Y. enterocolitica invariably produces urease which has been reported to enable biovar 1B and biovar 4 strains to survive in the acidic environment of the stomach [20, 21]. However, the role of urease in the survival of biovar 1A strains has not find more been investigated. The objective of this study was to determine the genetic organization of urease (ure) gene cluster, factors affecting urease activity, and the survival of biovar 1A strain of Y. enterocolitica in acidic pH in vitro. Methods Bacterial strains and growth conditions Y. enterocolitica biovar 1A (serovar O:6,30) isolated from the stools of a diarrheic patient and deposited

with Yersinia National Reference Laboratory and WHO Collaborating Center, Pasteur Institute (Paris) under reference number IP27403 was used to characterize ure gene complex and the enzyme urease. The details of other Y. enterocolitica strains used in this study namely serovars, source of isolation, country of origin, reference laboratory accession numbers and clonal groups have been reported previously [22]. Y. enterocolitica 8081 (bioserovar 1B/O:8) was obtained from M. Skurnik (Haartman Institute, Helsinki, Finland). Y. enterocolitica IP26329 (bioserovar 2/O:9), IP26249 (bioserovar 2/O:5,27), and IP134 (bioserovar 4/O:3) were obtained from E. Carniel (Yersinia National Reference Laboratory and WHO Collaborating Center, Pasteur Institute, France). All strains were grown overnight at 28°C

https://www.selleckchem.com/products/XL184.html in Luria broth (HiMedia, Mumbai, India). DNA extraction, primers and Polymerase Chain Reaction Genomic DNA was isolated from overnight grown cultures using DNeasy tissue kit (Qiagen GmbH) as reported earlier [14]. Urease gene sequences of Y. enterocolitica science biovar 1B

and biovar 4 with GenBank accession numbers L24101[23] and Z18865[24] respectively were used to design primers U1 and U2 using PrimerSelect 5.03 software (DNASTAR Inc., Madison, USA) such that the structural genes (ureA, ureB, ureC) may be amplified as one amplicon. As these primers failed to consistently amplify the ureABC region of biovar 1A strains, primers for amplification of each of the structural genes separately were designed from the following sequences in the database (accession numbers are given in parentheses): Y. enterocolitica biovar 1B (L24101, AM286415), Y. enterocolitica biovar 4 (Z18865), Y. aldovae (AY363680), Y. bercovieri (AY363681), Y. frederiksenii (AY363682), Y. intermedia (AY363683), Y. kristensenii (AY363684), Y. mollaretii (AY363685), Y. rohdei (AY363686), Y. pestis (AE017042, AL590842, AE009952, AF095636) and Y. pseudotuberculosis (U40842, BX936398). These sequences were also used to design primers for ure accessory (ureE, ureF, ureG, ureD) and urea transport (yut) genes. The most conserved regions for each of the genes were identified using MegAlign (DNASTAR) or ClustalW version 1.83 (accessible at http://​www.​ebi.​ac.​uk/​tools/​clustalW).

The maximum quality score was 6 point [40, 41]. The quality scores were showed in additional file 1. Statistical Analysis learn more The primary end points variables were defined as dichotomous data (e.g., remission rate of pain used variables as follows: the effective or the ineffective after treatment). We standardized the therapeutic results by obtaining the relative risk (RR). RR is defined as a ratio of risk of uncontrolled pain or adverse effects occurring in transdermal

fentanyl group versus sustained-release oral morphine group. To test for heterogeneity among the trials, Cochran’s χ2 test was used. P-value of more than 0.05 for the χ2-test indicated a lack of heterogeneity across the studies, so pooled estimation of the RRs of each study was calculated by the fixed effects model. Otherwise, the random effects model was used. An estimate of the potential publication bias was carried out by funnel plot, in Captisol mouse which the

