A well-constructed and properly operating influenza surveillance scheme is essential for public health. This study was conducted to evaluate the distribution of respiratory viruses in patients with influenza-like illness (ILI) through the first teaching hospital-based surveillance scheme for ILI in South Korea.Respiratory specimens were obtained from adult patients (≥18 years) who visited the emergency department (ED) with ILI from week 40, 2011 to week 22, 2012. Multiplex PCR was performed to detect respiratory viruses: influenza virus, adenovirus, coronavirus, respiratory syncytial virus, rhinovirus, human metapneumovirus, parainfluenza virus, bocavirus, and enterovirus.Among 1,983 patients who visited the ED with ILI, 811 (40.9%) were male. The median age of patients was 43 years. Influenza vaccination rate was 21.7%  during the 2011–2012 season. At least one comorbidity was found in 18% of patients. The positive rate of respiratory viruses was 52.1%  and the total number of detected viruses was 1,100. Influenza A virus was the dominant agent  in all age groups. The prevalence of human metapneumovirus was higher in patients more than 50 years old, while adenovirus was detected only in younger adults. In 58 (5.6%) cases, two or more respiratory viruses were detected. The co-incidence case was identified more frequently in patients with hematologic malignancy or organ transplantation recipients, however it was not related to clinical outcomes.This study is valuable as the first extensive laboratory surveillance of the epidemiology of respiratory viruses in ILI patients through a teaching hospital-based influenza surveillance system in South Korea. Surveillance for influenza is indispensable for the timely monitoring and response to outbreaks or epidemics of influenza The annual epidemics of respiratory viruses including influenza virus are various by countries and region. Large-scaled study on epidemiology of respiratory viruses is important to understand the situation of individual country. Previously, a retrospective analysis of the respiratory viruses was performed in patients with respiratory illnesses in South Korea This study involves the first extensive laboratory surveillance of ILI patients through the first teaching hospital-based influenza surveillance scheme in South Korea during the 2011–2012 influenza season. This study aimed to investigate the distribution and seasonality of respiratory viruses detected in adult patients with ILI who visited EDs at seven teaching hospitals.The study protocol was approved by the Institutional Review Board (IRB) in each hospital : Korea University Guro Hospital , Korea University Ansan Hospital (AS11047), Hallym University Kangnam Sacred Heart Hospital (2011-06-50), The Catholic University St. Vincent’s Hospital (VC11ONME0118), Inha University Hospital (11-1534), Yonsei University Wonju Christian Hospital (CR311025), and Chungbuk National University Hospital (2011-06-044). All patients gave written informed consent. The study was conducted in accordance with the principles and guidelines expressed in the Declaration of Helsinki.Hospital-based Influenza Morbidity and Mortality (HIMM) is the teaching hospital-based clinical and laboratory influenza surveillance scheme in South Korea. A total of seven teaching hospitals participated the scheme in the 2011–2012 influenza season: two hospitals in Seoul, two hospitals in Gyeonggi province (Ansan and Suwon), one hospital in Incheon, one hospital in Gangwon province (Wonju) and one hospital in Chungcheongbuk province (Cheongju). A nasopharyngeal or oropharyngeal flocked swab was obtained from each ILI patient. The flocked swab was placed in viral transport medium  immediately and stored at 4°C while being transferred to the laboratory at Korea University Guro Hospital, Seoul. Samples were then kept at −70°C until use.Total RNA was extracted from the viral transport medium containing the flocked swab using NucliSENS® easyMAG®  as per the manufacturer’s protocol. Reverse transcription was performed using the AccuPower® CycleScript RT PreMix  and random hexamer was used to synthesize cDNA. All cDNA samples were stored at −20°C until use. Multiplex PCR was performed using Seeplex® RV15 ACE Detection . To detect influenza A virus, influenza B virus, adenovirus, coronavirus OC43, coronavirus 229E/NL63, respiratory syncytial virus (RSV) A, RSV B, rhinovirus, human metapneumovirus, parainfluenza virus type 1, parainfluenza virus type 2, parainfluenza virus type 3, parainfluenza virus type 4, bocavirus, and enterovirus, each sample was tested in three tubes reactions. Briefly, 4 µL 5X RV15 ACE primer mix, 10 µL 2X Multiplex master mix, 3 µL 8-MOP solution, and 3 µL of the sample’s cDNA were mixed in a tube. After denaturation at 94°C for 15 minutes, a PCR reaction was performed using 40 cycles at 94°C for 30 seconds, 60°C for 90 seconds, 72°C for 90 seconds, followed by extension at 72°C for 10 minutes. Among the samples tested, those positive for influenza A virus were selected and were tested to differentiate subtype using Seeplex® Influenza A/B Onestep Typing . Briefly, 7.5 µL 8-MOP solution, 2.5 µL random hexamer, 10 µL 5X Flu A/B One-Step primer mix, 10 µL 5X OneStep RT-PCR buffer, 2 µL OneStep RT-PCR enzyme mix, 8 µL RNase-free water, and 10 µL sample RNA were mixed in the tube. Cycling conditions included a reverse transcription step at 50°C for 30 minutes and a denaturation step at 94°C for 15 minutes. The PCR reaction was performed using 45 cycles at 94°C for 30 seconds, 60°C for 90 seconds, 72°C for 60 seconds, followed by extension at 72°C for 10 minutes. Positive and negative controls were included in each PCR analysis. Electrophoresis of all PCR amplicons was performed on a 1% agarose gel.P value of <0.05 was considered to be statistically significant.Data are expressed as median with interquartile range (IQR) for continuous variables and count with percent for categorical variables. Fisher’s exact test was used to analyze categorical variables. Statistical analyses were carried out using SPSS 12.0  and a From week 40, starting Sep 25, 2011 to week 22, ending Jun 2, 2012, 1,983 patients with ILI visited EDs. The clinical characteristics of the patients are shown in Median time from initial symptom onset to sample collection was 1 day (IQR 1–3). Among 1,983 respiratory specimens from ILI patients, 1,033 samples (52.1%) were positive for at least one respiratory virus, and a total of 1,100 respiratory viruses were detected , Table 2Among 1,100 respiratory viruses, 254 (23.1%) were non-influenza respiratory viruses. Rhinovirus was detected in 86 patients (7.8%), and it circulated throughout the entire study period, excluding November and December. Human metapneumovirus was found in 61 (5.5%) patients. It appeared around late winter, and the epidemic peak presented during spring (week 15: Apr 8 to 14). Most RSV cases occurred in winter, but there were some episodes during spring. There was not a single detection of bocavirus or enterovirus.The epidemic peak of influenza was divergent according to age group . It presThe proportions of other respiratory viruses were different according to age group also. The prevalence of human metapneumovirus was higher in patients more than 50 years old: 3.7% (23/630) in 18–49 years group; 8.1% (38/470) in patients aged ≥50 years. Adenovirus was detected only in patients aged less than 50 years old.There were 58 cases (5.6%) in which two or more respiratory viruses were detected from respiratory specimens. In 52 cases, two respiratory viruses were detected concurrently . Five paP = 0.02) and organ transplantation recipients  had higher rate of co-incidence  during the 2011–2012 season. In addition, 23.1% of the respiratory viruses detected in respiratory specimens from adult patients with ILI were viruses other than influenza viruses.The respiratory virus detection rates in respiratory specimens from ILI patients ranged from 15.6% to 78.7%, depending on the study period, the characteristics of the study population including age, type of specimens, circulating viruses, and analysis method The co-incidence rates of respiratory viruses in ILI patients vary according to study (0.7–15.3%) This study has some limitations. First, the surveillance population was limited to adults who visited an ED with ILI. Second, the evaluation of the causative agents of ILI covered only 15 respiratory viruses, and other bacterial or viral pathogens which can cause acute febrile respiratory illnesses were not investigated. Third, there is a possibility that a low viral titer could not be detected by RT-PCR. Fourth, patients with acute febrile illness but without respiratory illness could be enrolled in this study because the recruitment depended on whether cases met the established ILI criteria or not. Finally, this study described only the result of laboratory data. However, this study is valuable for understanding the respiratory viral etiology of ILI patients during the 2011–2012 season. This study is noteworthy in that it is the first extensive laboratory surveillance of adult ILI patients through a teaching hospital-based influenza surveillance scheme in South Korea.


