Of note, the primer set employed for the assay with THI4 was position C (Fig. 1a) different from that (position B) for the assay for V5-tagged Pdc2p, and the pdc2Δ mutant was used instead of strain thi2Δ. We confirmed that the protein level of thi2p was also unchanged by the experimental conditions (data not shown). As expected,

V5-tagged Thi2p coimmunoprecipitated with all the THI genes except PDC5 (Fig. 1d). The association with the target DNA was also decreased by thiamin in the medium and the absence of Thi3p or, in this case, Pdc2p. These findings strongly suggest that both Pdc2p and 5FU Thi2p alone can bind target DNA and assist each other in recruitment to the THI promoters via interaction with Thi3p. We next intended to map the DNA-binding domain BKM120 order in Pdc2p. Pdc2p is 925 amino acids (aa) long with an internal (aa 407–581) transactivation domain and C-terminal (aa 668–889) Thi3p-interacting domain (Nosaka et al., 2008). In addition, Pdc2p possesses putative DNA-binding

domains at the N-terminus. We constructed plasmids to produce a truncated Pdc2p with a V5-tag and used them in ChIP assays. As expected from the presumed sequence, when the N-terminal 406 aa were removed, no association with THI genes and PDC5 was detected (Fig. 1e). Conversely, V5-tagged Pdc2p(1–406) coimmunoprecipitated with all the genes tested, although very weakly as compared with intact Pdc2p (Fig. 1e). In particular, its association with THI genes was markedly reduced. The Sitaxentan elimination of the first ten N- or C-terminal aa from Pdc2p(1–406) led to abolishment of the ChIP signal (data not shown). Thus, the 1–406 aa region is necessary for Pdc2p to bind with target

DNA. However, this region alone may not be adequate to exert full binding activity. Alternatively, because of a lack of the Thi3p-interacting domain (Nosaka et al., 2008), it is assumed that the recruitment of Pdc2p(1–406) to the promoter regions of THI genes was decreased. Meanwhile, we attempted to locate the promoter region responsible for the expression of PDC5. Although two ethanol-repression sequence (ERA) sites are recognized in the upstream region of PDC5 (Liesen et al., 1996), it is unclear whether these cis-acting elements are involved in the induction of PDC5 gene expression in response to thiamin starvation. We constructed a series of plasmids containing terminal and internal deletions of the PDC5 promoter and used the β-galactosidase activity to monitor their promoter activities. The results are summarized in Fig. 2. The LacZ gene with PDC5′s upstream region truncated at position −418 from the start codon conferred almost full promoter activity. However, the upstream region truncated at position −390 showed significantly less promoter activity, and that at position −345 barely showed any activity. Furthermore, the deletion of the upstream region from −397 to −346 almost completely abolished the activity.

3a). It was important to verify that the C-terminal HemA truncations encode functional enzymes and exhibit normal regulation. Plasmid-encoded, truncated, and tagged S. enterica hemA complemented an E. coli hemA mutant. Regulation in response to heme was tested by Western blot (Fig. 3b). To eliminate the possibility that a partial defect in the enzyme activity of the truncated proteins could affect the results of the test, an E. coli host that is wild type for hemA was used, and the plasmid-encoded proteins were specifically detected by an additional C-terminal FLAG tag. Truncated HemA exhibited

normal regulation in response to heme limitation. His-tagged C-terminally truncated HemA was RO4929097 concentration purified by Ni-NTA affinity chromatography. The purified protein was red in color, suggesting the presence of bound heme. The absorption spectrum of purified HemA protein (Fig. 1a) contains features characteristic of heme, including a prominent peak at 424 nm (the mTOR inhibitor Soret band). Upon reduction with Na-dithionite, the peak at 424 nm became sharper and shifted toward a longer wavelength (426 nm), and two other peaks appeared: one at 530 nm and another at 560 nm. The spectrum of reduced heme (hemin), which was used as a control, was very similar to that of the purified protein (data not shown). Three separate protein preparations

averaged 0.055 mol heme mol−1 protein monomer as determined by the pyridine hemochromogen assay. HemA1−412 [C170A]-His6 was purified according to the Mannose-binding protein-associated serine protease same protocol as that used for HemA1−412-His6. The C170A mutant protein was colorless, suggesting that it is unable to bind heme. The absence of heme was also demonstrated by its absorption spectrum, which lacks the peaks characteristic of heme-containing proteins (Fig. 1b). The HemA spectrum is that of a b-type heme; this class of molecules is attached noncovalently. Treatment with the strong denaturant, 6 M guanidine-HCl, removed a maximum of 7% of heme from HemA, and in two trials, failed

