The sequenced strain of S.

meliloti Rm1021 displays reduced biofilm formation on the microplate assay when grown in a rich medium compared with minimal medium (Fujishige et al., 2005). A nutritionally limited environment promotes learn more the transition from a planktonic to a sessile mode of life. Biofilm formation may therefore represent a strategy for survival of bacteria in nutritionally limited environments, because colonization of surfaces provides certain advantages, for example increased capture of nutrients that can be absorbed from the medium (Wimpenny & Colasanti, 1997). In contrast, nutrient abundance in the medium seems to favor biofilm formation in Pseudomonas (O’Toole & Kolter, 1998b; Yousef-Coronado et al., 2008), possibly by increasing bacterial population size and accumulation of autoinducers, which promote biofilm formation. In view of previous findings that the nutrient content of the growth medium regulates the development of biofilms by Pseudomonas species (O’Toole & Kolter, 1998a, b), the effects of various nutrients and environmental conditions on the biofilm formation ability of S. meliloti were tested (Rinaudi et al., 2006). The concentrations of sucrose, NVP-BKM120 purchase phosphate, and calcium were

positively correlated with biofilm formation, whereas extreme temperatures and pH values had a negative effect. These findings support the hypothesis that biofilm formation promotes the survival of non-spore-forming rhizobia in soil in the absence of a legume host. The key regulatory pathways in S. meliloti biofilm formation have been identified. The exoR and exoS–chvI two-component system controls many phenotypes, including biofilm formation. Wells et al. (2007) showed that this system affects succinoglycan production, prototrophy, nitrogen fixation, and motility, and also regulates attachment to abiotic surfaces. The exoR95 and exoS96 mutants showed a considerably increased biofilm formation, compared with the wild-type or the other strains tested. Rhizobium nod genes, and their products, Nod

factors, are essential for the development of nitrogen-fixing nodules on legume roots (Lerouge et al., 1990). Microscopic analysis revealed that Nod factors are critical for the establishment of a mature rhizobial biofilm (Fujishige et al., 2008). This is a new function for Nod factors, and is distinct Buspirone HCl from their established role as a morphogen-inducing legume nodule development. The dual functions of Nod factors, as structural components in biofilms and independently as precursors of host-specific morphogens, imply the existence of two different sets of control mechanisms, one dependent on flavonoids (plant-derived inducers of nod genes in S. meliloti) and the other independent of flavonoids, which regulate Nod factor production (Fujishige et al., 2008). Bacteria have various mechanisms for movement, including flagellar swimming, swarming, twitching, and gliding motility.

Effective antiretroviral (ARV) regimens for the treatment of HIV infection have increased life expectancy, and many individuals Y-27632 concentration infected with HIV now live for decades with chronic illness [1]. Long-term complications are emerging as the greatest challenges facing HIV-infected individuals. Atherosclerotic cardiovascular disease (CVD) is a leading comorbidity and cause of mortality among HIV-infected adults [2]. Several studies have shown that HIV-infected children, compared with their healthy peers, have higher rates of CVD risk factors, including dyslipidaemia, insulin resistance, obesity

and central adiposity [3-7]. HIV infection also results in prolonged chronic inflammation, thereby increasing CVD risk. Exogenous obesity, which is common among perinatally HIV-infected children and adolescents, can also contribute to CVD risk [8, 9]. For perinatally infected children, these exposures

start in utero and continue through critical periods of growth, puberty and development. Inflammation, which is now considered the primary mechanism leading to atherosclerosis, can initiate a complex sequence of events that eventually produce Olaparib concentration detectable arterial changes and symptomatic CVD [10]. A host of cellular pathways are activated through inflammation, with most being initiated through injury to the endothelium [10]. Factors associated with endothelial injury include oxidized cholesterol, hyperglycaemia, lifestyle (smoking), and familial/genetic risks [11]. In HIV-infected patients, the effects of chronic immune activation from HIV infection [12, 13] and potential oxidative stress (induced by mitochondrial dysfunction) caused by highly active antiretroviral therapy (HAART) also come into play [14, 15]. These factors initiate a cascade of events that can increase inflammation and produce changes in endothelial function and/or coagulation status. Although HIV-infected children carry risk factors that

are associated with premature atherosclerotic 6-phosphogluconolactonase CVD, it is currently difficult to ascertain whether the adverse CVD outcomes attributed to HIV infection in adults will be observed as HIV-infected children age. Emerging evidence from large, long-term and prospective studies on CVD risk in non-HIV-infected healthy children [16, 17] shows that risk factors tracked from early childhood are associated with adverse CVD outcomes in adulthood. Studies that show direct evidence of vascular inflammation may provide further proof of increased CVD risk that, in turn, may ultimately lead to new, preventive interventions for these children. C-reactive protein (CRP) is one of the best-studied measures of systemic inflammation and high levels can predict adverse CVD outcomes in adults [18]. A number of other biomarkers are associated with more specific changes in these inflammatory pathways in both HIV-infected and HIV-uninfected populations [19-21].

