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BACKGROUND: Clostridioides difficile infection (CDI) is a debilitating nosocomial disease. Postmenopausal women may have an increased risk of CDI, suggesting estrogen influence. Soybean products contain a representative estrogenic isoflavone, genistein. METHODS: The anti-inflammatory and antiapoptotic effects of genistein were determined using primary human cells and fresh colonic tissues. The effects of oral genistein therapy among mice and hamsters were evaluated. RESULTS: Within 10 days of CDI, female c57BL/6J mice in a standard environment (regular diet) had a 50% survival rate, while those with estrogen depletion and in an isoflavone-free environment (soy-free diet) had a 25% survival rate. Oral genistein improved their 10-day survival rate to 100% on a regular diet and 75% in an isoflavone-free environment. Genistein reduced macrophage inflammatory protein-1α (MIP-1α) secretion in fresh human colonic tissues exposed to toxins. Genistein inhibited MIP-1α secretion in primary human peripheral blood mononuclear cells, abolished apoptosis and BCL-2-associated X (BAX) expression in human colonic epithelial cells, and activated lysine-deficient protein kinase 1 (WNK1) phosphorylation in both cell types. The anti-inflammatory and antiapoptotic effects of genistein were abolished by inhibiting estrogen receptors and WNK1. CONCLUSIONS: Genistein reduces CDI disease activity by inhibiting proinflammatory cytokine expression and apoptosis via the estrogen receptor/G-protein estrogen receptor/WNK1 pathways.

BackgroundClostridium difficile infection (CDI) causes diarrhea and colitis. We aimed to find a common pathogenic pathway in CDI among humans and mice by comparing toxin-mediated effects in human and mouse colonic tissues.MethodUsing multiplex enzyme-linked immunosorbent assay, we determined the cytokine secretion of toxin A- and B-treated human and mouse colonic explants.ResultsToxin A and toxin B exposure to fresh human and mouse colonic explants caused different patterns of cytokine secretion. Toxin A induced macrophage inflammatory protein (MIP) 1α secretion in both human and mouse explants. Toxin A reduced the expression of chloride anion exchanger SLC26A3 expression in mouse colonic explants and human colonic epithelial cells. Patients with CDI had increased colonic MIP-1 α expression and reduced colonic SLC26A3 (solute carrier family 26, member 3) compared with controls. Anti-MIP-1 α neutralizing antibody prevented death, ameliorated colonic injury, reduced colonic interleukin 1β (IL-1β) messenger RNA expression, and restored colonic SLC26a3 expression in C. difficile-infected mice. The anti-MIP-1 α neutralizing antibody prevented CDI recurrence. SLC26a3 inhibition augmented colonic IL-1 β messenger RNA expression and abolished the protective effect of anti-MIP-1 α neutralizing antibody in mice with CDI.ConclusionMIP-1 α is a common toxin A-dependent chemokine in human and mouse colon. MIP-1 α mediates detrimental effects by reducing SLC26a3 and enhancing IL-1 β expression in the colon.

Clostridium difficile is an opportunistic pathogen that establishes in the colon when the gut microbiota are disrupted by antibiotics or disease. C. difficile infection (CDI) is largely caused by two virulence factors, TcdA and TcdB. Here, we report a 3.87-Å-resolution crystal structure of TcdB holotoxin that captures a unique conformation of TcdB at endosomal pH. Complementary biophysical studies suggest that the C-terminal combined repetitive oligopeptides (CROPs) domain of TcdB is dynamic and can sample open and closed conformations that may facilitate modulation of TcdB activity in response to environmental and cellular cues during intoxication. Furthermore, we report three crystal structures of TcdB-antibody complexes that reveal how antibodies could specifically inhibit the activities of individual TcdB domains. Our studies provide novel insight into the structure and function of TcdB holotoxin and identify intrinsic vulnerabilities that could be exploited to develop new therapeutics and vaccines for the treatment of CDI.

Background Clostridium difficile infection (CDI) is a debilitating nosocomial infection. C. difficile produces toxins A and B, which cause inflammation. Existing therapies have issues with recurrence, cost, and safety. We aim to discover a safe, effective, and economical nonmicrobiological therapeutic approach against CDI. Methods We included human primary peripheral blood mononuclear cells (PBMCs), fresh human colonic explants, and humanized HuCD34-NCG mice. Toxin A+B+ VPI 10463 and A−B+ ribotype 017 C. difficile strains were used. We used single-cell RNA profiling and high-throughput screening to find actionable toxin B–dependent pathways in PBMCs. Results Histamine 1 receptor–related drugs were found among the hit compounds that reversed toxin-mediated macrophage inflammatory protein (MIP) 1α expression in PBMCs. We identified loratadine as the safest representative antihistamine for therapeutic development. Loratadine inhibited toxin B–induced MIP-1α secretion in fresh human colonic tissues. Oral loratadine (10 mg/kg/d) maintained survival, inhibited intestinal CCl3 messenger RNA expression, and prevented vancomycin-associated recurrence in the VPI 10463–infected mice and ribotype 017-infected hamsters. Splenocytes from loratadine-treated mice conferred anti-inflammatory effects to the VPI 10463–infected T/B-cell-–deficient Rag −/− mice. Oral loratadine suppressed human MIP-1α expression in monocytes/macrophages in toxin B–expressing ribotype 017-infected humanized HuCD34-NCG mice. Conclusions Loratadine may be repurposed to optimize existing therapies against CDI. [ABSTRACT FROM AUTHOR]