to remove any (Supporting Information). The ability to retain noncovalently bound heme in the presence of strong denaturants has been documented for other proteins (Hargrove & Olson, 1996; Wójtowicz et al., 2009). Although these results demonstrate a strong association between heme and HemA, covalent binding cannot be inferred from this assay. Thiol reagents, which have been used to distinguish covalent heme-protein bonds, are incompatible with Ni-NTA. The nature of the association between heme and HemA was further examined using a different method. Heme-associated peroxidase activity, which can be measured by standard ECL reagents (a Western blot without the antibody; Dorward, 1993), detects heme-binding proteins (such as cytochrome c). Purified proteins were separated by SDS-PAGE and then assessed for heme-associated peroxidase activity.

The factors included in the fishbone diagram were brainstormed by the members of the team and were based on individual experience. The factors were not quantified. Of these reasons, the team specifically focused on ‘provider factors’ because among physicians there may be low awareness of venous thromboembolism evidence-based guidelines.[2] Several published studies have proposed multifaceted strategies

to change physician prescribing behaviour including education and incorporating the task into the physician’s workflow.[3, 4] Based on these strategies, the team brainstormed various interventions that could influence these ‘provider factors’ (Table 1). Create poster reminder to perform a DVT risk assessment. Conduct an in-service Protein Tyrosine Kinase inhibitor regarding the importance of DVT prophylaxis ABT-199 solubility dmso Nurse driven risk stratification and prophylaxis order Pharmacist

driven risk stratification and prophylaxis order Force function for DVT score and orders in the electronic medical record Computerized physician order entry Computerized DVT prophylaxis reminders The GIM team felt that the best intervention would be to embed the DVT risk-assessment tool and DVT orders into a standardized physician admission order-set and to educate users regarding the availability of the order-set. Users were not informed that the order-set was created to improve DVT prophylaxis rates. The team then created an aim statement that stated: ‘This Rucaparib project will increase the percentage of newly admitted GIM patients receiving optimal

DVT prophylaxis by developing a standardized medicine admission order-set with an embedded risk-assessment tool and DVT prophylaxis orders. The preliminary review indicated that there were 65 admitted GIM patients in a 1-month period. Of the 65 patient charts, VTE forms were completed by a physician in only two charts (3%). Of the 65 patients admitted, only 49 (75%) received appropriate prophylaxis. Two-month post-intervention data indicated that of 72 GIM patients audited in a 1-month period, the standardized admission orders were used 86% of the time and that 91% of the patients received optimal DVT prophylaxis. The number of patients receiving correct DVT prophylaxis increased from 75% to 91%. Chart review 1 year after the implementation of the order-set revealed that the increase in DVT prophylaxis was sustained at 95% even after the project was complete. Utilization of the embedded risk-assessment tool for DVT prophylaxis increased from 3% to 86% but declined to 64% at the 1-year review (Figure 2). However, the use of the DVT orders within the order-set remained high at 90%. Of the 72 patient charts audited at 2 months, patients were more likely to receive prophylaxis (94%) when the standardized order-set was completed versus when the orders were not completed (70%).

, 2005). The expression of virF, which encodes a positive regulator of type III secretion genes, is enhanced by the direct binding of CpxR to its promoter (Nakayama & Watanabe, 1998). In an interesting example of post-transcriptional regulation by the Cpx response, the protein levels of InvE, but not its mRNA abundance, are decreased in a cpxA mutant of Shigella sonnei, in which the Cpx response is presumably constitutively activated (Mitobe et al., 2005). In

Legionella pneumophila, CpxR has been shown to positively regulate the transcription of numerous components of the Icm/Dot type IV secretion system and its substrates, including selleck products the chaperone IcmR (Gal-Mor & Segal, 2003); the structural subunits IcmV, IcmW, DotA and LvgA (Vincent et al., 2006; Altman & Segal, 2008); and a host of newly identified Icm/Dot translocated substrates (Altman & Segal, 2008). Curiously, mutations in either cpxR or cpxA have no effect upon L. pneumophila intracellular growth within macrophages or amoebae (Gal-Mor & Segal, 2003). The benefit of Cpx regulation of type IV secretion in L. pneumophila therefore remains