This knowledge is required to evaluate the effect of differential flagellum expression on competition for nodulation. To this end, we obtained site-directed mutants in each of the flagellin-encoding Akt inhibitor gene clusters of B. japonicum, both in the background of the LP 3004 and LP 3008 strains, and tested their competitiveness. Strains are summarized in Supporting Information, Table S1. Bradyrhizobium japonicum was grown in Götz medium (Quelas et al., 2006) or HM salts with 0.1% yeast extract, 0.1%l-arabinose, and 0.1% sodium gluconate (Kanbe et al., 2007). For conjugation, PSY medium (Regensburger & Hennecke, 1983) was used. Swimming assays were performed in Götz agar (0.3% w/v) (Althabegoiti

et al., 2008). Escherichia coli was grown in Luria–Bertani (Sambrook UK-371804 mouse & Russell, 2001). Antibiotics were at the following concentrations (μg mL−1): streptomycin (Sm), 400 (B. japonicum) or 100 (E. coli); spectinomycin (Sp), 200; kanamycin, 150 (B. japonicum) or 25 (E. coli); ampicillin, 200; and gentamicin, 100 (B. japonicum) or 10 (E. coli). Deletion mutants were obtained and checked as described (Quelas et al., 2010) using the primers and plasmids indicated in Table S1. Strains LP 5843 and LP5844 (ΔfliC1-4) carried the nptII cassette in the replacement of bases 6 410 133–6 418 950, thus removing 8817 bp between bll5843 and bll5846 coding regions (Kaneko et al., 2002). Strains LP6865 and LP 6866 (ΔfliCI-II) carried the

Ω-Sm-Sp-interposon between bases 7 560 766 and 7 563 627, thus replacing 2861 bp of bll6865 and bll6866 coding regions. The double mutants LP6543 and LP 6644 had nptII between bases 6 410 133 and 6 418 950

of LP6865 and LP 6866, respectively. Rhizobia grown in liquid HM salts were vortexed for 5 min and centrifuged at 10 000 g for 30 min at 4 °C. The supernatant was incubated with 1.3% polyethylene glycol 6000 and 166 mM PtdIns(3,4)P2 NaCl for 2 h at 4 °C. Afterwards, this suspension was centrifuged at 11 000 g for 40 min at 4 °C and the resulting pellet was resuspended in phosphate-buffered saline. For analysis, the samples were boiled in Laemmli (1970) loading buffer for 10 min and then separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (Laemmli, 1970). Light microscopy was performed using a Nikon Eclipse E 200 microscope. Videos were recorded using a Nikon 518CU digital camera coupled to the microscope. Electron microscopy was performed as described elsewhere (Althabegoiti et al., 2008). Competitiveness was assayed using mixtures of LP 3004 or LP 3008 with the indicated mutant (Fig. S1). Each strain was at a concentration of approximately 106 rhizobia mL−1 in a modified N-free Fåhraeus plant nutrient solution contained in vermiculite pots (Lodeiro et al., 2000a; López-García et al., 2001, 2002). The pots were allowed to drain the excess solution through holes at the bottom to achieve 100% field capacity, and one plantlet was aseptically planted in each pot.

However, the expression levels of a transcriptional regulatory protein (MalR) and a hypothetical protein (GSU1247) in wild-type strain grown in 4 mM copper were about two- and fourfold lower than wild type grown without copper, respectively. The intracellular metabolites produced by Pseudomonas sp. TLC6-6.5-4 and the mutant strain CSM2 grown with or without copper was analyzed by GC-MS. A total of 44 compounds – organic acids, sugars, amino acids, nucleosides and lipids – were identified. To examine the overall metabolic changes, the relative metabolite concentrations

