pylori genes, including peptidyl-prolyl cis–trans isomerase (PPIase), which has been characterized as a virulent factor of Legionella pneumophila and Trypanosoma cruzi (Fischer et al., 1992; Pereira et al., 2002) and is implicated in the regulation of gastric epithelial cell growth and apoptosis (Basak et al., 2005). A total of 64 H. pylori strains were

cultured from gastric biopsies from adult patients. These included 22 cases from gastric cancer patients and 42 from superficial gastritis patients. All samples were obtained with informed consent under a protocol approved by the hospital ethical committee at the China Medical University. Helicobacter pylori were cultured at 5% O2/10% CO2/85% N2, 37oC on brain heart infusion agar (Difco) supplemented selleck kinase inhibitor with 7% sheep blood, 0.4% IsoVitaleX, amphotericin B (8 g mL−1), trimethoprim (5 g mL−1) and vancomycin (6 g mL−1). Helicobacter pylori colonies were identified based on their typical morphology, characteristic appearance on Gram staining, a positive urease test and

gene-specific PCR tests. Bacterial DNA was extracted with phenol–chloroformisoamyl alcohol by standard procedures and precipitated by the addition of 1/10 volume of ammonium acetate and 2.5 volume of cold ethanol. After centrifugation, the DNA pellet was washed with 70% ethanol and dissolved in TE buffer [10 Mm Tris-HCl (pH 8.3), 0.1 mmol L−1 EDTA] as we have described previously (Gong et al., 2005). The differences in gene content between the gastric cancer-associated H. pylori strain and the superficial gastritis-associated strain was determined using Selleck Palbociclib the PCR-Select™ DNA Substraction Kit (Clontech). To detect gastric cancer-specific Selleck Fludarabine genes, genomic DNA from gastric cancer strain, L301, was used as the tester DNA and genomic DNA from superficial gastritis strain, B975,

was used as the driver DNA. To detect genes that were less abundant or absent in gastric cancer-associated H. pylori strain, genomic DNA from B975 was used as the tester DNA and genomic DNA from L301 was used as the driver DNA. Two micrograms of either tester or driver DNAs were digested to completion with 60 U of AluI (New England Biolabs) for 16 h in 200 μL reaction volumes. The digested products were extracted with phenol, precipitated with ethanol and resuspended in 10 mM Tris-HCl, pH 7.5, at a final concentration of 200 ng μL−1. Two aliquots of the digested tester DNAs were ligated separately to two different adaptors (Adaptor 1, 5′-CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAGGT-3′ and 3′-GGCCCGTCCA-5′; Adaptor 2R, 5′-CTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAGGT-3′ and 3′-GCCGGCTCCA-5′) using T4 DNA ligase (New England Biolabs). Then, 1 μL of each adaptor-ligated tester DNAs was mixed with 2.0 μL of digested driver DNAs and 1.0 μL of 4 × hybridization buffer. The DNA fragments were denatured at 98 °C for 1.5 min, and allowed to anneal at 63 °C for 1.5 h.

In our previous study, we failed to identify the conditions under which Cls1 plays a major role in CL synthesis. The previously tested conditions were high salinity, continuous culture at a low/high temperature (30 and 42 °C), mildly acidic conditions (pH 5.0) and anaerobiosis (Tsai et al., 2011). Here, we further explored the conditions under which Cls1 plays a dominant role in CL production, and we tested the effect of stressors that would physically alter the cell membrane. The tested conditions were PD0325901 mouse a temperature shift (from 37 to 0, 4, 30, 42 and 48 °C over 15 min),

antibiotic treatment (at the MIC of oxacillin, vancomycin and nisin for 15 min), high osmotic pressure and acid stress. Our results indicated that the temperature shift and antibiotics did not affect CL accumulation in the tested strains (data not shown). Treatment with 4 M NaCl, 4 M KCl or 20% raffinose induced CL accumulation in the cls2 mutant (Ncls2), although the effect of 4 M KCl was relatively weak. This suggests that Cls1 can induce CL production in response to broad high osmolality stressors (Fig. 1). However, the CL level did not change significantly in WT and Ncls1 cells under conditions

