ShRNA inhibit gene expression of HBV strains with different genotypes in vitro The levels of cytoplasmic HBV pg/pc RNA (3.5 kb) and HBV DNA in cultured supernatants were

determined by realtime RT-PCR/PCR and presented in Figure 3. The pg/pc RNA level of five HBV strains with different genotypes were reduced by 58%~93% in B245(69%~93%), B376(59%~91%), B1581(67%~90%) and B1789(58%~88%) treatments, while the HBV DNA level observed in supernatants was decreased by 77%~99% in these shRNA plasmid treatments (B245: 83%~99%, B376: 79%~99%, B1581:88%~98%, B1789: 77%~99%). Figure 3 SiRNAs inhibit RNA and DNA expression of HBV strains with different genotypes in Huh7 cells. The histogram show the cytoplasmic HBV pg/pc RNA levels (A, B, C, D, E) and learn more extracellular buy RG7420 HBV DNA (F, G, H, I, J) of five HBV strains with genotypes Ae(N10), Ba(C4371), C1(Y1021), D1(Y10) and I1(W29) in treated shRNA plasmids, treated pSUPER vector, and non-treated Huh7 cells. In addition, the extracellular and intracellular antigen levels in Huh7 cells that were co-transfected with HBV and shRNA plasmids were also determined (Figure 4). In the shRNA-treated Huh7 cells, the average extracellular HBsAg expression level of all five HBV strains decreased by 1.66 ± 0.36 logs. The average intracellular HBsAg expression level decreased by 1.47 ± 0.33 logs, while the extracellular HBeAg levels decreased by 1.04 ± 0.23 logs, and the intracellular HBcAg levels by 1.71 ±

0.49 logs. The effect of the siRNA treatment on HBeAg levels was weaker than that on the HBsAg or HBcAg levels (P < 0.001, Figure 5). EVP4593 Figure 4 SiRNAs inhibit viral antigens expression of HBV strains with different genotypes in Huh7 cells. (A, B, C, D) Extracellular HBsAg, intracellular HBsAg, extracellular HBeAg, and intracellular HBcAg expression levels of HBV N10(Ae), respectively. (E, F, G, H) Extracellular HBsAg, intracellular

HBsAg, extracellular HBeAg, and intracellular HBcAg expression almost levels of HBV C4371(Ba), respectively. (I, J, K, L) Extracellular HBsAg, intracellular HBsAg, extracellular HBeAg, and intracellular HBcAg expression levels of HBV Y1021(C1), respectively. (M, N, O, P) Extracellular HBsAg, intracellular HBsAg, extracellular HBeAg, and intracellular HBcAg expression levels of HBV Y10(D1), respectively. (Q, R, S, T) Extracellular HBsAg, intracellular HBsAg, extracellular HBeAg and intracellular HBcAg expression levels of HBV W29(I1), respectively. Figure 5 Comparing the RNAi-induced silencing effect on different viral markers. Data were displayed the average antigen level of the 4 siRNAs reduced for five HBV strains. “”Ex”" = Extracellular and “”In”" = Intracellular. The Mann-Whitney test was used to assess the difference. An asterisk represents a statistical difference of P < 0.01 in comparison with the other markers (Ex HBeAg vs. Others P < 0.001, Ex HBsAg vs. In HBsAg P = 0.05, Ex HBsAg vs. In HBcAg P = 0.82, In HBsAg vs. In HBcAg P = 0.10.

Here check details we describe the in depth characterization of a broad host range PB1-like phage with a slight prevalence to clinical isolates. We used an artificial sputum medium to simulate the conditions in the CF lung and investigated the ability of phage JG024 to infect P. aeruginosa and multiply under these conditions. Results and Discussion Isolation and host range of phage JG024 Phages were isolated from sewage as described in Methods. We isolated 59 P. aeruginosa specific phages and used an initial set of 5 different P. aeruginosa strains as the laboratory strains PAO1, PA14 as well as three clinical isolates (BT2, PACF15 and MH19, Table 1) to test the host range. One phage, which was named JG024, was able to conduct

clear lysis on this set of bacterial strains. To determine the host range of JG024 in more detail, we used 19 clinical isolates from CF patients and from urinary tract infections as well as a collection of 100 environmental strains (Table 1). JG024 is able to infect 84% of all tested clinical isolates. HMPL-504 Furthermore, JG024 is even capable of infecting a P. aeruginosa mucA mutant

