, 1991; Kalpana et al., 1991; Chua et al., 2000). To confirm that this was not occurring, we rescued the genomic region flanking the EZ::TN transposon from the mutants and looked for a 9-bp target site duplication in the mutant DNA. Analysis Doxorubicin of the DNA sequence flanking the EZ::TN transposon at MEL and MER revealed that each insertion was flanked by the 9-bp duplication characteristic of the Tn5 insertion (Table 2) (Berg & Berg, 1983), confirming that the antibiotic-resistant transconjugants arose by transposition of the EZ::TN transposon into the host chromosome. The library was screened for auxotrophic mutants to demonstrate the usefulness of the modified EZ::TN5 transposome in mutant library construction.

Five hundred BF638R transposon mutants were replica plated onto minimal media with BGB324 order or without Casamino acids (0.5% w/v) (Baughn & Malamy, 2002). One of 500 transposon mutants screened failed to grow on minimal medium without Casamino acids, suggesting

that a gene in an amino acid biosynthesis pathway was disrupted (Mutant EZY6). The disrupted gene in the auxotrophic mutant was identified by the SRP-PCR (Fig. 3). The identification of the 19-bp inverted repeat on the amplified PCR products confirmed that isolated auxotrophic mutant was a ‘true’ transposon insertant. We also identified the transposon-disrupted gene using the alternative rescue cloning method described in ‘Materials and methods’. Both the methods independently indicated that EZY6 had a mutation in argC (acetylglutamyl phosphate reductase, BF638R_0529), a gene in the arginine biosynthesis pathway. We found that the SRP-PCR technique was faster and simpler than the rescue cloning method for identifying the disrupted gene. Selected mutants that grew slowly on minimal medium were also chosen for further study. The mutated genes were identified by SRP-PCR, and results are presented in Fig. 4. Phosphoribosylglycinamide formyltransferase Mutants had transposon insertions in two-component regulators (EZY7), cell division

proteins (EZY11), aminotransferase (EZY17), GMP biosynthesis pathway (EZY19), transport-related proteins (EZY21), and various other genes. The disrupted genes were scattered throughout the genome of BF638R (Fig. 4), confirming that the custom EZ::TN5 transposome described here can randomly insert the transposon into the B. fragilis chromosome. The utility of the customized EZ::TN5 transposon for generating mutants in BF 9343 (ATCC 25285), BF clinical isolates, and B. thetaiotaomicron (Pumbwe et al., 2006a) was examined. The transposome was prepared from BF638R-modified pYV03. The efficiencies of the transposition in the clinical strain BF14412 and B. thetaiotaomicron were 3.6 ± 0.67 × 103 and 6.3 ± 1.2 × 103, respectively, indicating that the system may be useful for some clinical strains of BF as well as B. thetaiotaomicron. No mutants were generated in BF 9343 or the clinical isolate BF7320.

A spaced HFS paradigm was used to induce non-decremental protein synthesis-dependent LTP in urethane-anesthetized rats (Messaoudi et al., 2002, 2007). As shown in Fig. 1, HFS resulted in a robust and stable increase in the slope of field excitatory postsynaptic potential (fEPSP) and amplitude of the population selleckchem spike (Fig. 1A–C). A second group of rats received HFS following

systemic (i.p.) injection of the competitive NMDAR antagonist, CPP. As previously shown (Williams et al., 1995; Messaoudi et al., 2002), LTP of the fEPSP and population spike was inhibited in CPP-treated rats. No changes in synaptic efficacy were observed in a third group of rats receiving LFS only. As a positive control for NMDAR-dependent gene regulation, we examined expression of immediate-early gene zif268 (also known as Egr1) in homogenate samples from microdissected dentate gyrus (Cole et al., 1989; Havik et al., 2003). At 2 h post-HFS, zif286 mRNA levels