of high osmotic pressure. We found that a low pH (4.6, 2.6 and 2.0) induced CL accumulation in Ncls2 cells (Fig. 1) more efficiently than mildly acidic conditions (pH 5.0: Tsai et al., 2011). The low-pH response was faster (< 15 min) than the osmotic stress response (Fig. 1). Importantly, the CL level in Ncls1 did not increase after 15 min of exposure to a pH of 2.6 or this website 2.0, resulting in a statistically significant difference compared with S. aureus N315 cells. This suggests that Cls2 function is impaired by this type of low-pH treatment. Cells of both types from overnight (Fig. 1a and b) and logarithmic-phase (Fig. 1c and d) cultures exhibited a similar tendency. The cls1 mutant exhibited 100-fold increased susceptibility

in the logarithmic phase upon a sudden change in pH from 7.4 to 2.6 (Fig. 2a, log phase). The cls1/cls2 double mutant was 10-fold Montelukast Sodium more susceptible compared with the cls1 mutant, but the survival of the cls2 single mutant was equal to that of the WT. Namely, survival against acute acid stress depends largely on cls1 and does not rely on cls2 when cls1 is available. The importance of cls1 in acute acid stress was also observed in an overnight culture, but the difference was not statistically significant. Acute acid stress is the first condition under which cls1 has been found to be physiologically important for S. aureus survival: the cls1 mutant was equal to the WT in terms of long-term survival under conditions of high salinity and susceptibility to antibiotics and antimicrobial peptides (Tsai et al., 2010, 2011), as well as extended incubation at pH 4.6 (Fig. 2b). We noticed that the increase in CL at a low pH in cls1 was very fast – within 5 min (Fig. 3).

When the calY gene deleted the intact signal peptide expressed in E. coli BL21 (DE3), a large amount of (His)6-camelysin (molecular mass approximately 25 kDa) was produced in the form of solution (Fig. 2a). The (His)6-camelysin was purified by affinity chromatography

on a HisTrap FF crude 1-mL Rapamycin column (Fig. 2b). A B. thuringiensis integration plasmid pKESX was constructed to integrate erm into the B. thuringiensis chromosome. Plasmid pKESX was transformed by electroporation into B. thuringiensis KCTF12. The transformants conferring both chloramphenicol sensitivity and erythromycin resistance were selected as calY replacement mutants. Proper gene replacements of several isolates were confirmed by PCR amplification with appropriate primers (Fig. 3a). When the temperature-sensitive plasmid was apparently recombined with the calY gene in the chromosome by a single cross-over, a recombinant strain was generated containing the whole sequence of pKESX in the chromosomal DNA, which conferred both Cell Cycle inhibitor chloramphenicol and erythromycin resistance. PCR analysis indicated that the plasmid pKESX was recombined with KCTF12 chromosome by a double cross-over, generating a 2.8-kb fragment containing the homologous arms and erm by the primer pair P7/P9 (Table 2). In contrast, the fragment was 2.1 kb with a template of KCTF12. At the same time, the primer pair P1/P2

(Table 2) was used to confirm that when the calY was replaced successfully by erm, only the 3-ends of the calY of about 56 bp were left, which could conveniently be used in the complementation mutants. The complementation

plasmid pKPC was electroporated into strain KCTF, and the transformants conferring chloramphenicol resistance were designated KCTFC. Transformants were confirmed by PCR amplification with chromosomal DNA as templates (Fig. 3b). The PCR analysis indicated that the plasmid pKPC was successfully electroporated into strain KCTF, thereby generating a 913-bp fragment containing the calY and its promoter in the plasmid, and a 1510-bp fragment containing the promoter of the calY and erm in the chromosome with the primer pair P11/P12 (Table 2). In contrast, the fragment was 913 bp in KCTF12, and 1510 bp in KCTFC with the primer pair P11/P12. Western blot analysis (Fig. 3c) confirmed that the level of expression others of camelysin was either deficient or successfully complemented. It also confirmed that the camelysin, which was replaced in the study, was a single copy in the chromosome of the B. thuringiensis. The global proteins of stationary phase KCTF12, KCTF and KCTFC cultures were analyzed and compared by SDS-PAGE (Fig. 4a). Strain KCTF12 produced a large protein band of metalloproteinase camelysin protein, suggesting that the expression of camelysin was very high in B. thuringiensis. As also shown in the SDS-PAGE, one protein band disappeared in KCTF. When the camelysin was complemented in KCTFC, the protein band reappeared.

mu opioid stimulation [by d-Ala, nMe-Phe, Glyol-enkephalin (DAMGO) microinjection] of either the core or shell of the NAc to amplify cue-triggered levels of motivation to pursue sucrose reward, measured with a Pavlovian-Instrumental Transfer (PIT) procedure, a relatively pure assay of incentive salience. Cue-triggered ‘wanting’ in PIT was enhanced by amphetamine or DAMGO microinjections equally, AUY-922 chemical structure and also equally at nearly all sites throughout the entire core and medial shell (except for a small far-rostral strip of shell). NAc dopamine/opioid stimulations specifically enhanced

