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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-1β, 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.

Oral administration of hydrogen water ameliorates Parkinson's disease (PD) in rats, mice, and humans. We previously reported that the number of putative hydrogen-producing bacteria in intestinal microbiota is low in PD compared to controls. We also reported that the amount of hydrogen produced by ingestion of lactulose is low in PD patients. The decreased hydrogen production by intestinal microbiota may be associated with the development and progression of PD. We measured the amount of hydrogen production using gas chromatography by seven bacterial strains, which represented seven major intestinal bacterial groups/genera/species. Blautia coccoides and Clostridium leptum produced the largest amount of hydrogen. Escherichia coli and Bacteroides fragilis constituted the second group that produced hydrogen 34- to 93-fold lower than B. coccoides. Bifidobacterium pseudocatenulatum and Atopobium parvulum constituted the third group that produced hydrogen 559- to 2164-fold lower than B. coccoides. Lactobacillus casei produced no detectable hydrogen. Assuming that taxonomically neighboring strains have similar hydrogen production, we simulated hydrogen production using intestinal microbiota that we previously reported, and found that PD patients produce a 2.2-fold lower amount of intestinal hydrogen compared to controls. The lower amount of intestinal hydrogen production in PD was also simulated in cohorts of two other countries. The number of hydrogen-producing intestinal bacteria may be associated with the development and progression of PD. Further studies are required to prove its beneficial effect.

After tendon injuries, biomechanical properties of the injured tendon are not fully recovered in most cases. Modulation of signaling pathways, which are involved in tendon development and tendon repair, is one of attractive modalities to facilitate proper regeneration of the injured tendon. The roles of TGF-β signaling in tendon homeostasis and tendon development have been elucidated. In contrast, the roles of Wnt/β-catenin signaling in tendon remain mostly elusive. We found that the number of β-catenin-positive cells was increased at the injured site, suggesting involvement of Wnt/β-catenin signaling in tendon healing. Activation of Wnt/β-catenin signaling suppressed expressions of tenogenic genes of Scx, Mkx, and Tnmd in rat tendon-derived cells (TDCs) isolated from the Achilles tendons of 6-week old rats. Additionally, activation of Wnt/β-catenin reduced the amounts of Smad2 and Smad3, which are intracellular mediators for TGF-β signaling, and antagonized upregulation of Scx induced by TGF-β signaling in TDCs. We found that Wnt/β-catenin decreased Mkx and Tnmd expressions without suppressing Scx expression in Scx-programmed tendon progenitors. Our studies suggest that Wnt/β-catenin signaling is a repressor for tenogenic gene expressions.

Abnormal activation of the Wnt/β-catenin signaling is implicated in the osteoarthritis (OA) pathology. We searched for a pre-approved drug that suppresses abnormally activated Wnt/β-catenin signaling and has a potency to reduce joint pathology in OA. We introduced the TOPFlash reporter plasmid into HCS-2/8 human chondrosarcoma cells to estimate the Wnt/β-catenin activity in the presence of 10 μM each compound in a panel of pre-approved drugs. We found that fluoxetine, an antidepressant in the class of selective serotonin reuptake inhibitors (SSRI), down-regulated Wnt/β-catenin signaling in human chondrosarcoma cells. Fluoxetine inhibited both Wnt3A- and LiCl-induced loss of proteoglycans in chondrogenically differentiated ATDC5 cells. Fluoxetine increased expression of Sox9 (the chondrogenic master regulator), and decreased expressions of Axin2 (a marker for Wnt/β-catenin signaling) and Mmp13 (matrix metalloproteinase 13). Fluoxetine suppressed a LiCl-induced increase of total β-catenin and a LiCl-induced decrease of phosphorylated β-catenin in a dose-dependent manner. An in vitro protein-binding assay showed that fluoxetine enhanced binding of β-catenin with Axin1, which is a scaffold protein forming the degradation complex for β-catenin. Fluoxetine suppressed LiCl-induced β-catenin accumulation in human OA chondrocytes. Intraarticular injection of fluoxetine in a rat OA model ameliorated OA progression and suppressed β-catenin accumulation.