to be determined. In contrast to these results, recent studies have suggested that in many pathogens, activation of the Cpx response is detrimental to virulence (Table 1). In several organisms, mutations in cpxA, which in many cases result in an accumulation of phosphorylated CpxR (Wolfe et al., 2008; Malpica and Raivio, in preparation), have been found to decrease expression of adhesins and adherence to host cells. For example, expression of the Src inhibitor EPEC BFP, the UPEC Pap pilus and invasin, a nonfimbrial adhesin produced by Yersinia pseudotuberculosis, is decreased in cpxA mutant strains (Hernday et al., 2004; Carlsson et al., 2007b; Vogt et al., 5-FU datasheet 2010). In addition, a Salmonella enterica serovar Typhimurium cpxA mutant has defects in host cell adherence, although the specific adhesin affected in this strain was not determined (Humphreys et al., 2004). The Cpx response therefore appears to have

a conserved role in the repression of adhesive structures. Expression of several virulence-associated protein secretion systems is also reduced by mutations in cpxA, including the EPEC and Yersinia enterocolitica type III secretion systems and the Haemophilus ducreyi LspB-LspA2 two-partner secretion system (Carlsson et al., 2007a; MacRitchie et al., 2008b; Labandeira-Rey et al., 2009). Accordingly, the S. Typhimurium and H. ducreyi cpxA mutants were also found to be less virulent in infection models (Humphreys et al., 2004; Spinola et al., 2010). As suggested earlier, this repression of adhesive structures and secretion systems by the Cpx response may be a pre-emptive mechanism to prevent further envelope protein misfolding.

p.m. precursor tolerance, 0.35 Da MS/MS (analysis of the tandem mass) fragment tolerance, carbamydomethyl LBH589 chemical structure cysteine (CAM) as fixed modification, and oxidized methionine as variable modification, allowing one missed cleavage. All spectra and database results were manually inspected in detail using the above software. Protein scores greater than 56 were accepted as statistically significant (P < 0.05), and the identification was considered positive when the protein score confidence interval (CI) was above 98%. In the case of MS/MS spectra, the total ion score CI was greater 95%. Similarity percentages

between V. tapetis isolates were calculated on the basis of protein profile similarities calculated between pairs of isolates using the simple matching co-efficient (Sneath & Sokal, 1973). bionumerics 5.1 2D software (Applied-Maths) was used to construct a maximum parismony tree based on the different protein content of V. tapetis isolates. Genomic DNA extraction and amplification of the 16S rRNA gene was performed as previously described (Beaz-Hidalgo et al., 2008). Sequences for five protein-coding housekeeping genes, atpA (α subunit of ATPase), pyrH (uridyl monophosphate kinase),

recA (recombinase A), rpoA (α subunit of RNA polymerase) and rpoD (RNA polymerase sigma factor), were performed according to Thompson et al. (2004, 2005, 2007) and Pascual et al. (2010). Sequencing reactions were performed with the GenomeLab DTCS-Quick Start kit (Beckman Coulter, click here Ireland). Clomifene Sequence data analysis was performed with the dnastarseqman program (Lasergene). The percentage of similarity of concatenated sequence of genes was calculated using the dnastarmegaling program (Lasergene). For maximum-likelihood (ML) analysis, the optimal model of nucleotide substitution was estimated with the program jmodeltest 0.1.1 (Posada, 2008) using the Akaike information criterion. The ML estimation was implemented in phyml (Guindon & Gascuel, 2003), using the GTR model as recommended by jmodeltest 0.1.1.

Bootstrap analyses were performed using 1000 replications. The three strains yielded different numbers of spots in 2-DE gels, despite loading the same quantities of protein. There were 729 (± 13 standard deviation), 681 (± 2) and 556 (± 6) spots for CECT 4600T, GR0202RD and HH6087, respectively (Fig. 1). Technical replicates showed a high degree of congruence (0.91 for CECT 4600T and GR0202RD, 0.85 for HH6087) (Fig. 1). Visual inspection of gels showed that the majority of proteins detected were localized in the acidic part of the pH range studied and they also showed similar or different protein profiles depending on a specific molecular weight region (Fig. 2). Thus, the high molecular weight region was very similar in all strains, whereas the low molecular weight region was more similar between CECT 4600T and GR0202RD strains than between CECT 4600T and HH6087 strains.

