A majority of the sequences (32 clones) exhibited high similarity (98.8–100% sequence identity) to bacteria of genus Aeromonas, accounting for nearly 3-MA chemical structure 80% of Gammaproteobacteria. The other nine sequences were related to the genera Beggiatoa, Pseudomonas, Dicheya, and Enterobacter (Table 1; Fig. 1b). Betaproteobacteria were less abundant than Alpha and Gamma classes of Proteobacteria. Of the 27 clones in the Betaproteobacteria

class (Fig. 1b), 20 were closely related to Burkholderiales (74.1% of Betaproteobacteria) and belonged to genera Roseateles, Aquincola, Ideonella, Piscinibacter, Coccomonas, Hydrogenophaga, Rhodoferax, and Janthinobacterium. An additional seven clones were grouped into Rhodocyclales and classified as Dechloromonas. Dechloromonas and Rhodoferax were the most abundant genera in this subgroup (Table 1). Fifteen clones grouped into Deltaproteobacteria, U0126 including five OTUs, were closely related to five different species of sulfate-reducing bacteria (SRB) (97–100% sequence identity). Of these, the sequences of five clones were closely

related to Desulfomicrobium norvegicum and Pelobacter propionicus, making them the dominant species of Deltaproteobacteria. In addition, other clones were assigned to Desulfomonile limimaris, Desulfobacterium catecholicum, and Desulfovibrio putealis. All three clones related to Epsilonproteobacteria showed high similarity to Sulfurospirillum halorespirans (99.9%

sequence identity) (Table 1; Fig. 1c). In total, the SRB occupied nearly 13.6% of Proteobacteria. Among non-Proteobacteria, the remaining 15 and 11 clones exhibited high similarity to the Firmicutes and CFB phyla (Fig. 1c), respectively. In Firmicutes, all 15 clones belonged to Clostridiales and the dominant genus was Clostridium (10 clones). Other genera Pembrolizumab in vivo included Cohnella (two clones), Acidaminobacter (two clones), and Acetobacterium (one clone). Of 11 clones grouped into the CFB phylum, four were closely related to genera Bacteroides (99.4% sequence identity) and Prevotella (97.9% sequence identity) in the Bacteroidales order, and others were distantly related to the genera Paludibacte, Prolixibacter, Wandonia, and Flavisolibacter (90–92% sequence identity). Finally, four clones represented sequences assigned to Fusobacteria; they were distantly related to Ilyobacter (91.2% sequence identity) in the order Fusobacteriales (Table 1; Fig. 1c). Furthermore, alignment of all 166 sequences showed that the number of single type sequences was 15, and the calculated coverage of the clone library was 90.97%. The rarefaction curve also tended to plateau (Fig. 2), indicating that this library was sufficient to detect a large majority of the endophytic bacterial diversity in the reed roots used in our research.

The status of layer IV in area 4 thus pertains to core organisational features of the cortex, its connections and evolution. “
“Cocaine stimuli often trigger relapse of drug-taking, even following periods of prolonged abstinence. Here, electrophysiological

recordings were made in rats (n = 29) to determine how neurons in the prelimbic (PrL) or infralimbic (IL) regions of the medial prefrontal cortex (mPFC) encode cocaine-associated stimuli and cocaine-seeking, and whether this find more processing is differentially altered after 1 month of cocaine abstinence. After self-administration training, neurons (n = 308) in the mPFC were recorded during a single test session conducted either the next day or 1 month later. Test sessions consisted of three phases during which (i) the tone–houselight stimulus previously paired with cocaine infusion during self-administration was randomly presented by the experimenter, (ii) rats responded on the lever previously associated with cocaine during extinction and (iii) the tone–houselight

was presented randomly between cocaine-reinforced MEK inhibitor responding during resumption of cocaine self-administration. PrL neurons showed enhanced encoding of the cocaine stimulus and drug-seeking behavior (under extinction and self-administration) following 30 days of abstinence. In contrast, although IL neurons encoded cocaine cues and cocaine-seeking, there were no pronounced changes

