Salmonella enterica serovar Typhimurium adhesion and cytotoxicity during epithelial cell stress is reduced by Lactobacillus rhamnosus GG

<p>Abstract</p> <p>Background</p> <p>Physiological stressors may alter susceptibility of the host intestinal epithelium to infection by enteric pathogens. In the current study, cytotoxic effect, adhesion and invasion of <it>Salmonella enterica </it>serovar Typhimurium (<it>S</it>. Typhimurium) to Caco-2 cells exposed to thermal stress (41°C, 1 h) was investigated. Probiotic bacteria have been shown to reduce interaction of pathogens with the epithelium under non-stress conditions and may have a significant effect on epithelial viability during infection; however, probiotic effect on pathogen interaction with epithelial cells under physiological stress is not known. Therefore, we investigated the influence of <it>Lactobacillus rhamnosus </it>GG and <it>Lactobacillus gasseri </it>on <it>Salmonella </it>adhesion and <it>Salmonella</it>-induced cytotoxicity of Caco-2 cells subjected to thermal stress.</p> <p>Results</p> <p>Thermal stress increased the cytotoxic effect of both <it>S</it>. Typhimurium (P = 0.0001) and nonpathogenic <it>E. coli </it>K12 (P = 0.004) to Caco-2 cells, and resulted in greater susceptibility of cell monolayers to <it>S</it>. Typhimurium adhesion (P = 0.001). Thermal stress had no significant impact on inflammatory cytokines released by Caco-2 cells, although exposure to <it>S</it>. Typhimurium resulted in greater than 80% increase in production of IL-6 and IL-8. Blocking <it>S</it>. Typhimurium with anti-ShdA antibody prior to exposure of <it>Salmonella </it>decreased adhesion (P = 0.01) to non-stressed and thermal-stressed Caco-2 cells. Pre-exposure of Caco-2 cells to <it>L. rhamnosus </it>GG significantly reduced <it>Salmonella</it>-induced cytotoxicity (P = 0.001) and <it>Salmonella </it>adhesion (P = 0.001) to Caco-2 cells during thermal stress, while <it>L. gasseri </it>had no effect.</p> <p>Conclusion</p> <p>Results suggest that thermal stress increases susceptibility of intestinal epithelial Caco-2 cells to <it>Salmonella </it>adhesion, and increases the cytotoxic effect of <it>Salmonella </it>during infection. Use of <it>L. rhamnosus </it>GG as a probiotic may reduce the severity of infection during epithelial cell stress. Mechanisms by which thermal stress increases susceptibility to <it>S</it>. Typhimurium colonization and by which <it>L. rhamnosus </it>GG limits the severity of infection remain to be elucidated.</p

Characterization of surface proteins of Cronobacter muytjensii using monoclonal antibodies and MALDI-TOF Mass spectrometry

<p>Abstract</p> <p>Background</p> <p><it>Cronobacter </it>spp. is a newly emerging pathogen that causes meningitis in infants and other diseases in elderly and immunocompromised individuals. This study was undertaken to investigate surface antigenic determinants in <it>Cronobacter </it>spp. using monoclonal antibodies (MAbs) and MALDI-TOF Mass spectrometry.</p> <p>Results</p> <p>Spleenocytes from mice that were immunized with heat-killed (20 min, 80°C) <it>Cronobacter </it>cells were fused with SP2 myeloma cells. Five desirable MAbs (A1, B5, 2C2, C5 and A4) were selected. MAbs A1, B5, 2C2 and C5 were of IgG2a isotype while A4 was an IgM. Specificity of the MAbs was determined by using immunoblotting with outer membrane protein preparations (OMPs) extracted from 12 <it>Cronobacter </it>and 6 non-<it>Cronobacter </it>bacteria. All MAbs recognized proteins with molecular weight ranging between 36 and 49 kDa except for one isolate (44) in which no OMPs were detected. In addition, MAbs recognized two bands (38-41 kDa) in four of the non-<it>Cronobacter </it>bacteria. Most of the proteins recognized by the MAbs were identified by MALDI-TOF peptide sequencing and appeared to be heterogeneous with the identities of some of them are still unknown. All MAbs recognized the same epitope as determined by an additive Index ELISA with their epitopes appeared to be conformational rather than sequential. Further, none of the MAbs recognized purified LPS from <it>Cronobacter </it>spp. Specificity of the MAbs toward OMPs was further confirmed by transmission electron microscopy.</p> <p>Conclusions</p> <p>Results obtained in this study highlight the immunological cross-reactivity among <it>Cronobacter </it>OMPs and their <it>Enterobacteriaceae </it>counterparts. Nevertheless, the identity of the identified proteins appeared to be different as inferred from the MALDI-TOF sequencing and identification.</p

