Although only the protein synthesis inhibitors resulted in increased tmRNA expression, a study by Luidalepp et al. (2005) indicated that disruption of trans-translation increased susceptibility to inhibitors of cell wall synthesis as well as to ribosome inhibitors. It was speculated that this reflected an impaired stress response in the trans-translation-deficient organism. However, the lack of a change in tmRNA expression in mycobacteria exposed to cell wall synthesis inhibitors suggested that any

stress response elicited by these agents in mycobacteria did not include trans-translation. The observed changes in tmRNA expression following ribosome inhibition with antimicrobial agents conflict somewhat with the findings of Moore PTC124 purchase & Sauer (2005), who reported that an increased requirement for trans-translation did not increase expression of tmRNA in E. coli. This suggested that bacteria have a significant tmRNA-SmpB reserve capacity. However, there was a key difference between the Moore & Sauer (2005) study and the antimicrobial agents studies presented here and elsewhere (Montero et al., 2006; Paleckova click here et al., 2006). In the Moore & Sauer (2005) study, an increase in trans-translation was directly, and canonically, triggered by overexpression of a transcript lacking a stop codon.

In the other studies, the primary effect of the antimicrobial agents was inhibition Cyclic nucleotide phosphodiesterase of ribosome function, most likely including inhibition of trans-translation. This suggested that the changes in tmRNA expression following exposure to ribosome inhibitors may not have been in response to increased trans-translation. Although ribosome inhibitors, such as erythromycin,

cause ribosome stalling (Rogers et al., 1990; Min et al., 2008), there is evidence that the state of the ribosome is fundamentally different to the stalling associated with triggering of trans-translation. For instance, tRNA is believed to still be able to access the A-site of ribosomes inhibited by agents such as aminoglycosides and macrolides (Walsh, 2003), although the A-site is believed to be vacant when trans-translation is triggered canonically (Moore & Sauer, 2007). Furthermore, there is evidence that translation complexes inhibited by macrolides can dissociate (suggested by the release of peptidyl-tRNA) in the absence of trans-translation (Tenson et al., 2003). Triggering of trans-translation may occur as an indirect effect of drug-associated ribosome dysfunction. For example, aminoglycosides and macrolides can cause translation errors such as frameshifts and stop codon readthrough (Martin et al., 1989; Schroeder et al., 2000; Thompson et al., 2004), which could lead to ribosomes reaching the end of a transcript without encountering a translation termination signal.

The key result from the project was the formulation of national pharmacy learning outcomes and exemplar standards (PhLOS) for all students graduating

from entry-level pharmacy programmes. These have been endorsed by both students and academics. Learning outcomes have been developed through a collaborative process for pharmacy programmes TSA HDAC price across Australia through harmonisation of the various expectations and regulatory requirements for pharmacy education programmes. Application of these learning outcomes and exemplar standards will ensure that all graduates of all entry-level pharmacy programmes will have achieved at least the same threshold, regardless of the university from which they graduate prior to entering their internship year. “
“Objectives  The study evaluated the compliance of community pharmacies with legal requirements as laid down by the drug regulatory framework in Pakistan. Methods  An exploratory cross sectional survey was conducted with a total of 371 randomly selected community pharmacies in three cities in Pakistan, namely Islamabad (n = 118), Peshawar (n = 120) and Lahore (n = 133). A questionnaire ICG-001 was developed and finalized by focus-group discussions and pilot

testing. The questionnaire included background information and a legal requirement scale consisting of six subscales: licensing requirements, premises requirements, storage requirements, documentation requirements, narcotics section requirements and prescription checking. The data were coded, entered and analysed using SPSS software (version

16). Kruskal–Wallis, Mann–Whitney and chi square tests were used for analysis. Key findings  The pharmacies were operating with one of the three licence types operating in Pakistan: type A (n = 96, 25.9%), type B (n = 186, new 50.1%) and type C (n = 89, 24.0%). A narcotics licence was issued to 133 (35.8%) pharmacies; licences of 66 (17.8%) pharmacies were expired while the validity of 87 (23.0%) licences could not be determined. Only 113 (30.5%) pharmacies were totally clean. Eighty percent of the pharmacies had a refrigerator for storage of medicines, but only 284 (76%) of the refrigerators were in working condition. Complete medicine purchase records with warranties were available at 210 (56.6%) pharmacies. Conclusions  None of the pharmacies completely complied with the legal requirements in terms of licensing, premises, storage, documentation, narcotics section, drug labelling and prescription checking. This speaks of poor regulation and control by health authorities on the sale and dispensing of medicines in Pakistan. This study will serve as a baseline for policy makers, managers, researchers and other stakeholders in developing designs for future interventions as well as for methods of accountability and control.

