Comprehensive analysis of insertion sequences within rRNA genes of CPR bacteria and biochemical characterization of a homing endonuclease encoded by these sequences.

The Candidate Phyla Radiation (CPR) represents an extensive bacterial clade comprising primarily uncultured lineages and is distinguished from other bacteria by a significant prevalence of insertion sequences (ISs) within their rRNA genes. However, our understanding of the taxonomic distribution and characteristics of these ISs remains limited. In this study, we used a comprehensive approach to systematically determine the nature of the rRNA ISs in CPR bacteria. The analysis of hundreds of rRNA gene sequences across 65 CPR phyla revealed that ISs are present in 48% of 16S rRNA genes and 82% of 23S rRNA genes, indicating a broad distribution across the CPR clade, with exceptions in the 16S and 23S rRNA genes of Candidatus (Ca.) Saccharibacteria and the 16S rRNA genes of Ca. Peregrinibacteria. Over half the ISs display a group-I-intron-like structure, whereas specific 16S rRNA gene ISs display features reminiscent of group II introns. The ISs frequently encode proteins with homing endonuclease (HE) domains, centered around the LAGLIDADG motif. The LAGLIDADG HE (LHE) proteins encoded by the rRNA ISs of CPR bacteria predominantly have a single-domain structure, deviating from the usual single- or double-domain configuration observed in typical prokaryotic LHEs. Experimental analysis of one LHE protein, I-ShaI from Ca. Shapirobacteria, confirmed that its endonuclease activity targets the DNA sequence of its insertion site, and chemical cross-linking experiments demonstrated its capacity to form homodimers. These results provide robust evidence supporting the hypothesis that the explosive proliferation of rRNA ISs in CPR bacteria was facilitated by mechanisms involving LHEs. IMPORTANCE: Insertion sequences (ISs) in rRNA genes are relatively limited and infrequent in most bacterial phyla. With a comprehensive bioinformatic analysis, we show that in CPR bacteria, these ISs occur in 48% of 16S rRNA genes and 82% of 23S rRNA genes. We also report the systematic and biochemical characterization of the LAGLIDADG homing endonucleases (LHEs) encoded by these ISs in the first such analysis of the CPR bacteria. This study significantly extends our understanding of the phylogenetic positions of rRNA ISs within CPR bacteria and the biochemical features of their LHEs.

Microbiome analysis of the restricted bacteria in radioactive element-containing water at the Fukushima Daiichi Nuclear Power Station.

A major incident occurred at the Fukushima Daiichi Nuclear Power Station following the tsunami triggered by the Tohoku-Pacific Ocean Earthquake in March 2011, whereby seawater entered the torus room in the basement of the reactor building. Here, we identify and analyze the bacterial communities in the torus room water and several environmental samples. Samples of the torus room water (1 × 109 Bq137Cs/L) were collected by the Tokyo Electric Power Company Holdings from two sampling points between 30 cm and 1 m from the bottom of the room (TW1) and the bottom layer (TW2). A structural analysis of the bacterial communities based on 16S rRNA amplicon sequencing revealed that the predominant bacterial genera in TW1 and TW2 were similar. TW1 primarily contained the genus Limnobacter, a thiosulfate-oxidizing bacterium. γ-Irradiation tests on Limnobacter thiooxidans, the most closely related phylogenetically found in TW1, indicated that its radiation resistance was similar to ordinary bacteria. TW2 predominantly contained the genus Brevirhabdus, a manganese-oxidizing bacterium. Although bacterial diversity in the torus room water was lower than seawater near Fukushima, ~70% of identified genera were associated with metal corrosion. Latent environment allocation-an analytical technique that estimates habitat distributions and co-detection analyses-revealed that the microbial communities in the torus room water originated from a distinct blend of natural marine microbial and artificial bacterial communities typical of biofilms, sludge, and wastewater. Understanding the specific bacteria linked to metal corrosion in damaged plants is important for advancing decommissioning efforts. IMPORTANCE: In the context of nuclear power station decommissioning, the proliferation of microorganisms within the reactor and piping systems constitutes a formidable challenge. Therefore, the identification of microbial communities in such environments is of paramount importance. In the aftermath of the Fukushima Daiichi Nuclear Power Station accident, microbial community analysis was conducted on environmental samples collected mainly outside the site. However, analyses using samples from on-site areas, including adjacent soil and seawater, were not performed. This study represents the first comprehensive analysis of microbial communities, utilizing meta 16S amplicon sequencing, with a focus on environmental samples collected from the radioactive element-containing water in the torus room, including the surrounding environments. Some of the identified microbial genera are shared with those previously identified in spent nuclear fuel pools in countries such as France and Brazil. Moreover, our discussion in this paper elucidates the correlation of many of these bacteria with metal corrosion.

