Symbiotic bacterial metabolites regulate gastrointestinal barrier function via the xenobiotic sensor PXR and Toll-like receptor 4.

Intestinal microbial metabolites are conjectured to affect mucosal integrity through an incompletely characterized mechanism. Here we showed that microbial-specific indoles regulated intestinal barrier function through the xenobiotic sensor, pregnane X receptor (PXR). Indole 3-propionic acid (IPA), in the context of indole, is a ligand for PXR in vivo, and IPA downregulated enterocyte TNF-α while it upregulated junctional protein-coding mRNAs. PXR-deficient (Nr1i2(-/-)) mice showed a distinctly "leaky" gut physiology coupled with upregulation of the Toll-like receptor (TLR) signaling pathway. These defects in the epithelial barrier were corrected in Nr1i2(-/-)Tlr4(-/-) mice. Our results demonstrate that a direct chemical communication between the intestinal symbionts and PXR regulates mucosal integrity through a pathway that involves luminal sensing and signaling by TLR4.

Targeting the PXR-TLR4 signaling pathway to reduce intestinal inflammation in an experimental model of necrotizing enterocolitis.

BackgroundThere is substantial evidence that signaling through Toll-like receptor 4 (TLR4) contributes to the pathogenesis of necrotizing enterocolitis (NEC). Pregnane X receptor (PXR), a xenobiotic sensor and signaling intermediate for certain host-bacterial metabolites, has been shown to negatively regulate TLR4 signaling. Here we investigated the relationship between PXR and TLR4 in the developing murine intestine and explored the capacity of PXR to modulate inflammatory pathways involved in experimental NEC.MethodsWild-type and PXR-/- mice were studied at various time points of development in an experimental model of NEC. In addition, we studied the ability of the secondary bile acid lithocholic acid (LCA), a known PXR agonist in liver, to activate intestinal PXR and reduce NEC-related intestinal inflammation.ResultsWe found a reciprocal relationship between the developmental expression of PXR and TLR4 in wild-type murine intestine, with PXR acting to reduce TLR4 expression by decreasing TLR4 mRNA stability. In addition, PXR-/- mice exhibited a remarkably heightened severity of disease in experimental NEC. Moreover, LCA attenuated intestinal proinflammatory responses in the early stages of experimental NEC.ConclusionThese findings provide proactive insights into the regulation of TLR4 in the developing intestine. Targeting PXR may be a novel approach for NEC prevention.

A New Bis(aquated) High Relaxivity Mn(II) Complex as an Alternative to Gd(III)-Based MRI Contrast Agent.

Disclosed here are a piperazine, a pyridine, and two carboxylate groups containing pentadentate ligand H2pmpa and its corresponding water-soluble Mn(II) complex (1). DFT-based structural optimization implied that the complex had pentagonal bipyramidal geometry where the axial positions were occupied by two water molecules, and the equatorial plane was constituted by the ligand ON3O donor set. Thus, a bis(aquated) disc-like Mn(II) complex has been synthesized. The complex showed higher stability compared with Mn(II)-EDTA complex [log KMnL = 14.29(3)] and showed a very high r1 relaxivity value of 5.88 mM-1 s-1 at 1.41 T, 25 °C, and pH = 7.4. The relaxivity value remained almost unaffected by the pH of the medium in the range of 6-10. Although the presence of 200 equiv of fluoride and bicarbonate anions did not affect the relaxivity value appreciably, an increase in the value was noticed in the presence of phosphate anion due to slow tumbling of the complex. Cell viability measurements, as well as phantom MR images using clinical MRI imager, consolidated the possible candidature of complex 1 as a positive contrast agent.

Pregnane X Receptor Activation Attenuates Inflammation-Associated Intestinal Epithelial Barrier Dysfunction by Inhibiting Cytokine-Induced Myosin Light-Chain Kinase Expression and c-Jun N-Terminal Kinase 1/2 Activation.

