Protein content was measured using a Bio-Rad protein assay kit. The sample was precipitated and dissolved in Reagent3 (Bio-Rad). Details are described in Supporting Information, Appendix S1. The solution was used to rehydrate an IPG ReadyStrip (7 cm, pH 3–10; Bio-Rad). The first-dimensional isoelectric focusing (IEF) was focused in three steps at 150 V (15 min), 150–4000 V (2 h), and 4000 V (8 h) using a Protean IEF cell (Bio-Rad). Equilibration and SDS-PAGE were performed according to the manufacturers’ instructions with 10% SDS-PAGE gel on a Mini-PROTEAN Tetra cell (Bio-Rad) at 150 V. The gel was stained with SYPRO Ruby Protein Gel Stain (Molecular Probes) following the manufacturer’s guidelines.

Relative fluorescence intensities http://www.selleckchem.com/Proteasome.html were calculated using Image J software (http://rsbweb.nih.gov/ij/). In-gel digestion and LC-MS/MS analysis of that were performed as previously described (Ogata et al., 2010) with some modifications (Appendix S1). Total RNAs were extracted from inoculated wood meal suspensions using Plant RNA Isolation Reagent (Invitrogen) and purified with

an RNeasy Plant Mini kit (Qiagen) according to the manufacturers’ instructions. A cDNA encoding BUNA2 was cloned by a series of PCR procedures using the primers listed in Table S1. The 3′-coding region of the gene was cloned by 3′-rapid amplification of cDNA ends (RACE) using a 3′-Full RACE core set (TaKaRa Bio) and primer BUNA2dF and sequenced. The 5′-coding region was cloned by 5′-RACE using a 5′-Full RACE core set (TaKaRa Bio) and 5′-phosphorylated primer 5phosBUNA2R and two nested primer sets, corresponding to 3′-RACE PCR fragments Osimertinib BUNA2F1–BUNA2R1 and BUNA2F2–BUNA2R2. Genomic DNA was isolated from P. sordida YK-624 mycelium using ISOPLANT II (Nippon Gene). TAIL-PCR was performed using the degenerate primers TAIL1-6, as described previously (Yamagishi et al.,

2007), to obtain the 5′ flanking region of bee2. Nested primers BUNA2R1, R2, and R3 were used as gene-specific primers. Inverse PCR was performed to further upstream of the 5′ flanking region using the primer sets bee2proF1–bee2proR1 and bee2proF2–bee2proR2 and the restriction enzyme SacII (New Uroporphyrinogen III synthase England Biolabs), as previously described (Ochman et al., 1988). The full-length 5′ flanking region of bee2 (1584 kb) was amplified using primer sets bee2proF1–bee2proR1. The procedure for constructing the MnP gene (mnp4) expression plasmid, pBUNA2pro-mnp4, is shown in Fig. S1, and details are described in Appendix S1. UV-64 protoplasts were prepared and then transformed with pPsURA5 and pBUNA2pro-mnp4 using standard techniques. The cotransformed clones were selected by PCR, as described previously (Sugiura et al., 2009), with the following modifications: primers bee2proF4 and mnp4R3 were designed to amplify the mnp4 gene fused with the bee2 promoter. Phanerochaete chrysosporium ME-446, P.

4). Cytochrome c was included as a positive control for a protein

with covalent attachment of heme, and its heme-associated selleck inhibitor peroxidase activity was detected at the expected position based on the mass of the protein (∼13 kDa), independent of treatment with thiol reagents. Both HemA1−412-His6 and HemA1−412 [C170A]-His6 were detected in Coomassie-stained gels at the predicted molecular mass of ∼46 kDa (Fig. 4, left lanes); however, peroxidase activity was only detected for the HemA1−412-His6 and only in unheated samples lacking both dithiothreitol and β-ME. Any one of three treatments, dithiothreitol, β-ME, or boiling, abolished the signal (Fig. 4, and data not shown), indicating that heme is not covalently bound. HemA1−412 [C170A]-His6 failed to produce a detectable signal under any of the conditions tested. Three bands are observed for the untreated wild-type sample. The smallest and most Screening Library price abundant band corresponds to HemA protein. The bands above it are likely aggregates as observed in other studies (Schroder et al., 1992; Verkamp et al., 1992; Schauer et al., 2002). According to one model, heme binding to HemA protein sensitizes

