Near-infrared in vivo imaging system for dynamic visualization of lung-colonizing bacteria in mouse pneumonia.

In vivo imaging of bacterial infection models enables noninvasive and temporal analysis of individuals, enhancing our understanding of infectious disease pathogenesis. Conventional in vivo imaging methods for bacterial infection models involve the insertion of the bacterial luciferase LuxCDABE into the bacterial genome, followed by imaging using an expensive ultrasensitive charge-coupled device (CCD) camera. However, issues such as limited light penetration into the body and lack of versatility have been encountered. We focused on near-infrared (NIR) light, which penetrates the body effectively, and attempted to establish an in vivo imaging method to evaluate the number of lung-colonizing bacteria during the course of bacterial pneumonia. This was achieved by employing a novel versatile system that combines plasmid-expressing firefly luciferase bacteria, NIR substrate, and an inexpensive, scientific complementary metal-oxide semiconductor (sCMOS) camera. The D-luciferin derivative "TokeOni," capable of emitting NIR bioluminescence, was utilized in a mouse lung infection model of Acinetobacter baumannii, an opportunistic pathogen that causes pneumonia and is a concern due to drug resistance. TokeOni exhibited the highest sensitivity in detecting bacteria colonizing the mouse lungs compared with other detection systems such as LuxCDABE, enabling the monitoring of changes in bacterial numbers over time and the assessment of antimicrobial agent efficacy. Additionally, it was effective in detecting A. baumannii clinical isolates and Klebsiella pneumoniae. The results of this study are expected to be used in the analysis of animal models of infectious diseases for assessing the efficacy of therapeutic agents and understanding disease pathogenesis. IMPORTANCE: Conventional animal models of infectious diseases have traditionally relied upon average assessments involving numerous individuals, meaning they do not directly reflect changes in the pathology of an individual. Moreover, in recent years, ethical concerns have resulted in the demand to reduce the number of animals used in such models. Although in vivo imaging offers an effective approach for longitudinally evaluating the pathogenesis of infectious diseases in individual animals, a standardized method has not yet been established. To our knowledge, this study is the first to develop a highly versatile in vivo pulmonary bacterial quantification system utilizing near-infrared luminescence, plasmid-mediated expression of firefly luciferase in bacteria, and a scientific complementary metal-oxide semiconductor camera. Our research holds promise as a useful tool for assessing the efficacy of therapeutic drugs and pathogenesis of infectious diseases.

Development of a novel bacterial production system for recombinant bioactive proteins completely free from endotoxin contamination.

Endotoxins, or lipopolysaccharides (LPS), are potent immunostimulatory molecules of critical concern in bacterial recombinant protein expression systems. The gram-negative bacterium Acinetobacter baumannii exhibits an interesting and unique phenotype characterized by the complete loss of LPS. In this study, we developed a novel system for producing recombinant proteins completely devoid of endotoxin contamination using LPS-deficient A. baumannii. We purified endotoxin-free functional green fluorescent protein, which reduced endotoxin contamination by approximately three orders of magnitude, and also purified the functional cytokine tumor necrosis factor (TNF)-α. Additionally, utilization of the Omp38 signal peptide of A. baumannii enabled the extracellular production of variable domain of heavy chain of heavy chain (VHH) antibodies. With these advantages, mNb6-tri-20aa, a multivalent VHH that specifically binds to the spike protein of severe acute respiratory syndrome coronavirus 2, was purified from the culture supernatant, and endotoxin contamination was reduced by a factor of approximately 2 × 105 compared with that in conventional expression systems. A virus neutralization assay demonstrated the functionality of the purified antibody in suppressing viral infections. Moreover, we applied our system to produce ozoralizumab, a multispecific VHH that binds to human TNF-α and albumin and are marketed as a rheumatoid arthritis drug. We successfully purified a functional antibody from endotoxin contamination. This system establishes a new, completely endotoxin-free platform for the expression of recombinant proteins, which distinguishes it from other bacterial expression systems, and holds promise for future applications.

Effects of quorum sensing-interfering agents, including macrolides and furanone C-30, and an efflux pump inhibitor on nitrosative stress sensitivity in Pseudomonas aeruginosa.

