In each of the sporting disciplines, except team events, a higher proportion of the study participants took energy drinks. In addition, a higher proportion of long

distance and middle distance runners, compared with short distance runners, indicated that they consumed energy drinks. The findings also suggest that a higher proportion of middle distance runners, long distance runners and selleck athletes who actively participate in both track and field events are more likely to consume energy drinks than athletes who participate in only team events and short distance disciplines. Most athletes in the team events group ABT-263 research buy (with the exception of athletes who run as a team in track events) did not drink energy drinks, perhaps because these team events, by their nature, require explosive reactions, coupled with maximum strength, power and techniques rather than sustained energy levels. Therefore consuming energy drinks can offer little or no assistance to athletes who participate in these team events with respect to athletic performance. Also, the duration and intensity of team events can influence the decisions of athletes not to consume energy drinks frequently and in great quantities. It is known

that middle and long distance events require sustained energy levels throughout the events (running at times between moderate to high intensity levels that could last for 40 minutes, an hour or beyond, with minimal or no rest intervals) compared with team events in which sustained energy periods for athletes are of short durations AZD2014 ic50 (with intermittent rest intervals), which may necessarily not require the consumption of energy drinks. Conclusions and suggestions for

further study Consumption of energy drinks is a popular practice among university student-athletes in Ghana, as 62.2% of the study participants reported that they drank at least a can of energy drink in the week prior to the study. Approximately 20.5% of the consumers who were all males drank between 3 and 4 cans per week. Most of the student-athletes who drank energy drinks indicated that the main reason why they drank energy drinks was to help replenish Benzatropine lost energy. Some athletes had wrong perceptions regarding the benefits of energy drinks which include its ability to help replace lost body fluids, improve one’s performance and reduce fatigue when participating in any physical activity. Obviously, these wrong perceptions are as a result of the ignorance of students about the proven positive benefits and negative effects of energy drinks. The results suggest the need to create awareness through health education to prevent the consumption of energy drinks in excessive quantities and correct some wrong perceptions that athletes have regarding the benefits of energy drinks.

Transposon Tn7 is also known to associate with integron class 2 and is therefore an important MGE [26]. We therefore also analysed the 65 strains for the presence of integron classes 1, 2, 3 and

4, conjugative plasmids, the tnpM gene of transposon Tn21 and the transposase of Tn7 transposon. Methods Sources of Vibrio cholerae strains Strains that were included in this study were obtained from distinct outbreaks occurring in different parts of Kenya between 1994 and 2007 as indicated in figure 1. For consistency, a distinct outbreak was defined as a gap of at least 2 months between the last GDC-0449 order known cholera case and a report of a new case in the same location. Archived isolates were initially subcultured on thiosulphate citrate bile salts sucrose agar (TCBS) and confirmation of strain identity was done by serology using polyvalent, anti-Ogawa, and anti-Inaba antisera (Denka Seiken, Tokyo, Japan). Haemolysis test was done by growing V. cholerae on 5% sheep blood nutrient agar plates incubated at 37°C overnight. Figure 1 Sources of V. cholera strains used for this study. The geographic locations from which the isolates were obtained are indicated using a

black dot. The number of the strains and the year of isolations are also indicated. All the strains from various regions regardless of the year of isolation had an identical profile for antibiotic Selleckchem CX5461 susceptibility profiles and for genes associated with Raf inhibitor resistance and virulence in V. cholerae. Antimicrobial susceptibility testing Antimicrobial susceptibility tests were performed using commercial cAMP discs following manufacturer’s instructions (Cypress diagnostics, Langdorp, Belgium). Susceptibility to β-lactam antibiotics was tested using ampicillin (10 μg) while susceptibility to cephalosporins was determined using cefixime (30 μg), cefotaxime (30 μg), cefepime (30 μg) cefoxitin (30 ug), cefuroxime (30 ug), ceftriaxone (30 ug), and ceftazidime (30 ug). Ciprofloxacin

