Meta-analysis of shotgun sequencing of gut microbiota in Parkinson's disease.

We aimed to identify gut microbial features in Parkinson's disease (PD) across countries by meta-analyzing our fecal shotgun sequencing dataset of 94 PD patients and 73 controls in Japan with five previously reported datasets from USA, Germany, China1, China2, and Taiwan. GC-MS and LC-MS/MS assays were established to quantify fecal short-chain fatty acids (SCFAs) and fecal polyamines, respectively. α-Diversity was increased in PD across six datasets. Taxonomic analysis showed that species Akkermansia muciniphila was increased in PD, while species Roseburia intestinalis and Faecalibacterium prausnitzii were decreased in PD. Pathway analysis showed that genes in the biosyntheses of riboflavin and biotin were markedly decreased in PD after adjusting for confounding factors. Five out of six categories in carbohydrate-active enzymes (CAZymes) were decreased in PD. Metabolomic analysis of our fecal samples revealed that fecal SCFAs and polyamines were significantly decreased in PD. Genes in the riboflavin and biotin biosyntheses were positively correlated with the fecal concentrations of SCFAs and polyamines. Bacteria that accounted for the decreased riboflavin biosynthesis in Japan, the USA, and Germany were different from those in China1, China2, and Taiwan. Similarly, different bacteria accounted for decreased biotin biosynthesis in the two country groups. We postulate that decreased SCFAs and polyamines reduce the intestinal mucus layer, which subsequently facilitates the formation of abnormal α-synuclein fibrils in the intestinal neural plexus in PD, and also cause neuroinflammation in PD.

Calcitriol ameliorates motor deficits and prolongs survival of Chrne-deficient mouse, a model for congenital myasthenic syndrome, by inducing Rspo2.

Signal transduction at the neuromuscular junction (NMJ) is compromised in a diverse array of diseases including congenital myasthenic syndromes (CMS). Germline mutations in CHRNE encoding the acetylcholine receptor (AChR) ε subunit are the most common cause of CMS. An active form of vitamin D, calcitriol, binds to vitamin D receptor (VDR) and regulates gene expressions. We found that calcitriol enhanced MuSK phosphorylation, AChR clustering, and myotube twitching in co-cultured C2C12 myotubes and NSC34 motor neurons. RNA-seq analysis of co-cultured cells showed that calcitriol increased the expressions of Rspo2, Rapsn, and Dusp6. ChIP-seq of VDR revealed that VDR binds to a region approximately 15 â€‹kbp upstream to Rspo2. Biallelic deletion of the VDR-binding site of Rspo2 by CRISPR/Cas9 in C2C12 myoblasts/myotubes nullified the calcitriol-mediated induction of Rspo2 expression and MuSK phosphorylation. We generated Chrne knockout (Chrne KO) mouse by CRISPR/Cas9. Intraperitoneal administration of calcitriol markedly increased the number of AChR clusters, as well as the area, the intensity, and the number of synaptophysin-positive synaptic vesicles, in Chrne KO mice. In addition, calcitriol ameliorated motor deficits and prolonged survival of Chrne KO mice. In the skeletal muscle, calcitriol increased the gene expressions of Rspo2, Rapsn, and Dusp6. We propose that calcitriol is a potential therapeutic agent for CMS and other diseases with defective neuromuscular signal transmission.

Development of machine learning models for predicting unfavorable functional outcomes from preoperative data in patients with chronic subdural hematomas.

Chronic subdural hematoma (CSDH) often causes neurological deterioration and is treated with hematoma evacuation. This study aimed to assess the feasibility of various machine learning models to preoperatively predict the functional outcome of patients with CSDH. Data were retrospectively collected from patients who underwent CSDH surgery at two institutions: one for internal validation and the other for external validation. The poor functional outcome was defined as a modified Rankin scale score of 3-6 upon hospital discharge. The unfavorable outcome was predicted using four machine learning algorithms on an internal held-out cohort (n = 188): logistic regression, support vector machine (SVM), random forest, and light gradient boosting machine. The prediction performance of these models was also validated in an external cohort (n = 99). The area under the curve of the receiver operating characteristic curve (ROC-AUC) of each machine learning-based model was found to be high in both validations (internal: 0.906-0.925, external: 0.833-0.860). In external validation, the SVM model demonstrated the highest ROC-AUC of 0.860 and accuracy of 0.919. This study revealed the potential of machine learning algorithms in predicting unfavorable outcomes at discharge among patients with CSDH undergoing burr hole surgery.