Long-term administration of certain macrolides is efficacious in patients with persistent pulmonary Pseudomonas aeruginosa infection, despite how limited the clinically achievable concentrations are, being far below their MICs. An increase in the sub-MIC of macrolide exposure-dependent sensitivity to nitrosative stress is a typical characteristic of P. aeruginosa. However, a few P. aeruginosa clinical isolates do not respond to sub-MIC of macrolide treatment. Therefore, we examined the effects of sub-MIC of erythromycin (EM) on the sensitivity to nitrosative stress together with an efflux pump inhibitor (EPI) phenylalanine arginyl ß-naphthylamide (PAßN). The sensitivity to nitrosative stress increased, suggesting that the efflux pump was involved in inhibiting the sub-MIC of macrolide effect. Analysis using efflux pump-mutant P. aeruginosa revealed that MexAB-OprM, MexXY-OprM, and MexCD-OprJ are factors in reducing the sub-MIC of macrolide effect. Since macrolides interfere with quorum sensing (QS), we demonstrated that the QS-interfering agent furanone C-30 (C-30) producing greater sensitivity to nitric oxide (NO) stress than EM. The effect of C-30 was decreased by overproduction of MexAB-OprM. To investigate whether the increase in the QS-interfering agent exposure-dependent sensitivity to nitrosative stress is characteristic of P. aeruginosa clinical isolates, we examined the viability of P. aeruginosa treated with NO. Although treatment with EM could reduce cell viability, a high variability in EM effects was observed. Conversely, C-30 was highly effective at reducing cell viability. Treatment with both C-30 and PAßN was sufficiently effective against the remaining isolates. Therefore, the combination of a QS-interfering agent and an EPI could be effective in treating P. aeruginosa infections.

PmrAB, the two-component system of Acinetobacter baumannii, controls the phosphoethanolamine modification of lipooligosaccharide in response to metal ions.

Acinetobacter baumannii is highly resistant to antimicrobial agents, and XDR strains have become widespread. A. baumannii has developed resistance to colistin, which is considered the last resort against XDR Gram-negative bacteria, mainly caused by lipooligosaccharide (LOS) phosphoethanolamine (pEtN) and/or galactosamine (GalN) modifications induced by mutations that activate the two-component system (TCS) pmrAB. Although PmrAB of A. baumannii has been recognized as a drug resistance factor, its function as TCS, including its regulatory genes and response factors, has not been fully elucidated. In this study, to clarify the function of PmrAB as TCS, we elucidated the regulatory genes (regulon) of PmrAB via transcriptome analysis using pmrAB-activated mutant strains. We discovered that PmrAB responds to low pH, Fe2+, Zn2+, and Al3+. A. baumannii selectively recognizes Fe2+ rather than Fe3+, and a novel region ExxxE, in addition to the ExxE motif sequence, is involved in the environmental response. Furthermore, PmrAB participates in the phosphoethanolamine modification of LOS on the bacterial surface in response to metal ions such as Al3+, contributing to the attenuation of Al3+ toxicity and development of resistance to colistin and polymyxin B in A. baumannii. This study demonstrates that PmrAB in A. baumannii not only regulates genes that play an important role in drug resistance but is also involved in responses to environmental stimuli such as metal ions and pH, and this stimulation induces LOS modification. This study reveals the importance of PmrAB in the environmental adaptation and antibacterial resistance emergence mechanisms of A. baumannii. IMPORTANCE: Antimicrobial resistance (AMR) is a pressing global issue in human health. Acinetobacter baumannii is notably high on the World Health Organization's list of bacteria for which new antimicrobial agents are urgently needed. Colistin is one of the last-resort drugs used against extensively drug-resistant (XDR) Gram-negative bacteria. However, A. baumannii has become increasingly resistant to colistin, primarily by modifying its lipooligosaccharide (LOS) via activating mutations in the two-component system (TCS) PmrAB. This study comprehensively elucidates the detailed mechanism of drug resistance of PmrAB in A. baumannii as well as its biological functions. Understanding the molecular biology of these molecules, which serve as drug resistance factors and are involved in environmental recognition mechanisms in bacteria, is crucial for developing fundamental solutions to the AMR problem.

