Therefore, these differences in the phylogenetic diversities suggest that CTI is spread among all different groups of proteobacteria and the large identity variation indicates the enzymatic differences or development with the same enzymatic function (Heipieper et al. 2003). The next step was to verify the physiological activity of a cis–trans isomerase of unsaturated

fatty acids in M. capsulatus Bath. The most important environmental factors tested so far for their ability to trigger cis–trans isomerase activity in Pseudomonas and Vibrio strains are increases in temperature and the presence of organic solvents (Heipieper et al., 2003). Both factors are known to increase the fluidity selleck chemicals llc of the membrane, which is discussed as being the major signal for an activation of the constitutively present CTI (Kiran et al., 2004, 2005). Therefore, in the first experiments, cells of M. capsulatus that were regularly grown at 45 °C were exposed to different temperatures and the effect on the fatty acid composition was measured. The membrane phospholipids of cells grown exponentially selleck chemicals at 45 °C contained the

following major fatty acids: C16:0, C16:1Δ9trans, C16:1Δ9cis, C16:1Δ10cis, C16:1Δ11cis and C17cyclo. This fatty acid pattern as well as the relative abundances of the fatty acids are in agreement with previous observations for this bacterium (Makula, 1978; Nichols et al., 1985; Bowman et al., 1991; Guckert et al., 1991). Table 2 summarizes the effect of different growth temperatures on the fatty acid composition of M. capsulatus. When the cells were exposed to 60 °C, a significant increase Urease in the trans/cis ratio of unsaturated fatty acids was observed within one hour, whereas no change occurred at the growth temperature of 45 °C or when the cells were exposed to a lower temperature of 30 °C (Fig. 1). This increase in the content of palmitelaidic acid (16:1transΔ9)

was caused by a decrease in the content of the corresponding isomer palmitoleic acid (16:1cisΔ9), whereas the abundance of the other forms of 16:1cis (16:1cisΔ10 and (16:1cisΔ11) that are known to be exclusively present in methanotrophic bacteria (Makula, 1978; Nichols et al., 1985; Bowman et al., 1991; Guckert et al., 1991) remained constant. This observation is in agreement with previous findings showing that double bonds located deeper in the phospholipid bilayer such as Δ10 or Δ11 cannot be converted by the cis–trans isomerase, which is a hydrophilic periplasmic protein. This enzyme can only reach double bonds at a certain depth in the membrane and could be ‘within reach’ of the active site of the enzyme, which is anchored at the membrane surface. Under the conditions tested, positions Δ10 and Δ11 would be ‘out of reach’ (Heipieper et al., 2001). These results provided an indication for the presence of a cis–trans isomerase of unsaturated fatty acids in M. capsulatus.

We thank Dr J.P. Euzéby for his advice on nomenclature. This work was supported by Priority Research Centers Program (#2010-0094020) and a National

Research Foundation grant (#2011-0016498) through the National Research Foundation of Korea, funded by the Ministry of Education, Science, and Technology, Republic of Korea. The GenBank accession numbers for the genome sequences of strains LMG 5135T and ATCC 51223T are AFWQ00000000 selleck screening library and AFWR00000000, respectively. Please note: Wiley-Blackwell is not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. “
“Monitoring of methanogenic communities in anaerobic digesters using molecular-based methods is very attractive but can be cost-intensive. A new and fast quantification method by microscopic image analysis was developed to accompany molecular-based methods. This digitalized method, called quantitative microscopic fingerprinting (QMF), enables quantification of active methanogenic cells (N mL−1) by their characteristic auto-fluorescence

based on coenzyme F420. QMF was applied to analyze the methanogenic www.selleckchem.com/products/bgj398-nvp-bgj398.html communities in three biogas plant samples, and the results were compared with the relative proportion of gene copy numbers obtained with the quantitative PCR (qPCR). Analysis of QMF demonstrated dominance of Methanomicrobiales and Methanobacteriales