CS+ ability to trigger phasic peaks of ‘wanting’ to obtain UCS, without altering baseline efforts when CS+ was absent. We conclude that dopamine/opioid stimulation

throughout nearly the entire NAc can causally amplify the reactivity of mesocorticolimbic circuits, and so magnify incentive salience or phasic UCS ‘wanting’ peaks triggered by a CS+. Mesolimbic amplification of incentive salience may explain why a particular cue encounter can become irresistibly tempting, even when previous encounters were successfully resisted before. “
“The pedunculopontine nucleus (PPN), part of the reticular activating system, modulates waking and paradoxical sleep. During waking see more and paradoxical sleep, EEG responses are characterized by low-amplitude, high-frequency oscillatory activity in the beta–gamma band range (∼20–80 Hz). We have previously reported that gamma band activity may be intrinsically generated by the membrane electroresponsiveness of PPN neurons, and that the neuronal ensemble generates different patterns of gamma activity in response to specific transmitters. This study attempted Protein kinase N1 to identify the voltage-gated calcium and potassium channels involved in the rising and falling phases of gamma oscillations in PPN neurons. We found that all rat (8–14 day)

PPN cell types showed gamma oscillations in the presence of TTX and synaptic blockers when membrane potential was depolarized using current ramps. PPN neurons showed gamma oscillations when voltage-clamped at holding potentials above −30 mV, suggesting that their origin may be spatially located beyond voltage-clamp control. The average frequency for all PPN cell types was 23 ± 1 Hz and this increased under carbachol (47 ± 2 Hz; anova df = 64, t = 12.5, P < 0.001). The N-type calcium channel blocker ω-conotoxin-GVIA partially reduced gamma oscillations, while the P/Q-type blocker ω-agatoxin-IVA abolished them. Both ω-CgTX and ω-Aga blocked voltage-dependent calcium currents, by 56 and 52% respectively. The delayed rectifier-like potassium channel blocker α-dendrotoxin also abolished gamma oscillations. In carbachol-induced PPN population responses, ω-agatoxin-IVA reduced higher, and ω-CgTx mostly lower, frequencies. These results suggest that voltage-dependent P/Q- and, to a lesser extent, N-type calcium channels mediate gamma oscillations in PPN.

One of these effectors corresponds

to SifA, a protein required for the formation of lysosomal glycoprotein (lgp)-containing structures (Sifs) in epithelial cells, which emerge from the vacuole (Stein et al., 1996; Boucrot et al., 2003). SifA binds to SseJ, host proteins SKIP (SifA kinesin-interacting protein), and RhoA family GTPases to cooperatively regulate the dynamics of SCV membrane in infected cells (Ohlson et al., 2008; Dumont et al., 2010). In addition, SopD2 corresponds to an SPI-2-regulated protein that promotes Sif Ruxolitinib formation, which contributes to virulence in mice (Ruiz-Albert et al., 2002; Brown et al., 2006; Schroeder et al., 2010). In S. Typhimurium, sopD2 encodes a protein sharing 42% identity with SopD, a known SPI-1-dependent Selleckchem Palbociclib effector that plays a major role in gastroenteritis in animal models of Salmonella infection (Jones et al., 1998). Mutation of sopD2 in S. Typhimurium led to a prolonged survival in infected mice compared with

survival in mice infected with the otherwise isogenic wild-type strain. Furthermore, in a competition index assay, the sopD2 mutant was recovered at a significantly lower level compared with the wild type after the two strains coinfected the same mouse, indicating a significant role of this effector in Salmonella pathogenesis (Brumell et al., 2003). Salmonella enterica serovar Typhi lacks several effector proteins that in S. Typhimurium are crucial for the pathogenicity of the serovar (Raffatellu et al., 2005), such as sopD2, which in S. Typhi is described as a pseudogene (Parkhill et al., 2001; McClelland et al., 2004). We suggest that sopD2 inactivation is involved in human host adaptation of S. Typhi. To evaluate this, Methane monooxygenase in this study we examined the effect of trans-complementation