it to proteolytic attack. The combined observations of a regulatory defect and absence of bound heme in purified C170A led us to predict that the mutant would exhibit increased stability over wild-type HemA. Isogenic strains expressing wild-type and C170A mutant HemA in a single copy from the native locus in the S. enterica chromosome were analyzed by Western blot after inhibition of protein synthesis (Fig. 5a). Wild-type HemA was

present at lower levels than the mutants and was detectable only at the initial time point. HemA[KK], included as a positive control, remained stable over the time course of the experiment. In support of the model, the C170A mutant was nearly as stable as HemA[KK] (Fig. 5b). Our current understanding of heme biosynthesis by the C5 pathway in bacteria involves two different regulatory mechanisms: feedback inhibition of enzyme activity by heme and post-translational control of enzyme abundance by proteolysis (Wang et al., 1997; Wang et al., 1999a). Furthermore, HemA enzyme from Chlorobium vibrioforme, as well as some eukaryotic enzymes, mostly expressed in E. coli, contains tightly bound heme (Vothknecht et al., 1996; Srivastava & Beale, 2005; Srivastava et al., 2005). This work focuses on S. http://www.selleck.co.jp/products/Decitabine.html enterica, the species in which regulation by proteolysis was discovered (Wang et al., 1999a). We were unsuccessful in previous attempts to use the T7 RNA polymerase system to overexpress Salmonella HemA, which is 94% identical to the E. coli enzyme. Jahn’s group succeeded with the E. coli HemA enzyme by coexpressing the protein with the chaperones DnaJK and GrpE (Schauer et al., 2002). Analysis of the purified E. coli enzyme showed (1) no copurifying prosthetic group detectable by spectroscopy and (2) no inhibition of enzyme activity by heme in vitro. This apparent difference between the Salmonella and E.

However, the protective efficacy of the Alpelisib datasheet protein remained to be evaluated. SS2 strain ZYS was isolated from the brain of a diseased piglet and expressed muramidase-released protein, extracellular protein factor and suilysin (Tang et al., 2006). All the field strains used in this study were derived from samples of different tissues (lung, brain, joint, heart and blood) of diseased pigs with pneumonia and systemic infection (meningitis, arthritis, endocarditis or septicaemia) from 16 provinces (municipalities) of China. All S. suis strains were maintained on tripticase soy agar (Difco Laboratories, Detroit, MI)

plus 10% bovine serum or cultured in Todd–Hewitt broth medium (THB) (Oxoid, Wesel, Germany) plus 10% bovine serum to mid-log phase (OD600 nm of 0.4) at 37 °C under aerobic conditions. Laboratory Escherichia coli strains DH5α and BL21 were used as nonadherent and noninvasive recipients of recombinant protein-containing plasmids pET-28a (+). The purified recombinant HP0272 (accession no. YP_001197640) was performed as described previously (Zhang et al., 2008) and then was resolved on a 10% v/v polyacrylamide vertical slab gel with a 4% v/v stacking gel. After electrophoresis, the gel was visualized by being stained with CBB R-250 and the band of interest was picked, digested with trypsin and then subjected to matrix-assisted laser desorption/ionization-time

of flight MS analysis as described (Fountoulakis & Langen, 1997; Kussmann et al., 1997). Peptide this website masses were searched against Pyruvate dehydrogenase lipoamide kinase isozyme 1 the NCBI database using the mascot program (http://www.matrixscience.com). All experimental protocols were approved by the Laboratory Animal Monitoring Committee of Hubei Province and were performed accordingly. Thirty-two 4–6-week-old female BALB/c mice were randomly assigned to four groups of eight each. The purified recombinant HP0272 (100 μg) was emulsified with Marcol 52 (ESSO)-based adjuvant and then applied to immunized mice in group 1 intraperitoneally.