The tryptic peptide mixture was eluted with 0.1% this website formic acid. LC-MS/MS analysis was performed using a Thermo Finnigan’s ProteomeX workstation LTQ linear ion trap MS (Thermo Electron, San Jose, CA) equipped with NSI sources (San Jose, CA) as reported previously (Heo et al., 2007). Briefly, 12 μL of peptide from the

in-gel digestion was injected and loaded onto a peptide trap cartridge (Agilent, Palo Alto, CA). Trapped peptides were eluted onto a 10 cm reversed-phase (RP) PicoFrit column packed in-house with 5 μm, 300 Å pore size C18, followed by gradient elution. Mobile phases consisted of H2O (A) and ACN (B) containing 0.1% v/v formic acid. The flow rate was maintained at 200 nL min−1. The gradient started at 2% B, reached 60% B in 50 min, 80% B in the next 5 min and 100% A in the final 15 min. Data-dependent acquisition (m/z 400–1800) was enabled, and each survey MS scan was followed

by five MS/MS scans with dynamic exclusion within 30 s. The spray voltage was 1.9 kV and the temperature of the ion transfer tube was set to 195 °C. The normalized collision energy was set to 35%. Tandem mass spectra were extracted, and the charge state was deconvoluted and deisotoped using sorcerer 3.4 beta2 (Sorcerer software 3.10.4, Sorcerer Web interface 2.2.0 r334 and Trans-Proteomic Pipeline 2.9.5). All MS/MS samples were analyzed using sequest (Thermo Finnigan, San Jose, CA; version v.27, rev. 11), which was set to search the NCBI database (L. Veliparib order monocytogenes, 6365 entries) with semiTrypsin as the digestion enzyme. SEQUEST search parameters set the fragment ion mass tolerance to 1.00 Da and the parent ion tolerance to 1.5 Da. Oxidation of methionine and the addition of iodoacetamide to cysteine were specified as fixed modifications. nearly To improve false-positive statistics, the decoy option was selected while searching data using the sorcerer program, consequently improving the results by reducing noise. scaffold (version Scaffold-02_04_00, Proteome Software Inc., Portland, OR) was used to validate MS/MS-based peptide and protein identifications. Identifications were accepted only if proteins had a probability >95.0% and

contained at least two identified peptides, as specified by the Peptide Prophet algorithm (Keller et al., 2002). Protein probabilities were assigned by the Protein Prophet algorithm (Nesvizhskii et al., 2003). Proteins containing similar peptides that could not be differentiated based on MS/MS analysis alone were grouped together in order to satisfy the principles of parsimony. The peptide false-positive rate (FPR) was calculated using the scaffold software. For each charge state, the incorrect assignments were tabulated to calculate the FPRi=[(#assigned incorrect at 95% probability)/(total# incorrect assigned)] × 100, with i being the charge state. An assignment was considered correct if associated with a protein that has 95% probability, according to the Protein Prophet algorithm (Hendrickson et al.

, 2009) to obtain pKT-cra, which was then transformed into the Δcra strain. Stationary-phase overnight cultures grown in YLB medium at pH 7.0 were diluted to 106 CFU mL−1 in PBS at pH 4.5 and incubated at 37 °C for 2 h. The cultures were serially diluted and plated onto YLB agar plates and colonies were counted after 20 h growth at 37 °C. Percent survival was calculated as described previously (Hu et al., 2009). All assays were repeated at least three selleck chemical times and the data were analyzed by Student’s

t-test. We applied 2D gel to screen proteins whose expression was induced or repressed at pH 4.5, which is a sublethal pH for YpIII (Hu et al., 2009); 21 proteins showed more than twofold changes in all three replicate experiments (Fig. 1). These proteins were identified by MALDI-TOF MS and are summarized in Table 1. Among these proteins, eight proteins involved in carbohydrate metabolism were up- or downregulated over twofold at pH 4.5 (Fig. 2a). It is worth noting that the three proteins that were involved in the beginning step of