"Space," as a fundamental conceptual term, has been frequently and widely applied within various disciplines, such as sociology, architecture, philosophy, math, art, and so forth. In pictorial art, the term "space" is often used as a lens to understand specific concepts or artworks. This study aims to explain and describe pictorial spatial construction in paintings by applying various approaches. However, in the field of formalist analysis over the past century, except for a few formalist critics who have engaged with pictorial space, few have attempted to use creative methodologies to systematically analyze how pictorial space is constructed in the painting, particularly in works by contemporary painters. Therefore, to fill in this lacuna in the research, this study utilizes new formal analytical methods to analyze and explain spatial construction in paintings. In terms of the method of analysis, this study analyzes a sample of Western contemporary paintings and utilizes the approaches of the overlap of the abstract and the figurative, parallel narratives existing in multiple spaces, and the juxtaposition of different material dimensions, respectively, to rethink the method of spatial construction in the painting. The result concludes that paintings are an essential research object within visual art. The construction of image space is rich and includes multiple layers of meaning. The overlapping of abstract space (sensibility) and figurative space (rationality), as well as the coexistence of this contradictory system, enables artists to discover more possibilities when creating works and effectively enrich visual effects and textures in their paintings. [ABSTRACT FROM AUTHOR]

Abstract Background Major Clostridium difficile virulence factors are the exotoxins TcdA and TcdB. Due to the large size and poor stability of the proteins, the active recombinant TcdA and TcdB have been difficult to produce. Results The toxin genes tcdA and tcdB were amplified by PCR using chromosomal DNA from a toxigenic strain as a template, and cloned into a shuttle vector pHis1522. The sequences of both tcdA and tcdB genes in the vector have been verified by DNA sequencing. The constructs were transformed into B. megaterium protoplasts and the protein expression was controlled under a xylose promoter. The recombinant toxins (rTcdA and rTcdB) were purified from bacterial crude extracts. Approximately 5 – 10 mg of highly purified recombinant toxins were obtained from one liter of bacterial culture. The resulting rTcdA and rTcdB had similar molecular masses to the native toxins, and their biological activities were found to be similar to their native counterparts after an extensive examination. Conclusion We have generated the full length and active recombinant TcdA and TcdB in Bacillus megaterium.

A polyurethane (PU) nanospiked gold electrode-based label-free electrochemical immunosensor for Clostridium difficile (C. difficile) toxin B detection was developed. Different from nanomaterial-modified electrodes, the PU nanospiked gold electrode was fabricated by soft lithography directly. The morphology of the nanospiked electrode was characterized by scanning electron microscope (SEM), and its good electrochemical performance was demonstrated by cyclic voltammetry (CV) as well as differential pulse voltammetry (DPV). Then, the PU nanospiked gold electrode-based immunosensor was developed by fixing antitoxin B single domain antibody on the electrode surface as the receptor. DPV was used as a detection technology for C. difficile toxin B detection. It revealed that the immunosensor has good specificity, repeatability, and stability. Even in a label-free style, the limit of detection for toxin B was 0.5 pg/mL. In the concentration range of 1–130 pg/mL, the linear regression equation ΔI = −1.216-[C] – 41.62 was found; the correlation coefficient R2 was 0.9839. Compared with the flat PU electrode-based immunosensor, the detection signal of the PU nanospiked gold electrode-based immunosensor was amplified about 6 times. Benefiting from its low cost and simple processing, the PU nanospiked gold electrode-based immunosensor can be used as a disposable electrochemical sensor for toxin B rapid detection. [ABSTRACT FROM AUTHOR]

The global prevalence of severe Clostridium difficile infection highlights the profound clinical significance of clostridial glucosylating toxins (1-4). Virulence is dependent on the autoactivation of a toxin cysteine protease (5-9), which is promoted by the allosteric cofactor inositol hexakisphosphate ([InsP.sub.6]) (10-17). Host mechanisms that protect against such exotoxins are poorly understood. It is increasingly appreciated that the pleiotropic functions attributed to nitric oxide (NO), including host immunity, are in large part mediated by S-nitrosylation of proteins (18,19). Here we show that C. difficile toxins are S-nitrosylated by the infected host and that S-nitrosylation attenuates virulence by inhibiting toxin self-cleavage and cell entry. Notably, [InsP.sub.6]- and inositol pyrophosphate ([InsP.sub.7])-induced conformational changes in the toxin enabled host S-nitrosothiols to transnitrosylate the toxin catalytic cysteine, which forms part of a structurally conserved nitrosylation motif. Moreover, treatment with exogenous [InsP.sub.6] enhanced the therapeutic actions of oral S-nitrosothiols in mouse models of C. difficile infection. Allostery in bacterial proteins has thus been successfully exploited in the evolutionary development of nitrosothiol-based innate immunity and may provide an avenue to new therapeutic approaches., C. difficile infection is the most prevalent cause of hospital-acquired infectious diarrhea and life-threatening colitis worldwide (1-4). Two large exotoxins, TcdA (308 kDa) and TcdB (270 kDa), are secreted from [...]