Varsha Singh

Lactobacillus acidophilus stimulates the expression of SLC26A3 via a transcriptional mechanism

Clinical efficacy of probiotics in treating various forms of diarrhea has been clearly established. However, mechanisms underlying antidiarrheal effects of probiotics are not completely defined. Diarrhea is caused either by decreased absorption or increased secretion of electrolytes and solutes in the intestine. In this regard, the electroneutral absorption of two major electrolytes, Na(+) and Cl(-), occurs mainly through the coupled operation of Na(+)/H(+) exchangers and Cl(-)/OH(-) exchangers. Previous studies from our laboratory have shown that Lactobacillus acidophilus (LA) acutely stimulated Cl(-)/OH(-) exchange activity via an increase in the surface levels of the apical anion exchanger SLC26A3 (DRA). However, whether probiotics influence SLC26A3 expression and promoter activity has not been examined. The present studies were, therefore, undertaken to investigate the long-term effects of LA on SLC26A3 expression and promoter activity. Treatment of Caco-2 cells with LA for 6-24 h resulted in a significant increase in Cl(-)/OH(-) exchange activity. DRA mRNA levels were also significantly elevated in response to LA treatment starting as early as 8 h. Additionally, the promoter activity of DRA was increased by more than twofold following 8 h LA treatment of Caco-2 cells. Similar to the in vitro studies, in vivo studies using mice gavaged with LA also showed significantly increased DRA mRNA ( approximately 4-fold) and protein expression in the colonic regions as assessed by Western blot analysis and immunofluorescence. In conclusion, increase in DRA promoter activity and expression may contribute to the upregulation of intestinal electrolyte absorption and might underlie the potential antidiarrheal effects of LA.

The probiotic Lactobacillus plantarum counteracts TNF-α-induced downregulation of SMCT1 expression and function

The major short-chain fatty acid (SCFA) butyrate is produced in the colonic lumen by bacterial fermentation of dietary fiber. Butyrate serves as primary fuel for the colonocytes and also ameliorates mucosal inflammation. Disturbed energy homeostasis seen in inflamed mucosa of inflammatory bowel disease patients has been attributed to impaired absorption of butyrate. Since sodium-coupled monocarboxylate transporter 1 (SMCT1, SLC5A8) has recently been shown to play a role in Na(+)-coupled transport of monocarboxylates, including SCFA, such as luminal butyrate, we examined the effects of proinflammatory TNF-α on SMCT1 expression and function and potential anti-inflammatory role of probiotic Lactobacillus species in counteracting the TNF-α effects. Rat intestinal epithelial cell (IEC)-6 or human intestinal Caco-2 cells were treated with TNF-α in the presence or absence of Lactobacilli culture supernatants (CS). TNF-α treatments for 24 h dose-dependently inhibited SMCT1-mediated, Na(+)-dependent butyrate uptake and SMCT1 mRNA expression in IEC-6 cells and SMCT1 promoter activity in Caco-2 cells. CS of L. plantarum (LP) stimulated Na(+)-dependent butyrate uptake (2.5-fold, P < 0.05), SMCT1 mRNA expression, and promoter activity. Furthermore, preincubating the cells with LP-CS followed by coincubation with TNF-α significantly attenuated the inhibitory effects of TNF-α on SMCT1 function, expression, and promoter activity. In vivo, oral administration of live LP enhanced SMCT1 mRNA expression in the colonic and ileal tissues of C57BL/6 mice after 24 h. Efficacy of LP or their secreted soluble factors to stimulate SMCT1 expression and function and to counteract the inhibitory effects of TNF-α on butyrate absorption could have potential therapeutic value.