Combing results from these two samples, we found that two genes, parathyroid hormone (PTH) and interleukin 21 receptor (IL21R), achieved consistent association results in both the discovery and replication samples. The PTH gene SNPs, rs9630182, rs2036417, and rs7125774, achieved p values of 1.10 × 10−4, 3.24 × 10−4, and 3.06 × 10−4, respectively, in the discovery sample; p values of 6.50 × 10−4, 5.08 × 10−3, and 5.68 × 10−3, respectively, in the replication sample; and combined p values of 3.98 × 10−7, 9.52 × 10−6, and 1.05 × 10−5, respectively, in the total sample. The IL21R gene SNPs, rs8057551, rs8061992, and rs7199138, achieved p values of 1.51 × 10−4, 1.53 × 10−4, and 3.88 × 10−4, respectively, in the discovery sample; p values of 2.36 × 10−3, 6.74 × 10−3, and 6.41 × 10−3, respectively, in the replication sample; and combined p values of 2.31 × 10−6, 8.62 × 10−6, and 1.41 × 10−5, respectively, in the total sample. The effect size of each SNP was approximately 0.11 SD estimated in the discovery sample. PTH and IL21R both have potential biologic functions important to bone metabolism. Overall, our findings provide some new clues to the understanding of the genetic architecture of osteoporosis. © 2010 American Society for Bone and Mineral Research.Bone mineral density (BMD) measured at the femoral neck (FN) is the most important risk phenotype for osteoporosis and has been used as a reference standard for describing osteoporosis. The specific genes influencing FN BMD remain largely unknown. To identify such genes, we first performed a genome-wide association (GWA) analysis for FN BMD in a discovery sample consisting of 983 unrelated white subjects. We then tested the top significant single-nucleotide polymorphisms (SNPs; 175 SNPs with  Osteoporosis is a serious public health problem associated with substantive morbidity and mortality, as well ESR1, COL1A1, VDR, LRP5, OPG, and CYP19A1. who had both the phenotype and genotype information. All the subjects were white US citizens of northern European origin living in Omaha, Nebraska, and its surrounding regions in the Midwest. Subjects with chronic diseases and conditions that potentially might affect bone mass, structure, or metabolism were excluded from the study to minimize the influence of known environmental and therapeutic factors on bone variation. The exclusion criteria have been detailed in an earlier publication. BMD measThe replication sample was derived from the Framingham Heart Study (FHS) SNP Health Association Resource (SHARe) Project, for which genotyping was conducted in over 9300 phenotyped subjects from three generations (including over 900 families). Details about and descriptions of the FHS were reported previously.,21 We hap < .0001), and 91,395 SNPs with minor allele frequencies (MAFs) of less than 5%. Therefore, the final analyses were restricted to 342,854 SNPs.For the discovery sample, genomic DNA was extracted from whole human blood using a commercial isolation kit  following the standard protocol. Genotyping was carried out at Vanderbilt Microarray Shared Resource using the Affymetrix Human Mapping 500K array set , as described in a previous publication. The finahttp://www.ncbi.nlm.nih.gov/projects/gap/cgibin/study.cgi?study_id=phs000007.v3.p2). The quality control was the same as that adopted for the discovery sample by excluding SNPs with a call rate of less than 95%, deviating from HWE (p < .0001), and with MAFs of less than 5%. There were 386,731 SNPs available for subsequent analyses.For the replication sample, genotyping was performed using approximately 550,000 SNPs . For details of the genotyping method, please refer to the FHS SHARe at the NCBI dbGaP Web site (p < 5 × 10−4 (175 SNPs) to test for associations with FN BMD. FBAT to quantify the overall evidence for association with BMD. The individual Z-score was weighted by the square root of the sample size of each study. We added the individual weighted Z-scores derived from each sample together and divided by the square root of the sum of the sample sizes to obtain an overall Z-score and an associated combined p value. was set in the replication stage to ensure that the overall significant association is robust across populations.Combining results from all sample sets by meta-analysis, we set the threshold for genome-wide significance at d Sabatti and Lencd Sabatti who prefr2) pattern and plot the haplotype block patterns.Haploview Version 4.1 was usedDifferent genotyping platforms were used in our GWA study and in previous BMD GWA studies.,14,17 Fop < 5 × 10−4 , rs2036417 (combined p = 9.52 × 10−6), and rs7125774 (combined p = 1.05 × 10−5), respectively. In particular, rs9630182 achieved the genome-wide significance level (p < 4.2 × 10−7). These three SNPs are highly correlated with one another  gene. The whole PTH gene, including these three SNPs (from upstream to downstream), was localized to a single block with a size of 125 kb (p > .05) , rs8061992 (combined p = 8.62 × 10−6), and rs7199138 (combined p = 1.41 × 10−5). Although these three SNPs did not reach genome-wide significance, they are clustered in a potential candidate gene, interleukin 21 receptor (IL21R). This gene is a cytokine receptor that is important to bone biology. The three SNPs are in strong LD with each other (r2 > 0.95) and are located in an LD block within intron 1 of IL21R  for the preceding 6 SNPs in er group . For PTHp = .012) and rs4870044 (p = .045) in ESR1, rs6469804 (p = .030) in OPG, rs3736228 (p = .048) in LRP5, and rs2010281 (p = .048) in MARK3 . Meanwhile, for SNPs that were not confirmed in our sample, we list the results in Supplemental Using the genotyped and imputed genotypes in our GWA discovery sample of 983 unrelated white persons, we examined the associations between FN BMD and the key SNPs identified in previous GWA studies.,14,17 Fiin MARK3 . MoreoveADAMTS18 (16q23) and TGFBR3 (1p22)—for spine or hip BMD previously.(RANKL (13q14), OPG (8q24), RANK (18q21), ESR1 (6q25), LRP5 (11q13), SOST (17q21), MARK3 (14q32), and SP7 (12q13). However, these loci in combination can explain only a small fraction of BMD variation, leaving the majority of the genetic factors that influence BMD variation unknown. In addition, most published GWA studies focused only on the genes or SNPs of top-ranking statistical significance, which may ignore some useful information. In this study, by using available GWA data sets from two white populations, we identified two susceptibility genes—PTH (11p15) and IL21R (16p11)—associated with FN BMD variation. These two genes were not in the top-significance list in either of the populations and were not identified by our previous GWA study on BMD.. Thus we hypothesized that those SNPs potentially might regulate PTH gene expression through intergenic transcription, although the real molecular mechanisms await further investigation.ormation.,34–36 Ho results.–41 Our selations.,43 As anelations.,45 In paIL21R has multiple functions. For example, IL21R plays an important role in the proliferation and differentiation of various immune cells, such as T cells and B cells. Studies have shown that B cells may participate in osteoclastogenesis.(IL21R induces the growth-promoting signals of its ligand, IL21, which might be involved in the maturation and function of myeloid cells.(IL21R and IL21 have been revealed to be involved in a variety of human diseases, including cancers, inflammatory bowel disease and Crohn's disease, and multiple autoimmune diseases. Especially, IL21R has been identified as associated with the activated phenotype of rheumatoid arthritis fibroblasts and correlates negatively with the destruction of cartilage and bone.(IL21R may be a new candidate gene for BMD.Cytokins are potent mediators regulating homeostasis of the immune system and pathophysiologic processes. As a member of the type I cytokine receptors, ogenesis. IL21R inid cells. IL21R anand bone. With thip < .05 was considered significant. We confirmed associations for several SNPs located in the previously well-studied candidate genes, such as ESR1, OPG, and LRP5 (We compared the results for the key SNPs identified in previous BMD GWA studies,14,17 wiand LRP5 . Howeverand LRP5 , which mIt is worth emphasizing that population stratification is unlikely to be a major concern in this GWA study. This is so first because we used EIGENSTRAT to perform GWA analyses in the discovery sample, which can control for potential population stratification effectively. Second, we used a family-based sample to perform replication analyses. Family-based samples are ideal for the follow-up validation of initial GWA findings because PTH and IL21R, that may influence FN BMD variation. Although additional functional studies are required to elucidate the detailed roles and potential functional variants of these loci, our findings provide some new insights into the understanding of the genetic architecture of BMD and osteoporosis.In summary, we identified two susceptibility bone mass candidate genes,