(5 μg), norfloxacin (10 μg) and nalidixic acid (30 μg) were used for testing susceptibility to the quinolones. Aztreonam (30 μg), a monobactam antibiotic, was also included in the assay. Aminoglycosides used in susceptibility tests included kanamycin (30 μg), amikacin (30 μg), streptomycin (30 μg), gentamicin (10 μg), neomycin (30 μg), and tobramycin (10 μg). Tetracycline antibiotics included minocycline (30 μg), doxycycline (30 μg) and tetracycline (30 μg). Other antibiotics included chloramphenicol (30 μg), furazolidone (50 μg), rifampicin (30 μg) and nitrofurantoin (30 μg). Sulphamethoxazole (25 μg), trimethoprim (5.2 μg) and sulphonamides (300 μg) were also tested. β-lactam and β-lactamase inhibitor combinations included augmentin (comprising 20 μg amoxicillin and 10 μg clavulanic acid) and a combination of piperacillin (100 μg) and tazobactam (10 μg). E.

RNA was collected by centrifugation at 18630 × g (4°C), washed with 70% ethanol and resuspended in water. Any contaminating DNA was removed by DNase digestion (Turbo-DNase, Ambion) according to the manufacturer’s instructions. Quality and quantification of ACP-196 mouse total bacterial mRNA extracted was 4SC-202 molecular weight assessed using the Experion system (Experion RNA Standard Sense Kit, Bio-Rad). Complementary DNA was synthesised from 1 μg total RNA using the Transcriptor First Strand cDNA Synthesis Kit (Roche) and random

hexamer primers (supplied) according to manufacturer’s instructions. Real-time and reverse-transcriptase PCR Real-Time PCR reactions were performed in the LightCycler version 1.5 (Roche Diagnostics) using either the LightCycler MasterPlus SYBR Green (Roche) or the Master SYBR Green kit (Roche). PCR master mixes (SYBR Green dye and FastStart Taq DNA polymerase were supplied) were prepared according to the manufacturer’s instructions. A four step experimental protocol was used: (i) activation (95°C for 15 min) (ii) amplification step

repeated for 45 cycles (95°C for 10 sec; primer-specific Tm for 10 sec, 72°C for 10 sec with a single fluorescence measurement) (iii) melting curve analysis (65°C-95°C with a heating rate of 0.1°C per second and a continuous fluorescence measurement) (iv) cooling step down to 40°C (see Table 1 for annealing temperatures). Refer to Table 5 for a complete list of NVP-LDE225 datasheet primer sequences used to analyse the genes of interest. RNA template and no-template controls were included to determine DNA contamination of RNA samples or PCR reactions. All PCR reactions as well as all biological experiments were done in triplicate. Relative quantification of gene expression was done using the REST-384 Version 1 software with PCR efficiency correction for individual real-time PCR transcripts [48]. SigA was used as the internal standard to normalise target gene expression levels in each RNA sample [59] as it has been shown that sigA expression Acyl CoA dehydrogenase remains constant

under various growth and stress conditions [60]. Table 5 Primer sequences used for the relative quantification of glutamine synthetase and glutamate dehydrogenase genes.* Gene Sense Primer (5′-3′) Antisense Primer (5′-3′) Product size (bp) Annealing Temperature (°C) glnA1 ATGTGCTGCTGTTCAAGT TGAAGGTGACGGTCTTGC 66 55 sigA GACTCGGTTCGCGCCTA CCTCTTCTTCGGCGTTG 64 55 msmeg_6272 TGATCCGCCACATCCTG GATGTAGGTGCCGATGC 65 56.5 msmeg_5442 AGATCATGCGGTTCTGTC GTGTATTCACCGATGTGCC 61 55 msmeg_4699 GTGAGGACTTCCGCACC CCGCTTGACGACGAATC 104 55 *The product size and annealing temperatures are also given. sigA was used as an internal control or housekeeping gene. Reverse transcriptase PCR reactions were carried out in the GeneAmp PCR System 9700 Reverse transcriptase PCR reactions were carried out in the GeneAmp PCR System 9700 (Applied Biosystems) using HotStar Taq DNA Polymerase (Qiagen) according to manufacturer’s instructions.