fructose metabolism Protein Tyrosine Kinase inhibitor (FruB-1, FruB-2, FruK) (Ow et al., 2007) were all upregulated by acid challenge (Fig. 2a and b). To further confirm the increased expression of fruBKA at acidic pH, we constructed translational lacZ fusions of fruB∷lacZ and fruA∷lacZ, which are located at the beginning and end of the fruBKA transcription unit (Fig. 3a). As seen in Fig. 3b, in accordance with our 2D gel results, higher β-galactosidase activities of both fruB∷lacZ and fruA∷lacZ fusions were observed at pH 4.5 than at pH 7.0, suggesting that expression of fruBKA is acid induced. Expression of the fruBKA operon encoding FruB, FruK and FruA was reported to be negatively controlled by a transcription factor Cra at physiological pH in several bacteria (Saier & Ramseier, 1996). This raised the question of whether the acid-induced fruBKA expression is mediated by Cra. To address this question, we constructed translational cra∷lacZ fusion and compared the β-galactosidase activities with or without acid challenge. β-Galactosidase

activities of cells challenged with acid were obviously lower than those without challenge, suggesting cra expression is repressed by acid (Fig. 4a). Furthermore, we constructed the cra deletion strain named Δcra and compared fruB and fruA Fenbendazole expressions in Δcra and YpIII wild-type strains. Expressions of fruB and fruA were both acid induced in YpIII wild-type strain (Fig. 4b and c). But there was no significant difference of β-galactosidase activities at pH 7.0 and at pH 4.5 in Δcra, although the values in Δcra were obviously higher than in YpIII, which confirmed the Cra regulates fruBKA expression in YpIII. Together, these results suggested that the acid induction of fruBKA expression is mediated by repressed expression of Cra at acidic pH. It was established that Cra acts as a global regulatory protein (Crasnier-Mednansky et al.

9) in patients with a CMV viral load >400 copies/mL. Unlike Deayton et al. [21], we found a significant association between baseline CMV DNA and the progression to other ODs. In the case of the significant

association between detectable CMV DNA in plasma and ODs or death, CMV reactivation can be considered as a marker of immune suppression and impaired CD4 cell function in patients positive for CMV IgG. Panagiotakis et al. observed that CMV DNAemia detected in the peripheral blood lymphocytes of patients with CD4 counts <200 cells/μL was correlated with a delayed increase Epacadostat solubility dmso in CD4 count after initiating HAART [24]. CMV is also considered to function as a cofactor as it interacts at the molecular or cellular level to promote HIV pathogenicity learn more and the progression of AIDS [25]. Moreover, CMV encodes a large number of immunomodulatory functions which modulate both the innate and the adaptive arms of the immune response

[26]. It seems that increased inflammation benefits CMV dissemination [26] and prostaglandins, such as tumour necrosis factor (TNF)-α, released during inflammation may contribute to CMV reactivation [27]. This mechanism could explain why asymptomatic CMV viraemia has been detected in critically ill immunocompetent patients and patients with septic shock [28,29]. It is therefore no surprise that the best prognostic performance of CMV DNA was achieved for CMV end-organ disease (AUC 0.81), and that the prognostic performance increased during the first 6 months. In the case of other ODs and death, the performance was acceptable (AUC 0.77 and 0.61, respectively) during the first 6 months, and then became of marginal acceptability. Our study has several limitations inherent to retrospective analyses of prospectively collected data; in particular, the limitation of the original threshold and the impossibility of serial measurements, which may have emphasized the difference between measuring constant detectable low levels of CMV DNA and increasing levels over time. This in turn would enable determination enough of the best cut-off CMV DNA level

in plasma to maximize its predictive value. The low frequency of CMV end-organ disease is also a limitation which may have resulted in a lack of power in the detection of factors associated with this event and a limitation in the number of adjustment factors in the Cox multivariate models. Despite this, the association between CMV viraemia and our end-points is strong and significant. We used a cohort of patients that encompassed most of the Swiss HIV-infected population and was representative of the patients encountered in Western clinics. Compared with previous studies, our cohort of patients was larger, represented a greater number of endpoint cases, covered the period after 2003 and used a newer and more sensitive PCR.