Prodigiosins (PGs) are a family of natural red pigments with anticancer activity, and one member of the family has entered clinical phase II trials. However, the anticancer mechanisms of PGs remain largely unclear. This study was designed to investigate the molecular basis of anticancer activity of UP, a derivative of PGs, in P388 cells. By introducing pharmacological inhibitors and utilizing a variety of analytical approaches including western blotting, flow cytometry and confocal laser microscopy, we found that UP inhibited proliferation of P388 via arresting cells at G2/M phase and inducing cells apoptosis, which was related to the activation of P38, JNK rather than ERK1/2 signaling. ROS regeneration and acidification in cells appear not involved in UP induced apoptosis. Furthermore, utilizing mass spectrometry, sucrose density gradient fractionation and immunofluorescence staining, we discovered that UP was apparently located at ribosome. These results together indicate that ribosome may be the potential target of UP in cancer cells, which opened a new avenue in delineating the anticancer mechanism of PGs. Prodigiosins (PGs) are a family of natural red pigments, structurally characterized by a common pyrrolylpyrromethene skeleton with varying side chains. PGs, originally isolated from Serratia by Amak in 1929, are composed of prodigiosin (PG), prodigiosin 25-C (PG 25-C), metacycloprodigiosin (MP), cycloprodigiosin (CPrG) and undecylprodigiosin (UP), etc. PGs are the secondary metabolites of various bacteria with various biological activities such as anti-microbial, anti-malarial, immunosuppressive and anticancer. The structures of PG and UP are shown in Increasing studies have suggested the anticancer activity of PGs. It has been reported that PGs induces apoptosis in haematopoietic, gastrointestinal, breast and lung cancer cells whereas non-toxic to non-malignant cells Growing studies have been conducted to reveal the molecular targets of PGs to gain insights into its anticancer efficacy, but the findings revealed great discrepancies in different cellular context or using individual compounds. PGs have been reported to trigger signaling pathways possibly through induction of DNA double-strand breaks and/or neutralization of pH gradients, which leads to cell cycle alternations and apoptosis. Janus tyrosine kinase 3 (Jak3) that associates with IL-2R upon activation was also suggested to be the molecular target for PGs in gastric cancer cells Saccharopolyspora sp.Nov, and found that this compound exhibited significant cytotoxic activity against five cancer cell lines, among which the impact on murine leukemia P388 was the most evident We have extracted UP from the fermentation broth of a sponge Mycale plumose-derived actinomycete UP and PG were provided by Professor Gu . Their purities were >99.5%. 4,6-diamidino-2-phenyllindile (DAPI), propidium iodide (PI). Fetal bovine serum (FBS) was purchased from GIBCO . An enhanced chemiluminescence (ECL) kit was purchased from PIERCE.U0126, SP60012, SB203580, SRB, DCFH-DA, BCECF AM, AO were all purchased from Beyotime Institute of Biotechnology . PARP, Cyt C, Caspase-3, Caspse-8, Caspase-9, β-actin, p-AKT, AKT, p-ERK, ERK, p-JNK, JNK, p-P38 and P38 antibody were purchased from Cell Signaling . Ribosomal protein S3 antibody was purchased from Santa Cruz Biotechnology .Cell lines were purchased from the Institute of Biochemistry and Cell Biology , and maintained according to the suppliers’ instructions. In brief, P388 cells were maintained in Dulbecco’s modified Eagle’s minimum essential medium (DMEM); A459 cells were cultured in F12K medium. Both media were supplemented with 10% fetal bovine serum. The cells were maintained in 5% CO2 at 37°C.3 cells/well in 96-well plates and then treated with UP for 72 h. In some experiments the cells were pretreated with NAC, iminazole or MAPK inhibitor for 1 h. The cells were incubated with 16% trichloroacetic acid (TCA) at 4°C for 1 h, after which, plates were washed five times with cold water, the excess water was drained off and the plates left to dry in air. SRB stain  was added to each well and left in contact with the cells for 30 min, then the cells were washed with 1% acetic acid for five times. The plates were dried and 150 µl of 10 mM Tris base (pH 10.5) was added to each well to solubilise the dye for 30 min. The absorbance (OD) of each well was read on a Microplate Reader  at 490 nm wavelength. The% of cell viability = (OD of treated cells/OD of control cells)× 100%.The cell proliferation was measured by SRB assay. P388 cells were cultured at 8×105 cells/dish, 24 h later, cells were treated with UP for 24 h. After that, the cells were harvested, fixed with 70% cold ethanol at 4°C for 12 h, incubated with RNase A (30 µg/ml) for 30 min at room temperature, and then stained with propidium iodide (50 µg/ml) at 4°C for 30 min. Cellular DNA was analyzed by flow cytometry .P388 cells were plated in 100 mm culture dish at 2×106 cells were washed with PBS and then lysed with RIPA  for 30 min on ice. The cell lysate was loaded for electrophoresis in 10% SDS-PAGE. The separated proteins were electrophoretically blotted onto NC membrane . The membranes were blocked in 5% non-fat milk diluted in TBS-T for 2 h at RT, then incubated overnight with antibodies for the PARP, Cyt C, Caspase-8, 9, 3, p-AKT, AKT, p-ERK, ERK, p-JNK, JNK, p-P38 and P38. Goat anti-rabbit IgG conjugated to horseradish peroxidase (1∶3000 dilution) were incubated for 1 h at RT. Between each incubation, the membranes were washed 3×5 min in TBS-T. Antibody binding was detected with enhanced chemiluminescence reagent.After UP (0.05 µM) treatment for 1 h or 24 h, 1×10P388 cells grown on coverslips were treated with UP (0.05 µM).at 37°C for 1 h. After three washes with PBS, cells were then incubated with DAPI. The coverslips were examined with a laser scanning confocal microscope  The living cultured cells were stained with acridine orange as described previously P388 cells were exposed to 0.05 µM of UP for 1 h. Cultured cells were washed with PBS, centrifuged and loaded with 10 µM 29, 79-bis-(carboxyethyl)-5(69)-Carboxyfluorescein acetoxymethyl ester (BCECF-AM) at 37°C for 30 min in PBS. After centrifugation, cells were re-suspended in PBS and analyzed by flow cytometry Intracellular oxidative stress was assessed by measuring intracellular oxidation of 2′, 7′-dichlorofluorescin (DCFH). The substrate is DCFH-DA which easily diffuses into the cell and is next deacetylated by cellular esterases to the more hydrophilic, nonfluorescent DCFH. ROS generation in the cell oxidizes DCFH to the fluorescent 2′, 7′-dichlorofluorescein (DCF). P388 cells were seeded, and incubated with UP (0.05 µM ) for 1 h, then harvested cells, washed, and loaded with DCFH (10 µM ) at 37°C for 30 min. Then the cells were harvested, washed, and loaded with DCFH (10 µM ) at 37°C for 30 min. Finally the cells were washed with PBS, and cellular fluorescence was acquired using flow cytometry with excitation at 488 nm and emission at 530 nm After incubation with UP (0.05 µM) for 1 h at 37°C, P388 cells were harvested and lysed with lysis buffer  for 30 min on the ice, centrifuged at 12000 g for 20 min at 4°C. An equal volume of loading buffer was added to supernatant. The samples were separated on 8% Native–PAGE, and the strip was cut according to the color of UP. The proteins binding with UP were analyzed by LC-ESI-LTQ (Thermo Finnigan LTQ) against NCBI mouse protein database To prepare cytoplasmic extract for ribosomal fractionation, P388 cells were treated with 0.05 µM UP for 1 h, then washed with ice-cold PBS twice and lysed in ice-cold RIPA for 30 min, then centrifuged at 10,000 ×g for 15 min to clear the resultant supernatant of nuclei, mitochondria and debris. Lysate protein solution was layered over 9 ml linear sucrose gradient solution (10–50%) in a 11.5 ml Sorvall centrifuge tube and centrifuged at 35,000 ×g for 3 h at 4°C in ultracentrifuge (Beckman Optima tm L-100 XP). Only UP without lysate on top of linear sucrose gradient solution was used as control P388 cells were inoculated in a six-well plate and cultured overnight. Cells were exposed to UP (0.05 µM) for 1 h. Cells were rinsed twice with phosphate-buffered saline (PBS) and fixed for 10 min with 4% formaldehyde. Next, the cells were washed twice with PBS and then permeabilized for 10 min with 0.1% Triton X-100, the cells were pre-incubated with PBS containing 1% BSA for 20–30 min and stained with ribosomal protein S3 antibody overnight at 4°C, followed by incubation with anti-mouse IgG-FITC for 30 min. The cells were then washed with PBS and examined using confocal laser-scanning microscopes μΜ, UP treatment resulted in a concentration-dependent inhibition in cell growth of P388 cells. The IC20 and IC50 on P388 cells were 0.0049 μΜ and 0.042 μΜ respectively  for 24 h showed accumulation in G2/M (40.73%) with a concomitant decrease in G0/G1 phase cells, suggesting a cell cycle arrest at G2/M phase. UP also induced a higher percentage of sub G0/G1 population, indicative of apoptosis occurrence. About 8.23% cells was apoptotic when treated with UP for 24 h  in contrWe next examined the mitochondrial release of Cyt C, the upstream pathway of caspase 9. After 24-h treatment with UP, the release of Cyt C in the cytosol was considerably increased, whereas Cyt C in the mitochondria decreased obviously . It has UP emits red fluorescence at 488 nm excitation, which can be visualized in living cells. P388 cells were stained with the DNA binding dye DAPI after 1 h exposure to 0.05 µM UP to examine the sub-cellular localization of UP. The cells were imaged under confocal laser microscopy. As shown in To determine the potential involvement of protein kinase pathways in G2/M arrest and apoptosis induced by UP, we probed the phosphorylation status of four major protein kinases which relate to cell proliferation in P388 cells after exposed to UP for 1 h. As shown in To further address whether ERK1/2, JNK1/2 or P38 was involved in UP-inhibited cell growth, cells were incubated with or without the inhibitor of ERK1/2, JNK1/2 and P38 respectively and then cotreated with 0.05 µM UP for 72 h. Cells proliferation was estimated by the SRB method. As shown in 2O2 suppressed cell proliferation was clearly reversed by NAC treatment. These