were analyzed in an unsupervised hierarchical cluster analysis (HCA) using Pearson correlation as the distance metric (Fig. S2). A more robust statistical method, one-way anova, was applied to examine the changes in relative metabolic levels, which identified selleck chemicals llc significant changes of 15 compounds (Fig. 3). Several sugars and amino acids such as glycerol-3-phosphate, alpha-d-glucopyranoside, l-proline and l-isoleucine decreased significantly in the CSM2 mutant compared with wild type see more grown without copper. However, these compounds significantly increased in wild type grown with 4 mM copper. In addition, the concentration of several organic

acids including phosphoric acid, butanedioic acid and hexadecanoic acid were significantly reduced in wild-type strain with copper exposure, whereas the concentration of these compounds was not altered in the CSM2 mutant compared with wild-type strain grown without copper. Transposon insertion in CSM2 mutant resulted in the down-regulation of the ABC transporter pathway compared with its up-regulation Fludarabine cost in wild-type strain in the presence of copper (Table 2). Besides ABC transporters, TCA cycle, protein digestion, and absorption and glyoxylate metabolism were affected by exposure to high levels of copper. ABC transporters (amino acid; organic

ion and oligosacchride) Protein digestion and absorption Glyoxylate and dicarboxylate metabolism In this study, the response of Pseudomonas sp. TLC6-6.5-4 to elevated copper ion concentrations was evaluated using morphological, transposon insertion, proteomic, and metabolomic analyses. Alternation in cell morphology is a visible indicator of bacterial adaptation to environmental stress (Justice et al., 2008). A significant reduction of bacterial cell size observed in the wild type in the presence of copper was similar to that of a lead-resistant Pseudomonas aeruginosa strain exposed to 0.8 M lead nitrate (Naik & Dubey, 2011). Pseudomonas outer-membrane has two major groups of lipoproteins with peptidoglycan binding lipoproteins and efflux porins (Remans et al., 2010). Bacterial shape is controlled by peptidoglycan and its associated lipoproteins (Pierce et al., 2011). It is likely that a peptidoglycan-binding lipoprotein or the efflux lipoprotein identified in this study may have a role in cell size regulation.

“
“In the brains of adult vertebrates, including humans, neurogenesis occurs in restricted niches where it maintains cellular turnover and cognitive plasticity. In virtually all species, however, aging is associated with a significant decline in adult neurogenesis. Moreover, an acceleration of neurogenic defects is observed in models of Alzheimer’s disease and other neurodegenerative diseases, suggesting an involvement in aging- and disease-associated cognitive deficits. To gain insights into when, how and why adult neurogenesis decreases

in the aging brain, we critically reviewed the scientific literature on aging of the rodent find more subventricular zone, the neurogenic niche of the adult forebrain. Our analysis revealed that deficits in the neurogenic pathway are largely established by middle age, but that there remains

striking ambiguity in the underlying mechanisms, especially at the level of stem and progenitor cells. We identify and discuss several challenging issues that have contributed to these key gaps in our current knowledge. In Selleck JAK inhibitor the future, addressing these issues should help untangle the interactions between neurogenesis, aging and aging-associated diseases. “
“Epilepsy is a common neurological disease. Understanding the mechanisms of epileptogenesis at the cellular and molecular levels may provide novel targets Ergoloid for preventing this disorder. Brain-derived neurotrophic factor (BDNF) and its receptor tropomyosin-related kinase type B (TrkB) are believed to be critical for epileptogenesis. Previous studies have revealed possible changes in the expression of full-length TrkB receptors (TrkB.FL) and truncated TrkB receptors (TrkB.T) in neurodegenerative disorders. In this study, we investigated alterations in TrkB receptor expression and TrkB signalling activity in a rat hippocampal neuronal model of spontaneous recurrent epileptiform discharges (SREDs) and the effects on the epileptiform discharges. To induce

epileptiform discharges, we established a model with Mg2+-free treatment. We found a dramatic upregulation of TrkB.T and a decrease in TrkB.FL in the SREDs model. Calpain contributed to the downregulation of TrkB.FL. The upregulation of TrkB.T required transcription and translation activity. Furthermore, BDNF induced the activation of TrkB.FL signalling. However, TrkB.FL signalling was inhibited in the SREDs model. Although calpain inhibitors prevented a decrease in TrkB.FL, they did not restrain the downregulation of TrkB.FL signalling activity in the model. However, a SREDs model with a translation inhibitor prevented the increase in TrkB.T and re-activated TrkB.FL signalling activity. Finally, we used electrophysiology to observe that a downregulation of TrkB.