in IL responsiveness following 30 days of abstinence. Importantly, cue-related changes do not represent a generalised stimulus-evoked discharge as PrL and IL neurons in control animals (n = 4) exhibited negligible recruitment by the tone–houselight stimulus. The results support the view that, following abstinence, neural encoding in the PrL but not IL may play a key role in enhanced cocaine-seeking, particularly following re-exposure to cocaine-associated cues. “
“Within most contemporary learning theories, reinforcement prediction error, the difference between the obtained and expected reinforcer value, critically influences associative learning. In some theories, this prediction error determines the momentary effectiveness of the reinforcer itself, such that the same Buspirone HCl physical event produces more learning when its presentation is surprising than when it is expected. In other theories, prediction error enhances attention to potential cues for that reinforcer by adjusting cue-specific associability parameters, biasing the processing of those stimuli so that they more readily enter into new associations in the future. A unique feature of these latter theories is that such alterations in stimulus associability must be represented in memory in an enduring fashion. Indeed, considerable data indicate that altered associability may be expressed days after its induction.

, 2012). Ammonium, nitrite, and nitrate were extracted from the soil with 2 M KCl and measured using a SAN++ Continuous Flow Analyzer (Skalar Analytical, The Netherlands). Total nitrogen, soil organic matter, Mn2+, and Mn4+ were measured according to standard methods (Bao, 2000). Soil pH was determined at a soil/water ratio of 1 : 2.5. All analyses were performed in triplicate on each sample. The in situ measurement of oxygen concentration was achieved by OXY Meter S/N 4164 with stainless electrode sensor (Unisense, Denmark) (Gundersen et al., 1998). Statistical analyses were performed using program spss for Windows. DNA in soil and sediment samples were extracted

from 0.25 g selleck chemical samples using the Powersoil DNA isolation kits (Mobio). DNA from enriched anammox biomass was extracted according to the method described previously (Schmid et al., Ulixertinib 2008). For the specific PCR amplification of the anammox hzsB gene,

newly designed primer pair of hzsB_396F and hzsB_742R was applied based on our new findings in anammox molecular mechanism (Kartal et al., 2011; Harhangi et al., 2012). The pmoA gene of n-damo bacteria was amplified using a nested approach (first-step primer pair A189_b-cmo682, followed by primer pair cmo182-cmo568) according to Luesken et al. (2011c). The 16S rRNA gene of n-damo was amplified using a nested approach (first-step primer pair 202F-1545R, followed by primer pair qP1F-qP2R) according to Juretschko et al. (1998) and Ettwig et al. (2009). The sequences of primers and thermal profiles were shown in Table 1. PCRs

were performed with the PerfeCTa SYBR Green FastMix (Quanta). 10 min at 95 °C, followed by 35 cycles of 60 s at 95 °C, 60 s at 59 °C and 45 sat 72 °C (PCR) 3 min at 95 °C, followed by 40 cycles of 30 s at 95 °C, 30 s at 59 °C and 30 sat 72 °C (qPCR) 10 min at 95 °C, followed by30 cycles of 60 s at 95 °C, 60 s at 63 °C and 45 sat 72 °C (PCR, qP1F – qP2R) 3 min at 95 °C, followed Methisazone by 40 cycles of 30 s at 95 °C, 30 s at 63 °C and 30 sat 72 °C (qPCR, qP1F – qP1R) PCR amplified fragments were cloned using the pGEM-T Easy cloning kit (Promega) according to the manufacturer’s instructions. Plasmid DNA was isolated with the GeneJET Plasmid Miniprep kit (Fermentas, Lithuania). Plasmids were digested with EcoRI enzyme, and the digestion products were examined for an insert with expected size by agarose (1%) gel electrophoresis. Selected clones were sequenced using primer of M13f targeting vector sequences adjacent to the multiple cloning sites. Phylogenetic analysis was performed using mega 5.0 software (Tamura et al., 2011) by neighbor-joining (NJ) with the Jukes-Cantor correction. Diversity indices, including Chaol, Shannon, and Simpson, were generated by DOTUR for each clone library (Schloss & Handelsman, 2005). Quantitative PCR was performed on a Bio-Rad iQ5 real-time PCR instrument (Bio-Rad) with a SYBR Green qPCR kit (Quanta).