Subsequent booster injections were given on the 14th day with the same antigen. Mice in group 2 immunized with commercially SS2-inactive vaccine (China) served as positive controls. Mice in group 3 were inoculated with phosphate-buffered saline (PBS) emulsified in the same adjuvant served as negative controls, and mice in group 4 were inoculated with PBS alone as blank controls. On the 10th day after the booster immunization, sera were obtained from each group by tail vein bleeding. All mice were then challenged with a lethal dose of 2 × 109 CFU of log-phase SS2 ZYS in 0.5 mL PBS. All mice were observed for 10 days for morbidity and mortality. Microtitre plates were coated overnight at 4 °C with 200 ng 100 μL−1 of purified recombinant HP0272 diluted in sodium carbonate buffer (pH 9.6). Having been treated with washing buffer (PBS, pH 7.2, containing 0.

This is a case report of a young man who presented as an emergency with type 1 diabetes, adrenal failure and primary hypothyroidism. It highlights the importance of considering the diagnosis of adrenal failure in an individual presenting with type 1 diabetes who does not respond as expected to initial treatment, and of looking for other autoimmune conditions at the initial presentation. JL, a 35-year-old gentleman, presented at emergency with a three-week history of feeling generally unwell. Specifically he had symptoms of malaise, tiredness and feeling faint. He had also noticed increased thirst, drinking more than 3L/day, urinary frequency, leg cramps and reduced exercise tolerance. For the preceding week

he had been troubled Volasertib manufacturer by nausea and vomiting to the extent that he was unable to eat but had been able to drink. The day prior to admission he had developed abdominal pain and diarrhoea. During this time DAPT manufacturer period he had lost weight but there were no other associated symptoms or signs. He had recently visited his GP for the treatment of oral thrush but a capillary blood glucose was normal at that

time. He had a past medical history of mild asthma and used Ventolin infrequently. There was a family history of autoimmune disease; a cousin with type 1 diabetes and an aunt with a ‘thyroid problem’. He worked as an engineer, was a non-smoker and drank six units of alcohol per week. On examination, he was noted to be thin, dehydrated with extensive oral thrush. He was tanned but there was no pigmentation of the buccal mucosa or palmar creases. His temperature

was 35.5oC. He was cardiovascularly stable with a pulse of 90bpm and blood pressure 141/61mmHg but he was unable to stand without feeling dizzy. Cardiovascular and respiratory examination was normal and his abdomen was soft but tender to light palpation with normal bowel sounds and no rebound or guarding. A capillary blood glucose was 20.7mmol/L. Arterial blood gases were done and were normal (pH 7.43, pCO2 4.2kPa, pO2 10.6kPa, BE -2.6). Urine dipstick was positive for ketones (+++) and glucose (+++). After the initial assessment the impression was that he had newly diagnosed diabetes but had not developed diabetic ketoacidosis, he was dehydrated and that his abdominal TCL symptoms may have been related to his diabetes but that a polyendocrine syndrome should be considered. An insulin infusion and intravenous fluids were commenced. Blood was sent for urea and electrolytes, glucose, thyroid function, liver function, calcium, amylase and cortisol. He was reviewed four hours later. At this time despite his glucose normalising and fluid resuscitation having occurred his pulse had increased and his blood pressure had dropped (Figure 1). He looked worse and did not feel any better despite appropriate treatment. An intravenous venous short synacthen test was performed with a baseline ACTH.