Volatile organic compounds from human skin as biomarkers of menstruation phase and severity of premenstrual syndrome: An exploratory pilot study.

Background and aim: Numerous women of reproductive age experience physical or mental discomfort during their natural menstrual cycle due to paramenstrual symptoms, such as premenstrual syndrome (PMS). To date, there is no established biomarker for the diagnosis of PMS. This study investigated the relationship between skin gas composition and menstruation cycles, and evaluated the possibility of skin gas composition as a biomarker of paramenstrual symptoms. Methods: We conducted an exploratory pilot study. Healthy Japanese women (aged 20-29 years) underwent blood and skin gas analyses on 1 day corresponding to menstruation, preovulatory, middle luteal, and late luteal phases. Skin gas was collected from the cubital fossa and armpit using a Passive Flux Sampler; samples were analyzed for 65 volatile organic compounds (VOCs) by gas chromatography-mass spectrometry (GC-MS). Non-parametric statistical analysis was performed to identify VOCs related to the menstrual cycle, levels of female hormones, and severity of PMS. Results: Fourteen women participated; of those, 12 completed the study. Regarding the relationship with the menstrual cycles, seven and four VOCs were significantly and marginally changed, respectively, at the cubital fossa during menstruation. Of those 11 compounds, 10 were also correlated with the levels of serum female hormones. At the armpit, five and three compounds were significantly and marginally changed, respectively, during menstruation. Of those eight compounds, five were also correlated with the levels of serum female hormones. In the study of PMS severity, analysis of the changes in VOCs suggested that ketones and fatty acids are increased during menstruation in the severe PMS group versus the mild PMS group. Conclusions: The results of this study suggest that certain VOCs emitted in skin gas related to the menstrual cycle, levels of female hormones, and severity of PMS. These findings may advance the metabolic understanding and development of diagnostic biomarkers for menstruation-related symptoms.

Lactobacillus paracasei KW3110 Prevents Inflammatory-Stress-Induced Mitochondrial Dysfunction in Mouse Macrophages.

Lactobacillus paracasei KW3110 (KW3110) has anti-inflammatory effects, including the prevention of blue light exposure induced retinal inflammation and ageing-related chronic inflammation in mice. The mechanism involves the promotion of anti-inflammatory cytokine interleukin (IL)-10 production by KW3110, leading to reduced pro-inflammatory cytokine IL-1ß production. Although various stress-induced mitochondrial damages are associated with excessive inflammatory responses, the effect of KW3110 on inflammatory-stress-induced mitochondrial damage remains unknown. In this study, we investigated the effect of KW3110 on inflammatory stress-induced mitochondrial damage using the murine macrophage-like cell line J774A.1. KW3110 treatment suppressed lipopolysaccharide (LPS)-induced mitochondrial dysfunction, including downregulation of membrane potential, induction of reactive oxygen species, and respiratory dysfunction. In addition, KW3110 prevented LPS-induced disruption of mitochondrial morphology including cristae structures. IL-10 treatment also ameliorated LPS-induced mitochondrial dysfunction and morphology disruption. These results suggest that KW3110 prevents LPS-induced mitochondrial dysfunction, potentially via promoting IL-10 production in mouse macrophages. We are the first to reveal a suppressive effect of lactic acid bacteria on mitochondrial morphology disruption in inflammatory-stressed macrophages. Our findings contribute to understanding inflammatory-stress-induced mitochondrial damage and developing food ingredients with preventive effects on mitochondrial-damage-derived inflammatory conditions.

Evaluation of efflux pump inhibitors of MexAB- or MexXY-OprM in Pseudomonas aeruginosa using nucleic acid dyes.