findings suggest that ROS generation is most likely not involved in UP-induced apoptosis.It is reported that PGs could induce the ROS production in cells Acridine orange is an “acid- tropic” weak base, which is taken up by living cells and accumulates in acidified compartments such as lysosomes In contrast, cell-permeable bases imidazole treatment markedly prevented the decrease in pHi. We then tested whether UP-caused growth inhibition could be rescued by imidazole. Concurrent to UP exposure, we weren’t able to identify any significant differences compared with cells treated, about only 3% recovery , suggestWe proceeded to identify the molecular targets of UP in p388 cells by identifying UP binding proteins using mass spectrometry analysis. UP migration can be easily observed with an unaided eye as a band in orange color in native PAGE . The gelFor further validation, P388 cells were incubated with UP at 0.05 µM for 1 h and ribosome was isolated by sucrose density gradient fractionation. Comparing with control group where UP was enriched at top of sucrose density solution, obvious layered color-bands were found in UP treated cells , supportsUp to date, the anticancer mechanism of PGs still have not been clarified in details. For example, Francisco R reported PG impacted mitochondria, exerted an uncoupling effect on the electronic chain, the cell nucleus was preserved from prodigiosin accesWe found that UP inhibited the cell proliferation, arrested the cell cycle at G2/M phase, led to activation of caspase 3, 8, 9, alterations of mitochondria membrane potential, release of Cyt C and cleavage of PARP. These results indicate that UP has cytotoxicity to P388 and induces P388 cells apoptosis involving in mitochondria apoptotic pathway at least. In order to elucidate the mechanism underlying apoptosis induced by UP, we first assessed the cell distribution of UP with its auto-fluorescence, the result showed that UP distributed in cytoplasm but not membrane or nucleus, which suggests us that UP may interact with molecules in cytoplasm to induce apoptosis.. However, in our experiments, UP wasn’t found to influence AKT activation of P388.The PI3K/Akt pathway regulates fundamental cellular functions such as cell growth, survival, and movement. Extravagant activation of the PI3K/Akt pathway has been reported to be associated with the development of cancer. Akt can also directly regulate the apoptotic machinery by phosphorylating and inactivating pro-apoptotic proteins such as BAD, in addition, Akt activity is required for G2/M phase transition and AKT inhibition often induces G2-M arrest MAP kinases are composed of serine/threonine protein kinases, including the extracellular signal-regulated kinases (ERK), the p38 MAPK, the c-Jun-NH2-terminal kinases (JNK), and MAP kinases signaling pathways regulate essentially all aspects of malignant cell behavior, proliferation, survival, migration and invasion PK signaling pathways. Ling Liu et al reported that NG-induced apoptosis of HepG2 cells was characteristic of intracellular ROS generation. Simultaneously NG treatment could lead to the activation of the phosphorylation of JNK and p38 but not ERK1/2. Our data demonstrated that UP induced intracellular ROS production in P388 cells. However, a ROS scavenger NAC could not reverse inhibition of proliferation caused by UP, although it obviously antagonized the ROS production by H2O2. These results indicate that generation of ROS is not implicated in apoptosis induced by UP.ROS is generated intracellularly as byproducts of normal aerobic metabolism or as second messengers in various signal transduction pathways or in response to environmental stress +/H+, Na+-dependent and-independent Cl−/HCO3− exchangers, vacuolar type H+-ATPase (V-ATPase) and others. Daigo Ya mamoto reported that the intracellular acidification of KPL-1 by cPrG.HCl treatment induced apoptosis and cycle arrest, which was strongly suppressed by imidazole, a cell-permeable base. It has been demonstrated that Bafilomycin A1, a potent selective inhibitor of vacuolar H+-ATPase The pHi within acidic organelles are responsible for a wide variety of important cellular functions, such as endocytosis, exocytosis and intracellular trafficking, as well as cell differentiation, cell growth and cell death. The pHi in transformed or cancerous cells generally remains neutral or even slightly more alkaline than normal cells Taking advantage of its autofluorescence feature, we noticed that UP is mainly distributed in cytoplasm. We further isolated the proteins binding to UP in native-PAGE gel and submitted to mass spectrometry analysis. 171 proteins from 951 detectable proteins were ribosome-related, suggesting that UP can likely bind to ribosome. We further validated the hypothesis by sucrose density gradient fractionation method, a conventional approach to isolate and study ribosome and by immunofluorescence staining to observe colocalization of UP and ribosome in p388 cells and A549 cells. Ribosomal proteins play multiple roles in coordinating protein biosynthesis to maintain cell homeostasis and survival. Recent evidence suggests that a number of ribosomal proteins have secondary functions independent of their involvement in protein biosynthesis. These proteins function as cell proliferation regulators and in some instances as inducers of cell death In summary, our results presented in this study exclude the involvement of ROS and acidification in UP induced apoptosis, in spite of tremendous literatures. Our data instead suggests that UP binds to ribosome, which may, at least partially, trigger the P38, JNK signaling and mitochondrial pathway apoptosis, eventually leading to inhibition of cell proliferation.The targeted ribosomal molecules needs to be further studied. Nevertheless, ribosome could possibly open a new avenue for exploring anticancer mechanism of PGs in the future.


The amygdaloid complex is a key brain structure involved in the expression of behaviors and emotions such as learning, fear, and anxiety. Brain diseases including depression, epilepsy, autism, schizophrenia, and Alzheimer's disease, have been associated with amygdala dysfunction. For several decades, neuroanatomical, neurophysiological, volumetric, and cognitive approaches have been the gold standard techniques employed to characterize the amygdala functionality. However, little attention has been focused specifically on the molecular composition of the human amygdala from the perspective of proteomics. We have performed a global proteome analysis employing protein and peptide fractionation methods followed by nano-liquid chromatography tandem mass spectrometry (nanoLC-MS/MS), detecting expression of at least 1820 protein species in human amygdala, corresponding to 1814 proteins which represent a nine-fold increase in proteome coverage with respect to previous proteomic profiling of the rat amygdala. Gene ontology analysis were used to determine biological process represented in human amygdala highlighting molecule transport, nucleotide binding, and oxidoreductase and GTPase activities. Bioinformatic analyses have revealed that nearly 4% of identified proteins have been previously associated to neurodegenerative syndromes, and 26% of amygdaloid proteins were also found to be present in cerebrospinal fluid (CSF). In particular, a subset of amygdaloid proteins was mainly involved in axon guidance, synaptic vesicle release, L1CAM interactome, and signaling pathways transduced by NGF and NCAM1. Taken together, our data contributes to the repertoire of the human brain proteome, serving as a reference library to provide basic information for understanding the neurobiology of the human amygdala. Therefore, the amygdala assessed in this study was the left one. The diagnosis was carried out on the right cerebral hemisphere. Following fixation in 10% formaldehyde for approximately 3 weeks, the brains was sectioned according to the recommendation guide proposed by BrainNet Europe  and homogenized in lysis buffer containing 7 M urea, 2 M thiourea, 4% (v/v) CHAPS, 50 mM DTT. The homogenates were spinned down at 100.000 × g for 1 h at 15°C. Protein concentration was measured in the supernatants with the Bradford assay kit (Bio-Rad). Prior to proteomic analysis, the amygdala samples were pooled (~200 μg of protein from each specimen).20 prior to mass spectrometric analysis.Protein material (~350 μg of protein) was precipitated using methanol/chloroform extraction. Pellet was dissolved in 6 M urea, 100 mM Tris, pH 7.8. Reduction was performed by addition of DTT to a final concentration of 10 mM and incubation at 25°C for 1 h. Subsequent alkylation by 30 mM iodoacetamide was performed for 1 h in the dark. An additional reduction step was performed by 30 mM DTT, allowing the reaction to stand at 25°C for 1h. Proteins were digested for 4 h with Lys-C (Promega) at 37°C . The mixture were then diluted to 0.6 M urea using MilliQ-water, and after trypsin addition (Promega) , the sample was incubated at 37°C for 18 h. Digestion was quenched by acidification with acetic acid. The digestion mixture was dried in a SpeedVac, reconstituted with 40 ul of 5 mM ammonium bicarbonate (ABC) pH 9.8, and injected to an Ettan LC system with a high pH stable X-Terra RP18 column  (Waters) at a flow rate of 40 μl/min. Peptides were eluted with a mobile phase B of 5–65% linear gradient over 35 min . Twenty fractions were collected  and loaded on 17 cm pH 3–10 nonlinear immobilized pH gradient strip in a focusing tray. IEF was performed on a Bio-Rad PROTEAN IEF system (Bio-Rad). Conditions for performing IEF were as follows: after rehydration of the IPG strip for 12 h at 50 V