Application of Benchtop NMR for Metabolomics Study Using Feces of Mice with DSS-Induced Colitis

Nuclear magnetic resonance (NMR)-based metabolomics, which comprehensively measures metabolites in biological systems and investigates their response to various perturbations, is widely used in research to identify biomarkers and investigate the pathogenesis of underlying diseases. However, further applications of high-field superconducting NMR for medical purposes and field research are restricted by its high cost and low accessibility. In this study, we applied a low-field, benchtop NMR spectrometer (60 MHz) employing a permanent magnet to characterize the alterations in the metabolic profile of fecal extracts obtained from dextran sodium sulfate (DSS)-induced ulcerative colitis model mice and compared them with the data acquired from high-field NMR (800 MHz). Nineteen metabolites were assigned to the 60 MHz 1H NMR spectra. Non-targeted multivariate analysis successfully discriminated the DSS-induced group from the healthy control group and showed high comparability with high-field NMR. In addition, the concentration of acetate, identified as a metabolite with characteristic behavior, could be accurately quantified using a generalized Lorentzian curve fitting method based on the 60 MHz NMR spectra.journal articl

SuperNova, a monomeric photosensitizing fluorescent protein for chromophore-assisted light inactivation

Takemoto, K., Matsuda, T., Sakai, N. et al. SuperNova, a monomeric photosensitizing fluorescent protein for chromophore-assisted light inactivation. Sci Rep 3, 2629 (2013). https://doi.org/10.1038/srep02629

Myc-induced nuclear antigen constrains a latent intestinal epithelial cell-intrinsic anthelmintic pathway

<div><p>Expulsion of parasitic gastrointestinal nematodes requires diverse effector mechanisms coordinated by a Th2-type response. The evolutionarily conserved JmjC protein; Myc Induced Nuclear Antigen (Mina) has been shown to repress IL4, a key Th2 cytokine, suggesting Mina may negatively regulate nematode expulsion. Here we report that expulsion of the parasitic nematode <i>Trichuris muris</i> was indeed accelerated in Mina deficient mice. Unexpectedly, this was associated not with an elevated Th2- but rather an impaired Th1-type response. Further reciprocal bone marrow chimera and conditional KO experiments demonstrated that retarded parasite expulsion and a normal Th1-type response both required Mina in intestinal epithelial cells (IECs). Transcriptional profiling experiments in IECs revealed anti-microbial α-defensin peptides to be the major target of Mina-dependent retention of worms in infected mice. In vitro exposure to recombinant α-defensin peptides caused cytotoxic damage to whipworms. These results identify a latent IEC-intrinsic anthelmintic pathway actively constrained by Mina and point to α-defensins as important effectors that together with Mina may be attractive therapeutic targets for the control of nematode infection.</p></div

A monoclonal antibody-based sandwich enzyme-linked immunosorbent assay for detection of secreted alpha-defensin

Paneth cells at the base of small intestinal crypts secrete alpha-defensins, which contribute to innate immunity and shape composition of enteric microbiota. Efforts to establish a relationship between secreted alpha-defensins and disease have been hampered by a lack of sensitive assays to quantify luminal alpha-defensins. Here we report on a highly sensitive sandwich enzyme-linked immunosorbent assay (ELISA) for the mouse Paneth cell alpha-defensin cryptdin-4 (Crp4) in varied sources, including luminal contents rinsed from stomach to distal colon and fecal pellets. One pair of monoclonal antibodies (mAbs), selected from 10 rat hybridomas secreting Crp4-specific mAbs, was optimized for Crp4 detection and specificity in the sandwich ELISA. In CD1 mice, luminal Crp4 levels increased gradually from 6.8 +/- 5.2 ng/ml in proximal small intestine to 54.3 +/- 10.3 ng/ml in distal small intestine, and the peptide was detected in colonic lumen and feces. Secreted Crp4 was reduced significantly in feces of IL10 null mice, a model of inflammatory bowel disease (IBD) when compared with wild-type controls. This Crp4 sandwich ELISA enables accurate determinations of luminal alpha-defensins as biomarkers of Paneth cell function and enteric integrity in diverse disease states such as IBD, infectious disease, graft versus host disease, and obesity in association with dysbiosis of the intestinal microbiota. (C) 2013 Elsevier Inc. All rights reserved

Efficient and simple genetic engineering of enteroids using mouse isolated crypts for investigating intestinal functions

Intestinal epithelial cells separate subepithelial tissues from luminal environment formed with food, incoming pathogens, and resident intestinal microbiota, etc., and elicit various intestinal function. Enteroid, a three-dimensional culture system of small intestinal epithelial cells, has been widely used for analyzing the intestinal function, further a transgenic enteroid was developed to investigate the mo-lecular mechanisms. However, conventional transgenic enteroid production method, which transfer gene into single stem cells, has limitations including low efficiency and time-consuming. Here we show that by gene transfer into small intestinal isolated crypts maintaining stem cell niche, a transgenic enteroid was obtained quickly and efficiently. Isolated crypts were transfected by lentiviral vector without separating into single cells, and transgenic enteroid composed of all lineages of intestinal epithelial cells was generated at day 7 with yield of 56%, maintaining the intestinal function in drug transport and innate immunity. Our efficient and simple transgenic enteroid generation method enables high-throughput investigation of intestinal epithelial cells and contributes to understanding intestinal function.(c) 2022 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

Functional analysis of an α-helical antimicrobial peptide derived from a novel mouse defensin-like gene

Gene-encoded antimicrobial peptides (AMPs) are an essential component of the innate immune system in many species. Analysis of β-defensin gene expression in mouse tissue using primers that were specific for conserved sequences located outside of the β-defensin translated region identified a novel small gene. The novel gene had an open reading frame of 114 basepairs and encoded a predicted protein of 37 amino acid residues. A search of the genome database revealed that the gene locus and the sequence of exon 1 of this novel gene were similar to subgroup 1 mouse β-defensins. A small peptide, K17 (FSPQMLQDIIEKKTKIL), derived from the amino acid sequence of this novel gene was synthesized. Circular dichroism (CD) spectroscopic analysis of chemically synthesized peptide demonstrated that the peptide exhibited random coil conformation in aqueous solution, but the peptide adopted helical conformation in the presence of trifluoroethanol or sodium dodecyl sulfate, a membrane mimicking environment. The peptide exhibited bactericidal activity against Salmonella enterica serovar Typhimurium (Gram negative) and Staphylococcus aureus (Gram positive); it was not cytotoxic in cultures of mammalian cells or hemolytic in cultures of erythrocytes. These results suggested that K17 may be a candidate therapeutic for the treatment of bacterial infection