In the mouse, each layer 4 barrel is composed of a central region of high density neuropil, containing the clustered VPM axonal arborizations surrounded by a cell-dense wall of layer 4 neurons that orient their dendritic and axonal arborizations into one specific barrel (Woolsey et al., 1975). Optical stimulation has been used to study the functional projection from VPM thalamus to barrel cortex, revealing prominent VPM glutamatergic input onto neurons located in layer 4, layer 5B and layer 6 (Bureau et al., 2006; Petreanu et al., 2009; Cruikshank et al., 2010). Thalamocortical POM

neurons also project to the primary somatosensory barrel cortex, terminating densely in layer 1 and layer 5A. Functional characterization of this projection has revealed a prominent POM input onto layer 5A barrel cortex neurons (Bureau et al., www.selleckchem.com/products/AG-014699.html 2006; Petreanu et al., 2009). In the rat, several subdivisions and additional parallel pathways have been characterized from the principal trigeminal and spinal trigeminal nuclei via different subdivisions of the VPM and POM thalamus (Deschenes, 2009). It has been suggested that Selumetinib purchase these parallel pathways in the rat process different aspects of whisker sensorimotor information (Yu et al., 2006).

However, in the mouse, little is currently known about the differential sensory information signalled by VPM and POM neurons, and further experimental work focusing on these issues will be of great importance. Progress has also been made toward defining the synaptic circuits within mouse S1 barrel cortex through simultaneous whole-cell recordings (Lefort et al., 2009) and glutamate uncaging Interleukin-2 receptor (Bureau et al., 2006; Xu & Callaway, 2009), providing complementary data to that obtained in rat (Lübke & Feldmeyer, 2007; Schubert et al., 2007). These studies have revealed several prominent synaptic pathways

for processing sensory information within a cortical barrel column (defined as the entire thickness of the cortex from layer 1 to layer 6 and laterally bounded by the extent of the layer 4 barrel). Specific investigation of the microcircuits in the C2 barrel column revealed that excitatory neurons in layer 4 dominate synaptic connectivity within this barrel column (Lefort et al., 2009). Layer 4 neurons signal to neurons located in all other cortical layers and they are therefore able to robustly transmit to the entire barrel column the tactile information received via the dense layer 4 innervation by VPM. Other prominent neocortical signalling pathways in the mouse C2 barrel column are from supragranular to infragranular layers, with an interesting elevated reciprocal connectivity between layer 2 and layer 5A (Bureau et al., 2006; Lefort et al., 2009). In vivo recordings from mouse barrel cortex neurons are beginning to shed light on how these neocortical microcircuits operate functionally during behavior (Crochet & Petersen, 2006; Poulet & Petersen, 2008; Gentet et al., 2010).

2% sequence similarity). DNA–DNA hybridization comparisons demonstrated a 64.8% DNA–DNA relatedness between strain E13T and A. flavithermus DSM 2641T. On the basis of phenotypic characteristics, phylogenetic data and DNA–DNA hybridization data, it was concluded that the isolate merited classification as a novel subspecies of A. flavithermus, for which the name Anoxybacillus flavithermus ssp. yunnanensis ssp. nov. is proposed. The type strain of this subspecies is E13T (=CCTCC AB2010187T=KCTC 13759T). Organic-solvent-tolerant bacteria are a relatively new subgroup of extremophiles.

They are able to overcome the toxic and destructive effects of organic solvents on account of their unique adaptive mechanisms. Ethanol (log Pow=−0.32) (Pow=partitioning coefficient n-octanol/water) is a low toxic compound when compared with extremely toxic solvents with a log Pow value between 1.5 and 4.0. Several mesophilic bacteria capable of Dabrafenib research buy tolerating high concentrations of ethanol have been investigated extensively. For example, Lactobacillus heterohiochii (a later heterotypic synonym of Lactobacillus

fructivorans) and Zymomonas mobilis exhibited tolerance to ethanol up to 18% (% value is in v/v) (Ingram 1990) and 13% (Liu & Qureshi, 2009), respectively. However, thermophilic bacteria rarely tolerate >2% ethanol (Rani & Seenayya, 1999; Burdette et al., click here 2002), primarily because the level of ethanol tolerance decreases drastically with increasing temperature (Georgieva et al., 2007). Recently, a mutant strain of Thermoanaerobacter ethanolicus 39E-H8 has been reported to survive