macleodii was acclimated for 7 days to iron-limited conditions, by daily transfer in AQUIL medium (Price et al., 1989) containing 5.4 nM of non-radioactive Fe-EDTA. The experiments were conducted with cells transferred into

freshly prepared AQUIL medium containing 5.4 nM of 55Fe-EDTA. Triplicate live incubations (20 mL) were performed in the dark, at 20 °C and under agitation. For triplicate controls, formaldehyde (FA, 2% final concentration) was added to the A. macleodii culture and kept for 1 h before the addition of 55Fe. A second set of experiments was performed with natural bacterial communities. In the NW Mediterranean Sea, seawater samples were collected five nautical miles offshore at Station POLA (42°28′300N – 03°15′500E, 90 m overall depth). CX5461 Seawater (2 L) was pumped at 5 m using a trace metal clean Teflon pump (ASTI) connected to an acid-washed PVC tube. The samples were stored in 1 L acid-washed PC bottles in the dark until arrival to the laboratory about 1 h later. In the Southern

Ocean, samples were collected during PD-0332991 in vivo the Kerguelen Ocean and Plateau Compared Study 2 cruise (KEOPS2, October–November 2011) at Station E-4W (48°45′900S – 71°25′500E, 1384 m overall depth). Samples were collected at 20 m using trace metal clean 12L modified Niskin bottles and further processed in a clean laboratory. At both sites, seawater was filtered at low pressure (< 200 mm Hg) through 0.8 μm acid-washed PC filters (47 mm; Millipore). Subsamples (100 mL) of filtered seawater were spiked with 55Fe at a final concentration of 15 nM. This concentration was chosen to limit isotope dilution as determined by saturation curves (data not shown) and to allow a maximum number of cells to obtain the critical amount of 55Fe for silver grain production (see 'Results and discussion' Section). Samples from the NW Mediterranean Sea were incubated on a rotary

shaking platform at in situ temperature (20 °C), in the dark, for periods ranging between 24 h and 7 days. Experiments only were carried out in triplicate. In the Southern Ocean, the PC bottles were incubated at 1% PAR irradiance in an on-deck incubator supplied with circulating surface seawater. For both sites, control treatments of the seawater samples were killed with formaldehyde and kept for 1 h before the addition of 55Fe. Following incubation with 55Fe, subsamples for the determination of the radioactivity incorporated into bacterial cells were collected on nitrocellulose filters (NC, 25 mm diameter, 0.2 μm pore size; Nuclepore), and subsamples for microscopic observations were collected on 0.2-μm PC filters (25 mm diameter; Millipore) (Fig. 1). To investigate the efficiency of eliminating extracellular 55Fe, two rinsing solutions and 0.2-μm-filtered seawater were tested. Subsamples of the A. macleodii culture (1 mL) and the natural seawater (10 mL) were filtered onto 0.2-μm NC filters. In the case of A.

aureus, and contributed considerably to biofilm formation in some clinical isolates (Otto, 2008). The dispersal stage involves the overproduction of proteases that are controlled by the quorum-sensing system agr, whereby single cell or cell clusters are detached from biofilms (Boles & Horswill, 2008). Besides, phenol-soluble modulins may also be involved (Otto, 2008). To date, several factors such as glucose (Mack et al., 1992), urea (Hjelm & Lundell-Etherden, 1991), Fe2+ (Deighton & Borland, 1993; Elci et al., 1995), EDTA (Banin et al., 2006), ethanol

(Knobloch et al., 2001), antibiotics (Rachid et al., 2000b; Hoffman et al., 2005; Yakandawala et al., 2007), anaerobiosis (Cramton et al., 2001), osmolarity and temperature (Rachid et al., 2000a), have been reported to affect S. aureus biofilm formation. Glucose plays an inductive role in the transcription of ica, while the detailed mechanism remains unknown selleck products (Dobinsky et al., 2003). Our study has revealed that the presence of thiols affected the glucose Cell Cycle inhibitor metabolism in S. aureus, and PIA biosynthesis was reduced significantly. Bacterial strains used in this study are listed in Supporting Information, Table S1. For routine cultivation, S. aureus and S. epidermidis strains were grown at 37 °C in tryptone

soy broth (TSB) medium (0.25% glucose, Oxido) with aeration at 37 °C. Bacterial cells at stationary phase were inoculated into 96-well polystyrene culture plates (Corning, Costar) with a dilution of 1 : 200 Ixazomib research buy for a 12-h cultivation to form biofilms as described previously (Lim et al., 2004). The supernatant was removed and the plates were washed twice with distilled water. The biofilms were then fixed and stained with staining buffer containing