Gastrointestinal disorders in Parkinson's disease and other Lewy body diseases.

Parkinson's disease (PD) is pathologically characterized by the abnormal accumulation of α-synuclein fibrils (Lewy bodies) in the substantia nigra and other brain regions, although the role of Lewy bodies remains elusive. Constipation usually precedes the motor symptoms in PD, which is in accordance with the notion that α-synuclein fibrils start from the intestinal neural plexus and ascend to the brain in at least half of PD patients. The gut microbiota is likely to be involved in intestinal and brain pathologies. Analyses of the gut microbiota in PD, rapid-eye-movement sleep behavior disorder, and dementia with Lewy bodies suggest three pathological pathways. First, Akkermansia, which is increased in PD, degrades the intestinal mucus layer and increases intestinal permeability, which triggers inflammation and oxidative stress in the intestinal neural plexus. Second, decreased short-chain fatty acids (SCFAs)-producing bacteria in PD reduce the number of regulatory T cells. Third, SCFAs also aggravate microglial activation with an unelucidated pathway. In addition, in dementia with Lewy bodies (DLB), which is another form of α-synucleinopathies, increased genera, Ruminococcus torques and Collinsella, may mitigate neuroinflammation in the substantia nigra by increasing secondary bile acids. Interventions for the gut microbiota and their metabolites may potentially delay or mitigate the development and progression of PD and other Lewy body diseases.

Machine learning models predict delayed hyponatremia post-transsphenoidal surgery using clinically available features.

PURPOSE: Delayed hyponatremia (DHN), a unique complication, is the leading cause of unexpected readmission after pituitary surgery. Therefore, this study aimed to develop tools for predicting postoperative DHN in patients undergoing endoscopic transsphenoidal surgery (eTSS) for pituitary neuroendocrine tumors (PitNETs). METHODS: This was a single-center, retrospective study involving 193 patients with PitNETs who underwent eTSS. The objective variable was DHN, defined as serum sodium levels < 135 mmol/L at ≥ 1 time between post operative days 3 and 9. We trained four machine learning models to predict this objective variable using the clinical variables available preoperatively and on the first postoperative day. The clinical variables included patient characteristics, pituitary-related hormone levels, blood test results, radiological findings, and postoperative complications. RESULTS: The random forest (RF) model demonstrated the highest (0.759 ± 0.039) area under the curve of the receiver operating characteristic curve (ROC-AUC), followed by the support vector machine (0.747 ± 0.034), the light gradient boosting machine (LGBM: 0.738 ± 0.026), and the logistic regression (0.710 ± 0.028). The highest accuracy (0.746 ± 0.029) was observed in the LGBM model. The best-performing RF model was based on 24 features, nine of which were clinically available preoperatively. CONCLUSIONS: The proposed machine learning models with pre- and post-resection features predicted DHN after the resection of PitNETs.

Gut microbiota in dementia with Lewy bodies.

Gut microbiota and fecal bile acids were analyzed in 278 patients with α-synucleinopathies, which were comprised of 28 patients with dementia with Lewy bodies (DLB), 224 patients with Parkinson's disease (PD), and 26 patients with idiopathic rapid eye movement sleep behavior disorder (iRBD). Similarly to PD, short-chain fatty acids-producing genera were decreased in DLB. Additionally, Ruminococcus torques and Collinsella were increased in DLB, which were not changed in PD. Random forest models to differentiate DLB and PD showed that high Ruminococcus torques and high Collinsella, which presumably increase intestinal permeability, as well as low Bifidobacterium, which are also observed in Alzheimer's disease, were predictive of DLB. As Ruminococcus torques and Collinsella are also major secondary bile acids-producing bacteria, we quantified fecal bile acids and found that the production of ursodeoxycholic acid (UDCA) was high in DLB. Increased UDCA in DLB may mitigate neuroinflammation at the substantia nigra, whereas neuroinflammation may not be critical at the neocortex. Theraeutic intervention to increase Bifidobacteirum and its metabolites may retard the development and progression of DLB.