Limitations of glycated albumin standardization when applied to the assessment of diabetes patients.

OBJECTIVES: Glycated albumin (GA) has potential value in the management of people with diabetes; however, to draw meaningful conclusions between clinical studies it is important that the GA values are comparable. This study investigates the standardization of the Norudia Glycated Albumin and Lucica Glycated Albumin-L methods. METHODS: The manufacturer reported imprecision was verified by performing CLSI-EP15-A3 protocol using manufacturer produced controls. The Japanese Clinical Chemistry Reference Material (JCCRM)611-1 was measured 20 times to evaluate the accuracy of both methods. GA was also measured in 1,167 patient samples and results were compared between the methods in mmol/mol and %. RESULTS: Maximum CV for Lucica was ≤0.6 % and for Norudia ≤1.8 % for control material. Results in mmol/mol and % of the JCCRM611-1 were within the uncertainty of the assigned values for both methods. In patient samples the relative difference in mmol/mol between the two methods ranged from -10.4 % at a GA value of 183 mmol/mol to +8.7 % at a GA value of 538 mmol/mol. However, the relative difference expressed in percentage units ranged from of 0 % at a GA value of 9.9 % to +1.7 % at a GA value of 30 %. CONCLUSIONS: The results in mmol/mol between the two methods for the patient samples were significantly different compared to the results in %. It is not clear why patient samples behave differently compared to JCCRM611-1 material. Valuable lessons can be learnt from comparing the standardization process of GA with that of HbA1c.

Study Profile of the Tsuruoka Metabolomics Cohort Study (TMCS).

The Tsuruoka Metabolomics Cohort Study (TMCS) is an ongoing population-based cohort study being conducted in the rural area of Yamagata Prefecture, Japan. This study aimed to enhance the precision prevention of multi-factorial, complex diseases, including non-communicable and aging-associated diseases, by improving risk stratification and prediction measures. At baseline, 11,002 participants aged 35-74 years were recruited in Tsuruoka City, Yamagata Prefecture, Japan, between 2012 and 2015, with an ongoing follow-up survey. Participants underwent various measurements, examinations, tests, and questionnaires on their health, lifestyle, and social factors. This study uses an integrative approach with deep molecular profiling to identify potential biomarkers linked to phenotypes that underpin disease pathophysiology and provide better mechanistic insights into social health determinants. The TMCS incorporates multi-omics data, including genetic and metabolomic analyses of 10,933 participants, and comprehensive data collection ranging from physical, psychological, behavioral, and social to biological data. The metabolome is used as a phenotypic probe because it is sensitive to changes in physiological and external conditions. The TMCS focuses on collecting outcomes for cardiovascular disease, cancer incidence and mortality, disability and functional decline due to aging and disease sequelae, and the variation in health status within the body represented by omics analysis that lies between exposure and disease. It contains several sub-studies on aging, heated tobacco products, and women's health. This study is notable for its robust design, high participation rate (89%), and long-term repeated surveys. Moreover, it contributes to precision prevention in Japan and East Asia as a well-established multi-omics platform.

Modified ESR-based photosafety test (ESR-PT) detecting singlet oxygen and free radical formation.

The electron spin resonance-based photosafety test (ESR-PT) was modified using a new parameter, photoreactivity index (PRI), to detect singlet oxygen and free radical photoproducts simultaneously. With this modification, the modified ESR-PT is expected to reduce the number of false negative results due to chemicals producing free radical photoproducts other than singlet oxygen. The assay performance of the modified ESR-PT was evaluated using 56 chemicals, including hydrophobic chemicals. When using the PRI cutoff value of 2.0 in the modified ESR-PT, the accuracy relative to photosafety reference data was 91.1%, and the applicability (100%) was better than the other non-animal photosafety test. Among the chemicals producing positive results, bithionol, fenticlor, and doxycycline HCl were considered positive based on the detection of free radical photoproducts, suggesting that these three chemicals may have phototoxic or photoallergic potential via radical reactions. Additionally, this finding demonstrated the fundamental advantage of the modified ESR-PT using ESR spectroscopy, which can detect radicals selectively and quantitatively. Accordingly, the new parameter PRI is effective for photosafety evaluations based on not only singlet oxygen but also free radical photoproducts generated from chemicals. Therefore, the modified ESR-PT has a great potential for a photosafety test method applicable to various chemicals.

