Follow-up time was defined as time between first fracture and subsequent click here fracture, death or end of the study period of 5 years. With respect to mortality, the follow-up time was defined as time between first fracture and death or end of the study period. Hazard ratios (HR) and 95% confidence intervals (95%CI) were reported. Two-tailed p < 0.05 was considered significant.

The Schoenfeld residuals were used to check the assumptions of proportionality. If violated, then we used the time-dependent Cox regression analysis to represent the profile of the HR over time. Linearity was checked for age. SPSS 15.0 for windows (SPSS Inc., Illinois, USA) was used to process the data. Results A total of 1,921 patients aged over 50 years were included, 1,433 women and 488 men. Women were significantly older than men (women 73.5 ± 11.5 years and men 67.1 ± 12.2 years, p < 0.001). The majority of the baseline fractures occurred at the ulna/radius (number of patients = 502, 26.1%), hip (number of patients = 469, 24.4%) and other (number of patients = 561, 29.2%; Table 1). The patients can be categorised buy GSK2126458 into the following four groups: patients who died without (n = 509) or after a subsequent NVF (n = 111) and patients still alive after 5 years of follow-up with (n = 227) or without a subsequent NVF (n = 1,074; Fig. 1) during a total of 7,685 patient-years. Clearly, the most common outcome 5 years

after a NVF is to be alive without a subsequent fracture (in 55.9% of patients;

Fig. 1). Fig. 1 Flowchart of patients included in the study Subsequent fractures During the 5-year follow-up period, 338 patients had 379 subsequent NVFs, indicating an AR of 17.6% (95%CI, 15.9–19.3; Fig. 1). Table 2 Vistusertib nmr mortality incidence: multivariable Cox regression analysis with time-dependent covariates Variable Hazard ratio 95%CI p value Sex men vs. women 1.74 1.44–2.10 <0.001 Age (per decade) 2.59 2.37–2.84 <0.001 Baseline fracture location (major vs. minor)         0 months 5.56 3.48–8.88 <0.001   12 months 2.44 1.90–3.14 <0.001   24 months 1.49 1.13–1.96 0.004   36 months 1.27 0.97–1.66 0.083   48 months 1.50 1.14–1.97 0.004   60 months 2.47 1.41–4.33 0.002 Patients with a subsequent fracture vs. patients without a subsequent fracture 1.65 1.33–2.05 <0.001 In univariable analysis, women sustained Leukocyte receptor tyrosine kinase significantly more subsequent fractures than men (19.3% vs. 12.7%, p = 0.001; HR, 1.54; 95%CI, 1.17–2.03). Also, increasing age (HR, per decade, 1.49; 95%CI, 1.36–1.64) and major baseline fracture location (HR 1.60; 95%CI, 1.29–1.98) contributed in univariable analysis to subsequent fracture risk (Fig. 2). Fig. 2 Kaplan–Meier curves stratified by sex (univariable analysis). A1–B1 Subsequent fracture incidence by baseline fracture location. C1–D1 Subsequent fracture incidence by age in groups. A2–B2 Mortality incidence according to baseline fracture location.

13.11.3) (alpha and beta), gentisate 1,2-dioxygenase (EC 1.13.11.4), homogentisate 1,2-dioxygenase (EC 1.13.11.5), protocatechuate 4,5-dioxygenase (EC 1.13.11.8) (alpha and beta), methyl-coenzyme

M reductase (EC 2.8.4.1) (alpha), methane monooxygenase (EC 1.14.13.25) (particulate: pMMO and soluble: sMMO). The metagenome reads were further compared to a protein sequence library for alkane monooxygenase (alkB) on the freely available Bioportal computer service [66]. The reference library for alkB was downloaded from Fungene (Functional gene pipeline & repository) version v6.1 [74], including only sequences with a score (bits saved) of 100 or more from the HMMER Hidden Markov Model search against NCBIs non-redundant protein database. We used blastX against the protein sequences of the enzyme library with a maximum expectation value of #selleck chemicals llc randurls[1|1|,|CHEM1|]# 10-20[67]. Maximum one alignment was reported. PCA analysis The PCA-plots were created using the vegan library in R [75–77]. The ordination was based on reads assigned at the phylum level in selleck chemicals MEGAN version 4 (“Not assigned” and “No hits” were excluded)