and 20°C, the run was started at 250 V for 15 min followed by rapid voltage ramping to 10,000 V without exceeding 50 A/strip. The IEF run was finished when the voltage reached 60,000 V. The voltage was held at 500 V until the run was stopped. The IEF run was performed at 20°C. The entire IPG strip was divided into 25 equal parts (~0.7 cm each) for in-gel reduction, alkylation, and digestion. In-gel tryptic digestion was performed with 20 ng/μl trypsin in 50 mM ABC for 18 h at 37°C. The resulting tryptic peptides were extracted with 5% formic acid, 50% acetonitrile, and 5% formic acid, 85% acetonitrile. Peptide mixtures were completely dried in a SpeedVac and resuspended in 20 μl 98% MilliQ-HHomo Sapiens . False discovery rate (FDR) was performed using a nonlinear fitting method . The column and the autosampler were maintained at a temperature of 40°C and 4°C respectively. A trap column Acclaim PepMap100  and an analytical column Acclaim PepMap RSLC  (Dionex) were used following the next gradient: 0–1 min (2% Buffer B), 1–110 min (2–30% Buffer B), 110–120 min (30–40% Buffer B), 120–125 min (40–90% Buffer B), 125–130 min (90% Buffer B), 130–132 min (90–2% Buffer B) and 132–150 min (2% Buffer B) . MS analysis was performed on a Q-TRAP 5500 system (ABSciex) with a NanoSpray® III ion source (ABSciex). The mass spectrometer was operated in positive ion mode at unit resolution. Each fraction was analyzed twice in technical replicates (90 LC-MS/MS runs). For MS analysis, Enhanced MS (EMS) and Enhanced Resolution (ER) scans were acquired at a scan rate of 10,000 Da/s and 250 Da/s respectively. The ER scan was used to confirm the charge state and confirm the mass assignment. For MS/MS analysis, survey scans were acquired from m/z 400 to 1000 with up to 6 precursors selected for MS/MS (Enhanced Product Ion scan) from m/z 230 to 1000 using dynamic exclusion, and the rolling collision energy was used to promote fragmentation. MS/MS data acquisition was performed using Analyst 1.5.2 (AB Sciex) and submitted to Protein Pilot™ Software (v.4.0.8085-ABSciex) using Paragon™ Algorithm (v.4.0.0.0)  was calculated using the ProtParam tool at Expasy Server (http://web.expasy.org/protparam/). Reactome Database (http://www.reactome.org)  that the association between the amygdala proteins and a specific pathway is explained by chance alone (only p < 0.01 were considered significant).The proteins identified in this study were classified by DAVID  Bioinformatics Resources (v6.7)  annotation code using DAVID software , cellular localization , organic acid metabolism , cellular aminoacids and derivative metabolism , and nucleobase, nucleoside, and nucleotide metabolism . Representative biological process categories from each cluster are shown in Figure p < 0.01) are shown in Supplementary Table With respect to the biological process and molecular function categories, an enrichment analysis has been performed against P-value: 9,0E-21), nucleotide binding , hydrolase activity , and ATP binding  were the most significantly enriched molecular functions in our dataset. Representative molecular function categories from each cluster are represented in Figure Respects to molecular function ontology, 12 clusters are significantly enriched in our human amygdala dataset  between 4 and 8 h, where proteins are reasonably stable  (Legrain et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


However, the detailed mechanisms of caffeic acid phenylethyl amide need further studies. In summary, this study demonstrated the protective potential of chronic treatment of caffeic acid phenylethyl amide against the metabolic consequences in diabetes mellitus.Caffeic acid phenyl ester is distributed wildly in nature and has antidiabetic and cardiovascular protective effects. However, rapid decomposition by esterase leads to its low bioavailability in vivo. In this study, chronic metabolic and cardiovascular effects of oral caffeic acid phenylethyl amide, whose structure is similar to caffeic acid phenyl ester and resveratrol, were investigated in ICR mice. We found that caffeic acid phenylethyl amide protected against diet or streptozocin-induced metabolic changes increased coronary flow and decreased infarct size after global ischemia-reperfusion in Langendorff perfused heart. Further study indicated that at least two pathways might be involved in such beneficial effects: the induction of the antioxidant protein MnSOD and the decrease of the proinflammatory cytokine TNF Diabetes mellitus (DM) is a metabolic disease with hyperglycemia and usually accompanied with many complications –5. Lifesβ-cell toxin that induces rapid and irreversible necrosis of β cells. It is widely used in making experimental animal models of type 1 DM [Streptozocin (STZ) is a pancreatic ype 1 DM . Since iype 1 DM , 8 and cype 1 DM  animals ype 1 DM –15. Highype 1 DM . Since sMany natural polyphenolic compounds are demonstrated to have anti-inflammatory, antioxidant, anticarcinogenic, antithrombotic, and cardiovascular protective effects –19. Resv2Cl2) (5 mL) was added to a mixture of caffeic acid (100 mg), R-NH2 (1.2 eq), and triethylamine (Et3N) (0.08 mL) in dimethylformamide (DMF) (1.0 mL). The mixture was stirred at 0°C for 30 min, then allowed to stir at room temperature for 12 h. This reaction mixture was evaporated under in vacuo, and the residue was partitioned between ethyl acetate (AcOEt) and H2O. Successively, the AcOEt layer was washed with 3 N aqueous HCl and 10% NaHCO3(aq), dried over MgSO4 and concentrated in vacuo. The residue was further purified by column chromatography with eluting solution  on silica gel . The final products (82–88% yield) were recrystallized from AcOEt to obtain pure crystals. 1H and 13C NMR spectra were recorded on a Bruker Avance 500 spectrometer. Electron impact mass spectrometries (EIMS) were determined on a Finnigan TSQ-46C mass spectrometer. IR spectra were recorded on a Nicolet Magna-IR 550 spectrophotometer.Beginning with caffeic acid, CAPA was obtained from the following amide binding coupling method. The solution of benzotriazol-1-yloxytris (dimethylamino)phosphonium hexafluorophosphate (BOP) (1.2 eq) in dichloromethane (CHνmax (cm−1): 3288, 1642, 1591, 1523, 1361, 1279, 1036, 975, 849. 1H NMR : δ 2.84 , 3.53 , 6.43 , 6.83 , 6.92 , 7.07 , 7.15–7.30 , 7.35 , 7.43 , 8.20 , 8.42 . EI-MS m/z(%): 283 , 178 (22), 163 (100).Caffeic acid phenylethyl amide: solid. mp 148-149°C. IR 4-week-old male BLTW: CD1(ICR) mice were acquired from BioLasco Taiwan Co., Ltd. and maintained at National Taiwan University College of Medicine Experimental Animal Center, in a temperature- and humidity-controlled (22±1°C and 60 ± 5%) environment with a strict 12 hour light-dark cycle and given free access to food and water. After the acclimatizing period (at least 3 days), mice with fasting plasma glucose levels higher than 130 mg/dL or lower than 70 mg/dL were excluded.Type 1 diabetic mice were induced by modifying the previous method . In brieCaffeic acid phenylethyl amide (10 mg/kg) or metformin (300 mg/kg) was given once a day orally. The investigation followed the University guidelines for the use of animals in experimental studies and conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health . The animal experiments were approved by the IACUC of National Taiwan University (IACUC no. 20100007). Additional 24.18% w/w palm oil and 2.56% w/w soybean oil were added to powder of Purina Laboratory Rodent Diet 5001 standard chow  and this high-fat diet mixture was reconstituted in small pellets. High fat diet and fructose-sweetened water (containing 20% fructose) were used to induce type 2 diabetes in ICR mice. Metabolizable energy of standard rodent chow, HF diet, and fructose water was 3.04 kcal/gm, 4.63 kcal/gm, and 0.8 kcal/mL, respectively.Mice were anesthetized with pentobarbital , and blood was withdrawn from the orbital venous plexus using a heparinized capillary tube. Blood samples were centrifuged at 10000 xg for 5 min, and plasmas were placed on ice or stored at −20°C until assay .An aliquot of plasma was added to glucose kit reagent  and incubated at 37°C for 5 min. The concentration of plasma glucose was then estimated via a spectrophotometer with samples run in duplicate . DetermiIntraperitoneal glucose tolerance test (IPGTT) and insulin tolerance test (ITT) were performed according to the method described previously , 26. BriAbout 50 mg of tissue sample was dissolved in 1 N KOH at 70°C for 30 min. Dissolved homogenate was neutralized by glacial acetic acid and incubated overnight in acetate buffer  containing amyloglucosidase . Samples were then analyzed by measuring glucosyl units using Trinder reaction. The reaction mixture was neutralized with 1 N NaOH .μm, and mounted onto slides.HEstaining was performed for histological analysis. Tissue sections were examined using a microscope and were photographed with a digital camera.Tissues were immersion-fixed in neutral 10% buffered formalin. Sections were paraffin-embedded, cut at 4 μg/mL streptomycin in a humidified atmosphere with 5% CO2 at 37°C. The cells were cultured at 90% confluence and then seeded onto 6 cm culture dishes to perform the experiments [α  or H2O2 (100 to 500 μM). Incubation with TNF-α was conducted for 60 min for IKK (IκB kinase) phosphorylation as described by others [ HepG2 cell lines were grown, as a monolayer, in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/mL penicillin G, and 100 eriments . Cells wy others .Cells were lysed in lysis buffer (20 mM HEPES (pH 7.6), 150 mM NaCl, 1% Triton X-100, 0.1% SDS) and total fat was extracted by Bligh and Dyer method . Brieflyα , MnSOD , p-p65 , p-IKKα/β , PEPCK (a kind gift from Professor DK Granner), and GLUT4 (R&D systems). To adjust for loading differences, blots were reprobed with a monoclonal antibody to β-actin . Densities of the obtained immunoblots were quantified using ImageQuant.Protein contents were measured by Western blotting using commercially available polyclonal antibodies specific for TNF3, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, and 2.5 CaCl2, 11 glucose and gassed with 95% O2-5% CO2 (pH 7.4). Hearts were allowed to beat spontaneously; basal coronary flow and heart rate were recorded after a 30-minute balance period. Then hearts were subjected to a 30-minute global ischemia  and 2-hour reperfusion. The infarct sizes were analyzed by TTC stain method and expressed as percentage of AAR.Isolated perfused mouse heart model was set as previously described with minor modification . In brien) of animals in the group as indicated in figures. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Dunnett's post-hoc test. P < 0.05 was regarded as statistically significant.Data were represented as the means±SEM for the number  for two weeks preserved the basal flow . After aα, a major proinflammatory cytokine, was significantly enhanced in livers of mice on a chronic high-fat and high-fructose diet  expression was increased in CAPA or metformin treated mice on high-fat and high-fructose diet  for 3 hours and then cultured for another 48 hours in fresh DMEM without H2O2 for triglyceride assay. The triglyceride levels rose with increasing H2O2 concentrations. Incubation with CAPA  1 h before H2O2 prevent triglycerides accumulation in HepG2 cells  mice. Our data show that CAPA improves lipid and glucose metabolism and basal coronary flow and protects myocardium from damage inflicted by high-fat and high-fructose diet or STZ. The induction of the antioxidant protein MnSOD and the decrease of NFWhite adipose tissue is now recognized as a secretary organ and secrets adipocytokines, such as RBP4 and adiponectin, which are involved in the regulation of glucose and lipids metabolism. Though it is reported that plasma adiponectin level is lower in obese, insulin resistant, or diabetic patients, in this study we found that high fat and fructose diet did not cause lower plasma adiponectin level. Liu et al.  observedYang et al.  showed tP < 0.05). Since oxidative stress is reported to induce triglycerides accumulation in hepatocytes and might play important role in hepatic insulin resistance [2O2 and found that CAPA decreased the triglycerides accumulation in HEPG2 after H2O2 stimulation. This observation is correlated with the prominent reduction of plasma triglycerides by CAPA in diet-induced diabetic mice. However, whether induction of MnSOD was required in this effect of CAPA needs further studies.However, it is noteworthy that CAPA preserved MnSOD in the liver at a higher level than metformin did  overexpression under control of its natural promoter in mice prevents high-fat diet-induced glucose intolerance, and this effect may result, in part, from prevention of high-fat diet-induced activation of the proinflammatory NF or Nrf1 . Since C


Intradermal priming with DNA prior to MVA boosts gives strong and broad immunogenicity, however that required 5 injections at each immunization. A higher concentration of DNA might allow a simpler administration.This double blind, placebo-controlled trial, enrolled 120 (12 placebo) HIV-uninfected volunteers, in Dar es Salaam and Mbeya. Two pools of DNA plasmids were used  boosted with MVA CMDR EnvE, GagA, PolA. Volunteers were randomized in three groups of 40, primed with either two injections of 300ug, one in each arm,  of DNA with combined plasmids (group IA) or two injections of 300ug with one pool in each arm  of DNA (group IIA) “simplified regimens” or five injections, 2 (pool1) and 3 (pool2) injections in the right and left arm respectively,  of DNA (IIIA) “standard regimen”. DNA/Placebo priming was administered by a needlefree (Biojector) device intradermally at weeks 0, 4 and 12. All volunteers were boosted intramuscularly with 10 pfu of recombinant MVA/placebo at weeks 30 and 46. The primary end point was the number of ELISpot responders to Gag and Env, 2 weeks post the last vaccination.There were no safety concerns. The response rate to Gag and/or Env was 27/32 (84%) in group IA vs 31/33 (94%) in group IIA (p=0.26). The response rate to Gag and/or Env when comparing the ‘simplified regimens’ vs ‘standard’ regimen was 58/65 (89%) vs 32/32 (100%) p=0.09. In responders the median magnitude (IQR) response to Gag was 165  SFC/million PBMC vs 210 , p=0.46 while the magnitude for Env was 150  vs 110  p=0.17 for the ‘simplified’ vs ‘standard’ regimens.The simplified HIV-1 DNA regimens primed as well as the standard regimen for cellular immune responses following boosting with with MVA.


Exposure within an environmental enrichment paradigm results in neurobiological adaptations and decreases the baseline of locomotor activity. The current study determined activation of DARPP-32 (dopamine- and cAMP-regulated phosphoprotein-32) and CREB (cAMP response element binding protein), and locomotor activity in rats raised in enriched (EC), impoverished (IC), and standard (SC) conditions following repeated administration of nicotine or saline. In the saline-control group, the basal phosphorylation state of DARPP-32 at Threonine-34 site (pDARPP-32 Thr34) in the prefrontal cortex (PFC) was lower in EC compared to IC and SC rats, which was positively correlated with their respective baseline activities. While nicotine  produced locomotor sensitization across all housing conditions when the nicotine-mediated locomotor activity was expressed as a percent change from their respective saline control, EC rats displayed greater sensitization to nicotine than IC and SC rats. Consistent with the behavioral findings, repeated nicotine injection increased pDARPP-32 Thr34 in PFC of EC and IC rats and in nucleus accumbens of EC rats; however, the magnitude of change from saline control in nicotine-induced enhancement of pDARPP-32 Thr34 in PFC was strikingly increased in EC rats relative to IC rats. Moreover, EC rats had lower basal phosphorylation levels of CREB at serine 133 in PFC and nucleus accumbens compared to IC and SC rats, whereas the nicotine-induced increase in phosphorylated CREB-Ser133 was more pronounced in PFC of EC rats relative to IC and SC rats. Collectively, these findings suggest innovative insights into advancing our understanding of the molecular mechanisms of enrichment-induced changes in the motivational effects of nicotine, and aiding in the identification of new therapeutic strategies for tobacco smokers. Convergent evidence suggests that environmental factors impact individual susceptibility to drugs of abuse Nicotine activates nicotinic acetylcholine receptors throughout the brain, thereby stimulating dopamine (DA) release within the mesocorticolimbic system 2+-dependent calcineurin by D2 receptors results in dephosphorylation of pDARPP-32 Thr34 Activation of the DA/D1 receptor/cAMP/protein kinase A (PKA) pathway increases phosphorylation of DA and cAMP-regulated phosphoprotein-32 (DARPP-32) at the site Threonine 34 (pDARPP-32 Thr34), but decreases phosphorylation of DARPP-32 at Threonine 75 (pDARPP-32 Thr75) All of the experimental procedures in the animals were performed according to the National Institute of Health guidelines in AAALAC accredited facilities. The experimental protocol for this study was approved by the Institutional Animal Care and Use Committee (IACUC) at the University of South Carolina under compliance with animal welfare assurance #A3049-01.ad libitum in a colony room in the Division of Laboratory Animal Resources at the University of South Carolina, which was maintained at 21±2°C, 50±10% relative humidity and on a 12-h light/dark cycle with lights on at 07:00 AM.Male Sprague-Dawley rats were obtained from Harlan Laboratories, Inc. . Rats arrived at the age of 21 days and were housed with food and water Guide for the Care and Use of Laboratory Animals. Rats were maintained in these environments until 53 days of age and throughout all experiments.Upon arrival, rats were assigned randomly to the EC, IC, or SC group using a previously published method The activity apparatuses were square (40×40 cm) chambers  that detect free movement of animals by infrared photocell interruptions. This equipment contained an infrared photocell grid (32 emitter/detector pairs) to measure locomotor activity with Digipro System Software . Each beam was spaced 2.5 cm apart and 7.0 cm above the chamber floor. The chambers were converted into round (∼40 cm diameter) compartments by adding clear Plexiglas inserts; photocell emitter/detector pairs were tuned by the manufacturer to handle the extra perspex width. Total horizontal activity measured all beam breaks in the horizontal plane with total rearing activity measuring all beam breaks in the vertical plane. All activity monitors were located in an isolated room away from the colony.3 to reduce irritation.An initial experiment was performed to assess the dose effects of acute nicotine on DARPP-32 phosphorylation in brain regions of EC and IC rats. EC and IC rats were administered saline (1 ml/kg) or nicotine  subcutaneously (s.c.). To determine whether enrichment results in differential changes in activation of DARPP-32 and CREB, and their behavioral response to repeated nicotine administration, an optimal dose of nicotine (0.35 mg/kg) was chosen based on results showing the dose effects of acute nicotine on DARPP-32 activity and the previous report demonstrating that nicotine at a similar dose (0.4 