A population-based urinary and plasma metabolomics study of environmental exposure to cadmium.

BACKGROUND: The application of metabolomics-based profiles in environmental epidemiological studies is a promising approach to refine the process of health risk assessment. We aimed to identify potential metabolomics-based profiles in urine and plasma for the detection of relatively low-level cadmium (Cd) exposure in large population-based studies. METHOD: We analyzed 123 urinary metabolites and 94 plasma metabolites detected in fasting urine and plasma samples collected from 1,412 men and 2,022 women involved in the Tsuruoka Metabolomics Cohort Study. Regression analysis was performed for urinary N-acetyl-beta-D-glucosaminidase (NAG), plasma, and urinary metabolites as dependent variables, and urinary Cd (U-Cd, quartile) as an independent variable. The multivariable regression model included age, gender, systolic blood pressure, smoking, rice intake, BMI, glycated hemoglobin, low-density lipoprotein cholesterol, alcohol consumption, physical activity, educational history, dietary energy intake, urinary Na/K ratio, and uric acid. Pathway-network analysis was carried out to visualize the metabolite networks linked to Cd exposure. RESULT: Urinary NAG was positively associated with U-Cd, but not at lower concentrations (Q2). Among urinary metabolites in the total population, 45 metabolites showed associations with U-Cd in the unadjusted and adjusted models after adjusting for the multiplicity of comparison with FDR. There were 12 urinary metabolites which showed consistent associations between Cd exposure from Q2 to Q4. Among plasma metabolites, six cations and one anion were positively associated with U-Cd, whereas alanine, creatinine, and isoleucine were negatively associated with U-Cd. Our results were robust by statistical adjustment of various confounders. Pathway-network analysis revealed metabolites and upstream regulator changes associated with mitochondria (ACACB, UCP2, and metabolites related to the TCA cycle). CONCLUSION: These results suggested that U-Cd was associated with metabolites related to upstream mitochondrial dysfunction in a dose-dependent manner. Our data will help develop environmental Cd exposure profiles for human populations.

Systematic Analysis of Diverse Polynucleotide Kinase Clp1 Family Proteins in Eukaryotes: Three Unique Clp1 Proteins of Trypanosoma brucei.

The Clp1 family proteins, consisting of the Clp1 and Nol9/Grc3 groups, have polynucleotide kinase (PNK) activity at the 5' end of RNA strands and are important enzymes in the processing of some precursor RNAs. However, it remains unclear how this enzyme family diversified in the eukaryotes. We performed a large-scale molecular evolutionary analysis of the full-length genomes of 358 eukaryotic species to classify the diverse Clp1 family proteins. The average number of Clp1 family proteins in eukaryotes was 2.3 ± 1.0, and most representative species had both Clp1 and Nol9/Grc3 proteins, suggesting that the Clp1 and Nol9/Grc3 groups were already formed in the eukaryotic ancestor by gene duplication. We also detected an average of 4.1 ± 0.4 Clp1 family proteins in members of the protist phylum Euglenozoa. For example, in Trypanosoma brucei, there are three genes of the Clp1 group and one gene of the Nol9/Grc3 group. In the Clp1 group proteins encoded by these three genes, the C-terminal domains have been replaced by unique characteristics domains, so we designated these proteins Tb-Clp1-t1, Tb-Clp1-t2, and Tb-Clp1-t3. Experimental validation showed that only Tb-Clp1-t2 has PNK activity against RNA strands. As in this example, N-terminal and C-terminal domain replacement also contributed to the diversification of the Clp1 family proteins in other eukaryotic species. Our analysis also revealed that the Clp1 family proteins in humans and plants diversified through isoforms created by alternative splicing.

Metabolomics profiles alterations in cigarette smokers and heated tobacco product users.

