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The detailed mechanism of the process during bone healing of drill-hole injury has been elucidated, but a crucial factor in regulating drill-hole healing has not been identified. The transcription factor p53 suppresses osteoblast differentiation through inhibition of osterix expression. In present study, we demonstrate the effects of p53 deficiency on the capacity of MSCs and osteoblasts during drill-hole healing.Mesenchymal stromal cells (MSCs) and osteoblasts were collected from bone marrow and calvaria of p53 knockout (KO) mice, respectively. The activities of cell mobility, cell proliferation, osteoblast differentiation, and wound healing of MSCs and/or osteoblasts were determined by in vitro experiments. In addition, bone healing of drill-hole injury in KO mice was examined by micro-CT and immunohistological analysis using anti-osterix, Runx2, and sclerostin antibodies.KO MSCs stimulated cell mobility, cell proliferation, and osteoblast differentiation. Likewise, KO osteoblasts enhanced cell proliferation and wound healing. KO MSCs and osteoblasts showed high potency in the inflammation and callus formation phases compared to those from wild-type (WT) mice. In addition, increased expression of osterix and Runx2 was observed in KO MSCs and osteoblasts that migrated in the drill-hole. Conversely, sclerostin expression was inhibited in KO mice. Eventually, KO mice exhibited high repairability of drill-hole injury, suggesting a novel role of p53 in MSCs and osteoblasts in improving bone healing.p53 Deficiency promotes bone healing of drill-hole injury by enhancing the bone-regenerative ability of MSCs and osteoblasts.

Corpora amylacea (CA) is usually present in benign prostatic ducts and acini, and its presence is considered suggestive of negative or low-risk prostate cancer. The clinicopathological definition of CA among prostate cancer cells (CAPCCs)-described as CA entirely surrounded by invasive cancer cells-has not been discussed. As intraductal carcinoma of the prostate (IDC-P) is a well-known adverse prognostic factor in prostate cancer, this study aimed to elucidate the relationship between CAPCC and IDC-P. We enrolled 366 patients who underwent robotic-assisted radical prostatectomies between 2012 and 2018 at Aichi Medical University Hospital. All surgical specimens were independently reviewed by two genitourinary pathologists. The median age of the patients was 68.5 years; the median serum prostate-specific antigen was 6.49 ng/mL. IDC-P was observed in 143 (39.1%) patients, while the presence of CAPCC was observed in 47 cases (12.8%). Patients with CAPCC were associated with more advanced clinical and pathological T stages, as well as Gleason scores, than those without CAPCC (p=0.018, p

Background Validation and standardization of methodologies for microbial community measurements by high-throughput sequencing are needed to support human microbiome research and its industrialization. This study set out to establish standards-based solutions to improve the accuracy and reproducibility of metagenomics-based microbiome profiling of human fecal samples. Results In the first phase, we performed a head-to-head comparison of a wide range of protocols for DNA extraction and sequencing library construction using defined mock communities, to identify performant protocols and pinpoint sources of inaccuracy in quantification. In the second phase, we validated performant protocols with respect to their variability of measurement results within a single laboratory (that is, intermediate precision) as well as interlaboratory transferability and reproducibility through an industry-based collaborative study. We further ascertained the performance of our recommended protocols in the context of a community-wide interlaboratory study (that is, the MOSAIC Standards Challenge). Finally, we defined performance metrics to provide best practice guidance for improving measurement consistency across methods and laboratories. Conclusions The validated protocols and methodological guidance for DNA extraction and library construction provided in this study expand current best practices for metagenomic analyses of human fecal microbiota. Uptake of our protocols and guidelines will improve the accuracy and comparability of metagenomics-based studies of the human microbiome, thereby facilitating development and commercialization of human microbiome-based products.

The programmed cell death 1 (PD1)/PD1 ligand (PD-L1) axis plays an important role in tumor cell escape from immune control and has been most extensively investigated for therapeutic purposes. However, PD-L1 immunohistochemistry is still not used widely for diagnosis. We review the diagnostic utility of PD-L1 (by clone SP142) immunohistochemistry in large-cell lymphomas, mainly consisting of classic Hodgkin lymphoma (CHL) and diffuse large B-cell lymphoma (DLBCL). Neoplastic PD-L1 (nPD-L1) expression on Hodgkin and Reed-Sternberg cells is well-established among prototypic CHL. Of note, EBV+ CHL often poses a challenge for differential diagnosis from peripheral T-cell lymphoma with EBV+ non-malignant large B-cells; their distinction is based on the lack of PD-L1 expression on large B-cells in the latter. The nPD-L1 expression further provides a good diagnostic consensus for CHL with primary extranodal disease conceivably characterized by a combined pathogenesis of immune escape of tumor cells and immunodeficiency. Compared with CHL, the nPD-L1 expression rate is much lower in DLBCL, highlighting some specific subgroups of intravascular large B-cell lymphoma, primary mediastinal large B-cell lymphoma, and EBV+ DLBCL. They consist of nPD-L1-positive and -negative subgroups, but their clinicopathological significance remains to be elucidated. Microenvironmental PD-L1 positivity on immune cells may be associated with a favorable prognosis in extranodal DLBCL. PD-L1 (by SP142) immunohistochemistry has helped us to understand the immune biology of lymphoid neoplasms possibly related by immune escape and/or immunodeficiency. However, knowledge of these issues remains limited and should be clarified for diagnostic consensus in the future.

The programmed death 1 (PD1)/PD1 ligand (PD-L1) axis plays an important role in the pathogenesis of Epstein-Barr virus-positive diffuse large B cell lymphoma, not otherwise specified (EBV+ DLBCL, NOS). Here, we describe PD-L1 expression by EBV+ DLBCL, NOS in order to evaluate its possible contribution to the pathogenesis of this tumor. The study included 57 cases of EBV+ DLBCL, NOS. The median patient age was 69 years and 95% (n = 54) were aged > 45. Extranodal lesions were present in 39 (69%) at initial diagnosis. PD-L1 expression (mAb SP142-positive staining) was present in more than 5% of tumor cells in only six cases (11%), in clear contrast to the 77% reported in cases aged under 45 years. Among the PD-L1+ cases, three were nodal lesions. All six PD-L1+ cases progressed in the 3 years after diagnosis and four of the six patients died of the disease within 2 years. PD-L1+ cases had significantly shorter PFS (P = 0.002) and relatively short OS (P = 0.26), compared with PD-L1− cases. EBV+ DLBCL, NOS in the elderly infrequently expressed PD-L1 and had poor prognosis. PD-L1 expression in EBV+ DLBCL, NOS of the elderly sheds light on the pathogenetic role of immune senescence.