and the clinical isolate BT73, which both showed the same mucoid phenotype. mucA mutants produce large amounts of the exopolysaccharide alginate and mutations in mucA are critical for the conversion of non-mucoid to mucoid P. aeruginosa variants in the lung of CF patients [20, 21]. Additionally, we determined the host range of the phage JG024 with a collection of 100 P. aeruginosa environmental strains isolated from different rivers (Oker, Aller, Weser) in Lower Saxony, Germany. The results showed that JG024 was able to infect PI3K inhibitor 50% of the strains. Interestingly, phage JG024 showed a clear lysis for only 45% of the 50 lysed environmental isolates but was able to conduct clear lysis on 68% of the 19 lysed clinical isolates. Table 1 Strains and phages used in this study. Bacterial strain or phage Phenotype or genotype Reference PAO1 wild type [48] PA14 wild type

[49] FRD1 mucoid CF isolate [34] PAO1 ΔmucA PAO1 mucA::aacC1-gfp GmR Sabrina Thoma, this laboratory, unpublished PAO1 ΔpilA pilA inactivated by allelic displacement; tagged with eGFP, TcR, GmR [50] PAO1 ΔfliM fliM inactivated by allelic displacement; tagged with eGFP, TcR, GmR [50] PAO1 ΔalgC PAO1 Progesterone algC ::aacC1-gfp GmR Julia Garbe, this laboratory, unpublished BT2, BT72, BT73, RN3, RN43, RN45, NN84 clinical CF isolates Medical Highschool Hannover, Germany PACF15, PACF21, PAKL1, PAKL4, PACF60, PACF61, PACF62, PACF63 clinical CF isolate Gerd Döring, Tübingen, Germany Nr. 18, 19, 26, 29 urinary tract infection isolate Michael Hogardt, München, Germany Environmental strains   Katherina Selezska, HZI Braunschweig, Germany JG024 wild type PAO1 LPS specific lytic bacteriophage this study Family affiliation of JG024 To determine family affiliation of phage JG024, we determined the nature of the nucleic acids and the morphology of the phage to assign the family by comparison [22].

Species identification and subtyping of Brucella isolates is very important for epidemiologic surveillance and investigation of outbreaks in Brucella-endemic regions [3, 4]. Recent studies have confirmed that multiple-locus variable-number tandem-repeat analysis (MLVA) is a useful tool for identifying and genotyping

Brucella strains and the resultant data can be used for epidemiological trace-back investigations [3, 5–8]. In efforts to better improve surveillance and evaluate the power of epidemiological trace-back in China, the MLVA-16 scheme was used to type a collection of 105 B. melitensis isolates from 18 different regions throughout China. (This study was presented in part at the 5th Brucellosis International Research Conference of the American Society for Microbiology, Buenos Aires, Wnt assay Argentina, 2011.) Results Typing and clustering of B. melitensis isolates by MLVA-16 Using the complete MLVA-16 assay (including panel 1, 2A and 2B loci), the 105 B. melitensis isolates were clustered in 69 different genotypes with 17 clusters and 52 singleton genotypes (Figure 1). The corresponding diversity index for panels 1, 2A, and 2B were 0.37, 0.11, and 0.98 respectively. The overall discriminatory

index of MLVA-16 in this population was 0.99. Using panel 1, the present population clustered into five known genotypes Phosphoglycerate kinase and a new genotype. learn more The five known genotypes were included in the previously named the ‘East Mediterranean’ group with genotypes 42 (83 strains), 43(5 strains), 45(3 strains), 58(4 strains) and 63(8 strains). All were included in the previously recognized ‘East Mediterranean’

group. Two strains from Guangdong, isolated in 2008, had the genotype (1-5-3-13-2-1-3-2), labeled as CN-1. The two strains were a single-locus variant (SLV) to genotype 42(1-5-3-13-2-2-3-2). To date the genotype associated with CN-1 has not been reported from any other check details country. Figure 1 Dendrogram based on the MLVA-16 genotyping assay showing relationships of the 105 B. melitensis isolates. MLVA type: panel 1 and panel 2 genotypes in this article; key: serial number for the isolate in the Brucella2010 MLVA database http://​mlva-u-psud.​fr/​; strain: strain name in the laboratory in which the DNA extraction was done; province/year: province and year of isolation; panel1, panel 2A, panel 2B: genotypes corresponding to each isolates in the database for each set of loci; The actual biotyping result is indicated in the species-biovar column. Greater diversity among the Chinese B. melitensis isolates was apparent when the eight additional markers encompassing panel 2A and 2B were included. The number of strains populating a cluster ranged from two (eight clusters) to six.