Cell line studies show that HMGB1 is click here strongly up-regulated in breast cancer, colon cancer, melanoma, pancreatic cancer and prostate cancer; upregulated HMGB1 activates TLR2 and TLR4 expressed on immune cells and induces cancer progression and metastasis [20]. We previously reported elevated expression of S100 proteins in melanoma cell lines

relative to normal melanocyte lines. S100 proteins released by melanoma cells stimulated melanoma cells as well as PBLs and acted as an autocrine tumor growth factor [50]. S100A4 is responsible for metastasis and is an indicator of poor prognosis for patients with Epoxomicin datasheet breast cancer [51]. However, although this inflammatory protein is associated with metastatic cancer cells, in the tumor microenvironment it is also expressed by macrophages, lymphocytes and fibroblasts. Elevated interstitial fluid levels of S100A4 in tumors [52] suggest that stromal cells in the tumor microenvironment externalize S100A4, which then activates TLR signals. Recent studies reveal that S100A8 and S100A9 produced by primary tumors can activate serum amyloid A (SAA) 3 in lung tissue prior to pulmonary metastasis. SAA3 has a role in the accumulation of myeloid cells and acts as a positive-feedback regulator for secretion of S100 proteins. SAA3 is a ligand for TLR4 in lung endothelial cells and macrophages. The activation of TLR4

facilitates migration Akt inhibitor of cancer cells from the primary tumor to lung

tissue by creating a tumor microenvironment [53]. Blocking the S100-TLR4 cascade therefore might be an effective strategy for the prevention of pulmonary metastasis. Nucleic Acid Fragments Act as DAMPs During tumor expansion, nucleic acids released from necrotic cancer cells or adjacent injured normal epithelial cells act as DAMPs. Kariko et al. [54] demonstrated that TLR3 expressed in DCs was activated by mRNA released from necrotic cells; subsequent TLR signals upregulated DC maturation, leading to IFN-α secretion. This upregulation could Carnitine dehydrogenase be abolished by pretreatment of necrotic cells with RNase. The mRNA released by cancer cells circulates in the blood [55] and its serum levels have been correlated with disease outcome [56]. In our studies, TLR3 expression was upregulated (24.6–121.3% in mean value) in melanoma cells incubated 12 h with purified total RNA from normal PBL or allogeneic melanoma cells (Fig. 2), and TLR activation promoted melanoma cell migration [5]. Thus, RNA derived from melanoma cells can act as a TLR3 ligand and facilitate migration of melanoma cells, without support from immune cells. Fig. 2 TLR3 ligation and subsequent TLR3 mRNA expression in melanoma cells incubated with purified total RNA from normal donor PBLs or allogeneic melanoma cells. When ME7 and ME1 human melanoma cells were incubated 12 h with total RNA from normal PBL and ME5 melanoma cells, mean TLR3 mRNA expression increased 24.6–121.

It should be taken into account, too, that the light path in typical measuring chambers (usually 1–2 cm) is much smaller than that in the culture vessel (5–10 cm), so that the light intensity reaching every single cell is VX-680 higher due to less self-shading of the cells. The use of O2 electrodes of the Clark type is a common technology which will not be

explained here in detail. It should be noted, however, that Clark-type electrodes can easily be converted to H2 electrodes just by applying a different potential. Details of assembling and using these electrodes are given in references Wang (1980), Kuroda et al. (1991), and Takeshita et al. (1993). An easy method to analyze in vivo H2-production rates of illuminated C. reinhardtii cells without a H2 electrode will

be described here. This technique is suitable to determine the real H2-evolution rate of the cells, which can be only roughly concluded from the accumulation of H2 in the gas phase of the incubation TSA HDAC flask or in a gas trap (see below). For this purpose, a 2-ml sample of the main culture is taken with a syringe by piercing through the septum and gently injected through the rubber seal of an 8–10 ml headspace bottle as described above, which has been gassed with Ar before. The little vessels are then placed in the light. The cell NSC23766 suspension has to be rocked or stirred so that the cells do not settle. A shaking water bath made from plexiglas standing on top a light source is optimal. After 10 min, a volume of the gas phase is analyzed with a gas chromatograph to determine H2 concentration. Then, the cells are incubated for 1 h, and H2 is detected again. The difference of the H2 concentration in the beginning and after 60 min is the amount of H2 that has been produced by the cells. It should be noted that the 10 min of pre-incubation is applied to let the cells adapt to the system, which will differ from the incubation conditions of the main culture in some aspects. Furthermore, during the