Epstein-Barr virus (EBV) is prevalent among healthy individuals, and is implicated in numerous reactive and neoplastic processes in the immune system. The authors originally identified a series of senile or age-related EBV-associated B-cell lymphoproliferative disorders (LPD) bearing a resemblance to immunodeficiency-associated ones. These LPDs may be associated with immune senescence and are now incorporated into the revised 4th edition of 2017 WHO lymphoma classification as EBV-positive (EBV+) diffuse large B-cell lymphoma (DLBCL), not otherwise specified (NOS). These EBV+ B-cells often have a Hodgkin/Reed-Sternberg (HRS)-like appearance and are shared beyond the diagnostic categories of mature B-cell neoplasms, mature T-cell neoplasms, classic Hodgkin lymphoma, and immunodeficiency-associated LPD. In addition, peculiar new diseases, such as EBV+ mucocutaneous ulcer and EBV+ DLBCL affecting the young, were recognized. On the other hand, lymphoma classification is now evolving in accord with deeper understanding of the biology of programmed death ligand 1 (PD-L1). Assessing PD-L1 positivity by staining with the anti-PD-L1 monoclonal antibody SP142 provides new insight by discriminating between immune evasion and senescence or immunodeficiency. The aim of the present review is to briefly summarize the diagnostic use of immunostaining with SP142 in malignant lymphomas and/or LPDs that feature tumor and nonmalignant large B-cells harboring EBV.

Composite lymphoma is a well-known diagnostic entity exhibiting the synchronous occurrence of two or more distinct types of lymphomas in the same specimen. Here we report two patients, a 14-year-old female (Case 1) and a 45-year-old male (Case 2), with mediastinal composite lymphoma, comprising nodular sclerosis classic Hodgkin lymphoma (NSCHL) and primary mediastinal large B-cell lymphoma (PMBL). Both patients had a mediastinal mass, and manifested two different histologic components in the same biopsy, one characteristic of NSCHL and the other PMBL. The NSCHL areas included Hodgkin and Reed-Sternberg (HRS) cells with typical immunophenotypic features (CD30-positive and CD20-negative), whereas the sheets of large tumor cells characteristic of PMBL were strongly and uniformly CD20-positive. Interestingly, although both cases showed neoplastic PD-L1 (nPD-L1) positivity on the HRS cells of NSCHL, they differed regarding nPD-L1 expression on the PMBL tumor cells. In Case 1, the nPD-L1-negative PMBL component was anatomically situated outside the NSCHL lesion. On the other hand, in Case 2, the nPD-L1-positive PMBL component was characterized by transitional or continuous areas with the NSCHL component. These findings suggested that nPD-L1 expression may define two subtypes of PMBL that are more similar to or distinct from classic Hodgkin lymphoma.

Although several reports have highlighted neoplastic PD-L1 (nPD-L1) expression in classic Hodgkin lymphoma (CHL), some have addressed associations between its expression and detailed histopathologic features. Here we describe four cases of syncytial variant of CHL (SV-CHL), with and without Epstein-Barr virus (EBV) association, and highlight the diagnostic utility of PD-L1 (clone SP142) immunohistochemistry. The patients were a 61-year-old male, 45-year-old male, 85-year-old female, and 89-year-old female. All presented with cervical or axillary lymphadenopathy, which on biopsy had the established histopathologic features of SV-CHL with a biphasic pattern of cohesive sheets of large tumor cells and typically scattered distribution of Hodgkin and Reed-Stenberg (HRS) cells. These tumor cells showed identical immunophenotypic findings for CD15, CD30, Fascin, PAX5, OCT2, BOB1 and EBV harboring, regardless of location. The exception was absent or decreased expression of nPD-L1 from tumor cells in the confluent sheets, contrasting with HRS cell positivity in typical areas of CHL. These findings offer the first suggestion of possible downregulation of nPD-L1 expression in association with the histopathologic progression of CHL. The results may be relevant for recognizing 'confluent' sheets in the diagnostic workup for SV-CHL.

Bacillus Calmette-Guérin (BCG) instillation is the gold standard for the treatment of patients with pT1 bladder cancer but causes severe adverse effects. Few predictive factors have been established for intravesical recurrence and/or stage progression in bladder cancer. We analysed 138 patients who underwent transurethral resection of bladder tumour and were pathologically confirmed to have stage pT1 bladder cancer. Of these, 72 patients (52.2%) received intravesical BCG instillation, 12 patients (8.7%) demonstrated stage progression, and five patients (3.6%) died of the disease. The number of patients who received BCG instillation was more in the group with multifocal tumours than that in the group with unifocal tumours (p=0.0034). Among 53 patients (38.4%) who demonstrated cytokeratin 5/6 (CK5/6) expression, 15 patients (28.3%) showed CK5/6 expression in more than 10% of tumour cells and 38 patients (71.7%) showed CK5/6 expression in 1-10% of tumour cells. CK5/6 expression was observed in both invasive and non-invasive components in 15 patients (28.3%), only in invasive components in 30 patients (56.6%), and only in non-invasive components in eight patients (15.1%). Furthermore, CK5/6 expression was observed in tumour cells only in front of the invasive component and stroma in 24 patients. The proportion of CK5/6-expressing tumour cells in the invasive component was significantly higher than that in the non-invasive component (p0.001). The follow-up period for patients who received BCG tended to be shorter than that in the non-BCG patients. The CK5/6-positive group displayed significantly shorter recurrence-free survival (RFS) than the CK5/6-negative group (p=0.0412). Importantly, CK5/6 expression was a significant predictive factor of inferior RFS in the BCG instillation group (p=0.0197). In contrast, CK5/6 expression was not significantly associated with RFS in the non-BCG instillation group (p=0.841). Thus, CK5/6 expression can be a predictive marker for RFS in patients with pT1 bladder cancer and can provide critical information for patient care.

