Diversity analysis of thermophilic hydrogenogenic carboxydotrophs by carbon monoxide dehydrogenase amplicon sequencing using new primers

The microbial H2-producing (hydrogenogenic) carbon monoxide (CO)-oxidizing activity by the membrane-associated CO dehydrogenase (CODH)/energy-converting hydrogenase (ECH) complex is an important metabolic process in the microbial community. However, the studies on hydrogenogenic carboxydotrophs had to rely on inherently cultivation and isolation methods due to their rare abundance, which was a bottleneck in ecological study. Here, we provided gene-targeted sequencing method for the diversity estimation of thermophilic hydrogenogenic carboxydotrophs. We designed six new degenerate primer pairs which effectively amplified the coding regions of CODH genes forming gene clusters with ECH genes (CODHech genes) in Firmicutes which includes major thermophilic hydrogenogenic carboxydotrophs in terrestrial thermal habitats. Amplicon sequencing by these primers using DNAs from terrestrial hydrothermal sediments and CO-gas-incubated samples specifically detected multiple CODH genes which were identical or phylogenetically related to the CODHech genes in Firmictes. Furthermore, we found that phylogenetically distinct CODHech genes were enriched in CO-gas-incubated samples, suggesting that our primers detected uncultured hydrogenogenic carboxydotrophs as well. The new CODH-targeted primers provided us with a fine-grained (~ 97.9% in nucleotide sequence identity) diversity analysis of thermophilic hydrogenogenic carboxydotrophs by amplicon sequencing and will bolster the ecological study of these microorganisms

Biome-specific distribution of Ni-containing carbon monoxide dehydrogenases

Ni-containing carbon monoxide dehydrogenase (Ni-CODH) plays an important role in the CO/CO₂-based carbon and energy metabolism of microbiomes. Ni-CODH is classified into distinct phylogenetic clades, A–G, with possibly distinct cellular roles. However, the types of Ni-CODH clade used by organisms in different microbiomes are unknown. Here, we conducted a metagenomic survey of a protein database to determine the relationship between the phylogeny and biome distribution of Ni-CODHs. Clustering and phylogenetic analyses showed that the metagenome assembly-derived Ni-CODH sequences were distributed in ~ 60% Ni-CODH clusters and in all Ni-CODH clades. We also identified a novel Ni-CODH clade, clade H. Biome mapping on the Ni-CODH phylogenetic tree revealed that Ni-CODHs of almost all the clades were found in natural aquatic environmental and engineered samples, whereas those of specific subclades were found only in host-associated samples. These results are comparable with our finding that the diversity in the phylum-level taxonomy of host-associated Ni-CODH owners is statistically different from those of the other biomes. Our findings suggest that while Ni-CODH is a ubiquitous enzyme produced across diverse microbiomes, its distribution in each clade is biased and mainly affected by the distinct composition of microbiomes

Preoperative Use of Alpha-1 Receptor Blockers in Male Patients Undergoing Extracorporeal Shock Wave Lithotripsy for a Ureteral Calculus

In this retrospective single-center cohort study, we investigated the impact of preoperative use of an alpha-1 adrenergic receptor (AR) blocker on the outcome of single-session extracorporeal shock wave lithotripsy (SWL) in 193 male patients who underwent SWL for a single ureteral calculus between 2006 and 2016. We reviewed their medical records to obtain the data on the preoperative use of alpha-1 AR blockers. The primary outcome was treatment success after single-session SWL. We performed a multivariable logistic regression analysis adjusting for clinically important confounders to examine the association between preoperative use of alpha-1 AR blockers and the treatment success of SWL. Among the 193 patients, 15 (7.8%) were taking an alpha-1 AR blocker preoperatively. A multivariable analysis showed that preoperative use of an alpha-1 AR blocker was a significant negative predictor for treatment success of SWL (adjusted odds ratio 0.17; 95% confidence intervals, 0.04-0.74). Our findings suggest that the preoperative use of an alpha-1 AR blocker was a negative predictor of treatment success of SWL in male patients with a single ureteral calculus. Clinicians should pay more attention to the preoperative drug use in determining an appropriate stone therapy modality

Detection of anaerobic carbon monoxide-oxidizing thermophiles in hydrothermal environments.