standard error (SE) of log RR of each study was plotted against its log RR. An asymmetric plot suggested a possible publication bias. All analyses were performed strictly with RevMan software (version 4.2.8, Cochrane). P value less than 0.05 was considered as significant in difference. Results Characteristics of selected trials 578 trials were examined in the preliminary review; 32 of them were considered eligible and included in the analysis. The data extracted from 32 trials were shown in additional file 1[8–39]. A total of 2651 cancer pain patients were treated in all selected trials, 1296 with transdermal fentanyl, and 1355 with sustained-release oral morphine. 30 of selected trials were included in the analysis of clinical efficacy; and 31, 31 and 28 of selected trials were included in the analysis

of constipation, nausea/vomiting and vertigo/somnolence. Only 6 trials supplied data about QOL evaluated in Metalloexopeptidase different criteria [9, 14, 17, 32–34]. Sustained-release oral morphine was Morphine Hydrochloride-Southwest Pharm in 8 of selected trials [8, 16, 19, 25, 27, 29, 32, 33]. Trials were excluded from the analysis for one or more of the following reasons: uncorrelated, review, case report, no valid data, no followed-up time, and non-cancer pain. Trials applied either numerical rating scale or visual analogue scales for assessing cancer pain. The criterion of remission of cancer pain was described as follow. Five categories of pain relief: category 0, no remission (pain didn’t release); category 1, mild remission (pain released one quarter); category 2, moderate remission (pain released a half); category 3, obvious remission (pain released three quarters); category 4, complete remission (pain disappeared). Pain can be controlled denotes that patients gain category 2 or above of pain relief.

baumannii might also be easily isolated from nature. Recently, 10 phages were obtained from wastewater using 125 clinical isolates of A. baumannii as indicator hosts [20, 23]. These phages were designated AB1 to AB9 and AB11. Examination by transmission electron microscopy suggested that phages AB1-7 and AB9 belonged to the

Podoviridae family, and phages AB8 and AB11 belonged to the Myoviridae family. Two of the 10 phages, AB1 and AB2, were further characterized at 35°C and 37°C, respectively. Based on morphology and genomic analysis, the two phages Selleckchem R406 were classified as new members of the ϕKMV-like phages [20, 23]. In this study, the phage ZZ1, which is specific to A. baumannii, was isolated from fishpond water, which further confirmed that phages specific to A. baumannii are waiting to be exploited as an abundant natural resource. The ability of phage ZZ1 to form clear plaques on lawns of AB09V is indicative of lytic phage, and a large burst size with a short latent period is further suggestive of the lytic nature of phage ZZ1. Morphologically, ZZ1 exhibits features similar to the Myoviridae family (order Caudovirales), which, broadly, are tailed phages with icosahedral head symmetry and contractile tail structures. Genome analysis of ZZ1 showed that it is fairly similar to four other Acinetobacter phages (Acj9, Acj61, Ac42, and 133).

In a recent review by Petrov et al. [18], the four Acinetobacter P5091 molecular weight phages were initially assigned to the “T4-like Viruses” genus. Each of these Acinetobacter phages has a unique set of ORFs that occupy ~35% of the genome. That is, each represents a different type of T4-related phage genome [18]. The genome size of the phage ZZ1 (166,682 bp) is also close to the genome size of T4-like phages. These genomes vary between ~160,000 and

~250,000 bp [18]. Therefore, the above features confirmed that the phage http://www.selleck.co.jp/products/Nutlin-3.html ZZ1 is most likely a new member of the T4-like virus family of Acinetobacter phages. However, according to the 2011 Virus Taxonomy List (current) from the International Committee for the Taxonomy of Viruses (http://​www.​ncbi.​nlm.​nih.​gov/​ICTVdb/​index.​htm.), only the Acinetobacter phage 133 can be searched and classified in the unassigned genus of the Myoviridae family, most likely because the phage is inadequately characterized. At the very least, the current sequence database for the Myoviridae phages should prove to be a rich source of genetic markers for bioprospecting and a mine of reagents for basic research and biotechnology. Our future research will focus on further detailed analysis of the whole ZZ1 genome to understand the genetic characteristics of this phage. The main aim of this study was the isolation and characterization of a lytic bacteriophage with potential for prophylactic/therapeutic use. Therefore, the antibacterial activity of the phage against its different host cells was the focus of our research.