, 2012). Ammonium, nitrite, and nitrate were extracted from the soil with 2 M KCl and measured using a SAN++ Continuous Flow Analyzer (Skalar Analytical, The Netherlands). Total nitrogen, soil organic matter, Mn2+, and Mn4+ were measured according to standard methods (Bao, 2000). Soil pH was determined at a soil/water ratio of 1 : 2.5. All analyses were performed in triplicate on each sample. The in situ measurement of oxygen concentration was achieved by OXY Meter S/N 4164 with stainless electrode sensor (Unisense, Denmark) (Gundersen et al., 1998). Statistical analyses were performed using program spss for Windows. DNA in soil and sediment samples were extracted

from 0.25 g mTOR inhibitor samples using the Powersoil DNA isolation kits (Mobio). DNA from enriched anammox biomass was extracted according to the method described previously (Schmid et al., find more 2008). For the specific PCR amplification of the anammox hzsB gene,

newly designed primer pair of hzsB_396F and hzsB_742R was applied based on our new findings in anammox molecular mechanism (Kartal et al., 2011; Harhangi et al., 2012). The pmoA gene of n-damo bacteria was amplified using a nested approach (first-step primer pair A189_b-cmo682, followed by primer pair cmo182-cmo568) according to Luesken et al. (2011c). The 16S rRNA gene of n-damo was amplified using a nested approach (first-step primer pair 202F-1545R, followed by primer pair qP1F-qP2R) according to Juretschko et al. (1998) and Ettwig et al. (2009). The sequences of primers and thermal profiles were shown in Table 1. PCRs

were performed with the PerfeCTa SYBR Green FastMix (Quanta). 10 min at 95 °C, followed by 35 cycles of 60 s at 95 °C, 60 s at 59 °C and 45 sat 72 °C (PCR) 3 min at 95 °C, followed by 40 cycles of 30 s at 95 °C, 30 s at 59 °C and 30 sat 72 °C (qPCR) 10 min at 95 °C, followed by30 cycles of 60 s at 95 °C, 60 s at 63 °C and 45 sat 72 °C (PCR, qP1F – qP2R) 3 min at 95 °C, followed Decitabine purchase by 40 cycles of 30 s at 95 °C, 30 s at 63 °C and 30 sat 72 °C (qPCR, qP1F – qP1R) PCR amplified fragments were cloned using the pGEM-T Easy cloning kit (Promega) according to the manufacturer’s instructions. Plasmid DNA was isolated with the GeneJET Plasmid Miniprep kit (Fermentas, Lithuania). Plasmids were digested with EcoRI enzyme, and the digestion products were examined for an insert with expected size by agarose (1%) gel electrophoresis. Selected clones were sequenced using primer of M13f targeting vector sequences adjacent to the multiple cloning sites. Phylogenetic analysis was performed using mega 5.0 software (Tamura et al., 2011) by neighbor-joining (NJ) with the Jukes-Cantor correction. Diversity indices, including Chaol, Shannon, and Simpson, were generated by DOTUR for each clone library (Schloss & Handelsman, 2005). Quantitative PCR was performed on a Bio-Rad iQ5 real-time PCR instrument (Bio-Rad) with a SYBR Green qPCR kit (Quanta).

, 2012). Ammonium, nitrite, and nitrate were extracted from the soil with 2 M KCl and measured using a SAN++ Continuous Flow Analyzer (Skalar Analytical, The Netherlands). Total nitrogen, soil organic matter, Mn2+, and Mn4+ were measured according to standard methods (Bao, 2000). Soil pH was determined at a soil/water ratio of 1 : 2.5. All analyses were performed in triplicate on each sample. The in situ measurement of oxygen concentration was achieved by OXY Meter S/N 4164 with stainless electrode sensor (Unisense, Denmark) (Gundersen et al., 1998). Statistical analyses were performed using program spss for Windows. DNA in soil and sediment samples were extracted

from 0.25 g CH5424802 cell line samples using the Powersoil DNA isolation kits (Mobio). DNA from enriched anammox biomass was extracted according to the method described previously (Schmid et al., buy RAD001 2008). For the specific PCR amplification of the anammox hzsB gene,