BACKGROUND: Heated tobacco products (HTPs) have gained global popularity, but their health risks remain unclear. Therefore, the current study aimed to identify plasma metabolites associated with smoking and HTP use in a large Japanese population to improve health risk assessment. METHODS: Metabolomics data from 9,922 baseline participants of the Tsuruoka Metabolomics Cohort Study (TMCS) were analyzed to determine the association between smoking habits and plasma metabolites. Moreover, alterations in smoking-related metabolites among HTP users were examined based on data obtained from 3,334 participants involved from April 2018 to June 2019 in a follow-up survey. RESULTS: Our study revealed that cigarette smokers had metabolomics profiles distinct from never smokers, with 22 polar metabolites identified as candidate biomarkers for smoking. These biomarker profiles of HTP users were closer to those of cigarette smokers than those of never smokers. The concentration of glutamate was higher in cigarette smokers, and biomarkers involved in glutamate metabolism were also associated with cigarette smoking and HTP use. Network pathway analysis showed that smoking was associated with the glutamate pathway, which could lead to endothelial dysfunction and atherosclerosis of the vessels. CONCLUSIONS: Our study showed that the glutamate pathway is affected by habitual smoking. These changes in the glutamate pathway may partly explain the mechanism by which cigarette smoking causes cardiovascular disease. HTP use was also associated with glutamate metabolism, indicating that HTP use may contribute to the development of cardiovascular disease through mechanisms similar to those in cigarette use.

Microenvironmental Analysis and Control for Local Cells under Confluent Conditions via a Capillary-Based Microfluidic Device.

Sophisticated functions of biological tissues are supported by small biological units of cells that are localized within a region of 100 µm scale. The cells in these units secrete molecules to form their microenvironment to play a vital role in biological functions. Various microfluidic devices have been developed to analyze the microenvironment but were not designed for cells in a culture dish in a confluent condition, a typical setup for cell and tissue cultivation. This study presents a novel glass capillary-based microfluidic device for studying confluent cells in a culture dish. The multiple capillaries allow the device to confine the local flow in 100 µm or smaller scale to form two adjacent regions with different chemical properties; it can simultaneously perform local cell stimulation and collect secreted molecules from the stimulated cells. Cell removal was achieved upon trypsin stimulation from a limited area (3.8 × 10-3 ± 1.0 × 10-3 mm2), which corresponded to 7.6 ± 2.0 cells, using the mouse skeletal myoblast cell line (C2C12 cells) in a confluent condition. Microenvironmental analysis was demonstrated by measuring the secreted tumor necrosis factor alpha (TNF-α) collected from the microenvironment of the stimulated and unstimulated mouse leukemic monocyte cell line (RAW264 cells) to track temporal changes in the TNF-α production. The TNF-α secreted from stimulated cells was approximately four-fold higher than that from unstimulated cells in 90 min. This device enables local cell stimulation and the collection of secreted molecules for cells under confluent conditions, which contributes to the analysis of the cellular microenvironment.

A phase 3, randomized, double-blind, clinical study to evaluate the long-term safety and efficacy of gefapixant in Japanese adult participants with refractory or unexplained chronic cough.

BACKGROUND: In two phase 3, global clinical trials (COUGH-1 and COUGH-2), the P2X3-receptor antagonist gefapixant significantly reduced objective 24-h cough frequency in participants with refractory or unexplained chronic cough (RCC or UCC) at a dosage of 45 mg twice daily (BID), with an acceptable safety profile. The primary objective of this phase 3, randomized, double-blind, parallel-group study was to assess the safety and tolerability of gefapixant in Japanese participants with RCC or UCC (ClinicalTrials.gov, NCT03696108; JAPIC-CTI, 184154). METHODS: Participants aged ≥20 years with chronic cough lasting ≥4 months and a diagnosis of RCC or UCC despite treatment in accordance with Japanese Respiratory Society guidelines were randomized 1:1 to receive gefapixant 15 or 45 mg BID for 52 weeks. The primary objective was to evaluate the safety and tolerability of gefapixant, including adverse events (AEs) and discontinuations due to AEs. Cough-specific quality of life was assessed using the Leicester Cough Questionnaire as a secondary objective. RESULTS: Of 169 randomized and treated participants, 63% were female and mean age was 58 years. Adverse events were reported by 79 (94%) and 82 (96%) participants in the 15- and 45-mg BID groups, respectively. Most treatment-related AEs were taste related. Discontinuations due to AEs occurred in 6 (7%) and 17 (20%) participants receiving gefapixant 15 or 45 mg BID, respectively. There were no serious treatment-related AEs or deaths. Leicester Cough Questionnaire total scores improved from baseline through Week 52. CONCLUSIONS: Gefapixant had an acceptable safety profile, with no serious treatment-related AEs in Japanese participants.