Short chain fatty acids-producing and mucin-degrading intestinal bacteria predict the progression of early Parkinson's disease.

To elucidate the relevance of gut dysbiosis in Parkinson's disease (PD) in disease progression, we made random forest models to predict the progression of PD in two years by gut microbiota in 165 PD patients. The area under the receiver operating characteristic curves (AUROCs) of gut microbiota-based models for Hoehn & Yahr (HY) stages 1 and 2 were 0.799 and 0.705, respectively. Similarly, gut microbiota predicted the progression of Movement Disorder Society-Unified Parkinson's Disease Rating Scale (MDS-UPDRS) III scores in an early stage of PD with AUROC = 0.728. Decreases of short-chain fatty acid-producing genera, Fusicatenibacter, Faecalibacterium, and Blautia, as well as an increase of mucin-degrading genus Akkermansia, predicted accelerated disease progression. The four genera remained unchanged in two years in PD, indicating that the taxonomic changes were not the consequences of disease progression. PD patients with marked gut dysbiosis may thus be destined to progress faster than those without gut dysbiosis.

Molecular Hydrogen Enhances Proliferation of Cancer Cells That Exhibit Potent Mitochondrial Unfolded Protein Response.

Molecular hydrogen ameliorates pathological states in a variety of human diseases, animal models, and cell models, but the effects of hydrogen on cancer have been rarely reported. In addition, the molecular mechanisms underlying the effects of hydrogen remain mostly unelucidated. We found that hydrogen enhances proliferation of four out of seven human cancer cell lines (the responders). The proliferation-promoting effects were not correlated with basal levels of cellular reactive oxygen species. Expression profiling of the seven cells showed that the responders have higher gene expression of mitochondrial electron transport chain (ETC) molecules than the non-responders. In addition, the responders have higher mitochondrial mass, higher mitochondrial superoxide, higher mitochondrial membrane potential, and higher mitochondrial spare respiratory capacity than the non-responders. In the responders, hydrogen provoked mitochondrial unfolded protein response (mtUPR). Suppression of cell proliferation by rotenone, an inhibitor of mitochondrial ETC complex I, was rescued by hydrogen in the responders. Hydrogen triggers mtUPR and induces cell proliferation in cancer cells that have high basal and spare mitochondrial ETC activities.

Altered gut microbiota in Parkinson's disease patients with motor complications.

INTRODUCTION: Parkinson's disease (PD) is associated with gut dysbiosis. However, whether gut dysbiosis can cause motor complications is unclear. METHODS: Subjects were enrolled from four independent movement disorder centers in Japan. We performed 16S ribosomal RNA gene sequence analysis of gut microbiota. Relative abundance of gut microbiota and relationships between them and clinical characteristics were statistically analyzed. Analysis of co-variance (ANCOVA) was used to assess altered gut microbiota associated with wearing-off or dyskinesia. RESULTS: We enrolled 223 patients with PD. Wearing-off was noted in 47.5% of patients and dyskinesia in 21.9%. We detected 98 genera of bacteria. Some changes in the gut microbiota were observed in patients with PD and motor complications. After Bonferroni correction, patients with wearing-off showed decreased relative abundance of Lachnospiraceae Blautia (p < 0.0001) and increased relative abundance of Lactobacillaceae Lactobacillus (p < 0.0001), but patients with dyskinesia no longer showed significant changes in the gut microbiota. Adjustment with two models of confounding factors followed by ANCOVA revealed that age (p < 0.0001), disease duration (p = 0.01), and wearing-off (p = 0.0004) were independent risks for the decreased relative abundance of Lachnospiraceae Blautia, and wearing-off (p = 0.009) was the only independent risk factor for the increased relative abundance of Lachnospiraceae Lactobacillus. CONCLUSION: Relative abundance of Lachnospiraceae Blautia and Lactobacillaceae Lactobacillus was significantly decreased and increased, respectively, in the gut microbiota of PD patients with motor complications. This indicates that an altered gut microbiota is associated with the development of motor complications in patients with advanced PD.

Effects of Pesticide Intake on Gut Microbiota and Metabolites in Healthy Adults.