transfer of the cells, some air (i.e., O2) might have entered the cell suspension, and the cells might have been shaded to some extent. the During the pre-incubation, the algae will stabilize their H2 metabolism. The first analysis of the H2 concentration after the 10 min duration is important to take into account the H2 which has been produced during this pre-incubation phase and the gas which was introduced into the reaction vessel by the algal suspension. In the active H2-producing phase of S-deprived C. reinhardtii cultures, significant amounts of H2 are dissolved in the medium of the cells. The above point should also be kept in mind when carrying out in vitro hydrogenase activity assays with S-depleted algae.

Animal models and cell culture systems have provided indications that lactobacilli are able to counteract alterations in paracellular permeability evoked

by cytokines, chemicals, peptides, infections or stress [36]. A paper by Seth et al. [37] reported that the administration of live and heat inactivated L.GG, bacterial supernatants and peculiar L.GG purified soluble proteins to Caco-2 cells treated by hydrogen peroxide that destroys TER and increases permeability, caused the secretion of proteins of this strain effective against the insult. In our study, the administration of viable and heat killed L.GG as well as its conditioned medium, caused only a slight and not significant increase in TER after 90 min from exposure without any effects on lactulose flux and zonulin release. By opposite, in Caco-2 cells treated with gliadin, the addition of viable L.GG, but also L.GG-HK learn more and L.GG-CM, significantly restored cell barrier function. Also the MX69 mw single and total polyamine levels diminished significantly when Caco-2 cells were exposed to gliadin in combination with viable and heat killed L.GG. Recently, our group reported that the administration of viable, heat killed L.GG and L.GG homogenate to DLD-1 and HGC-27 cell lines significantly reduced neoplastic proliferation as well as polyamine content and biosynthesis [19, 20, 38]. As regards the protective effects

of some probiotics against gliadin, our findings are in line with data in literature [39] and

different mechanisms could be evoked to explain the effects exerted by L.GG, Cell Penetrating Peptide not only as viable bacteria, but also when they were heat inactivated or their conditioned medium was used. Firstly, L.GG might inhibit gliadin-induced damage in Caco-2 cells by hydrolyzing gliadin similarly to other live probiotic bacteria as in the VSL3# probiotic preparation [40]. These strains showed the ability to colonize the human stomach and duodenum, where the hydrolysis of gliadin epitopes may be relevant for decreasing the abnormal secretion of zonulin and the initial step of immune response to gliadin [41, 42]. Secondly, the peculiar set of peptidases shown by L.GG was probably able to inhibit the gliadin-induced damage to Caco-2 cells breaking up wheat gliadin into small harmless peptide products [43]. Thirdly, L.GG might modulate directly the function of epithelial cells. It has already been reported that different probiotic strains, probiotic bacterial lysates or conditioned medium increase epithelial barrier function as measured by TER [44]. In addition, L.GG might protect the epithelium from the gliadin insult by direct action on the cells. One interesting Tipifarnib in vitro finding of the present study is that viable L.GG per se was able to significantly increase ZO-1, Claudin-1 and Occludin expression after 6 h of exposure. Even if the gliadin effects on TJ expression were significant only after 24 h, the co-administration of viable L.