in relation to the total methanogenic community in digesters operating at high ammonia concentrations, which corresponded to the results established by qPCR. Absolute microbial counts by QMF and the numbers obtained by qPCR were not always comparable. On the other hand, the restricted morphological analysis by QMF was enhanced by the capability of qPCR to identify microbes. Consequently, dual investigations of both methods are proposed to improve monitoring of anaerobic digesters. For a rough estimation of the methanogenic composition Montelukast Sodium in anaerobic digesters, the QMF method seems to be a promising approach for the rapid detection of microbial changes. “
“The Gram-negative bacterium, Vibrio parahaemolyticus, is a major cause of seafood-derived food poisoning throughout the world. The pathogenicity of V. parahaemolyticus is attributed to several virulence factors, including two type III secretion systems (T3SS), T3SS1 and T3SS2. Herein, we compare the virulence of V. parahaemolyticus POR strains, which harbor a mutation in the T3SS needle apparatus of either system, to V. parahaemolyticus CAB strains, which harbor mutations in positive transcriptional regulators of either system. These strains are derived from the clinical RIMD 2210633 strain. We demonstrate that each mutation affects the virulence of the bacterium in a different manner.

All clinical specimens were stored at −70 °C for the duration of the study. DNA from culture samples was prepared by a simple

boiling method (Merritt et al., 2006). Culture samples obtained from the diagnostic laboratory were subcultured on nutrient agar and incubated at 37 °C overnight. DNA extraction from culture samples was done as described with some modifications. A single colony from the overnight culture was picked using a flamed wire loop and suspended in 100 μL of sterile distilled water. The bacterial suspension was then boiled at 100 °C for 10 min followed by centrifugation at 13 000 g for 1 min and the supernatant containing the DNA was aliquoted and stored at −20 °C for the course of the study. Extraction of DNA from blood samples was performed according to the protocol provided with the Qiagen Blood Mini Amp Kit (Qiagen). Three sets of primers were designed, each one targeting groEL (chaperonin) (gro1 and gro2) of Burkholderia genus, mprA (serine metalloprotease) check details (mpr1 and mpr2) gene of B. pseudomallei and zmpA (zinc metalloprotease) (zmp1 and zmp2) gene of B. cepacia, respectively (Table 1, Patent Ref: PI 20083144). All gene sequences were obtained from the National Centre for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov), and analyzed using the blast and clustalw programs to reveal the conserved as well

as unique regions of the targeted genes. The GenBank accession numbers for groEL, mprA and zmpA were AF287633, AF254803 and AY143552, respectively. The primers were designed with selleck kinase inhibitor similar melting temperatures to enable conversion of standard PCR to multiplex PCR in future. Each of the sequences was then analyzed using blast to ascertain the specificity of

the primers for the possibility of cross-reaction with other closely related organisms. The primer sequences were also analyzed for the presence of secondary structures using the oligo analyzer software. Primers that satisfactorily fulfilled the basic criteria were chosen and synthesized by Helix Biotech (Sigma Proligo, France). All PCR reactions these were set up in 0.5-μL flat cap Eppendorf microcentrifuge tubes. Optimization parameters included MgCl2 concentration, annealing temperature and the number of PCR cycles. MgCl2 concentrations were optimized using 1.0 mM, 1.5 mM and 2.5 mM and the annealing temperature was set at 52 °C (predicted, based on melting temperature of primers) and number of cycles randomly at 35. The annealing temperature was then optimized using gradient PCR at temperatures ranging from 50 to 60 °C. Finally, PCR cycles were optimized using 25, 30 and 35 cycles. The rest of the parameters were followed within the range recommended by standard PCR protocol: 1 × buffer, 0.2 μM of each of the primers, 200 μM of dNTP, 1.25 U of Taq DNA Polymerase recombinant and 5 ng μL−1 of DNA for 50 μL of final reaction volume. PCR reactions were performed using a BioRad DNA thermal cycler.

As with the DR task, aged monkeys are slower to learn the task and perform more poorly Selleckchem Cyclopamine as delay intervals are increased (Shamy et al., 2011). Behavioral flexibility is another frontal-dependent cognitive process that is compromised with aging. This has been studied using a variety of tasks, notably extradimensional set-shifting and reversal tasks in humans (Ridderinkhof et al., 2002; Marschner et al., 2005; Weiler et al., 2008), monkeys (Bartus et al., 1979; Lai et al., 1995; Voytko, 1999; Moore et al., 2003) and rats (Stephens et al., 1985; Barense et al., 2002; Schoenbaum et al., 2002; Nicolle & Baxter, 2003; Mizoguchi et al., 2010). Interestingly,