Development of a Reporter System for In Vivo Monitoring of gamma-Secretase Activity in Drosophila

The gamma-secretase complex represents an evolutionarily conserved family of transmembrane aspartyl proteases that cleave numerous type-I membrane proteins, including the beta-amyloid precursor protein (APP) and the receptor Notch. All known rare mutations in APP and the gamma-secretase catalytic component, presenilin, which lead to increased amyloid betapeptide production, are responsible for early-onset familial Alzheimer\u27s disease. beta-amyloid protein precursor-like (APPL) is the Drosophila ortholog of human APP. Here, we created Notch- and APPL-based Drosophila reporter systems for in vivo monitoring of gamma-secretase activity. Ectopic expression of the Notch- and APPL-based chimeric reporters in wings results in vein truncation phenotypes. Reporter-mediated vein truncation phenotypes are enhanced by the Notch gain-of-function allele and suppressed by RNAi-mediated knockdown of presenilin. Furthermore, we find that apoptosis partly contributes to the vein truncation phenotypes of the APPL-based reporter, but not to the vein truncation phenotypes of the Notch-based reporter. Taken together, these results suggest that both in vivo reporter systems provide a powerful genetic tool to identify genes that modulate gamma-secretase activity and/or APPL metabolism

Self-directed growth of AlGaAs core-shell nanowires for visible light applications

Al(0.37)Ga(0.63)As nanowires (NWs) were grown in a molecular beam epitaxy system on GaAs(111)B substrates. Micro-photoluminescence measurements and energy dispersive X-ray spectroscopy indicated a core-shell structure and Al composition gradient along the NW axis, producing a potential minimum for carrier confinement. The core-shell structure formed during the growth as a consequence of the different Al and Ga adatom diffusion lengths.Comment: 20 pages, 7 figure

Effect of FYM, foliar feeding of nitrogen and deficit irrigation on drip irrigated coriander (Coriandrum sativum L.)

A field experiment was conducted to study the water use, nitrogen (N) uptake and economics of coriander (Coriandrum sativum L.) under organic enrichment, foliar feeding of N and fertigation. Drip irrigation at 80% ETc + foliar spray of 25% N (15 kg ha-1) + fertigation (45 kg ha-1) gave the highest plant height, branch plant-1, umbel plant-1, umbelets umbel-1, seed umbel-1, and test weight compared to irrigation at 80% ETc and at 60% ETc (upto flowering stage) + 80% ETc (reproductive stage) with or without foliar spray. Increased yield parameters were attributed to the highest seed yield and B:C ratio of 1.90 using 265.74 mm water. Water use efficiency (WUE) was also the highest. This treatment also recorded the highest N uptake of 52.6 kg ha-1. Further, addition of 10 t ha-1 FYM in addition to recommended nitrogen dose (60 kg ha-1) gave higher yield attributes viz., branch plant-1, umbel plant-1, seed umbel-1 and test weight as compared to recommended level of fertilizers. Enhanced yield attributes thus increased seed yield, N removal, WUE and B:C ratio. &nbsp

Effect of drip irrigation and bioregulators on yield, economics and water use of fenugreek (Trigonella foenum-graecum)

The field experiment was conducted during winter seasons of 2009–10 and 2010–11 to assess the effect of drip irrigation levels and bioregulators (thiourea and Thioglycollic acid) on fenugreek. Increasing levels of drip irrigation from 0.6 to 1.0 ETc enhanced yield attributes viz., branches plant-1, pods plant-1, pod length, seeds pod-1 and test weight. Increased yield attributes with 1.0 ETc thus, enhanced seed yield (15.53 q ha-1) and stover yield (31.54 q ha-1). However highest B:C ratio (2.11) was recorded with 0.8 ETc irrigation level and highest water use efficiency of 4.83 kg ha-1 mm with 0.6 ETc. Recommended surface irrigation treatment recorded 13.02 q ha-1 seed yield with using 540 mm of water. Whereas, fenugreek under drip irrigation at 0.6, 0.8 and 1.0 ETc used 278.16, 370.88 and 463.60 mm of water, respectively. Seed treatment of fenugreek before sowing with bio-regulator thiourea (500 ppm) for 4 h recorded higher yield attributes viz., pods plant-1, pod length and test weight, as well as, seed yield of 14.85 q ha-1 against 14.36 q ha-1 where no seed treatment was done. Thus, in turn fetched higher water use efficiency (4.00 kg ha-1 mm) and B:C ratio (2.08). Further, foliar spray with 200 ppm thioglycollic acid at vegetative and seed formation stages recorded highest seed yield of 15.01 q ha-1 over 100 ppm thioglycollic acid spray (14.59 q ha-1) or no spray treatment (14.21 q ha-1). Increased yield thus enhanced water use efficiency (3.93-4.05 kg ha-1 mm) and B:C ratio (2.06-2.11) in thioglycollic acid treated crop. &nbsp

Loss-of-function of triacylglycerol lipases are associated with low flour rancidity in pearl millet [Pennisetum glaucum (L.) R. Br.]