It is well known that recipients of kidney transplants are at an increased risk of developing malignant or premalignant cutaneous neoplasms (MPCNs) after transplantation. However, the pathogenesis of MPCNs after kidney transplant has not been well-studied in Asian populations. This study aimed to describe the clinicopathologiccharacteristics of MPCNs in an Asian population. We retrospectively reviewed the medical records of 1956 patients who received kidney transplants at two hospitals in Japan, between 2003 and 2019. Among these patients, 24 developed 50 MPCN lesions, including 14 squamous cell carcinoma (SCC, 28%), 23 Bowen’s disease (BD, 46%), 11 actinic keratosis (AK, 22%), and two basal cell carcinoma (BCC, 4%). No patient had malignant melanoma. The duration from transplantation to the diagnosis was significantly longer for SCC than for BD or AK (P=0.021, 0.036, respectively). Seven patients had multiple MPCNs in sun-exposed areas of skin. Among the 50 MPCNs, 40 (80%) were located in sun-exposed areas, and 10 (20%) were located in sun-protected areas. MPCNs in sun-exposed skin were frequently accompanied by dermal solar elastosis (90%, 36/40). We found high-risk human papillomavirus (HR-HPV) infections in two anogenital lesions (100%, 2/2). In contrast, HR-HPV infections were not detected in any extragenital lesions (0%, 0/30). Our results suggested that, among Japanese recipients of kidney transplant, MPCNs in sun-exposed skin areas may be associated with immunosuppression and ultraviolet exposure.

Serotonin (5-HT) in the central nervous system regulates a variety of biological functions, from the basic homeostatic control to higher brain functions, by acting on fourteen known receptor subtypes. However, it is still usually unclear which receptor subtype is responsible for a specific function due to the lack of highly selective ligands for most of these receptors. Although 5-HT receptor knockout mice are useful, the brain-wide distribution of various receptors makes it difficult to dissect receptor functions in specific and brain regions and cell types. Recent advances in CRISPR/Cas9-mediated in vivo genome editing technology may overcome this problem. In this study, we constructed a viral vector expressing a single guide (sg)RNA targeting Htr1a (sgHtr1a) and Cre recombinase under the control of a neuron-specific promoter. Injection of the viral vector into the dorsal raphe nucleus (DRN) of Cre-dependent Cas9 knock-in mice induced Cre-dependent Cas9 expression mainly in DRN serotonin and GABA neurons. Mismatch cleavage assay and Sanger sequencing showed insertion or deletion formation at the target site. 5-HT1A receptor agonist-induced hypothermia was attenuated and antidepressant effect of a selective serotonin reuptake inhibitor (SSRI) was enhanced by microinjection of the viral vector expressing sgHtr1a into the DRN of Cre-dependent Cas9 knock-in mice. These results suggest that this in vivo CRISPR/Cas9-mediated 5-HT receptor gene knockout strategy provides a reliable and low-cost method for elucidating 5-HT receptor functions in specific cell types and brain regions. Further, we demonstrate that the neuronal 5-HT1A receptor in the DRN regulates body temperature and antidepressant effect of SSRI.

Brown adipose tissue (BAT) is a key organ that produces heat and dissipates energy, and may be clinically relevant to the treatment of obesity and diabetes. Tetrahydrobiopterin (BH4) is an essential co-factor of tyrosine hydroxylase, a rate-limiting enzyme of catecholamine biosynthesis including that of norepinephrine (NA) and nitric oxide (NO) synthase. Although both NA and NO are well-known factors in BAT differentiation, the role of BH4 in the differentiation of BAT is poorly understood. We investigated the role of BH4 in differentiation of BAT using a mouse model of BH4 deficiency, the Hph-1 mouse. Hph-1 neonatal mice exhibit dysplasia of BAT as well as attenuated thermogenesis-related gene expressions (UCP1, Dio2, Pgc1a) compared with control mice. As differentiation capacity is considered to be at maximum during late gestation, we administered BH4 to fetal Hph-1 mice via placental transfer using intraperitoneal injection to pregnant Hph-1 mice for 6 days just before birth. Maternal BH4 supplementation ameliorated dysplasia of neonatal BAT and restored thermogenesis-related genes expression. Intriguingly, offspring of the maternal BH4-supplementation group showed less weight gain, ameliorated glucose intolerance, and improved cold tolerance after growth under high fat chow diet. We then evaluated the direct effect of BH4 on brown adipocyte differentiation using brown pre-adipocytes isolated from control and Hph-1 mice. Compared with control mice, brown pre-adipocytes from Hph-1 mice showed reduced differentiation capacity, while BH4 supplementation in brown pre-adipocytes from Hph-1 mice ameliorated differentiation capacity nitric oxide-dependently. Taking these findings together, BH4 plays an integral role in the differentiation of BAT, especially in the fetal period, and may represent a novel target for prevention of metabolic disorders such as obesity and diabetes. Disclosure H. Minamino: None. Y. Fujita: None. Y. Oguri: None. T. Goto: None. A. Ohashi: None. F. Furuya: None. N. Isomura: None. T. Kohei: None. Y. Li: None. S. Kawarasaki: None. T. Kawada: None. H. Hasegawa: None. N. Inagaki: Research Support; Self; Astellas Pharma Inc., Drawbridge Health, Japan Tobacco Inc., Kissei Pharmaceutical Co., Ltd., Kyowa Hakko Kirin Co., Ltd., Life Scan Japan, Nippon Boehringer Ingelheim Co. Ltd., Novartis Pharma K.K., Novo Nordisk Pharma Ltd.,, Sanofi K.K., Sanwa Kagaku Kenkyusho, Terumo Medical Corporation. Speaker’s Bureau; Self; Astellas Pharma Inc., Merck & Co., Inc., Mitsubishi Tanabe Pharma Corporation, Nippon Boehringer Ingelheim Co. Ltd., Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited.