Carboxydotrophic anaerobic thermophiles have been isolated from various hydrothermal environments and are considered to be important carbon monoxide (CO) scavengers or primary producers. However, the ecological factors that influence the distribution, abundance and CO-oxidizing activities of these bacteria are poorly understood. A previous study detected the carboxydotrophic bacteria Carboxydothermus spp. in a hot spring sample and found that they constituted up to 10% of the total bacterial cells. In this study, we investigated environmental features, potential microbial CO-oxidation activities and the abundance of Carboxydothermus spp. in various hot springs to determine environmental factors that affect CO oxidizers and to see whether Carboxydothermus spp. are common in these environments. We detected potential microbial CO-oxidation activities in samples that showed relatively high values of total organic carbon, total nitrogen, oxidation-reduction potential and soil-water content. The abundance of Carboxydothermus spp. did not correlate with the presence of potential microbial CO-oxidation activities; however, Carboxydothermus spp. were detected in a wide range of environments, suggesting that these bacteria are widely distributed in spite of the relatively low population size. This study implies that thermophilic CO oxidizers occur in a wide range of environments and oxidize CO in somewhat oxidative environments rich in organic matter

Case report: An N-of-1 study using amplitude modulated transcranial alternating current stimulation between Broca's area and the right homotopic area to improve post-stroke aphasia with increased inter-regional synchrony

Over one-third of stroke survivors develop aphasia, and language dysfunction persists for the remainder of their lives. Brain language network changes in patients with aphasia. Recently, it has been reported that phase synchrony within a low beta-band (14–19 Hz) frequency between Broca's area and the homotopic region of the right hemisphere is positively correlated with language function in patients with subacute post-stroke aphasia, suggesting that synchrony is important for language recovery. Here, we employed amplitude-modulated transcranial alternating current stimulation (AM-tACS) to enhance synchrony within the low beta band frequency between Broca's area and the right homotopic area, and to improve language function in a case of chronic post-stroke aphasia. According to an N-of-1 study design, the patient underwent short-term intervention with a one-time intervention of 15 Hz-AM-tACS with Broca's and the right homotopic areas (real condition), sham stimulation (sham condition), and 15 Hz-AM-tACS with Broca's and the left parietal areas (control condition) and long-term intervention with sham and real conditions (10 sessions in total, each). In the short-term intervention, the reaction time and accuracy rate of the naming task improved after real condition, not after sham and control conditions. The synchrony between the stimulated areas evaluated by coherence largely increased after the real condition. In the long-term intervention, naming ability, verbal fluency and overall language function improved, with the increase in the synchrony, and those improvements were sustained for more than a month after real condition. This suggests that AM-tACS on Broca's area and the right homotopic areas may be a promising therapeutic approach for patients with poststroke aphasia

Periodontal Tissue Regeneration Using Fibroblast Growth Factor -2: Randomized Controlled Phase II Clinical Trial

Background: The options for medical use of signaling molecules as stimulators of tissue regeneration are currently limited. Preclinical evidence suggests that fibroblast growth factor (FGF)-2 can promote periodontal regeneration. This study aimed to clarify the activity of FGF-2 in stimulating regeneration of periodontal tissue lost by periodontitis and to evaluate the safety of such stimulation. Methodology/Principal Findings: We used recombinant human FGF-2 with 3% hydroxypropylcellulose (HPC) as vehicle and conducted a randomized double-blinded controlled trial involving 13 facilities. Subjects comprised 74 patients displaying a 2- or 3-walled vertical bone defect as measured ?3 mm apical to the bone crest. Patients were randomly assigned to 4 groups: Group P, given HPC with no FGF-2; Group L, given HPC containing 0.03% FGF-2; Group M, given HPC cotaining 0.1% FGF-2; and Group H, given HPC Containing 0.3% FGF-2. Each patient underwent flap operation during which we administered 200 μL of the appropriate investigational drug to the bone defect. Before and for 36 weeks following administration, patients underwent periodontal tissue inspections and standardized radiography of the region under investigation. As a result, a significant difference (p = 0.021) in rate of increase in alveolar bone height was identified between Group P (23.92%) and Group H (58.62%) at 36 weeks. The linear increase in alveolar bone height at 36 weeks in Group P and H was 0.95 mm and 1.85 mm, respectively (p = 0.132). No serious adverse events attribute to the investigational drug were identified. Conclusions: Although no statistically significant differences were noted for gains in clinical attachment level and alveolar bone gain for FGF-2 groups versus Group P, the significant difference in rate of increase in alveolar bone height (p = 0.021) between Groups P and H at 36 weeks suggests that some efficacy could be expected from FGF-2 in stimulating regeneration of periodontal tissue in patients with periodontitis