and grow weakly in up to 8% ethanol at 60 °C (Burdette et al., 2002). Ethanol tolerance (maintain viability) as high as 10% has been reported in Geobacillus thermoglucosidasius M10EXG (Fong et al., 2006). There is no report of thermophilic bacterial strains capable of active growth in 8% ethanol, or growth in concentrations above 10%. In the search for new thermophilic ethanol-tolerant bacteria, samples taken from hot springs were screened by ethanol enrichment, resulting in the isolate E13T. It exhibits a unique and remarkable ability to Vildagliptin preferably grow in the presence of ethanol (up to 8%) at high temperature and is able to tolerate 13% ethanol at 60 °C. The phylogenetic 16S rRNA gene sequence analysis revealed that strain E13T is affiliated with the recently established genus Anoxybacillus (Pikuta et al., 2000). At present, the genus Anoxybacillus comprises 15 species with validly published names. Only Anoxybacillus kamchatkensis contains two subspecies (Gul-Guven et al., 2008). None of these Anoxybacillus strains is reported to tolerate ethanol. On the basis of phenotypic features as well as molecular studies, we propose to classify the strain E13T as a novel subspecies, Anoxybacillus flavithermus ssp. yunnanensis ssp. nov.

2 to 5.5. This result supports an involvement of LCP proteins in a late step of WTA synthesis in S. aureus. As LCP proteins in B. subtilis are essential, it could be that the staphylococcal LCP triple mutant is only viable because of compensatory mutations, which remains to be verified. However, it is also possible that the functions of LCP proteins in S. aureus are not identical to those in B. subtilis, www.selleckchem.com/products/epacadostat-incb024360.html because differences

have been found in the WTA synthesis pathways of these closely related bacteria (Brown et al., 2010). Also, in contrast to S. aureus, WTA-deficient strains in B. subtilis have significantly decreased growth rates and lost their rod shape, indicating potential differences in the roles of WTA ligases in B. subtilis and S. aureus cell division (Weidenmaier et al., 2004; D’Elia et al., 2006). Measurement of CWSS expression in an S. aureus SA113ΔtarO (ΔtagO) mutant (Weidenmaier et al., 2004), with the reporter plasmid psas016p-luc+, revealed that inhibition of the first step of WTA synthesis induces the CWSS (Fig. 4b). This result is in conflict to the observations by Campbell et al., (2011) who showed that inhibition of TarO (TagO) by subinhibitory concentrations of tunicamycin

does not induce the CWSS. They suggested that CWSS induction is triggered by the sequestration of Ruxolitinib the lipid carrier rather than WTA deficiency (Campbell et al., 2011, 2012). However, our analysis of the tarO (tagO) mutant indicates that further research is required to reveal the actual trigger of CWSS

induction. Deletion of LCP proteins increased basal expression levels of CWSS genes via the VraSR two-component system. The LCP triple mutant showed very high basal expression of the CWSS, close to levels triggered by antibiotic stress. The LCP double and single mutants, however, still responded to cell wall stress by further upregulating the CWSS. Promoter regions required for VraR-dependent induction of the LCP genes and sas016 shared low overall nucleotide similarity, but all contained fragments of the predicted CesR-like binding consensus or the VraR-binding motif of the vraSR operon and all were in close proximity to the −35 box of the gene’s promoter. Hyper susceptibility of the triple mutant to bacitracin, the virtual absence of WTA and partial restoration of WTA levels by complementation with each of the single LCP Teicoplanin proteins, as well partial complementation of its growth defect by TarO (TagO) inhibition, support the hypothesis that S. aureus LCP proteins have WTA ligase functions, as suggested by Kawai and colleagues for B. subtilis (Kawai et al., 2011). An enzymatic analysis of all three LCP proteins will be required to confirm their specific WTA ligase functions, substrates and products. We thank C. Weidenmaier for providing the tarO mutant strain. The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 241446 (project ANTIRESDEV).

Gene inactivation in Y. enterocolitica was performed by plasmid insertion through homologous recombination

using the conjugative suicide vector pDS132 (Philippe et al., 2004). Plasmids for generating inv and flhDC null mutants selleck chemical were constructed using PCR-generated intragenic DNA fragments. A 900-bp fragment of inv was amplified using the primers inv1 (5′-TCTCTAGAGTGCGCTTCCCAGTAAAGTC-3′) and inv900 (5′-TCGAGCTCGCCAAACTTCCCCACTCTC-3′), and a 300-bp fragment of flhDC was amplified using primers flhDCXba (5′-TCTAGATCATATTTGCTTTTAGCACAACG-3′) and flhDCSac (5′-TCGAGCTCTCTTTTCTTAGACGCACTACCG-3′). The PCR-generated DNA fragments were digested with XbaI and SacI and ligated to XbaI/SacI-digested pDS132. The resulting constructs pDSinv and pDSfdc were transferred from E. coli S17-1 λpir to Y. enterocolitica strain Ye9N by biparental conjugation. Strains harboring plasmids integrated into the chromosome were recovered by selecting for CmR. The insertion mutant strains obtained by this strategy were designated DC2 (inv) and DN1 (flhDC). Correct integration at the inv or flhDC loci was confirmed by PCR (data not shown). Swimming assays were performed using tryptone broth (TB) motility agar plates consisting of 10 g tryptone L−1 and 0.3% Bacto agar.

