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Background: This study investigated whether directly measured small dense low-density lipoprotein cholesterol (D-sdLDL-C) can predict long-term coronary artery disease (CAD) events compared with low-density lipoprotein cholesterol (LDL-C), non-high-density lipoprotein cholesterol (non-HDL-C), apolipoprotein B (apoB), and estimated small dense low-density lipoprotein cholesterol (E-sdLDL-C) determined by the Sampson equation in patients with stable CAD., Methods: D-sdLDL-C measured at Showa University between 2010 and 2022, and E-sdLDL-C were evaluated in 790 male and 244 female patients with stable CAD. CAD events, defined as sudden cardiac death, onset of acute coronary syndrome, and/or need for coronary revascularization, were monitored for 12 years. Cutoff lipid levels were determined by receiver operating characteristic curves., Results: CAD events were observed in 238 male and 67 female patients. The Kaplan-Meier event-free survival curves showed that patients with D-sdLDL-C ≥32.1 mg/dL (0.83 mmol/L) had an increased risk for CAD events (P = 0.007), whereas risk in patients with E-sdLDL-C ≥36.2 mg/dL (0.94 mmol/L) was not increased. In the group with high D-sdLDL-C, the multivariable-adjusted hazard ratio (HR) was 1.47 (95% CI, 1.15-1.89), and it remained significant after adjustment for LDL-C, non-HDL-C, or apoB and in patients treated with statins. HRs for high LDL-C, non-HDL-C, or apoB were not statistically significant after adjustment for high D-sdLDL-C. Higher D-sdLDL-C was associated with enhanced risk of high LDL-C, non-HDL-C, and apoB (HR 1.73; 95% CI, 1.27-2.37)., Conclusions: Higher D-sdLDL-C can predict long-term recurrence of CAD in stable CAD patients independently of apoB and non-HDL-C. D-sdLDL-C is an independent risk enhancer for secondary CAD prevention, whereas E-sdLDL-C is not.UMIN-CTR Clinical Trial Number: UMIN000027504., (© Association for Diagnostics & Laboratory Medicine 2024.)

Objectives: Anaemia, a common comorbidity of RA, is related to high disease activity and poor prognosis. It is unknown which biologic/targeted synthetic (b/ts)-DMARDs are optimal for patients with anaemia and RA in regulating anaemia and controlling disease activity., Methods: We investigated the change in haemoglobin (Hb) levels, drug retention rates and disease activities after the administration of b/ts-DMARDs with different modes of action [TNF inhibitors (TNFis), immunoglobulin fused with cytotoxic T-lymphocyte antigen (CTLA-4-Ig), IL-6 receptor inhibitors (IL-6Ris) and Janus kinase inhibitors (JAKis)] in patients with RA stratified by baseline Hb levels using the multicentre observational registry for patients with RA in Japan (ANSWER cohort)., Results: A total of 2093 patients with RA were classified into three groups based on tertiles of the baseline Hb levels (Hblow, anaemic; Hbint, intermediate; Hbhigh, non-anaemic). IL-6Ri increased Hb levels in all groups (the mean change at 12 months in Hblow was +1.5 g/dl, Hbint +0.7 g/dl and Hbhigh +0.1 g/dl). JAKis increased the Hb level in patients with anaemia and RA and retained or decreased the Hb level in non-anaemic patients (the mean change at 12 months in Hblow was +0.6 g/dl, Hbint 0 g/dl and Hbhigh -0.3 g/dl). In patients with anaemia and RA, overall adjusted 3-year drug retention rates were higher in JAKi followed by IL-6Ri, CTLA4-Ig and TNFi (78.6%, 67.9%, 61.8% and 50.8%, respectively). Change of disease activity at 12 months was not different among different b/ts-DMARDs treatments., Conclusion: IL-6Ri and JAKi can effectively treat patients with anaemia and RA in a real-world setting., (© The Author(s) 2023. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The first steps in chitin degradation in marine bacteria involve chitinase, which produces N,N'-diacetylchitobiose (GlcNAc)2 from chitin. Moreover, in Vibrio bacteria, chitinase activity is enhanced by heterodisaccharide β-N-acetyl-d-glucosaminyl-(1,4)-d-glucosamine (GlcNAc-GlcN) produced from (GlcNAc)2 by chitin oligosaccharide deacetylase (COD). However, the role of COD in other marine bacteria, such as Shewanella, remains unexplored. This study investigates GlcNAc-GlcN's impact on chitinase gene expression and enzyme production in S. baltica ATCC BAA-1091, drawing parallels with Vibrio parahaemolyticus RIMD2210633. Using real-time quantitative PCR, the study assesses the upregulation of chitinase gene expression in S. baltica in response to GlcNAc-GlcN, informed by COD's known ability to produce GlcNAc-GlcN from (GlcNAc)2. In Vibrio, GlcNAc-GlcN considerably upregulates chitinase gene expression. This study posits a similar regulatory mechanism in S. baltica, with preliminary investigations indicating COD's capacity to produce GlcNAc-GlcN. This study highlights the importance of exploring GlcNAc-GlcN's regulatory role in chitin metabolism across diverse marine bacteria. The potential induction of chitinase production in S. baltica suggests broader ecological implications. Further research is crucial for a comprehensive understanding of chitin utilization and regulatory pathways in marine bacterial genera., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

The extended tubular shape of root hairs is established by tip growth and concomitant hardening. Here, we demonstrate that a syntaxin of plants (SYP)123-vesicle-associated membrane protein (VAMP)727-dependent secretion system delivers secondary cell wall components for hardening the subapical zone and shank of Arabidopsis (Arabidopsis thaliana) root hairs. We found increased SYP123 localization at the plasma membrane (PM) of the subapical and shank zones compared with the tip region in elongating root hairs. Inhibition of phosphatidylinositol (PtdIns)(3,5)P2 production impaired SYP123 localization at the PM and SYP123-mediated root hair shank hardening. Moreover, root hair elongation in the syp123 mutant was insensitive to a PtdIns(3,5)P2 synthesis inhibitor. SYP123 interacts with both VAMP721 and VAMP727. syp123 and vamp727 mutants exhibited reduced shank cell wall stiffness due to impaired secondary cell wall component deposition. Based on these results, we conclude that SYP123 is involved in VAMP721-mediated conventional secretion for root hair elongation as well as in VAMP727-mediated secretory functions for the delivery of secondary cell wall components to maintain root hair tubular morphology., Competing Interests: Conflict of interest statement. The authors declare that they have no competing interests., (© The Author(s) 2023. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