We did find an increased prevalence of carotid lesions among HIV-infected men compared with HIV-uninfected men in our sample. Our findings are slightly different from those of the previous detailed analysis of carotid IMT data from the MACS [13], which included more men and adjusted for different confounders BMN 673 solubility dmso in the analysis. Antiretroviral therapy is associated with insulin resistance, diabetes, and hyperlipidaemia, all of which contribute to the development of CVD [33-35]. Results from previous studies of the association between antiretroviral therapy

and CVD have been inconsistent, with some showing no association [36, 37] and others showing an association [2, 38]. A large retrospective study of Veterans Affairs patients [36] showed no increase in CVD mortality related to antiretroviral therapy. Interestingly, a large prospective study of treatment interruptions based on CD4 cell count revealed NU7441 ic50 that individuals who were on antiretroviral therapy continuously had a lower incidence of major CVD than individuals who had structured interruptions in their therapy [39]. Antiretroviral therapy has not consistently been associated with subclinical CVD assessed by IMT or CAC. In a previous analysis from the MACS Cardiovascular Substudy focused on IMT, low CD4 T-cell count, but not antiretroviral

therapy, was positively associated with an increased prevalence of carotid lesions [13]. There was, however, a trend towards an association between PI use and carotid lesions in men. A small AIDS Clinical Trials Group (ACTG) study assessed subclinical CVD using IMT and revealed no atherogenic effect of HIV status or prolonged PI therapy [40]. An analysis of the MACS Cardiovascular Substudy focused on CAC revealed that increasing IMP dehydrogenase age was most strongly associated with both the prevalence and the extent of CAC, and long-term HAART use was associated

with a decreased extent of calcification among individuals who had calcification [13]. In our study, current PI use was associated with carotid lesion presence, but not the other measurements of subclinical CVD. CAC and IMT provide valuable information about early atherosclerotic changes to identify subclinical CVD. These tests are not currently recommended as screening tools in asymptomatic individuals, but may be helpful in individuals with intermediate CVD risk in whom additional information may influence treatment decisions. Both CAC and IMT have been prospectively associated with the development of CVD. Data from the large, prospective Multiethnic Study of Atherosclerosis revealed that CAC is a better predictor of coronary heart disease while IMT is a better predictor of stroke [41]. Noncalcified plaques, which are not measured by CAC, are more likely to rupture and cause acute myocardial infarction. However, individuals with more calcified plaques (higher CAC) are also more likely to have more noncalcified plaques.

The nucleotide and amino acid sequences were compared with the EMBL, SwissProt and GenBank databases. blast searches were carried out at the NCBI (http://www.ncbi.nlm.nih.gov/BLAST/). DNA sequences were analysed using the sci-ed software package. Sequence alignments were performed with the clustalw2 program of the EBI (http://www.ebi.ac.uk/Tools/clustalw2/),

and visualized with the jalview 2.6.1 software (Waterhouse et al., 2009). Total RNA was extracted from late-exponential phase E1 cells cultivated on acetate, n-dodecane, n-hexadecane, Belinostat cost n-octadecane and n-eicosane using the TRIzol reagent (Amersham Pharmacia) and method. To prepare DNA-free RNA, 15 μg of total RNAs was treated with 5 U of RNase-free DNase I (Fermentas) according to the supplier’s protocol. The quantity and the quality of the recovered RNAs were verified by means of spectrophotometry (Nanodrop 1000) and agarose gel electrophoresis. First-strand cDNA synthesis of 2 μg of total RNA in a final volume of 20 μL was carried out with RevertAid M-MuLV Reverse Transcriptase (Fermentas), using random hexamers. For real-time PCR, 1 μL of cDNA was mixed

with Power SYBR Green PCR Master Mix (Applied Biosystems), 5 pmol of forward primer and 5 pmol of reverse primer in a final volume of 20 μL in three replicates. No-template controls were included. The primers for the 16S rRNA gene and for AZD5363 nine selected ORFs were designed using the primer express software (Applied Biosystems). Real-time PCR was carried out with the ABI Prism 7000 Sequence Detection System (Applied Biosystems) with the following protocol: 45 cycles at 95 °C for 15 s, followed by 60 °C for 1 min. The

specificity of the amplifications was verified at the end of the PCR run through use of the abi prism dissociation curve analysis software. The expression levels of investigated genes were normalized to 16S rRNA gene levels and were correlated to the amounts of the corresponding transcripts in samples grown on acetate. The normalized relative transcript levels were obtained by the method (Livak & Schmittgen, 2001). The expression aminophylline vectors for complementation studies were constructed applying the PCR products amplified by alkBPromF and rubCFLAG primers from Dietzia spp. The PCR fragments were EcoRI digested and ligated between the HindII/EcoRI sites of the streptomycin cassette-carrying pNV18Sm shuttle vector (Szvetnik et al., 2010). The plasmid pNV18Sm-E1BRF obtained was introduced into either wild-type or ΔBR cells, while pNV18Sm expressing AlkB-Rubs of four other Dietzia spp. was introduced into ΔBR cells (Table 1). Control transformations with pNV18Sm vectors were also included. The growth kinetics of each cell line on n-eicosane was determined as described above.