Animal experiments have indicated that pesticides may affect gut microbiota, which is responsible for the production of short-chain fatty acids (SCFAs) and polyamines. Here, we present a preliminary observation of the relationship between pesticide exposure and fecal SCFAs and polyamines in Japanese adults. In total, 38 healthy adults aged 69 ± 10 years (mean ± SD) were recruited and subjected to stool and spot urine tests. Urinary dialkylphosphates (DAP), 3-phenoxybenzoic acid, and glyphosate were assayed as pesticide exposure markers of organophosphorus insecticide (OP), a pyrethroid insecticide, and glyphosate, respectively. Significant negative correlations (p < 0.05, Spearman's rank correlation coefficient) were found between urinary DAP, fecal acetate (r = −0.345), and lactate (r = −0.391). Multiple regression analyses revealed that urinary DAP was a significant explanatory variable of fecal acetate concentration (p < 0.001, ß = −24.0, SE = 4.9, t = −4.9) with some vegetable intake (adjusted R-square = 0.751). These findings suggest that OP exposure is independently associated with lower fecal acetate levels, which may contribute to human health in middle-aged and older adult groups. Given that the human gut environment has long-term effects on the host, studies on wide-range age groups, including children, are necessary.

Intestinal Collinsella may mitigate infection and exacerbation of COVID-19 by producing ursodeoxycholate.

The mortality rates of COVID-19 vary widely across countries, but the underlying mechanisms remain unelucidated. We aimed at the elucidation of relationship between gut microbiota and the mortality rates of COVID-19 across countries. Raw sequencing data of 16S rRNA V3-V5 regions of gut microbiota in 953 healthy subjects in ten countries were obtained from the public database. We made a generalized linear model (GLM) to predict the COVID-19 mortality rates using gut microbiota. GLM revealed that low genus Collinsella predicted high COVID-19 mortality rates with a markedly low p-value. Unsupervised clustering of gut microbiota in 953 subjects yielded five enterotypes. The mortality rates were increased from enterotypes 1 to 5, whereas the abundances of Collinsella were decreased from enterotypes 1 to 5 except for enterotype 2. Collinsella produces ursodeoxycholate. Ursodeoxycholate was previously reported to inhibit binding of SARS-CoV-2 to angiotensin-converting enzyme 2; suppress pro-inflammatory cytokines like TNF-α, IL-1ß, IL-2, IL-4, and IL-6; have antioxidant and anti-apoptotic effects; and increase alveolar fluid clearance in acute respiratory distress syndrome. Ursodeoxycholate produced by Collinsella may prevent COVID-19 infection and ameliorate acute respiratory distress syndrome in COVID-19 by suppressing cytokine storm syndrome.

Short-Chain Fatty Acid-Producing Gut Microbiota Is Decreased in Parkinson's Disease but Not in Rapid-Eye-Movement Sleep Behavior Disorder.

Gut dysbiosis has been repeatedly reported in Parkinson's disease (PD) but only once in idiopathic rapid-eye-movement sleep behavior disorder (iRBD) from Germany. Abnormal aggregation of α-synuclein fibrils causing PD possibly starts from the intestine, although this is still currently under debate. iRBD patients frequently develop PD. Early-stage gut dysbiosis that is causally associated with PD is thus expected to be observed in iRBD. We analyzed gut microbiota in 26 iRBD patients and 137 controls by 16S rRNA sequencing (16S rRNA-seq). Our iRBD data set was meta-analyzed with the German iRBD data set and was compared with gut microbiota in 223 PD patients. Unsupervised clustering of gut microbiota by LIGER, a topic model-based tool for single-cell RNA sequencing (RNA-seq) analysis, revealed four enterotypes in controls, iRBD, and PD. Short-chain fatty acid (SCFA)-producing bacteria were conserved in an enterotype observed in controls and iRBD, whereas they were less conserved in enterotypes observed in PD. Genus Akkermansia and family Akkermansiaceae were consistently increased in both iRBD in two countries and PD in five countries. Short-chain fatty acid (SCFA)-producing bacteria were not significantly decreased in iRBD in two countries. In contrast, we previously reported that recognized or putative SCFA-producing genera Faecalibacterium, Roseburia, and Lachnospiraceae ND3007 group were consistently decreased in PD in five countries. In α-synucleinopathy, increase of mucin-layer-degrading genus Akkermansia is observed at the stage of iRBD, whereas decrease of SCFA-producing genera becomes obvious with development of PD.IMPORTANCE Twenty studies on gut microbiota in PD have been reported, whereas only one study has been reported on iRBD from Germany. iRBD has the highest likelihood ratio to develop PD. Our meta-analysis of iRBD in Japan and Germany revealed increased mucin-layer-degrading genus Akkermansia in iRBD. Genus Akkermansia may increase the intestinal permeability, as we previously observed in PD patients, and may make the intestinal neural plexus exposed to oxidative stress, which can lead to abnormal aggregation of prion-like α-synuclein fibrils in the intestine. In contrast to PD, SCFA-producing bacteria were not decreased in iRBD. As SCFA induces regulatory T (Treg) cells, a decrease of SCFA-producing bacteria may be a prerequisite for the development of PD. We propose that prebiotic and/or probiotic therapeutic strategies to increase the intestinal mucin layer and to increase intestinal SCFA potentially retard the development of iRBD and PD.