BACKGROUND: Increased expression of efflux pumps is an important mechanism of antibiotic resistance in Pseudomonas aeruginosa, and treatment with inhibitors of active efflux pumps seems an attractive strategy to combat with multidrug resistance. Assays using ethidium bromide (EtBr), which accumulates by binding to nucleic acids, are often employed to assess the efficacy of efflux pump inhibitors (EPIs). However, few studies have reported on assays using other nucleic acid dyes. OBJECTIVE: We used different classes of EPIs for MexAB- or MexXY-OprM to measure the accumulation of various fluorescent dyes, including SYBR Safe, AtlasSight, and GelGreen. METHODS: Escherichia coli MG1655ΔacrBΔtolC strain harboring plasmids carrying the mexAB-oprM (pABM) or mexXY-oprM (pXYM) genes of P. aeruginosa were constructed. Then, the accumulation of the above-mentioned nucleic acid dyes and EtBr was measured to assess the efflux ability in the presence and absence of EPIs (MexAB-OprM-specific inhibitor of pyridopyrimidine derivative [ABI-PP], berberine, non-specific inhibitor of phenylalanine-arginine ß-naphthylamide [PAßN], and protonophore of carbonyl cyanide m-chlorophenyl hydrazone [CCCP]). RESULTS: Decreased accumulations of nucleic acid dyes were observed in strains with pABM or pXYM compared with the parental strain. ABI-PP or berberine addition significantly increased the accumulation of any nucleic acids in the strains with the specific pumps. PAßN or CCCP addition showed increased accumulation of almost all dye in strains with pABM or pXYM. However, the inhibition patterns of EPIs differed according to the nucleic acid dyes used. CONCLUSIONS: Accumulation assays for EPIs were suitable to evaluate EPI candidates using various nucleic acid dyes.

Dectin-2 mediates phagocytosis of Lactobacillus paracasei KW3110 and IL-10 production by macrophages.

Lactic acid bacteria (LAB) are most generally used as probiotics and some strains of LAB are known to have anti-inflammatory effects. A specific strain of lactic acid bacteria, Lactobacillus paracasei KW3110 (KW3110), activates macrophages to produce interleukin-10 (IL-10), an anti-inflammatory cytokine; however, the biological mechanism remains unclear. In this study, we showed that the amount of incorporated KW3110 into a macrophage cell line, RAW 264.7, was higher than other genetically related strains using fluorescence microscopy. RNA-seq analysis indicated that treatment of macrophages with KW3110 induced Dectin-2 gene expression, which is a pattern recognition receptor, recognizing α-mannose. In addition, antibody treatment and knock down of Dectin-2, or factors downstream in the signaling pathway, decreased the amount of incorporated KW3110 and IL-10 production. Substantial lectin array analysis also revealed that KW3110 had higher binding affinities to lectins, which recognize the carbohydrate chains comprised of α-mannose, than two other LAB. In conclusion, KW3110 is readily incorporated into macrophages, leading to IL-10 production. Dectin-2 mediated the phagocytosis of KW3110 into macrophages and this may be involved with the characteristic carbohydrate chains of KW3110.

Long-term intake of Lactobacillus paracasei KW3110 prevents age-related circadian locomotor activity and changes in gut metabolism in physiologically aged mice.

Aging involves age-progressive loss of physiological functions in organs and tissues. We previously showed that Lactobacillus paracasei KW3110 suppressed age-related inflammation and prevented age-related retinal ganglion cell (RGC) loss. As RGCs mediate biological behaviors associated with responses to ambient light, we assessed whether L. paracasei KW3110 affects circadian locomotor activities in physiologically aged mice. The ratio of locomotor activity during the nighttime (active phase) to daytime (inactive phase) significantly decreased in physiologically aged mice compared with young mice: intake of L. paracasei KW3110 prevented this decrease. We also performed metabolomics analysis of cecal contents using both capillary electrophoresis and liquid chromatography time-of-flight mass spectrometry to better understand the benefical effects for aging of L. paracasei KW3110 through a gut retina axis, since our previous study showed that L. paracasei KW3110 mitigated not only age-related expansions of intestinal inflammatory immune cells but age-related alternation of gut microbiome composition. Principal component analysis showed clear changes in metabolites between physiologically aged mice fed a diet containing L. paracasei KW3110 and age-matched control mice. Furthermore, we found that intake of L. paracasei KW3110 mitigated age-related changes in some fatty acids compared with age-matched control mice. Taken together, L. paracasei KW3110 might regulate age-related alternation of metabolites in cecal contents, potentially leading to suppression of age-related decline in physiological functions, including impairment of circadian locomotor activities.

