Cerebral palsy (CP) results in non-progressive damage to the central nervous system, leading to functional disorders of the gastrointestinal tract and requiring enteral nutrition via gastrostomy in some patients. The aim of the study was to assess the impact of enteral nutrition on intestinal inflammation expressed by stool calprotectin and intestinal permeability determined by fecal zonulin and IFABP, and to determine whether CP affects these parameters. The study group consisted of 30 children with CP, fed enterally (Cerebral Palsy Enteral Nutrition-CPEN), and two reference groups: 24 children with CP, fed orally with a standard diet (CPC-Cerebral Palsy Controls) and 24 healthy children (HC-healthy controls). The differences between these groups and between the combined CP groups (CPG and CPEN + CPC) and HC were analyzed. Fecal zonulin, calprotectin, and intestinal fatty acid-binding protein 2 (IFABP2) levels were determined by ELISA. The concentrations of fecal calprotectin and zonulin were significantly higher in the CPEN group than in the CPC group (p = 0.012, p = 0.025). When comparing the CPG (n = 53) with the HC group (n = 24), statistically significant differences were observed for calprotectin (p = 0.000018, higher in the CPG) and IFABP (p = 0.021, higher in HC). Enteral nutrition was associated in our cohort with increased fecal calprotectin and zonulin. Children with cerebral palsy presented with increased fecal calprotectin but not increased intestinal permeability expressed by stool zonulin.

The cGAS/STING sensor system drives innate immune responses to intracellular microbial double-stranded DNA (dsDNA) and bacterial cyclic nucleotide second messengers (e.g., c-di-AMP). STING-dependent cell-intrinsic responses can increase resistance to microbial infection and speed pathogen clearance. Correspondingly, STING activation and signaling are known to be targeted for suppression by effectors from several bacterial pathogens. Whether STING responses are also positively regulated through sensing of specific bacterial effectors is less clear. We find that STING activation through dsDNA, by its canonical ligand 2\'-3\' cGAMP, or the small molecule DMXAA is potentiated following intracellular delivery of the AB5 toxin family member pertussis toxin from Bordetella pertussis or the B subunit of cholera toxin from Vibrio cholerae. Entry of pertussis toxin or cholera toxin B into mouse macrophages triggers markers of endoplasmic reticulum (ER) stress and enhances ligand-dependent STING responses at the level of STING receptor activation in a manner that is independent of toxin enzymatic activity. Our results provide an example in which STING responses integrate information about the presence of relevant ER-transiting bacterial toxins into the innate inflammatory response and may help to explain the in vivo adjuvant effects of catalytically inactive toxins.

Vibrio mimicus bacteria have caused sporadic cases and outbreaks of cholera-like diarrhea throughout the world, but the association of lineages with such events is unexplored. Genomic analyses revealed V. mimicus lineages carrying the virulence factors cholera toxin and toxin coregulated pilus, one of which has persisted for decades in China and the United States.

UNASSIGNED: Cholera is a bacterial diarrheal disease caused by pathogen bacteria Vibrio cholerae, which produces the cholera toxin (CT). In addition to improving water sanitation, oral cholera vaccines have been developed to control infection. Besides, rehydration and antibiotic therapy are complementary treatment strategies for cholera. ToxT regulatory protein activates transcription of CT gene, which is enhanced by bicarbonate (HCO3-).
UNASSIGNED: This review delves into the genomic blueprint of V. cholerae, which encodes for α-, β-, and γ- carbonic anhydrases (CAs). We explore how the CAs contribute to the pathogenicity of V. cholerae and discuss the potential of CA inhibitors in mitigating the disease\'s impact.
UNASSIGNED: CA inhibitors can reduce the virulence of bacteria and control cholera. Here, we reviewed all reported CA inhibitors, noting that α-CA from V. cholerae (VchCAα) was the most effective inhibited enzyme compared to the β- and γ-CA families (VchCAβ and VchCAγ). Among the CA inhibitors, acyl selenobenzenesulfonamidenamides and simple/heteroaromatic sulfonamides were the best VchCA inhibitors in the nM range. It was noted that some antibacterial compounds show good inhibitory effects on all three bacterial CAs. CA inhibitors belonging to other classes may be synthesized and tested on VchCAs to harness cholera.