and to level I SEED subsystems extracted from MG-RAST (“No hits” was excluded) [68, 69]. All metagenome data were given as percent of total reads. Symmetric scaling, for both parameters and sites, was used in the plot. The geochemical parameters [25] were fitted onto the ordination using the envfit function. The lengths of arrows for the fitted parameters were automatically adjusted to the physical size of the plot, and can therefore not be compared across plots. To account for the different measuring units, all geochemical parameters were normalized by dividing with the standard deviation and subtracting the smallest number from all numbers in each row. Rarefaction analysis Rarefaction analysis was performed in MEGAN version Fossariinae 4 [68, 69]. The MEGAN program uses an LCA-algorithm

to bin reads to taxa based on their blast-hits. This results in a rooted tree. The leaves in this tree are then used as OTUs in the rarefaction analysis. The program randomly chooses 10%, 20% … 100% of the total number of reads as subsets. For each of these random subsets the number of leaves (hit with at least 5 reads (min-support)) was determined. This sub sampling is repeated 20 times for each percentage and then the average value is used for each percentage. The analysis was done for all taxa (including Bacteria, Archaea, Eukaryota, viruses and unclassified sequences) at the genus level, and at the most detailed level (typically species or strain) of the NCBI taxonomy in MEGAN. Comparison of the metagenomes Comparison tables of absolute numbers for different bacterial and archaeal taxonomic (NCBI) levels for the seven metagenomes were extracted from MEGAN [68, 69]. Likewise, comparison tables of absolute numbers of reads assigned to SEED subsystems in the seven metagenomes were extracted from MG-RAST [72, 73].

Exponentially growing cells were seeded into 96-well plates and preincubated for

24 h. Then the medium was replaced with the fresh RPMI 1640 medium containing 0.01 to 50 μg/mL of gemcitabine or GEM-ANPs or ANPs. Samples were sterilized by 60 Co radiations before exposure to cells. Cell activity after 72 h of further culture was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay (MTT) with optical density at 490 nm (OD490 nm) using a micro plate reader (EL×800, BioTek, Winooski, VT, USA) (n = 5). A blank control group without medication was used as control. The inhibition rate was calculated as follows: where ODc and ODt are the OD490 nm values of the control group and the treatment group, respectively. The half maximal inhibitory concentration (IC50) was calculated with the Bliss method [16, 17]. Cell cycle analysis by flow cytometry After exposure to different samples for 72 h, Bindarit ic50 PANC-1 cells were released by treatment with trypsin, washed with phosphate buffered solution (0.01 M, pH 7.4), and fixed in ice-cold 95% ethanol. After centrifugation at 252×g for 5 min, the cells were pretreated

with 1 mL Triton X-100 and centrifuged at 252×g for 5 min. A further treatment Dactolisib cost with 1 mL RNase was performed at 37°C for 10 min. Then the DNA of cells was stained with 1 mL propidium iodide. Cell cycle variation after different treatment was analyzed with a FACS flow cytometer (FACS Calibur, Becton-Dickinson, Franklin Lakes, NJ, USA) using the Cell Quest software. All experiments were performed in triplicate. Drug distribution and toxic side effect assessment in vivo Animals Male Sprague–Dawley (SD) rats, 4 to 5 weeks old, (Shanghai SLAC Laboratory Animal Co., Ltd., Shanghai, China) were housed in sterilized cages and fed with autoclaved food and water ad libitum. Athymic nude male mice, 6 to 8 weeks old, were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. and housed in a specific pathogen-free animal facility. All animal procedures were approved by the institutional animal care committee, the Science and Selleck Y-27632 Technology Commission of Shanghai Municipality.

All guidelines met the ethical standards required by law and also complied with the guidelines for the use of experimental animals in China. Drug distribution Ceramide glucosyltransferase A total of 30 clean laboratory SD rats, with an average weight of 200 g, were randomly divided into three groups as follows: Group A: 110-nm GEM-ANPs Group B: 406-nm GEM-ANPs Group C: pure gemcitabine Samples were sterilized by 60 Co radiations and dispersed into 1 mL saline before injection. After being anesthetized with 10% chloral hydrate by intraperitoneal injection (3.0 mL/kg), SD rats were injected with the solution through the femoral vein. The amount of the injection in the 110-nm GEM-ANP group, 406-nm GEM-ANP group, and gemcitabine group was converted from gemcitabine (90 mg/kg, n = 10).