Efficacy and safety of ipragliflozin in Japanese patients with type 2 diabetes and inadequate glycaemic control on sitagliptin.

AIMS: To assess the efficacy, safety and tolerability of ipragliflozin 50 mg once daily added to sitagliptin 50 mg once daily monotherapy in Japanese patients with type 2 diabetes (T2D). MATERIALS AND METHODS: The results of two clinical trials are reported. In both trials, patients had glycated haemoglobin (HbA1c) levels of 7.0% to 10.0% on sitagliptin 50 mg once daily 2 weeks prior to addition of ipragliflozin or placebo. In one trial (Trial 843), patients were randomized 1:1 to addition of blinded ipragliflozin 50 mg once daily (n = 73) or placebo (n = 70) for 24 weeks; the primary endpoint was efficacy (change in HbA1c at Week 24). In the other trial (Trial 849), open-label ipragliflozin 50 mg once daily was added for 52 weeks (n = 77); the primary objective was to assess safety/tolerability. RESULTS: In Trial 843, baseline characteristics were similar between groups (mean age 60.5 years, HbA1c 8.0%); after 24 weeks, adding ipragliflozin provided significantly greater reduction in HbA1c compared to placebo: least squares mean difference -0.77% (95% confidence interval -0.98, -0.57; P <0.001). In Trial 843, the incidences of adverse events (AEs) overall and prespecified AEs of clinical interest (symptomatic hypoglycaemia, urinary tract infection, genital infection, hypovolaemia, and polyuria/pollakiuria) were similar between groups. In Trial 849, specific AEs with incidence ≥5% were nasopharyngitis, pollakiuria, back pain, thirst, constipation, influenza and arthralgia; drug-related AEs reported in ≥2 patients were pollakiuria, thirst and constipation. CONCLUSIONS: Ipragliflozin 50 mg once daily added on to sitagliptin 50 mg once daily monotherapy provided significant improvement in glycaemic control and was generally well tolerated in Japanese patients with T2D. ClinicalTrials.gov: NCT02577003, NCT02564211.

A randomized, placebo-controlled trial to assess the efficacy and safety of sitagliptin in Japanese patients with type 2 diabetes and inadequate glycaemic control on ipragliflozin.

AIMS: To investigate the efficacy, safety and tolerability of sitagliptin 50 mg once daily added to ipragliflozin 50 mg once daily monotherapy in Japanese patients with type 2 diabetes (T2D). MATERIALS AND METHODS: Japanese patients with T2D and glycated haemoglobin (HbA1c) 7.0% to 10.0% while treated with ipragliflozin 50 mg once daily were randomized 1:1 to additional treatment with sitagliptin 50 mg once daily (N = 70) or matching placebo (N = 71) for 24 weeks. The primary efficacy endpoint was change in HbA1c at Week 24. Secondary efficacy endpoints were changes in 2-hour post-meal glucose (PMG), total PMG 0- to 2-hour area under the curve (AUC0-2h ), and fasting plasma glucose (FPG). RESULTS: Baseline characteristics were similar in the two groups (mean age 55.5 years, mean baseline HbA1c 8.0%). After 24 weeks, the addition of sitagliptin provided significantly greater reduction in HbA1c compared to placebo (least squares [LS] mean difference -0.83% [95% confidence interval -1.05, -0.62]; P <0.001). Significant reductions were also observed in all secondary endpoints: LS mean differences from placebo in changes in 2-hour PMG, total PMG AUC0-2h , and FPG were -42.5 mg/dL, -67.0 mg·h/dL and -11.2 mg/dL, respectively (all P <0.001). The incidence of adverse events (AEs) overall and incidence of predefined AEs of clinical interest (symptomatic hypoglycaemia, urinary tract infection, genital infection, hypovolaemia and polyuria/pollakiuria) were similar in the two groups. CONCLUSIONS: In Japanese patients with T2D, sitagliptin 50 mg once daily added to ipragliflozin 50 mg once daily monotherapy provided significant improvement in glycaemic control and was generally well tolerated. ClinicalTrials.gov: NCT02577016.