The programmed death 1 (PD1)/PD1 ligand (PD-L1) axis plays an important role in the pathogenesis of Epstein-Barr virus-positive diffuse large B cell lymphoma, not otherwise specified (EBV+ DLBCL, NOS). Here, we describe PD-L1 expression by EBV+ DLBCL, NOS in order to evaluate its possible contribution to the pathogenesis of this tumor. The study included 57 cases of EBV+ DLBCL, NOS. The median patient age was 69 years and 95% (n = 54) were aged 45. Extranodal lesions were present in 39 (69%) at initial diagnosis. PD-L1 expression (mAb SP142-positive staining) was present in more than 5% of tumor cells in only six cases (11%), in clear contrast to the 77% reported in cases aged under 45 years. Among the PD-L1+ cases, three were nodal lesions. All six PD-L1+ cases progressed in the 3 years after diagnosis and four of the six patients died of the disease within 2 years. PD-L1+ cases had significantly shorter PFS (P = 0.002) and relatively short OS (P = 0.26), compared with PD-L1- cases. EBV+ DLBCL, NOS in the elderly infrequently expressed PD-L1 and had poor prognosis. PD-L1 expression in EBV+ DLBCL, NOS of the elderly sheds light on the pathogenetic role of immune senescence.

We report the complete genome sequence of Flavonifractor plautii JCM 32125T (=VPI 0310T). The genome consists of a single circular chromosome of 3,985,392 bp (G+C content, 60.9%) and was predicted to contain 3 complete sets of rRNA genes, 63 tRNA genes, and 3,764 protein-coding sequences.

We report a complete genome sequence of Collinsella aerofaciens JCM 10188T (=VPI 1003T). The genome consists of a circular chromosome (2,428,218 bp with 60.6% G+C content) and two extrachromosomal elements. The genome was predicted to contain 5 sets of rRNA genes, 58 tRNA genes, and 2,079 protein-encoding sequences.

We announce the complete genome sequence of Megamonas funiformis JCM 14723T (YIT 11815T). The genome consists of a circular chromosome (2,522,577 bp, 31.5% G+C content) and a plasmid of 46,189 bp (29.4% G+C content). The genome was predicted to contain 6 rRNA operons, 53 tRNA genes, and 2,440 protein-coding sequences.

We report a complete genome sequence of Blautia producta JCM 1471T. The genome consists of a single circular chromosome of 6,197,116 bp with a G+C content of 45.7%. The genome was annotated as containing 5 complete sets of rRNA genes, 70 tRNA genes, and 5,516 protein-coding sequences.

Nuclear protein, lamin A, which is a component of inner membrane on nucleoplasm, plays a role in nuclear formation and cell differentiation. The expression of mutated lamin A, termed progerin, causes a rare genetic aging disorder, Hutchinson-Gilford progeria syndrome, which shows abnormal bone formation with the decrease in a number of osteoblasts and osteocytes. However, exact molecular mechanism how progerin exerts depressive effects on osteogenesis has not been fully understood. Here, we created mouse lamin A dC50 cDNA encoding progerin that lacks 50 amino acid residues at C-terminus, transfected it in mouse preosteoblast-like MC3T3-E1 cells, and examined the changes in osteoblast phenotype. When lamin A dC50-expressed cells were cultured with differentiation-inductive medium, alkaline phosphatase (ALP) activity and mRNA levels of major osteoblast markers, type I collagen (Col1), bone sialoprotein (BSP), dentine matrix protein 1 (DMP1), and Runx2 were significantly decreased, and no mineralized nodules were detected as seen in control cells expressing empty vector. In the culture with mineralization-inductive medium, mRNA levels of BSP, osteocalcin, DMP1, Runx2, and osterix were strongly decreased parallel with loss of mineralization in lamin A dC50-expressed cells, while mineralized nodules appear at 21 days in control cells. Furthermore, lamin A dC50 expression was depressed nuclear localization of β-catenin with the decrease of GSK-3β phosphorylation level. These results suggest that lamin A dC50 depresses osteoblast differentiation in both early and late stages, and it negatively regulates β-catenin activity interacting with GSK-3β in cytoplasm.

Lamin A/C is a component of the nuclear lamina, which is involved in cellular proliferation and differentiation. However, the mechanism by which lamin A regulates osteoblast differentiation is not well understood. In this study, we investigated lamin A/C expression during osteoblast differentiation in a preosteoblastic cell line, MC3T3-E1. Real-time PCR analysis showed that lamin A/C mRNA expression was upregulated during BMP-2 induced osteoblast differentiation. Treatment with the estrogen receptor antagonist, fulvestrant, inhibited osteoblast differentiation and the upregulation of lamin A/C mRNA and protein expressions in the presence of BMP-2. These results clearly demonstrated that lamin A/C expression correlates with osteoblast differentiation. To determine the roles of lamin A expression in osteoblast differentiation, MC3T3-E1 cells were transfected with a vector overexpressing lamin A. Results showed that lamin A overexpression promoted osteoblast differentiation and calcification by inducing the expression of alkaline phosphatase, type 1 collagen, BSP, osteocalcin, and DMP-1 in the presence of BMP-2. Furthermore, lamin A overexpression partially restored osteoblastic capacity in the presence of fulvestrant by increasing the expression of BSP, osteocalcin, and DMP-1. These results suggest that lamin A plays important roles in maintaining the osteoblast differentiation and function.