Dasatinib may cause pleural, pericardial and peritoneal effusions; additionally interaction with platelet function is discussed to explain higher rates of gastrointestinal bleeding observed Ilomastat research buy in clinical practice. Bosutinib is associated with significant gastrointestinal toxicity (diarrhea) and hepatotoxicity. Serious AE observed with Ponatinib are an alarming high rate of arterial thrombosis, and cardiovascular events as well as hepatotoxicity. Differences in the safety profiles of these TKI seem to be at least partially explained

by the additional inhibition of other signaling pathways apart BCR-ABL [c-Kit, Src family kinases, PDGFR, and others]. However, it should be kept in mind that TKI treatment of CML has to be administered

lifelong and knowledge about potential long-term risks and efficacy, especially for the second generation TKI Dasatinib, Nilotinib and Bosutinib, is still limited. Whether risks associated with Ponatinib treatment can be tolerated is currently under discussion again. Not only from a regulatory perspective careful attention on recommended Selleck Talazoparib risk minimization measures as defined in the product information is at the end essential to avoid treatment complications that may completely jeopardize the sought treatment success. Orphan drug status of TKI The orphan regulation aims at fostering drug development for serious or life-threatening diseases with a prevalence of less than 5 in 10.000 people in the EU. A sponsor may apply for orphan designation any time prior to an application for marketing authorization (VS-4718 usually even before clinical development). The orphan drug status then needs to be confirmed during the marketing authorization procedure. The most important incentive

of the regulation is ten year market exclusivity Chlormezanone for an orphan medicinal product with respect to similar medicinal products. Neither EMA nor EU member states can authorize a product, which is regarded similar with respect to chemical structure and mode of action and therapeutic indication. Generics, by definition, fulfill all of these criteria. Imatinib is the paradigm of targeted therapy with its target, the Philadelphia chromosome, occurring in two rare forms of cancer, CML and acute lymphatic leukemia (ALL) which remain rare in spite of recent advances for treatment. Other cancers, e.g. renal cell carcinoma, was recently reported to exceed the prevalence threshold of 5 in 10.000 people so that no further orphan designations are expected. Orphan similarity and market exclusivity In addition to the incentive of the a.m. ten year market exclusivity intended by the European orphan regulation [19] there may be a probably unintended additional incentive.

Body weight, complete blood count, and serum biochemistry were monitored before and after dosing (Day 0 and Day 7). Postmortem observation of the gastrointestinal tract, liver, kidney, spleen, lung and heart were performed and organ weights were measured. No body weight or organ weight loss was noted (Figure 4A and B). No adverse effects on liver and kidney indices were noted (Figure 4C-D). In addition, no changes in red and white blood cells plasma indices

were noted at the efficacy doses tested (Additional file 1: Table S1 and Table S2). TAI-1 shows no adverse effect under efficacious oral dose levels. Figure 4 7-day toxicology study of TAI-1 in mice shows no significant change in body weight, organ weight, and plasma indices. C.B-17 SCID mice (n = 8) were orally administered TAI-1 for 7 days and body weights (A) and organ Wnt inhibitor weights (B) were measured. Liver (C) and kidney (D) plasma indices were determined. Safety studies of TAI-1 The clinical application of anticancer drugs is often limited by their non-specific target activity leading to organ toxicity

and other side effects. To evaluate the preliminary safety profile of TAI-1, we investigated the inhibitory potential of TAI-1 against normal cell lines, against a panel of kinases, and also on its binding to hERG, a known target for cardiac toxicity. To determine the cancer cell specificity of TAI-1, normal cell lines were tested. In normal fibroblast (WI-38), renal tubule cells (RPTEC), umbilical vein cells (HuVEC) and aortic smooth muscle (HAoSMC) cell lines, TAI-1 mTOR inhibitor review had a GI50 of more than 1000 times that of cancer cell GI50 (Table 2), showing a high therapeutic index. When screened against

a panel of known kinases, TAI-1 has no inhibitory effects against these targets MycoClean Mycoplasma Removal Kit (Figure 5A), confirming the specificity of TAI-1 to Hec1 and against these kinases targets. Figure 5 TAI-1 does not https://www.selleckchem.com/screening/ion-channel-ligand-library.html inhibit a number of kinases and hERG at below 10 μM. (A) Inhibition of kinases were performed with 10 μM TAI-1 with standard assays. (B) hERG inhibition was determined with 10 μM TAI-1. Results show good cardiac safety of TAI-1. We have tested TAI-1 with the hERG assay, which assesses the most common mechanism involved in drug-induced prolongation of QT interval, which increases the risk of ventricular tachyarrhythmia through the inhibition of potassium ion flow and may lead to sudden cardiac death [13, 14]. The hERG channel assay revealed a competition IC50 1000 times that of cancer cell GI50 (Figure 5B), suggesting that this compound has little potential of cardiac toxicity through the hERG channel at the therapeutic doses. In summary, TAI-1 exhibits high specificity to cancer cells and to target and shows no cardiac toxicity by hERG.