newly designed primer pair of hzsB_396F and hzsB_742R was applied based on our new findings in anammox molecular mechanism (Kartal et al., 2011; Harhangi et al., 2012). The pmoA gene of n-damo bacteria was amplified using a nested approach (first-step primer pair A189_b-cmo682, followed by primer pair cmo182-cmo568) according to Luesken et al. (2011c). The 16S rRNA gene of n-damo was amplified using a nested approach (first-step primer pair 202F-1545R, followed by primer pair qP1F-qP2R) according to Juretschko et al. (1998) and Ettwig et al. (2009). The sequences of primers and thermal profiles were shown in Table 1. PCRs

were performed with the PerfeCTa SYBR Green FastMix (Quanta). 10 min at 95 °C, followed by 35 cycles of 60 s at 95 °C, 60 s at 59 °C and 45 sat 72 °C (PCR) 3 min at 95 °C, followed by 40 cycles of 30 s at 95 °C, 30 s at 59 °C and 30 sat 72 °C (qPCR) 10 min at 95 °C, followed by30 cycles of 60 s at 95 °C, 60 s at 63 °C and 45 sat 72 °C (PCR, qP1F – qP2R) 3 min at 95 °C, followed check details by 40 cycles of 30 s at 95 °C, 30 s at 63 °C and 30 sat 72 °C (qPCR, qP1F – qP1R) PCR amplified fragments were cloned using the pGEM-T Easy cloning kit (Promega) according to the manufacturer’s instructions. Plasmid DNA was isolated with the GeneJET Plasmid Miniprep kit (Fermentas, Lithuania). Plasmids were digested with EcoRI enzyme, and the digestion products were examined for an insert with expected size by agarose (1%) gel electrophoresis. Selected clones were sequenced using primer of M13f targeting vector sequences adjacent to the multiple cloning sites. Phylogenetic analysis was performed using mega 5.0 software (Tamura et al., 2011) by neighbor-joining (NJ) with the Jukes-Cantor correction. Diversity indices, including Chaol, Shannon, and Simpson, were generated by DOTUR for each clone library (Schloss & Handelsman, 2005). Quantitative PCR was performed on a Bio-Rad iQ5 real-time PCR instrument (Bio-Rad) with a SYBR Green qPCR kit (Quanta).

A survey conducted in 2005, indicated an increased number of paediatric dentists were using the 1-appointment IPT technique in the United States compared with a 1997 survey[23]. In our study, the CH-IPT and 3Mix-MP overall success rates at the 12–29 month recall were 94% and 78%, respectively. These results are consistent with previous studies where the success rates of IPT with follow-up periods from 3 months–7 years ranged from 84–100% regardless of the type of base material or final restoration[4-9, 24-27]. The slightly lower success rate in our study may have resulted from inclusion criteria that accepted only mandibular

primary molars in which the diagnosis of pathology is easier compared with the maxillary teeth where overlapping permanent tooth buds can complicate their evaluation. Other studies included

maxillary and mandibular primary molars[4, 5, 7, 8, 24, PD0325901 cell line 25] and some included both primary and permanent molars[6, 27]. Our data indicate that both techniques yielded similar success rates. Marchi et al.[24] found that the most frequent cause of IPT failure at the 6–12 month follow-up was the clinical observation of a fistula (2 of 27 teeth; 7.41%), suggesting misdiagnosis of the pulpal condition. In contrast, we did not find any clinical signs or symptoms of irreversible pulpitis or pulp necrosis at our 6–11 month recall. We did find a fistula or abnormal mobility in two teeth in the 3Mix-MP check details group at the 12–29 month recall. In our study, almost all findings of overall failure