DOCK2 is involved in the host genetics and biology of severe COVID-19

「コロナ制圧タスクフォース」COVID-19疾患感受性遺伝子DOCK2の重症化機序を解明 --アジア最大のバイオレポジトリーでCOVID-19の治療標的を発見--. 京都大学プレスリリース. 2022-08-10.Identifying the host genetic factors underlying severe COVID-19 is an emerging challenge. Here we conducted a genome-wide association study (GWAS) involving 2, 393 cases of COVID-19 in a cohort of Japanese individuals collected during the initial waves of the pandemic, with 3, 289 unaffected controls. We identified a variant on chromosome 5 at 5q35 (rs60200309-A), close to the dedicator of cytokinesis 2 gene (DOCK2), which was associated with severe COVID-19 in patients less than 65 years of age. This risk allele was prevalent in East Asian individuals but rare in Europeans, highlighting the value of genome-wide association studies in non-European populations. RNA-sequencing analysis of 473 bulk peripheral blood samples identified decreased expression of DOCK2 associated with the risk allele in these younger patients. DOCK2 expression was suppressed in patients with severe cases of COVID-19. Single-cell RNA-sequencing analysis (n = 61 individuals) identified cell-type-specific downregulation of DOCK2 and a COVID-19-specific decreasing effect of the risk allele on DOCK2 expression in non-classical monocytes. Immunohistochemistry of lung specimens from patients with severe COVID-19 pneumonia showed suppressed DOCK2 expression. Moreover, inhibition of DOCK2 function with CPYPP increased the severity of pneumonia in a Syrian hamster model of SARS-CoV-2 infection, characterized by weight loss, lung oedema, enhanced viral loads, impaired macrophage recruitment and dysregulated type I interferon responses. We conclude that DOCK2 has an important role in the host immune response to SARS-CoV-2 infection and the development of severe COVID-19, and could be further explored as a potential biomarker and/or therapeutic target

The whole blood transcriptional regulation landscape in 465 COVID-19 infected samples from Japan COVID-19 Task Force

「コロナ制圧タスクフォース」COVID-19患者由来の血液細胞における遺伝子発現の網羅的解析 --重症度に応じた遺伝子発現の変化には、ヒトゲノム配列の個人差が影響する--. 京都大学プレスリリース. 2022-08-23.Coronavirus disease 2019 (COVID-19) is a recently-emerged infectious disease that has caused millions of deaths, where comprehensive understanding of disease mechanisms is still unestablished. In particular, studies of gene expression dynamics and regulation landscape in COVID-19 infected individuals are limited. Here, we report on a thorough analysis of whole blood RNA-seq data from 465 genotyped samples from the Japan COVID-19 Task Force, including 359 severe and 106 non-severe COVID-19 cases. We discover 1169 putative causal expression quantitative trait loci (eQTLs) including 34 possible colocalizations with biobank fine-mapping results of hematopoietic traits in a Japanese population, 1549 putative causal splice QTLs (sQTLs; e.g. two independent sQTLs at TOR1AIP1), as well as biologically interpretable trans-eQTL examples (e.g., REST and STING1), all fine-mapped at single variant resolution. We perform differential gene expression analysis to elucidate 198 genes with increased expression in severe COVID-19 cases and enriched for innate immune-related functions. Finally, we evaluate the limited but non-zero effect of COVID-19 phenotype on eQTL discovery, and highlight the presence of COVID-19 severity-interaction eQTLs (ieQTLs; e.g., CLEC4C and MYBL2). Our study provides a comprehensive catalog of whole blood regulatory variants in Japanese, as well as a reference for transcriptional landscapes in response to COVID-19 infection