The aim of this multi-centre retrospective study was to clarify the prognostic factors for respiratory-related death in patients with interstitial lung disease (ILD) complicated rheumatoid arthritis (RA). Patient background data, treatment regimen, and disease activity indicators of RA and ILD at baseline, 6 months after the diagnosis of ILD, and at the last follow-up visit were extracted. A total of 312 patients with RA-ILD (17 patients who died from respiratory-related causes and 295 survivors) were included. Patients who died from respiratory-related causes had an older median age, a higher proportion of being male, and a higher anti-cyclic citrullinated peptide antibody positivity rate than survivors (p = .0001, .038, and .016, respectively); they also had significantly higher baseline serum levels of Krebs von den Lungen-6 (KL-6) than survivors (p = .013). Patients who died from respiratory-related causes showed significantly greater changes in serum KL-6 levels between the 6-month time point and the last visit [ΔKL-6 (6 months - last)] than survivors (p = .011). Multivariate analysis showed that the ΔKL-6 (6 months - last) corrected by disease duration was a predictor of respiratory-disease-related death in patients with RA-ILD (p < .0001). Long-term increase in serum KL-6 levels is associated with respiratory-disease related death in patients with RA-ILD., (© Japan College of Rheumatology 2022. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objectives: This multicenter, retrospective study evaluated the effectiveness of add-on methotrexate (MTX) or iguratimod (IGU) in patients with rheumatoid arthritis exhibiting an inadequate response to Janus kinase inhibitors (JAKis)., Methods: Forty-five patients were treated with new additional MTX (n = 22) or IGU (n = 23) and followed for 6 months. Patients' background is as follows: age, 59.2 years; disease activity score of 28 joints with C-reactive protein (DAS28-CRP), 3.4; clinical disease activity index, 15.7; biological disease-modifying antirheumatic drug (DMARD)-switched cases, 77.8%; first JAKi cases, 95.6%; and JAKi treatment: tofacitinib (n = 25), baricitinib (n = 17), upadacitinib (n = 2), and peficitinib (n = 1) for 9.6 months., Results: Thirty-five patients continued the combination therapy for 6 months without a significant change in concomitant glucocorticoid or other conventional synthetic DMARDs. DAS28-CRP (MTX, 3.6 to 2.6, p < 0.05; IGU, 3.3 to 2.1, p < 0.001) and clinical disease activity index (MTX, 16.7 to 8.8, p < 0.05; IGU, 14.6 to 6.5, p < 0.01) improved significantly from baseline. Using the 2019 European League Against Rheumatism criteria, 45.4% (MTX) and 39.1% (IGU) achieved moderate or good response and 40.9% (MTX) and 39.1% (IGU) achieved American College of Rheumatology 20% improvement criteria., Conclusions: Adding MTX or IGU to inadequate responders of JAKi can be considered as a complementary treatment., (© Japan College of Rheumatology 2022. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Starch is stored temporarily in the leaves during the day but degraded during the night. In this study, we investigated the relationship between diurnal changes in starch content in rice leaf blades and the mRNA levels of β-amylase genes. In addition to the known plastid-type β-amylases OsBAM2 and OsBAM3, OsBAM4, and OsBAM5 were also identified as plastid targeted proteins. In the leaf blades, starch contents, which reached its maximum at the end of day, showed two periods of marked decrease: from 18:00 to 21:00 and from 24:00 to 6:00. The expression of OsBAM2, OsBAM3, OsBAM4, and OsBAM5 was maintained at a low level from 18:00 to 21:00 but increased strongly after midnight. Furthermore, β-amylase activity gradually increased after 21:00, reaching a maximum during the early morning. These results suggest that in rice leaf blades, β-amylase plays an important role in starch degradation by being highly active from midnight to dawn., (© The Author(s) 2023. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Objective: B-cell activating factor (BAFF) is implicated in SLE pathogenesis. Blocking BAFF signalling has contributed to reducing glucocorticoid dosage and preventing organ damage. However, clinical characteristics of patients who may benefit from this therapy are not yet fully elucidated. Therefore, we identified patients with high BAFF-bioactivity to investigate their clinical characteristics and BAFF-producing cells., Methods: We established the reporter cell for BAFF and investigated the clinical characteristics of SLE patients with high BAFF-bioactivity. We identified BAFF-expressing kidney cells using publicly available scRNA-seq data and immunohistological analysis. SLE patients were stratified based on the bioactivity of BAFF and type-I IFN (IFN-I) to identify associated characteristic clinical manifestations., Results: SLE patients, especially patients with LN, had significantly higher serum BAFF-bioactivity than healthy controls (HC) and non-LN patients. Additionally, single-cell-RNA-seq data and immunohistological analysis of kidney samples from LN patients revealed that BAFF is expressed in glomerular macrophages and mesangial cells. Notably, BAFF bioactivity was elevated in the urine of LN patients compared with that of non-LN patients, while no IFN-I bioactivity was detected in the urine. Furthermore, SLE stratification based on bioactivities of serum BAFF and IFN-I revealed the clinical characteristics of patients: high BAFF represented patients with LN and high IFN-I represented patients with blood and skin manifestations., Conclusions: Monitoring urinary BAFF-bioactivity may be valuable in diagnosing LN. Furthermore, stratification based on serum BAFF and IFN-I bioactivities may allow the identification of appropriate patients for biologics targeting BAFF and IFN-I., (© The Author(s) 2022. Published by Oxford University Press on behalf of the British Society for Rheumatology.)

Nontypeable Haemophilus influenzae (NTHi) is considered a major pathogen underlying middle ear infection. This study aimed to investigate the impact of IL-17 on chronic otitis media (COM) induced by NTHi in mice. NTHi was inoculated into the tympanic bulla with eustachian tubal obstruction. Middle ear effusions (MEEs) and tissues were collected on days 3, 14, and at 1, 2, and 6 months after injection. The expression of interleukin-17A (IL-17A) in MEEs was significantly elevated compared to that in the control group at the translational and transcriptional levels during the experiments. The quantities of IL-17-producing γδ T cells were significantly increased compared to that in the control group during COM, but that of Th17 cells did not. Depletion of γδ T cells by anti-γδ T-cell receptor (TCR) monoclonal antibody (mAb) administration significantly decreased the bacteria counts and the concentrations of IL-1β, IL-6, IL-17A, TNF-α, and IL-10 in MEEs. Our results suggest that IL-17 may play an important role in prolonging the inflammation in the middle ear in COM and that IL-17-producing γδ T cells may contribute to the exacerbated inflammatory response in the middle ear. In this study, anti-γδ TCR mAb administration was found to improve chronic middle ear inflammatory conditions., (© The Author(s) 2023. Published by Oxford University Press on behalf of FEMS.)

Streptomyces incarnatus NRRL8089 produces the antiviral, antifungal, antiprotozoal nucleoside antibiotic sinefungin. To enhance sinefungin production, multiple mutations were introduced to the rpoB gene encoding RNA polymerase (RNAP) β-subunit at the target residues, D447, S453, H457, and R460. Sparse regression analysis using elastic-net lasso-ridge penalties on previously reported H457X mutations identified a numeric parameter set, which suggested that H457R/Y/F may cause production enhancement. H457R/R460C mutation successfully enhanced the sinefungin production by 3-fold, while other groups of mutations, such as D447G/R460C or D447G/H457Y, made moderate or even negative effects. To identify why the rif cluster residues have diverse effects on sinefungin production, an RNAP/DNA/mRNA complex model was constructed by homology modeling and molecular dynamics simulation. The 4 residues were located near the mRNA strand. Density functional theory-based calculation suggested that D447, H457, and R460 are in direct contact with ribonucleotide, and partially positive charges are induced by negatively charged chain of mRNA., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Attenuation of the secondary injury of spinal cord injury (SCI) can suppress the spread of spinal cord tissue damage, possibly resulting in spinal cord sparing that can improve functional prognoses. Granulocyte colony-stimulating factor (G-CSF) is a haematological cytokine commonly used to treat neutropenia. Previous reports have shown that G-CSF promotes functional recovery in rodent models of SCI. Based on preclinical results, we conducted early phase clinical trials, showing safety/feasibility and suggestive efficacy. These lines of evidence demonstrate that G-CSF might have therapeutic benefits for acute SCI in humans. To confirm this efficacy and to obtain strong evidence for pharmaceutical approval of G-CSF therapy for SCI, we conducted a phase 3 clinical trial designed as a prospective, randomized, double-blinded and placebo-controlled comparative trial. The current trial included cervical SCI [severity of American Spinal Injury Association (ASIA) Impairment Scale (AIS) B or C] within 48 h after injury. Patients are randomly assigned to G-CSF and placebo groups. The G-CSF group was administered 400 μg/m2/day × 5 days of G-CSF in normal saline via intravenous infusion for five consecutive days. The placebo group was similarly administered a placebo. Allocation was concealed between blinded evaluators of efficacy/safety and those for laboratory data, as G-CSF markedly increases white blood cell counts that can reveal patient treatment. Efficacy and safety were evaluated by blinded observer. Our primary end point was changes in ASIA motor scores from baseline to 3 months after drug administration. Each group includes 44 patients (88 total patients). Our protocol was approved by the Pharmaceuticals and Medical Device Agency in Japan and this trial is funded by the Center for Clinical Trials, Japan Medical Association. There was no significant difference in the primary end point between the G-CSF and the placebo control groups. In contrast, one of the secondary end points showed that the ASIA motor score 6 months (P = 0.062) and 1 year (P = 0.073) after drug administration tend to be higher in the G-CSF group compared with the placebo control group. Moreover, in patients aged over 65 years old, motor recovery 6 months after drug administration showed a strong trend towards a better recovery in the G-CSF treated group (P = 0.056) compared with the control group. The present trial failed to show a significant effect of G-CSF in primary end point although the subanalyses of the present trial suggested potential G-CSF benefits for specific population., (© The Author(s) (2021). Published by Oxford University Press on behalf of the Guarantors of Brain.)