The estimated prevalence of EAH in the 24-hour running race (R3) was 8.3% from 12 ultra-runners. One ultra-runner (EAH-B-R3) developed EAH, as his plasma [Na+] dropped from 137 mmol/l pre-race to 133 mmol/l post-race. No ultra-runner showed pre-race or post-race hypernatremia. The estimated prevalence of EAH in the multi-stage MTB race was 7.1% from 14 MTBers. One MTBer (EAH-C-R4) developed EAH, as his plasma [Na+] dropped from 142 mmol/l pre-race

to 134 mmol/l post-race. No MTBer developed pre-race or post-race hypernatremia. Table 3 Characteristics of the three cases (EAH-A-R2, EAH-B-R3, EAH-C-R4) with exercise-associated hyponatremia (n = 3)   EAH-A-R2 EAH-B-R3 EAH-C-R4 Type of race 24-h MTB race 24-h RUN race Multi-stage MTB race Age (years) 39 38 42 Body height (m) 196 168 177 BMI (kg/m 2 ) 23.4 18.8 23.6 Pre-race body mass (kg) 90.0 54.6 73.9 Post-race body mass (kg) 88.2 53.2 71.7 Δ body mass (kg) –1.8 –1.4 –2.2 Δ body mass (%) –2.0 –2.6 –3.0 MI-503 molecular weight Pre-race plasma sodium (mmol/l) 138.0 137.0 142.0 Post-race plasma sodium (mmol/l) 129.0 133.0 134.0 Δ haematocrit (%) –7.6 –9.4 3.8 Δ plasma potassium (mmol/l) Cyclosporin A 32.6 –29.2 3.6 Δ plasma osmolality (mosmol/kg H 2 O) –0.7 –1.1 1.7 Pre-race urine specific gravity (g/ml)

1.015 1.010 1.007 Post-race urine specific gravity (g/ml) 1.025 1.025 1.028 Δ urine osmolality (mosmol/kg H 2 O) 338:9 163.5 228.0 Δ urine potassium (mmol/l) 323.2 90.5 1282.0 Δ urine sodium (mmol/l) 108.3 25.9 –71.4 Δ K/Na in urine (%) 103.1 51.2 4737.0 Δ Transtubular potassium gradient (%) 1262.5 611.4 4340.0 Years as active cyclist or runner 5 15 5 Number of finished ultra-marathons 4 30 2 Total training hours weekly, h 12 13 10 Training cycle or run hours weekly, h 10 30 10 Training intensity, b/min 140 130 140 The intake of NSAIDs was reported by 3 (25%)

of 12 ultra-runners and by no cyclist (from 41) in any race. Regarding symptoms associated with race performance in R1, most of ultra-MTBers without EAH noted in the post-race questionnaires muscle weakness (41.7%), problems Farnesyltransferase with antidiuresis (33.3%), and breathing problems (33.3%). Muscle weakness (46.7%), problems with antidiuresis (40%), headache (26.7%), and breathing problems (26.7%) were the most reported post-race symptoms associated with race performance in R2 by finishers without EAH. Chills (50.8%), stomach pain (33.3%) and irritability (33.3%) were the most noted post-race symptoms associated with race performance in R3 by ultra-runners without EAH. MTBers without EAH reported muscle weakness (50%), swelling (42.9%) and myalgia (35.7%) in R4. Subjects who exhibited hyponatremia reported no intake of NSAIDs during the study period. The ultra-MTBer (EAH-A-R2) reported muscle weakness.