Tetrahydrobiopterin (BH4) is a common coenzyme of phenylalanine-, tyrosine-, and tryptophan hydroxylases, alkylglycerol monooxygenase, and NO synthases (NOS). Synthetic BH4 is used medicinally for BH4-responsive phenylketonuria and inherited BH4 deficiency. BH4 supplementation has also drawn attention as a therapy for various NOS-related cardio-vascular diseases, but its use has met with limited success in decreasing BH2, the oxidized form of BH4. An increase in the BH2/BH4 ratio leads to NOS dysfunction. Previous studies revealed that BH4 supplementation caused a rapid urinary loss of BH4 accompanied by an increase in the blood BH2/BH4 ratio and an involvement of probenecid-sensitive but unknown transporters was strongly suggested in these processes. Here we show that OAT1 and OAT3 enabled cells to take up BP (BH4 and/or BH2) in a probenecid-sensitive manner using rat kidney slices and transporter-expressing cell systems, LLC-PK1 cells and Xenopus oocytes. Both OAT1 and OAT3 preferred BH2 and sepiapterin as their substrate roughly 5- to 10-fold more than BH4. Administration of probenecid acutely reduced the urinary exclusion of endogenous BP accompanied by a rise in blood BP in vivo. These results indicated that OAT1 and OAT3 played crucial roles: (1) in determining baseline levels of blood BP by excluding endogenous BP through the urine, (2) in the rapid distribution to organs of exogenous BH4 and the exclusion to urine of a BH4 excess, particularly when BH4 was administered, and (3) in scavenging blood BH2 by cellular uptake as the gateway to the salvage pathway of BH4, which reduces BH2 back to BH4.

Brown adipose tissue (BAT) is a key organ that produces heat and increases energy expenditure; its regulatory factors could be clinically useful in the treatment of obesity and diabetes. Tetrahydrobiopterin (BH4) is an essential co-factor of tyrosine hydroxylase and nitric oxide synthase. Our group recently showed that BH4 activates BAT function and regulates systemic energy metabolism using Hph-1 mice, a mouse model of BH4 deficiency in which GTP-cyclohydrolase I (GTPCH I), a rate-limiting enzyme of BH4, synthesis is deficient. Interestingly, we found that dysplasia of BAT already has been confirmed in the neonatal period. We investigated the role of BH4 in the development and differentiation of BAT using the mouse model of BH4 deficiency. Compared with control mice, Hph-1 neonatal mice exhibited lower weight of BAT as well as attenuated thermogenesis-related gene expressions (UCP1, Dio2, Pgc1a). We also evaluated the role of BH4 in brown adipocyte differentiation using brown pre-adipocytes isolated from control and Hph-1 mice. Compared with control mice, brown pre-adipocytes from Hph-1 mice reduced differentiation capacity measured by accumulated lipid droplets and thermogenesis-related gene expressions. In addition, inhibition of GTPCH I by 2,4-Diamino-6-Hydroxypyrimidine (DAHP) reduced differentiation capacity in brown pre-adipocytes. These data strongly suggest that BH4 is required for brown adipocyte differentiation. Furthermore, supplementation of BH4 in brown pre-adipocytes from Hph-1 mice dose-dependently increased accumulation of lipid droplets, thermogenesis-related gene expressions, and nitric oxide production. Taken together, BH4 plays an integral role in developmental differentiation of BAT, and has therapeutic potential for metabolic disorders such as obesity and diabetes. Disclosure H. Minamino: None. Y. Fujita: None. Y. Oguri: None. T. Goto: None. A. Ohashi: None. F. Furuya: None. N. Isomura: None. S. Kawarasaki: None. T. Kawada: None. H. Hasegawa: None. N. Inagaki: Research Support; Self; Astellas Pharma Inc., Boehringer Ingelheim Pharmaceuticals, Inc., Daiichi Sankyo Company, Limited, Japan Tobacco Inc., Kyowa Hakko Kirin Co., Ltd., Merck Sharp & Dohme Corp., Mitsubishi Tanabe Pharma Corporation, Novartis Pharmaceuticals Corporation, Ono Pharmaceutical Co., Ltd., Sanofi, Sumitomo Dainippon Pharma Co., Ltd., Takeda Pharmaceutical Company Limited.

Serotonin (5-HT) in the central nervous system regulates a variety of biological functions, from the basic homeostatic control to higher brain functions, by acting on fourteen known receptor subtypes. However, it is still usually unclear which receptor subtype is responsible for a specific function due to the lack of highly selective ligands for most of these receptors. Although 5-HT receptor knockout mice are useful, the brain-wide distribution of various receptors makes it difficult to dissect receptor functions in specific and brain regions and cell types. Recent advances in CRISPR/Cas9-mediated in vivo genome editing technology may overcome this problem. In this study, we constructed a viral vector expressing a single guide (sg)RNA targeting Htr1a (sgHtr1a) and Cre recombinase under the control of a neuron-specific promoter. Injection of the viral vector into the dorsal raphe nucleus (DRN) of Cre-dependent Cas9 knock-in mice induced Cre-dependent Cas9 expression mainly in DRN serotonin and GABA neurons. Mismatch cleavage assay and Sanger sequencing showed insertion or deletion formation at the target site. 5-HT

A novel, strictly anaerobic, short rod-shaped bacterium, designated strain TBC1T, was isolated from methanogenic granular sludge in a full-scale mesophilic upflow anaerobic sludge blanket reactor treating high-strength starch-based organic wastewater. Cells of this strain were 2–4 µm long and 0.4–0.6 µm wide. They were non-motile and Gram-stain-negative. The optimum growth temperature was 30–37 °C, with a range of 20–40 °C. The optimum pH for growth was around pH 7.0, while growth occurred in a range of pH 6.5–9.0. Strain TBC1T grew chemo-organotrophically on a narrow range of carbohydrates under anaerobic conditions. Yeast extract was required for its growth. The major fermentative end products from glucose, supplemented with yeast extract, were acetate, malate, propionate, formate and hydrogen. Doubling time under optimal growth conditions was estimated to be 1 day. The DNA G+C content of strain TBC1T was 49.2 mol% as determined by HPLC. Major cellular fatty acids were C16 : 0, C18 : 0, C16 : 1 ω9c and C18 : 1 ω9c. Based on its 16S rRNA gene sequence, strain TBC1T was shown to represent a distinct lineage at the family level in the phylum Bacteroidetes . Among previously described species of this phylum, Mucilaginibacter boryungensis BDR-9T ( Sphingobacteriaceae ) displayed the highest sequence similarity (85.9 %) with strain TBC1T. Phylogenomic analyses using 38–83 single copy marker genes also supported the novelty of strain TBC1T at the family level. Based on its characteristics, strain TBC1T (=JCM 30898T=DSM 100618T) is considered to be the type strain of a novel species of a new genus, Lentimicrobium saccharophilum gen. nov., sp. nov. A new family, Lentimicrobiaceae fam. nov., is also proposed encompassing the strain and related environmental 16S rRNA gene clone sequences.