In animal gonads, transposable elements are actively repressed to preserve genome integrity through the PIWI-interacting RNA (piRNA) pathway. In mice, piRNAs are abundantly expressed in male germ cells, and form effector complexes with three distinct PIWIs. The depletion of individual Piwi genes causes male-specific sterility with no discernible phenotype in female mice. Unlike mice, most other mammals have four PIWI genes, some of which are expressed in the ovary. Here, purification of PIWI complexes from oocytes of the golden hamster revealed that the size of the PIWIL1-associated piRNAs changed during oocyte maturation. In contrast, PIWIL3, an ovary-specific PIWI in most mammals, associates with short piRNAs only in metaphase II oocytes, which coincides with intense phosphorylation of the protein. An improved high-quality genome assembly and annotation revealed that PIWIL1- and PIWIL3-associated piRNAs appear to share the 5'-ends of common piRNA precursors and are mostly derived from unannotated sequences with a diminished contribution from TE-derived sequences, most of which correspond to endogenous retroviruses. Our findings show the complex and dynamic nature of biogenesis of piRNAs in hamster oocytes, and together with the new genome sequence generated, serve as the foundation for developing useful models to study the piRNA pathway in mammalian oocytes., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Objective: this prospective cohort study aimed to assess the safety and efficacy of bevacizumab combined with chemotherapy in Japanese patients with relapsed ovarian, fallopian tube or primary peritoneal cancer., Methods: in this study, 40 Japanese patients with relapsed ovarian, fallopian tube or primary peritoneal cancer selected to receive bevacizumab with chemotherapy were enrolled. Patients in poor general condition were excluded. Each patient was monitored prospectively for adverse events, administration status, disease status and survival. Treatment was continued until intolerable adverse events or disease progression. The primary endpoint was safety., Results: bevacizumab plus platinum-based chemotherapy was performed for 30 patients (median cycle; 16.5), while bevacizumab plus non-platinum chemotherapy was performed for 10 patients (median cycle; 5.5). Among bevacizumab-related adverse events, hypertension occurred in 80% of patients, proteinuria in 83%, mucositis in 25%, bleeding in 20%, thromboembolic events in 5.0% and fistula in 2.5%. Gastrointestinal perforation or other life-threatening lethal adverse events were not observed. Response rate and median progression-free survival were 73% and 19.3 months for patients with bevacizumab plus platinum-based chemotherapy, and 30% and 3.9 months for patients with bevacizumab plus non-platinum chemotherapy, respectively. There was no correlation between response rate and occurrence of adverse events such as hypertension or proteinuria., Conclusion: bevacizumab combined with chemotherapy was tolerable and effective for Japanese patients with relapsed ovarian cancer, fallopian tube cancer or primary peritoneal cancer. Hypertension and proteinuria are frequently occurred and managed properly for continuing treatment., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: To date, only few large studies are available concerning the safety and diagnostic concordance rates of outpatient flexible hysteroscopy. In our institution, outpatient hysteroscopy has been routinely and educationally applied Kosuke Tsuji to intrauterine lesions; thus, we retrospectively investigated the institution's outpatient flexible hysteroscopy cases., Methods: A total of 1591 cases of outpatient flexible hysteroscopy conducted at our institution in 2012-2016 were retrospectively analyzed in terms of their clinical background, complications and diagnostic concordance rates., Results: A total of 1591 cases included 546 cases of benign tumors (317 endometrial polyps, 168 myomas and 61 endometrial hyperplasia), 361 cases of atypical endometrial hyperplasia, 571 cases of endometrial cancers and 113 cases of other diagnoses. No major complications, including uterine perforation, occurred. However, one patient (0.06%) was diagnosed with septic shock caused by intrauterine infection that required prolonged immunosuppressive drug administration. Meanwhile, 335 patients diagnosed with benign tumors through outpatient flexible hysteroscopy underwent operation, and the diagnostic concordance rate was 74.6% (250 cases). However, this rate included 14 cases (4.2%) diagnosed with malignant tumors postoperatively. In preoperative endometrial cancer cases, the sensitivity and specificity for cervical invasion diagnosis were 39.4 and 90.8%, respectively. In addition, only one patient manifested positive ascites cytology intraoperatively, possibly caused by outpatient hysteroscopy., Conclusions: Outpatient flexible hysteroscopy is highly safe, with a slight negligible effect on ascites cytology. However, the diagnosis should be determined by multidisciplinary approaches, as hysteroscopy alone can miss malignancy., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

TAFRO syndrome is a newly proposed disease that is characterised by thrombocytopenia, anasarca, fever, reticulin fibrosis (or renal dysfunction), and organomegaly. Generally, high doses of corticosteroids are recommended for the initial treatment of TAFRO syndrome; however, some patients experience prolonged refractory thrombocytopenia after initiating such therapies. If corticosteroid treatment alone is ineffective, additional immunosuppressive therapies such as cyclosporine A are recommended. Since long-term use of immunosuppressive therapies with TAFRO syndrome sometimes causes serious infection, it is important to recognise the time to recovery from thrombocytopenia. In this study, we investigated how long it took to recover from thrombocytopenia, to aid clinicians in decision-making regarding the need to strengthen treatment for prolonged thrombocytopenia. Here, we describe three of our patients with TAFRO syndrome exhibiting prolonged thrombocytopenia. We also investigated the median period to recovery from this complication (defined as the time to increase the platelet count above 50,000/µL) after the initiation of high-dose corticosteroid treatment in our 3 cases and 38 peer-reviewed cases. We found that it took our patients 61 days to recover from thrombocytopenia; in comparison, our investigation of the 38 peer-reviewed case reports revealed a median recovery time of 47.5 days among previously reported patients. We showed the time to recovery from thrombocytopenia in patients with TAFRO syndrome for the first time. Our findings ought to be useful for decision-making among clinicians regarding the administration of other immunosuppressive treatments in addition to corticosteroid.

Objectives: To clarify changes in the incidence of cervical lesions in rheumatoid arthritis (RA) patients with advanced treatment and the impact of cervical lesions on the patients' quality of life (QOL). Methods : Incidence of radiographic cervical lesions in 1333 RA patients in 2015 was compared with that in our 1999 survey. The association between cervical lesions and QOL evaluated using three different patient-based questionnaires was also analyzed. Results: The incidence of atlantoaxial subluxation (AAS), vertical subluxation (VS), and subaxial subluxation (SAS) in 2015 decreased by 50%, 75%, and 5%, respectively, compared to the 1999 survey. Although QOL, evaluated using the Japanese Orthopedic Association Cervical Myelopathy Evaluation Questionnaire (JOACMEQ; specific to myelopathy), deteriorated as the cervical lesion progressed, there was no association between cervical lesion progression and QOL evaluated using the Short Form-8™ (SF-8™; comprehensive health-related QOL). Cervical lesion progression was also associated with QOL deterioration evaluated using the Health Assessment Questionnaire Disability Index (HAQ-DI; specific to RA), but age and disease duration had stronger influences. Conclusion: The incidence of cervical lesions decreased in 2015 compared to 1999. Cervical lesion progression may be associated with QOL deterioration due to myelopathy. Age and disease duration have more impact on disease-specific QOL.