in the HFS-treated dentate gyrus were significantly elevated 2.8-fold above the contralateral, control dentate gyrus (Fig. 1D). This increase was abolished in the CPP group and absent in the LFS group. These results confirmed generation of stable NMDAR-dependent LTP associated with robust changes in gene expression. Microarray expression profiling was performed to screen for LTP-regulated miRNAs 2 h post-HFS. MirVana-purified RNA from the HFS-treated and contralateral control dentate gyrus from two animals was differentially hybridized to rat miRNA chips (MiRat_8.0_060307) representing all miRNA transcripts Everolimus cost listed in Sanger miRBase Release 8.0. Figure 2A

shows miRNAs exhibiting mean changes of at least 20%. By this arbitrary criterion 10 miRNAs showed increased expression (rno-miRNA-28, -103, -107, -125a, -132, -151*, -212, -320, -485, -543) and 11 miRNAs showed decreased expression (rno-miRNA-17, -19b, -21, -23a, -23b, -138, -181b, -219, -247, -338, -494), of a total of 237 probes on the Dynein chip. Real-time RT-PCR analysis was used for independent validation and further study of three candidate regulated miRNAs (Fig. 2B). In agreement with the array data, miR-132 and miR-212 levels were significantly elevated, while miR-219 levels were significantly decreased at 2 h post-HFS in treated dentate gyrus relative to untreated control dentate gyrus. This regulation was HFS dependent, as no changes in miRNA expression were observed in rats receiving LFS only. We anticipated that blockade of LTP by CPP would eliminate or reduce the changes in miRNA expression. Instead, each of the three miRNAs exhibited enhanced expression when HFS was applied in the presence of CPP. Thus, miR-132 levels were elevated from 1.38-fold in the HFS group to 1.83-fold in the HFS + CPP group, miR-212 levels increased from 1.26- to 1.59-fold, and miR-219 levels flipped from a decrease of 0.68-fold in the HFS group to an increase of 1.27-fold in the HFS + CPP group (Fig. 2C).

A spaced HFS paradigm was used to induce non-decremental protein synthesis-dependent LTP in urethane-anesthetized rats (Messaoudi et al., 2002, 2007). As shown in Fig. 1, HFS resulted in a robust and stable increase in the slope of field excitatory postsynaptic potential (fEPSP) and amplitude of the population MK-2206 manufacturer spike (Fig. 1A–C). A second group of rats received HFS following

systemic (i.p.) injection of the competitive NMDAR antagonist, CPP. As previously shown (Williams et al., 1995; Messaoudi et al., 2002), LTP of the fEPSP and population spike was inhibited in CPP-treated rats. No changes in synaptic efficacy were observed in a third group of rats receiving LFS only. As a positive control for NMDAR-dependent gene regulation, we examined expression of immediate-early gene zif268 (also known as Egr1) in homogenate samples from microdissected dentate gyrus (Cole et al., 1989; Havik et al., 2003). At 2 h post-HFS, zif286 mRNA levels

in the HFS-treated dentate gyrus were significantly elevated 2.8-fold above the contralateral, control dentate gyrus (Fig. 1D). This increase was abolished in the CPP group and absent in the LFS group. These results confirmed generation of stable NMDAR-dependent LTP associated with robust changes in gene expression. Microarray expression profiling was performed to screen for LTP-regulated miRNAs 2 h post-HFS. MirVana-purified RNA from the HFS-treated and contralateral control dentate gyrus from two animals was differentially hybridized to rat miRNA chips (MiRat_8.0_060307) representing all miRNA transcripts Daporinad purchase listed in Sanger miRBase Release 8.0. Figure 2A