lesions of area 9 in marmoset monkeys affected extradimensional set-shifting performance, whereas lesions of the orbital PFC affected see more reversal performance (Dias et al., 1996). These data suggest that these tasks rely on different brain structures within the PFC. Extradimensional set-shifting refers to the problem of switching attention between cues that are in different perceptual dimensions in order to perform the task correctly. An example

of this is to train a rat to use light cues to determine which arm to select in a maze, and then shift the relevant cue to an auditory stimulus. When the extradimensional set-shifting occurs, the rat must shift its strategy and follow the sound cue in order to select the correct baited arm (e.g., Insel et al., 2012). In contrast, reversal learning refers to adapting a behavior to the changing contingencies required to reach a goal. For example, a rat can initially learn to press a lever in a ‘light-on’ condition to receive reward. After a reversal, the rat must adapt its behavior to press during the ‘light-off’ condition (e.g., Nomura et al., 2004). In parallel to the age-related cognitive deficits discussed above, aging is also associated with changes in attentional processes (Gazzaley & D’Esposito, 2007; Prakash et al., 2009; Hedden et al., 2012). This is accompanied by a greater susceptibility to distraction or interference during the delay period of a working memory task in humans (Bowles & Salthouse, 2003; Campbell et al., 2012) and monkeys (Bartus & Dean,

1979; Cyclin-dependent kinase 3 Prendergast et al., 1998). Additionally, fMRI studies in older adults have reported that there is increased activity in brain regions mediating distraction (Milham et al., 2002; Stevens et al., 2008), and in cases where task-irrelevant stimuli are presented (Gazzaley et al., 2005). One of the most consistent finding in the literature on aging brain is a decline in the volume of the PFC of humans, monkeys and rodents. This decline is one of the earliest changes detected, and for almost fifty years it was thought that the decrease in frontal lobe volume was the result of cell loss (Haug, 1986; Peters, 2002). The early reports of cell loss, however, turned out to be an error resulting from differential shrinkage of young and aged tissue during processing (Haug et al.

As with the DR task, aged monkeys are slower to learn the task and perform more poorly learn more as delay intervals are increased (Shamy et al., 2011). Behavioral flexibility is another frontal-dependent cognitive process that is compromised with aging. This has been studied using a variety of tasks, notably extradimensional set-shifting and reversal tasks in humans (Ridderinkhof et al., 2002; Marschner et al., 2005; Weiler et al., 2008), monkeys (Bartus et al., 1979; Lai et al., 1995; Voytko, 1999; Moore et al., 2003) and rats (Stephens et al., 1985; Barense et al., 2002; Schoenbaum et al., 2002; Nicolle & Baxter, 2003; Mizoguchi et al., 2010). Interestingly,

lesions of area 9 in marmoset monkeys affected extradimensional set-shifting performance, whereas lesions of the orbital PFC affected Crizotinib concentration reversal performance (Dias et al., 1996). These data suggest that these tasks rely on different brain structures within the PFC. Extradimensional set-shifting refers to the problem of switching attention between cues that are in different perceptual dimensions in order to perform the task correctly. An example

of this is to train a rat to use light cues to determine which arm to select in a maze, and then shift the relevant cue to an auditory stimulus. When the extradimensional set-shifting occurs, the rat must shift its strategy and follow the sound cue in order to select the correct baited arm (e.g., Insel et al., 2012). In contrast, reversal learning refers to adapting a behavior to the changing contingencies required to reach a goal. For example, a rat can initially learn to press a lever in a ‘light-on’ condition to receive reward. After a reversal, the rat must adapt its behavior to press during the ‘light-off’ condition (e.g., Nomura et al., 2004). In parallel to the age-related cognitive deficits discussed above, aging is also associated with changes in attentional processes (Gazzaley & D’Esposito, 2007; Prakash et al., 2009; Hedden et al., 2012). This is accompanied by a greater susceptibility to distraction or interference during the delay period of a working memory task in humans (Bowles & Salthouse, 2003; Campbell et al., 2012) and monkeys (Bartus & Dean,

1979; PTK6 Prendergast et al., 1998). Additionally, fMRI studies in older adults have reported that there is increased activity in brain regions mediating distraction (Milham et al., 2002; Stevens et al., 2008), and in cases where task-irrelevant stimuli are presented (Gazzaley et al., 2005). One of the most consistent finding in the literature on aging brain is a decline in the volume of the PFC of humans, monkeys and rodents. This decline is one of the earliest changes detected, and for almost fifty years it was thought that the decrease in frontal lobe volume was the result of cell loss (Haug, 1986; Peters, 2002). The early reports of cell loss, however, turned out to be an error resulting from differential shrinkage of young and aged tissue during processing (Haug et al.