A randomized, placebo-controlled study to evaluate the efficacy and safety of adding omarigliptin to insulin therapy in Japanese patients with type 2 diabetes and inadequate glycaemic control.

AIM: To evaluate the efficacy and safety of adding the once-weekly oral dipeptidyl peptidase-4 inhibitor omarigliptin to treatment of Japanese patients with type 2 diabetes and inadequate glycaemic control on insulin monotherapy. MATERIALS AND METHODS: In a 52-week clinical trial, Japanese patients on insulin monotherapy were randomized to once-weekly omarigliptin 25 mg (N = 123) or placebo (N = 61) for a 16-week, double-blind, placebo-controlled period. After Week 16, patients continued or switched to omarigliptin for a 36-week open-label period. RESULTS: From a mean baseline of approximately 8.8%, the Week 16 least squares mean changes in HbA1c were -0.61% (omarigliptin) and 0.29% (placebo); the between-group difference was -0.90% (p < .001). At Week 52, the mean change from baseline in HbA1c was -0.57% in both the group on omarigliptin for 52 weeks and the group on omarigliptin for 36 weeks (switched from placebo at Week 16). During the first 16 weeks of treatment, the incidences of adverse events (AEs), serious AEs, drug-related AEs and discontinuation from trial medication because of an AE were similar in both groups. A slight increase in incidence of symptomatic hypoglycaemia was observed in the omarigliptin group (n = 13 [10.6%]) compared with placebo (n = 4 [6.6%]). No severe hypoglycaemia was reported during the study. No new safety signals emerged with treatment beyond Week 16 through Week 52. CONCLUSION: The addition of once-weekly omarigliptin to insulin therapy for up to 52 weeks was generally well tolerated and provided clinically meaningful improvement in glycaemic control throughout the trial period. ClinicalTrials.gov: NCT02906709.

Development of a new photosafety test method based on singlet oxygen generation detected using electron spin resonance.

Photosafety evaluations of chemicals used in consumer products, such as pharmaceuticals and cosmetics, are very important. Currently, two non-animal tests for photosafety evaluations, the in vitro 3T3 neutral red uptake phototoxicity test (NRU PT) and the reactive oxygen species (ROS) assay, are used to detect photoreactive chemicals. However, these two tests are difficult to apply to hydrophobic chemicals. In the present study, we attempted to develop a new photosafety test method, named the electron spin resonance-based photosafety test (ESR-PT), that would be applicable even to hydrophobic chemicals based on the detection of singlet oxygen generation after irradiation using ESR spectroscopy with 4-hydroxy-2,2,6,6-tetramethyl-piperidine as a spin trap reagent. To achieve a quantitative evaluation, the singlet oxygen formation (SOF) value, which can be calculated as the increment in relative intensity after irradiation of the test mixture normalized by the increment in relative intensity after irradiation of the vehicle control solution, was calculated. The performance of the ESR-PT was evaluated by testing all the proficiency chemicals of the ROS assay plus additional chemicals, including hydrophobic chemicals and chemicals that tested false negative in the 3T3-NRU PT and ROS assay. SOF values were successfully calculated for all the chemicals tested including the hydrophobic chemicals, and the accuracy of the ESR-PT using a tentative cutoff value of 2.8 against the photosafety information was 100%. Therefore, the SOF value could be an effective parameter for photosafety evaluations, suggesting that the newly developed ESR-PT is a promising non-animal test applicable even to hydrophobic chemicals.

Total RNA and genomic DNA of Lactobacillus gasseri OLL2809 induce interleukin-12 production in the mouse macrophage cell line J774.1 via toll-like receptors 7 and 9.

BACKGROUND: Lactobacillus gasseri OLL2809 can highly induce interleukin (IL)-12 production in immune cells. Even though beneficial properties of this strain for both humans and animals have been reported, the mechanism by which the bacteria induces the production of IL-12 in immune cells remains elusive. In this study, we investigated the mechanism of induction of IL-12 using a mouse macrophage cell line J774.1. RESULTS: Inhibition of phagocytosis of L. gasseri OLL2809, and myeloid differentiation factor 88 and Toll-like receptors (TLRs) 7 and 9 signalling attenuated IL-12 production in J774.1 cells. Total RNA and genomic DNA of L. gasseri OLL2809, when transferred to the J774.1 cells, also induced IL-12 production. The difference in the IL-12-inducing activity of Lactobacilli is attributed to the susceptibility to phagocytosis, but not to a difference in the total RNA and genomic DNA of each strain. CONCLUSION: We concluded that total RNA and genomic DNA of phagocytosed L. gasseri OLL2809 induce IL-12 production in J774.1 cell via TLRs 7 and 9, and the high IL-12-inducing activity of L. gasseri OLL2809 is due to its greater susceptibility to phagocytosis.