Lactobacillus acidophilus upregulates intestinal NHE3 expression and function

A major mechanism of electroneutral NaCl absorption in the human ileum and colon involves coupling of Na(+)/H(+) and Cl(-)/HCO(3)(-) exchangers. Disturbances in these mechanisms have been implicated in diarrheal conditions. Probiotics such as Lactobacillus have been indicated to be beneficial in the management of gastrointestinal disorders, including diarrhea. However, the molecular mechanisms underlying antidiarrheal effects of probiotics have not been fully understood. We have previously demonstrated Lactobacillus acidophilus (LA) to stimulate Cl(-)/HCO3- exchange activity via an increase in the surface levels and expression of the Cl(-)/HCO3- exchanger DRA in vitro and in vivo. However, the effects of LA on NHE3, the Na(+)/H(+) exchanger involved in the coupled electroneutral NaCl absorption, are not known. Current studies were, therefore, undertaken to investigate the effects of LA on the function and expression of NHE3 and to determine the mechanisms involved. Treatment of Caco2 cells with LA or its conditioned culture supernatant (CS) for 8-24 h resulted in a significant increase in Na(+)/H(+) exchange activity, mRNA, and protein levels of NHE3. LA-CS upregulation of NHE3 function and expression was also observed in SK-CO15 cells, a human colonic adenocarcinoma cell line. Additionally, LA treatment increased NHE3 promoter activity, suggesting involvement of transcriptional mechanisms. In vivo, mice gavaged with live LA showed significant increase in NHE3 mRNA and protein expression in the ileum and colonic regions. In conclusion, LA-induced increase in NHE3 expression may contribute to the upregulation of intestinal electrolyte absorption and might underlie the potential antidiarrheal effects of probiotics.

Lactobacillus acidophilus attenuates downregulation of DRA function and expression in inflammatory models

Probiotics, including Lactobacilli, are commensal bacteria that have been used in clinical trials and experimental models for the prevention and treatment of diarrheal disorders. Our previous studies have shown that Lactobacillus acidophilus (LA) and its culture supernatant (CS) stimulated Cl(-)/HCO3 (-) exchange activity, acutely via an increase in the surface levels of downregulated in adenoma (DRA, SLC26A3) and in long-term treatments via increasing its expression involving transcriptional mechanisms. However, the role of LA in modulating DRA activity under inflammatory conditions is not known. Current in vitro studies using human intestinal epithelial Caco-2 cells examined the efficacy of LA or its CS in counteracting the inhibitory effects of interferon-γ (IFN-γ) on Cl(-)/HCO3 (-) exchange activity. Pretreatment of cells with LA or LA-CS for 1 h followed by coincubation with IFN-γ significantly alleviated the inhibitory effects of IFN-γ on Cl(-)/HCO3 (-) exchange activity. In the in vivo model of dextran sulfate sodium-induced experimental colitis (3% in drinking water for 7 days) in C57BL/6J mice, administration of live LA (3 × 10(9) colony-forming units) via oral gavage attenuated colonic inflammation. LA administration also counteracted the colitis-induced decrease in DRA mRNA and protein levels. Efficacy of LA or its secreted soluble factors in alleviating inflammation and inflammation-associated dysregulation of DRA activity could justify their therapeutic potential in inflammatory diarrheal diseases.

Regulation of intestinal serotonin transporter expression via epigenetic mechanisms: role of HDAC2

The serotonin (5-HT) transporter (SERT) facilitates clearance of extracellular 5-HT by its uptake and internalization. Decreased expression of SERT and consequent high 5-HT levels have been implicated in various diarrheal disorders. Thus, appropriate regulation of SERT is critical for maintenance of 5-HT homeostasis in health and disease. Previous studies demonstrated that SERT is regulated via posttranslational and transcriptional mechanisms. However, the role of epigenetic mechanisms in SERT regulation is not known. Current studies investigated the effects of histone deacetylase (HDAC) inhibition on SERT expression and delineated the mechanisms. Treatment of Caco-2 cells with the pan-HDAC inhibitors butyrate (5 mM) and trichostatin (TSA, 1 μM) decreased SERT mRNA and protein levels. Butyrate- or TSA-induced decrease in SERT was associated with decreased activity of human SERT (hSERT) promoter 1 (upstream of exon 1a), but not hSERT promoter 2 (upstream of exon 2). Butyrate + TSA did not show an additive effect on SERT expression, indicating that mechanisms involving histone hyperacetylation may be involved. Chromatin immunoprecipitation assays demonstrated enrichment of the hSERT promoter 1 (flanking nt -250/+2) with tetra-acetylated histone H3 or H4, which was increased (~3-fold) by butyrate. Interestingly, specific inhibition of HDAC2 (but not HDAC1) utilizing small interfering RNA decreased SERT mRNA and protein levels. The decrease in SERT expression by HDAC inhibition was recapitulated in an in vivo model. SERT mRNA levels were decreased in the ileum and colon of mice fed pectin (increased availability of butyrate) compared with controls fed a fiber-free diet (~50-60%). Our results identify a novel role of HDAC2 as a regulator of SERT gene expression in intestinal epithelial cells.