The inflammatory bowel diseases (IBDs) are chronic inflammatory disorders with a complex etiology. IBD is thought to arise in genetically susceptible individuals in the context of aberrant interactions with the intestinal microbiota and other environmental risk factors. Recently, the pregnane X receptor (PXR) was identified as a sensor for microbial metabolites, whose activation can regulate the intestinal epithelial barrier. Mutations in NR1I2, the gene that encodes the PXR, have been linked to IBD, and in animal models, PXR deletion leads to barrier dysfunction. In the current study, we sought to assess the mechanism(s) through which the PXR regulates barrier function during inflammation. In Caco-2 intestinal epithelial cell monolayers, tumor necrosis factor-α/interferon-γ exposure disrupted the barrier and triggered zonula occludens-1 relocalization, increased expression of myosin light-chain kinase (MLCK), and activation of c-Jun N-terminal kinase 1/2 (JNK1/2). Activation of the PXR [rifaximin and [[3,5-Bis(1,1-dimethylethyl)-4-hydroxyphenyl]ethenylidene]bis-phosphonic acid tetraethyl ester (SR12813); 10 µM] protected the barrier, an effect that was associated with attenuated MLCK expression and JNK1/2 activation. In vivo, activation of the PXR [pregnenolone 16α-carbonitrile (PCN)] attenuated barrier disruption induced by toll-like receptor 4 activation in wild-type, but not Pxr-/-, mice. Furthermore, PCN treatment protected the barrier in the dextran-sulfate sodium model of experimental colitis, an effect that was associated with reduced expression of mucosal MLCK and phosphorylated JNK1/2. Together, our data suggest that the PXR regulates the intestinal epithelial barrier during inflammation by modulating cytokine-induced MLCK expression and JNK1/2 activation. Thus, targeting the PXR may prove beneficial for the treatment of inflammation-associated barrier disruption in the context of IBD.

Protective effect of naringenin against experimental colitis via suppression of Toll-like receptor 4/NF-κB signalling.

Naringenin, one of the most abundant flavonoids in citrus, grapefruits and tomatoes, has been used as a traditional anti-inflammatory agent for centuries. However, the molecular mechanism of naringenin in intestinal inflammation remains unknown so far. The present study investigated a molecular basis for the protective effect of naringenin in dextran sulphate sodium-induced murine colitis. Pre-administration of naringenin significantly reduced the severity of colitis and resulted in down-regulation of pro-inflammatory mediators (inducible NO synthase (iNOS), intercellular adhesion molecule-1 (ICAM-1), monocyte chemoattractant protein-1 (MCP-1), cyclo-oxygenase-2 (Cox2), TNF-α and IL-6 mRNA) in the colon mucosa. The decline in the production of pro-inflammatory cytokines, specifically TNF-α and IL-6, correlated with a decrease in mucosal Toll-like receptor 4 (TLR4) mRNA and protein. Phospho-NF-κB p65 protein was significantly decreased, which correlated with a similar decrease in phospho-IκBα protein. Consistent with the in vivo results, naringenin exposure blocked lipopolysaccharide-stimulated nuclear translocation of NF-κB p65 in mouse macrophage RAW264.7 cells. In addition, in vitro NF-κB reporter assays performed on human colonic HT-29 cells exposed to naringenin demonstrated a significant inhibition of TNF-α-induced NF-κB luciferase expression. Thus, for the first time, the present study indicates that targeted inhibition of the TLR4/NF-κB signalling pathway might be an important mechanism for naringenin in abrogating experimental colitis.

AIEE activated Pyrene-Dansyl coupled FRET probe for discriminating detection of lethal Cu2+ and CN-: Bio-Imaging, DNA binding studies and prompt prognosis of Menke's disease.

In present work a pyrene-dansyl dyad functionalized chemoreceptor, DPNS is unveiled towards ultrasensitive chromo-fluorogenic detection of heavy and transition metal ions (HTMs) like Cu2+ and pernicious CN-. It demonstrated distinct chromogenic responses; colorless to faint yellow (Cu2+), intense yellow (CN-) from contaminant aqueous sources. Cu2+ instigated alteration in DPNS fluorescence from feeble emission to sparkling green with LOD: 37.75 × 10-9 M, cyan emission for CN- having LOD 61.51 × 10-8M. In particular, chemical scaffold of DPNS consists of -C = N, O = S = O donor entitities that escalates overall polarity thereby providing an excellent binding pocket for simultaneous Cu2+ and CN- recognition with distinct photophysical signaling. Impressively, presence of two fluorophoric moieties triggers FRET, CHEF phenomenon. The conceivable host:guest interactive pathway is manifested by LMCT- FRET-PET-CHEF, C = N isomerization for Cu2+ and ICT-H-bonding for CN-. An exquisite experimental and theoretical corroboration further strengthened the recognition phenomenon. In addition owing to pyrene excimer formation, DPNS exhibits AIEE with increasing water fraction. Notably, DPNS could successfully undergo intracellular tracking of Cu2+ in Tecoma Stans, Peperomia Pellucida. DPNS•••Cu2+ adduct displayed significant intercalative DNA binding activity rationalized by spectral investigation, competitive EB binding, viscosity study. The overall findings, excellent properties endows DPNS a potential contender towards discriminative detection of Cu2+ and CN- like toxic industrial contaminants.