shows miRNAs exhibiting mean changes of at least 20%. By this arbitrary criterion 10 miRNAs showed increased expression (rno-miRNA-28, -103, -107, -125a, -132, -151*, -212, -320, -485, -543) and 11 miRNAs showed decreased expression (rno-miRNA-17, -19b, -21, -23a, -23b, -138, -181b, -219, -247, -338, -494), of a total of 237 probes on the IMP dehydrogenase chip. Real-time RT-PCR analysis was used for independent validation and further study of three candidate regulated miRNAs (Fig. 2B). In agreement with the array data, miR-132 and miR-212 levels were significantly elevated, while miR-219 levels were significantly decreased at 2 h post-HFS in treated dentate gyrus relative to untreated control dentate gyrus. This regulation was HFS dependent, as no changes in miRNA expression were observed in rats receiving LFS only. We anticipated that blockade of LTP by CPP would eliminate or reduce the changes in miRNA expression. Instead, each of the three miRNAs exhibited enhanced expression when HFS was applied in the presence of CPP. Thus, miR-132 levels were elevated from 1.38-fold in the HFS group to 1.83-fold in the HFS + CPP group, miR-212 levels increased from 1.26- to 1.59-fold, and miR-219 levels flipped from a decrease of 0.68-fold in the HFS group to an increase of 1.27-fold in the HFS + CPP group (Fig. 2C).

g. the anxiety-prone nature of bLRs or drug addiction proclivity of bHRs). “
“Postnatal brain development continues throughout adolescence into young adulthood. In particular, synapse strengthening and elimination are prominent

processes during adolescence. However, molecular data of this relatively late stage of synaptic development are sparse. In this study, we used iTRAQ (isobaric tag for relative and absolute quantification)-based proteomics and electron microscopy to investigate the molecular composition of a synaptic membrane fraction from adolescent postnatal day (P)34 and P44 and adult (P78) rat medial prefrontal cortex. Differential expression of proteins was most prominent between early adolescence and young adulthood (35%, P34–P78), with an over-representation of cell-membrane proteins during adolescent development selleck chemicals llc (between P34 and P44), and synaptic vesicle proteins between late adolescence and young adulthood (P44–P78). Indicative of the critical period of development, we found that, between P34 and P44, a substantial number of proteins was differentially expressed

(14%), much more than during the period after adolescence, i.e. between P44 and P78 (5%). A striking observation was the developmental non-stoichiometric regulation of distinct classes of proteins from the synaptic vesicle and the presynaptic release machinery. Electron microscopy demonstrated a small change in the number of docked vesicles between P34 and P44, but not in the total number of synaptic vesicles and in the size of the vesicle cluster. We conclude that the molecular composition Akt inhibitor of synapses, and more specifically the synaptic release machinery, of the medial prefrontal cortex changes drastically during adolescent development. “
“The protective impact of exercise on neurodegenerative processes has not been confirmed, and the mechanisms underlying the benefit of exercise have not been determined in human Parkinson’s disease or in chronic animal disease models.

This research examined the long-term neurological, behavioral, and mechanistic consequences of endurance Ribonuclease T1 exercise in experimental chronic parkinsonism. We used a chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced mouse model of Parkinson’s disease with moderate neurodegeneration and examined the effects of treadmill exercise on movement and balance coordination, changes in dopamine neuron biomarkers, mitochondrial functions, and neurotrophic factor activities in the nigrostriatal system. The exercise results were compared with those of the control and sedentary chronic parkinsonian animals. After 18 weeks of exercise training in the chronic parkinsonian mice, we observed a significant deterrence in the loss of neuronal dopamine-producing cells and other functional indicators.

Hyphomicrobium sulfonivorans S1T was grown in a batch culture on dimethylsulfone as described previously by Boden et al. (2011) and R. sulfidophilum was grown photoorganoautotrophically on DMS according to McDevitt et al. (2002). Sagittula stellata was grown in

steady-state chemostats at a range of dilution rates (D) between 0.01 and 0.15 h−1 on fructose (12 mM) and between 0.01 and 0.10 h−1 on succinate (2 mM) with or without the addition of DMS (1 mM). Kinetic parameters were determined as described previously (Boden et al., 2010). Five volume changes at each steady state occurred before the kinetic parameters were determined. Cells were harvested for enzyme assays by centrifugation at 13 000 g for 30 min at 4 °C. selleck chemical Doxorubicin solubility dmso Cells were washed and resuspended in 50 mM PIPES-HCl, pH 7.4, containing 50 mM magnesium sulfate. If not used immediately, cells were snap-frozen in liquid nitrogen and stored at −80 °C. Spectrophotometric enzyme assays were routinely conducted at 30 °C in an Ultrospec 3100pro UV/Visible Spectrophotometer

