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In this study, a three-dimensional electromagnetic thermal fluid simulation program was developed to simulate arc movement numerically while considering metal vapor generated from a molten metal bridge along with an electrode opening under external magnetic field application. The change in the physical properties of the arc and the increment rate of the arc voltage per unit length under different electrode opening velocities were analyzed by applying an external magnetic field with different intensities. It was found that the current path of the arc became dispersed when the electrode opening velocity was high because the metal vapor generated from the molten metal bridge remained dominant and highly concentrated. Before the completion of the electrode opening process, the difference in the increment rate of the arc voltage per unit length at different electrode opening velocities became more significant when an external magnetic field was applied. This was because under high and low electrode opening velocities, the cross-sectional area of the arc became narrow and broad, respectively.

In computer-aided diagnosis systems for lung cancer, segmentation of lung nodules is important for analyzing image features of lung nodules on computed tomography (CT) images and distinguishing malignant nodules from benign ones. However, it is difficult to accurately and robustly segment lung nodules attached to the chest wall or with ground-glass opacities using conventional image processing methods. Therefore, this study aimed to develop a method for robust and accurate three-dimensional (3D) segmentation of lung nodule regions using deep learning. In this study, a nested 3D fully connected convolutional network with residual unit structures was proposed, and designed a new loss function. Compared with annotated images obtained under the guidance of a radiologist, the Dice similarity coefficient (DS) and intersection over union (IoU) were 0.845 ± 0.008 and 0.738 ± 0.011, respectively, for 332 lung nodules (lung adenocarcinoma) obtained from 332 patients. On the other hand, for 3D U-Net and 3D SegNet, the DS was 0.822 ± 0.009 and 0.786 ± 0.011, respectively, and the IoU was 0.711 ± 0.011 and 0.660 ± 0.012, respectively. These results indicate that the proposed method is significantly superior to well-known deep learning models. Moreover, we compared the results obtained from the proposed method with those obtained from conventional image processing methods, watersheds, and graph cuts. The DS and IoU results for the watershed method were 0.628 ± 0.027 and 0.494 ± 0.025, respectively, and those for the graph cut method were 0.566 ± 0.025 and 0.414 ± 0.021, respectively. These results indicate that the proposed method is significantly superior to conventional image processing methods. The proposed method may be useful for accurate and robust segmentation of lung nodules to assist radiologists in the diagnosis of lung nodules such as lung adenocarcinoma on CT images.

Objective: To provide data on the molecular characteristics of extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae clinical isolates in Japan. Methods: A total of 100 clinical isolates of ESBL-producing K. pneumoniae collected throughout Japan between June and July 2018 were studied. ESBL genes were analyzed using PCR and DNA sequencing. Transferability of ESBL genes was investigated by conjugation experiments. Plasmid replicon types, virulence genes (rmpA, rmpA2, iucA, iroB, and peg-344) associated with hypervirulent K. pneumoniae (hvKp), and capsule types were detected using PCR. Genotyping was performed using multilocus sequence typing. Results: All ESBL-producing isolates carried blaCTX-M genes. The most predominant CTX-M-type identified was CTX-M-15 (n = 55). We identified 24 sequence types (STs) among the CTX-M-15 producers, with ST25 (n = 8) being the most common. Most of the transconjugants carrying blaCTX-M-15 contained the FIIk replicon. Of the 100 ESBL-producing isolates, 31 were hvKp defined by the presence of the virulence genes. These ESBL-producing hvKp isolates belonged to eight STs (STs 23, 25, 36, 65, 86, 268, 412, and 4492), with five capsule types (K1, K2, K20, K57, and undefined). Conclusions: CTX-M-15 was the predominant ESBL among K. pneumoniae isolates from Japan. This study shows that ESBL-producing hvKp strains comprising various clones are emerging in Japan.

Abstract Background The aim of this in vitro study was to examine the possible enhancement of the biofilm peeling effect of a sonic toothbrush following the use of an antimicrobial mouth rinse. Methods The biofilm at a noncontact site in the interdental area was treated by sound wave convection with the test solution or by immersion in the solution. The biofilm peeling effect was evaluated by determining the bacterial counts and performing morphological observations. A Streptococcus mutans biofilm was allowed to develop on composite resin discs by cultivation with stirring at 50 rpm for 72 h. The specimens were then placed in recesses located between plastic teeth and divided into an immersion group and a combination group. The immersion group was treated with phosphate buffer, chlorhexidine digluconate Peridex™ (CHX) mouth rinse or Listerine® Fresh Mint (EO) mouth rinse. The combination group was treated with CHX or EO and a sonic toothbrush. Results The biofilm thickness was reduced by approximately one-half compared with the control group. The combination treatment produced a 1 log reduction in the number of bacteria compared to the EO immersion treatment. No significant difference was observed in the biofilm peeling effect of the immersion group compared to the control group. Conclusions The combined use of a sonic toothbrush and a mouth rinse enhanced the peeling of the biofilm that proliferates in places that are difficult to reach using mechanical stress.