Int J Sport Nutr 1996,6(1):14–23.PubMed 67. Graham TE, Spriet LL: Metabolic, catecholamine, and exercise performance responses to various doses of caffeine. J Appl Physiol 1995,78(3):867–874.PubMed 68. Butts KN, Crowell D: Effect of caffeine ingestion on cardiorespiratory endurance in men and women. Res Q Exerc Sport 1985, 56:301–305. 69. Matsuo T, Yoshioka M, Suzuki M: Capsaicin in diet does not affect glycogen contents in the liver and skeletal muscle of rats before and after exercise.

J Nutr Sci Vitaminol (Tokyo) 1996,42(3):249–256. 70. Lim K, Kim KM, Yoshioka M: Effects of capsaicin on carbohydrate and fat metabolism in exercise rats. Korean Journal of Physical Education 1995, 34:248–256. 71. Hyllegard R, Mood DP, Morrow JR: Interpreting Research in Sport and Exercise Science. St. Louis, MO: Mosby-Year Book, Inc 1996. Vorinostat Competing interests The authors declare that they have no competing Selleckchem Androgen Receptor Antagonist interests.

Authors’ contributions AAW was the primary author of the manuscript and played an important role in data collection and assessment. AG-881 supplier TJH, EDR, PBC, and KMH played an important role in data collection and manuscript preparation. JRS and TWB played an important role in study design and manuscript preparation. JTC was the senior author and played an important role in the grant procurement, study design, data analysis and interpretation, and manuscript preparation. All authors have read and approved the final manuscript.”
“Background Delayed BCKDHA onset muscle soreness (DOMS) is muscle pain and discomfort experienced approximately one to three days after exercise [1]. DOMS is thought to be a result of microscopic muscle fiber tears and is more common after eccentric exercise (the muscle must lengthen or remain the same length against a weight) rather than concentric exercise (the muscle can shorten against a weight load). While DOMS is not a disease

or disorder, it can be painful and is a concern for athletes because it can limit further exercise in the days following an initial training [2]. In most cases, DOMS will resolve spontaneously within 3 to 7 days. There is some evidence that ibuprofen, naproxen, and massage may accelerate the resolution of DOMS [2]. Treatment strategies have often integrated multiple therapeutic approaches such as cryotherapy, ultrasound, compression therapy, stretching and deep tissue massage [3–7]. In addition, several dietary supplements have been tested in the treatment of DOMS including protein, vitamin C, proteases (enzymes), phosphatidylserine, chondroitin sulfate, and fish oil, all with variable success [2, 8–14]. There is no clear consensus in the extant literature on a method or discipline that can effectively relieve pain following eccentric exercise. The test product in this study was BounceBack™ capsules; a proprietary dietary supplement combination containing proteolytic enzymes, curcumin, phytosterols from unsaponifiable avocado and soybean oils, vitamin C, and resveratrol.

In this study, we demonstrated the utility of Luc-DENV for measuring neutralization and enhancing antibodies. Using three identified neutralizing mAbs, Luc-based assay showed well correlation with the PRNT-based assay. 4G2 and 2B8 are both IgG1 isotype mAbs, and 2A10G6 belongs to IgG2a isotype. 2B8 recognizes the domain III of DENV E protein and inhibit viral binding, while 2A10G6 and 4G2 inhibit fusion. All three mAbs were active in inhibiting plaque forming and see more Luc expression in Luc-DNEV infected Vero cells. The value of PRNT50 and LRNT50 are well correlated (R2 > 0.95). The

