8 mM IPTG (Sigma, St. Louis, MO). After 4 h shaking at 37 °C, cells were harvested by centrifugation at 9300 g for 2 min at 4 °C. The precipitations were resuspended in 80 mL ice-cold buffer A containing 50 mM Tris base, 50 mM EDTA, 50 mM NaCl, 0.5 mM dithiothreitol and 5% glycerol, and then disrupted using a high-pressure cracker (JNBIO,

Guangzhou, China). Protein purification was performed according to the method of Sambrook & Russell (2006). Protein concentration was measured with the Bicinchoninic Acid Protein Assay Kit (Beijing CellChip Biotechnology, China). The band position, molecular weight, and distribution of initial expression products and purified proteins Selleckchem Crenolanib were estimated by SDS-PAGE. Western blotting was performed as described by Li et al. (2011). After the proteins were separated by SDS-PAGE and electrotransferred to polyvinylidene fluoride membranes, the membranes were blocked by 5% (w/v) nonfat dry milk in phosphate-buffered saline (PBS) overnight at 4 °C. The membranes were washed three times with TBST buffer (20 mM Tris–HCl, 150 mM NaCl, 0.05% Tween-20), and incubated with mouse anti-His antibody for 1 h, followed by Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Southern Biotech, Birmingham, check details AL) diluted 1 : 5000 for 1 h. The membranes were developed using the DAB Horseradish Peroxidase Color Development Kit (Beyotime, Shanghai, China). The immunization and challenge assay was performed

in mice, based on the International Guiding Principles for Biomedical Research Involving Animals – 1985. Fossariinae A highly virulent ExPEC strain PCN033 (Tan et al., 2012) was chosen for challenge. Twenty-four female BALB/c mice (Hubei Center for Disease Control and Prevention, China) were evenly assigned to three groups. Mice in Groups 1 and 2 were injected intraperitoneally twice at 1-week intervals with 200 μL 50 μg purified OmpC and OmpF, respectively, mixed with 50% (v/v) Imject Alum adjuvant. Mice in Group 3 were injected with 50% (v/v) Imject Alum adjuvant in PBS as a control. Two weeks after the second injection, the immunized and control mice were challenged by intraperitoneal inoculation with 200 μL PBS containing 2.5 × 107 CFU of log-phase ExPEC PCN033.

To determine antibody responses, sera were obtained by tail vein bleeding prior to each injection and challenge. The mortality in each group of mice was monitored daily for 7 days after challenge. Titers of recombinant protein-specific total IgG and two IgG subclasses (IgG1 and IgG2a) in mouse sera were examined by ELISA as described by Zhang et al. (2009). A 96-well plate was coated with purified products of 500 ng 100 μL−1 per well in sodium carbonate buffer overnight at 4 °C. The plate was washed three times with PBST (PBS supplemented with 0.05% Tween-20). After saturation with 0.5% nonfat dry milk in PBST for 2 h at 37 °C, the plate was washed three times with PBST and subsequently incubated with serially diluted mouse serum (initially in 1 : 100) for 30 min.

In this two-alternative forced choice (2-AFC) task, subjects had to indicate for one half of the CS set (10 CS+ and 10 CS−) first, whether a stimulus had been paired with a shock or not during conditioning and second, whether the shock had been administered to the right or left index finger. A d’ sensitivity measure (Green & Swets, 1966) was calculated for recognising a CS belonging to the correct affective category and for reporting the correct hand if a CS+ had been presented. For statistical evaluation of subjects’ performance, the d’ values were tested against 0 with one-sample t-tests. (ii) With the other Forskolin solubility dmso half of the CS set, a complete pair comparison

was performed, involving the presentation of all possible pairs of 20 CS and resulting in 190 comparison trials. This CS pair comparison task involved the subsequent presentation of two click-tones with a temporal delay of 750 ms. Subjects had to decide which one of the two stimuli they found more pleasant (2-AFC). The statistical analysis was restricted to comparisons of pairs from different affective categories. The mean percentage of preference for the CS− (or rejection Z-VAD-FMK molecular weight of the CS+) was tested against chance level (50%) to determine whether subjects were able to differentiate CS+ and CS− on a more implicit

level of processing. (iii) The third task involved the affective priming of positive and negative adjectives with the CS, which constituted an indirect measure of stimulus valence (e.g. Spruyt et al., 2007). Forty positive and 40 negative adjectives were selected from a set established by Kissler et al. (2007), who provided valence and arousal ratings from a reference group (n = 45). The words did not differ with respect to mean word length (negative adjectives, 7.2 characters;