1), but the cells appeared as visible clumps at 10–20 days after they were introduced into the fluids (Fig. 2). Xylella fastidiosa cell densities in grapevine xylem fluid were higher than those in the other tested xylem fluids by 20 days after inoculation (Fig. 1), but the

cell densities increased by 20 days in all xylem fluids. Bacterial cells grown in each xylem fluid were then inoculated to PD3 medium and confirmed to be X. fastidiosa species by specific PCR (data not shown). These data showed that X. fastidiosa can grow in the pure xylem fluid of citrus and grapevine in vitro. The percentage of aggregated cells of X. fastidiosa in grapevine xylem fluid was similar to that in PD3 medium, but significantly higher than that seen in citrus xylem fluid (Fig. 3). The bacterial cells aggregated to form tight clumps Androgen Receptor signaling pathway Antagonists in the xylem fluid of grapefruit, orange, and lemon. In contrast, bacterial cells were loosely clumped in grapevine xylem fluid (Fig. 2). Bacteria cells were more loosely clumped in PD3 medium than in the xylem fluids (Fig. 2). After 20 days of culturing, X. fastidiosa cells in the grapevine xylem fluid formed more biofilm than those in the citrus xylem fluid (Fig. 4). Of 111 selected

genes from X. fastidiosa tested in a DNA macroarray, 27 genes were differentially expressed in grapevine xylem fluid vs. citrus xylem fluid (Table 1). Most had a higher expression in the grapevine xylem fluid, but two genes had selleck screening library a higher expression in the citrus xylem fluid. Using RT-PCR, several genes putatively involved in virulence were validated based on differential expression in the xylem fluid of grapevine vs. citrus (Fig. 5). rRNA was detected at similar levels in bacteria grown

in each of the xylem fluids. No RNA was detected in the water and pure xylem fluid controls. The observation that X. fastidiosa cells growing in a pure xylem fluid from citrus and grapevine and appearing as visible clumps at 20 days after introduction into the fluid was consistent with previous studies using a mixture (1 : 1) of PD3 and xylem fluid (Bi et al., 2007). Xylella fastidiosa cells have been reported elsewhere to grow in 100% grapevine xylem fluid (Andersen et al., 2007; Zaini et al., Methocarbamol 2009), and in the present study, xylem fluid of citrus supported the growth of a PD strain of X. fastidiosa, although this strain does not cause disease in citrus. This supports the hypothesis that citrus may serve as an asymptomatic reservoir for X. fastidiosa in southern California (Perring et al., 2001; Bi et al., 2007). Biofilm formation is a major factor in X. fastidiosa virulence (Marques et al., 2002), and our measurements of enhanced biofilm formation in grapevine xylem fluid are consistent with the recent report of Zaini et al. (2009). The observation that more biofilm was formed in the grapevine xylem fluid than in the citrus xylem fluids (Fig. 4) would be compatible with the observation that infections of citrus species by X.

, 2001). For

each transformant that disrupted a gene in the selleck products current library that had not been disrupted in the previous library, the genomic position of the transposon was confirmed by performing two sets of PCR amplifications, analyzed on agarose gels stained with ethidium bromide, as described (French et al., 2008). The first set used a transposon-specific primer paired with a gene-specific primer. The presence of a PCR product of the predicted size indicated that the transposon was at the expected location, provided that the same PCR product was absent when using the parental wild-type strain as template. For the second PCR amplification, two gene-specific primers were used that would flank the site of the transposon. If the expected product was obtained with wild-type DNA as template but no product was obtained with the transformant DNA as template, it was concluded that the gene was disrupted

and that the transformant lacked a second, intact copy of the gene. For some transformants, the PCR amplifications confirmed the location of the transposon but also detected the presence of an intact copy of the gene. In these cases, the transformant culture was subcloned and the two PCR reactions were performed again on each subclone. Before subcloning, cell aggregates were Epigenetic inhibitor chemical structure disrupted by sonicating in a sonifier (model Teicoplanin 250/450; Branson, Danbury, CT) at a power level of 5 and a duty cycle of 10% for 20 s. These conditions maximally increased the CFU of the cultures.

