression of LDLR was decreased, suggesting that defective LDL clearance contributed to the development of hypercholesterolemia. Curiously, the liver was enlarged, with a larger number of Ki-67-positive cells. These results demonstrate that transient upregulation of SS stimulates cholesterol biosynthesis as well as lipoprotein production, providing the first in vivo evidence that SS plays a regulatory role in cholesterol metabolism through modulation of HMG-CoA reductase activity and cholesterol biosynthesis.

Obesity is a major health problem in industrialized societies, and fatty liver disease (hepatic steatosis) is common in obese individuals. Oxidative stress originating from increased intracellular levels of fatty acids has been implicated as a cause of hepatocellular injury in steatosis, although the precise mechanisms remain to be elucidated. p53, widely known as a tumor suppressor, has been shown often to be activated in stressed cells, inducing cell cycle arrest or death. Here we demonstrate that p53 is involved in the molecular mechanisms of hepatocellular injury associated with steatosis. We found that p53 in the nucleus is induced in the liver from two mouse models of fatty liver disease, ob/ob and a transgenic mouse model that overexpresses an active form of sterol regulatory element-binding protein-1 in the liver (TgSREBP-1), the one with obesity and the other without obesity. This activation of the p53 pathway leads to the elevation of p21 mRNA expression, which can be considered an indicator of p53 activity, because ob/ob mice lacking p53 generated by targeting gene disruption exhibited the complete restoration of the p21 elevation to wild type levels. Consistent with these results, the amelioration of hepatic steatosis caused by Srebp-1 gene disruption in ob/ob mice lowered the p21 expression in a triglyceride content-dependent manner. Moreover, p53 deficiency in ob/ob mice resulted in a marked improvement of plasma alanine aminotransferase levels, demonstrating that p53 is involved in the mechanisms of hepatocellular injury. In conclusion, we revealed that p53 plays an important role in the pathogenesis of fatty liver disease.

Effects of the clumping of bacterial spores on their heat resistance as a result of heat treatment were investigated. Spore suspensions of Bacillus cereus, Bacillus coagulans and Bacillus licheniformis were heated at 85 degrees C. Survivor curves of the three strains showed tailing in all treatments after 30 min. As the treatment time increased, the formation of spore clumps increased in all strains after 20 min. Relative hydrophobicity of the spore surface increased as a result of heat treatment. The effect of spore concentration on the inactivation of the B. licheniformis spores was investigated, and surviving curves showed no tailing below a concentration of 4.9 log CFU/ml.