This is most likely because these Ironman triathletes did not overdrink and no fluid overload occurred. Noakes et al.[38] described that fluid overload as a consequence of excessive drinking, correlated with both a decrease in serum [Na+ and an increase in body mass. This has also been confirmed by Noakes et al.[39] and Speedy et al.[40]

where Ironman athletes with less weight loss showed a lower serum [Na+. This leads us to the conclusion that in the present Ironman triathletes no fluid overload occurred and therefore no disturbance of the body fluid homeostasis or of any other dimension could E1 Activating inhibitor be determined. Fluid overload, as a consequence of excessive drinking, is the main risk factor in the pathogenesis of exercise-associated hyponatremia (EAH) [38, 41, 42]. Regarding the ‘Position Statement’ of the ‘International Marathon Medical Directors Association’ [43] which recommends drinking ad libitium between 0.4 and 0.8 L/h during a race the present Ironman triathletes behaved correctly by drinking only in response to their thirst. Like in the reports of Hew-Butler et al.[44], Speedy et al.[45], Selleck TGF beta inhibitor and Noakes [46] describing no correlation between sodium intake, post-race serum [Na+ and the change in serum [Na+, we also

found no correlation between these parameters and therefore can confirm their findings. Kavouras [47] and Shireffs [48] described that in case of dehydration body mass decreases while urine specific buy Staurosporine gravity increases. In the present Ironman athletes, body mass significantly decreased by 3.2% and urine specific gravity significantly increased by 1.33% indicating dehydration following their definition [47, 48]. Decrease in the circumferences of the lower limb but not of the upper limb A further finding was that the circumferences of the thigh and the calf decreased by 2.7% and 2.4%, respectively, whereas the circumference of the upper arm remained unchanged. This indicates that the estimated skeletal muscle mass at the lower limbs became reduced. Since the change in the estimated skeletal muscle mass showed no association with the change in plasma urea, we presume that no substantial

degradation of myofibrillar proteins must have occurred, and the loss in estimated skeletal muscle mass might be due to a depletion of intramyocellular stored energy, such as muscle glycogen and intramyocellular lipids [49]. We furthermore found a relationship between the change in estimated skeletal muscle mass and the change in body mass. This finding confirms recent findings where Ironman triathletes lost skeletal muscle mass [36]. However, it was unexpected that the decrease in estimated skeletal muscle mass showed no association with the decrease in the lower leg volume. However, the reduction in limb circumference could also be due to a reduction in interstitial fluid. The decrease in the lower leg volume might also suggest an action of the ‘muscle pump’ during exercise helping to clear pre-race swelling.

aeruginosa, S. aureus, and E. coli cultures were reduced approximately 1 log in comparison with bacteria cultured in the absence of NPs. Figure 3 Inhibitory effect of NO/THCPSi NPs (0.1 mg/mL) on bacterial cultures. E. coli (blue bars), S. aureus (yellow bars), and P. aeruginosa (green bars) after 24 h of incubation in TSB medium

(37°C, initial bacteria density 104 CFU/mL; n = 3; mean ± standard deviation shown). Further experiments showed that growth inhibition by NO/THCPSi NPs against planktonic S. aureus was evident as early as 2 to 4 h after NP treatment (Figure 4). After 2 h, the bacterial counts were reduced by 0.52 log compared to the control (bacteria only), and after 4 h, a further reduction occurred (1.04 log). In contrast,

glucose/THCPSi NPs supported S. aureus proliferation at the same incubation times. Growth inhibition of S. aureus was sensitive to the dose of NO/THCPSi NPs applied (Figure 4). When higher concentrations of NO/THCPSi NPs were CYT387 mw applied, the S. aureus bacterial load decreased by 1.3 log. It should be noted that a by-product of increasing NP concentration is glucose supplementation, which may be reflected by the increase in bacterial density in cultures treated with glucose/THCPSi NPs. Cultures treated with NO/THCPSi NPs, however, showed no such upward trend in bacterial growth WZB117 rate, suggesting that the release of NO was able to counter any influence wrought by additional glucose provided by NO/THCPSi NPs. Therefore, these results indicate that the