To construct plasmid pENA9, a 0.25-kb DNA fragment containing the phaR promoter region was amplified by PCR. EPZ015666 datasheet After digestion with EcoRI and HindIII, the DNA fragment was inserted into pLC4 (Ray et al., 1985), which carries the xylE reporter gene. To construct plasmid pENA10, a 2-bp mutation was introduced into the −35 region of the putative σA-like promoter of phaR using a two-step PCR method (Higuchi et al., 1988). The resulting DNA fragment was digested with EcoRI plus HindIII and cloned into pLC4. Disruptions of the chromosomal phaC, sigB, sigH, spo0A, spo0F, or sigF gene of B. thuringiensis by integrations of plasmid pRN5101-derived pENA1, pENA2, pENA3, pENA4, pENA5, or pENA6, respectively,

through a Campbell-like single-crossover recombination were performed as described previously (Fedhila et al., 2002). The correct integrations were verified by PCR. Construction of the abrB deletion mutant BNA7, in which the abrB gene was replaced with the kanamycin resistance gene kan via a two-step gene replacement event, was performed according to the method described previously (Arnaud et al., 2004). The correct replacement of the chromosomal abrB sequence was verified by PCR. To construct the abrB spo0A double mutant BNA8, the pRN5101-derived plasmid pENA4 was introduced into

the abrB deletion mutant BNA7. Chromosomal integration of pENA4 through a single-crossover recombination was carried out as described above. The correct integration was verified Roscovitine supplier by PCR. The PHB contents of B. thuringiensis cells were determined by GC as described Liothyronine Sodium previously (Tseng et al., 2006) as well as using the method involving spectrophotometric determination of crotonic acid generated from digestion of PHB with concentrated sulfuric acid (Law & Slepecky, 1961). The sample preparation was carried out according to the method described in the literature (Tian et al., 2005), but with a slight modification as described previously (Chen et

al., 2009). Thin sections were examined on a JEM 2000EXII TEM (JEDL Inc.). Transformation of B. thuringiensis cells by electroporation was carried out as described previously(Bone & Ellar, 1989). Total RNA was isolated from B. thuringiensis according to the previously described method (Zuber & Losick, 1983). Northern blotting and primer extension analysis were performed using standard methods (Sambrook & Russell, 2001). An established method was used for spectrophotometric measurement of XylE (catechol 2,3-dioxygenase) activity (Ray et al., 1985). Protein concentrations were determined using the BCA protein assay method according to the instructions of the manufacturer (Pierce Biotechnology Inc.). Measurement of the PHB contents of B. thuringiensis revealed that this bacterium gradually accumulated PHB in the stationary phase after growth in LB medium (Fig. 1a). To demonstrate that the B. thuringiensis homolog of the B.

Our findings advance the literature by defining factors that are independently associated with reported difficulty taking ART/nonadherence to ART when a broad range of personal, socioeconomic, treatment-related NU7441 purchase and disease-related characteristics are considered. Such information will assist clinicians to target individuals with higher likelihood of experiencing difficulty taking ART. Many past studies investigating nonadherence to cART have investigated a smaller number of factors than assessed in our study, making it difficult to be certain which factors are truly independently associated

with nonadherence to cART. Our study also provides data on reported difficulty taking ART in a best-practice context, given that Australia has been recognized as having a best-practice population health response to the HIV epidemic [32]. The findings of our study are potentially limited by the cross-sectional nature of the available data and the use of a proxy

variable to assess factors associated with nonadherence to cART. Given the cross-sectional nature of the data, we are unable to assess causal relationships or determine which factors are associated with long-term reported difficulty taking ART. The use of a proxy variable for adherence behaviour means that we cannot be certain which independently associated factors are associated with concerning levels of nonadherence; IAP inhibitor however, we believe that our proxy variable is providing relevant information to the study of factors associated with nonadherence to cART, given that our proxy variable was found to be associated with self-reported nonadherence and reporting a detectable viral load, and that our findings broadly agree with the existing literature about nonadherence to cART. A further potential limitation of the current study is its use of self-report Myosin data. However, self-report measures have been widely used in adherence

studies [23] and have been shown to correlate with more objective measures of adherence such as those provided by medication event monitoring caps and pharmacy records [21,22,33]. We expect the results of our study to be highly generalizable to the broader Australian population of PLWH and HIV-positive men who have sex with men. The generalizability of our findings to heterosexual and injecting drug user populations of PLWH is limited because of the demographics of the Australian population of PLWH [34]. Given the multitude of factors found to be independently associated with reported difficulty taking ART, our study reaffirms the dynamic nature of adherence behaviour and highlights how important it is that adherence discussions and interventions remain an integral component of the clinical management of HIV infection. We thank the 1106 HIV-positive Australians who completed the HIV Futures 6 survey and shared their experiences of living with HIV in Australia.