Objective: The purpose of this research was to develop a deep-learning model to assess radiographic finger joint destruction in RA., Methods: The model comprises two steps: a joint-detection step and a joint-evaluation step. Among 216 radiographs of 108 patients with RA, 186 radiographs were assigned to the training/validation dataset and 30 to the test dataset. In the training/validation dataset, images of PIP joints, the IP joint of the thumb or MCP joints were manually clipped and scored for joint space narrowing (JSN) and bone erosion by clinicians, and then these images were augmented. As a result, 11 160 images were used to train and validate a deep convolutional neural network for joint evaluation. Three thousand seven hundred and twenty selected images were used to train machine learning for joint detection. These steps were combined as the assessment model for radiographic finger joint destruction. Performance of the model was examined using the test dataset, which was not included in the training/validation process, by comparing the scores assigned by the model and clinicians., Results: The model detected PIP joints, the IP joint of the thumb and MCP joints with a sensitivity of 95.3% and assigned scores for JSN and erosion. Accuracy (percentage of exact agreement) reached 49.3-65.4% for JSN and 70.6-74.1% for erosion. The correlation coefficient between scores by the model and clinicians per image was 0.72-0.88 for JSN and 0.54-0.75 for erosion., Conclusion: Image processing with the trained convolutional neural network model is promising to assess radiographs in RA., (© The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Rheumatology.)

Cilia are organelles that serve as cellular antennae. Intraflagellar transport particles containing the IFT-A and IFT-B complexes mediate bidirectional trafficking of ciliary proteins. Particularly, in concert with the BBSome complex, IFT particles play an essential role in trafficking of ciliary G-protein-coupled receptors (GPCRs). Therefore, proteins interacting with the IFT components are potential regulators of ciliary protein trafficking. We here revealed that an uncharacterized protein, C11ORF74, interacts with the IFT-A complex via the IFT122 subunit and is accumulated at the distal tip in the absence of an IFT-A subunit IFT139, suggesting that at least a fraction of C11ORF74 molecules can be transported towards the ciliary tip by associating with the IFT-A complex, although its majority might be out of cilia at steady state. In C11ORF74-knockout (KO) cells, the BBSome components cannot enter cilia. However, trafficking of Smoothened or GPR161, both of which are ciliary GPCRs involved in Hedgehog signalling and undergo BBSome-dependent trafficking, was not affected in the absence of C11ORF74. In addition, C11orf74/B230118H07Rik- KO mice demonstrated no obvious anatomical abnormalities associated with ciliary dysfunctions. Given that C11ORF74 is conserved across vertebrates, but not found in other ciliated organisms, such as nematodes and Chlamydomonas, it might play limited roles involving cilia., (© The Author(s) 2018. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Glucose-dependent insulinotropic polypeptide (GIP) exhibits direct cardiovascular actions in addition to its well-known insulinotropic effect. However, the role of GIP in peripheral artery disease remains unclear. In this study, we evaluated the effects of GIP against peripheral arterial remodeling in mouse models. The genetic deletion of GIP receptor (GIPR) led to exaggerated neointimal hyperplasia after transluminal femoral artery wire injury. Conversely, chronic GIP infusion suppressed neointimal hyperplasia and facilitated endothelial regeneration. The beneficial effects of GIP were abrogated by inhibiting nitric oxide (NO) synthase, suggesting a possible mechanism mediated by NO. In cultured human umbilical vein endothelial cells (HUVECs), GIP elevated cytosolic calcium levels without affecting intracellular cAMP levels. Furthermore, GIP dose-dependently increased NO production, whereas this effect was abolished by inhibiting AMP-activated protein kinase (AMPK). GIP induced AMPK phosphorylation, which was abrogated by inhibiting phospholipase C and calcium-calmodulin-dependent protein kinase kinase but not by adenylate cyclase or liver kinase B1, suggesting the existence of a calcium-mediated GIPR signaling pathway. These effects of GIP were retained in severe hyperglycemic Leprdb/ Leprdb mice and in high-glucose-cultured HUVECs. Overall, we demonstrated the protective effects of GIP against peripheral arterial remodeling as well as the involvement of a calcium-mediated GIPR signaling pathway in vascular endothelial cells. Our findings imply the potential vascular benefits of multiple agonists targeting G protein-coupled receptors, including GIPR, which are under development for the treatment of type 2 diabetes.

Variation in the shell coiling, or chirality, of land snails provides an opportunity to investigate the potential for "single-gene" speciation, because mating between individuals of opposite chirality is believed not possible if the snails mate in a face-to-face position. However, the evidence in support of single-gene speciation is sparse, mostly based upon single-gene mitochondrial studies and patterns of chiral variation between species. Previously, we used a theoretical model to show that as the chiral phenotype of offspring is determined by the maternal genotype, occasional chiral reversals may take place and enable gene flow between mirror image morphs, preventing speciation. Here, we show empirically that there is recent or ongoing gene flow between the different chiral types of Japanese Euhadra species. We also report evidence of mating between mirror-image morphs, directly showing the potential for gene flow. Thus, theoretical models are suggestive of gene flow between oppositely coiled snails, and our empirical study shows that they can mate and that there is gene flow in Euhadra . More than a single gene is required before chiral variation in shell coiling can be considered to have created a new species.

Objective: Interval debulking surgery (IDS) after neoadjuvant chemotherapy (NAC) is currently one of the preferred treatment options for advanced ovarian, fallopian tube or peritoneal cancer. This study was conducted to evaluate the clinical efficacy and safety of dose-dense paclitaxel plus carboplatin therapy (ddTC therapy) as NAC for these cancers., Patients and Methods: A retrospective study was conducted in 25 patients with Stage III/IV ovarian, fallopian tube or peritoneal cancer who received ddTC therapy as NAC. For ddTC therapy, paclitaxel (80 mg/m2) was administered intravenously on Days 1, 8 and 15 and carboplatin (AUC 6.0 mg/ml × min) was administered intravenously on Day 1 every 3 weeks. IDS was performed after three cycles of ddTC therapy, and ddTC therapy was also continued after surgery., Results: With ddTC therapy as NAC, the response rate was 92% and disease progression did not occur in any patient. Grade 4 hematologic toxicity and ≥Grade 3 non-hematologic toxicity both occurred in 8% of the patients, but no patient discontinued NAC because of adverse events. When IDS was performed, the complete surgery rate was 64% and the optimal surgery rate was 96%. ≥Grade 3 perioperative complications occurred in 16% of the patients, but there were no perioperative deaths. Median overall survival was 35.7 months and median progression-free survival was 17.7 months., Conclusion: This study showed that ddTC therapy was considerably effective and tolerable as NAC. The complete surgery rate was high with IDS, and perioperative complications were acceptable., (© The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2] is a low-abundance phospholipid known to be associated with a wide variety of physiological functions in plants. However, the localization and dynamics of PI(3,5)P2 in plant cells remain largely unknown, partially due to the lack of an effective fluorescent probe. Using Arabidopsis transgenic plant expressing the PI(3,5)P2-labeling fluorescent probe (tagRFP-ML1N*2) developed based on a tandem repeat of the cytosolic phosphoinositide-interacting domain (ML1N) of the mammalian lysosomal transient receptor potential cation channel, Mucolipin 1 (TRPML1), here we show that PI(3,5)P2 is predominantly localized on the limited membranes of the FAB1- and SNX1-positive late endosomes, but rarely localized on the membranes of plant vacuoles or trans-Golgi network/early endosomes of cortical cells of the root differentiation zone. The late endosomal localization of tagRFP-ML1N*2 is reduced or abolished by pharmacological inhibition or genetic knockdown of expression of genes encoding PI(3,5)P2-synthesizing enzymes, FAB1A/B, but markedly increased with FAB1A overexpression. Notably, reactive oxygen species (ROS) significantly increase late endosomal levels of PI(3,5)P2. Thus, tandem ML1N-based PI(3,5)P2 probes can reliably monitor intracellular dynamics of PI(3,5)P2 in Arabidopsis cells with less binding activity to other endomembrane organelles., (© The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Chitin oligosaccharide deacetylase (COD) from bacteria that have been examined so far typically comprise two carbohydrate-binding domains (CBDs) and one polysaccharide deacetylase domain. In contrast, Shewanella baltica ATCC BAA-1091 COD (Sb-COD) has only one CBD, yet exhibits chitin-binding properties and substrate specificities similar to those of other CODs.