Various carbapenemases have been identified in the Enterobacteriaceae. However, the induction and corresponding regulator genes of carbapenemase NmcA has rarely been detected in the Enterobacter cloacae complex (ECC). The NmcA-positive isolate ECC NR1491 was first detected in Japan in 2013. It was characterized and its induction system elucidated by evaluating its associated regulator genes nmcR, ampD, and ampR. The isolate was highly resistant to all β-lactams except for third generation cephalosporins (3GC). Whole-genome analysis revealed that blaNmcA was located on a novel 29-kb putatively mobile element called EludIMEX-1 inserted into the chromosome. The inducibility of β-lactamase activity by various agents was evaluated. Cefoxitin was confirmed as a strong concentration-independent β-lactamase inducer. In contrast, carbapenems induced β-lactamase in a concentration-dependent manner. All selected 3GC-mutants harboring substitutions on ampD (as ampR and nmcR were unchanged) were highly resistant to 3GC. The ampD mutant strain NR3901 presented with a 700 × increase in β-lactamase activity with or without induction. Similar upregulation was also observed for ampC and nmcA. NR1491 (pKU412) was obtained by transforming the ampR mutant (135Asn) clone plasmid whose expression increased by ∼100×. Like NR3901, it was highly resistant to 3GC. Overexpression of ampC, rather than nmcA, may have accounted for the higher MIC in NR1491. The ampR mutant repressed nmcA despite induction and it remains unclear how it stimulates nmcA transcription via induction. Future experiments should analyze the roles of nmcR mutant strains.

Memory is initially labile but can be consolidated into stable long-term memory (LTM) that is stored in the brain for extended periods. Despite recent progress, the molecular and cellular mechanisms underlying the intriguing neurobiological processes of LTM remain incompletely understood. Using the Drosophila courtship conditioning assay as a memory paradigm, here, we show that the LIM homeodomain (LIM-HD) transcription factor Apterous (Ap), which is known to regulate various developmental events, is required for both the consolidation and maintenance of LTM. Interestingly, Ap is involved in these 2 memory processes through distinct mechanisms in different neuronal subsets in the adult brain. Ap and its cofactor Chip (Chi) are indispensable for LTM maintenance in the Drosophila memory center, the mushroom bodies (MBs). On the other hand, Ap plays a crucial role in memory consolidation in a Chi-independent manner in pigment dispersing factor (Pdf)-containing large ventral-lateral clock neurons (l-LNvs) that modulate behavioral arousal and sleep. Since disrupted neurotransmission and electrical silencing in clock neurons impair memory consolidation, Ap is suggested to contribute to the stabilization of memory by ensuring the excitability of l-LNvs. Indeed, ex vivo imaging revealed that a reduced function of Ap, but not Chi, results in exaggerated Cl- responses to the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) in l-LNvs, indicating that wild-type (WT) Ap maintains high l-LNv excitability by suppressing the GABA response. Consistently, enhancing the excitability of l-LNvs by knocking down GABAA receptors compensates for the impaired memory consolidation in ap null mutants. Overall, our results revealed unique dual functions of the developmental regulator Ap for LTM consolidation in clock neurons and LTM maintenance in MBs.

Although rodents have been widely used for experimental models of spinal cord diseases, the details of the growth curves of their spinal canal and spinal cord, as well as the molecular mechanism of the growth of adult rat spinal cords remain unavailable. They are particularly important when conducting the experiments of cervical spondylotic myelopathy (CSM), since the disease condition depends on the size of the spinal canal and the spinal cord. Thus, the purposes of the present study were to obtain accurate growth curves for the spinal canal and spinal cord in rats; to define the appropriate age in weeks for their use as a CSM model; and to propose a molecular mechanism of the growth of the adult spinal cord in rats. CT myelography was performed on Lewis rats from 4 weeks to 40 weeks of age. The vertical growth of the spinal canal at C5 reached a plateau after 20 and 12 weeks, and at T8 after 20 and 16 weeks, in males and females, respectively. The vertical growth of the C5 and T8 spinal cord reached a plateau after 24 weeks in both sexes. The vertical space available for the cord (SAC) of C5 and T8 did not significantly change after 8 weeks in either sex. Western blot analyses showed that VEGFA, FGF2, and BDNF were highly expressed in the cervical spinal cords of 4-week-old rats, and that the expression of these growth factors declined as rats grew. These findings indicate that the spinal canal and the spinal cord in rats continue to grow even after sexual maturation and that rats need to be at least 8 weeks of age for use in experimental models of CSM. The present study, in conjunction with recent evidence, proposes the hypothetical model that the growth of rat spinal cord after the postnatal period is mediated at least in part by differentiation of neural progenitor cells and that their differentiation potency is maintained by VEGFA, FGF2, and BDNF.