01; 95% CI 0.76–1.35). Smoking was learn more also not associated with increased risk of AMI (HR 1.01; 95% CI 0.78–1.30). In addition to HCV, factors associated with CVD in multivariate analysis were greater age (HR: 1.65; 95% CI 1.54–1.76; P<0.001) and hypertension (HR 1.48; 95% CI 1.28–1.75; P<0.001). Type 2 diabetes mellitus again was associated with increased risk of CVD in unadjusted analysis (HR 1.56; 95% CI 1.32–1.85) but not in the adjusted model (HR 1.05; 95% CI 0.88–1.25). Duration of ART was not associated with

CVD in the adjusted or unadjusted models. Our data show that, in the HAART era, HCV coinfection is independently associated with a significantly increased risk of CVD and a trend towards an increased risk of AMI among HIV-infected patients. In the general population, Kalantar-Zadeh et al. [32] found HCV infection to be associated with higher all-cause and cardiovascular mortality among dialysis patients. Conversely, Arcari et al. found no association between HCV infection Compound Library supplier and AMI in young military recruits [33]. The finding is, however, hardly reassuring given the presumed level of physical fitness of the cohort. Our data are consistent with a recently published analysis comparing a large cohort of 82 083 HCV-monoinfected veterans with 89 582 HCV-negative control subjects. Despite

a favourable risk profile – younger age, lower lipid levels and lower prevalence of hypertension – HCV infection was associated with a higher risk of coronary artery disease after adjustment for traditional risk factors (HR 1.25; 95% CI 1.20–1.30) SSR128129E [34]. The current study suggests that these findings regarding HCV infection and cardiovascular disease also extend to patients with HIV infection. To date, there have been limited and contradictory findings on the role of HCV coinfection on the cardiovascular risk of HIV-infected patients. Analysis of the D:A:D cohort data recently found similar rates of AMI between HIV/HCV-coinfected and HIV-monoinfected patients, as in our cohort: 3.32 (95% CI

2.96–3.69) and 2.73 (95% CI 2.17–3.29) per 1000 patient-years, respectively; the difference was not statistically significant [14]. Conversely, in a cross-sectional analysis of a cohort of 395 HIV-infected patients with current or past alcohol abuse, Freiberg et al. [29] found that coinfection with HCV was associated with self-reported history of cardiovascular disease. This study was limited by the small sample size and had other limitations, including self-report of the outcome variable and several other covariates, and the fact that all study subjects had alcohol problems, reducing the generalizability of the study findings. Accordingly, the current study addresses a knowledge gap and provides important data germane to HIV treatment in the light of the high prevalence of HCV coinfection.

However, the measured values of TPP+ distribution also indicated that ionophores had only a minor influence on membrane potential. Some acidification of the cytoplasm occurred, but the total protonmotive force was

only decreased by about 20%. In contrast, the ATP pool fell by 75%. It should be noted that the experiments were performed with late-exponential or stationary-phase cells to reflect the conditions that pertain predominantly in the rumen (Hobson & Wallace, 1982). It seems improbable that the mechanisms differ in more active bacteria, as in mid-exponential phase, although the magnitude of the gradients and pools may be different. Russell and his colleagues SB203580 molecular weight have made similar observations with other species of ruminal bacteria. The high apparent intracellular concentrations of Na+ and K+ were similar to those measured in S. bovis (Russell, 1987). Ruminal bacteria have been described as mildly halophilic, based on their requirements of Na+ for growth (Caldwell et al., 1973; Caldwell & Hudson, 1974). The membrane potential fell by < 10% when monensin