(Amersham). Each reaction was conducted in a sevenfold replicate against a blank. Cell-free extracts were prepared by three passages through a French pressure cell (120 MPa), with debris removed by centrifugation (13 000 g, 30 min, 4 °C). Protein was quantified using the method of Bradford (1976). DMS dehydrogenase PD184352 (CI-1040) activity was assayed using a modification of the method of McDevitt et al. (2002). Two milliliters of 500 mM Tris-HCl, pH 8.0, 300 μL of 35 mM phenazine methosulfate and 300 μL of 100 μM 2,6-dichlorophenolindophenol (DCPIP) were placed in a 3 mL modified Thunberg cell (Baumberger, 1933) and degassed by bubbling with oxygen-free nitrogen for 10 min before adding 100 μL cell-free extract (containing 5–10-mg protein). Sixty microliters of 100 mM DMS solution in ethanol was placed in the bulb of the side-arm and the cell was assembled. The cell was evacuated on ice for 10 min before sealing and the reaction was initiated by pouring

the contents of the side-arm into the main chamber. The A600 nm was monitored and the rate of reduction of DCPIP was determined using the millimolar extinction coefficient for the oxidized form of 21.5 mM−1 cm−1. Cell-free extracts prepared from R. sulfidophilum SH1 grown photoorganoautotrophically with DMS as an energy source were used as a positive control (McDevitt et al., 2002). An alternative assay was performed using 300 μL of 3 mM potassium ferricyanide in place of DCPIP solution and reduction was monitored at 420 nm with a millimolar extinction coefficient of 1.0 mM−1 cm−1. DMSO reductase was assayed in the same way using a reaction mixture comprising 150 μL of 1.0 M Tris-HCl, pH 7.6, 20 μL of cell-free extract and 1.03 mL of MilliQ water in the main chamber of the cell. These were degassed in situ before adding 1.

HIV-infected patients were enrolled consecutively from two different urban teaching hospitals in Seoul,

South Korea between March 2012 and September 2012. Participants completed a detailed NP assessment of six cognitive domains commonly affected by HIV. The Frascati criteria were used for diagnosing HAND. Four key questions, the International HIV Dementia Scale (IHDS) and Montreal Cognitive Assessment PD98059 (MoCA)-K were also assessed as potential tools for screening for HAND. Among the 194 participants, the prevalence of HAND was 26.3%. Asymptomatic neurocognitive impairment and minor neurocognitive disorder accounted for 52.9 and 47.1% of the patients with HAND, respectively. In multivariate analysis, haemoglobin (Hb) level ≤ 13 g/dL (P = 0.046) and current use of a protease inhibitor-based

regimen (P = 0.031) were independent risk factors for HAND. The sensitivity and specificity of the IHDS were 72.6 and 60.8%, and those of MoCA-K were 52.9 and 73.4%, respectively. The IHDS (P < 0.001) and MoCA-K (P < 0.001) were both useful for screening for HAND. Among NP tests, the sensitivity and specificity of the Grooved Pegboard Test were 90.2 and 72.0%, and those of the Wisconsin Card Sorting Test were 61.2 and 84.4%, respectively. HAND is a prevalent comorbidity in HIV-infected Koreans. Active screening and diagnosis with effective tools, such as the IHDS, MoCA-K and Grooved Pegboard Test, could be used to identify this important complication. "
“The combination of HIV, chronic HBV infection and pregnancy presents unique management questions. Referral to the local STI571 in vivo designated