The effect of Mfa1 fimbriae of Porphyromonas gingivalis on the progression of bone resorption remains unclear, especially compared with another fimbriae, FimA. We investigated the effect of Mfa1 on osteoclastogenesis together with FimA. We also investigated the role of Toll-like receptors (TLRs) in Mfa1 recognition during osteoclast differentiation. Receptor activator of nuclear factor κβ ligand (RANKL)-prestimulated RAW264 cells were used to examine the effects of purified Mfa1 fimbriae. The number of osteoclasts was examined by tartrate-resistant acid phosphate (TRAP) staining, osteoclast activation was investigated by bone resorption assays, and gene expression of differentiation markers was examined by quantitative real-time PCR. Transfection of Tlr2 and Tlr4 siRNAs into RAW264 cells was also employed and their role in Mfa1 recognition was investigated. Mfa1 effectively induced the formation of TRAP-positive multinucleated cells and activated osteoclasts. Mfa1 also increased gene expression of Acp5, Mmp9, and Ctsk in RANKL-prestimulated RAW264 cells compared with the control. The osteoclastogenesis induced by Mfa1 was significantly decreased in cells transfected with Tlr2 or Tlr4 siRNAs compared with control siRNA. Our results revealed the role of Mfa1 fimbriae in osteoclastogenesis that may contribute to the partial elucidation of the mechanisms of periodontal disease progression and the development of new therapeutic strategies.

Breast cancer comprised at least 21.8% of the overall cancer among young adult (YA) women and became the leading cancer in this group in Japan, with 50% adolescent and YAs being diagnosed and 15–44-year-old women showing excellent 5-year survival. Surgical-chemoradiation therapy often results in excellent survivorship with an increased incidence of treatment-induced subfertility. Therefore, adding fertility preservation (FP) to the primary cancer treatment is necessary. Herein, we reported a series of cases of YA women with breast cancer who opted for FP, where their option was tailored accordingly. To date, the selection of oocytes, embryos and ovarian tissue is widely available as an FP treatment. PGT could reduce the risk of BRCA mutation transmission amongst BRCA carriers before pregnancy planning. Otherwise, gonadotropin-releasing hormone analog has no gonadoprotective effect and thus should not be considered as an FP option.

Self-assembled supramolecular structures in living cells and their dynamics underlie various cellular events, such as endocytosis, cell migration, intracellular transport, cell metabolism, and gene expression. Spatiotemporally regulated association/dissociation and generation/degradation of assembly components is one of the remarkable features of biological systems. The significant advancement in DNA nanotechnology over the last few decades has enabled the construction of various-shaped nanostructures via programmed self-assembly of sequence-designed oligonucleotides. These nanostructures can further be assembled into micrometer-sized structures, including ordered lattices, tubular structures, macromolecular droplets, and hydrogels. In addition to being a structural material, DNA is adopted to construct artificial molecular circuits capable of activating/inactivating or producing/decomposing target DNA molecules based on strand displacement or enzymatic reactions. In this review, we provide an overview of recent studies on artificially designed DNA-based self-assembled systems that exhibit dynamic features, such as association/dis­sociation of components, phase separation, stimulus responsivity, and DNA circuit-regulated structural formation. These biomacromolecule-based, bottom-up approaches for the construction of artificial molecular systems will not only throw light on bio-inspired nano/micro engineering, but also enable us to gain insights into how autonomy and adaptability of living systems can be realized.

Abstract We developed and validated a childhood eating disorder outcome scale based on outcomes associated with body mass index standard deviation score (BMI-SDS). This prospective observational study included 131 children with eating disorders (aged 5–15 years). Participants’ outcomes scales were completed at the first visit and at 1, 3, 6, and 12 months. The scale evaluated 12 outcomes: body weight change (BW), eating attitude (EA), fear of being fat (FF), body image distortion (BD), menstruation (ME), perceived physical condition (PC), attending school (AS), disease recognition by school (RS), family function (FA), disease recognition by parent (RP), social adaptation (SA), and relationships with friends (RF). Responses to all items were on a four-point Likert scale. Exploratory factor analysis was used to determine the number of factors based on the 12 outcomes. The relation between outcome scale scores and BMI-SDS over the 12-month follow-up period was analyzed. Two types of factors were extracted: disease-specific factors (EA, FF, BD) and biopsychosocial factors (BW, PC, AS, FA, SA, RF). Three items (ME, RS, RP) were excluded because they showed no significant loading effect. There was a significant negative correlation between the outcome scale and BMI-SDS, and changes in outcome scale scores from baseline to 12 months were significantly associated with improvement in BMI-SDS. We developed a childhood eating disorder outcome scale characterized by disease-specific and biopsychosocial factors. Biopsychosocial management combined with a therapeutic approach for disease-specific symptoms may support body weight recovery for children with eating disorders.

A nematode Caenorhabditis elegans (C. elegans) is a filter feeder, which draws a suspension of bacteria and separates bacteria from the solvent by using pharyngeal pumping motions and specific mouth parts. This mechanism has not been fully understood. We investigated the mechanism of filtering of bacteria in the pharynx of C. elegans by visualization by fluorescent particles and dyed E. coli. We succeeded in quantifying the selectivity of bacteria-sized particles by C. elegans. The most accumulated particles were those of 0.5 μm in diameter. The quantity of accumulated particles of 0.2 μm or 1.0 μm in diameter was about one third of that of particles of 0.5 μm in diameter. The least accumulated particles were those of 0.05 μm in diameter. These results suggest that the pharyngeal structures of C. elegans would be suitable for eating bacteria because the size of bacteria ingested by C. elegans worms is about 0.5 μm in diameter. We also succeeded in visualizing pharyngeal structures and pumping motions and flow in the pharynx. We found that there were phase differences in the motions among procorpus, metacorpus and isthmus. This result suggests filtering would occur at the two tips of procorpus and isthmus by the phase differences. We found that bacteria-sized particles and bacteria were flowed and trapped in the channels, which existed along the central lumen from tip of procorpus to isthmus. From our results, we proposed the novel mechanism of filtering of bacteria through the channels for flowing and trapping. In future, this selective filtering mechanism of C. elegans would be applied to development of microfluidic filtration devices for medical and biological equipment. Keywords: Nematode, C. elegans, Pharynx, Pumping, Filtering, Selectivity