Measurement and Microstructural Evaluation of Creep-Induced Changes in Magnetic Properties of a 410 Stainless Steel

There is a compelling desire by power generating plants to continue running existing stations and components for several more years, despite many of them have surpassed their design service life. The idea is to avoid premature retirement, on the basis of the so-called design life, because actual useful life could often be well in excess of the design life. This can most readily be achieved by utilizing nondestructive monitoring methods to monitor the degradation of the microstructure, either when a station is down for maintenance or preferably when it is under operation. This study evaluates the use of quasi static hysteresis measurements as a possible procedure to evaluate creep in a 410 martensitic stainless steel, a material utilized in power plant components. The creep rupture tests were conducted at stresses of 100 and 200 MPa, temperatures of 500°C and 620°C. and the times varied between 48 and 120 hours. Following the creep tests all specimens were evaluated magnetically and then metallurgically by optical and scanning electron microscopy, x-ray diffraction (XRD) and by energy dispersive spectroscopy (EDS). The microstructural changes were compared with the magnetization changes. It was determined that the changes in the hysteresis curves were clearly detectable and correlated with the creep-induced damage.

Measuring and modeling of corrosion in structural steels using magnetoelastic sensors

Magnetic measurements were performed on steel cables subjected to a magnetic field and the response measured without contact using Faradays law, to estimate the effect of temperature and corrosion on magnetic properties of structural steel. Magnetic measurements were compared with electrochemical measurements to correlate corrosion quantitatively in terms of mass loss. The magnetic measurements obtained for structural steel are discussed in this paper. The results obtained are helpful in bounding the achievable sensitivity for conventional magnetoelastic corrosion sensing and for suggesting the need for alternate techniques. Also in this paper the Jiles–Atherton model, a phenomenological model, is proposed to model physical properties of structural steel and magnetite (corrosion product). Results from hot rolled steel, a low carbon steel, were simulated using the Jiles model to understand and correlate the measured and simulated curves. The hysteresis curves for magnetite, one of the most prevalent corrosion products and the only ferromagnetic component, were obtained to simulate the effect of corrosion products on the magnetic measurements of corroded structural steel.

Temperature compensation and scalability of hysteretic/anhysteretic magnetic-property sensors

Non-destructive/noncontact stress sensors based on magnetoelastic principles have numerous applications for monitoring cable stress. However, their widespread use is hampered by problems related to temperature compensation and to scaling of calibration data to large cables, which is the domain of greatest interest. In this paper, experimental magnetic property and thermal response data are obtained and analyzed to address these problems. The magnetic property data include initial curves, major hysteresis loops, and anhysteresis curves, all obtained quasistatically. The thermal response data are presented in the form of a dimensionless correlation which is valid over a range of Reynolds numbers. Comparison of data indicates the feasibility of using absolute magnetic property measurements as the basis for direct calibration of large sensors.

Simulation of surface heating effects and effective permeabilities using a Jiles-Atherton model

The hysteretic, nonlinear character of ferromagnetic steels, coupled with pronounced stress and temperature sensitivities, make detailed magnetoelastic sensor design and calibration very difficult at present, particularly under possible high repetition rates during disaster scenarios, and in large cables (/spl delta//R<1,Bi<0.1, where /spl delta/ is the magnetic skin depth and Bi is the thermal Biot number) with inhomogeneous stress and thermal fields. The desire also exists to associate bulk magnetic material parameters with microstructural features to enable generally useful correlations to be developed. The Jiles-Atherton class of thermodynamic models potentially fit these requirements, and we explore their use for investigating field thermomagnetic effects in A36/1018 steels. Model development is supported by microstructural characterization of the anhysteretic via magnetic force microscopy and tunneling electron microscope imaging.

Quantitative validation testing of magnetoelastic corrosion sensing for bridge cables

Magnetic measurements were performed on steel cables subjected to a magnetic field and the response measured without contact using Faradays law, to estimate the effect of temperature and corrosion on magnetic properties of structural steel. Magnetic measurements were compared with electrochemical measurements to correlate corrosion quantitatively in terms of mass loss. The results obtained from the present work are helpful in bounding the achievable sensitivity for conventional magnetoelastic corrosion sensing and for suggesting the need for alternate techniques.