Protective effect of Bifidobacterium longum BB536 against nausea caused by pirfenidone in a mouse model of pellagra.

Pellagra is caused by abnormal intake and/or use of nicotinic acid and is known in part to be induced by the use of medications such as isoniazid or pirfenidone. We previously investigated atypical phenotypes of pellagra, such as nausea, using a mouse model of pellagra and found that gut microbiota play an important role in the development of these phenotypes. Here, we investigated the effect of Bifidobacterium longum BB536 on pellagra-related nausea caused by pirfenidone in our mouse model. Our pharmacological data indicated that pirfenidone (PFD) causes modulation of the gut microbiota profile, which appeared to play an important role in the development of pellagra-related nausea. A gut microbiota-mediated protective effect of B. longum BB536 against nausea caused by PFD was also identified. Finally, the urinary ratio of nicotinamide/N-methylnicotinamide was shown to be a biomarker of pellagra-like adverse effects induced by PFD, and it may contribute to the prevention of these effects in patients with idiopathic pulmonary fibrosis.

Targeted amplicon sequencing of wastewater samples for detecting SARS-CoV-2 variants with high sensitivity and resolution.

Wastewater-based epidemiology (WBE) is a promising approach for monitoring the spread of SARS-CoV-2 within communities. Although qPCR-based WBE is powerful in that it allows quick and highly sensitive detection of this virus, it can provide limited information about which variants are responsible for the overall increase or decrease of this virus in sewage, and this hinders accurate risk assessments. To resolve this problem, we developed a next generation sequencing (NGS)-based method to determine the identity and composition of individual SARS-CoV-2 variants in wastewater samples. Combination and optimization of targeted amplicon-sequencing and nested PCR allowed detection of each variant with sensitivity comparable to that of qPCR. In addition, by targeting the receptor binding domain (RBD) of the S protein, which has mutations informative for variant classification, we could discriminate most variants of concern (VOC) and even sublineages of Omicron (BA.1, BA.2, BA.4/5, BA.2.75, BQ.1.1 and XBB.1). Focusing on a limited domain has a benefit of decreasing the sequencing reads. We applied this method to wastewater samples collected from a wastewater treatment plant in Kyoto city throughout 13 months (from January 2021 to February 2022) and successfully identified lineages of wild-type, alpha, delta, omicron BA.1 and BA.2 as well as their compositions in the samples. The transition of these variants was in good agreement with the epidemic situation reported in Kyoto city during that period based on clinical testing. These data indicate that our NGS-based method is useful for detecting and tracking emerging variants of SARS-CoV-2 in sewage samples. Coupled with the advantages of WBE, this method has the potential to serve as an efficient and low cost means for the community risk assessment of SARS-CoV-2 infection.

Near full-automation of COPMAN using a LabDroid enables high-throughput and sensitive detection of SARS-CoV-2 RNA in wastewater as a leading indicator.

Wastewater-based epidemiology (WBE) is a promising tool to efficiently monitor COVID-19 prevalence in a community. For WBE community surveillance, automation of the viral RNA detection process is ideal. In the present study, we achieved near full-automation of a previously established method, COPMAN (COagulation and Proteolysis method using MAgnetic beads for detection of Nucleic acids in wastewater), which was then applied to detect SARS-CoV-2 in wastewater for half a year. The automation line employed the Maholo LabDroid and an automated-pipetting device to achieve a high-throughput sample-processing capability of 576 samples per week. SARS-CoV-2 RNA was quantified with the automated COPMAN using samples collected from two wastewater treatment plants in the Sagami River basin in Japan between 1 November 2021 and 24 May 2022, when the numbers of daily reported COVID-19 cases ranged from 0 to 130.3 per 100,000 inhabitants. The automated COPMAN detected SARS-CoV-2 RNA from 81 out of 132 samples at concentrations of up to 2.8 × 105 copies/L. These concentrations showed direct correlations with subsequently reported clinical cases (5-13 days later), as determined by Pearson's and Spearman's cross-correlation analyses. To compare the results, we also conducted testing with the EPISENS-S (Efficient and Practical virus Identification System with ENhanced Sensitivity for Solids, Ando et al., 2022), a previously reported detection method. SARS-CoV-2 RNA detected with EPISENS-S correlated with clinical cases only when using Spearman's method. Our automated COPMAN was shown to be an efficient method for timely and large-scale monitoring of viral RNA, making WBE more feasible for community surveillance.

COPMAN: A novel high-throughput and highly sensitive method to detect viral nucleic acids including SARS-CoV-2 RNA in wastewater.

During the coronavirus disease 2019 (COVID-19) pandemic, wastewater-based epidemiology (WBE) attracted attention as an objective and comprehensive indicator of community infection that does not require individual inspection. Although several severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection methods from wastewater have been developed, there are obstacles to their social implementation. In this study, we developed the COPMAN (Coagulation and Proteolysis method using Magnetic beads for detection of Nucleic acids in wastewater), an automatable method that can concentrate and detect multiple types of viruses from a limited volume (∼10 mL) of wastewater. The COPMAN consists of a high basicity polyaluminum chloride (PAC) coagulation process, magnetic bead-based RNA purification, and RT-preamplification, followed by qPCR. A series of enzymes exhibiting a high tolerance to PCR inhibitors derived from wastewater was identified and employed in the molecular detection steps in the COPMAN. We compared the detectability of viral RNA from 10-mL samples of virus-spiked (heat-inactivated SARS-CoV-2 and intact RSV) or unspiked wastewater by the COPMAN and other methods (PEG-qPCR, UF-qPCR, and EPISENS-S). The COPMAN was the most efficient for detecting spiked viruses from wastewater, detecting the highest level of pepper mild mottle virus (PMMoV), a typical intrinsic virus in human stool, from wastewater samples. The COPMAN also successfully detected indigenous SARS-CoV-2 RNA from 12 samples of wastewater at concentrations of 2.2 × 104 to 5.4 × 105 copies/L, during initial stages of an infection wave in the right and the left bank of the Sagami River in Japan (0.65 to 11.45 daily reported cases per 100,000 people). These results indicate that the COPMAN is suitable for detection of multiple pathogens from small volume of wastewater in automated stations.