Colistin is used to treat infectious diseases in humans and livestock; it has also been used as a feed additive for livestock for approximately 50 years. Since the mcr-1 plasmid-mediated colistin resistance gene was discovered in China in 2015, it has been detected worldwide, mainly in livestock. In this study, we investigated the prevalence and characteristics of mcr-mediated colistin-resistant Escherichia coli in livestock and farmers in Japan. We collected fecal samples from 295 healthy livestock (202 cattle and 93 swine) and 62 healthy farmers from 72 livestock farms (58 cattle farms and 14 swine farms) between 2013 and 2015. Twenty-eight mcr-1-harboring E. coli strains were isolated from 25 livestock (six cattle and 19 swine) and three farmers (two cattle farmers and one swine farmer). The prevalence rates of mcr-1-harboring E. coli in livestock and farmers were 8.47 and 4.84%, respectively. Of the 28 strains, the resistance genes of three were transferable via the mcr-1-coding plasmids to E. coli J53 at low frequencies (10−7–10−8). Six strains coharbored mcr-1 with CTX-M β-lactamases (CTX-M-14, CTX-M-27, or CTX-M-156). Of the isolates obtained from livestock and farmers in four farms (farms C, I, N, and P), nine strains had the same genotypical characteristics (sequence types and pulsed-field gel electrophoresis band patterns), plasmid characteristics (incompatibility group and plasmid transferability), and minimum inhibitory concentrations. Thus, the findings suggested that clonal strains could spread among livestock and farmers within farms. To our knowledge, this is the first study to detect clonal relatedness of mcr-1-mediated colistin-resistant E. coli in livestock and farmers. It is suggested that farmers are at a higher risk of acquiring mcr-1-harboring strains, calling for our attention based on the One Health concept.

Background. Identifying the factors related to low bone mineral density (BMD) can have significant implications for preventing hip fractures. The correlation between ascending aortic calcification and BMD has never been reported. Therefore, the purpose of the current study is to confirm the hypothesis that ascending aortic calcification can be used as a predictive factor for low BMD and to find a radiographic sign to show it. Method. Plain film and computed tomography (CT) images of the thorax were obtained from 91 patients with hip fractures. Using the images, the calcification line of the ascending aorta adjacent to the aortic arch was evaluated. A prominent calcification line confirmed by both plain film and CT was classified as +2. A line which was ambiguous on plain film but confirmed by CT was classified as +1. Cases with no calcification were categorized as 0 (control). We compared the classified score with the BMD and calculated the kappa coefficient to measure intraobserver reliabilities for this radiographic finding. Results. Twenty-eight patients showed a +2 line, twenty-four patients showed a +1 line, and thirty-nine patients showed 0 lines. The median BMD of each group was 0.37 for the +2 line, 0.45 for the +1 line, and 0.51 for the 0 line. The BMD for the +2 group was significantly lower than the others. The kappa coefficient was approximately 0.6 (p

Deep-sea water (DSW) contains multiple minerals and is widely used as drinking water, for cosmetic purposes, and as seasoning. In this study, several types of extract-added water with different levels of hardness (200, 300, 500) were prepared from DSW collected off the coast of Muroto City, Kochi Prefecture. We administrated it to obese mice for two months and tested it for several effects. Although there was no anti-obesity effect for any hardness level in obese mice, the cognitive functions of each DSW-extract-added water-treated group were significantly improved compared to control obese mice in the water maze test. Time-to-fall by the rota-rod test was also dramatically improved in the DSW-extract-added water-treated groups. The levels of triglycerides and blood urea nitrogen were significantly decreased in DSW-extract-added water-treated obese mice. However, these results did not depend on the hardness. Hardness levels of 200 or 300 of DSW-extract-added water had greater effects on cognitive function and serum scores compared to a level of 500. We analyzed DSW using inductively coupled plasma atomic emission spectroscopy and inductively coupled plasma mass spectrometry. High concentrations of magnesium and potassium were detected, but sodium was not detected at very high concentrations. Although the detailed mechanisms of its effects are not yet understood, chronic intake of DSW-extract-added water may have a beneficial effect on health.