2% crystal violet and 4% formaldehyde for 5 min. The stained biofilms were washed again to remove the unbound stain and allowed to dry at room temperature for the determination of A490 nm (ELX800 Universal Microplate Reader, Bio-Tek). Staphylococcus aureus NCTC8325 was grown in TSB medium to stationary phase at 37 °C with shaking. Stationary phase cells (250 μL) were inoculated into 50 mL fresh TSB, TSB supplemented with 10 mM dithiothreitol, TSB supplemented with 20 mM cysteine and TSB supplemented with 40 mM β-mercaptoethanol (BME) respectively. The cell densities were determined by measuring OD600 nm (DU730 Nucleic Acid/Protein Analyzer, Beckman Coulter) per 60 min for 8 h. The primary attachment assay was preformed as described previously (Knobloch et al., 2001). Staphylococcus aureus NCTC8325 was precultivated in TSB, TSB supplemented with 10 mM dithiothreitol or TSB supplemented with 20 mM BME to stationary phase. Bacterial cells were then diluted into the respective medium with appropriate densities in 24-well cell culture plates, and incubated at 37 °C for an hour.

aureus, and contributed considerably to biofilm formation in some clinical isolates (Otto, 2008). The dispersal stage involves the overproduction of proteases that are controlled by the quorum-sensing system agr, whereby single cell or cell clusters are detached from biofilms (Boles & Horswill, 2008). Besides, phenol-soluble modulins may also be involved (Otto, 2008). To date, several factors such as glucose (Mack et al., 1992), urea (Hjelm & Lundell-Etherden, 1991), Fe2+ (Deighton & Borland, 1993; Elci et al., 1995), EDTA (Banin et al., 2006), ethanol

(Knobloch et al., 2001), antibiotics (Rachid et al., 2000b; Hoffman et al., 2005; Yakandawala et al., 2007), anaerobiosis (Cramton et al., 2001), osmolarity and temperature (Rachid et al., 2000a), have been reported to affect S. aureus biofilm formation. Glucose plays an inductive role in the transcription of ica, while the detailed mechanism remains unknown Selleckchem AG14699 (Dobinsky et al., 2003). Our study has revealed that the presence of thiols affected the glucose http://www.selleckchem.com/products/epacadostat-incb024360.html metabolism in S. aureus, and PIA biosynthesis was reduced significantly. Bacterial strains used in this study are listed in Supporting Information, Table S1. For routine cultivation, S. aureus and S. epidermidis strains were grown at 37 °C in tryptone

soy broth (TSB) medium (0.25% glucose, Oxido) with aeration at 37 °C. Bacterial cells at stationary phase were inoculated into 96-well polystyrene culture plates (Corning, Costar) with a dilution of 1 : 200 ADAM7 for a 12-h cultivation to form biofilms as described previously (Lim et al., 2004). The supernatant was removed and the plates were washed twice with distilled water. The biofilms were then fixed and stained with staining buffer containing

2% crystal violet and 4% formaldehyde for 5 min. The stained biofilms were washed again to remove the unbound stain and allowed to dry at room temperature for the determination of A490 nm (ELX800 Universal Microplate Reader, Bio-Tek). Staphylococcus aureus NCTC8325 was grown in TSB medium to stationary phase at 37 °C with shaking. Stationary phase cells (250 μL) were inoculated into 50 mL fresh TSB, TSB supplemented with 10 mM dithiothreitol, TSB supplemented with 20 mM cysteine and TSB supplemented with 40 mM β-mercaptoethanol (BME) respectively. The cell densities were determined by measuring OD600 nm (DU730 Nucleic Acid/Protein Analyzer, Beckman Coulter) per 60 min for 8 h. The primary attachment assay was preformed as described previously (Knobloch et al., 2001). Staphylococcus aureus NCTC8325 was precultivated in TSB, TSB supplemented with 10 mM dithiothreitol or TSB supplemented with 20 mM BME to stationary phase. Bacterial cells were then diluted into the respective medium with appropriate densities in 24-well cell culture plates, and incubated at 37 °C for an hour.