A novel obligately anaerobic bacterium, designated strain TC1T, was isolated from methanogenic granular sludge in a full-scale mesophilic upflow anaerobic sludge blanket reactor treating high-strength starch-based wastewater. Cells had a multicellular filamentous morphology, stained Gram-negative and were non-motile. The filaments were flexible, generally >100 μm long and 0.3–0.4 μm wide. Growth of the isolate was observed at 25–43 °C (optimum 37 °C) and pH 6.0–8.5 (optimum pH 7.0). Strain TC1T grew chemo-organotrophically on a range of carbohydrates under anaerobic conditions. Yeast extract was required for growth. The major fermentative end products of glucose, supplemented with yeast extract, were acetate, lactate, succinate, propionate, formate and hydrogen. Co-cultivation with the hydrogenotrophic methanogen Methanospirillum hungatei DSM 864T enhanced growth of the isolate. The DNA G+C content was determined experimentally to be 42.1 mol%. The major cellular fatty acids were anteiso-C15 : 0, iso-C15 : 0 and iso-C17 : 0 3-OH. Based on 16S rRNA gene sequence analysis, strain TC1T belonged to the class Anaerolineae in the phylum Chloroflexi, in which Ornatilinea apprima P3M-1T was its closest phylogenetic relative (88.3 % nucleotide identity). Phylogenomic analyses using 38 and 83 single-copy marker genes also supported the novelty of strain TC1T at least at the genus level. Based on phylogenetic, genomic and phenotypic characteristics, we propose that strain TC1T represents a novel species of a new genus, for which we suggest the name Flexilinea flocculi gen. nov., sp. nov. The type strain of Flexilinea flocculi is strain TC1T ( = JCM 30897T = CGMCC 1.5202T).

Glucocorticoids (GCs) affect the proliferation of growth plate chondrocytes. In this study, we investigated the role of the GC-inducible promyelocytic leukemia zinc finger (PLZF) gene in chondrocyte differentiation by using the chondrogenic cell line ATDC5. PLZF overexpression suppressed cell cycle progression (p < 0.01) and promoted differentiation into hypertrophic chondrocytes by inducing mRNA expression of alkaline phosphatase (p < 0.01), and the cyclin-dependent kinase (CDK) inhibitor p21 (p < 0.01). In contrast, PLZF knockdown impaired differentiation into hypertrophic chondrocytes and promoted cell cycle progression (p < 0.01). Treatment with the GC analogue dexamethasone (10(-6) M) suppressed cell cycle progression in ATDC5 cells. PLZF shRNA attenuated dexamethasone-induced cell cycle arrest (p < 0.01) by downregulating the mRNA expression of the CDK inhibitors p21 and p57 (p < 0.01). These results clearly indicated that PLZF promoted differentiation into hypertrophic chondrocytes and mediated dexamethasone-induced cell cycle arrest by regulating CDK inhibitors.

Workflows for microbiome community profiling by high-throughput sequencing are prone to sample mix-ups and cross-contamination due to the complexity of the procedures and large number of samples typically analyzed in parallel. We employed synthetic 16S rRNA gene spike-in controls to establish a method for tracking of sample identity and detection of cross-contamination in microbiome community profiling assays based on 16S rRNA gene amplicon sequencing (16S-seq). Results demonstrated that combinatorial sample tracking mixes (STMs) can be reliably resolved by Illumina sequencing and faithfully represent their sample of origin. In a single-blinded experiment, addition of STMs at low levels was shown to be sufficient to unambiguously identify and resolve swapped samples. Using artificial admixtures of individually SMT-tagged samples, we further established the ability to detect and quantify cross-contamination down to a level of approximately 1%. The utility of our technique was underscored through detection of an unplanned case of cross-contamination that occurred during this study. By enabling detection of sample mix-ups and cross-contamination throughout 16S-seq workflows, the present technique thus assures provenance of sequence data on a per-sample basis. The method can be readily implemented in standard 16S-seq workflows and its routine application is expected to enhance the reliability of 16S-seq data.

The ocular surface is exposed to many chronic inflammatory stimuli, and goblet cell hyperplasia of the conjunctiva occurs in many situations. We report two cases of epithelial goblet cell hyperplasia with nonspecific chronic inflammation which occurred on the internal canthus of elderly people. These cases shared the same clinicopathological features that mimicked neoplastic lesion macroscopically, but are composed of nonspecific inflammatory changes pathologically. Immunostaining of the tissue showed few IgG4+ plasma cells, and no neoplastic changes were observed. Both cases arose in elderly patients over the age of 80 years. Pathogenesis and clinical significance of the lesion is unclear, but it might be age related. Recognition of this diagnosis might help us avoid overdiagnosis of malignancy and to reach the correct diagnosis.

We report here a high-quality draft genome sequence of Terrimicrobium sacchariphilum strain NM-5T, a facultative anaerobic, mesophilic, fermentative bacterium belonging to the class Spartobacteria of the phylum Verrucomicrobia. The genome comprises 4,751,807 bp in three contigs and has a G+C content of 60.19%. Annotation predicted 4,175 protein-coding sequences and 54 RNAs.

We report here a high-quality draft genome sequence of Terrimicrobium sacchariphilum strain NM-5 T , a facultative anaerobic, mesophilic, fermentative bacterium belonging to the class Spartobacteria of the phylum Verrucomicrobia . The genome comprises 4,751,807 bp in three contigs and has a G+C content of 60.19%. Annotation predicted 4,175 protein-coding sequences and 54 RNAs.