resulted from radiographic failure except in one tooth in the 3Mix-MP group that was a clinical failure (abnormal mobility) but exhibited radiographic success with canal obliteration. Most of our findings are consistent with Farooq et al.[7] who found all clinical failures exhibited radiographic failure, but not all radiographic failures had clinical signs and symptoms. In our study, bifurcation or inter-radicular radiolucency was the Abiraterone in vitro most frequent failure seen at the 6–11 month recall, whereas internal root resorption and bifurcation radiolucency were the most frequent failures observed at the 12–29 month recall. This finding is consistent with a previous study by Falster et al. in which the majority of their failures were from interradicular lesions noted at the 12–24 month recall, whereas internal root resorption was found in one tooth at the 18-month recall[4]. Precise radiographic and careful clinical diagnoses are essential to the high success rate of IPT. Pain and sensitivity are important clinical symptoms for proper diagnosis. It is difficult to obtain precise information related to these symptoms from children, however. Thus, parents, participation may help paediatric dentists more precisely make the pulpal diagnosis. The failures observed in our study could be explained by the difficulty in the diagnosis of pulpal status based on the child and parent’s report of symptoms.

Mice were kept at 21 ± 2 °C under a reversed 12 : 12-h light/dark cycle (lights off at 08:30 h, < 0.5 lux; lights on at 20 : 30 h; 170 lux at the bottom of the cage, unless otherwise stated). Standard food pellets (Scanbur, Sollentuna, Sweden) and water were available ad libitum. All experiments were performed according to the Finnish Act on the Use of Animals for Experimental Purposes, and were approved by the Animal Experiment Committee of the State Provincial Office of Southern Finland. All

experiments were carried out in accordance with Roxadustat purchase the European Communities Council Directive of 24 November 1986 (86/609/EEC), and with the Guidelines laid down by the NIH in the USA regarding the care and use of animals for experimental procedures. The total number of animals used in this study was 116. For the enzyme activity studies, histamine and 1-methylhistamine assays, and the in situ

hybridization assay, the mice were housed in groups of three, which may have potentially confounded the results. Although this issue has not been addressed specifically, we interpreted the data of Mistlberger & Skene (2004) and Paul & Schwartz (2007) to mean that, in the presence of light the effect of social cues should be small or absent. Importantly, the housing conditions were identical for all mice in the analysis. The chemical reagents used were as follows: Ketalar, Domitor, and Antisedane (Pfizer Animal Health, New York, USA); dental cement (Candulor, Wangen, Germany); buprenorphine (Temgesic; Reckitt Benckiser, Slough, UK); deoxyadenosine 5′-triphosphate, [α-33P] click here (NEG312H; NEN Research Products, PerkinElmer, Waltham, MS, USA); 3-methylhistamine, CaCl2, NaCl, KCl, KH2PO4, and methanol (Merck, Whitehouse Station, NJ, USA); 1-methylhistamine, aminoguanidine hydrochloride, citric acid, Denhardt’s solution, dithiothreitol, Celastrol histamine dihydrochloride,

H3PO4, l-histidine, NaH2PO4, NaN3, NaOH, N-lauroylsarcosine, o-phthalaldehyde, polyethylene glycol 300, pyridoxal phosphate, pargyline hydrochloride, HClO4, β-mercaptoethanol, phenylmethanesulfonyl fluoride, S-adenosyl-methionine, and sodium salt of octanesulphonic acid (Sigma, St Louis, MO, USA); MgCl2 and H3BO3 (Riedel-deHaёn, Seelze, Germany); formamide (Amresco, Solon, OH, USA); RNA (Roche, Basel, Switzerland); and dextran sulphate (Amersham, Amersham, UK). Thirty male 8–9-week-old C57BL/6J mice were kept in groups of three under a 12 : 12-h light/dark cycle. Mice were killed by decapitation at zeitgeber time (ZT) 4, 8, 12 (lights on), 16, 20 and 24 (lights off). The brains were removed, and rapidly frozen at −80 °C. Coronal 20-μm sections were cut with a Leica CM3050S cryostat (Leica, Wetzlar, Germany), and mounted onto SuperFrost slides (ThermoScientific, Portsmouth, USA). The section levels corresponding to the TMN region (−2.2 mm to −2.8 mm relative to bregma) were determined according to the mouse brain stereotaxic atlas (Paxinos & Franklin, 2004). The sections were stored at −20 °C until analysis.