Analysis of real-world data and a mouse model indicates that pirfenidone causes pellagra.

Background: Pirfenidone (PFD) is widely used in patients with idiopathic pulmonary fibrosis (IPF) and its adverse effects, such as nausea and photosensitivity, are well known. Many patients with IPF have reduced doses or even cessation of PFD because of its side-effects. No solutions have been found for these side-effects because the current mechanistic insights are insufficient. Methods: Using the results of real-world data analysis from the US Food and Drug Administration Adverse Events Reporting System, we hypothesised that PFD-related symptoms may be similar to pellagra. Reverse translational experiments using female BALB/c mice were performed to validate and estimate this hypothesis. Niacin and its metabolite responses were compared between patients with IPF treated with PFD and those treated without PFD. Results: The pellagra hypothesis was translated from real-world data analysis. Pharmacological and comprehensive genetic investigations showed that PFD caused pellagra-related nausea and photosensitivity in a mouse model, which may have been mediated by the actions of nicotinamide N-methyltransferase (NNMT). Higher NNMT substrate responses were observed in urine from patients and mice with PFD than in those without PFD. Conclusions: PFD may cause pellagra or pellagra-like symptoms such as photosensitivity. Further studies are required to investigate whether niacin prevents pellagra-like symptoms caused by PFD in patients with IPF.

Analysis of the gut microbiome to validate a mouse model of pellagra.

Pellagra is caused by an abnormal intake and/or use of niacin, but its phenotypes are diverse. The phenotypes of pellagra can also be atypical, such as nausea. We previously reported a mouse model of pellagra-related nausea. However, the mechanism of this model is unclear. In this study, we found that the gut microbiota, which is thought to be a source of niacin, played an important role in the development of pellagra-related nausea in germ-free mice. We also investigated the gut microbiome. We compared urinary niacin metabolite levels and the dermal response between mice fed a normal diet and those fed a low-niacin diet to investigate the putative trigger of pellagra. Epoxyeicosatrienoic and hydroxyeicosatetraenoic acid levels were higher in mice fed a low-niacin diet compared with those fed a normal diet. Furthermore, histological studies indicated a dermatological response to the low-niacin diet. Interestingly, higher levels of oxidised fatty acids in response to the germ-free state were also observed. These findings indicate successful establishment of our newly established mouse model of pellagra via the gut microbiota. We believe that this model could enable the discovery of the putative cause of pellagra and phenotypes of pellagra that have not been recognised yet.

The significance of NAD + metabolites and nicotinamide N-methyltransferase in chronic kidney disease.

Dysregulation of nicotinamide adenine dinucleotide (NAD +) metabolism contributes to the initiation and progression of age-associated diseases, including chronic kidney disease (CKD). Nicotinamide N-methyltransferase (NNMT), a nicotinamide (NAM) metabolizing enzyme, regulates both NAD + and methionine metabolism. Although NNMT is expressed abundantly in the kidney, its role in CKD and renal fibrosis remains unclear. We generated NNMT-deficient mice and a unilateral ureter obstruction (UUO) model and conducted two clinical studies on human CKD to investigate the role of NNMT in CKD and fibrosis. In UUO, renal NNMT expression and the degraded metabolites of NAM increased, while NAD + and NAD + precursors decreased. NNMT deficiency ameliorated renal fibrosis; mechanistically, it (1) increased the DNA methylation of connective tissue growth factor (CTGF), and (2) improved renal inflammation by increasing renal NAD + and Sirt1 and decreasing NF-κB acetylation. In humans, along with CKD progression, a trend toward a decrease in serum NAD + precursors was observed, while the final NAD + metabolites were accumulated, and the level of eGFR was an independent variable for serum NAM. In addition, NNMT was highly expressed in fibrotic areas of human kidney tissues. In conclusion, increased renal NNMT expression induces NAD + and methionine metabolism perturbation and contributes to renal fibrosis.

Effect of niacin supplementation on nausea-like behaviour in an isoniazid-induced mouse model of pellagra.

Niacin deficiency causes pellagra, the symptoms of which include dermatitis, diarrhoea and dementia. Investigating the mechanism underlying these phenotypes has been challenging due to the lack of an appropriate animal model. Here, we report a mouse model of pellagra-related nausea induced by feeding mice a low-niacin diet and administering isoniazid (INH), which is thought to induce pellagra. Mice fed a normal or low-niacin diet received INH (0·3 or 1·0 mg/mg per animal, twice daily, 5 d), and nausea was evaluated based on pica behaviour, which considered the rodent equivalent of the emetic reflex. Furthermore, the effect of therapeutic niacin administration on nausea was evaluated in this model. Urinary and hepatic metabolite levels were analysed by LC coupled with MS. INH-induced pica was observed in mice fed a low-niacin diet but not in those fed a normal diet. Levels of urinary metabolites, such as 1-methyl-2-pyridone-5-carboxamide, kynurenic acid and xanthurenic acid, were significantly reduced in the mice treated with INH compared with those that did not receive INH. Furthermore, niacin supplementation prevented pica and restored the levels of some metabolites in this mouse model. Our findings suggest that INH-related nausea is pellagra-like. We also believe that our newly established method for quantifying pica is a useful tool for investigating the mechanisms of pellagra-related nausea.