specialist should be undertaken to ensure that all aspects of care are addressed, including: the effects of HBV/HIV on pregnancy; effects of pregnancy on the course of coinfection; drug management for both HBV and HIV; and Protein tyrosine phosphatase PMTCT for both viruses. The prevalence of HBV coinfection in pregnant women tends to reflect that of the adult population (Europe/Africa 4–10%) [[3][[4][#[5]][6]]165] and is 40% higher than that found in the general population (HIV positive vs. HIV uninfected: RR 1.40; 95% CI 1.16–1.69) [6]. Up to one-third of hepatitis B surface antigen (HBsAg) are wild type [hepatitis B e antigen (HBeAg)-positive] and, depending on region, up to 6% are coinfected with HDV. Rates of HBV/HIV coinfection vary with race and ethnicity so that changing immigration patterns in Western countries with traditionally low prevalence may significantly influence rates at a regional level (e.g. 6% among Asian women in the USA vs. 0.6% in white women) [7]. The same is true for injection drug use (prevalence <0.1% in north-west Europe compared to 1–4% in southern Europe) and sexual transmission (prevalence higher in men who have sex with men). Although plausible because of higher levels of HBV DNA in coinfected women, there is no evidence of increased MTCT in coinfection over mono-infection.

Combining this evidence with expert opinion led to the development of 10 final Australian and New Zealand recommendations. The recommendations relate to pain measurement, and the use of analgesic medications in patients with and without co-morbidities and during

pregnancy www.selleckchem.com/products/VX-770.html and lactation. The recommendations reflect the clinical practice of the majority of the participating rheumatologists (mean level of agreement 7.24–9.65). Ten Australian and New Zealand evidence-based recommendations regarding the management of pain by pharmacotherapy in adults with optimally treated IA were developed. They are supported by a large panel of rheumatologists, thus enhancing their utility in everyday clinical practice. “
“Thiopurines have been a cornerstone of medical

management of patients with inflammatory bowel disease (IBD) and many rheumatological disorders. The thiopurines are metabolized to their end products, 6-methymercaptopurine (6MMP) and the 6-thioguanine nucleotides (6TGN), with 6TGN being responsible for thiopurine efficacy by causing apoptosis BMN673 and preventing activation and proliferation of T-lymphocytes. In IBD, conventional weight-based dosing with thiopurines leads to an inadequate response in many patients. Utilizing measurement of these metabolites and then employing dose optimization strategies has led to markedly improved outcomes in IBD. Switching between thiopurines as well as the addition of low-dose allopurinol can overcome adverse events and elevate 6TGN levels into the therapeutic window. There is a paucity of data on thiopurine metabolites in rheumatological diseases and further research is required. The thiopurines, 6-mercaptopurine (6MP) and its pro-drug azathioprine (AZA), have been a cornerstone of medical management of patients with inflammatory bowel disease (IBD) for over 30 years. They are well established in treatment algorithms for induction, maintenance and as steroid-sparing agents. Thiopurines have also been

used extensively in the management of rheumatological oxyclozanide disorders such as rheumatoid arthritis (RA), psoriasis and psoriatic arthritis, systemic lupus erythematous (SLE) and systemic vasculitis. In the IBD population, thiopurines are conventionally administered according to a weight-based dosing regimen. Up to 70% of patients do not respond to the standard dose of thiopurine therapy,[1] and up to 40% experience some sort of adverse event.[2] Recent advances enabling measurement of thiopurine metabolites have allowed clinicians to optimize the dose of thiopurines, leading to significant increases in numbers of patients achieving steroid-free clinical remission without the need for treatment escalation or change. Here we review the literature underpinning the measurement of thiopurine metabolites and the efficacy of thiopurine optimization. The pro-drug AZA is converted by glutathione to 6MP.

, 2011; Thakur & Sanyal, 2011; Fig. 1a). Clustered KTs are found in S. pombe as well except at metaphase where multiple foci of KT proteins were observed (Goshima et al., 1999; Tanaka et al., 2009; Jakopec et al., 2012). Although the exact nature of KT architecture in yeasts is uncertain, various genetic and biochemical studies indicate the presence of functional homologs of Torin 1 several KT proteins at distinct layers of a human KT in these yeasts (Table 1). Determination of relative positions of different proteins at the