Pearl millet is an important cereal crop of semi-arid regions since it is highly nutritious and climate resilient. However, pearl millet is underutilized commercially due to the rapid onset of hydrolytic rancidity of seed lipids post-milling. We investigated the underlying biochemical and molecular mechanisms of rancidity development in the flour from contrasting inbred lines under accelerated aging conditions. The breakdown of storage lipids (triacylglycerols; TAG) was accompanied by free fatty acid accumulation over the time course for all lines. The high rancidity lines had the highest amount of FFA by day 21, suggesting that TAG lipases may be the cause of rancidity. Additionally, the high rancidity lines manifested substantial amounts of volatile aldehyde compounds, which are characteristic products of lipid oxidation. Lipases with expression in seed post-milling were sequenced from low and high rancidity lines. Polymorphisms were identified in two TAG lipase genes (PgTAGLip1 and PgTAGLip2) from the low rancidity line. Expression in a yeast model system confirmed these mutants were non-functional. We provide a direct mechanism to alleviate rancidity in pearl millet flour by identifying mutations in key TAG lipase genes that are associated with low rancidity. These genetic variations can be exploited through molecular breeding or precision genome technologies to develop elite pearl millet cultivars with improved flour shelf life

Mutant polycystin-2 induces proliferation in primary rat tubular epithelial cells in a STAT-1/p21-independent fashion accompanied instead by alterations in expression of p57KIP2 and Cdk2

<p>Abstract</p> <p>Background</p> <p>Autosomal Dominant Polycystic Kidney Disease (ADPKD) is characterized by the formation of multiple fluid-filled cysts that destroy the kidney architecture resulting in end-stage renal failure. Mutations in genes <it>PKD1 </it>and <it>PKD2 </it>account for nearly all cases of ADPKD. Increased cell proliferation is one of the key features of the disease. Several studies indicated that polycystin-1 regulates cellular proliferation through various signaling pathways, but little is known about the role played by polycystin-2, the product of <it>PKD2</it>. Recently, it was reported that as with polycystin-1, polycystin-2 can act as a negative regulator of cell growth by modulating the levels of the cyclin-dependent kinase inhibitor, p21 and the activity of the cyclin-dependent kinase 2, Cdk2.</p> <p>Methods</p> <p>Here we utilized different kidney cell-lines expressing wild-type and mutant <it>PKD2 </it>as well as primary tubular epithelial cells isolated from a PKD transgenic rat to further explore the contribution of the p21/Cdk2 pathway in ADPKD proliferation.</p> <p>Results</p> <p>Surprisingly, over-expression of wild-type <it>PKD2 </it>in renal cell lines failed to inactivate Cdk2 and consequently had no effect on cell proliferation. On the other hand, expression of mutated <it>PKD2 </it>augmented proliferation only in the primary tubular epithelial cells of a rat model but this was independent of the STAT-1/p21 pathway. On the contrary, multiple approaches revealed unequivocally that expression of the cyclin-dependent kinase inhibitor, p57^KIP2, is downregulated, while p21 remains unchanged. This p57 reduction is accompanied by an increase in Cdk2 levels.</p> <p>Conclusion</p> <p>Our results indicate the probable involvement of p57^KIP2on epithelial cell proliferation in ADPKD implicating a new mechanism for mutant polycystin-2 induced proliferation. Most importantly, contrary to previous studies, abnormal proliferation in cells expressing mutant polycystin-2 appears to be independent of STAT-1/p21.</p

LHC Magnet Tests: Operational Techniques and Empowerment for Successful Completion

The LHC magnet tests operation team developed various innovative techniques, particularly since early 2004, to complete the superconductor magnet tests by Feb. 2007. Overall and cryogenic priority handling, rapid on-bench thermal cycling, rule-based goodness evaluation on round-the-clock basis, multiple, mashed web systems are some of these techniques applied with rigour for successful tests completion in time. This paper highlights these operation empowerment tools which had a pivotal role for success. A priority handling method was put in place to enable maximum throughput from twelve test benches, having many different constraints. For the cryogenics infrastructure, it implied judicious allocation of limited resources to the benches. Rapid On-Bench Thermal Cycle was a key strategy to accelerate magnets tests throughput, saving time and simplifying logistics. First level magnet appraisal was developed for 24 hr decision making so as to prepare a magnet further for LHC or keep it on standby. Web based systems (Tests Management and E-Traveller) were other essential ideas to track & coordinate various stages of tests handled by different teams