Biodivers Conserv Poschlod P, WallisDeVries MF (2002) The historical and socioeconomic perspective of check details calcareous grasslands: lessons from the distant and recent past. Biol Conserv 104:361–376CrossRef Poschlod P, Baumann A, Karlík P (2009) Origin and development of CP673451 price grasslands in Central Europe. In: Veen P, Jefferson R, de Smidt J, van der Straaten J (eds) Grasslands in Europe of high nature value. KNNV Publishing, Zeist, pp 15–26 Possingham HP,

Wilson KA (2005) Turning up the heat on hotspots. Nature 436:919–920PubMedCrossRef Rácz IA, Déri E, Kisfali M, Batiz Z, Varga K, Szabó G, Lengyel S (2013) Early changes of Orthopteran assemblages after grassland restoration: a comparison of space-for-time substitution versus repeated measures monitoring. Biodivers Conserv. doi:10.​1007/​s10531-013-0466-8 Schmitt T (2007) Molecular biogeography of Europe: Pleistocene cycles and postglacial trends. Front Zool SGC-CBP30 4:11PubMedCrossRef Thompson JD (2005) Plant evolution in the Mediterranean. Oxford University Press, New YorkCrossRef Valkó O, Török P, Matus G, Tóthmérész

B (2012) Is regular mowing the most appropriate and cost-effective management maintaining diversity and biomass of target forbs in mountain hay meadows? Flora 207:303–309CrossRef Veen P, Jefferson R, de Smidt J, van der Straaten J (eds) (2009) Grasslands in Europe of high nature value. KNNV Publishing, Zeist Vrahnakis MS, Janišová M, Rūsiņa S, Török P, Venn S, Dengler J (in press): The European Dry Grassland Group (EDGG): stewarding Europe’s most diverse habitat type. In: Baumbach H, Pfützenreuter S (eds) Steppenlebensräume Europas: Gefährdung, Erhaltungsmaßnahmen und Schutz. Thüringer Ministerium

für Landwirtschaft, Forsten, Umwelt und Naturschutz, Erfurt Wallace AR (1860) On the zoological geography of the Malay archipelago. Biol J Linn Soc 4:172–184 WallisDeVries MF, van Swaay CAM (2009) Grasslands as habitats for butterflies in Europe. In: Veen P, Jefferson R, de Smidt J, van der Straaten J (eds) Grasslands in Europe of high nature value. KNNV Publishing, Zeist, pp 27–34 WallisDeVries MF, Poschlod P, Willems JH (2002) Challenges for the conservation of calcareous grasslands in north-western LY294002 Europe: integrating the requirements of flora and fauna. Biol Conserv 104:265–273CrossRef Weiss N, Zucchi H, Hochkirch A (2013) The effects of grassland management and aspect on Orthoptera diversity and abundance: site conditions are as important as management. Biodivers Conserv. doi:10.​1007/​s10531-012-0398-8 Wellstein C, Chelli S, Campetella G, Bartha S, Galiè M, Spada F, Canullo R (2013) Intraspecific phenotypic variability of plant functional traits in contrasting mountain grasslands habitats. Biodivers Conserv. doi:10.​1007/​s10531-013-0484-6 Wiezik M, Svitok M, Wieziková A, Dovčiak M (2013) Shrub encroachment alters composition and diversity of ant communities in abandoned grasslands of western Carpathians. Biodivers Conserv. doi:10.

All of these phenomena suggested that either an https://www.selleckchem.com/products/qnz-evp4593.html unknown mechanism is present in the cell

to tightly control DNA phosphorothioation, Epoxomicin nmr or that over-expression of some of the proteins to override the regulation could be detrimental to the cells. We propose that the dosage of the Dnd proteins in the cells may not exceed the tolerable limit, and that the Dnd proteins must be balanced so as to be expressed in a highly coordinated manner in the cells. Therefore, simultaneous and/or unbalanced over-expression of one or even all four (dndB-E)of the dnd genes could seriously harm the cells, leading to inhibition of growth. The present study, showing that strongly induced expression of DndD and DndC, but not the other Dnd proteins, by the addition of thiostrepton, strongly suggests that these two proteins are the key determinants for the phenomenon. Being an IscS-like protein, DndA [21] was suggested to provide sulfur via its L-cysteine desulfurase activity and to catalyze iron-sulfur cluster assembly of DndC [22], probably by generating a persulfide (perhaps with the cysteine residue(s) in DndC

or DndD) in the modification process. As such IscS-like proteins are also often required, as multi-functional proteins, for many other metabolic pathways [21], the detrimental effect by over-expression of DndC and DndD could be attributed to deprivation of DndA which is vital for primary metabolism. Thus, the fact that DndA function could not be substituted by Silibinin other IscS homologs, at least in S. lividans analyzed here, might be due to a failure of proper persulfide formation, which could subsequently ARN-509 datasheet be delivered to target the DNA via DndC or DndD (not DndE because of its apparent lack of a .cysteine residue mediating persulfide formation). The exact mechanism of negative role of the over-expressed DndC and DndD proteins to cell viability remains, however, to be determined. Conclusion Genetic determination of the Dnd phenotype diagnostic for DNA sulfur modification in S. lividans was unambiguously attributed to a