ABSTRACT Carbapenemase-producing Enterobacteriaceae represent a serious public health threat worldwide. Carbapenemase genes, harbored on a transferable plasmid, have been isolated globally with distinct geographical features. Klebsiella pneumoniae, included in Enterobacteriaceae, also produces carbapenemase and often shows hypervirulence. Overlapping carbapenem resistance and hypervirulence in K. pneumoniae have been reported, but such strains have not yet been found in Japan. Here, we screened 104 carbapenemase-producing K. pneumoniae isolates collected from 37 hospitals and outpatient clinics in Japan between September 2014 and July 2015. PCR and DNA sequencing demonstrated IMP-1 in 21 isolates and IMP-6 in 83 isolates, 77 of which coharbored CTX-M-2. Most of the isolates showed low MICs toward imipenem and meropenem but high MICs toward penicillin and cephalosporins. Conjugation experiments with an Escherichia coli J53 recipient showed that most of the plasmids in IMP-6 producers were transferable, whereas only one-half of the plasmids in IMP-1 producers were transferable. PCR-based replicon typing and multiplex PCR identified five isolates belonging to the CG258 non-tonB79 cluster and no isolate belonging to the CG258-tonB79 cluster or sequence type 307 (ST307). Four K1-ST23 isolates, 10 K2-ST65 isolates, and 7 K2-ST86 isolates were detected that harbored virulence genes. The resistance genes in 85 isolates were transferable, but the virulence genes were not transferred. These results demonstrate the acquisition of IMP-type carbapenemase genes and CTX-M-type genes among hypervirulence isolates in Japan, warranting further attention and countermeasures. In this study, we have determined the molecular characteristics and epidemiology of IMP-6 producers that coharbored various CTX-M genes in Japan. IMPORTANCE Carbapenems serve as a last resort for the clinical treatment of multidrug-resistant infections. Therefore, the rapid spread of carbapenemase-producing strains represents a serious public health threat, further limiting antibiotic choices. The current findings of hypervirulent carbapenemase-producing Klebsiella pneumoniae clinical isolates in Japan demonstrate the potential broad spread and transfer of these genes, necessitating close surveillance.

Introduction: The ongoing outbreak of novel coronavirus disease 2019 (COVID-19) is a worldwide problem. Although diagnosing COVID-19 in fracture patients is important for selecting treatment, diagnosing early asymptomatic COVID-19 is difficult. We describe herein a rare case of femoral intertrochanteric fracture concomitant with early asymptomatic novel COVID-19. Case presentation: An 87-year-old Japanese woman was transferred to our emergency room with a right hip pain after she fell. She had no fever, fatigue, or respiratory symptoms on admission and within the 14 days before presenting to our hospital, and no specific shadow was detected in chest X-ray. However, chest computed tomography (CT) was performed considering COVID-19 pandemic, and showed ground-glass opacities with consolidation in the dorsal segment of the right lower lung field. Then, qualitative real-time reverse-transcriptase-polymerase-chain-reaction (RT-PCR) was carried out and turned out to be positive. She was diagnosed right femoral intertrochanteric fracture with concomitant COVID-19 infection. Conservative treatment was applied to the fracture due to infection. After admission, fever and oxygen demand occurred but she recovered from COVID-19. Throughout the treatment period, no cross-infection from the patient was identified in our hospital. Conclusion: This case highlights the importance of considering chest CT as an effective screening method for infection on hospital admission in COVID-19-affected areas, especially in trauma patients with early asymptomatic novel COVID-19.

Ruxolitinib, a Janus kinase inhibitor, considerably improves symptoms of patients with polycythemia vera and primary or secondary myelofibrosis. However, its association with the development of infectious complications is a concern. Herein, we report the case of an 80-year-old man with primary myelofibrosis who developed disseminated tuberculosis during treatment with ruxolitinib at 15 mg twice daily and prednisone at 5 mg. We also reviewed the literature on patients who developed tuberculosis during treatment with ruxolitinib. There are 13 case reports of patients who developed tuberculosis during treatment with ruxolitinib, including our case. Disseminated tuberculosis manifestations were observed in 84.6 % of the patients and 50 % of them died. Although the interferon-gamma release assay was performed for seven of the patients with six positive results at the time of tuberculosis diagnosis, none were tested before the commencement of ruxolitinib. We suggest taking a history of tuberculosis and screening for and treating latent tuberculosis before administering ruxolitinib, especially in areas where the risk of tuberculosis is high.

Invasive Streptococcus agalactiae (GBS) infections are increasingly common among neonates and the elderly. Therefore, GBS surveillance for better antibiotic treatment and prophylaxis strategies are needed. We retrospectively evaluated the clinical aspects of invasive infections and the phenotypic and genetic diversity of infectious isolates from Nara, Japan, collected between 2007 and 2016, by using information from hospital records. GBS strains collected from the blood and cerebrospinal fluid cultures were evaluated for capsular types, multi-locus sequence typing (MLST), antibiotic susceptibility, antibiotics resistance gene, and pulsed-field gel electrophoresis. Forty GBS isolates (10 from children and 30 from adults) were analyzed, and the distribution of molecular serotype and allelic profiles varied between children and adults. We found the rates of early-onset disease in neonates with birth complications to be higher than that of previous reports, indicating that there could be relevance between complications at birth and early-onset disease. Standard antibiotic prophylaxis strategies may need to be reconsidered in patients with birth complications. In adults, the mean age of the patients was 68 years (male: 63%). Primary bacteremia was the most common source of infection. In the neonates, six had early-onset diseases and four had late-onset diseases. The most frequently identified strains were molecular serotype Ia ST23 (40%) and molecular serotype Ib ST10 (20%) in children and molecular serotype Ib ST10 (17%), molecular serotype VI ST1 (13%), and molecular serotype V ST1 (13%) in adults. Levofloxacin-resistant molecular serotype Ib strains and molecular serotypes V and VI ST1 were common causes of GBS infection in adults but were rarely found in children. Furthermore, pulsed-field gel electrophoresis in our study showed that specific clone isolates, that tend to have antibiotics resistance were widespread horizontally for a decade. Continuous surveillance and molecular investigation are warranted to identify the transmission route and improve antibiotic treatment strategies.