Whereas this finding suggests that mannosucrose might be a better compatible solute than trehalose, this would need experimental support. Despite trehalose

synthesis was osmoregulated in R. tropici CIAT 899, our data suggest that HMPL-504 cost trehalose alone cannot account for the higher osmotolerance of this strain. Thus, osmoadaptation in R. tropici CIAT 899 (and most PLX3397 molecular weight soil microorganisms) is probably a complex process involving many physiological and biochemical response mechanisms, not yet fully elucidated. Although trehalose, without doubt, participates in some way to alleviate osmotic stress, there is increasing evidence that trehalose is primarily a stress metabolite designed to ensure cell survival. In fact, trehalose synthesis in E. coli is under the control of the general stress factor σS, which is responsible for the expression of genes induced upon entry of stationary phase [38]. In S. meliloti, trehalose synthesis is under the control of the general stress factor RpoE2 [46], which is also necessary for desiccation resistance [47]. Thus, it may be possible that NaCl-induced synthesis of trehalose and mannosucrose in

the isolated soil strains are also involved in drought tolerance. This will be investigated in a future work. In this work, we showed the presence of otsA within the genome of the four studied Rhizobium strains, suggesting P005091 purchase that trehalose synthesis in these strains occurs at least via OtsAB. In addition, by using [1/6-13C]mannitol as a carbon

source, we showed that in R. tropici CIAT 899 both trehalose moieties, as well as the β-glucan units, where RG7420 clinical trial derived directly from mannitol. This finding, together with in silico analysis of rhizobial genomes, suggests that R. tropici takes up mannitol via a sorbitol/mannitol ABC transporter. Subsequently, mannitol is converted to fructose (by a mannitol 2-dehydrogenase) and the latter one into glucose, the trehalose precursor, by a xylose isomerase. In the case of mannose, the in silico analysis suggest that R. tropici incorporates it through a phosphotransferase system, yielding mannose-6-phosphate, but it cannot convert mannose-6-phosphate into fructose-6-phosphate, as it may lack the mannose-6-phosphate isomerase. This metabolic reconstruction would explain why R. tropici CIAT 899 cannot synthesize trehalose from mannose. Methods Bacterial strains and growth conditions Bacterial strains used in this study were R. gallicum bv. gallicum 8a3, R. leguminosarum bv. phaseoli 31c3, R. etli 12a3, Agrobacterium sp. 10c2 (in this work renamed as A. tumefaciens 10c2) [23, 24], and R. tropici CIAT 899T [15]. The reference strain R. tropici CIAT 899T belongs to the CIAT (International Center for Tropical Agriculture, Colombia) culture collection. It is able to form effective symbiosis with P. vulgaris and Leucaena trees [15] and to tolerate high temperature, low pH, and salinity [25, 26]. Rhizobial strains were routinely grown in complex TY medium [48] at 28°C.

However, in the human intestine, low oxygen tension permits E. coli to grow by fermentation or respiration using an alternative

electron acceptor. As nitrate is readily available in the human intestine (14 μmol/kg [36]) and can be readily utilized by intestinal bacterial flora including E. coli [37, 38] we examined succinate selection using this alternate electron receptor. Interestingly, host nitrate synthesis can be stimulated in response to infections caused by gastroenteric pathogens [38]. To test if selection for loss of RpoS can occur under low oxygen conditions, cultures were grown in anaerobic jars (see Methods). GS-1101 cell line We first compared the anaerobic growth of wild type and aerobically-selected Suc++ www.selleckchem.com/products/rg-7112.html mutants on glucose and succinate plates. Wild type EDL933 grew as well as an isogenic rpoS knockout Y27632 mutant and derivative Suc++ mutants on glucose, while the rpoS and Suc++ mutants grew much better than wild type on succinate under both aerobic and anaerobic conditions (Figure 2). The growth of Suc++ mutants was similar to that of the

control rpoS null mutant under all conditions tested. Figure 2 Growth of EDL933 and derivative Suc ++ mutants on M9 glucose (Glu) and succinate (Suc) media. Colony size (diameter) was determined under a light microscope at 40× magnification. All VTEC strains were then tested for selection on succinate under anaerobic conditions. As under aerobic conditions, Suc++ mutants could be selected from all tested strains, except for CL3, R82F2 and N99-4390. Most (87%) of the Suc++ had