of S. Typhi with sopD2 from S. Typhimurium (sopD2STM) and its effect on reducing invasion of the epithelial cell line. Salmonella enterica serovar Typhi and S. Typhimurium strains used in this study are described in Table 1. Strains were routinely grown in Luria–Bertani (LB) at 37 °C with vigorous shaking, or anaerobically grown by adding 500 μL of sterile mineral oil as a barrier to oxygen before invasion assays in cultured human cells. When required, the medium was supplemented with chloramphenicol (20 μg mL−1). Comparative sequence analyses were made with the complete genome sequences of S. Typhi strains CT18 (AL513382) and Ty2 (AE014613), and the serovar Typhimurium (AE006468.1). The sequences were analyzed using blast, alignment and phylogeny tools available at http://www.ncbi.nlm.nih.gov/ and vector nt suite v.8 software (Invitrogen). PCR amplifications of S. Typhimurium 14028s sopD2 gene were performed using an Eppendorf thermal cycler and Taq DNA polymerase (Invitrogen). The reaction mixture contained 1 × PCR buffer, 1.

While it seems clear that drug-resistant microorganisms often have point mutation(s) in drug-target molecule genes (Walsh, 2000), no reports have yet described how and why such mutations occur in clinically important drug-resistant bacteria. We have reported that oxygen can buy CHIR-99021 induce DNA damage, causing mutations in rpoB and Rif resistance, in a strict anaerobe (Takumi et al., 2008). In the environment, there are numerous mutagens capable of damaging DNA and inducing mutation. Clinical drugs, such as some used in cancer therapy, may also be mutagenic (Kunz & Mis, 1989). Cigarette smoke also

contains many mutagenic chemicals (Fujita & Kamataki, 2001; Yim & Hee, 2001). Environmental microorganisms, especially indigenous microorganisms, may frequently be exposed to mutagens. Pseudomonas aeruginosa is an indigenous bacterium and emerging drug resistance in this bacterium is a growing concern (Jalal & Wretlind, C646 molecular weight 1998; Mouneimne et al., 1999; Akasaka et al., 2001; Wydmuch et al., 2005). In this study, we exposed P. aeruginosa to mutagens that are known to induce point mutation. The environmental concentrations of mutagens are similar or even higher than those we have used in the present experiments. The concentration of EMS in the Viracept case was 2.3 mg mL-1. (Gerber & Toelle, 2009), that of 1,6-DNP in soot was 0.41–0.71 μg g−1 (Schauer et al., 2004), and that of BCNU was 4 μg mL−1 in human plasma and

3.3 mg mL−1 in injection fluid (Petros et al., 2002). We have set the exposure time at 24 h because indigenous bacteria may be exposed Reverse transcriptase to these mutagens continuously in

the environment. We selected Rif and CPFX, because the emergence of microorganisms resistant to Rif and to CPFX is a growing concern (Jalal & Wretlind, 1998; Wydmuch et al., 2005). In addition, both antibacterial agents have obvious target molecules and mutations related to these target molecules are known to confer drug resistance (Campbell et al., 2001; Mariam et al., 2004). Pseudomonas aeruginosa is inherently relatively resistant to Rif (Yee et al., 1996), but has been susceptible to high concentrations of Rif. At the same time, the emergence of Rif-resistant M. tuberculosis is also a growing concern (Yee et al., 1996; Murphy et al., 2006). CPFX has been highly effective in treating P. aeruginosa infections, but recently, CPFX-resistant P. aeruginosa has become a growing problem (Jalal & Wretlind, 1998). Rif- and CPFX-resistant P. aeruginosa emerged after exposure to EMS and MNU. Meanwhile, BCNU induced Rif resistance, and 1,6-DNP induced CPFX resistance. NNN did not increase Rif- or CPFX-resistant P. aeruginosa. While BP induced mutation in S. Typhimurium TA100, Rif- or CPFX-resistant P. aeruginosa did not result. Susceptibility to BP differs considerably among strains (Jemnitz et al., 2004). We supposed that the P. aeruginosa was not susceptible to the mutagenic action of BP metabolites.

A near 100% specificity has been reported

with the combination of both techniques.[10] Although we cannot rule out the possibility of the IHA result being a false negative, we believe that artemisinin treatment delayed our patient’s seroconversion to 7.5 months, reflecting an absence of active ova-directed immune response at 5.5 months and likely selleck chemical a low level of ova production at this time. To our knowledge, seroconversion after 6 months has not been previously reported, and thus, it may be reasonable to consider longer seroconversion windows for returning travelers exposed to other active antiparasitic medications. In conclusion, returning travelers with Schistosoma infection can be asymptomatic and late seroconversion (>6 months) may occur, as was the case in our patient. In these circumstances, a negative serology should not exclude the diagnosis. Epidemiological history of fresh water contact as well as previous antiparasitic treatment is highly relevant. Invasive