respective KTs by ‘single molecule high-resolution colocalization’ demonstrates that axial localization of proteins at the KT at distinct phases of mitosis in S. cerevisiae (Joglekar et al., 2009) and humans is largely conserved (Wan et al., 2009; Fig. 1b). However, such studies are yet to be carried out in S. pombe and C. albicans. Nevertheless, the difference in the cross-linking time of KT proteins of C. albicans with CEN chromatin indicates a structural similarity between C. albicans (Sanyal et al., 2004; Roy et al., 2011; Thakur & Sanyal, 2011) and metazoans KTs. Dynamics of assembly of KT proteins is dissimilar in yeasts and metazoans. In metazoans, only the CEN-specific histone H3 variant and an inner KT-associated super-complex, commonly

known as constitutive centromere-associated network, remain localized at the KT throughout the cell cycle (Foltz et al., this website 2006; Liu et al., 2006; Okada et al., 2006). Localization/delocalization dynamics of middle and outer KT proteins is specific to stages of the cell cycle. For example, a middle KT protein

and a MT interacting protein are loaded at the KT at late interphase and delocalize from the KT Adenosine triphosphate during transition of late anaphase to telophase in metazoans (Liu et al., 2006; Cheeseman & Desai, 2008; Cheeseman et al., 2008). In contrast, proteins from all layers of a KT exhibit constitutive localization at the CEN in S. cerevisiae (Meluh et al., 1998; Goshima & Yanagida, 2000) and C. albicans (Sanyal & Carbon, 2002; Roy et al., 2011; Thakur & Sanyal, 2011). All the outer KT proteins of S. pombe localize at the CEN only during mitosis except one component, which remains localized at the KT throughout the cell cycle (Liu et al., 2005; Sanchez-Perez et al., 2005). Organization of CENs in different fungi including several yeast species can be classified into three categories: point, large regional and small regional CENs (Roy & Sanyal, 2011; Sanyal, 2012). S. cerevisiae has short point CENs (< 400 bp) with conserved DNA motifs for protein binding, and thus, they are genetically defined (Fitzgerald-Hayes et al., 1982; Hieter et al., 1985). In contrast, S. pombe has longer regional CENs (≥ 40 kb) consisting of repetitive as well as unique DNA elements (Clarke et al., 1986; Nakaseko et al., 1987; Fishel et al., 1988; Takahashi et al., 1992; Steiner et al., 1993; Baum et al., 1994; Wood et al., 2002). C.

, 2011; Thakur & Sanyal, 2011; Fig. 1a). Clustered KTs are found in S. pombe as well except at metaphase where multiple foci of KT proteins were observed (Goshima et al., 1999; Tanaka et al., 2009; Jakopec et al., 2012). Although the exact nature of KT architecture in yeasts is uncertain, various genetic and biochemical studies indicate the presence of functional homologs of BI 6727 price several KT proteins at distinct layers of a human KT in these yeasts (Table 1). Determination of relative positions of different proteins at the

respective KTs by ‘single molecule high-resolution colocalization’ demonstrates that axial localization of proteins at the KT at distinct phases of mitosis in S. cerevisiae (Joglekar et al., 2009) and humans is largely conserved (Wan et al., 2009; Fig. 1b). However, such studies are yet to be carried out in S. pombe and C. albicans. Nevertheless, the difference in the cross-linking time of KT proteins of C. albicans with CEN chromatin indicates a structural similarity between C. albicans (Sanyal et al., 2004; Roy et al., 2011; Thakur & Sanyal, 2011) and metazoans KTs. Dynamics of assembly of KT proteins is dissimilar in yeasts and metazoans. In metazoans, only the CEN-specific histone H3 variant and an inner KT-associated super-complex, commonly

known as constitutive centromere-associated network, remain localized at the KT throughout the cell cycle (Foltz et al., buy AZD6738 2006; Liu et al., 2006; Okada et al., 2006). Localization/delocalization dynamics of middle and outer KT proteins is specific to stages of the cell cycle. For example, a middle KT protein