Patients diagnosed with MI before HAART initiation were excluded. The analysis was conducted in four steps. First, we calculated the incidence [with 95% confidence PD0325901 concentration intervals (CIs)] of the first

hospitalization with MI, comparing periods before and after first initiation of abacavir treatment. We then fitted a Cox’s regression model to compute the incidence rate ratio for the first hospitalization with MI, as an estimate of relative risk controlling for confounding. We assessed the proportional-hazards assumption with plots and tests based on smoothed-scaled Schoenfeld residuals. In these analyses exposure to abacavir treatment was introduced as a time-dependent variable from date Epigenetics inhibitor of first exposure to abacavir until end of study. Secondly, we performed an analysis in which time on and time off abacavir were included in the same model. For abacavir-exposed patients, time on this medication was calculated as the period from the initiation of abacavir until 6 months after its discontinuation, and time off abacavir was calculated from 6 months after its discontinuation until either reinitiation of abacavir therapy or the end of the observation period (in accordance with the DAD study). All treatment periods were included

in these analyses. Thirdly, we undertook an analysis in which the start date of abacavir therapy was introduced as two time-dependent variables: (1) date of initiation of abacavir therapy as a part of a triple nucleoside reverse transcriptase Tau-protein kinase inhibitor (NRTI) regimen (mainly trizivir) not containing a PI or an NNRTI; and (2) date of

initiation of abacavir therapy as part of a PI- or an NNRTI-containing regimen. These analyses were performed because PI-sparing HAART regimens may have been preferred for treatment of HIV-infected patients with increased risk of heart disease. Fourthly, because abacavir is used as a second-line drug in many settings, we performed an analysis in which the start date of abacavir therapy was introduced as two other time-dependent variables: (1) start date of abacavir therapy in cases in which it was initiated <2 years after the start of HAART; (2) start date of abacavir in cases in which it was initiated 2 or more years after the start of HAART. The cut-off of 2 years was chosen because most HAART-naïve patients who were due to initiate the recommended regimen in Denmark (abacavir, lamivudine and efavirenz) were first started on zidovudine and subsequently switched to abacavir. This was done in an attempt to lower the risk of hypersensitivity reactions. We calculated the number of patients initiating abacavir treatment within 2 years after starting HAART vs.

5 billion. Conclusions Unnecessary spending on pharmacy charges has the potential to outstrip the estimated cost of medicines wastage in the UK. The cost-effectiveness of restricted prescription lengths for the cheaper, mostly generic medications merits an urgent re-examination. “
“Objective  Problem drinking is an increasing concern buy PS-341 to many governments worldwide including those of England and New Zealand. Screening and brief

intervention (SBI) is effective at reducing alcohol consumption and preventing escalation of hazardous drinking patterns into harmful drinking or dependence. Community pharmacy has been suggested as a potential site from which to provide readily accessible SBI services. This paper explores the views of 40 pharmacists on the prospect of providing SBI for alcohol health promotion purposes, focusing particularly upon potential barriers and incentives to provision of these services. The aim was to explore the views of community pharmacists toward the development of SBI for risky drinkers through semi-structured interviews. Methods  Qualitative, tape-recorded interviews conducted with 22 English pharmacists and 18 New Zealand pharmacists. Data collection continued until theme INCB018424 solubility dmso saturation

occurred. Transcribed interviews were thematically analysed. Key findings  Pharmacists considered there was a place for alcohol health promotion in community pharmacy. However, not all participants were positive about this potential new role and some expressed apprehension about implementing SBI services due to concerns about offending or alienating customers. Other barriers included lack of experience and confidence, problems faced with other health promotion initiatives, time, privacy and remuneration.