Late diagnosis

see more was defined as having a CD4 count <350 cells/μL or clinical AIDS (a CDC category C event) at the time of the first reported positive HIV test. Data were derived from the national case surveillance. In the national case surveillance, a high percentage (73%) of CD4 cell count data were missing. Moreover, CD4 values were significantly more often reported for patients with a poor clinical status, which would have led to an overestimation of the percentage of late presenters if only those patients were included in the analysis. Therefore, we performed a multiple imputation of the missing CD4 values. To do this we estimated the probability of low (<350 cells/μL) vs. high (≥350 cells/μL) CD4 count depending on age at diagnosis, date of diagnosis, transmission risk group, CDC status (A, B, C or unknown) and residence in big cities using a logistic regression. Based on this probability, missing data were imputed in 100 realizations of the estimated probability to reduce the reporting bias. This

procedure, including an error estimation, is described elsewhere [18, 19] and is based on the assumption that, given the explanatory variables, the missing data are missing at random (MAR). Late presentation for care was defined as having a CD4 count <350 cells/μL or clinical AIDS at the first contact at a treatment centre participating in the ClinSurv cohort. Of note, from 2001 to 2008 the German HIV treatment Selleckchem SP600125 guidelines unanimously recommended starting antiretroviral

treatment only for patients with a CD4 count <200 cells/μL. All centres are able to monitor disease progression and initiate ART if needed, which is in accordance with the most Protein tyrosine phosphatase recent European consensus definition of HIV care [16]. Patients who re-initiated therapy (e.g. non-first-line) and patients without valid CD4 cell count data were excluded. Patients with a viral load of <500 HIV-1 RNA copies/mL (which represented the initial limit of detection) at the initiation of ART were suspected of having already been on treatment and were excluded. All analyses were performed in stata, version 11 (StataCorp LP, TX, USA). Tests used for comparison of demographic data included Student’s t-test and the Kruskal–Wallis test. Univariate and multivariate logistic regression models were used to analyse risk factors for late presentation for HIV diagnosis and treatment. Transmission risk groups including MSM (reference), IDUs, heterosexual migrants from high-prevalence countries (migrants), heterosexuals from low-prevalence countries (heterosexuals) and persons with unknown transmission risk (unknown) were introduced to the model as categories.

The partial acdS gene (approximately 810 bp) this website from Mesorhizobium sp. MAFF303099 was amplified by PCR using primers F (5′-GGCAAGGTCGACATCTATGC-3′) (Duan et al., 2009) and R2 (5′-GCATCGATTTGCCCTCATAG-3′). The amplified sequence was used as a DNA hybridization probe that was constructed using the DIG-High Prime DNA Labeling Kit (Roche Applied Science, Germany), according to the manufacturer’s instructions. About 2 μg of total DNA was digested with the restriction enzyme BamHI and used for Southern hybridization as described by Sambrook & Russell (2001). The hybridization process was carried using Dig Easy Hyb hybridization buffer (Roche Applied Science) at 42 °C, followed by washes at 25 and 68 °C. After membrane

treatment with anti-Dig Fab fragments (Roche Applied Science) and posterior washing, the hybridization signals were detected using the CDP-star chemiluminescent method (Roche Applied Science). Membranes were then sealed in folders and exposed to X-ray film (Kodak). To assess Mesorhizobium ciceri UPM-Ca7T acdS gene expression in root nodules, an RT-PCR analysis amplification was conducted using the chickpea Mesorhizobium