Meta-Analysis of Gut Dysbiosis in Parkinson's Disease.

BACKGROUND: PD may begin with the intestinal accumulation of α-synuclein fibrils, which can be causally associated with gut dysbiosis. The variability of gut microbiota across countries prevented us from identifying shared gut dysbiosis in PD. OBJECTIVES: To identify gut dysbiosis in PD across countries. METHODS: We performed 16S ribosomal RNA gene sequencing analysis of gut microbiota in 223 patients with PD and 137 controls, and meta-analyzed gut dysbiosis by combining our dataset with four previously reported data sets from the United States, Finland, Russia, and Germany. We excluded uncommon taxa from our analyses. For pathway analysis, we developed the Kyoto Encyclopedia of Genes and Genomes orthology set enrichment analysis method. RESULTS: After adjusting for confounding factors (body mass index, constipation, sex, age, and catechol-O-methyl transferase inhibitor), genera Akkermansia and Catabacter, as well as families Akkermansiaceae, were increased, whereas genera Roseburia, Faecalibacterium, and Lachnospiraceae ND3007 group were decreased in PD. Catechol-O-methyl transferase inhibitor intake markedly increased family Lactobacillaceae. Inspection of these bacteria in 12 datasets that were not included in the meta-analysis revealed that increased genus Akkermansia and decreased genera Roseburia and Faecalibacterium were frequently observed across countries. Kyoto Encyclopedia of Genes and Genomes orthology set enrichment analysis revealed changes in short-chain fatty acid metabolisms in our dataset. CONCLUSIONS: We report that intestinal mucin layer-degrading Akkermansia is increased and that short-chain fatty acid-producing Roseburia and Faecalibacterium are decreased in PD across countries. © 2020 International Parkinson and Movement Disorder Society.

Molecular hydrogen upregulates heat shock response and collagen biosynthesis, and downregulates cell cycles: meta-analyses of gene expression profiles.

Molecular hydrogen exerts its effect on multiple pathologies, including oxidative stress, inflammation, and apoptosis. However, its molecular mechanisms have not been fully elucidated. In order to explore the effects of molecular hydrogen, we meta-analysed gene expression profiles modulated by molecular hydrogen. We performed microarray analysis of the mouse liver with or without drinking hydrogen water. We also integrated two previously reported microarray datasets of the rat liver into meta-analyses. We used two categories of meta-analysis methods: the cross-platform method and the conventional meta-analysis method (Fisher's method). For each method, hydrogen-modulated pathways were analysed by (i) the hypergeometric test (HGT) in the class of over-representation analysis (ORA), (ii) the gene set enrichment analysis (GSEA) in the class of functional class scoring (FCS), and (iii) the signalling pathway impact analysis (SPIA), pathway regulation score (PRS), and others in the class of pathway topology-based approach (PTA). Pathways in the collagen biosynthesis and the heat-shock response were up-regulated according to (a) HGT with the cross-platform method, (b) GSEA with the cross-platform method, and (c) PRS with the cross-platform method. Pathways in cell cycles were down-regulated according to (a) HGT with the cross-platform method, (b) GSEA with the cross-platform method, and (d) GSEA with the conventional meta-analysis method. Because the heat-shock response leads to up-regulation of collagen biosynthesis and a transient arrest of cell cycles, induction of the heat-shock response is likely to be a primary event induced by molecular hydrogen in the liver of wild-type rodents.