Control larvae were injected with 0.9% NaCl. To determine whether DHA confers a protective effect to Burkholderia-infected larvae, a single dose of DHA (50 mM) was administrated 6 h postinfection. To determine intracellular bacterial numbers, hemolymph was obtained from three infected

larvae by puncturing the larval abdomen with a sterile needle. The out-flowing hemolymph was immediately transferred into a sterile Eppendorf tube containing a few crystals of phenylthiourea to prevent melanization. Then, hemolymph was serially diluted in 0.9% DMXAA mw NaCl and plated on LB agar. Colonies were counted after incubation at 37 °C for 24 h. Three larvae per treatment for each time point (10 and 21 h postinfection) were cryopreserved, sliced and homogenized in 1 mL of Trizol reagent (Sigma–Aldrich). Whole animal RNA was extracted according to the manufacturer’s protocol. RNA was treated with Turbo DNase (Ambio, Applied Biosystems) according to manufacturer’s recommendations and quantified spectrophotometrically (NanoDrop ND-1000). Quantitative real-time reverse transcription PCR (RT–PCR) was performed BIBW2992 clinical trial with the 7500 real-time PCR system (Applied Biosystems), according to the protocols of the manufacturer. Briefly, cDNA was synthesized from 200 ng of total RNA using Taqman kit (Roche, Applied Biosystems) and then analyzed with Power SYBR Green

master mix (Applied Biosystems), using primers to amplify the genes encoding antimicrobial peptides: gallerimycin (Altincicek & Vilcinskas, 2006), galliomycin (Wojda et al., 2009), inducible metalloproteinase inhibitor (IMPI) (Altincicek & Vilcinskas, 2006), lysozyme (Altincicek & Vilcinskas, 2006) and the housekeeping gene actin (Altincicek & Vilcinskas, 2006). All samples were analyzed in triplicate, and the amount of mRNA detected normalized to control actin mRNA values. Relative quantification of genes expression was calculated by using the ∆∆CT method (Livak & Schmittgen, 2001). All experiments were performed a minimum of three times. Relative comparisons were done between corrected values with anova test for significance.

A P-value Mannose-binding protein-associated serine protease < 0.05 was considered statistically significant. The antibacterial activity of eight LCUFAs with various carboxyl lengths (18 carbons or more) was quantitatively evaluated against B. cenocepacia K56-2. The growth was monitored for 24 h at 37 °C in the presence of 20 mM of each LCUFA by measuring the OD640 nm. Of the eight lipids tested, only 3 [Petroselinic acid 18:1 (n-6), DHA 22:6 (n-3) and nervonic acid 24:1 (n-9)] showed growth inhibition effects. DHA caused the greatest growth inhibition (50–60%) compared with the control (Fig. 1), so it was selected for further studies. A control assay with 2.7% ethanol had no effect on the growth of B. cenocepacia K56-2 (Fig. 1). DHA against B. cenocepacia K56-2 recorded a MIC range of 40–50 mM, determined after 24 h by the reference broth microdilution method.

The greater role of community pharmacy has been continuously recognised to be vital to the operation of the National Health Service (NHS). The current role pharmacy plays needs to be significantly improved and amplified to conform to the ever changing healthcare environment and this can be achieved through practice research. Successful change

management is required to ensure community pharmacists’ (CPs) engagement in practice research is facilitated to directly improve patient outcomes, better pharmacy practice, expand the pharmacy profession and demonstrate pharmacy’s integral role within the recent NHS reforms.1 This cross-sectional study aimed to determine what CPs thought was meant by practice research and their current level of engagement, if any, in practice research. A structured PI3K inhibitor questionnaire (piloted and

amended accordingly), was posted for self-completion with follow up telephone interviews. The surveyed CPs were randomly selected from five random Primary Care Trust (PCT) areas in different geographical locations across England: Bedfordshire, Cornwall & Isles of Scilly, Richmond & Twickenham, Wakefield and Warwickshire. For the first phase of the study, the structured questionnaire allowed CPs to convey their responses by way of close ended questions (including Likert scales and multiple choice questions) and some open ended questions. The telephone interviews were used to further explore CPs’ attitudes and reasoning towards practice research. Data from the postal questionnaires AZD2014 chemical structure were entered and analysed using statistical software and telephones interviews were analysed using thematic and coding analysis.2 The study was approved by the Kingston University Ethics Committee. Following the data collection period, a total