ROTEMを用いた小児特発性ネフローゼ症候群患者の急性期における包括的全血凝固線溶能

Background: Venous thromboembolism is a rare, serious complication of idiopathic nephrotic syndrome (INS) in childhood. The mechanisms responsible for the hypercoagulable state in the acute phase of INS are poorly understood, however. This study aimed to assess overall coagulation and fibrinolytic function in pediatric patients with INS. Methods: Global coagulation and fibrinolysis were examined in whole blood samples from 22 children with initial onset INS (initial-group), 22 children with relapsed INS (relapse-group), and 15 control pediatric patients using rotational thromboelastometry (ROTEM®). In the initial-group, blood samples were obtained before (week 0) and 1-4 weeks after initiation of corticosteroid therapy. EXTEM and FIBTEM were used to assess coagulation and fibrinolysis, respectively. Clot time (CT), clot formation time (CFT), maximum clot firmness (MCF), and α-angle were determined as coagulation parameters, and lysis index at 30 and 60 min (LI30 and LI60, respectively) were assessed as fibrinolytic parameters. Results: CT was significantly shortened, and MCF and α-angle were significantly greater than controls at week 0 and week 1 both in the initial-group and the relapse-group. MCF correlated with serum albumin (r = 0.70, p < 0.001) and fibrinogen level (r = 0.68, p < 0.001). The fibrinolytic parameters (LI30 and LI60) in the initial-group were stable and higher than those in controls at all time points (p < 0.01). Conclusions: We have shown that the hypofibrinolytic defect did not improve with effective NS treatment at the early 4-week time-point. Additionally, a likely pre-thrombotic state was evident in the period before initial onset and 1 week after corticosteroid therapy in pediatric INS.博士（医学）・乙第1522号・令和4年3月15日© 2021. The Author(s), under exclusive licence to International Pediatric Nephrology Association.This version of the article has been accepted for publication, after peer review (when applicable) and is subject to Springer Nature’s AM terms of use, but is not the Version of Record and does not reflect post-acceptance improvements, or any corrections. The Version of Record is available online at: http://dx.doi.org/10.1007/s00467-021-05366-4.発行元が定める登録猶予期間終了の後、本文を登録予定（2023.01

Structural and phylogenetic diversity of anaerobic carbon-monoxide dehydrogenases

Anaerobic Ni-containing carbon-monoxide dehydrogenases (Ni-CODHs) catalyze the reversible conversion between carbon monoxide and carbon dioxide as multi-enzyme complexes responsible for carbon fixation and energy conservation in anaerobic microbes. However, few biochemically characterized model enzymes exist, with most Ni-CODHs remaining functionally unknown. Here, we performed phylogenetic and structure-based Ni-CODH classification using an expanded dataset comprised of 1942 non-redundant Ni-CODHs from 1375 Ni-CODH-encoding genomes across 36 phyla. Ni-CODHs were divided into seven clades, including a novel clade. Further classification into 24 structural groups based on sequence analysis combined with structural prediction revealed diverse structural motifs for metal cluster formation and catalysis, including novel structural motifs potentially capable of forming metal clusters or binding metal ions, indicating Ni-CODH diversity and plasticity. Phylogenetic analysis illustrated that the metal clusters responsible for intermolecular electron transfer were drastically altered during evolution. Additionally, we identified novel putative Ni-CODH-associated proteins from genomic contexts other than the Wood–Ljungdahl pathway and energy converting hydrogenase system proteins. Network analysis among the structural groups of Ni-CODHs, their associated proteins and taxonomies revealed previously unrecognized gene clusters for Ni-CODHs, including uncharacterized structural groups with putative metal transporters, oxidoreductases, or transcription factors. These results suggested diversification of Ni-CODH structures adapting to their associated proteins across microbial genomes