Mucosal immunity-mediated modulation of the gut microbiome by oral delivery of probiotics into Peyer's patches.

Methods capable of maintaining gut microbiota homeostasis to prevent bacterial translocation and infection under external threats are critical for multiple facets of human health but have been rarely reported. Here, we describe the elicitation of mucosal immunity to modulate the gut microbiota by oral delivery of living probiotics into Peyer's patches. Probiotics are individually camouflaged within a yeast membrane, on which the embedded ß-glucan can facilitate the phagocytosis of microfold cells that locate in the intestinal epithelium. The delivery of probiotics into lymphoid follicles after oral ingestion promotes robust mucosal immune responses and notably upgrades the production of secretory immunoglobulin A. The provoked immunity positively regulates the gut microflora, which, in turn, retains gut homeostasis and provides defense against environmental attacks. In two murine models of gut barrier impairment, oral administration with camouflaged probiotics effectively prevents the breakdown of intestinal barrier and evidences limited bacterial translocation and systemic inflammation.

Anticancer activity and cell death mechanism of Pt(II) complexes: Their in vitro bio-transformation to Pt(II)-DNA adduct formation and BSA binding study by spectroscopic method.

Pt(II) complex cis-[Pt(PEA)(OH2)2] X2, C-2 (where, PEA = 2-Pyridylethylamine and X  = ClO4- or NO3-) was synthesized by hydrolysis of cis-[Pt(PEA)Cl2] C-1. Glutathione (GSH) and DL-penicilamine (DL-pen) substituted complexes cis-[Pt(PEA)(GSH)],C-3 and cis-[Pt(PEA)DL-pen)]X C-4 were synthesized and characterized by spectroscopic methods. Kinetic studies were traced on complex C-2 with the thiols, GSH and DL-pen. Pt(II)-Sulfur adduct formation mechanisms of the substituted products C-3 and C-4 were established from the kinetic investigation. At pH 4.0, C-2 - thiols interactions follow two consecutive steps: the first step is dependent, and the second is independent of [thiol]. The association equilibrium constant (KE), substitution rate constants for both steps (k1 & k2), and activation parameters (ΔH‡ and ΔS‡) have been assessed to propose the mechanism. Agarose gel electrophoresis mobilization pattern of DNA with complexes was performed to visualize the interaction nature. CT-DNA and BSA binding activities of the complexes have been executed by electronic, fluorescence spectroscopy, and viscometric titration methods. Evaluation of thermodynamic parameters (ΔH0, ΔS0, and ΔG0) from BSA binding constants was executed to propose the driving forces of interaction between these species. A molecular docking study was performed to evaluate the binding mode of complexes with BDNA strands. Anticancer activity of the complexes C-1 to C-4 was explored on both A549 and HEp-2 cell lines, compared with approved anticancer drugs cisplatin, carboplatin, and oxaliplatin. All these complexes were tested by NBT assay on normal cell line skeletal muscle cells (L6 myotubes) to observe the adverse effects compared to recognized anticancer medications. The ultimate aim is to explore the role of anticancer agents on cell death mechanism, which has been performed by flow-cytometer on HEp-2 cell lines.

Orphan nuclear receptors as targets for drug development.

Orphan nuclear receptors regulate diverse biological processes. These important molecules are ligand-activated transcription factors that act as natural sensors for a wide range of steroid hormones and xenobiotic ligands. Because of their importance in regulating various novel signaling pathways, recent research has focused on identifying xenobiotics targeting these receptors for the treatment of multiple human diseases. In this review, we will highlight these receptors in several physiologic and pathophysiologic actions and demonstrate how their functions can be exploited for the successful development of newer drugs.

Association of tobacco smoke-infused water (tuibur) use by Mizo people and risk of Helicobacter pylori infection.