Amyotrophic lateral sclerosis (ALS) is an extremely complex neurodegenerative disease involving different cell types, but motoneuronal loss represents its main pathological feature. Moreover, compensatory plastic changes taking place in parallel to neurodegeneration are likely to affect the timing of ALS onset and progression and, interestingly, they might represent a promising target for disease-modifying treatments. Therefore, a simplified animal model mimicking motoneuronal loss without the other pathological aspects of ALS has been established by means of intramuscular injection of cholera toxin-B saporin (CTB-Sap), which is a targeted neurotoxin able to kill motoneurons by retrograde suicide transport. Previous studies employing the mouse CTB-Sap model have proven that spontaneous motor recovery is possible after a subtotal removal of a spinal motoneuronal pool. Although these kinds of plastic changes are not enough to counteract the functional effects of the progressive motoneuron degeneration, it would nevertheless represent a promising target for treatments aiming to postpone ALS onset and/or delay disease progression. Herein, the mouse CTB-Sap model has been used to test the efficacy of mitochondrial division inhibitor 1 (Mdivi-1) as a tool to counteract the CTB-Sap toxicity and/or to promote neuroplasticity. The homeostasis of mitochondrial fission/fusion dynamics is indeed important for cell integrity, and it could be affected during neurodegeneration. Lesioned mice were treated with Mdivi-1 and then examined by a series of behavioral test and histological analyses. The results have shown that the drug may be capable of reducing functional deficits after the lesion and promoting synaptic plasticity and neuroprotection, thus representing a putative translational approach for motoneuron disorders.

BACKGROUND: Ulcerative colitis (UC) is a type of inflammatory bowel disease associated with persistent inflammation. Animal studies proved the efficacy of metformin in UC.
OBJECTIVE: To investigate the potential role of metformin and its protective pathways in patients with UC.
METHODS: This is a randomized, controlled, and double-blinded clinical trial that included 60 participants with mild to moderate UC and was divided randomly into two groups (n = 30). For 6 months, the mesalamine group received 1 g of mesalamine three times daily (t.i.d.). For six months, the metformin group received mesalamine 1 g t.i.d. and metformin 500 mg twice daily. A gastroenterologist evaluated patients at baseline and 6 months after starting the treatment in order to measure serum levels of zonulin, sphingosine 1 phosphate (S1P), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α). Biopsies from the colon were used to measure gene expression of zonula occuldin-1 (ZO-1), signal transducer and activator of factor-3 (STAT-3), and intracellular adhesion molecule-1 (ICAM-1). The numeric pain rating scale (NRS) and partial Mayo score were also assessed for each patient.
RESULTS: When compared to the mesalamine group, the metformin group demonstrated a statistical decrease in serum IL-6, zonulin, TNF-α, SIP, gene expression of ICAM-1 and STAT-3, and a significant increase in colonic ZO-1 when compared to the mesalamine group. The metformin group also showed a significant decrease in NRS and partial Mayo score index in comparison with the mesalamine group.
CONCLUSIONS: Metformin may be a promising additional therapy for UC patients. Trial registration identifier: NCT05553704.

Vibrio cholerae O1 causes the diarrheal disease cholera, and the small intestine is the site of active infection. During cholera, cholera toxin is secreted from V. cholerae and induces a massive fluid influx into the small intestine, which causes vomiting and diarrhea. Typically, V. cholerae genomes are sequenced from bacteria passed in stool, but rarely from vomit, a fluid that may more closely represents the site of active infection. We hypothesized that V. cholerae O1 population bottlenecks along the gastrointestinal tract would result in reduced genetic variation in stool compared to vomit. To test this, we sequenced V. cholerae genomes from 10 cholera patients with paired vomit and stool samples. Genetic diversity was low in both vomit and stool, consistent with a single infecting population rather than coinfection with divergent V. cholerae O1 lineages. The amount of single-nucleotide variation decreased from vomit to stool in four patients, increased in two, and remained unchanged in four. The variation in gene presence/absence decreased between vomit and stool in eight patients and increased in two. Pangenome analysis of assembled short-read sequencing demonstrated that the toxin-coregulated pilus operon more frequently contained deletions in genomes from vomit compared to stool. However, these deletions were not detected by PCR or long-read sequencing, indicating that interpreting gene presence or absence patterns from short-read data alone may be incomplete. Overall, we found that V. cholerae O1 isolated from stool is genetically similar to V. cholerae recovered from the upper intestinal tract.
OBJECTIVE: Vibrio cholerae O1, the bacterium that causes cholera, is ingested in contaminated food or water and then colonizes the upper small intestine and is excreted in stool. Shed V. cholerae genomes from stool are usually studied, but V. cholerae isolated from vomit may be more representative of where V. cholerae colonizes in the upper intestinal epithelium. V. cholerae may experience bottlenecks, or large reductions in bacterial population sizes and genetic diversity, as it passes through the gut. Passage through the gut may select for distinct V. cholerae mutants that are adapted for survival and gut colonization. We did not find strong evidence for such adaptive mutations, and instead observed that passage through the gut results in modest reductions in V. cholerae genetic diversity, and only in some patients. These results fill a gap in our understanding of the V. cholerae life cycle, transmission, and evolution.