6,665-bp DNA region carrying five dnd genes, with dndB-E constituting an operon and dndA transcribed divergently. Mutations in each of four dnd genes (dndA, C, D, and E) abolished the Dnd phenotype while mutation of dndB aggravated the Dnd phenotype. The Dnd phenotype of all mutants could be restored by complementation with the corresponding dnd gene, suggesting that they are essential for DNA sulfur modification. The fact that the cells ceased growth by overdosage of DndC or DndD in vivo suggests that the frequency of DNA phosphorothioate modification is under strict control in the native host. Methods Bacterial strains and plasmids These are described in Additional file 1. Methods and techniques Standard methods for culturing cells, DNA cloning, PCR, Southern hybridization, and Western blotting were according to [23] in E. coli and [24] in Streptomyces.

Before the assay, cells were collected with non-enzymatic Cell Dissociation Solution (Sigma-Aldrich, Poland), centrifuged, resuspended in DMEM (with no FBS), counted in a Burker counting chamber (Roth, Germany) #KU55933 concentration randurls[1|1|,|CHEM1|]# in light microscopy with trypan, and diluted to the desired concentration. The cells were used immediately in the migration assay. Migration chamber preparation Fibronectin assay: 8-μm insert membranes (Falcon

BD Biosciences, USA) were sterilely covered with fibronectin (100 μg/ml, Falcon BD Biosciences). Both sides of the membrane were covered with 20 μl of the fibronectin suspension and incubated for 30 min at 37°C. Fibronectin was removed and the inserts were washed three times with sterile water. Subsequently, both sides of the membrane were immersed in a 0.1% albumin solution and incubated for 15 min. The inserts were washed three times with sterile water and dried. The prepared inserts were not stored, but used immediately

after preparation. Matrigel assay: according to the manufacturer’s instructions, the 8-μm insert membranes (Falcon BD Biosciences) were covered with matrigel diluted 1:4 with DMEM under sterile conditions, with cooling. Only the upper side of the membrane was covered with 10 μl of the matrigel suspension (i.e. approx. 7 μg/cm2 of the membrane) and slowly dried (overnight RG7112 in a covered plate) at 37°C. Such prepared inserts can be stored at -20°C. If frozen, they were defrosted at 37°C, and rehydrated with DMEM for 2 hours, and directly applied in the migration Prostatic acid phosphatase assay. Migration assay The cells were suspended in DMEM with no FBS, and applied to the upper section of the migration chamber, with 1 × 105 Hs294T cells/insert in both

the fibronectin and the matrigel assay, 4 × 105 B16 cells/insert in the matrigel assay, and 5 × 105 B16 cells/insert in the fibronectin assay. All preparations (bacteriophages, LPS, PBS basic control) were correlated and added at the same final volumes of PBS (125–135 μl), both in the upper and the lower sections of the migration chamber. All the preparations and cells in the upper section were completed with DMEM and with FBS-containing medium to 0.5 ml in the lower section (according to the manufacturer’s instructions). Final concentrations of the bacteriophage preparations were 1.5–2.5 × 109 pfu/ml containing 10 U/ml residual LPS. Concentration of the attracting agent, FBS, in the lower section of the migration chamber was 7.3–7.5%. The migration was carried out at 37°C with CO2. The time of migration was initially optimised and was 2 h for B16 on fibronectin, 7–8 h for B16 on matrigel, 1 h 20 min for Hs294T on fibronectin, and 4.5–5 h for Hs294T on matrigel.