The study aims to understand the influence of environmental and lifestyle factors and more specifically the role of tobacco smoke-infused water (tuibur) on Helicobacter pylori infection. It was a cross-sectional study to measure the epidemiological risk factors associated with H. pylori infection among the tribal population in Northeast India. Endoscopic samples were collected from the antrum region of the stomach from 863 participants with gastritis. H. pylori infection was confirmed in 475 samples by the rapid urease test and PCR-based methods. Information on demographic and lifestyle factors was collected using a validated and standardized questionnaire. Logistic regression was used to estimate odds ratio (OR) and 95% confidence interval (CI) for the association between the various factors and H. pylori. The use of tuibur was associated with an increased OR of H. pylori infection (OR = 3.32, 95% Cl = 1.95-5.83). Tobacco chewers (OR = 1.49, 95% Cl = 1.06-2.09), smokers (OR = 1.81, 95% Cl = 1.26-2.61), and alcohol consumers (OR = 1.81, 95% Cl = 1.19-2.76) were also infected with H. pylori. The results were not attenuated after adjusting for major well-known risk factors of H. pylori infection. The habit of tuibur consumption may be a contributing factor to the high prevalence of H. pylori infection and in turn, may contribute to the high prevalence of gastritis among the Mizo population.

Pathogenic microbes contaminating mobile phones in hospital environment in Northeast India: incidence and antibiotic resistance.

BACKGROUND: The present study attempts to identify and determine the pattern of drug susceptibility of the microorganisms present in mobile phones of health care workers (HCWs) and non-HCWs in a hospital environment. Mobile phones of 100 participants including both genders were randomly swabbed from nine different wards/units and the bacterial cultures were characterized using VITEK 2 system. RESULTS: Forty-seven mobile phones were culture positive and a total of 57 isolates were obtained which consisted of 28 Gram-positive organisms and 29 Gram-negative organisms. The predominating organisms were Acinetobacter baumannii and Staphylococcus hominis. Among all the isolates from the mobile phones of HCW and non-HCWs, five isolates had ESBL and three isolates had colistin resistance. Incidentally, MRSA was not found on the mobile phones tested. The isolated organisms showed 100% susceptibility to linezolid, daptomycin, vancomycin, imipenem, meropenem, gentamicin, amikacin, ciprofloxacin and tigecycline, while high resistance was shown against benzylpenicillin (75.0%), cefuroxime and cefuroxime axetil (56.5%). Non-HCWs' mobile phones were more contaminated as compared to HCWs (P = 0.001) and irrespective of individuals' gender or toilet habits, both Gram-positive and Gram-negative organisms were present on the mobile phones. CONCLUSION: This study reports for the first time that the mobile phones of non-health care workers harbour more bacterial diversity and are more prone to cause transmission of pathogens. This study can serve to educate the public on personal hand hygiene practices and on maintaining clean mobile phones through antiseptic measures.

Adverse Effects of Proton Pump Inhibitors on Platelet Count: A Case Report and Review of the Literature.

Proton pump inhibitors (PPIs) are the most effective and preferred class of drugs used to treat peptic ulcer disease, gastroesophageal reflux disease, and other diseases associated with increased production of gastric acid. PPIs in general have an excellent long-term safety profile and are well-tolerated. However, studies have shown some adverse reactions (e.g., osteoporosis, Clostridium difficile-associated diarrhea, Vitamin B12 and iron deficiency, and acute interstitial nephritis) on long-term PPI use. Thrombocytopenia attributed to use of PPIs has been described in a few case reports and a retrospective study. In this case report, we describe a case of PPI-induced thrombocytopenia. In our patient, thrombocytopenia immediately developed after the initiation of PPI on two separate occasions and resolved after its discontinuation. The strong association found in our case implies the potential role of PPI in causing this rare but serious adverse reaction. Based on this case report and the observation from other studies, a PPI-induced adverse event should be considered as a possible etiology for new-onset idiopathic thrombocytopenia.

Polymorphism in mtDNA control region of Mizo-Mongloid Breast Cancer samples as revealed by PCR-RFLP analysis.

Mutations in mitochondrial D-loop region of DNA (mtDNA) may serve as a potential sensor for cellular DNA damage and marker for cancer development. We investigated the restriction fragment length polymorphism (RFLP) pattern of the D-loop region in the blood samples of breast cancer patients among Mizoram population. Significant differences were observed among breast cancer and healthy blood samples in the RFLP pattern using AluI, HaeIII and RsaI enzymes. Polymorphic information content (PIC - 0.258), band informativeness (∑Ib - 3.283) and marker index (MI - 0.006) were highest in the case of RsaI enzyme. Our data suggest that the RsaI polymorphic site in the mitochondrial control region is an informative marker for breast cancer development in Mizo population.

Xenobiotic Receptor-Mediated Regulation of Intestinal Barrier Function and Innate Immunity.