Overexpression of the MexXY Multidrug Efflux System Correlates with Deficient Pyoverdine Production in Pseudomonas aeruginosa.

Multidrug-resistant Pseudomonas aeruginosa poses a serious problem due to hospital- and healthcare-associated infections. A major drug resistance mechanism of P. aeruginosa involves active efflux via resistance nodulation cell division (RND)-type multidrug efflux pumps of which MexXY is increasingly recognized as a primary determinant of aminoglycoside resistance in P. aeruginosa. MexXY overexpression is often observed in drug-resistant P. aeruginosa clinical isolates. MexXY deficiency increased pyoverdine production in all four P. aeruginosa strains we tested. MexXY-overproducing multidrug-resistant P. aeruginosa PA7 exhibited the greatest effect among the strains. Complementation with a MexXY-expressing plasmid restored low-level pyoverdine production in a MexXY-deficient P. aeruginosa mutant from PA7, indicating that MexXY expression decreases pyoverdine production. Because P. aeruginosa produces pyoverdine to acquire iron, MexXY-deficient mutants might be more susceptible to iron deficiency than MexXY-producing strains or might require extra iron. High-risk clones of multidrug-resistant P. aeruginosa reportedly tend to be MexXY overproducers but defective pyoverdine producers. This study suggests that P. aeruginosa reduces production of a virulence factor after acquiring a drug resistance factor.

A web-based survey on various symptoms of computer vision syndrome and the genetic understanding based on a multi-trait genome-wide association study.

A variety of eye-related symptoms due to the overuse of digital devices is collectively referred to as computer vision syndrome (CVS). In this study, a web-based survey about mind and body functions, including eye strain, was conducted on 1998 Japanese volunteers. To investigate the biological mechanisms behind CVS, a multi-trait genome-wide association study (GWAS), a multivariate analysis on individual-level multivariate data, was performed based on the structural equation modeling methodology assuming a causal pathway for a genetic variant to influence each symptom via a single common latent variable. Twelve loci containing lead variants with a suggestive level of significance were identified. Two loci showed relatively strong signals and were associated with TRABD2B relative to the Wnt signaling pathway and SDK1 having neuronal adhesion and immune functions, respectively. By utilizing publicly available eQTL data, colocalization between GWAS and eQTL signals for four loci was detected, and a locus on 2p25.3 showed a strong colocalization (PPH4 > 0.9) on retinal MYT1L, known to play an important role in neuronal differentiation. This study suggested that the use of multivariate questionnaire data and multi-trait GWAS can lead to biologically reasonable findings and enhance our genetic understanding of complex relationships among symptoms related to CVS.

Water-slider method reduced colonic polyp fragmentation compared with conventional suction retrieval: A prospective randomized controlled study.

OBJECTIVES: After resecting colonic polyps, retrieval through the scope channel may lead to polyp fragmentation, making pathologic evaluation challenging. An easy and reliable method for complete polyp retrieval is needed. METHODS: We developed the water-slider method (WSM), in which the resected polyp is surrounded by water from an auxiliary water channel during suctioning. We prospectively randomized patients who underwent endoscopic resection for colonic polyps in our institute into WSM and non-WSM groups, and evaluated the polyp fragmentation rate. RESULTS: Analysis of the data regarding small polyps (≤10 mm in size) revealed that the WSM group had a significantly lower polyp fragmentation rate (8.2%) than the non-WSM group (23.8%, p < 0.001). Polyp retrieval time did not differ significantly between groups. The rate of a clear-cut end on neoplastic polyps was significantly higher in the WSM group (63.8%) than in the non-WSM group (50.0%; p = 0.029). CONCLUSIONS: The WSM achieved a significantly lower polyp fragmentation rate, allowing for more accurate histologic evaluation than the conventional method. The WSM did not influence the polyp retrieval time.

Lactic acid bacterium, Lactobacillus paracasei KW3110, suppresses inflammatory stress-induced caspase-1 activation by promoting interleukin-10 production in mouse and human immune cells.