Mass-spectrometry based assays in structural biology studies measure either intact or digested proteins. Typically, different mass spectrometers are dedicated for such measurements: those optimized for rapid analysis of peptides or those designed for high molecular weight analysis. A commercial trapped ion mobility-quadrupole-time-of-flight (TIMS-Q-TOF) platform is widely utilized for proteomics and metabolomics, with ion mobility providing a separation dimension in addition to liquid chromatography. The ability to perform high-quality native mass spectrometry of protein complexes, however, remains largely uninvestigated. Here, we evaluate a commercial TIMS-Q-TOF platform for analyzing noncovalent protein complexes by utilizing the instrument\'s full range of ion mobility, MS, and MS/MS (both in-source activation and collision cell CID) capabilities. The TIMS analyzer is able to be tuned gently to yield collision cross sections of native-like complexes comparable to those previously reported on various instrument platforms. In-source activation and collision cell CID were robust for both small and large complexes. TIMS-CID was performed on protein complexes streptavidin (53 kDa), avidin (68 kDa), and cholera toxin B (CTB, 58 kDa). Complexes pyruvate kinase (237 kDa) and GroEL (801 kDa) were beyond the trapping capabilities of the commercial TIMS analyzer, but TOF mass spectra could be acquired. The presented results indicate that the commercial TIMS-Q-TOF platform can be used for both omics and native mass spectrometry applications; however, modifications to the commercial RF drivers for both the TIMS analyzer and quadrupole (currently limited to m/z 3000) are necessary to mobility analyze protein complexes greater than about 60 kDa.

OBJECTIVE: Patients with chronic obstructive pulmonary disease (COPD) frequently exhibit an inability to maintain postural balance. However, the contribution of increased intestinal permeability or leaky gut to the postural imbalance in COPD is not known.
METHODS: We measured plasma zonulin, a marker of leaky gut, with relevance to postural balance in male controls (n = 70) and patients with mild (n = 67), moderate (n = 66), and severe (n = 58) COPD. We employed a short physical performance battery to evaluate postural balance in supine, tandem, and semi-tandem positions. We also measured handgrip strength (HGS), gait speed, plasma c-reactive proteins (CRP), and 8-isoprostanes as potential mechanistic connections between postural imbalance and leaky gut.
RESULTS: COPD patients demonstrated higher plasma zonulin, CRP, and 8-isoprostanes levels and lower balance, HGS, and gait speed than controls (all p < 0.05). These findings were more robust in patients with moderate and severe than mild COPD. In addition, plasma zonulin exhibited significant potential in diagnosing poor balance, low HGS, and gait speed in COPD patients (all p < 0.05). We also found significant correlations of plasma zonulin with CRP and 8-isoprostanes, providing heightened inflammation and oxidative stress as mechanistic connections between leaky gut and postural imbalance.
CONCLUSIONS: Plasma zonulin may be helpful in evaluating postural imbalance in COPD patients. Repairing intestinal leaks can be a therapeutic target to improve postural control in COPD.

Chlamydia trachomatis (Ct) is the most common sexually transmitted bacterial infection worldwide, potentially leading to severe pathologies including pelvic inflammatory disease, ectopic pregnancy, and tubal infertility if left untreated. Current strategies, including screening and antibiotics, have limited effectiveness due to high rates of asymptomatic cases and logistical challenges. A multiepitope prophylactic vaccine could afford long-term protection against infection. Immunoinformatic analyses were employed to design a multiepitope Chlamydia vaccine antigen. B- and T-cell epitopes from five highly conserved and immunogenic Ct antigens were predicted and selected for the vaccine design. The final construct, adjuvanted with cholera toxin A1 subunit (CTA1), was further screened for immunogenicity. CTA1-MECA (multiepitope Chlamydia trachomatis antigen) was identified as antigenic and nonallergenic. A tertiary structure was predicted, refined, and validated as a good quality model. Molecular docking exhibited strong interactions between the vaccine and toll-like receptor 4 (TLR4). Additionally, immune responses consistent with protection including IFN-γ, IgG + IgM antibodies, and T- and B-cell responses were predicted following vaccination in an immune simulation. Expression of the construct in an Escherichia coli expression vector proved efficient. To further validate the vaccine efficacy, we assessed its immunogenicity in mice. Immunization with CTA1-MECA elicited high levels of Chlamydia-specific antibodies in mucosal and systemic compartments.