Periodontitis is one of the diabetic complications due to its high morbidity and severity in patients with diabetes. The prevention of periodontitis is especially important in diabetic patients because the relationship between diabetes and periodontitis is bidirectional. Here, we evaluated the impacts of glucagon-like peptide-1 (GLP-1) receptor agonist liraglutide on the amelioration of periodontitis. Five-wk-old Male Sprague–Dawley (SD) rats (n=30) were divided into 3 groups: normal, periodontitis, and periodontitis with liraglutide treatment groups. Periodontitis was induced by ligature around the maxillary second molar in SD rats. Half of the rats were administered liraglutide for 2 weeks. Periodontitis was evaluated by histological staining, gene expressions of inflammatory cytokines in gingiva, and microcomputed tomography. Periodontitis increased inflammatory cell infiltration, macrophage accumulation, and gene expressions of tumor necrosis factor-α and inducible nitric oxide synthase in the gingiva, all of which were ameliorated by liraglutide. Liraglutide decreased M1 macrophages but did not affect M2 macrophages in periodontitis. Moreover, ligature-induced alveolar bone resorption was ameliorated by liraglutide. Liraglutide treatment also reduced osteoclasts on the alveolar bone surface. These results highlight the beyond glucose-lowering effects of liraglutide on the treatment of periodontitis.

Streptococcus pneumoniae is an important causative organism of respiratory tract infections. Although periodontal bacteria have been shown to influence respiratory infections such as aspiration pneumonia, the synergistic effect of S. pneumoniae and Porphyromonas gingivalis, a periodontopathic bacterium, on pneumococcal infections is unclear. To investigate whether P. gingivalis accelerates pneumococcal infections, we tested the effects of inoculating P. gingivalis culture supernatant (PgSup) into S. pneumoniae-infected mice. Mice were intratracheally injected with S. pneumoniae and PgSup to induce pneumonia, and lung histopathological sections and the absolute number and frequency of neutrophils and macrophages in the lung were analyzed. Proinflammatory cytokine/chemokine expression was examined by qPCR and ELISA. Inflammatory cell infiltration was observed in S. pneumoniae-infected mice and S. pnemoniae and PgSup mixed-infected mice, and mixed-infected mice showed more pronounced inflammation in lung. The ratios of monocytes/macrophages and neutrophils were not significantly different between the lungs of S. pneumoniae-infected mice and those of mixed-infected mice. PgSup synergistically increased TNF-α expression/production and IL-17 production compared with S. pneumoniae infection alone. We demonstrated that PgSup enhanced inflammation in pneumonia caused by S. pneumoniae, suggesting that virulence factors produced by P. gingivalis are involved in the exacerbation of respiratory tract infections such as aspiration pneumonia.

Nucleic acid nanotechnology has great potential for future therapeutic applications. Here the authors build protein-driven RNA nanostructures that can function within mammalian cells and regulate the cell fate.

Abstract In vitro gliding assay of microtubules (MTs) on kinesins has provided us with valuable biophysical and chemo-mechanical insights of this biomolecular motor system. Visualization of MTs in an in vitro gliding assay has been mainly dependent on optical microscopes, limited resolution of which often render them insufficient sources of desired information. In this work, using high speed atomic force microscopy (HS-AFM), which allows imaging with higher resolution, we monitored MTs and protofilaments (PFs) of tubulins while gliding on kinesins. Moreover, under the HS-AFM, we also observed splitting of gliding MTs into single PFs at their leading ends. The split single PFs interacted with kinesins and exhibited translational motion, but with a slower velocity than the MTs. Our investigation at the molecular level, using the HS-AFM, would provide new insights to the mechanics of MTs in dynamic systems and their interaction with motor proteins.