A strain of lactic acid bacteria, Lactobacillus paracasei KW3110 (KW3110), activates M2 macrophages with anti-inflammatory reactions and mitigates aging-related chronic inflammation and blue-light exposure-induced retinal inflammation in mice. However, the mechanism underlying the anti-inflammatory effects of KW3110 remains unclear. In this study, we investigated the anti-inflammatory effects of KW3110 using both mouse and human immune cells and evaluated the suppressive effect of KW3110 on the inflammatory reactions of the cells stimulated with lipopolysaccharide and adenosine 5'-triphosphate (LPS/ATP). KW3110 treatment induced anti-inflammatory cytokine interleukin (IL)-10 production in the supernatants of murine macrophage-like cells, J774A.1, and suppressed IL-1ß production in the supernatants of LPS/ATP-stimulated cells. The influence of KW3110 on the production of these cytokines was inhibited by pre-treatment with phagocytosis blocker or transfection with siRNAs for IL-10 signaling components. KW3110 treatment also suppressed activation of caspase-1, an active component of inflammasome complexes, in LPS/ATP-stimulated J774A.1 cells, and its effect was inhibited by transfection with siRNAs for IL-10 signaling components. In addition to the effects of KW3110 on J774A.1 cells, KW3110 treatment induced IL-10 production in the supernatants of human monocytes, and KW3110 or IL-10 treatment suppressed caspase-1 activation and IL-1ß production in the supernatants of LPS/ATP-stimulated cells. These results suggest that KW3110 suppresses LPS/ATP stimulation-induced caspase-1 activation and IL-1ß production by promoting IL-10 production in mouse and human immune cells. Our findings reveal a novel anti-inflammatory mechanism of LAB and the effect of KW3110 on caspase-1 activation is expected to contribute to constructing future preventive strategies for inflammation-related disorders using food ingredients.

Lactobacillus paracasei KW3110 Suppresses Inflammatory Stress-Induced Premature Cellular Senescence of Human Retinal Pigment Epithelium Cells and Reduces Ocular Disorders in Healthy Humans.

Lactobacillus paracasei KW3110 (KW3110) has anti-inflammatory effects and mitigates retinal pigment epithelium (RPE) cell damage caused by blue-light exposure. We investigated whether KW3110 suppresses chronic inflammatory stress-induced RPE cell damage by modulating immune cell activity and whether it improves ocular disorders in healthy humans. First, we showed that KW3110 treatment of mouse macrophages (J774A.1) produced significantly higher levels of interleukin-10 as compared with other lactic acid bacterium strains (all p < 0.01). Transferring supernatant from KW3110- and E. coli 0111:B4 strain and adenosine 5'-triphosphate (LPS/ATP)-stimulated J774A.1 cells to human retinal pigment epithelium (ARPE-19) cells suppressed senescence-associated phenotypes, including proliferation arrest, abnormal appearance, cell cycle arrest, and upregulation of cytokines, and also suppressed expression of tight junction molecule claudin-1. A randomized, double-blind, placebo-controlled parallel-group study of healthy subjects (n = 88; 35 to below 50 years) ingesting placebo or KW3110-containing supplements for 8 weeks showed that changes in critical flicker frequency, an indicator of eye fatigue, from the week-0 value were significantly larger in the KW3110 group at weeks 4 (p = 0.040) and 8 (p = 0.036). These results suggest that KW3110 protects ARPE-19 cells against premature senescence and aberrant expression of tight junction molecules caused by chronic inflammatory stress, and may improve chronic eye disorders including eye fatigue.

A Specific Strain of Lactic Acid Bacteria, Lactobacillus paracasei, Inhibits Inflammasome Activation In Vitro and Prevents Inflammation-Related Disorders.