positive adjectives, 7.5 characters) or arousal (negative, mean ± SD, 5.85 ± 1.97; positive, 5.83 ± 2.2), but were significantly different in terms of valence ratings (negative, 1.67 ± 0.81; positive, 7.86 ± 1.11). Each of the 40 click-like tones was presented twice, once as a prime for a negative and once for a positive adjective, resulting in 80 priming trials, half of which were congruent (CS− and positive adjective, CS+ and negative adjective) and half of which Thalidomide were incongruent (CS+ and positive adjective, CS− and negative adjective). Each trial consisted of the presentation of a CS tone that was followed by the adjective with an inter-stimulus interval of 300 ms (cf. Hermans et al., 2003). Subjects had to decide whether the adjective’s meaning was positive or negative in an evaluative decision task and were instructed to respond as fast and as accurately as possible to the presented words. We restricted the analysis to correct responses and further excluded reaction times (RTs) that were above or below 2 SD of the individual mean, rejecting 7.01% of the trials.

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

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

with Power SYBR Green PCR Master Mix (Applied Biosystems), 5 pmol of forward primer and 5 pmol of reverse primer in a final volume of 20 μL in three replicates. No-template controls were included. The primers for the 16S rRNA gene and for buy RO4929097 nine selected ORFs were designed using the primer express software (Applied Biosystems). Real-time PCR was carried out with the ABI Prism 7000 Sequence Detection System (Applied Biosystems) with the following protocol: 45 cycles at 95 °C for 15 s, followed by 60 °C for 1 min. The

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

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

and visualized with the jalview 2.6.1 software (Waterhouse et al., 2009). Total RNA was extracted from late-exponential phase E1 cells cultivated on acetate, n-dodecane, n-hexadecane, PD98059 mouse n-octadecane and n-eicosane using the TRIzol reagent (Amersham Pharmacia) and method. To prepare DNA-free RNA, 15 μg of total RNAs was treated with 5 U of RNase-free DNase I (Fermentas) according to the supplier’s protocol. The quantity and the quality of the recovered RNAs were verified by means of spectrophotometry (Nanodrop 1000) and agarose gel electrophoresis. First-strand cDNA synthesis of 2 μg of total RNA in a final volume of 20 μL was carried out with RevertAid M-MuLV Reverse Transcriptase (Fermentas), using random hexamers. For real-time PCR, 1 μL of cDNA was mixed

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

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

The researchers in charge of evaluating the biopsies, interpreting the clinical data, or calculating and analysing the reference standard all performed each function without knowledge of the results of the other evaluations. Overall, results are presented as medians (percentile 25, percentile 75) for continuous variables and as frequencies and percentages for categorical data. Analysis of normality was performed with the Kolmogorov–Smirnov test. Categorical data and proportions were analysed using the χ2 test or Fisher’s exact test as required. The Student t-test was used to compare the means of the two groups with normal

distributions and the Mann–Whitney test to compare variables with nonnormal distributions. An analysis of variance (anova) adjusted with the Bonferroni this website test was used

to compare the means of three or more groups with normal distributions. Multiple association tests were performed using univariate logistic regression and forward stepwise logistic regression analyses to identify the independent variables associated with the primary endpoint (advanced fibrosis; F≥3). In the last analysis we included all variables that were statistically significant (P<0.05) in the univariate analysis. A forward stepwise logistic regression analysis was conducted with P-values for entry and exit of 0.05 and 0.10, respectively. We developed a new index for advanced fibrosis (F≥3) diagnosis using a logistic probability function that we have called HGM-3. We evaluated the diagnostic values of HGM-3 by calculating find more the areas under the receiver operating characteristic curves (AUC-ROCs) for the estimation and validation groups. For purposes of comparison, we also evaluated four simple reported models consisting of routine parameters to predict

liver fibrosis: (a) HGM-1 and HGM-2 [21], (b) FIB-4 [17], (c) APRI [16] and (d) Forns’ indexes [15]. We evaluated the diagnostic value of these indexes by comparing the calculated AUC-ROCs [22,23] for all patients included in this study. Moreover, we evaluated new cut-offs for the HGM-3 index Oxaprozin according to a sensitivity (Se) of 95% for the low cut-off used to predict liver biopsies without advanced fibrosis (F<3); and a specificity (Sp) of 95% for the high cut-off used to predict liver biopsies with advanced fibrosis (F≥3). We calculated the Se, Sp, positive predictive value and negative predictive value for each cut-off point to evaluate the diagnostic accuracy. We also calculated the diagnostic odds ratio (DOR) which expresses the strength of the association between the test result and disease: it is the ratio of the odds of a positive result in a person with the target condition compared to a person without the condition [24]. A DOR of 1 suggests the test provides no diagnostic evidence.