The molecular basis for the regulation of the intestinal barrier is a very fertile research area. A growing body of knowledge supports the targeting of various components of intestinal barrier function as means to treat a variety of diseases, including the inflammatory bowel diseases. Herein, we will summarize the current state of knowledge of key xenobiotic receptor regulators of barrier function, highlighting recent advances, such that the field and its future are succinctly reviewed. We posit that these receptors confer an additional dimension of host-microbe interaction in the gut, by sensing and responding to metabolites released from the symbiotic microbiota, in innate immunity and also in host drug metabolism. The scientific evidence for involvement of the receptors and its molecular basis for the control of barrier function and innate immunity regulation would serve as a rationale towards development of non-toxic probes and ligands as drugs.

Pregnane X Receptor Regulates Pathogen-Induced Inflammation and Host Defense against an Intracellular Bacterial Infection through Toll-like Receptor 4.

The nuclear pregnane X receptor (PXR) plays a central role in regulating xenobiotic metabolism. We now report a novel role for PXR as a critical negative regulator of innate immunity after infection. Pxr(-/-) mice exhibited remarkably elevated pro-inflammatory cytokine and chemokine production following infection with Listeria monocytogenes (Lm). Despite the more robust innate immune response, Pxr(-/-) mice were highly susceptible to Lm infection. Surprisingly, disruption of the Toll-like receptor 4 (TLR4) but not TLR2 signaling restored the inflammation to normal levels and the ability to clear Lm in Pxr(-/-) mice. Mechanistically, the heightened inflammation in Pxr(-/-) mice resulted in the death of inflammatory monocytes that led to the enhanced susceptibility to Lm infection. These data demonstrated that PXR regulated pathogen-induced inflammation and host defense against Lm infection through modulating the TLR4 pathway. In summary, we discovered an apical role for PXR in regulating innate immunity. In addition, we uncovered a remarkable negative impact of the TLR4 pathway in controlling the quality of the inflammatory response and host defense against a gram-positive bacterial infection.

Alleviation of gut inflammation by Cdx2/Pxr pathway in a mouse model of chemical colitis.

Pregnane X Receptor (PXR), a master regulator of drug metabolism and inflammation, is abundantly expressed in the gastrointestinal tract. Baicalein and its O-glucuronide baicalin are potent anti-inflammatory and anti-cancer herbal flavonoids that undergo a complex cycle of interconversion in the liver and gut. We sought to investigate the role these flavonoids play in inhibiting gut inflammation by an axis involving PXR and other potential factors. The consequences of PXR regulation and activation by the herbal flavonoids, baicalein and baicalin were evaluated in vitro in human colon carcinoma cells and in vivo using wild-type, Pxr-null, and humanized (hPXR) PXR mice. Baicalein, but not its glucuronidated metabolite baicalin, activates PXR in a Cdx2-dependent manner in vitro, in human colon carcinoma LS174T cells, and in the murine colon in vivo. While both flavonoids abrogate dextran sodium sulfate (DSS)-mediated colon inflammation in vivo, oral delivery of a potent bacterial ß-glucuronidase inhibitor eliminates baicalin's effect on gastrointestinal inflammation by preventing the microbial conversion of baicalin to baicalien. Finally, reduction of gastrointestinal inflammation requires the binding of Cdx2 to a specific proximal site on the PXR promoter. Pharmacological targeting of intestinal PXR using natural metabolically labile ligands could serve as effective and potent therapeutics for gut inflammation that avert systemic drug interactions.

Pregnane X receptor activation induces FGF19-dependent tumor aggressiveness in humans and mice.

The nuclear receptor pregnane X receptor (PXR) is activated by a range of xenochemicals, including chemotherapeutic drugs, and has been suggested to play a role in the development of tumor cell resistance to anticancer drugs. PXR also has been implicated as a regulator of the growth and apoptosis of colon tumors. Here, we have used a xenograft model of colon cancer to define a molecular mechanism that might underlie PXR-driven colon tumor growth and malignancy. Activation of PXR was found to be sufficient to enhance the neoplastic characteristics, including cell growth, invasion, and metastasis, of both human colon tumor cell lines and primary human colon cancer tissue xenografted into immunodeficient mice. Furthermore, we were able to show that this PXR-mediated phenotype required FGF19 signaling. PXR bound to the FGF19 promoter in both human colon tumor cells and "normal" intestinal crypt cells. However, while both cell types proliferated in response to PXR ligands, the FGF19 promoter was activated by PXR only in cancer cells. Taken together, these data indicate that colon cancer growth in the presence of a specific PXR ligand results from tumor-specific induction of FGF19. These observations may lead to improved therapeutic regimens for colon carcinomas.