Although peripheral nerves can regenerate, clinical outcomes after peripheral nerve injuries are not always satisfactory, especially in cases of severe or proximal injuries. Further, autologous nerve grafting remains the gold standard for the reconstruction of peripheral nerves, although this method is still accompanied by issues of donor-site morbidity and limited supply. Cell therapy is a potential approach to overcome these issues. However, the optimal cell type for promoting axon regeneration remains unknown. Here, we report a novel experimental model dedicated to elucidation of the axon-promoting effects of candidate cell types using simple and standardized techniques. This model uses rat sciatic nerves and consists of a 25 mm-long acellular region and a crush site at each end. The acellular region was made by repeated freeze/thaw procedures with liquid nitrogen. Importantly, the new model does not require microsurgical procedures, which are technically demanding and greatly affect axon regeneration. To test the actual utility of this model, red fluorescent protein-expressing syngeneic Schwann cells (SCs), marrow stromal cells, or fibroblasts were grafted into the acellular area, followed by perfusion of the rat 2 weeks later. All types of grafted cells survived well. Quantification of regenerating axons demonstrated that SCs, but not the other cell types, promoted axon regeneration with minimum variability. Thus, this model is useful for differentiating the effects of various grafted cell types in axon regeneration. Interestingly, regardless of the grafted cell type, host SCs migrated into the acellular area, and the extent of axon regeneration was strongly correlated with the number of SCs. Moreover, all regenerating axons were closely associated with SCs. These findings suggest a critical role for SCs in peripheral nerve axon regeneration. Collectively, this novel experimental model is useful for elucidating the axon-promoting effects of grafted cells and for analyzing the biology of peripheral nerve axon regeneration.

Almost all cases of carbapenemase-producing Enterobacteriaceae infections in Japan are caused by blaIMP-positive Enterobacteriaceae (especially blaIMP-6) and infections caused by other types of carbapenemase-producing Enterobacteriaceae are quite rare. We examined drug resistance genes co-harboring with blaIMP-6 and their inoculum size effects. We screened β-lactamase genes, plasmid-mediated quinolone resistance (PMQR) genes, and aminoglycoside-modifying enzyme genes by PCR and performed sequencing for 14 blaIMP-6-positive Enterobacteriaceae. Further, all PMQR-positive isolates were submitted to conjugation and inoculum effect evaluation. Our data showed that 13 of the 14 isolates harbored CTX-M-2 and one co-harbored CTX-M-2 and CTX-M-1 as extended-spectrum β-lactamases. All isolates carried one or more PMQRs; aac(6')-Ib-cr was the most prevalent (92.8%), and was followed by oqxA (64.3%), qnrS (50%), oqxAB (21.4%), and qnrB (14.3%). However, Klebsiella pneumoniae contains chromosomal OqxAB. Inoculum size effects were significant in all strains for meropenem, 13 strains for imipenem, 7 for levofloxacin, and 3 for amikacin. We observed that 11 of the experimental strains (100%), 8 strains (72.7%), and 1 strain showed inoculum size effects for meropenem, imipenem, and amikacin, respectively. However, four strains harbored qnr genes and two strains harbored qnr genes and QRDR mutations concurrently; no inoculum size effect was seen for levofloxacin. The blaIMP-6-positive Enterobacteriaceae that we studied was found to harbor at least one plasmid-mediated drug resistance gene. The inoculum size effect for carbapenems was thought to be mainly due to IMP-6-type metallo-β-lactamase; however qnrB and qnrS also had a minimal impact on the inoculum size effect for levofloxacin.

Porphyromonas gingivalis Mfa1 fimbriae are thought to act as adhesion factors and to direct periodontal tissue destruction but their immunomodulatory actions are poorly understood. Here, we investigated the effect of Mfa1 stimulation on the immune and metabolic mechanisms of gingival fibroblasts from periodontal connective tissue. We also determined the role of Toll-like receptor (TLR) 2 and TLR4 in Mfa1 recognition. Mfa1 increased the expression of genes encoding chemokine (C-X-C motif) ligand (CXCL) 1, CXCL3, intercellular adhesion molecule (ICAM) 1 and Selectin endothelium (E) in gingival fibroblasts, but did not have a significant effect on genes that regulate metabolism. Mfa1-stimulated up-regulation of genes was significantly suppressed in Tlr4 siRNA-transfected cells compared with that in control siRNA-transfected cells, which indicates that recognition by TLR4 is essential for immunomodulation by Mfa1. Additionally, suppression of Tlr2 expression partially attenuated the stimulatory effect of Mfa1. Overall, these results help explain the involvement of P. gingivalis Mfa1 fimbriae in the progression of periodontal disease.

Interleukin (IL)-35 is an immunosuppressive cytokine mainly produced by regulatory T cells. IL-35 mediates immunological functions by suppressing the inflammatory immune response. However, the role of IL-35 in bone-destructive diseases remains unclear, especially in terms of osteoclastogenesis. Therefore, the current study investigated the synergistic effect of IL-35 on osteoclastogenesis that is involved the pathogeneses of periodontitis and rheumatoid arthritis. Osteoclastic differentiation and osteoclastogenesis of RAW264 (RAW) cells induced by receptor activator of nuclear factor (NF)-κB ligand (RANKL) and IL-35 were evaluated by tartrate-resistant acid phosphate staining, hydroxyapatite resorption assays, and quantitative polymerase chain reaction. The effect of IL-35 on RANKL-stimulated signaling pathways was assessed by Western blot analysis. Costimulation of RAW cells by RANKL and IL-35 induced osteoclastogenesis significantly compared with stimulation by RANKL alone. Phosphorylations of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase tended to be increased by RANKL and IL-35 compared with RANKL or IL-35 alone. Additionally, the osteoclastogenesis induced by RANKL and IL-35 was suppressed by inhibition of ERK. In this study, IL-35 and RANKL induced osteoclastogenesis synergistically. Previous reports have shown that IL-35 suppresses the differentiation of osteoclasts. Therefore, IL-35 might play dual roles of destruction and protection in osteoclastogenesis.