and a MT interacting protein are loaded at the KT at late interphase and delocalize from the KT Ketotifen during transition of late anaphase to telophase in metazoans (Liu et al., 2006; Cheeseman & Desai, 2008; Cheeseman et al., 2008). In contrast, proteins from all layers of a KT exhibit constitutive localization at the CEN in S. cerevisiae (Meluh et al., 1998; Goshima & Yanagida, 2000) and C. albicans (Sanyal & Carbon, 2002; Roy et al., 2011; Thakur & Sanyal, 2011). All the outer KT proteins of S. pombe localize at the CEN only during mitosis except one component, which remains localized at the KT throughout the cell cycle (Liu et al., 2005; Sanchez-Perez et al., 2005). Organization of CENs in different fungi including several yeast species can be classified into three categories: point, large regional and small regional CENs (Roy & Sanyal, 2011; Sanyal, 2012). S. cerevisiae has short point CENs (< 400 bp) with conserved DNA motifs for protein binding, and thus, they are genetically defined (Fitzgerald-Hayes et al., 1982; Hieter et al., 1985). In contrast, S. pombe has longer regional CENs (≥ 40 kb) consisting of repetitive as well as unique DNA elements (Clarke et al., 1986; Nakaseko et al., 1987; Fishel et al., 1988; Takahashi et al., 1992; Steiner et al., 1993; Baum et al., 1994; Wood et al., 2002). C.

Samples were incubated at 37 °C for 20 min shaking (200 r.p.m.). Surviving cells were enumerated by serial dilution in PBS and subsequent plating onto BH agar. For colony forming unit (CFU) enumeration, overnight cultures (2 × 109 CFU mL−1) were washed twice in PBS and serially diluted to approximately 2 × 107 CFU mL−1. A further 1 Ku0059436 in 10 dilution into the desired growth media was performed resulting in an inoculum of approximately 2 × 106 CFU mL−1. Counts were taken every 2 h over an 8 h period by serial dilution in PBS and enumeration on BHI agar. All agar plates were incubated

at 37 °C. For concurrently running OD600 nm readings, a sample was removed at the same time points and measured using a spectrophotometer. RNA extraction from stationary phase cells was carried out using the Macaloid method (Raya et al., 1998). The reverse transcriptase PCR was run using 4 μL random primer p(dN)6, 2 μL RNA, and 2 μL DEPC water (Sigma) at 65 °C for 10 min and put directly on ice. To these samples, 32 μL of a mastermix was added containing: 1 μL Expand Reverse Transcriptase (Roche), 8 μL 5× Buffer (Roche), 4 μL 100 mM dTT (Roche), 1 μL dNTP mix (dATP, dCTP, dGTP, dTTP – 10 mM), and 18 μL DEPC water. This reaction was run at 30 °C for 10 min, 42 °C for 3 h, and held at 4 °C. cDNA was confirmed through PCR using L142 and U141 primers and the wild-type

L. monocytogenes extracted DNA as a positive control. Quantitative real-time PCR was used for transcriptional analysis. The Universal Probe Library Assay Design Center (https://www.roche-applied-science.com/sis/rtpcr/upl/index.jsp?id=UP030000)

was Epacadostat cell line used to design PCR primers that correspond to a specific probe in the library. Primer sequences, synthesized by MWG, and corresponding probes are listed in Online Resources (Table S1). The 16S rRNA gene was used as a housekeeping gene to compensate for any variability in the initial amount of PTK6 starting total RNA. Amplification reactions consisted of 2.5 μL of cDNA, 6.4 μL of 2× FastStart TaqMan Probe Master (Roche), primers (900 nM), and probe mix (250 nM). RNase-free water was added to bring the total volume of the reaction to 10 μL. Reactions were run in duplicate on 384-well plates using the LightCycler 480 System (Roche). Negative control reactions, without cDNA, were also included on the plate. Thermal cycling conditions were carried out according to manufacturer’s instructions (Roche), and the method (Livak & Schmittgen, 2001) was used to calculate the relative changes in gene expression from the qRT-PCR experiments. Zinc uptake systems have been described in numerous bacteria and include the high-affinity systems znuABC of E. coli and ycdHIycA of B. subtilis (Patzer & Hantke, 1998, 2000; Gaballa et al., 2002). For the most part, these systems are under the control of the zinc uptake regulator Zur, a homolog of which (ZurR) has been identified in L. monocytogenes (Dalet et al.