techniques for diagnosis should not be routinely considered, especially in asymptomatic patients. Final diagnosis can be difficult, and thus if suspicion is strong, an empirical therapeutic test should be considered. We thank Dr Carlos Chaccour for his input on the development of MI-503 in vitro this manuscript. We also thank Prof Paul Miller for help with the language editing of the manuscript. The authors state they have no conflicts of interest to declare. “
“Background. Hepatitis A vaccination is recommended to people traveling to countries where the disease is endemic. Until recently, people originating from developing countries were considered to be “naturally” immunized. Because of improving socioeconomic conditions, hepatitis A incidence has decreased ZD1839 cost in most previously highly endemic countries during the last three decades, especially in the younger age groups. Methods. We analyzed hepatitis

A seroprevalence of 989 travelers who had been born and lived at least 1 year in a developing country, wanted to travel to a hepatitis A endemic area, and consulted at the vaccination center of the Institut Pasteur of Paris between September 1, 2008 and February 28, 2010. Results. Hepatitis A serology results were available for 646 subjects. Overall seroprevalence was 82.4%. A total of 90, 82.6, 81.2, 68.4, 56.9, and 50% of people of sub-Saharan African, Near and Middle Eastern, North African, Asian, Latin American, and Eastern European origin had hepatitis A antibodies, respectively. The difference in seroprevalence according to the continent of origin, age, and length of stay in an endemic country was significant (p < 0.0001). More than 75% of seronegatives and less than 50% of seropositives were younger than 36 years. Almost three quarters of the positive group (while less than half of the negative group) lived longer than 18 years in a developing country.

A near 100% specificity has been reported

with the combination of both techniques.[10] Although we cannot rule out the possibility of the IHA result being a false negative, we believe that artemisinin treatment delayed our patient’s seroconversion to 7.5 months, reflecting an absence of active ova-directed immune response at 5.5 months and likely STA-9090 a low level of ova production at this time. To our knowledge, seroconversion after 6 months has not been previously reported, and thus, it may be reasonable to consider longer seroconversion windows for returning travelers exposed to other active antiparasitic medications. In conclusion, returning travelers with Schistosoma infection can be asymptomatic and late seroconversion (>6 months) may occur, as was the case in our patient. In these circumstances, a negative serology should not exclude the diagnosis. Epidemiological history of fresh water contact as well as previous antiparasitic treatment is highly relevant. Invasive

techniques for diagnosis should not be routinely considered, especially in asymptomatic patients. Final diagnosis can be difficult, and thus if suspicion is strong, an empirical therapeutic test should be considered. We thank Dr Carlos Chaccour for his input on the development of selleck chemical this manuscript. We also thank Prof Paul Miller for help with the language editing of the manuscript. The authors state they have no conflicts of interest to declare. “
“Background. Hepatitis A vaccination is recommended to people traveling to countries where the disease is endemic. Until recently, people originating from developing countries were considered to be “naturally” immunized. Because of improving socioeconomic conditions, hepatitis A incidence has decreased L-gulonolactone oxidase in most previously highly endemic countries during the last three decades, especially in the younger age groups. Methods. We analyzed hepatitis

A seroprevalence of 989 travelers who had been born and lived at least 1 year in a developing country, wanted to travel to a hepatitis A endemic area, and consulted at the vaccination center of the Institut Pasteur of Paris between September 1, 2008 and February 28, 2010. Results. Hepatitis A serology results were available for 646 subjects. Overall seroprevalence was 82.4%. A total of 90, 82.6, 81.2, 68.4, 56.9, and 50% of people of sub-Saharan African, Near and Middle Eastern, North African, Asian, Latin American, and Eastern European origin had hepatitis A antibodies, respectively. The difference in seroprevalence according to the continent of origin, age, and length of stay in an endemic country was significant (p < 0.0001). More than 75% of seronegatives and less than 50% of seropositives were younger than 36 years. Almost three quarters of the positive group (while less than half of the negative group) lived longer than 18 years in a developing country.