Phosphoinositides play an important role in various membrane trafficking events in eukaryotes. One of them, however, phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2], has not been studied widely in plants. Using a combination of fluorescent reporter proteins and the PI(3,5)P2-specific inhibitor YM202636, here we demonstrated that in Arabidopsis thaliana, PI(3,5)P2 affects various membrane trafficking events, mostly in the post-Golgi routes. We found that YM201636 treatment effectively reduced PI(3,5)P2 concentration not only in the wild type but also in FAB1A-overexpressing Arabidopsis plants. In particular, reduced PI(3,5)P2 levels caused abnormal membrane dynamics of plasma membrane proteins, AUX1 and BOR1, with different trafficking patterns. Secretion and morphological characteristics of late endosomes and vacuoles were also affected by the decreased PI(3,5)P2 production. These pleiotropic defects in the post-Golgi trafficking events were caused by the inhibition of PI(3,5)P2 production. This effect is probably mediated by the inhibition of maturation of FAB1-positive late endosomes, thereby impairing late endosome function. In conclusion, our results imply that in Arabidopsis, late endosomes are involved in multiple post-Golgi membrane trafficking routes including not only vacuolar trafficking and endocytosis but also secretion., (© The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Two kinds of oligosaccharides, N-acetylraffinosamine (RafNAc) and N-acetylplanteosamine (PlaNAc), were synthesized from N-acetylsucrosamine and melibiose using the transgalactosylation activity of Aspergillus niger α-galactosidase. RafNAc and PlaNAc are novel trisaccharides in which d-glucopyranose residues in raffinose (Raf) and planteose are replaced with N-acetyl-d-glucosamine. These trisaccharides were more stable in acidic solution than Raf. RafNAc was hydrolyzed more rapidly than Raf by α-galactosidase of green coffee bean. In contrast, RafNAc was not hydrolyzed by Saccharomyces cerevisiae invertase, although Raf was hydrolyzed well by this enzyme. These results indicate that the physicochemical properties and steric structure of RafNAc differ considerably from those of Raf.

It is believed that the plastids in green plants lost peptidoglycan (i.e., a bacterial cell wall-containing d-amino acids) during their evolution from an endosymbiotic cyanobacterium. Although wall-like structures could not be detected in the plastids of green plants, the moss Physcomitrella patens has the genes required to generate peptidoglycan (Mur genes), and knocking out these genes causes defects in chloroplast division. Here, we generated P patens knockout lines (∆Pp-ddl) for a homolog of the bacterial peptidoglycan-synthetic gene encoding d-Ala:d-Ala ligase. ∆Pp-ddl had a macrochloroplast phenotype, similar to other Mur knockout lines. The addition of d-Ala-d-Ala (DA-DA) to the medium suppressed the appearance of giant chloroplasts in ∆Pp-ddl, but the addition of l-Ala-l-Ala (LA-LA), DA-LA, LA-DA, or d-Ala did not. Recently, a metabolic method for labeling bacterial peptidoglycan was established using ethynyl-DA-DA (EDA-DA) and click chemistry to attach an azide-modified fluorophore to the ethynyl group. The ∆Pp-ddl line complemented with EDA-DA showed that moss chloroplasts are completely surrounded by peptidoglycan. Our findings strongly suggest that the moss plastids have a peptidoglycan wall containing d-amino acids. By contrast, no plastid phenotypes were observed in the T-DNA tagged ddl mutant lines of Arabidopsis thaliana., (© 2016 American Society of Plant Biologists. All rights reserved.)

Diacylglycerol (DG) lipase, which hydrolyses 1-stearoyl-2-arachidonyl-sn-glycerol to produce an endocannabinoid, 2-arachidonoylglycerol, was purified from the soluble fraction of rat brain lysates. DG lipase was purified about 1,200-fold by a sequential column chromatographic procedure. Among proteins identified by mass spectrometry analysis in the partially purified DG lipase sample, only DDHD domain containing two (DDHD2), which was formerly regarded as a phospholipase A1, exhibited significant DG lipase activity. Rat DDHD2 expressed in Chinese hamster ovary cells showed similar enzymatic properties to partially purified DG lipase from rat brain. The source of DG lipase activity in rat brain was immunoprecipitated using anti-DDHD2 antibody. Thus, we concluded that the DG lipase activity in the soluble fraction of rat brain is derived from DDHD2. DDHD2 is distributed widely in the rat brain. Immunohistochemical analysis revealed that DDHD2 is expressed in hippocampal neurons, but not in glia., (© The Authors 2016. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Fluvirucins are 14-membered macrolactam polyketides that show antifungal and antivirus activities. Fluvirucins have the β-alanine starter unit at their polyketide skeletons. To understand the construction mechanism of the β-alanine moiety in fluvirucin biosyntheses, we have identified the biosynthetic cluster of fluvirucin B2 produced from Actinomadura fulva subsp. indica ATCC 53714. The identified gene cluster contains three polyketide synthases, four characteristic β-amino acid-carrying enzymes, one decarboxylase, and one amidohydrolase. We next investigated the activity of the adenylation enzyme FlvN, which is a key enzyme for the selective incorporation of a β-amino acid substrate. FlvN showed strong preference for l-aspartate over other amino acids such as β-alanine. Based on these results, we propose a biosynthetic pathway for fluvirucin B2.

Neural activity plays roles in the later stages of development of cortical excitatory neurons, including dendritic and axonal arborization, remodeling, and synaptogenesis. However, its role in earlier stages, such as migration and dendritogenesis, is less clear. Here we investigated roles of neural activity in the maturation of cortical neurons, using calcium imaging and expression of prokaryotic voltage-gated sodium channel, NaChBac. Calcium imaging experiments showed that postmigratory neurons in layer II/III exhibited more frequent spontaneous calcium transients than migrating neurons. To test whether such an increase of neural activity may promote neuronal maturation, we elevated the activity of migrating neurons by NaChBac expression. Elevation of neural activity impeded migration, and induced premature branching of the leading process before neurons arrived at layer II/III. Many NaChBac-expressing neurons in deep cortical layers were not attached to radial glial fibers, suggesting that these neurons had stopped migration. Morphological and immunohistochemical analyses suggested that branched leading processes of NaChBac-expressing neurons differentiated into dendrites. Our results suggest that developmental control of spontaneous calcium transients is critical for maturation of cortical excitatory neurons in vivo: keeping cellular excitability low is important for migration, and increasing spontaneous neural activity may stop migration and promote dendrite formation., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Objectives: We report the long-term outcome of joint-preserving surgery by combining metatarsal osteotomies for shortening for forefoot deformity in patients with rheumatoid arthritis (RA)., Methods: Forty-three patients (57 feet) aged 41.7-70.8 years (mean, 57.7 years) underwent a combination of first tarsometatarsal fusion and distal realignment (modified Lapidus procedure), shortening oblique osteotomies of the bases of metatarsals 2-4, and fifth ray osteotomy (modified Coughlin procedure). Patients were followed up for 64-108 months (mean, 76.6 months)., Results: Average postoperative Foot Function Index scores for pain, disability, and activity were 10.3, 19.9, and 16.2, respectively. Average Japanese Society for Surgery of the Foot RA foot and ankle score improved significantly from 52.1 points preoperatively to 90.3 points postoperatively. Postoperatively, 41% of patients reported some forefoot stiffness, but showed no disability. Residual deformity and callosity were absent in all patients. Average hallux valgus and intermetatarsal angles decreased postoperatively from 48.5° to 8.6° and from 15.2° to 4.6°, respectively. Nonunion in two metatarsals, hardware breakage in three, and mild infection in one were identified during follow-up., Conclusions: With good perioperative medical management of RA, surgical repositioning of the metatarsophalangeal joints by proximal metatarsal shortening and consequent relaxing of the surrounding soft tissue shows successful long-term results.