Despite the low correlation of fungal biomass and bioluminescence at late time points after infection in the cortisone acetate and RB6-8C5 treatments, a good correlation between the PRN1371 molecular weight increase in the fungal biomass and the bioluminescence was observed under the cyclophosphamide regimen. Under this treatment, although the growing hyphae were responsible for diffuse parenchyma lesions, the accessibility of oxygen remains possible in the absence of inflammation. At late time points, an ongoing increase of the

luminescence signal reflects the increase of biomass. Therefore, cyclophosphamide Savolitinib molecular weight immunosuppression seems best suited to follow the effect of antifungal drug

treatment on clearance of fungal infections. This study additionally allowed gaining new insight concerning the impact of different immune effector cells in the defense against invasive aspergillosis. Alveolar macrophages (AM) were assumed to play an important role in clearance of conidia from tissues and provide a “”first-line of defense”" against A. fumigatus infections [3]. AM are thought to trigger the recruitment of immune effector cells this website to the site of infection after recognition and phagocytosis of conidia [27] through the release of inflammatory and chemotactic mediators. Due to the importance of AM in conidial host defense, we expected that their reduction by the clodrolip

treatment would increase the susceptibility of mice to IA. This assumption was not confirmed experimentally. Intranasally clodrolip-treated mice showed a 80% reduction in the number and viability of AM [28, 29], but a 2.6 fold increase in the number of BAL fluid neutrophils, one day post-infection. A significant increase in the neutrophil number in BAL fluid of macrophage-depleted (clodrolip-treated) mice 24 hours Isotretinoin after instillation of Pseudomonas aeruginosa has already been reported. However, in this work macrophage-deficient mice showed impaired bacterial clearance [30]. In contrast, in our model, neutrophil migration into the airways of macrophage-depleted infected mice is likely to have prevented conidial germination per se. Supporting this idea, we found that the thoracic region or BAL fluid of AM-depleted animals only showed a slight increase in bioluminescence above control levels (Figure 3). This finding correlated with survival data and histopathological findings, demonstrating an absence of conidial germination in AM-depleted mice.

Two weak-intensity infrared bands measured in the middle of infrared region located at 1,365 and 1,639 cm-1 are due to the bending vibrations of the hydroxyl groups (-OH), which are associated on the surface of nanospheres. The spectrum exhibited strong infrared 17-AAG cost absorption bands around 1,090 cm-1 which originate from the Si-O-Si asymmetric and symmetric stretching [8, 20]. The band at around 792 cm-1 is assigned to the Si-OH stretching. An intense sharp band at

473 cm-1 is attributed to the Tb-O-Si stretching vibrational mode. Furthermore, the intensity and broadening of the bands indicated a large number of OH groups and Si-OH molecules present on the surface. This could play an important role including biocompatibility in biological systems, functionality, and high colloidal stability under different conditions

[24]. These results corroborate with the analysis of FE-TEM micrographs, EDX, and XRD analysis which confirmed that silica had been successfully encapsulated on the surface of Tb(OH)3 molecules. Figure 6 FTIR spectrum of the prepared luminescent ACP-196 nmr mesoporous Tb(OH) 3 @SiO 2 core-shell nanosphere. Optical properties Figure 7 illustrates the optical absorption spectra of the as-synthesized luminescent mesoporous Tb(OH)3@SiO2 core-shell nanospheres. As shown in Figure 7, the absorption spectra were measured in ethanol and deionized water in similar concentrations. The absorption spectra in ethanol displayed an intense band located at 228 nm with a middle intensity band around 306 nm. The absorption at 228 nm originates from the silica parts, which agrees with the spectra of previous observations [25–28], and the middle intensity absorption band at 308 nm likely originates from the terbium hydroxide [26–28]. The spectrum displayed some small intensity absorption transitions in visible region which correspond to the forbidden 4f8-4f75d transitions of Tb3+ ion usually weak in silica matrices.

These prominent levels of terbium ions observed are assigned to the SB203580 mouse appropriate electronic transitions as 7F6 → 5G4 (304 nm), 7F6 → 5L10 (335 nm), and 7F6 → 5G6 (382 nm) [26–28]. The absorption spectrum confirms the formation of Tb(OH)3 nanoparticles along with silica surface in the core-shell nanospheres about [27]. The addition of silica layer is marked by a pronounced scattering and sharpening of the absorption peak, and weak terbium hydroxide absorption transitions are appearing in the Tb(OH)3@SiO2 colloid. Obviously, the silica-surface-modified terbium hydroxide nanoparticles is screened by the strong scattering from the silica colloid. These results can be corroborated visually by the loss of the characteristic light-yellow color to a dirty-white-colored solution with fine colloidal dispersion after silica adsorption on the terbium hydroxide surface.