The researchers in charge of evaluating the biopsies, interpreting the clinical data, or calculating and analysing the reference standard all performed each function without knowledge of the results of the other evaluations. Overall, results are presented as medians (percentile 25, percentile 75) for continuous variables and as frequencies and percentages for categorical data. Analysis of normality was performed with the Kolmogorov–Smirnov test. Categorical data and proportions were analysed using the χ2 test or Fisher’s exact test as required. The Student t-test was used to compare the means of the two groups with normal

distributions and the Mann–Whitney test to compare variables with nonnormal distributions. An analysis of variance (anova) adjusted with the Bonferroni Selleck GDC0449 test was used

to compare the means of three or more groups with normal distributions. Multiple association tests were performed using univariate logistic regression and forward stepwise logistic regression analyses to identify the independent variables associated with the primary endpoint (advanced fibrosis; F≥3). In the last analysis we included all variables that were statistically significant (P<0.05) in the univariate analysis. A forward stepwise logistic regression analysis was conducted with P-values for entry and exit of 0.05 and 0.10, respectively. We developed a new index for advanced fibrosis (F≥3) diagnosis using a logistic probability function that we have called HGM-3. We evaluated the diagnostic values of HGM-3 by calculating click here the areas under the receiver operating characteristic curves (AUC-ROCs) for the estimation and validation groups. For purposes of comparison, we also evaluated four simple reported models consisting of routine parameters to predict

liver fibrosis: (a) HGM-1 and HGM-2 [21], (b) FIB-4 [17], (c) APRI [16] and (d) Forns’ indexes [15]. We evaluated the diagnostic value of these indexes by comparing the calculated AUC-ROCs [22,23] for all patients included in this study. Moreover, we evaluated new cut-offs for the HGM-3 index Tyrosine-protein kinase BLK according to a sensitivity (Se) of 95% for the low cut-off used to predict liver biopsies without advanced fibrosis (F<3); and a specificity (Sp) of 95% for the high cut-off used to predict liver biopsies with advanced fibrosis (F≥3). We calculated the Se, Sp, positive predictive value and negative predictive value for each cut-off point to evaluate the diagnostic accuracy. We also calculated the diagnostic odds ratio (DOR) which expresses the strength of the association between the test result and disease: it is the ratio of the odds of a positive result in a person with the target condition compared to a person without the condition [24]. A DOR of 1 suggests the test provides no diagnostic evidence.

Eighty-four per cent of patients had a successful virological

response, and those who failed did not develop resistance. The IQ for boosted atazanavir is high, resulting in rare treatment failure without resistance mutations. This study showed that the protein-binding-adjusted IQ of atazanavir is close to those measured for lopinavir and darunavir used once daily in first-line treatment. Finally the selection of resistance in the case of virological failure (plasma viral load >400 HIV-1 RNA copies/mL) to atazanavir/ritonavir used in first-line therapy seems uncommon, as it is for all boosted PIs. Previous studies have shown that suboptimal plasma levels of protease inhibitors (PIs) are associated with virological treatment failure with the emergence of resistance mutations and that this effect can be selleck chemical further elucidated by determination of the protein-binding-adjusted inhibitory quotient (IQ) [1–3]. The IQ is defined as the ratio between the plasma trough concentration of a drug and the susceptibility of the virus in the patient to that drug. This is typically expressed as the plasma protein-corrected in vitro inhibitory concentration for 50% inhibition (IC50) and/or for 90% inhibition (IC90) [4,5]. The effect of protein binding on the activity of PIs must be taken into consideration when determining IC50 or IC90in vivo, as most are more than 90% bound to