SMXA-5 mice are a high-fat diet-induced type 2 diabetes animal model established from non-diabetic SM/J and A/J mice. By using F2 intercross mice between SMXA-5 and SM/J mice under feeding with a high-fat diet, we previously mapped a major diabetogenic QTL (T2dm2sa) on chromosome 2. We then produced the congenic strain (SM.A-T2dm2sa (R0), 20.8-163.0 Mb) and demonstrated that the A/J allele of T2dm2sa impaired glucose tolerance and increased body weight and body mass index in the congenic strain compared to SM/J mice. We also showed that the combination of T2dm2sa and other diabetogenic loci was needed to develop the high-fat diet-induced type 2 diabetes. In this study, to narrow the potential genomic region containing the gene(s) responsible for T2dm2sa, we constructed R1 and R2 congenic strains. Both R1 (69.6-163.0 Mb) and R2 (20.8-128.2 Mb) congenic mice exhibited increases in body weight and abdominal fat weight and impaired glucose tolerance compared to SM/J mice. The R1 and R2 congenic analyses strongly suggested that the responsible genes existed in the overlapping genomic interval (69.6-128.2 Mb) between R1 and R2. In addition, studies using the newly established R1A congenic strain showed that the narrowed genomic region (69.6-75.4 Mb) affected not only obesity but also glucose tolerance. To search for candidate genes within the R1A genomic region, we performed exome sequencing analysis between SM/J and A/J mice and extracted 4 genes (Itga6, Zak, Gpr155, and Mtx2) with non-synonymous coding SNPs. These four genes might be candidate genes for type 2 diabetes caused by gene-gene interactions. This study indicated that one of the genes responsible for high-fat diet-induced diabetes exists in the 5.8 Mb genomic interval on mouse chromosome 2.

In past years, the canonical Wnt/β-catenin signaling pathway has emerged as a critical regulator of cartilage development and homeostasis. FRZB, a soluble antagonist of Wnt signaling, has been studied in osteoarthritis (OA) animal models and OA patients as a modulator of Wnt signaling. We screened for FDA-approved drugs that induce FRZB expression and suppress Wnt/β-catenin signaling. We found that verapamil, a widely prescribed L-type calcium channel blocker, elevated FRZB expression and suppressed Wnt/β-catenin signaling in human OA chondrocytes. Expression and nuclear translocation of β-catenin was attenuated by verapamil in OA chondrocytes. Lack of the verapamil effects in LiCl-treated and FRZB-downregulated OA chondrocytes also suggested that verpamil suppressed Wnt signaling by inducing FRZB. Verapamil enhanced gene expressions of chondrogenic markers of ACAN encoding aggrecan, COL2A1 encoding collagen type II α1, and SOX9, and suppressed Wnt-responsive AXIN2 and MMP3 in human OA chondrocytes. Verapamil ameliorated Wnt3A-induced proteoglycan loss in chondrogenically differentiated ATDC5 cells. Verapamil inhibited hypertrophic differentiation of chondrocytes in the explant culture of mouse tibiae. Intraarticular injection of verapamil inhibited OA progression as well as nuclear localizations of β-catenin in a rat OA model. We propose that verapamil holds promise as a potent therapeutic agent for OA by upregulating FRZB and subsequently downregulating Wnt/β-catenin signaling.

Achondroplasia (ACH) is one of the most common skeletal dysplasias with short stature caused by gain-of-function mutations in FGFR3 encoding the fibroblast growth factor receptor 3. We used the drug repositioning strategy to identify an FDA-approved drug that suppresses abnormally activated FGFR3 signaling in ACH. We found that meclozine, an anti-histamine drug that has long been used for motion sickness, facilitates chondrocyte proliferation and mitigates loss of extracellular matrix in FGF2-treated rat chondrosarcoma (RCS) cells. Meclozine also ameliorated abnormally suppressed proliferation of human chondrosarcoma (HCS-2/8) cells that were infected with lentivirus expressing constitutively active mutants of FGFR3-K650E causing thanatophoric dysplasia, FGFR3-K650M causing SADDAN, and FGFR3-G380R causing ACH. Similarly, meclozine alleviated abnormally suppressed differentiation of ATDC5 chondrogenic cells expressing FGFR3-K650E and -G380R in micromass culture. We also confirmed that meclozine alleviates FGF2-mediated longitudinal growth inhibition of embryonic tibia in bone explant culture. Interestingly, meclozine enhanced growth of embryonic tibia in explant culture even in the absence of FGF2 treatment. Analyses of intracellular FGFR3 signaling disclosed that meclozine downregulates phosphorylation of ERK but not of MEK in FGF2-treated RCS cells. Similarly, meclozine enhanced proliferation of RCS cells expressing constitutively active mutants of MEK and RAF but not of ERK, which suggests that meclozine downregulates the FGFR3 signaling by possibly attenuating ERK phosphorylation. We used the C-natriuretic peptide (CNP) as a potent inhibitor of the FGFR3 signaling throughout our experiments, and found that meclozine was as efficient as CNP in attenuating the abnormal FGFR3 signaling. We propose that meclozine is a potential therapeutic agent for treating ACH and other FGFR3-related skeletal dysplasias.