Systemic sclerosis (SSc) presents stiffness of extremities due to sclerosis of the tissue especially at fingers, hands, and forearms. Here we report the case of a patient with diffuse cutaneous SSc who was administered anti-interleukin-6 receptor antibody tocilizumab (TCZ). Skin condition of SSc is evaluated by pinching the skin according to the Rodnan skin score, but sometimes tissue atrophy results in overestimation of the condition. To understand how the extremities softened after initiation of TCZ, we observed mobility of extremities. Range of motion (ROM) of joints was measured every four months after initiation of TCZ. The patient presented not only reduction of Rodnan score but also amelioration of mobility of extremities. The Rodnan skin score reduced from 35 to 7 within sixteen months, and ROM of most joints except ankle was expanded.

Plant shoot organs such as stems, leaves and flowers are derived from specialized groups of stem cells organized at the shoot apical meristem (SAM). Organogenesis involves two major processes, namely cell proliferation and differentiation, whereby the former contributes to increasing the cell number and the latter involves substantial increases in cell volume through cell expansion. Co-ordination between the above processes in time and space is essential for proper organogenesis. To identify regulatory factors involved in proper organogenesis, heavy-ion beam-irradiated de-etiolated (det) 3-1 seeds have been used to identify striking phenotypes in the A#26-2; det3-1 mutant. In addition to the stunted plant stature mimicking det3-1, the A#26-2; det3-1 mutant exhibited stem thickening, increased floral organ number and a fruit shape reminiscent of clavata (clv) mutants. DNA sequencing analysis demonstrated that A#26-2; det3-1 harbors a mutation in the CLV3 gene. Importantly, A#26-2; det3-1 displayed cracks that randomly occurred on the main stem with a frequency of approximately 50%. Furthermore, the double mutants clv3-8 det3-1, clv1-4 det3-1 and clv2-1 det3-1 consistently showed stem cracks with frequencies of approximately 97, 38 and 35%, respectively. Cross-sections of stems further revealed an increase in vascular bundle number, cell number and size in the pith of clv3-8 det3-1 compared with det3-1. These findings suggest that the stem inner volume increase due to clv mutations exerts an outward mechanical stress; that in a det3-1 background (defective in cell expansion) resulted in cracking of the outermost layer of epidermal cells., (© The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Development of the cerebral cortex depends partly on neural activity, but the identity of the ion channels that might contribute to the activity-dependent cortical development is unknown. KCNK channels are critical determinants of neuronal excitability in the mature cerebral cortex, and a member of the KCNK family, KCNK9, is responsible for a maternally transmitted mental retardation syndrome. Here, we have investigated the roles of KCNK family potassium channels in cortical development. Knockdown of KCNK2, 9, or 10 by RNAi using in utero electroporation impaired the migration of late-born cortical excitatory neurons destined to become Layer II/III neurons. The migration defect caused by KCNK9 knockdown was rescued by coexpression of RNAi-resistant functional KCNK9 mutant. Furthermore, expression of dominant-negative mutant KCNK9, responsible for the disease, and electrophysiological experiments demonstrated that ion channel function was involved in the migration defect. Calcium imaging revealed that KCNK9 knockdown or expression of dominant-negative mutant KCNK9 increased the fraction of neurons showing calcium transients and the frequency of spontaneous calcium transients. Mislocated neurons seen after KCNK9 knockdown stayed in the deep cortical layers, showing delayed morphological maturation. Taken together, our results suggest that dysfunction of KCNK9 causes a migration defect in the cortex via an activity-dependent mechanism.

Root hairs are fast-growing tubular protrusions on root epidermal cells that play important roles in water and nutrient uptake in plants. The tip-focused polarized growth of root hairs is accomplished by the secretion of newly synthesized materials to the tip via the polarized membrane trafficking mechanism. Here, we report the function of two different types of plasma membrane (PM) Qa-SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors), SYP123 and SYP132, in the growth of root hair in Arabidopsis. We found that SYP123, but not SYP132, localizes in the tip region of root hairs by recycling between the brefeldin A (BFA)-sensitive endosomes and the PM of the expanding tip in an F-actin-dependent manner. The vesicle-associated membrane proteins VAMP721/722/724 also exhibited tip-focused localization in root hairs and formed ternary SNARE complexes with both SYP123 and SYP132. These results demonstrate that SYP123 and SYP132 act in a coordinated fashion to mediate tip-focused membrane trafficking for root hair tip growth.

Background: The pathophysiology of intra-abdominal adhesions has not been studied extensively. The aim of this study was to elucidate the molecular mechanisms underlying adhesion formation in a murine model and in patients undergoing hepatectomy., Methods: Partial hepatectomy was performed using bipolar forceps in mice. Wild-type mice, antibodies to CD4 and interferon (IFN) γ, IFN-γ, natural killer T (NKT) cells and plasminogen activator inhibitor (PAI) 1 knockout (KO) mice were used. Recombinant hepatocyte growth factor (HGF) was tested for its ability to prevent adhesions. Liver specimens were obtained during surgery from patients undergoing hepatectomy. Adhesion formation was evaluated using a scoring system that ranged from 0 (no adhesions) to 5 (severe adhesions). Levels of IFN-γ and PAI-1 mRNA, and protein concentration of PAI-I were measured, and fluorescence immunostaining was performed., Results: Adhesion formation depended on IFN-γ produced by NKT cells, and NKT KO mice developed few adhesions (mean(s.d.) 1·7(0·3) versus 4·6(0·4) in wild-type mice; P = 0·037). In wild-type mice, the level of PAI-1 mRNA increased after hepatectomy, followed by a decrease in the tissue plasminogen activator (tPA) mRNA level. Adhesion formation was inhibited completely in PAI-1 KO mice (0(0) versus 4·1(0·8) in wild-type mice; P = 0·002). HGF inhibited formation of abdominal adhesions after hepatectomy by reducing IFN-γ and PAI-1 levels, and increasing tPA levels compared with those in mice treated with phosphate-buffered saline (P < 0·001, P = 0·002 and P = 0·035 respectively). In human liver specimens, NKT cells accumulated in the liver after hepatectomy, and PAI-1 expression was increased 5·25-fold (P = 0·030)., Conclusion: IFN-γ is a key molecule for abdominal adhesion formation after hepatectomy, acting via the reciprocal balance of PAI-1 and tPA. This molecular mechanism may also regulate adhesion formation in patients following hepatectomy. HGF inhibited formation of adhesions by regulating IFN-γ and PAI-1, suggesting that it may be an important target for prevention of adhesions after hepatectomy. SURGICAL RELEVANCE: Postoperative intra-abdominal adhesions can be asymptomatic or cause significant morbidity and mortality. Adhesion formation after hepatectomy has not been studied extensively. In the present study, the molecular mechanisms underlying intra-abdominal adhesions after hepatectomy were investigated in a murine model and in patients. Interferon (IFN) γ produced by natural killer T cells is a key molecule for adhesion formation after hepatectomy in mice, acting via the reciprocal balance between plasminogen activator inhibitor (PAI) 1 and tissue plasminogen activator, the pivotal factors in fibrinolytic activity. This mechanism was also involved in the regulation of adhesions in human tissue samples. Hepatocyte growth factor (HGF) strongly inhibited adhesion formation by regulating IFN-γ and PAI-1. These results indicate that IFN-γ and PAI-1 are possible therapeutic targets, and HGF could prevent postoperative adhesion formation after hepatectomy., (© 2014 BJS Society Ltd. Published by John Wiley & Sons Ltd.)