NO released form the NO/THCPSi NPs is an effective Erastin in vitro antimicrobial agent against medically relevant Gram-positive and Gram-negative bacteria. Figure 4 Time-based inhibition of S. aureus by NO/THCPSi NPs. S. aureus was treated with glucose/THCPSi NPs (blue columns) and NO/THCPSi NPs (orange columns) at different NP concentrations after (a) 2 h and (b) 4 h (initial bacteria density 104 CFU/mL). Statistically significant inhibition as compared with control (*P < 0.05, **P < 0.01; n = 3; mean ± standard deviation shown). Figure 5 shows the SEM images and EDX spectra of E. coli treated with NO/THCPSi NPs compared with an untreated control. Single NPs and NP aggregates were evident in the SEM images on the bacteria and on the background surface. The presence of the NO/THCPSi NPs on the surface of the cell membrane of the E. coli was confirmed by the EDX results, which showed a peak characteristic for Si (Figure 5c). Figure 5 SEM images and EDX spectra of NO/THCPSi NP-treated E. coli . (a) SEM image of NO/THCPSi NP-treated E. coli, (b) SEM image of the E. coli only, (c) EDX spectrum of NO/THCPSi NP-treated E. coli, and (d) EDX spectrum of untreated E. coli as a control. EDX analysis performed on bacterial surface (yellow overlay). NPs on the bacterial surface and settled on the background are indicated by red arrows. Anti-biofilm efficacy of NO/THCPSi NPs S. epidermidis biofilms were exposed to the NO/THCPSi NPs at a concentration of 0.

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pastoris,

but their expression levels remained low (below 280 mg/l). It is known that codon optimization is a useful strategy to increase the yield of target protein during heterogeneous expression. Many antimicrobial peptides, such as plectasin [30], NZ2114 [31] and AgPlectasin [32], were expressed Selleck CHIR99021 with high production through codon-usage optimization in our laboratory. In addition, Divercin V41, a class IIa bacteriocins was also expressed through this system [33]. These cases encouraged us to use codon optimization to break through the bottleneck of low yield in heterologous expression of EntA. The total protein level in the supernatant reached 180 mg/l with the activity of 51,200 AU/ml at 24 h of induction

in 5-L fermenter level (Figure 2C) after the gene was optimized. Although the yield of target peptide was still low, and even lower than 280 mg/l as the highest result of expression in case of enterocin L50 in P. pastoris [28], it was much higher than that of Pediocin PA-1 (0.4 mg/l), Enterocin P (0.006 mg/l), Divercin V41 (23 mg/l) and EntA (0.027 mg/l) expressed in E. coli and L. lactis [14,22,33]. Furthermore, the production of rEntA increased 2.99-times compared with its native sequence expressed in P. pastoris (45.1 mg/l), which indicated codon optimization is a good tool to enhance expression efficiency and level Selleckchem STI571 in P. pastoris, and at the same time, it also left a large room to improve in future work at the similar aim and technical scheme. However, the maximal activity of rEntA in the supernatant was reached at an early stage (24 h) of induction

(Figure 2C). This is similar to previous results in which the highest level of rEntA was reached at 36 h. An even earlier peak of rEntA at 6 h was observed in other yeasts such as Kluyveromyces lactis and Hansenula polymorpha [18]. Obviously, its final successful application suffered from this strong decomposition in the supernatant at an earlier period of expression related to the possible disruption of rEntA to host cells and the proteolysis of the target protein. The latter situation was reported in “collagen-like” bacteriocin with a high cleavage by collagenase [29]. However, the triclocarban exact mechanism of the above described early degradation and its solution should be further studied. A series of methods, such as ion exchange chromatography (SP and CM FF), hydrophobic exchange chromatography (Phenyl HP), and gel filtration (Superose 12), were attempted to purify rEntA in this study. Only gel filtration could purify rEntA with a yield of 3.02 mg/l (Figure 2F) after attempts with SP FF, CM FF, and phenyl HP in which almost all rEntA was lost in the penetration peak (data not shown) due to unknown reasons.