Other pharmacists were more positive, seeing potential in terms of remaining competitive. MG-132 research buy Facilitators included a public health campaign to raise awareness of problem drinking, having appropriate screening tools available and training for pharmacists. Conclusion  There appears to be potential for alcohol SBI services in community pharmacy, and interventions designed to reduce barriers and enhance incentivisation need to be implemented and evaluated. “
“The objective of this article was to assess if Australian pharmacy staff prevent potential adverse reactions in warfarin patients requesting over-the-counter (OTC) analgesia. Mystery shoppers entered 170 pharmacies across Australia to request OTC analgesia for a hypothetical patient with a wrist injury who currently takes warfarin following a heart valve replacement. The request was made to the first pharmacist or non-pharmacist staff member to approach the mystery shopper. The interaction was audio-taped and assessed by a pharmacist. The OTC analgesic recommended was assessed for the potential to cause an adverse bleeding event.

, 2009). Previously characterised adra2a-, adra2c- and adra2a/2c-ko mice (Hein et al., 1999) were crossed to GAD65-GFP mice to generate adra2a-ko GAD65-GFP, adra2c-ko GAD65-GFP, adra2a/2c-ko GAD65-GFP mice.

To label pyramidal neurons and interneurons, GAD65-GFP+ embryos from timed pregnant E14.5 dams were electroporated with a pRIX plasmid expressing a red fluorochrome (TOM+) under the regulation of the ubiquitin promoter in the ventricular zone (VZ) of the lateral pallium. For details of the construct see Dayer et al., 2007. After in utero electroporation, dams were killed at E17.5 by intraperitoneal (i.p.) pentobarbital injection (50 mg/kg), pups were killed by decapitation and brains were dissected. Cortical slices (200 μm thick) were cut on a Vibratome

(Leica VT100S; Nussloch, Germany), washed in a dissection medium (minimum essential medium, 1×; Tris, 5 mm; and penicillin–streptomycin, 0.5%) for 5 min, placed on porous nitrocellulose Pictilisib datasheet I-BET-762 cost filters (Millicell-CM; Millipore. Zug, Switzerland) in 60-mm Falcon Petri dishes and kept in neurobasal medium (Invitrogen, Lucerne, Switzerland) supplemented with B27 (Invitrogen), 2%; glutamine, 2 mm; sodium pyruvate, 1 mm; N-acetyl-cysteine, 2 mm; and penicillin–streptomycin, 1%. Drugs were obtained from Tocris (Abingdon, UK): medetomidine, cirazoline, guanfacine and isoproterenol hydrochloride (all diluted in H2O; stock 100 mm) and (R)-(+)-m-nitrobiphenyline oxalate (diluted in DMSO; stock 50 mm). Animals were deeply anesthetised with pentobarbital injected i.p (50 mg/kg), and killed

by intracardiac perfusion of 0.9% saline followed by cold 4% paraformaldehyde (PFA; pH 7.4). Brains were post-fixed over-night in PFA at 4 °C Lonafarnib manufacturer and coronal sections were cut on a Vibratome (Leica VT100S; Nussloch, Germany; 60-μm-thick sections) and stored at 4 °C in 0.1 m phosphate-buffered saline (PBS). For free-floating immunohistochemistry, sections were washed three times with 0.1 m PBS, incubated overnight at 4 °C with a primary antibody diluted in PBS with 0.5% bovine serum albumin (BSA) and 0.3% Triton X-100, washed in PBS, incubated with the appropriate secondary antibody for 2 h at room temperature, counterstained in Hoechst 33258 (1 : 10 000) for 10 min and then mounted on glass slides with Immu-Mount™ (Thermo Scientific, Erembodegem, Belgium). Primary antibodies were the following: rabbit anti-calretinin (1 : 1000; Swant, Switzerland), mouse anti-parvalbumin (1 : 5000; Swant), rat anti-somatostatin (1 : 100; Millipore, Zug, Switzerland), rabbit anti-NPY (1 : 1000; Immunostar, Losone, Switzerland), rabbit anti-VIP (1 : 1000; Immunostar) and mouse anti-reelin (1 : 1000; Medical Biological Laboratories, Nagoya, Japan). Secondary Alexa-568 antibodies (Molecular Probes, Invitrogen, Lucerne, Switzerland) raised against the appropriate species were used at a dilution of 1 : 1000. E17.5 cortical slices from GAD65-GFP+ pups electroporated at E14.