Some strains of lactic acid bacteria (LAB) have anti-inflammatory effects, but the mechanism underlying the alleviation of inflammation by LAB is not fully understood. In this study, we examined the inhibitory effect of a certain strain of LAB, Lactobacillus paracasei, on inflammasome activation, which is associated with various inflammatory disorders. Using bone marrow-derived macrophages from BALB/c mice, we found that L. paracasei, but not L. rhamnosus, suppressed NLRP3 inflammasome activation and inhibited subsequent caspase-1 activation and IL-1ß secretion. L. paracasei also had inhibitory effects on AIM2 and NLRC4 inflammasome activation as well as the NLRP3 inflammasome. These inhibitory effects of L. paracasei on inflammasome activation were dependent on autocrine IL-10 induced by L. paracasei-stimulated macrophages. Furthermore, IL-10 production by L. paracasei-stimulated macrophages was involved with phagocytosis and the NOD2 signaling pathway in macrophages. In addition to in vitro studies, oral administration of L. paracasei in C57BL/6 mice reduced monosodium urate crystal-induced peritoneal inflammation in vivo. Moreover, continuous intake of L. paracasei in C57BL/6 mice alleviated high fat diet-induced insulin resistance and aging-induced expression of biomarkers for T cell senescence. Taken together, we demonstrated that L. paracasei inhibits inflammasome activation in vitro and exhibits an anti-inflammatory function in vivo. These results indicate that LAB that have inhibitory effects on inflammasome activation might contribute to the alleviation of inflammation-related disorders.

[Immunosenescence: The Forefront of Infection and Trophic Control].

Recently, aging is becoming an important social problem in many developed countries including Japan. It is socially and universally important to unveil the impact of aging and extend healthy life expectancy. Here we show our recent finding that dedicator of cytokinesis 11 (DOCK11, also known as Zizimin2) may be involved in immunosenescence of B cells. DOCK11 was identified as a guanine nucleotide exchange factor for a small GTPase called cell division cycle 42. Expression of DOCK11 is restricted to lymphoid tissues, and becomes downregulated with age. Thus we examined the involvement of DOCK11 in immunosenescence of B-1a B cells as an example. B-1a cells are the main source of antibodies at steady state, and function as the first line of defense against infection. Although DOCK11 was expressed by B-1a cells, the expression levels declined with age. Furthermore, production of anti-pneumococcal immunoglobulin M antibodies was suppressed in aged mice, and was recovered by adoptive transfer with B-1a cells in a DOCK11-dependent manner. Thus DOCK11 may be involved in immunosenescence of B-1a cells.

13-(2-Methylbenzyl) Berberine Is a More Potent Inhibitor of MexXY-Dependent Aminoglycoside Resistance than Berberine.

We previously showed that berberine attenuates MexXY efflux-dependent aminoglycoside resistance in Pseudomonas aeruginosa. Here, we aimed to synthesize berberine derivatives with higher MexXY inhibitory activities. We synthesized 11 berberine derivatives, of which 13-(2-methylbenzyl) berberine (13-o-MBB) but not its regiomers showed the most promising MexXY inhibitory activity. 13-o-MBB reduced the minimum inhibitory concentrations (MICs) of various aminoglycosides 4- to 128 fold for a highly multidrug resistant P. aeruginosa strain. Moreover, 13-o-MBB significantly reduced the MICs of gentamicin and amikacin in Achromobacter xylosoxidans and Burkholderia cepacia. The fractional inhibitory concentration indices indicated that 13-o-MBB acted synergistically with aminoglycosides in only MexXY-positive P. aeruginosa strains. Time-kill curves showed that 13-o-MBB or higher concentrations of berberine increased the bactericidal activity of gentamicin by inhibiting MexXY in P. aeruginosa. Our findings indicate that 13-o-MBB inhibits MexXY-dependent aminoglycoside drug resistance more strongly than berberine and that 13-o-MBB is a useful inhibitor of aminoglycoside drug resistance due to MexXY.

Spodiobacter cordis gen. nov. sp. nov., a member of the family Flavobacteriaceae isolated from patients with infective endocarditis.