Clathrin-mediated endocytosis (CME) proceeds through a series of morphological changes of the plasma membrane induced by a number of protein components. Although the spatiotemporal assembly of these proteins has been elucidated by fluorescence-based techniques, the protein-induced morphological changes of the plasma membrane have not been fully clarified in living cells. Here, we visualize membrane morphology together with protein localizations during CME by utilizing high-speed atomic force microscopy (HS-AFM) combined with a confocal laser scanning unit. The plasma membrane starts to invaginate approximately 30 s after clathrin starts to assemble, and the aperture diameter increases as clathrin accumulates. Actin rapidly accumulates around the pit and induces a small membrane swelling, which, within 30 s, rapidly covers the pit irreversibly. Inhibition of actin turnover abolishes the swelling and induces a reversible open-close motion of the pit, indicating that actin dynamics are necessary for efficient and irreversible pit closure at the end of CME.

Release of growth hormone (GH) from the somatotroph is regulated by binding GH-releasing hormone (GHRH) to its cognate receptor (GHRHR), one of the members of the G protein–coupled receptor (GPCR) superfamily. Proteins bound to the carboxy (C)-terminus of GPCR have been reported to regulate intracellular trafficking and function of the receptor; however, no functionally significant protein associated with GHRHR has been reported. We have identified a protein interacting with C-kinase 1 (PICK1) as a binding partner of GHRHR. In vitro binding assay revealed the PDZ-domain of PICK1 and the last four amino acid residues of GHRHR were prerequisite for the interaction. Further, in vivo association of these proteins was confirmed. Immunostaining data of a stable cell line expressing GHRHR with or without PICK1 suggested the C-terminus of GHRHR promoted cell surface expression of GHRHR and PICK1 affected the kinetics of the cell surface expression of GHRHR. Furthermore, cAMP production assay showed the C-terminus of GHRHR is involved in the regulation of receptor activation, and the interaction of GHRHR with PICK1 may influence intensities of the signal response after ligand stimulation. Thus, the interaction of the C-terminus of GHRHR with PICK1 has a profound role in regulating the trafficking and the signaling of GHRHR. [Supplementary Figure: available only at http://dx.doi.org/10.1254/jphs.12287FP] Keywords:: protein interacting with C-kinase 1 (PICK1), growth hormone–releasing hormone receptor (GHRHR), interaction, trafficking, signaling

Recombination-dependent DNA replication, which is a central component of viral replication restart, is poorly understood in Firmicutes bacteriophages. Phage SPP1 initiates unidirectional theta DNA replication from a discrete replication origin (oriL), and when replication progresses, the fork might stall by the binding of the origin binding protein G38P to the late replication origin (oriR). Replication restart is dependent on viral recombination proteins to synthesize a linear head-to-tail concatemer, which is the substrate for viral DNA packaging. To identify new functions involved in this process, uncharacterized genes from phage SPP1 were analyzed. Immediately after infection, SPP1 transcribes a number of genes involved in recombination and replication from P(E2) and P(E3) promoters. Resequencing the region corresponding to the last two hypothetical genes transcribed from the P(E2) operon (genes 44 and 45) showed that they are in fact a single gene, re-annotated here as gene 44, that encodes a single polypeptide, named gene 44 product (G44P, 27.5 kDa). G44P shares a low but significant degree of identity in its C-terminal region with virus-encoded RusA-like resolvases. The data presented here demonstrate that G44P, which is a dimer in solution, binds with high affinity but without sequence specificity to several double-stranded DNA recombination intermediates. G44P preferentially cleaves Holliday junctions, but also, with lower efficiency, replicated D-loops. It also partially complemented the loss of RecU resolvase activity in B. subtilis cells. These in vitro and in vivo data suggest a role for G44P in replication restart during the transition to concatemeric viral replication.

Condensin is required for chromosome dynamics and diverse DNA metabolism. How condensin works, however, is not well understood. Condensin contains two structural maintenance of chromosomes (SMC) subunits with the terminal globular domains connected to coiled-coil that is interrupted by the central hinge. Heterotrimeric non-SMC subunits regulate SMC. We identified a novel fission yeast SMC hinge mutant, cut14-Y1, which displayed defects in DNA damage repair and chromosome segregation. It contains an amino acid substitution at a conserved hinge residue of Cut14/SMC2, resulting in diminished DNA binding and annealing. A replication protein A mutant, ssb1-418, greatly alleviated the repair and mitotic defects of cut14-Y1. Ssb1 protein formed nucleolar foci in cut14-Y1 cells, but the number of foci was diminished in cut14-Y1 ssb1-418 double mutants. Consistent with the above results, Ssb1 protein bound to single-strand DNA was removed by condensin or the SMC dimer through DNA reannealing in vitro. Similarly, RNA hybridized to DNA may be removed by the SMC dimer. Thus, condensin may wind up DNA strands to unload chromosomal components after DNA repair and prior to mitosis. We show that 16 suppressor mutations of cut14-Y1 were all mapped within the hinge domain, which surrounded the original L543 mutation site.