A near 100% specificity has been reported

with the combination of both techniques.[10] Although we cannot rule out the possibility of the IHA result being a false negative, we believe that artemisinin treatment delayed our patient’s seroconversion to 7.5 months, reflecting an absence of active ova-directed immune response at 5.5 months and likely Selumetinib in vitro a low level of ova production at this time. To our knowledge, seroconversion after 6 months has not been previously reported, and thus, it may be reasonable to consider longer seroconversion windows for returning travelers exposed to other active antiparasitic medications. In conclusion, returning travelers with Schistosoma infection can be asymptomatic and late seroconversion (>6 months) may occur, as was the case in our patient. In these circumstances, a negative serology should not exclude the diagnosis. Epidemiological history of fresh water contact as well as previous antiparasitic treatment is highly relevant. Invasive

techniques for diagnosis should not be routinely considered, especially in asymptomatic patients. Final diagnosis can be difficult, and thus if suspicion is strong, an empirical therapeutic test should be considered. We thank Dr Carlos Chaccour for his input on the development of selleck chemicals this manuscript. We also thank Prof Paul Miller for help with the language editing of the manuscript. The authors state they have no conflicts of interest to declare. “
“Background. Hepatitis A vaccination is recommended to people traveling to countries where the disease is endemic. Until recently, people originating from developing countries were considered to be “naturally” immunized. Because of improving socioeconomic conditions, hepatitis A incidence has decreased SB-3CT in most previously highly endemic countries during the last three decades, especially in the younger age groups. Methods. We analyzed hepatitis

A seroprevalence of 989 travelers who had been born and lived at least 1 year in a developing country, wanted to travel to a hepatitis A endemic area, and consulted at the vaccination center of the Institut Pasteur of Paris between September 1, 2008 and February 28, 2010. Results. Hepatitis A serology results were available for 646 subjects. Overall seroprevalence was 82.4%. A total of 90, 82.6, 81.2, 68.4, 56.9, and 50% of people of sub-Saharan African, Near and Middle Eastern, North African, Asian, Latin American, and Eastern European origin had hepatitis A antibodies, respectively. The difference in seroprevalence according to the continent of origin, age, and length of stay in an endemic country was significant (p < 0.0001). More than 75% of seronegatives and less than 50% of seropositives were younger than 36 years. Almost three quarters of the positive group (while less than half of the negative group) lived longer than 18 years in a developing country.

In the

LH, single-labeled orexin-ir cells and cells double-labeled for both orexin and Fos, here called orexin/Fos-ir, were counted within an area defined by the presence of the orexin-ir cells. Therefore number, not density, of orexin-ir and orexin/Fos-ir cells per section is reported here. Double-labeled cells in the VTA and LH were not included in measures of Fos-ir cells to provide non-dopaminergic or non-orexinergic cell phenotype-specific insights. Measurements EPZ-6438 research buy from each tissue section were averaged across sections to create one measurement per subregion per hamster. With data from so many subregions within each hamster, one goal of our statistical approach was to simplify the data and present it at a circuit level by identifying clusters of regions that showed similar patterns of Fos expression across animals. To do so, we used a combination of factor analysis and descriptive correlational analyses to complement AZD2014 previous functional and anatomical findings. Factor analysis, with principal axis factoring and a promax rotation, identified two clusters of subregions. Cluster 1 included Cg1, PrL, IL, AcbC, AcbSh, MePD, MePV, IF, PN, PBP and Tail, and we refer

to regions in this cluster as mesocorticolimbic. Cluster 2 included DM/PeF, LH, VMHM and VMHL, and we refer to regions in this cluster as hypothalamic. We then computed the correlations among the regions within each cluster as well as between the two clusters. The average within-cluster correlation was

0.34 in the mesocorticolimbic cluster and 0.42 in the hypothalamic cluster, based on 55 and six correlations, respectively. These indicate that Fos expression levels in subregions within the same cluster were consistently correlated with one another. We also examined correlations between regions falling into the two different clusters, and here the average of the 44 between-cluster correlations was 0.05, supporting the idea that Fos responses in these two clusters are relatively independent. Fos-ir cell density was next analysed with multilevel modeling treating animal as the upper-level sampling unit and brain region as the lower-level sampling unit. In this analysis, the cluster the region belonged to (mesocorticolimbic vs. hypothalamic) was treated as a within-subject variable, and age (juvenile vs. adult) and swab (blank vs. VS) were treated as between-subject Mannose-binding protein-associated serine protease independent variables. Multilevel modeling provides a more powerful analysis than a traditional repeated measures anova because it allows for analysis even if data from all subregions were unavailable for each hamster (as was the case in two juvenile and one adult hamsters due to poor quality tissue sections). The error structure was modeled to impose the traditional homoscedasticity assumption used in anova. Our hypotheses predicted that swab (blank vs. VS) will differentially affect Fos expression in adults and juvenile animals in some subregions.