Luc-based assay was readily applied in evaluation of clinical samples from vaccinated animals and infected patients. ADE infection of DENV has been well demonstrated in vitro and in vivo, and represents one of the major impediments against vaccine development. Previously, different methods based on infection rate [27, 28], progeny viral yield [29], and number of infectious centers [30, 31] have been reported to measure the ADE activity in FcR expressing cells including K562, U937 or THP-1 cells. The FACS analysis has been commonly

used to Selleckchem AUY-922 quantify the infection rate in C6/36 cells, Raji B, and human peripheral blood mononuclear cells [32, 33]. Progeny viral yield can be detected either by conventional plaque assay or NS1-based ELISA [34], ELISPOT [19], and real-time RT-PCR [32]. Recently, Tideglusib molecular weight Moi et al.[35] successfully established stable BHK-21 cell lines that express FcRIIA, which facilitate both neutralization and ADE assay. The plaque based assay determined the infectious particles released from virus-infected cells, whereas the RLU based assay described in this study offered

a simple method which detected viral protein expression in cells. Linear correlation was established between the two assays for both neutralization and ADE assays (Figure 1D and Figure 2B). The newly developed PIK3C2G assay method is comparable to the traditional plaque assay, with some unique advantages. First, this Luc-based assay is more substantial and time saving. The conventional plaque test used 12-well plates and 5–7 days observation for the plaque forming, the new test is compared performing the same protocol involved 24-well plates and cost no more than 2 days. Second, this new assay method has a more wide-range scope of application with high repetitiveness and reliability. Luc-DENV replicates well in multiple cells including BHK-21, K562, Vero and THP-1 and A549 cells, and luciferase activity can also be detected stably in various cells. Neutralization and ADE assays can be performed in the same cells [34]. Third, this new assay method is easy to adapt for a high-throughput manner [9], which is of critical importance for large-scale clinical samples assays during clinical trials of dengue vaccine.

The importance of ClpV

for secretion of hemolysin co-regulated protein (Hcp) has been demonstrated in both V. cholerae V52 and P. aeruginosa[9, 11]. In most T6SSs, Hcp and valine-glycine repeat protein G see more (VgrG) are exported by the secretion machinery under normal laboratory cultural conditions. This is not the case for V. cholerae O1 strain N16961, and therefore it was suggested that the T6SS of V. cholerae O1 strains was functionally inactive [12]. Our recent studies showed, however, that the T6SS of V. cholerae O1 strains can be activated when the bacteria are grown under high osmolarity conditions, resulting in the secretion of Hcp into the culture medium [13]. In the same study, Hcp secretion was shown to require the presence of VipA [13]. Here, residues within the previously identified VipB-binding domain of VipA (aa 104–113) [6] were exchanged to alanine as a means to identify key residues important for the interaction. To determine the biological consequences of a diminished VipA-VipB interaction in V. cholerae O1 strain A1552, the mutants were assessed for their ability to bind to and stabilize VipB, promote secretion of Hcp, and compete against E. coli in a competition assay. Results Substitutions within the large α-helix of

VipA negatively impacts on VipA/VipB complex formation To analyze the V. cholerae VipA-VipB interaction in detail, we undertook a mutagenesis-based approach. Our previous results using a yeast 2-hybrid assay (Y2H) showed that a deletion within the first part Fedratinib of the conserved

α-helical domain of VipA (mutant Δ104-113) abolished its binding to VipB [6], while a deletion within the second part (mutant Δ114-123) did not (Bröms, unpublished) (Figure 1). To validate these results by an independent approach, we here used an E. coli bacterial 2-hybrid assay (B2H) for which the amount of β-galactosidase production is directly proportional to the strength of a protein-protein interaction [14]. Similar to the positive control MglA-SspA [15], VipA and VipB were found to interact efficiently in this system (Figure 2A). Deletions within the AZD8186 price conserved α-helical domain of VipA (mutants Δ104-113 and Δ114-123) abolished its interaction U0126 chemical structure to VipB in B2H (Figures 1 and 2A), suggesting that residues within region 104–123 contribute to VipB binding. To identify the key residues important for this interaction, we generated alanine substitutions, focusing on the first part of the putative α-helix (residues 104–113), since this region was shown to be crucial for VipB binding regardless of the protein-protein interaction assay used (Figure 1). Importantly, according to Psipred V2.5 (http://​bioinf.​cs.​ucl.​ac.​uk/​psipred/​), none of the substitutions were predicted to affect the stability of the α-helix.