symbiosis system. This strain was used because it would give useful information about ACC deaminase expression and regulation in chickpea mesorhizobia, giving further insights into the ecology of these agronomical important strains. Three chickpea plants were grown and inoculated with M. ciceri UPM-Ca7T as described by Nascimento et al. (2012a). Briefly, chickpea seeds were surface sterilized and sown in pots containing sterilized vermiculite. buy Y-27632 The plants were grown in a growth chamber (Walk-in fitoclima, Aralab, Portugal) and irrigated with nitrogen-free nutrient solution (Broughton & Dilworth, 1971). After 3 weeks of plant growth, nodules were collected and treated for posterior RNA

extraction as described by Cabanes et al. (2000). Total RNA was extracted using the RNeasy Mini Kit (Qiagen, see more Germany) according to the manufacturer’s suggested protocol. After extraction, about 800 ng of total RNA was treated with 3U DNase I (Roche Applied Science) according to the enzyme manufacturer’s protocol. The conversion of total RNA to cDNA was conducted using RevertAid H Minus Reverse Transcriptase (Fermentas) as suggested by the manufacturer. Amplification of the acdS gene by PCR from the cDNA product was performed using primers F2 and R6 with the conditions previously described. ACC deaminase activity was assessed in Mesorhizobium type strains and in chickpea Mesorhizobium isolates growing in free-living conditions (Table 1). Determination of ACC deaminase activity in cells was performed following the method described by Duan et al. (2009). Strains were grown in TY at 28 °C for 2 days, and cells were collected by centrifugation and washed twice with 0.1 M Tris-HCl (pH 7.5). Cells were then re-suspended in M9 minimal medium containing 5 mM ACC. The bacterial suspension was incubated with shaking (150 r.p.m.

Both this database and pharmacy dispensing records were checked to identify discrepancies.

The inpatient regimen was considered correct if it matched the outpatient regimen. For those patients not followed at the hospital HIV clinic, admission data were also checked to rule out transcription errors. Drug–drug interactions were checked for contraindicated or not recommended combinations using national and international SRT1720 solubility dmso HIV websites [9–11]. If an error or interaction was detected, the pharmacist phoned the attending physician or nurse or added a footnote with a recommendation to the computerized prescription, so that the attending physician could see it the following day. The acceptance of the pharmacist’s recommendations was also reviewed during the following days. If the error was not corrected within 48 h of the recommendation, the prescription was classed as not accepted. Data were entered into an Access 2.0 database (Microsoft Corp., Redmond, WA, USA). For the descriptive analysis, qualitative variables were expressed as percentages and frequencies; quantitative variables were expressed as the mean (standard deviation [SD]). Fisher’s exact test was used to analyse contingency tables. Odds ratios (ORs) for risk factors associated with HAART-related problems

were analysed using a generalized estimating equation model. This multivariate model takes into account the correlation between different admissions belonging to the same patient. The statistical analysis was performed Calpain see more using stata (StataCorp. 2007, Stata Statistical Software, Release 10; Stata Corporation, College Station, TX, USA). Over a 1-year period, we reviewed the prescriptions for 247 admissions of 189 HIV-infected patients who received antiretroviral therapy. Forty-one patients were admitted more than once during the study period. Table 1

summarizes the demographic characteristics of these patients. The distribution of admissions by service was as follows: infectious diseases unit, 135 (54.7%); other medical units, 58 (23.5%); surgery services, 38 (15.4%); intensive care units, nine (3.6%); and units with surgical and nonsurgical patients, seven (2.8%). A total of 60 antiretroviral drug-related problems were identified in 41 patients (21.7% of the admitted patients had at least one antiretroviral problem). The types of HAART-related errors found are shown in Table 2. The most common was drug–drug interaction (33.3%), not only between antiretroviral agents, but also between antiretrovirals and other drugs. Atazanavir was the drug most commonly involved in interactions. The second most common problem was incorrect dose (16.7%), and the third most common was dose omission (15%), followed by lack of dosage reduction in patients with renal or hepatic impairment (11.7%), omission of one or more antiretroviral medications (10%), addition of an alternative antiretroviral drug (8.3%) and incorrect schedule according to outpatient treatment (5%).