The efficacy and safety of tazobactam/ceftolozane in Japanese patients with uncomplicated pyelonephritis and complicated urinary tract infection.

We report efficacy and safety results for a combination of a novel cephalosporin class antibiotic and a ß-Lactamase inhibitor, tazobactam/ceftolozane (1:2) at a dose of 1.5 g intravenously every 8 h in Japanese patients with uncomplicated pyelonephritis and complicated urinary tract infection. This study design was a nonrandomized, multicenter, open-label trial, and the treatment period was 7 days. Of 115 patients enrolled in this study, 114 received tazobactam/ceftolozane, and 90 were included in the efficacy analyses. Ninety-nine isolates (bacterial count ≥105 CFU/mL) were identified by urine culture. The main baseline uropathogens were Escherichia coli (80 isolates), Klebsiella pneumoniae (8 isolates), and Proteus mirabilis (3 isolates). Of these, 13 isolates were ESBL-producers. The favorable per-patient microbiological response rate at 7 days after the final administration of tazobactam/ceftolozane was 80.7% (71/88). The response rate in uncomplicated pyelonephritis was 90.0% (36/40), complicated pyelonephritis 63.6% (14/22), and complicated cystitis 80.8% (21/26). The favorable clinical response rate was 96.6% (86/89), and composite response rate (based on microbiological and clinical response) was 80.7% (71/88). The eradication rate by uropathogen was 83.5% (66/79) in E. coli, 42.9% (3/7) in K. pneumoniae, and 100% (3/3) in P. mirabilis. The incidence of drug-related adverse events was 17.5% (20/114 patients). The most common drug-related adverse events were diarrhea and alanine aminotransferase increased in 5.3% (6/114 patients each). Drug-related serious adverse events and deaths were not observed. These results support the safety and efficacy of tazobactam/ceftolozane and suggest it will be a useful treatment for uncomplicated pyelonephritis and complicated urinary tract infection.

The importance of nonstop treatment after delivery for pregnant women with type 2 diabetes.

BACKGROUND: There are no reports of very long follow-up studies of pregnant women with type 2 diabetes after delivery. Here we describe cases of Japanese women whom we treated for 20 to 50 years after deliveries to investigate the relationship between blood glucose control and diabetic complications. METHODS: In Japan, the prevalence of type 1 diabetes is very low, and we have very few long-term follow-up cases with type 1 diabetes. Therefore, we chose to describe subjects with type 2 diabetes only. We present data on a total of 80 deliveries, 68 cases, treated by one of us (Y.O.) for more than a 50-year period. They are divided into 4 groups based on duration of treatment after delivery: more than 50 years (1 delivery, 1 patient), 40 to 49 years (13 deliveries, 11 patients), 30 to 39 years (19 deliveries, 16 patients), and 20 to 29 years (47 deliveries, 40 patients). Their present average ages in these 4 groups are 77, 72.4, 65.9, and 55.5 years, respectively. Their average HbA1c levels at last visit, in May 2014, are 8.2%, 7.6%.,7.2%, and 8.3%, respectively. RESULTS: Despite elevated HbA1c levels, they had relatively few complications: 40% (no retinopathy), 43.8% (simple retinopathy), and 12.5% (treated with photocoagulation); 67.5% (no albuminuria), 26.3% (albuminuria), and 6.3% (treated with renal transplantation or hemodialysis). Therefore, even if glycemic control is not ideal, nonstop treatment of Japanese women for type 2 diabetes after deliveries is effective to prevent diabetic complications. CONCLUSIONS: Long-term attention to care of diabetes after pregnancy may be preventive of diabetic complications in other populations as well.

Systematic characterization of artificial small RNA-mediated inhibition of Escherichia coli growth.