plasma proteins [6–9]. Atazanavir, the first once-daily administered PI approved

for the treatment of HIV-1 infection, is recommended for use in antiretroviral treatment-naïve and -experienced patients [10–12]. Few studies, SP600125 however, have explored the virological and pharmacological parameters on virological response when combination atazanavir/ritonavir is administered to treatment-naïve patients [13,14]. This study retrospectively analysed 100 treatment-naïve patients who received two nucleoside reverse transcriptase inhibitors (NRTIs) and atazanavir 300 mg plus ritonavir 100 mg once daily. Quantification of plasma viral load (pVL) was performed at baseline and at weeks 12 and 24 using the Amplicor Monitor® assay (Cobas 1.5, Roche Diagnostics, Basel, Progesterone Switzerland), which has a lower detection limit of 50 HIV-1 RNA copies/mL. The reverse transcriptase and protease gene sequences were determined by population sequencing, according to the Agence Nationale de Recherches sur le SIDA (ANRS – the French National Agency for AIDS Research) consensus method, with an ABI 3100 Genetic Analyzer (PE Applied Biosystems, Foster City, CA, USA). The sequences were analysed with Seqscape software (PE Applied Biosystems), and the differences in the amino acid sequences with respect to the sequence of wild-type virus strain HXB2 were noted. Resistance was defined according to the current ANRS algorithm (http://www.hivfrenchresistance.org/2009/Algo-2009.pdf). Pharmacokinetic study was performed in a subgroup of 43 HIV-infected patients.

5 M sucrose+10 mM potassium phosphate, and (5) 272 mM sucrose+7 mM sodium phosphate+1 mM MgCl2. Electroporation was performed using a Bio-Rad Gene Pulser with field strength settings from 5 to 20 kV cm−1 and a Bio-Rad Pulse Controller Plus with resistance settings of 200–400Ω. A 2.1-kb fragment containing the rpsL gene was generated by PCR with primers rpsLup-F and rpsLdn-R (Table 2 and Fig. 1), using genomic DNA of the spontaneous streoptomycin resistance mutant (SR1) as template. The PCR amplicon was cloned into the pGEM-T easy

vector (Promega) to generate pSR1-rpsL. To introduce the silent CYC202 mouse point mutations used to identify true transformants, plasmid pSR1-rpsL was used as template for inverse PCR using the phosphorylated primers

rpsL-WM-F (containing the silent point mutations) and rpsL-WM-R (Table 2 and Fig. 1). Then the PCR reaction was purified and digested with DpnI to eliminate the template plasmid pSR1-rpsL. The PCR fragment, which actually was a linearized plasmid, was then self-ligated and transformed into E. coli. The plasmid containing the expected silent point mutations was confirmed by sequencing and designated as pWM-rpsL. Using the resulting plasmid as template, the 2.1-kb fragment with the introduced point mutations was generated with primer INCB024360 datasheet pair rpsLup-F/rpsLdn-R (Table 2 and Fig. 1). It has been reported in other bacteria that spontaneous mutations in the rpsL gene can confer streptomycin resistance (Shima et al., 1996; Bjorkman et al., 1998; Barnard et al., 2010). To generate a selective marker for testing the transformability of V. parvula PK1910, we isolated spontaneous streptomycin-resistant mutants and sequenced the rpsL gene of these mutants. From the Isotretinoin eight

clones randomly selected for sequencing, all carried a single point mutation at codon 43 of the rpsL gene, among which five had a change from AAG to AAC (named SR1) while three from AAG to AAT (named SR2). These mutations resulted in exactly the same substitution of the wild-type lysine (K) by asparagine (N) at codon 43. This indicates that it is the K43N mutation in RpsL that confers streptomycin resistance in V. parvula. Analysis of the draft sequence of V. parvula PK1910 revealed a type I restriction system, suggesting a potential transformation barrier for foreign DNA. Thus, to avoid complications with the restriction system, we chose to use the chromosomal DNA from the isogenic rpsL mutant strain as transforming DNA to optimize transformation conditions. Several factors have been reported to affect the efficiency of electroporation-mediated transformation, including cultivation conditions, composition of the electroporation buffer, and electroporation conditions.