Moraxella catarrhalis is a Gram-negative aerobic diplococcus that is currently the third most frequent cause of bacterial acute otitis media (AOM) in children. In this study, we developed an experimental murine AOM model by inoculating M. catarrhalis in the middle ear bulla and studied the local response to this inoculation, and modulation of its course by the pili of M. catarrhalis. The pili-positive and pili-negative M. catarrhalis showed differences in bacterial clearance and infiltration of inflammatory cells in the middle ear. Pili-negative M. catarrhalis induced a more delayed and prolonged immune response in the middle ear than that of pili-positive M. catarrhalis. TLR2, -4, -5 and -9 mRNA expression was upregulated in neutrophils that infiltrated the middle ear cavity during AOM caused by both pili-positive and pili-negative bacteria. TLR5 mRNA expression and TLR5 protein in the neutrophils were induced more robustly by pili-positive M. catarrhalis. This immune response is likely to be related to neutrophil function such as toll-like 5-dependent phagocytosis. Our results show that mice may provide a useful AOM model for studying the role of M. catarrhalis. Furthermore, we show that pili play an important role in enhancing M. catarrhalis clearance from the middle ear that is probably mediated through neutrophil-dependent TLR5 signaling., (© 2013 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Aspergillus oryzae has been utilized for over 1000 years in Japan for the production of various traditional foods, and a large number of A. oryzae strains have been isolated and/or selected for the effective fermentation of food ingredients. Characteristics of genetic alterations among the strains used are of particular interest in studies of A. oryzae. Here, we have sequenced the whole genome of an industrial fungal isolate, A. oryzae RIB326, by using a next-generation sequencing system and compared the data with those of A. oryzae RIB40, a wild-type strain sequenced in 2005. The aim of this study was to evaluate the mutation pressure on the non-syntenic blocks (NSBs) of the genome, which were previously identified through comparative genomic analysis of A. oryzae, Aspergillus fumigatus, and Aspergillus nidulans. We found that genes within the NSBs of RIB326 accumulate mutations more frequently than those within the SBs, regardless of their distance from the telomeres or of their expression level. Our findings suggest that the high mutation frequency of NSBs might contribute to maintaining the diversity of the A. oryzae genome.

A 47-year-old female patient with Behçet's disease had been treated with colchicine, prednisolone, cyclosporine A, and infliximab. Because she relapsed, however, treatment with tocilizumab, a humanized anti-interleukin 6 receptor antibody, was started. This treatment suppressed the patient's clinical manifestations, including ocular attacks, for 1 year and improved her visual acuity. This experience indicates that tocilizumab may constitute a therapeutic option for refractory Behçet's disease.

MicroRNAs (miRNAs) are a class of noncoding small RNAs that act as negative regulators of gene expression. To identify miRNAs that may regulate human cell immortalization and carcinogenesis, we performed comparative miRNA array profiling of human normal and SV40-T antigen immortalized cells. We found that miR-296 was upregulated in immortalized cells that also had activation of telomerase. By an independent experiment on genomic analysis of cancer cells we found that chromosome region (20q13.32), where miR-296 is located, was amplified in 28/36 cell lines, and most of these showed enriched miR-296 expression. Overexpression of miR-296 in human cancer cells, with and without telomerase activity, had no effect on their telomerase function. Instead, it suppressed p53 function that is frequently downregulated during human cell immortalization and carcinogenesis. By monitoring the activity of a luciferase reporter connected to p53 and p21(WAF1) (p21) untranslated regions (UTRs), we demonstrate that miR-296 interacts with the p21-3'UTR, and the Hu binding site of p21-3'UTR was identified as a potential miR-296 target site. We demonstrate for the first time that miR-296 is frequently upregulated during immortalization of human cells and contributes to carcinogenesis by downregulation of p53-p21(WAF1) pathway.

Objectives: Macrolide antibiotics are used for the treatment of immunological disorders such as psoriasis. However, few studies have investigated the immunoregulatory efficacy of macrolides in bacterial superantigen-stimulated immune cells., Methods: The suppressive efficacies of azithromycin, clarithromycin, roxithromycin and prednisolone were evaluated in vitro against the concanavalin A- or toxic shock syndrome toxin 1 (TSST-1)-induced proliferation of peripheral-blood mononuclear cells (PBMCs) obtained from nine healthy subjects. The concentrations of six cytokines in a PBMC-culture medium were measured using bead-array procedures followed by flow cytometry. Cellular c-jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) activity were measured using cell-based ELISA procedures., Key Findings: Azithromycin, clarithromycin and roxithromycin inhibited the proliferation of both the concanavalin A- and superantigen-stimulated PBMCs dose-dependently. The effect of azithromycin was the strongest, with IC50 values of less than 5 µg/ml. Furthermore, the suppressive efficacy of prednisolone against concanavalin A- or TSST-1-stimulated PBMCs was significantly promoted in combination with 5 µg/ml azithromycin (P < 0.002). The concentrations of TNF-α, interleukin (IL)-2, -4, -5 and -10 in the supernatant of concanavalin A- or TSST-1-stimulated PBMCs cultured for 72 h decreased by 65-98% in the presence of 5 µg/ml azithromycin. The stimulation of PBMCs with concanavalin A or TSST-1 increased cellular JNK and ERK activity, and 5 µg/ml azithromycin significantly attenuated the increased activity of JNK in the TSST-1-stimulated cells and ERK in the concanavalin A- and TSST-1-stimulated PBMCs, respectively (P < 0.05)., Conclusions: Azithromycin suppresses mitogen- or superantigen-induced proliferation of PBMCs by possibly inhibiting both cellular JNK and ERK activity., (© 2011 The Authors. JPP © 2011 Royal Pharmaceutical Society.)

Objectives: For patients with advanced melanoma, no treatment options are available at present that provide either sufficient response rates or a significant prolongation of overall survival. The present study examines the effects of two inorganic and six organic arsenic compounds on cell proliferation and cell invasion of melanoma cells in vitro., Methods: The effects of arsenic compounds on proliferation of human melanoma A375 cells and murine melanoma B16F10 cells were examined by MTT assay and 5-bromo-2'-deoxyuridine (BrdU) incorporation assay, and the effects of the compounds on cell invasion were examined by the Boyden chamber invasion assay. The amounts of active matrix metalloproteinase (MMP)-2 and pro-MMP-2 in the culture supernatant of A375 cells were determined by an MMP-2 activity assay system., Key Findings: Arsenate and arsenic trioxide (As(2) O(3) ) inhibited the proliferation of A375 and B16F10 cells significantly at concentration ranges of 0.1-20µg/ml (P<0.001), while the organic compounds arsenobetaine, arsenocholine, dimethylarsinic acid, methylarsonic acid, tetramethylarsonium and trimethylarsine oxide did not show any inhibitory effects even at 20µg/ml. Cell invasion of A375 and B16F10 cells through a layer of collagen IV was significantly inhibited by 0.1-20 µg/ml of arsenate or As(2) O(3) (P<0.05), while the organic compounds did not inhibit cell invasion. Arsenate or As(2) O(3) at 0.2-10µg/ml significantly inhibited the amount of active MMP-2 and pro-MMP-2 secreted into the A375 cell culture supernatant (P<0.05)., Conclusions: Our findings show that the inorganic arsenic compounds arsenate and As(2) O(3) inhibit cell proliferation and prevent the invasive properties of melanoma cells, possibly by decreasing MMP-2 production from the cells., (© 2011 The Authors. JPP © 2011 Royal Pharmaceutical Society.)