No gene was considered to be disrupted unless the PCR data indicated that at least one subclone had the transposon at the expected site with no intact copy of the gene. Rarely, the PCR data indicated that all subclones of a transformant had an intact copy of the gene that was disrupted by the transposon. The presence of both a disrupted and an intact copy of the gene suggested gene duplication. In these cases, the identity of the PCR products was confirmed by performing another PCR amplification. The products from the first amplification that had used primers that flanked the insertion site of the transposon were used as template, and an internal set of primers was used for amplification. In cases where there was doubt regarding the results, the PCR products were also sequenced. A total of 1210 different minitransposon insertion sites were mapped. Thus, the library is smaller than the original Tn4001T library for which 1856 different insertion sites were mapped (French et al., 2008). Combined, the libraries provide excellent coverage, with, on average, a transposon insertion site every 300 bp in the 960-kb genome of M. pulmonis.

Weekly weighing indicated no weight loss in TMZ-treated rats (Fig. S1). Bromodeoxyuridine (BrdU; Sigma) was injected intraperitoneally at a dose of 200 mg/kg (concentration, 15 mg/mL) to mark the dividing cells in the dentate gyrus. In the first experiment RG7422 cell line (Fig. 1A), the overall effect of TMZ on adult hippocampal neurogenesis was examined in naïve adult rats. To evaluate the effect of chemotherapy on a larger population of cells generated

during and surviving past the drug treatment, we injected BrdU multiple times during the first treatment cycle (three daily injections) – BrdU was injected first, and this was followed by a TMZ injection at least 2 h later. Each BrdU injection labeled the population of cells that were in S-phase during the 2 h for which BrdU remains systemic. In all further experiments, BrdU was injected only once, to enable more straightforward determination of the age of the labeled cell population. In the next two experiments this website (Fig. 1B and C), BrdU was injected at different time points with regard to both drug treatment and learning/training, to verify the expected reduction in the number of BrdU-labeled cells caused by TMZ, and to examine possible changes in this reduction. The rats in the first three experiments (Fig. 1A–C) were all euthanised 21 days after the (last) BrdU injection. In the last experiment (Fig. 1D), we assessed the effects of long-term chemotherapy on the size of the proliferating cell population. For

this, BrdU was injected after a total of four cycles of drug treatment, and rats were euthanised 7 days later. It is acknowledged that the repeated injections might act as a stressor, and thus affect the outcome of the experiments. However, the number Adenosine triphosphate of injections was the same for rats treated with saline and for those treated with TMZ. In addition, in male rats, stress facilitates rather than impairs learning (Maeng et al., 2010). To assess learning and memory, we used different variations of classical eyeblink conditioning, a type of learning for which the neural basis is well known, and learning does not require

physical activity or exploration. In eyeblink conditioning, a neutral conditioned stimulus (CS) is repeatedly paired with aversive stimulation of the eyelid [unconditioned stimulus (US)]. As a result, the subject learns to blink the eyelid shut in response to the CS. In the trace variant of this task, the CS precedes the US, but the two stimuli do not overlap. In the VLD and delay variants, the CS onset precedes the US, and the two stimuli overlap and coterminate. To study the effects of chemotherapy on hippocampus-dependent associative learning, we trained TMZ/saline-treated rats in trace eyeblink conditioning (Fig. 1B). The same rats were then trained in standard delay eyeblink conditioning, a hippocampus-independent task, to ensure that possible learning deficits observed during trace conditioning were not caused by an overall inability to learn an eyeblink conditioned response.

Weekly weighing indicated no weight loss in TMZ-treated rats (Fig. S1). Bromodeoxyuridine (BrdU; Sigma) was injected intraperitoneally at a dose of 200 mg/kg (concentration, 15 mg/mL) to mark the dividing cells in the dentate gyrus. In the first experiment BMN 673 (Fig. 1A), the overall effect of TMZ on adult hippocampal neurogenesis was examined in naïve adult rats. To evaluate the effect of chemotherapy on a larger population of cells generated

during and surviving past the drug treatment, we injected BrdU multiple times during the first treatment cycle (three daily injections) – BrdU was injected first, and this was followed by a TMZ injection at least 2 h later. Each BrdU injection labeled the population of cells that were in S-phase during the 2 h for which BrdU remains systemic. In all further experiments, BrdU was injected only once, to enable more straightforward determination of the age of the labeled cell population. In the next two experiments CHIR99021 (Fig. 1B and C), BrdU was injected at different time points with regard to both drug treatment and learning/training, to verify the expected reduction in the number of BrdU-labeled cells caused by TMZ, and to examine possible changes in this reduction. The rats in the first three experiments (Fig. 1A–C) were all euthanised 21 days after the (last) BrdU injection. In the last experiment (Fig. 1D), we assessed the effects of long-term chemotherapy on the size of the proliferating cell population. For