OBJECTIVE: The purpose of this study was to explore the experiences of municipal public health nurses in the wake of the March 2011 massive earthquake and tsunami and resulting nuclear accident in Fukushima, Japan, from the time of the disaster until December 2013. DESIGN AND SAMPLE: Thirty-two public health nurses working in three cities in Fukushima prefecture were divided into four focus groups and took part in interviews, which were analyzed using a qualitative descriptive method. RESULTS: Two major themes were extracted: (1) experiences of difficulties and dilemmas, and (2) professional challenges and the meaning of excellence as a public health nurse. Subjects recounted their experiences based on the timeline of events. The process of overcoming various dilemmas-between prescribed roles and actual needs on the ground, being both civil servants and private citizens with families, and having to be publicly accountable while lacking adequate information-caused participants to reexamine the meaning of excellence in the practice of public health. CONCLUSION: The strenuous and complex demands of extended disaster management caused subjects to grow professionally. Helping them process their emotions should also help these nurses give focus to their posttraumatic growth, and strengthen their sense of professionalism. Language: en

Endothelial nitric oxide synthase (eNOS) dysfunction induces insulin resistance and glucose intolerance. Tetrahydrobiopterin (BH4) is an essential cofactor of eNOS that regulates eNOS activity. In the diabetic state, BH4 is oxidized to 7,8-dihydrobiopterin, which leads to eNOS dysfunction owing to eNOS uncoupling. The current study investigates the effects of BH4 on glucose metabolism and insulin sensitivity in diabetic mice. Single administration of BH4 lowered fasting blood glucose levels in wild-type mice with streptozotocin (STZ)-induced diabetes and alleviated eNOS dysfunction by increasing eNOS dimerization in the liver of these mice. Liver has a critical role in glucose-lowering effects of BH4 through suppression of hepatic gluconeogenesis. BH4 activated AMP kinase (AMPK), and the suppressing effect of BH4 on gluconeogenesis was AMPK-dependent. In addition, the glucose-lowering effect and activation of AMPK by BH4 did not appear in mice with STZ-induced diabetes lacking eNOS. Consecutive administration of BH4 in ob/ob mice ameliorated glucose intolerance and insulin resistance. Taken together, BH4 suppresses hepatic gluconeogenesis in an eNOS-dependent manner, and BH4 has a glucose-lowering effect as well as an insulin-sensitizing effect in diabetic mice. BH4 has potential in the treatment of type 2 diabetes.

Aims Epstein–Barr virus (EBV)-positive diffuse large B-cell lymphoma (DLBCL) not otherwise specified is defined as monoclonal EBV+ B-cell proliferation affecting patients without any known immunosuppression. Non-neoplastic EBV+ cells proliferating in or adjacent to EBV− DLBCL were reported recently, but their clinical significance is unclear. Thus, the aim of this study was to investigate the prognostic impact of EBV+ cells in DLBCL. Methods and results We compared the clinicopathological characteristics of 30 EBV+ DLBCL patients and 29 and 604 EBV− DLBCL patients with and without EBV+ bystander cells (median age of onset 71, 67 and 62 years, respectively). Both EBV+ DLBCL patients and EBV− DLBCL patients with EBV+ bystander cells tended to have high and high–intermediate International Prognostic Index scores (60% and 59%, respectively), as compared with only 46% of EBV− DLBCL patients without EBV+ bystander cells. EBV− DLBCL patients with EBV+ bystander cells showed a significantly higher incidence of lung involvement than those without EBV+ bystander cells (10% versus 2%, P < 0.05). Furthermore, EBV+ DLBCL patients and EBV− DLBCL patients with EBV+ bystander cells had a poorer prognosis than patients without any detectable EBV+ cells [median overall survival (OS) of 100 months and 40 months versus not reached, P < 0.01]. Notably, EBV+ DLBCL patients and EBV− DLBCL patients with EBV+ bystander cells treated with rituximab showed overlapping survival curves (OS, P = 0.77; progression-free survival, P = 1.0). Conclusions EBV− DLBCL with bystander EBV+ cells has similar clinical characteristics to EBV+ DLBCL. DLBCL with EBV+ bystander cells may be related to both age-related and microenvironment-related immunological deterioration.

We report here a high-quality draft genome sequence of the type strain (JL) of Tepidanaerobacter syntrophicus , an obligately anaerobic and moderately thermophilic bacterium, which is able to perform syntrophic lactate degradation with hydrogenotrophic methanogens. The genome comprises 2.43 Mb in 9 scaffolds, with a G+C content of 38.6%.

Tetrahydrobiopterin (BH 4 ) is synthesized de novo in particular cells, but in the case of a systemic or local BH 4 deficiency, BH 4 supplementation therapy is applied. BH 4 -responsive PKU has also been effectively treated with BH 4 supplementation. However, the rapid clearance of the supplemented BH 4 has prevented the therapy from being widely accepted. Deposition of BH 4 after supplementation involves oxidation of BH 4 to dihydrobiopterin (BH 2 ) and subsequent conversion to BH 4 by the salvage pathway. This pathway is known to be almost ubiquitous in the body. However, the mechanism for the redistribution and exclusion of BH 4 across the plasma membrane remains unclear. The aim of this work was to search for the key transporter of the uptake precursor of the salvage pathway. Based on the observed sensitivity of pterin transport to nitrobenzylthioinosine (NBMPR), we examined the ability of ENT1 and ENT2, representative equilibrative nucleoside transporters, to transport sepiapterin (SP), BH 2 or BH 4 using HeLa cell and Xenopus oocyte expression systems. hENT2 was capable of transporting the pterins with an efficiency of SP>BH 2 >BH 4 . hENT1 could also transport the pterins but less efficiently. Non-transfected HeLa cells and rat aortic endothelial cells were able to incorporate the pterins and accumulate BH 4 via uptake that is likely mediated by ENT2 (SP>BH 2 >BH 4 ). When exogenous BH 2 was given to mice, it was efficiently converted to BH 4 and its tissue deposition was similar to that of sepiapterin as reported (Sawabe et al., 2004). BH 4 deposition after BH 2 administration was influenced by prior treatment with NBMPR, suggesting that the distribution of the administered BH 2 was largely mediated by ENT2, although urinary excretion appeared to be managed by other mechanisms. The molecular basis of the transport of SP, BH 2 , and BH 4 across the plasma membrane has now been described for the first time: ENT2 is a transporter of these pterins and is a plausible gateway to the salvage pathway of BH 4 biosynthesis, at least under conditions of exogenous pterin supplementation. The significance of the gateway was discussed in terms of BH 2 uptake for BH 4 accumulation and the release for modifying the intracellular BH 2 /BH 4 ratio.