was added to S. bovis, although intracellular pH was affected to a greater extent (Russell, 1987). The protonmotive force of a ruminal Peptostreptococcus was unaffected by monensin, yet the ATP pool fell by two-thirds (Chen & Russell, 1989). It therefore appears that it is not the collapse of transmembrane ion gradients that causes the toxic effect of ionophores on intact bacteria, but the energy expenditure required to support the increased energy demand of homoeostatic Selleck CP868596 mechanisms maintaining the gradients. Any extra demands induced by adding different cations may therefore have an influence on the efficacy of an ionophore, even if the ion is not translocated by the ionophore. In conclusion,

it may be possible to enhance the efficacy of ionophores by adding salts of mineral cations to the diet. However, the spectrum of antibacterial activity against different species, upon which ionophore depends for its nutritional effects, may well be different Selleckchem Verteporfin when the added cations are present, depending on the ion gradients present in different species. Thus, the nutritional effects of the ionophores (Chen & Russell, 1989) may not be the same at different cation concentrations. The present results also have implications for mechanisms by which ruminal bacteria may become resistant to ionophores. Adaptive resistance to ionophores involves changes in the permeability of the cell envelope (Newbold et al., 1992; Callaway & Russell, 1999), which may well affect changes in transmembrane ion gradients. One of the fears concerning the use of antimicrobials in livestock production is that transmissible resistance factors will arise and by transfer to human pathogens will render antibiotic therapy ineffective (Goodrich et al., 1984). However, there is no evidence that such resistance arises by exposure to ionophores such as monensin (Russell & Houlihan, 2003; Phillips, 2007).

Data were analysed by first subtracting the background and then normalizing the signals using a LOWESS filter (Locally-weighted Regression). For the analysis of zif268, 500 ng of total RNA was used as an input to generate cDNA using the SuperScript III First Strand Synthesis System (Invitrogen, Carlsbad, CA, USA). The cDNA was diluted 10-fold, and 5 μL was used as template for semiquantitative real-time RT-PCR together

with the iQSYBR Green Supermix (Bio Rad, Hercules, CA, USA). Samples were assayed in triplicate and normalized to polyubiquitine (Alme et al., 2007). Primers used to detect zif268 and polyubiquitine: forward and reverse zif268 (5′-AACAACCCTACGAGCACCTG-3′ and 5′-AGGCCACTGACTAGGCTGAA-3′),

and forward and reverse polyubiquitine (5′-GGCAAGACCATCACCCTAGA-3′ Afatinib purchase and 5′-GCAGGGTTGACTCTTTCTGG-3′). Changes in mature miRNA levels were determined using the TaqMan® microRNA Reverse Transcription kit and TaqMan® microRNA Assays (Applied Biosystems, Foster City, CA, USA) according to manufacturer’s selleck chemicals llc protocol. cDNA (15 μL) was generated from 30 ng of total RNA, and 5 μL of a threefold dilution was used for real-time PCR reactions. Three small RNAs (y1, snoRNA, Rnu6B) and one miRNA were considered for normalization in an initial set of samples. In the end, Y1 and miR-16 were selected for normalization based on their sufficiently high and stable levels of expression among samples. The TaqMan assays used are listed in Table S1. To amplify precursor sequences, the forward and reverse primers were designed to bind within the stem portion of the precursor miRNA.

To amplify the primary transcript specifically, at least one primer was placed outside the precursor in the 5′ or 3′ flanking sequence. The precursor primers will amplify both precursor and longer transcripts, whereas primers annealing in the primary transcript will amplify the long primary sequence only. Precursor and primary sequences were obtained from the miRNA registry and UCSC Genome Browser, respectively (Griffiths-Jones, 2004; Kuhn et al., 2007). Primers were designed using Primer3 (Rozen & Skaletsky, 2000). Oligonucleotide Cediranib (AZD2171) sequences used for priming and PCR are listed in Tables S2 and S3. Semiquantitative real-time RT-PCR of precursors and primary transcripts was carried out according to Jiang et al. (2005) and Schmittgen et al. (2008), with minor modifications. Briefly, total RNA was treated with RNase-free DNase (Ambion), and 500 ng of RNA was reverse-transcribed using a mix of gene-specific reverse miR primers (final concentration 10 μm) and the SuperScript III First Strand Synthesis System (Invitrogen). An initial step of 80°C for 1 min was added to the SuperScript III protocol to denature the hairpin structures. cDNA was diluted 20-fold, and 5 μL was used in each PCR reaction using the iQ SYBR Green Supermix (Bio Rad).