Strains were grown overnight in TB medium at 25 °C and a 4-μL aliquot was spotted onto the plates, which were then incubated at 25 °C. After overnight Cell Cycle inhibitor incubation, the plates were photographed and the swimming zones were evaluated. Bacteria were grown overnight in LB medium at 25 °C. HEp-2 human epithelial cells were cultured in 12-well plates containing Dulbecco’s modified Eagle’s medium (Sigma) supplemented with 5% heat-inactivated fetal bovine serum (Sigma). Monolayers (5 × 105 HEp-2 cells) were infected at a multiplicity of infection of 10 bacteria per epithelial cell and incubated in a 5% CO2 atmosphere at 37 °C. After 90 min, the medium was removed and the HEp-2 cells were washed three times with phosphate-buffered saline (pH 7.2) to remove nonadherent bacteria. The cells

were then lysed with 1% Triton X-100 for 5 min and the number of CFU corresponding to Florfenicol the total number of cell-associated bacteria was determined by plating on LB medium. Adhesion (adherence) was calculated as the percentage of cell-associated bacteria. To determine the number of intracellular bacteria, infected and washed HEp-2 cells were incubated for a further 90 min in fresh cell culture medium containing 100 μg mL−1 gentamicin to eliminate extracellular bacteria. The cells were then washed (two times) to remove the antibiotic and lysed with 1% Triton X-100 for 5 min to release intracellular bacteria, and the number of CFU was determined by plating on LB medium. Invasion (invasiveness) was calculated as the percentage of gentamicin-resistant (i.e. intracellular) bacteria.

When we considered factors that could change over follow-up, a greater number of CD4 counts < 350 cells/μL and a greater proportion of CD4 counts < 350 cells/μL were both strong predictors of more rapid ART uptake. Furthermore, those with a higher average CD4 cell count over follow-up or a higher CD4 percentage were less likely to start ART, whereas those with higher viral loads were more likely to start. Later calendar year of follow-up was also associated with more rapid ART uptake.

Many of these factors remained significantly associated with ART uptake after adjustment for confounding factors (right-hand side of Table 2). In particular, Dabrafenib mw compared with male heterosexuals, female heterosexuals were 13% more likely to start ART [adjusted relative hazard (aRH) 1.13], whereas injecting drug users (IDUs) were 47% less likely to do so (aRH 0.53). Epacadostat Compared with those of White ethnicity, those of unknown ethnicity were less likely to start ART (aRH 0.69). A previous AIDS diagnosis (aRH 1.14), older age (aRH per 10-year increment 1.15) and later calendar year of follow-up (aRH per later year 1.20) were all independently associated with more rapid ART uptake. The

results from the multivariable analysis confirmed that patients with a greater number of CD4 count measurements < 350 cells/μL (aRH per additional count 1.18) and those who had a lower CD4 count over follow-up (aRH per 50 cells/μL higher average CD4 count

0.57) remained more likely to start ART. The association with the first CD4 count < 350 cells/μL was reversed in this final analysis (aRH Histidine ammonia-lyase per 50 cells/μL higher 1.18), suggesting that ART was most likely to be started in individuals experiencing a more rapidly declining (i.e. a high nadir and a low follow-up value) CD4 count. In addition to the associations with the CD4 cell count itself, individuals with higher CD4 percentage values (aRH per 5% higher 0.90) were less likely to initiate HAART, whereas those with higher viral loads (aRH per log10 copies/mL higher 1.06) were more likely to initiate HAART. The benefits of starting ART once the CD4 count has fallen below 350 cells/μL have become more evident over recent years, with guidelines evolving to reflect this. Unfortunately, most analyses that have been performed to assess adherence to guidelines are complicated by the fact that many patients only present for the first time once their CD4 cell count has already fallen below the recommended threshold [9-12]. Our results, which demonstrate that only 9.0% of patients with a CD4 count < 350 cells/μL from 2004 to 2008 remained untreated when last seen in 2008, suggest that clinics are successfully following guidelines.