In treating hereditary deficiency of tetrahydrobiopterin (BH(4)), supplementation with BH(4) might be the ultimate choice of therapy. Oral administration of BH(4) has been believed to be inefficient owing to poor absorption of BH(4) in the intestine. In this study, we found a considerable amount of BH(4) as well as its oxidized pterins in the ingredients of intestinal lumen of mice when they were served food that did not contain significant amounts of biopterin. Ligation of the biliary duct led to significant decrease in luminal biopterin. Supplementation of BH(4) either by intraperitoneal administration of sepiapterin or of 6RBH(4) ((6R)-L-erythro-5,6,7,8-tetrahydrobiopterin) increased the BH(4) content in the intestinal lumen with a slight delay after the rise of blood BH(4). In these mice, biopterin appeared in the large intestine, caecum and colon, 2 h after the administration. The appearance of BH(4) in the large intestine was accompanied by a large amount of pterin (2-amino-4-hydroxypteridine). The amounts of biopterin + pterin that appeared in the large intestine after intraperitoneal administration of BH(4) were not greater than those found after oral administration at the same dose. When the mice were treated with a large dose of antibiotics prior to the BH(4) administration, the amount of biopterin increased in the caecum but the amount of pterin decreased greatly. These results suggested that a large proportion of BH(4) administered moved to the large intestine, where most biopterin was decomposed presumably by enteric bacteria. Nonetheless, most of the orally administered biopterin was taken up by the small intestine and the amount of biopterin reaching the large intestine was almost the same as that which appeared after direct injection of 6RBH(4) into the peritoneal cavity.

Monoamines exert diverse functions in various cells in peripheral organs as well as in the central nervous system. 5-Hydroxy-l-tryptophan (5-HTP) has been simply regarded as a precursor of serotonin, and it is believed that the biological significance of 5-HTP is essentially ascribable to the production of serotonin. Systemic treatment with 5-HTP is often applied to patients with low serotonin levels in the brain. Here we show that endogenous and exogenous 5-HTP but not serotonin induced the development of microvilli in the gut villi epithelium. In contrast, serotonin but not 5-HTP regulated phagocytosis by macrophages. 5-HTP specifically induced actin remodeling and decreased phosphorylation of extracellular signal-regulated kinase (ERK) in the gut, whereas serotonin stimulated actin remodeling and increased ERK phosphorylation in macrophages. Functionally, inhibition of ERK activity promoted the development of microvilli in the gut and ameliorated phagocytosis by macrophages. Thus, 5-HTP and serotonin contribute to distinct cell-type-specific functions via common mediators. Our study might create an opportunity to explore the effects of exogenously applied 5-HTP in humans.

We generated a high-quality draft genome sequence of Bacteroidales strain 6E, a strict anaerobe newly isolated from Japanese rice paddy field soil. The genome consists of 61 contigs, with a total size of 4,436,542 bp and mean G+C content of 45.4%. Annotation predicted 3,620 protein-coding and 54 RNA genes.

Members of the class Anaerolineae in the bacterial phylum Chloroflexi are widespread in a range of ecosystems but remain poorly understood. We present here the draft genome sequences of the type strains of five Anaerolineae species, Anaerolinea thermolimosa IMO-1, Bellilinea caldifistulae GOMI-1, Leptolinea tardivitalis YMTK-2, Levilinea saccharolytica KIBI-1, and Longilinea arvoryzae KOME-1.

We report the draft genome sequence of Anaerolineae bacterium strain TC1, newly isolated from a methanogenic wastewater treatment system. The assembly contains 16 contigs in 3 scaffolds representing 3,510,630 bp in total with a G+C content of 41.35%. The genome is predicted to contain 2,793 protein-coding genes and 56 RNAs.

Glucocorticoids (GCs) regulate proliferation and differentiation in cultured mesenchymal cells through the modulation of various molecules. However, the relationship between growth factor signaling and GCs in differentiating chondrocytes has not been elucidated. In this study, we examined the effects of Wnt/β-catenin signaling on chondrocyte differentiation and the effects of a GC analogue, dexamethasone (Dex), on Wnt/β-catenin signaling activity by using a chondrocyte progenitor cell line ATDC5. Western blot analysis and TCF/LEF-optimized promoter EGFP (TOPEGFP) assay showed that both β-catenin protein levels and TCF/LEF transcription were up-regulated during insulin–transferrin–sodium selenite (ITS)-induced chondrogenic differentiation. Morphological analysis showed that TCF/LEF transcription activity was most prominent in cartilage nodule-like structures. Furthermore, a β-catenin mutant with constitutive transcriptional activity (ΔN90) showed increased Alcian blue staining intensity and mRNA expression of Sox9, Col2a, aggrecan, Col10, and alkaline phosphatase, even in the absence of ITS stimulation. In contrast, Dex suppressed formation of ITS-induced cartilage nodule-like structures, TCF/LEF-mediated transcription, and β-catenin protein levels. Real-time PCR analysis showed that Dex increased the mRNA expression levels of secreted frizzled-related protein 1 (sFRP1) and Axin2. Furthermore, treatment with a sFRP1 inhibitor or the ΔN90 β-catenin mutant transfection attenuated Dex-induced suppression of cartilage matrix production by increasing Sox9 mRNA levels. These results suggest that Dex inhibits chondrocyte differentiation via down-regulation of Wnt/β-catenin signaling, which promotes chondrocyte differentiation in ATDC5 cells.