Two gram-negative, catalase-negative, oxidase-positive strains (PAGU 1467T and PAGU 1468) isolated from patients with infective endocarditis were investigated to determine their taxonomic status. 16S rRNA gene sequence analysis indicated that the two strains were members of the Bergeyella-Chryseobacterium-Riemerella branch of the family Flavobacteriaceae. Strains PAGU 1467T and PAGU 1468 were highly related to each other (98.8% 16S rRNA gene sequence similarity). Phylogenetically closely-related species to PAGU 1467T comprised Bergeyella zoohelcum (95.0% 16S rRNA gene sequence similarity), Riemerella anatipestifer (94.3%) and Cloacibacterium normanense (94.3%). The major fatty acids of the two isolates were iso-C15:0 , iso-C17:0 3-OH and iso-C15:0 3-OH. The presence of C16:0 3-OH and iso-C15:0 2-OH allowed these isolates to be distinguished from B. zoohelcum. Menaquinone MK-6 was the only respiratory quinone in these organisms; this is a consistent characteristic of the family Flavobacteriaceae. The guanine-plus-cytosine content of the genomic DNA was 42.0%, which is higher than that of other close phylogenetic relatives. On the basis of their phenotypic properties and genetic distinctiveness, isolates PAGU 1467T and PAGU 1468 were classified within the novel genus Spodiobacter, as Spodiobacter cordis gen. nov., sp. nov., which is also the type species. The type strain of S. cordis is PAGU 1467T ( = CCUG 65564T = NBRC 109998T ).

Incidence of osteonecrosis and insufficiency fracture of the hip and knee joints based on MRI in 300 renal transplant patients.

INTRODUCTION: The purpose of this study was to investigate the incidence of necrosis and insufficiency fracture of the hip and knee using magnetic resonance imaging (MRI) in renal transplant patients. METHODS: We examined 300 consecutive renal transplant patients using MRI of the hips and knees (T1-weighted coronal MRI) preoperatively and at 3 and 6 months postoperatively. Oblique axial-plane T1-weighted MRI and short-tau inversion-recovery T2-weighted MRI were performed parallel to the femoral neck when distinguishing the osteonecrosis of the femoral head (ONFH) from subchondral insufficiency fracture (SIF) of the femoral head was difficult. RESULTS: ONFH was observed in 3 hips (3 cases, 1.0%). Osteonecrosis of the knee was observed in 2 knees (1 case, 0.3%). SIF of the femoral head was observed in 5 hips (3 cases, 1.0%). Bilateral simultaneous onset of SIF of the femoral head was observed in 2 cases (0.7%). Insufficiency fracture of the knee was observed in 2 knees (1 case, 0.3%). CONCLUSIONS: When it was difficult to distinguish ONFH from SIF of the femoral head, oblique axial-plane T1-weighted MRI and the short-tau inversion-recovery T2-weighted MRI parallel to the femoral neck were useful to establish the correct diagnosis.

Description of Paraclostridium bifermentans subsp. muricolitidis subsp. nov., emended description of Paraclostridium bifermentans (Sasi Jyothsna et al., 2016), and creation of Paraclostridium bifermentans subsp. bifermentans subsp. nov.

Taxonomic studies of strain PAGU 1678T , an obligately anaerobic, gram-positive, spore-forming bacterium isolated from biobreeding rat feces, were performed. This strain has been demonstrated to have the ability to exacerbate pathosis in a mouse model of dextran sulfate sodium-induced ulcerative colitis. Phylogenetic analysis based on the 16S rRNA gene showed high homology with Paraclostridium bifermentans. To clarify the correct taxonomic position of strain PAGU 1678T , a comparative taxonomic study using P. bifermentans PAGU 2008T (═JCM 1386T ) and the closely related bacterial species P. benzoelyticum PAGU 2068T (═LMG 28745T ) was carried out. Despite the close similarity of 16S rRNA gene sequences, DNA-DNA hybridization between strain PAGU 1678T and P. bifermentans PAGU 2008T was 60.03% on average, average nucleotide identity was 96.17%, and it was shown to have different genomic sequences. Biochemically, strain PAGU 1678T could be differentiated from P. bifermentans PAGU 2008T by H2 S production. Furthermore, strain PAGU 1678T was characterized by the presence of two phospholipids with different polarity on polar lipid analysis. In addition, strain PAGU 1678T differed from P. bifermentans PAGU 2008T in findings on whole-cell protein analysis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry. On the basis of these biochemical and genetic characteristics, a novel subspecies of P. bifermentans with the name Paraclostridium bifermentans subsp. muricolitidis subsp. nov. is here proposed, with PAGU 1678T (═CCUG 72489T ═NBRC 113386T ) as the type strain, which automatically creates P. bifermentans subsp. bifermentans subsp. nov. JCM 1386T (═ATCC 638T ═DSM 14991T ).