2% gluconate and grown at 30°C. FM-images of samples taken at time points as indicated were generated Metabolism inhibitor after

staining with Nile red in red channel (top rows) or without staining in green channel (bottom rows) or. Note, individual PHB granules of PhaP5 or eYfp-PhaP5-expressing cells near cell poles or at mid cell were not resolved in FM images as in TEM images (Figure 6). Bar 3 μm. As in the case of PhaM, no difference in number, size and localization of PHB granules was observed for the over-expressed eYfp-PhaP5 fusion in learn more comparison to over-expression of PhaP5 alone. Growth and accumulation of PHB were similar in the recombinant strains as in the wild type. However, when the time-course of PHB granule formation and localization was investigated by TEM-analysis Selleckchem Alisertib remarkable differences to the wild type were observed for the PhaP5 over-expressing strains (Figure 6): PHB granules were formed in aggregated clusters of in average 2–6 granules in most cells near both cell poles of the rod-shaped cells. These clusters could not be resolved

by FM-analysis (Nile red staining) and resulted in the impression of only two (large) PHB granules near the cell poles (Figure 7). The number of individual granules visible in TEM images was increased but the diameter was decreased compared to wild type granules. In most cells, the PHB granule clusters or at least individual PHB granules of a cluster were clearly detached from the nucleoid region (see arrowheads in Figure 6). In conclusion, over-expression of PhaP5 has an impact on number, size and localization

of PHB granules and leads to detachment of the granules from the nucleoid. This can be explained by binding of over-expressed PhaP5 to PhaM molecules thus preventing PhaM from binding to DNA and/or to Orotic acid PhaC. Alternatively, a competitive displacement of PhaM molecules from PHB granules surface by over-expressed PhaP5 could be responsible for the phenotype. Number and localization of PHB granules in a ∆phaP5 strain were, however, not significantly changed in comparison to wild type (data not shown). Conclusions Our data clearly show that formation and localization of PHB granules occurs not randomly but is specifically controlled in R. eutropha. Other examples of species with non-random localization of PHB granules are Rhodospirillum rubrum[33], Haloquadrata walsbyi, Azotobacter vinelandii, Beijerinckia indica[34], Caryophanon latum[35] and Hyphomicrobium facile (supplementary material of [32]). However, we do not know whether attachment of PHB or PHA granules to the DNA is a general feature of PHB or PHA accumulating bacteria. PHB granules in R. eutropha are attached to the nucleoid via PhaM. Our conclusion is supported by previous TEM analysis of others if the “dark-stained mediation elements” are interpreted as denatured chromosomal DNA [36, 37].

These

medication records JNK-IN-8 in vitro were reviewed for the dispensing of bisphosphonates, calcium supplements and vitamin D during the follow-up period. After the study period, pharmacists received comparable information on patients who were originally assigned to the control group. This study was not covered by the Medical Research Involving Human Subjects Act (WMO) since the patients were not directly exposed to the intervention, and approval by an ethical committee was not required. Outcome measurements All patients were followed up from baseline until the start of eFT508 concentration osteoporosis prophylaxis or the end of the study period (the date of second data extraction), whichever came first. The primary endpoint was a dispensing of a bisphosphonate. Secondary endpoints were the dispensing of other prophylactic osteoporosis drugs (calcium supplements or vitamin D)

and a dispensing of any prophylactic osteoporosis drug as a composite endpoint (bisphosphonate, calcium supplements or vitamin D, only the first event was counted). Statistical analyses We assumed an event rate of 10 % in the control group over 6 months and an increase to 20 % in the intervention group [18, 21]. With a two-sided alpha of 0.05 and 90 % power, a total sample size of 584 patients was estimated which was increased to 695 patients. Chi-square tests or Fisher’s exact tests were used to determine baseline differences between the comparison groups for categorical variables and independent sample t selleck products tests for continuous variables (p < 0.05). Cox proportional hazard models were used to estimate hazard ratios (HRs) for the start of osteoporosis prophylaxis during the follow-up period by comparing the intervention group to the control group. Hazard ratios were adjusted for covariates that were unevenly distributed between the intervention group and control group (p < 0.05). Cytidine deaminase Patients who did not receive any prescription of glucocorticoids during the follow-up period were

censored at 1 day after baseline. In subgroup analyses, results were stratified by gender, the number of prednisone equivalents (DDDs) received in the 6 months before baseline (67.5–134, 135–270, >270) and age categories (≤70, >70 years) for the primary and composite endpoint. Finally, a Kaplan–Meier plot was used to visualize the time to start of bisphosphonate use after baseline and the proportion of patients being newly treated for GIOP during the study period. This plot was stratified by the randomised intervention. All analyses were performed using SAS, version 9.1. Results During the first data extraction period, 735 patients were selected from the participating pharmacies. Of these patients, 31 (4.2 %) were not eligible for bisphosphonate prophylaxis according to the Dutch guideline.