A new screening system for artificial small RNAs (sRNAs) that inhibit the growth of Escherichia coli was constructed. In this system, we used a plasmid library to express RNAs of ∼120 nucleotides, each with a random 30-nucleotide sequence that can recognize its target mRNA(s). After approximately 60,000 independent colonies were screened, several plasmids that inhibited bacterial growth were isolated. To understand the inhibitory mechanism, we focused on one sRNA, S-20, that exerted a strong inhibitory effect. A time-course analysis of the proteome of S-20-expressing E. coli and a bioinformatic analysis were used to identify potential S-20 target mRNAs, and suggested that S-20 binds the translation initiation sites of several mRNAs encoding enzymes such as peroxiredoxin (osmC), glycyl-tRNA synthetase α subunit (glyQ), uncharacterized protein ygiM, and tryptophan synthase ß chain (trpB). An in vitro translation analysis of chimeric luciferase-encoding mRNAs, each containing a potential S-20 target sequence, indicated that the translation of these mRNAs was inhibited in the presence of S-20. A gel shift analysis combined with the analysis of a series of S-20 mutants suggested that S-20 targets multiple mRNAs that are responsible for inhibiting E. coli growth. These data also suggest that S-20 acts like an endogenous sRNA and that E. coli can utilize artificial sRNAs.

[Evaluation of a Newly Developed Reagent "Lias Auto P-FDP" on Coapresta 2000 and Clinico-Pathological Analysis of Discrepant Samples in FDP Value].

Patients with disseminated intravascular coagulation (DIC) exhibit increased levels of fibrin/fibrinogen degradation products (FDP), and FDP levels are carefully monitored as a marker of fibrinolysis during the diagnosis of DIC and other fibrinolysis-related conditions. Although FDP levels can be measured using automatic analyzers, it is reported that measured FDP values differ depending on the reagent used. Recently, a newly developed reagent for measuring FDP levels (Lias Auto P-FDP) was launched. In this study, we used Lias Auto P-FDP in combination with the Coapresta 2000 automatic analyzer and evaluated its reactivity. We confirmed the reproducibility of the measurements obtained with the Lias Auto P-FDP reagent when the manufacturer's instructions were followed and that the Lias Auto P-FDP reagent can be used with the Coapresta 2000. In the reactivity test, the Lias Auto P-FDP reagent exhibited stronger reactivity with low molecular weight FDP than the reagent we currently use (BL2 P-FDP). Moreover, the samples that exhibited non-specific reactions to the BL2 P-FDP reagent did not display similar reactions to the Lias auto P-FDP reagent. In the clinico-pathological analysis of divergent value between Lias Auto P-FDP and BL2 P-FDP, seven cases were highly discrepant value of FDP. Interestingly, we found the description of pleural fluid and/or ascites in 85.7% cases. In conclusion, we confirmed that the Lias Auto P-FDP reagent can be used in combination with the Coapresta 2000. In addition, the reactivity of FDP varied depending on the reagent used. It is important to understand the characteristics of each FDP reagent and which automatic analyzers they can be used with. [Original].

Dopamine D1 Receptor-Mediated Transmission Maintains Information Flow Through the Cortico-Striato-Entopeduncular Direct Pathway to Release Movements.

In the basal ganglia (BG), dopamine plays a pivotal role in motor control, and dopamine deficiency results in severe motor dysfunctions as seen in Parkinson's disease. According to the well-accepted model of the BG, dopamine activates striatal direct pathway neurons that directly project to the output nuclei of the BG through D1 receptors (D1Rs), whereas dopamine inhibits striatal indirect pathway neurons that project to the external pallidum (GPe) through D2 receptors. To clarify the exact role of dopaminergic transmission via D1Rs in vivo, we developed novel D1R knockdown mice in which D1Rs can be conditionally and reversibly regulated. Suppression of D1R expression by doxycycline treatment decreased spontaneous motor activity and impaired motor ability in the mice. Neuronal activity in the entopeduncular nucleus (EPN), one of the output nuclei of the rodent BG, was recorded in awake conditions to examine the mechanism of motor deficits. Cortically evoked inhibition in the EPN mediated by the cortico-striato-EPN direct pathway was mostly lost during suppression of D1R expression, whereas spontaneous firing rates and patterns remained unchanged. On the other hand, GPe activity changed little. These results suggest that D1R-mediated dopaminergic transmission maintains the information flow through the direct pathway to appropriately release motor actions.