of 53 postal questionnaires out of 323 were returned else (response rate of 16.4%). 49% (26/53) of CPs claimed that they had engaged in some form of research in the past where 50% of this cohort (13/26) considered audits to be a form of research activity. Of those that had not engaged in research in the past, 51.9% (14/27) of CPs were interested in engaging in research in the future. Overall, 67.9% (36/53) of CPs wished to engage in research in the future, of which 55.5% (20/36) expressed that they required training to facilitate their engagement. 12 CPs from the surveyed population were interviewed. Thematic analysis revealed the following themes; research reflecting on day-to-day practice, community pharmacy as an appropriate setting for research, improving health outcomes and achieving benefits as a driver for engagement, sharing best practice, time pressures and busy schedules and lack of management support and training. Suggestions were made as to how CPs could be encouraged to engage in practice research, which included better communication, support and training and change management.

, 2001a,b; Garcia-Osta et al., VX-770 in vitro 2006; Nikitin, 2007), neuroinflammation

(Cardinaux et al., 2000; Ejarque-Ortiz et al., 2007; Straccia et al., 2011; Fields & Ghorpade, 2012), neurogenesis, and neuronal proliferation and differentiation (Cortés-Canteli et al., 2002; Calella et al., 2007; Aguilar-Morante et al., 2011), whereas its role in neuronal survival/apoptosis remains unclear. In fact, C/EBP β induces the expression of genes involved in brain injury and inflammatory processes; it is upregulated after ischemic injury and in a mouse model of hippocampal kainate excitotoxicity, as well as in adult hippocampal neurogenesis (Cortés-Canteli et al., 2004, 2008, 2011; Sandhir & Berman, 2010; Rininger et al., 2012). In cortical neurons, C/EBP β expression is induced after hypoxic stress, supporting neuronal survival by inhibiting p53 (Halterman et al., 2008). On the other hand, C/EBP β induces apoptosis in neuroblastoma through p53 activation (Cortés-Canteli et al., 2002). In primary cultures of rat cerebellar granule neurons (CGNs), high Ca2+ influx through N-methyl-d-aspartate (NMDA) receptors increases BMS-777607 in vitro nuclear C/EBP β levels and induces excitotoxic neuronal death (Marshall et al., 2003). However, no studies so far have studied the expression of all C/EBP β isoforms in survival/apoptotic conditions. To fill this gap, we used neuronal primary cultures and induced apoptosis, in order

to study the role of C/EBP β isoforms in neuronal survival/death. Primary cultures of CGNs were prepared from 7-day-old Wistar Han Outbred Rat pups derived from a local animal house (Gallo et al., 1987). All animal experiments were authorized

by a local bioethical committee (Protocol no. 17-72-1212), and experiments were carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC). Animal health and comfort were veterinarily controlled. For all experiments presented here, a total number of 72 pups were used. Briefly, animals were rapidly anesthetized with an ice-cold treatment, and killed by decapitation; cerebella were removed and dissected from their meninges in Krebs’ buffer containing 0.3% bovine serum albumin (BSA). Cerebella were then dissociated with trypsin at 37 °C for 15 min, and triturated by use of a Pasteur pipette, in a 0.125 mg/mL CYTH4 DNaseI/0.52 mg/mL soybean trypsin inhibitor solution. Dissociated cells were collected by centrifugation, resuspended in Basal Medium Eagle (Invitrogen, DH Breda, NL, USA), supplemented with 2 mm glutamine, 100 μm gentamicin sulfate, 10% inactivated fetal bovine serum (Invitrogen), and 25 mm KCl, and plated in plastic dishes, previously coated with poly-l-lysine (0.01 mg/mL), at a density of 2.2 × 106 cells per 35-mm dish. After incubation for 16 h at 37 °C in a 95% air/5% CO2 (v/v) atmosphere, 10 μm cytosine arabinofuranoside was added to reduce proliferation of non-neuronal cells.