this, BrdU was injected after a total of four cycles of drug treatment, and rats were euthanised 7 days later. It is acknowledged that the repeated injections might act as a stressor, and thus affect the outcome of the experiments. However, the number Phosphatidylinositol diacylglycerol-lyase of injections was the same for rats treated with saline and for those treated with TMZ. In addition, in male rats, stress facilitates rather than impairs learning (Maeng et al., 2010). To assess learning and memory, we used different variations of classical eyeblink conditioning, a type of learning for which the neural basis is well known, and learning does not require

physical activity or exploration. In eyeblink conditioning, a neutral conditioned stimulus (CS) is repeatedly paired with aversive stimulation of the eyelid [unconditioned stimulus (US)]. As a result, the subject learns to blink the eyelid shut in response to the CS. In the trace variant of this task, the CS precedes the US, but the two stimuli do not overlap. In the VLD and delay variants, the CS onset precedes the US, and the two stimuli overlap and coterminate. To study the effects of chemotherapy on hippocampus-dependent associative learning, we trained TMZ/saline-treated rats in trace eyeblink conditioning (Fig. 1B). The same rats were then trained in standard delay eyeblink conditioning, a hippocampus-independent task, to ensure that possible learning deficits observed during trace conditioning were not caused by an overall inability to learn an eyeblink conditioned response.

The antibiotic stock solutions were prepared by dissolving them in sterile distilled water at concentrations of 256 μg mL−1 (ampicillin, aztreonam, cefotaxime, cefoxitin, ceftazidime, cephalothin, oxacillin, and piperacillin) and serial

dilution (1 : 2) with TSB (pH 7.3). The strains of S. aureus KACC13236, S. aureus CCARM 3080, S. Typhimurium KCCM 40253, and S. Typhimurium CCARM 8009 were anaerobically cultured in TSB at pH 5.5 and 7.3 to obtain planktonic and biofilm cells. In accordance with the CLSI procedure, the planktonic and biofilm cells grown in Bleomycin molecular weight TSB at pH 5.5 and 7.3 were incubated in the diluted antibiotic solutions for 18 h at 37 °C to evaluate the susceptibility of cells to antibiotics. Minimum inhibitory concentrations (MICs) were determined at concentrations at which there was no visible growth. The susceptible (S), intermediate (I), and resistant

(R) strains were defined based on MIC values of < 4 μg mL−1, between 4 and 8 μg mL−1, and more than 16 μg mL−1, respectively (Hamilton-Miller & Shah, 1996). The numbers of planktonic and biofilm cells were Trichostatin A ic50 estimated using the plate count method. For planktonic cell counts, the cell suspensions were collected and the remaining non-adherent cells were rinsed by flooding the plate surface with 10 mL of 0.1% sterile BPW. For biofilm cell counts, the attached cells were collected with a cell scraper (Thermo Scientific Nunc, Rochester, NY) and suspended by sonication at 20 kHz for 10 min in 20 mL of 0.1% sterile BPW. The collected cells were serially diluted (1 : 10) with 0.1% sterile BPW and the proper dilutions were plated on trypticase soy agar (TSA). The agar plates were incubated at 37 °C for 48 h PI-1840 for enumeration of planktonic and biofilm cells. Each planktonic or biofilm culture (0.5 mL) was mixed with 1 mL of RNAprotect Bacteria Reagent (Qiagen, Hilden, Germany) and centrifuged at 5000 g for 10 min. The collected cells were used for RNA extraction according to the RNeasy® Mini Handbook (Qiagen). The collected cells were disrupted in a buffer containing guanidine isothiocyanate

and lysozyme, mixed with ethanol to adjust proper binding conditions, and then loaded into an RNeasy mini column for RNA isolation. The cDNA was synthesized as described previously (Xu et al., 2010), according to the QuantiTect Reverse Transcription protocol (Qiagen). In brief, the RNA sample was mixed with a master mixture containing Quantiscript Reverse Transcriptase, Quantiscript RT Buffer, RT Primer Mix and RNase-free water, incubated at 42 °C for 15 min, and then immediately incubated at 95 °C for 3 min to inactivate the Quantiscript Reverse Transcriptase. The custom-synthesized oligonucleotide primers using IDT (Integrated DNA Technologies Inc., Coralville, IA) were used in this study (Tables 1 and 2). The PCR mixture (20 μL) containing 2× QuantiTect SYBR Green PCR Master (10 μL), 60 pmol primer (0.6 μL), cDNA (2 μL), and RNase-free water (6.