reduced catalase activity. We sequenced the rpoS region of 15 Suc++ mutants isolated Aspartate from EDL933 and found mutations in rpoS, resulting in impaired RpoS function, in 13 mutants while the rpoS gene in the other two Suc++ mutants remained unchanged (data not shown). Expression of virulence-related traits, RDAR and cell adherence Mutations in rpoS may affect virulence factor expression in pathogenic strains [39, 40]. To test this, we examined two virulence-related traits, the RDAR morphotype and cell adherence. Extracellular components, such as curli fimbriae and cellulose, are correlated with biofilm formation and virulence in Salmonella sp. and E. coli strains [41–43]. The expression of curli and cellulose can be visualized by staining with Congo Red dye to produce a red, dry and rough morphotype (RDAR) [43, 44]. Biosynthesis of both curli and cellulose is positively regulated by RpoS through a transcriptional regulator CsgD in E. coli K12 [45, 46]. However, to our knowledge, the role of RpoS in expression of RDAR has not been previously tested in pathogenic E. coli isolates. Wild type EDL933 exhibited a more pronounced RDAR morphotype than an isogenic rpoS null deletion mutant and Suc++ mutants (Figure 3A), suggesting that RpoS is important for RDAR development.

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The three B. thailandensis E264 PIs, PI-E264-1,

-2, and -3, correspond to B. thailandensis GI1, Bt GI13, and Bt GI12, respectively, as described Screening Library high throughput by Yu et al [24], although the range of genes in the PIs described here differ slightly due to our criteria for inclusion. Similarly, PI-668-1 corresponds to GI15c from B. pseudomallei MSHR668 in Tuanyok et al [4]. As mentioned above, no PIs were detected in B. pseudomallei 1106a/b, although phage-like remnants were found in these strains. Overall, seventeen of the 24 identified regions were located on chromosome I of the respective bacterial strain, and all but five were putative prophages (i.e., most likely to be active prophages containing all of the prophage-like elements described in the Materials and Methods). Of the seven regions located on chromosome II, one (PI-E264-3) was classified as a putative prophage, while the remaining six were designated prophage-like. Paired strains B. pseudomallei 1710a/b and B. pseudomallei 1106a/b The two pairs B. pseudomallei 1710a/b and B. pseudomallei 1106a/b represent

two bacterial strains isolated at different time points from the same two infected patients, isolated from the primary infection (a) and the relapse (b). We hypothesized that difference/s in sequence relating to the relapse or host selection would be detected, either in the form of SNPs/indels or as variation in the phage harbored within each strain. Three PIs were identified in each of the B. pseudomallei BGB324 cell line 1710 strains. PI-1710a/b-1 is immediately followed by PI-1710a/b-2 on chromosome I, separated by a tRNA pseudogene in each strain. This region is described

as GI6b in Tuanyok et al. [4]. PI-1710a/b-3 is located further downstream on chromosome I. All three regions are nearly identical, averaging 98.4, 97.7, and 96.6% identity over 98.2, 97.1, and 96.2% of length (for -1, -2, and -3, respectively). PI-1710a-1 and PI-1710b-1 are 41.3 and 41.4 kb in length, respectively, and both are bounded by tRNA-Pseudo-2 and a 23 bp exact repeat of the 3′ end of this tRNA. Both PI-1710a-2 and PI-1710b-2 are 60.6 kb in size and are bounded by tRNA-Pro-2 and a 49 bp exact repeat. The Rho prophage-like regions in both strains (PI-1710a-3, PI-1710b-3) are defined by the presence of a phage integrase at the 3′ end by tRNA-Thr-3, with several viral-like proteins immediately upstream, but no repeat region could be identified to Luminespib cost define the 5′ end. Both are 62.8 kb. Since the three prophage islands are nearly identical between B. pseudomallei 1710a and B. pseudomallei 1710b, from here on we will only refer to B. pseudomallei 1710b and associated prophage islands. These results indicate that the prophage in Bp 1710a/b were not excised and did not experience any significant changes even after passage through a host. By the definitions set forth for prophage islands given in this work, no PIs were identified in either of the B. pseudomallei 1106 strains. Tuanyok et al.