Table 1 LEC (fundamental charge units) at some relevant atoms in the cone apices shown in Figure 2 b,c Sites 1 2 3 Maximum One-pentagon −0.071e +0.014e −0.059e +0.042e Two-pentagon −0.055e −0.067e −0.066e +0.076e The

maximum value occurs at the zigzag edge of each system. Figure 6 depicts the LEC for the two types of CNC structures, showing that the non-equilibrium of the charge distribution is www.selleckchem.com/products/sgc-cbp30.html restricted to the apex and edge regions: electric GSK2126458 price neutrality is found at all the other surface sites. The values found for the LEC at the apex regions are found to be independent of the size of the cones whereas this is not true for the edge states. When the number of atoms of the CNC structure is even, the edge-state LEC exhibits the same symmetry of the cone. For odd N C , the Fermi

level is occupied by a single electron, and then, the LEC at Selleckchem Vistusertib the edge states reflects the breaking of symmetry. Figure 6 Electric charge distribution in neutral CNCs. (Color Online) For a single-pentagon cone with 245 atoms (a) and for two-pentagon cone with 246 atoms (b). The values of electric charges for some sites are given in Table 1. Absorption spectra We have also calculated the absorption coefficient for the CND and CNC structures, for different photon polarizations. Figure 7 shows the results for the absorption coefficients α x and α y , for polarization perpendicular to the cone axis, and α z for parallel polarization. Calculated results are shown for a nanodisk composed of 5,016 atoms, a single-pentagon nanocone

with 5,005 atoms, and a two-pentagon nanocone with 5,002 atoms. For the case of large CNDs, the spectra present the general features observed for the absorption of a graphene monolayer. In the infrared region, the absorption coefficient of a graphene monolayer is expected to be strictly constant [27], whereas for higher energies the spectrum shows a strong interband absorption peak coming from Leukocyte receptor tyrosine kinase transitions near the M point of the Brillouin zone of graphene [28]. The main difference for a finite CND is a departure from a completely frequency-independent behavior for low energies, where the absorption coefficient shows oscillations as a function of the photon energy instead of a constant value. This is a consequence of the border states that are manifested as a peak in the total DOS at the Fermi energy [24, 29]. For CNCs, the general behavior is the same as for nanodisks, except for the dependence of the absorption on the photon polarization, in particular for low energies. Furthermore, the main absorption peaks for different polarizations occur when the photon energy is equal to the energy between the two DOS van Hove-like peaks (cf. Figure 4). Notice that the overlap integral s≠0 leads to an energy shift of the main resonant absorption peak given by δ≈2s 2|t|/(1−s 2)≈100 meV.

Assessment of adverse events All subjects were

questioned about adverse events (AEs) of treatment at each visit, and all adverse events reported were analyzed regardless of the investigators’ assessments of causality. The Medical Dictionary for Regulatory Activities (MedDRA, Version 8.1J) was used to categorize reported adverse events. Statistical analysis All the data analyses were performed by statisticians from Ono under the supervision and confirmation of data analyses by one of the authors (Ohashi, Y.). The intention-to-treat selleck products (ITT) population comprised all Selleck Eltanexor patients who received at least one dose of study medication and who attended at least one follow-up visit for any observation Fedratinib clinical trial of efficacies. The ITT population was used for all fracture and height analyses. Safety analyses population comprised all patients who received at least one dose of study medication

in either treatment group. A per-protocol (PP) approach was used as a primary approach to analyze the bone turnover markers because they can change rapidly by protocol violations, interruption of study therapy, or concurrent illness. The PP approach excluded protocol violators who took less than 75% study drug, who took prohibited medications during the course of the trial, or who violated the protocol in a significant manner as specified in the data analysis plan, and patients who took study drug for less than 12 months. This population included all patients in the ITT population, except those with a protocol deviation deemed to have a significant impact on the efficacy variables, i.e., major deviations regarding the inclusion/exclusion criteria, patients with insufficient compliance (<75% of the study medication), documentation of forbidden concomitant

medication that could bias the fracture results, and patients lacking an assessable baseline and follow-up for X-ray assessments for less than 12 months. The risk of patients with new morphometric vertebral fractures at 24 months, as the primary endpoint, was analyzed by testing the superiority of minodronate group to the placebo group by the time-to-event curves (Kaplan–Meier method), the event being the first new incident Astemizole vertebral fracture. The primary hypothesis was tested using an ITT analysis that was modified to include all subjects randomized, who had taken at least one dose of study drug, and attended at least one follow-up visit. A Cox regression model was used to estimate the relative risk of vertebral fracture and its 95% confidence interval in minodronate group and placebo group. Log-rank test was used to determine the superiority of the minodronate group to the placebo group. The power calculation was based on the predictive risk of vertebral fracture. For the study to achieve a power of 90% to detect the superiority, a sample size of 290 subjects per group was required.