Aims: To investigate the attractant effect of 4-O-(N-acetyl-β-D-glucosaminyl)-D-glucosamine (GlcNAc-GlcN) in the chemotaxis of Vibrio bacteria that produce carbohydrate esterase (CE) family 4 chitin oligosaccharide deacetylase (COD), an enzyme that catalyzes the production of GlcNAc-GlcN from N,N'-diacetylchitobiose (GlcNAc)(2)., Methods and Results: The chemotactic effect of disaccharides from chitin on several strains of Vibrio bacteria was investigated using an agar gel lane-migration method. The results demonstrated that GlcNAc-GlcN functions as an effective chemoattractant in the CE family 4 COD-producing vibrios, Vibrio parahaemolyticus and Vibrio alginolyticus. In contrast, this phenomenon was not observed in Vibrio nereis or Vibrio furnissii, which lack genes encoding this enzyme. From transmission electron microscope observation of V. parahaemolyticus cells following the chemotaxis assay, GlcNAc-GlcN appears to stimulate polar flagellum rotation., Conclusions: GlcNAc-GlcN is a specific chemoattractant for the CE family 4 COD-producing vibrios, V. parahaemolyticus and V. alginolyticus., Significance and Impact of the Study: It was clarified for the first time that GlcNAc-GlcN functions as a signalling molecule in the chemotaxis of Vibrio bacteria that have an ability to produce CE family 4 COD, which generate GlcNAc-GlcN from (GlcNAc)(2)., (© 2011 The Authors. Letters in Applied Microbiology © 2011 The Society for Applied Microbiology.)

A 65-year-old woman who had suffered from chronic graft-versus-host disease (GVHD) presented with extensive purpura and was diagnosed with acquired hemophilia A. Because she was refractory to corticosteroids and her condition was complicated with diabetes mellitus, glaucoma, and hypoglobulinemia, she was treated with tocilizumab. Tocilizumab treatment increased the activity of factor VIII in a rapid and sustained manner, leading to a reduction of the prednisolone dose. Tocilizumab may thus be an optional treatment modality for acquired hemophilia A.

Background: Plasma concentrations of small dense (sd)-LDL are associated with the prevalence of cardiovascular events. However, the special equipment and long assay times required for sd-LDL measurement have hindered its clinical application. Herein, we report development of a simple homogeneous assay for sd-LDL-cholesterol (C) adaptable to autoanalyzers., Materials and Methods: We identified suitable surfactants and phospholipases by screening for those selective for the sd-LDL fraction (d 1.044-1.063 kg/L) and for the dissociation of other lipoproteins, including large buoyant LDL (lb-LDL). Principal characteristics of this assay were compared with ultracentrifugal isolation of LDL subfractions and with our previous heparin-magnesium precipitation assay for sd-LDL. We measured sd-LDL-C concentrations in 460 healthy, normolipidemic individuals., Results: We used a polyoxyethylene benzylphenyl ether derivative to dissociate triglyceride-rich lipoproteins and HDLs, whereas sphingomyelinase proved most effective for dissociation of lb-LDL from LDL owing to the higher sphingomyelin content in the lb-LDL subfractions. A polyoxyethylene styrenephenyl ether derivative protected sd-LDL against the dissociative actions of sphingomyelinase and cholesterol oxidase/esterase during an initial incubation step. Next, polyoxyethylene alkyl ether dissociated sd-LDL-C and the cholesterol released from sd-LDL were subsequently measured by using cholesterol oxidase/esterase. The homogeneous method correlated excellently with ultracentrifugation for sd-LDL-C (y = 0.99x-0.09, R(2) = 0.91, n = 60) and exhibited within-run precision CVs <1.1%. The distribution of sd-LDL-C was skewed, and the central 95% of sd-LDL-C concentrations ranged from 0.24 to 0.88 mmol/L (9.4-34.0 mg/dL)., Conclusions: The homogeneous assay allows reproducible measurement of sd-LDL-C within 10 min and appears promising in further investigations of the clinical significance of sd-LDL-C.

Objective: SSc is an autoimmune disease characterized by fibrosis of the skin and internal organs. Although the aetiology remains uncertain, many reports have suggested that IL-6 is involved in SSc pathogenesis. Tocilizumab, an anti-IL-6 receptor antibody, is an anti-arthritis medicine that works through the blockade of IL-6 functions. To examine the effect of tocilizumab on SSc, we administered tocilizumab to two SSc patients., Methods: Two dcSSc patients were administered tocilizumab at 8 mg/kg once a month for 6 months. One patient had pulmonary fibrosis assessed by CT and spirometry, and the other had chronic renal failure caused by scleroderma renal crisis. Their skin condition was monitored with a Vesmeter and the modified Rodnan total skin score (mRTSS). Skin biopsies were obtained before and after the tocilizumab treatment to investigate the histological changes., Results: After tocilizumab treatment, both patients showed softening of the skin with reductions of 50.7 and 55.7% in the total z-score of Vesmeter hardness and 51.9 and 23.0% in the mRTSS, respectively. Histological examination showed thinning of the collagen fibre bundles in the dermis. The creatinine clearance in the patient with chronic renal failure improved from 38 to 55 ml/min. However, the fibrotic changes in the lung in the other patient remained unchanged., Conclusions: In the two cases of SSc that we report here, softening of the skin was observed during the treatment with tocilizumab.

Interferon gamma (IFN-gamma) production is a critical step of antituberculosis (anti-TB) immune response. The purpose of this study was to determine the influences of biologics, including the interleukin (IL)-6 receptor-inhibitor tocilizumab (TCZ), and tumor necrosis factor (TNF) antagonists infliximab (INF) and etanercept (ETA), on Mycobacterium tuberculosis (MTB) antigen-induced IFN-gamma production. MTB antigen (ESAT-6 and CFP-10)-induced IFN-gamma-releasing assay was performed with or without addition of biologics (TCZ, ETA, and INF) using whole blood from patients with active TB. ETA and INF inhibited IFN-gamma production in a dose-dependent manner. In whole blood from TB patients, ESAT-6 stimulated significant production of IFN-gamma (1.30 +/- 1.95 IU/ml), and TCZ did not inhibit IFN-gamma production (1.56 +/- 1.88 IU/ml). IFN-gamma production by ESAT-6 was inhibited by ETA and INF (0.98 +/- 1.74, 0.75 +/- 1.66 IU/ml, respectively). CFP-10 stimulated significant production of IFN-gamma (1.46 +/- 1.60 IU/ml), and TCZ did not inhibit IFN-gamma production (1.51 +/- 1.77 IU/ml). IFN-gamma production by CFP-10 was inhibited by ETA and INF (0.91 +/- 0.99, 0.72 +/- 0.88 IU/ml, respectively). TCD did not inhibit MTB antigen-induced IFN-gamma production. As IFN-gamma production is important in antimycobacterial host defenses, the minimal influence of TCZ on IFN-gamma-releasing assay suggests a low risk of latent TB infection reactivation during tocilizumab therapy.

The function of cytochrome c(554) of Vibrio parahaemolyticus has not yet been determined. We have determined the physicochemical properties and crystal structure of cytochrome c(554) at 1.8 A in order to help elucidate its function. The physicochemical properties and the tertiary structure of cytochrome c(554) resemble those of dimeric cytochrome c(552) from Pseudomonas nautica, but the Vibrio genus contains no gene for nitrite reductase, cytochrome cd(1), in its genome DNA. These results raise the possibility that both cytochromes denote an electron to an electron carrier and accept an electron from same electron carrier.

A genomic DNA sequence and cDNA encoding a putative chitin synthase were isolated from the white rot basidiomycete Lentinula edodes. The gene, named LeChs2, consists of a 2,598-bp open reading frame interrupted by 14 introns and encodes a putative protein of 866 amino acid residues. The data obtained in this study suggest that LeChs2 belongs to the class II chitin synthases.

The gill browning of Lentinula edodes fruit-bodies during preservation is thought to be due to melanin biosynthesis catalyzed by tyrosinase. We isolated a genomic DNA sequence and cDNA encoding a putative tyrosinase from the white rot basidiomycete Lentinula edodes (shiitake mushroom). The gene, named Letyr, consists of a 1,854-bp open reading frame interrupted by eight introns, and encodes a putative protein of 618 amino acid residues with an estimated molecular mass of 68 kDa. Amino acid residues known to be involved in copper-binding domains were conserved in the deduced amino acid residues of LeTyr. Transcriptional and translational expression of Letyr in the gills of the fruit-body increased during preservation after harvest. This correlation between Letyr expression and fruit-body preservation suggests that tyrosinase gene expression contributes to gill browning.