Interestingly, our own predictions of enzyme localization Pirfenidone nmr using signalp 3.0 (Bendtsen et al., 2004) and lipop v. 1.0 (Juncker et al., 2003), as well as the locatep database (Zhou et al., 2008) indicate that EF2863 is a secreted protein, whereas the leader peptide of EF0114 seems to have no signal peptidase I cleavage site, meaning that this protein may be N-terminally anchored to the cell membrane. Different localization of the two endoglycosidases may reflect different physiological roles. Proteins with high-mannose N-linked glycans are frequently found in human

glycoproteins (Fujiwara et al., 1988, Furukawa et al., 1989). Even though the release of nutrients from these glycoproteins

seems to be a physiologically important role of enzymes such as EfEndo18A, one may speculate about additional physiological roles such as modulation of the host immune system. Interestingly, it has been shown that EfEndo18A from E. faecalis V583 is up-regulated in blood and urine (Vebo et al., 2009, 2010), where E. faecalis frequently causes infection. The prevalence of endoglycosidases that exploit, alter or inactivate host glycoproteins may give pathogenic bacteria selleck chemical an advantage during infection. This work was supported by grant 183637/S10 from the Research Council of Norway. We thank Britt Dahl for technical assistance during the cloning experiments. “
“The Pectobacterium atrosepticum strain SCRI1043 genome contains two complete prophage

sequences. One, ECA41, is Mu-like and is able to integrate into, and excise from, Rucaparib nmr various genomic locations. The other, ECA29, is a P2 family prophage, and is also able to excise from the genome. Excision of both prophages is rare and we were unable to induce lysis of cultures. Deletion of the entire prophages, both separately and in combination, did not affect the growth rate or the secretion of plant cell wall-degrading enzymes, but swimming motility was decreased. The virulence of prophage deletion strains in the potato host was decreased. Lysogenization of a bacterial host by temperate bacteriophages can alter bacterial physiology. Most dramatically, this manifests itself as lysogenic conversion, where a previously avirulent strain becomes a serious pathogen. Enterohaemorrhagic Escherichia coli and Vibrio cholerae are prime examples, where Stx phage and CTXΦ provide the Shiga toxin and cholera toxin genes, respectively (O’Brien et al., 1984; Waldor & Mekalanos, 1996). Phage-encoded functions are diverse. Bor and Lom, carried by phage λ, are involved in resistance to the host immune system and cell adhesion, respectively (Barondess & Beckwith, 1990; Pacheco et al., 1997); SopE is an effector protein secreted by the Type III secretion system in Salmonella that activates human Rho GTPases (Hardt et al.

In two of these pools, the dorsomedial nucleus (DMN) and the dorsolateral nucleus (DLN), dimorphic motoneurons are intermixed with non-dimorphic neurons innervating anal and external urethral sphincter muscles. As motoneurons in these nuclei EX 527 molecular weight are reportedly linked by gap junctions, we examined immunofluorescence labeling for the gap junction-forming protein connexin36 (Cx36) in male and female mice and rats. Fluorescent Cx36-labeled puncta occurred in distinctly greater amounts in the DMN and DLN of male rodents than in other spinal cord regions. These puncta were localized to motoneuron somata, proximal dendrites, and neuronal appositions, and were distributed

either as isolated or large patches of puncta. In both rats and mice, Cx36-labeled puncta were associated with nearly all (> 94%) DMN and DLN motoneurons. The density of Cx36-labeled puncta increased dramatically from postnatal days 9 to 15, unlike the developmental decreases in these puncta observed in other central

nervous system regions. In females, Cx36 labeling of puncta in the DLN was similar to that in PLX4032 males, but was sparse in the DMN. In enhanced green fluorescent protein (EGFP)–Cx36 transgenic mice, motoneurons in the DMN and DLN were intensely labeled for the EGFP reporter in males, but less so in females. The results indicate the presence of Cx36-containing gap junctions in the sexually dimorphic DMN and DLN of both male and female rodents, suggesting coupling of not only sexually dimorphic but also non-dimorphic motoneurons in these nuclei. “
“Lateral hypothalamus (LH) orexin neurons are essential for the expression of a cocaine place preference. However, the afferents that regulate the activity of these orexin neurons during reward behaviors are not completely understood. Using tract tracing combined with Fos staining, we examined LH afferents for Fos induction during cocaine preference in rats.

We found that the ventral bed nucleus of the stria terminalis (vBNST) was a major input to the LH orexin cell field that was significantly Fos-activated during cocaine conditioned place preference (CPP). Inactivation of the vBNST with baclofen plus muscimol blocked expression of old cocaine CPP. Surprisingly, such inactivation of the vBNST also increased Fos induction in LH orexin neurons; as activity in these cells is normally associated with increased preference, this result indicates that a vBNST–orexin connection is unlikely to be responsible for CPP that is dependent on vBNST activity. Because previous studies have revealed that vBNST regulates dopamine cells in the ventral tegmental area (VTA), which is known to be involved in CPP and other reward functions, we tested whether vBNST afferents to the VTA are necessary for cocaine CPP.