mg/kg) produces locomotor sensitization in both EC and IC rats All animals were habituated to the locomotor activity chambers for two 60-min sessions, once/day with no injection. Twenty-four hours after the second habituation session, all rats were habituated to the locomotor chambers for 30 min prior to injection, and then injected subcutaneously with saline and placed into the activity chambers for 60-min to measure baseline activity.The behavioral sensitization procedure began 24 h after the saline baseline measurement. All rats received a 30-min habituation period in the testing chamber prior to nicotine (0.35 mg/kg) or saline injection as reported previously Brains from rats acutely or repeatedly treated with nicotine were removed by rapid decapitation 20 min after the last injection. Brains were dissected in a chilled matrix. PFC, nucleus accumbens (NAc) and striatum were sonicated immediately on ice in a homogenization buffer containing 20 mM HEPES, 0.5 mM EDTA, 0.1 mM EGTA, 0.4 M NaCI, 5 mM MgCI2, 20% glycerol, 1 mM PMSF, phosphatase inhibitor cocktails I  and protease inhibitors , as described previously Proteins were separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) for 55–65 min at 150 V, and transferred to Immobilon-P transfer membranes  in transfer buffer  using a Mini Trans-Blot Electrophoretic Transfer Cell  for 110 min at 75 V. Transfer membranes were then incubated with blocking buffer (5% dry milk powder in PBS containing 0.5% Tween 20) for 1 h at room temperature and then incubated overnight at 4°C in blocking buffer with primary antibodies against the following proteins: DARPP32 , pDARPP32 Thr34 , pDARPP32 Thr75 , CREB , pCREB , respectively.Blots were washed 10 min×3 times with wash buffer (PBS containing 0.5% Tween 20) at room temperature, and then incubated for 1 h in blocking buffer including one of the following secondary affinity-purified, horseradish peroxidase-conjugated antibodies: anti-rabbit IgG , anti-mouse IgG . Blots were then washed 3 times for 10 min per wash. Blots on the transfer membranes were detected using enhanced chemiluminescence (ECL-plus) and developed on Hyperfilm . After detection and quantification of these proteins, each blot was stripped in a Re-blot plus mild antibody stripping solution  for 20 min at room temperature and reprobed for detection of β-tubulin . β-tubulin was used to monitor protein loading among samples. Multiple autoradiographs were obtained using different exposure time, and immunoreactive bands within the linear range of detection were quantified by densitometric scanning using Scion image software .p<0.05.Locomotor activity data are presented as the mean ± standard error of the mean (SEM) number of beam breaks and were analyzed by mixed-factor analyses of variance (ANOVA) with housing condition  and treatment  as between-group factors, and with day and/or time as within-subject factors. Tukey’s post-hoc tests were performed where appropriate. A housing × day × time (3×2×12) mixed factorial ANOVA was used to analyze data from the 2 habituation days, and a housing × time (3×12) factorial ANOVA was conducted on the saline baseline day. The pre-injection habituation part of the experiment was analyzed using a housing × treatment × day × time (3×2×8×12) ANOVA. The effect of repeated nicotine injection on total horizontal activity was analyzed using a housing × treatment × day × time (3×2×8×12) factorial ANOVA, with housing and treatment as between-group factors, and day and time as within-subject factors. Additionally, nicotine-mediated sensitization in EC, IC and SC groups was analyzed by linear regression. To evaluate the effects of repeated nicotine administration on the activity of signaling proteins ; separate two-way ANOVAs (housing condition × treatment) were performed on PFC, NAc, and striatum with environment and treatment as between-group factors. Simple effect comparisons were made for post hoc analyses. To determine whether a relationship existed between locomotor activity and immunoreactivity of DARPP-32, pDARPP-32 Thr34, and pDARPP-32 Thr75, separate Pearson correlations were conducted. All statistical analyses were performed using SPSS . Differences were considered significant at  = 5.2, p<0.05), but not in PFC and striatum compared to saline control. However, EC rats treated with nicotine (0.3 mg/kg) showed an increase in pDARPP-32 Thr34 in PFC  = 7.5, p<0.05), NAc  = 9.5, p<0.05) and striatum  = 10.2, p<0.05). Following 0.8 mg/kg nicotine administration, EC rats had a significant increase of pDARPP-32 Thr34 in PFC  = 12.1, p<0.01) and in NAc  = 8.1, p<0.01), but not in striatum. In addition, nicotine (0.3 mg/kg) increased pDARPP-32 Thr75 in striatum  = 7.9, p<0.01) in EC rats. Acute nicotine had no effects on total DARPP-32 in EC and IC rats. These findings suggest that nicotine dose-dependently increases pDARPP-32 Thr34 levels most prominently in the EC-reared condition and that 0.3 mg/kg acute dose of nicotine is the optimal dose to elicit the most robust changes in pDARPP-32 Thr34 among the measured brain regions of EC and IC rats.To determine whether environmental enrichment regulates DARPP-32 signaling in response to nicotine in a dose-dependent manner, we examined the acute effects of nicotine  on pDARPP-32 Thr34, pDARPP-32 Thr75 and total DARPP-32 in PFC, NAc and striatum in EC and IC rats . In EC gIn the saline control groups, the levels of pDARPP-32 Thr34 were lower in all regions of EC rats relative to IC rats, whereas no significant differences were noted in the levels of pDARPP-32 Thr75 between EC and IC rats. These results suggest environmental enrichment diminishes the basal level of phosphorylated DARPP-32 at Thr34 site.F = 46.48, p<0.001), day  = 317.41, p<0.001) and time  = 148.57, p<0.001), and a significant housing condition × day × time interaction  = 2.48, p<0.001). All animals showed the most activity at the first 10 min of the habituation session, the activity decreased over the following 20 min, and all groups achieved asymptote for the remaining 30 min of the session . On the third day, total activity was recorded for all groups after a saline injection to determine baseline activity prior to the induction of the sensitization phase of the experiment  = 79.02, p<0.001) and time  = 108.96, p<0.001), and a housing condition × time interaction  = 4.09, p<0.001). In general, all animals showed the most activity during the first 10 min of the saline baseline day, acquired asymptotic levels of activity more quickly, and showed a lower asymptote compared to that of the habituation sessions (ps>0.05) during the habituation and saline baseline sessions.Habituation refers to a progressive decrease in locomotor activity following repeated exposure to a particular context. Novelty-elicited locomotor behavior was measured during the two days of habituation to the locomotor chambers for EC, IC, and SC rats. The total activity that occurred for 60 min during the two habituation days is shown in  session . EC ratsperiment . The housessions . None of = 333.90, p<0.001), treatment  = 5.16, p<0.05), day  = 298.97, p<0.001), time  = 47.77, p<0.001) and a significant day × housing condition interaction  = 7.75, p<0.001). There were no main effects of treatment and interactions containing this factor. EC rats exhibited less locomotor activity than did IC and SC rats across the pre-injection habituation sessions .All animals were placed into locomotor chambers for 30 min prior to the activity measurement to produce within-session habituation of activity in response to the context prior to nicotine or saline injection. Total activity in saline control and nicotine-treated group during the 30 min habituation period across the 15-day treatment was recorded as shown in  = 104.29, p<0.001), treatment  = 333.57, p<0.001) and day  = 139.97, p<0.001). A significant housing condition × treatment × day interaction  = 2.76, p<0.05) was found. As shown in  = 20.97, p<0.01), IC  = 27.94, p<0.01) and SC  = 33.11, p<0.01). There was no significant difference across the slopes of the three housing groups, but a significant difference in intercepts or elevations was noted  = 22.48, p<0.001). Thus, nicotine injection produced a robust behavioral sensitization in EC, IC and SC rats.To determine whether differential housing environments alter nicotine-mediated locomotor sensitization, we analyzed locomotor activity during daily administration of nicotine (0.35 mg/kg) or saline during Days 1–15 for EC, IC, and SC rats . A housi = 80.77, p<0.001) and day  = 2.52, p<0.05). Also, a significant housing condition × day interaction  = 1.84, p<0.001) was found, indicating repeated overall difference in total activity among the different housing groups. Post hoc tests (Bonferroni t-test) showed that EC rats were significantly less active than were IC or SC rats across treatment days (p<0.001), but no difference between IC and SC rats was found (p>0.05). In nicotine-treated groups, the two-way ANOVA analysis revealed significant main effects of housing condition  = 32.57, p<0.001) and day  = 243.45, p<0.001), but post hoc tests showed no difference between the IC and SC groups. There was a significant housing condition × day interaction  = 5.51, p<0.001), suggesting EC rats have lower nicotine-mediated locomotor activity across treatment days. Overall, all rats treated with nicotine displayed greater locomotor activity on Days 3–15 than on Day 1 (ps<0.05).Based on the significant three-way interaction, separate two-factor ANOVAs were conducted. In saline-treated groups, a housing condition × day ANOVA analysis revealed significant main effects of housing condition  = 108.16, p<0.001), treatment  = 243.44, p<0.001), day  = 209.67, p<0.001) and time  = 231.97, p<0.001). In addition, there was a significant housing condition × treatment × day interaction  = 3.66; p<0.05). When the data were expressed as a percentage change relative to the respective saline controls, on Day 1 acute nicotine increased locomotor activity (243%) in EC  and (116%) in IC, but decreased activity (20%) in SC rats  = 3.43, p<0.05) and a trend of housing × treatment interaction  = 2.87, p =  0.06), but no significant effect of treatment (p<0.01), but no difference between the IC and SC groups was found. Following repeated nicotine administration, the level of pDARPP-32 Thr34 in PFC was increased in the EC  = 8.32, p<0.05) and IC  = 3.12, p<0.05) compared to the respective saline controls. No difference between SC-Sal and SC-Nic groups was found. In NAc, two-way ANOVA on the levels of pDARPP-32 Thr34 revealed a main effect of housing condition  = 4.21, p<0.05), and no significant effect of treatment or their interaction (p<0.05), but not in IC and SC rats (ps>0.05). In striatum, no difference in pDARPP-32 Thr34 level was found among EC, IC and SC rats with nicotine or saline injection  = 21.22, p < 0.001) and treatment  = 98.64, p < 0.001), and a significant housing condition × treatment interaction  = 4.69, p < 0.05) were found. Post hoc analysis revealed that the ratio of pCREB /CREB was lower in EC-Sal than in IC-Sal  = 11.44, p< 0.05) and SC-Sal  = 58.00, p<0.001), indicating that environmental enrichment decreases the basal levels of pCREB. Repeated nicotine administration significantly increased pCREB levels in PFC of EC  = 88.57, p<0.001), IC  = 29.57, p<0.01), and SC  = 21.35, p<0.01) compared to the respective saline control groups.We examined whether environmental enrichment changed CREB and pCREB in the PFC, NAc, and striatum in the EC, IC, and SC groups. As shown in  = 9.34, p<0.05) was found. There was no significant effect of treatment and their interaction. The ratio of pCREB /CREB was lower in EC-Sal than in IC-Sal  = 5.92, p<0.05) and SC-Sal  = 4.89, p<0.05). Repeated nicotine administration increased the level of pCREB in EC-Nic rats  = 6.71, p<0.05) but not in IC-Nic and SC-Nic rats. In striatum, no differences in pCREB and total CREB levels were found among the EC, IC and SC rats with nicotine or saline injection  or pDARPP-32 Thr75 protein levels . However, pDARPP-32 Thr34 protein levels were correlated positively with mean total horizontal activity . Correlations were also examined in the nicotine-treated rats; however, no statistically significant correlations were found (data not shown). Together, these results suggest that pDARPP-32 Thr34 levels in the PFC are responsible for EC reductions in locomotor basal levels and possibly involved in repeated nicotine-induced locomotor sensitization.To determine whether the basal level of DARPP-32 activity was associated with the results of behavior tests, the correlation of locomotor activity and DARPP-32 activity was examined. The current findings demonstrate that an enriched housing environment alters the levels of phosphorylated DARPP-32 and CREB under control conditions  and following nicotine administration. Specifically, the effects of enrichment on activity of DARPP-32 and CREB are robustly found in PFC relative to NAc and striatum. The fact that the basal phosphorylation state of DARPP-32 at Thr34 site in PFC is positively correlated with locomotor activity in EC, IC, and SC rats under saline control conditions, suggests that the PFC DARPP-32 phosphorylation at Thr34 may play an important role in enriched environment-induced changes in locomotion. Acute nicotine injection produces increased levels of pDARPP-32 Thr34 in EC rats in a dose-dependent manner. Although repeated nicotine administration produced locomotor sensitization in EC, IC, and SC rats, when the nicotine-mediated activity was expressed as a percent change from the respective saline controls EC rats displayed greater sensitization to nicotine than IC and SC rats. In addition, the magnitude of change from saline control in nicotine-induced enhancement of pDARPP-32 Thr34 and pCREB in PFC was strikingly increased in EC rats relative to IC and SC rats, indicating a potential mechanism through which environmental enrichment could reduce locomotor effects of nicotine. Together, the findings demonstrate a novel role of PFC pDARPP-32 Thr34 in enriched environment-induced neuroplasticity and behavioral changes associated with repeated nicotine administration.Relative to the IC condition, the complexity of an enriched environment paradigm comprises multiple components: a large space, physical exercise, novel objects, and social cohorts. We acknowledge that one limitation of the current study is that the results do not address which of these components, or combination of components, is specifically responsible for the environmental-induced behavioral and neurochemical changes. To examine this issue completely, several variations of “control” conditions would be needed, including manipulations to study the effects of cages size, numbers of social partners, presence of novel objects, amount of exercise, and so on, which is beyond the scope of the current study. In this study, we chose a social control condition with only one other animal in a small cage because this represents the NIH standard housing condition, which is the most typical housing condition used across various laboratories and allows for cross-comparisons with results published in the literature. However, some studies have used other controls, such as single-housed animals with novel objects or social caged animals (n = 8–10 per cage) without novel objects. For example, in a recent study The most important finding from the study is that EC rats show diminished basal levels of pDARPP-32 Thr34 in PFC and NAc relative to IC and SC rats under saline control conditions, indicating that PKA-mediated Thr34 pathway is quite sensitive to enrichment-induced plasticity. The Thr34 phosphorylation levels are regulated by both dopaminergic and glutamatergic receptor stimulation signaling in vitro conditions , nicotine at a low concentration decreases Thr34 levels, whereas nicotine at a high concentration increases Thr34 and decreases Thr75 levels Acute nicotine regulated the phosphorylation levels of DARPP-32 at Thr34 and Thr75 sites in a dose-dependent manner. While acute nicotine (0.1 mg/kg) only increased Thr34 levels in NAc of EC rats and had no effects on Thr75, the high dose of nicotine (0.8 mg/kg) increased Thr34 levels in PFC and NAc levels in EC rats. However, nicotine (0.3 mg/kg) obviously increased Thr34 levels in all regions and Thr75 levels in striatum in EC rats, suggesting an enhanced maximal action of nicotine on the phosphorylation of DARPP-32. Under in vivo voltammetry assay has demonstrated that basal DA clearance in medial PFC is lower in EC rats than IC rats under saline control condition, whereas systemic acute nicotine injection only increases the DA clearance in EC rats, but not in IC rats Repeated nicotine administration eliminated the basal difference in pDARPP-32 Thr34 observed between the EC and IC rats and increased pDARPP-32 Thr34 in the PFC of EC rats relative to IC rats. Nicotine also elicited Thr34 increases in the NAc of EC rats, despite being not as robust as in the PFC. This implicates that the processes mediating the lower basal levels of pDARPP-32 Thr34 in the PFC of EC rats do not prevent repeated nicotine stimulation from regulating DARPP-32 signaling. Rather, compared to their respective saline controls, the magnitude of change in nicotine-induced Thr34 level in PFC is greater in EC than in IC and SC rats. DA D1 receptor activation has been demonstrated to increase Thr34 levels In addition to DARPP-32, this study also determined the effects of enrichment on CREB activity following repeated saline and nicotine administration. Enrichment-mediated changes in phosphorylated CREB at Ser133 are in parallel with the changes of pDARPP-32 Thr34 levels, which are associated with PKA activation Our results demonstrated that EC rats had greater nicotine-mediated increases in pDARPP-32 Thr34 and pCREB, which were robustly found in the PFC relative to the NAc, and striatum. PFC, specifically prelimbic and infralimbic cortices as well as nucleus accumbens have been implicated in addiction, extinction, and relapse and have been hypothesized to constitute a series circuit underlying behavioral changes in response to psychostimulants The baseline locomotor activity in all saline control animals was correlated positively with the respective basal levels of pDARPP-32 Thr34 in PFC, but not Thr75 and total DARPP-32, suggesting that the basal phosphorylation state of Thr34 in PFC is associated with changes in basal locomotor activity in housing conditions. Notably, because EC rats have lower baseline levels of ambulatory activity relative to IC and SC rats, it is important to point out how to express environment-dependent differences in drug effects when the saline-treated control groups differ. In the current study, the nicotine-mediated locomotor activities in EC, IC, and SC groups on Days 1 and 15 were expressed as percent changes from the respective saline controls; EC rats actually showed greater locomotor sensitization to nicotine than IC and SC rats. However, when the nicotine-induced activities in the three housing groups were expressed as absolute values, EC rats had less sensitivity to nicotine-induced sensitization than that in IC and SC rats In conclusion, the current study has begun to identify the neurobiological mechanism of enriched environment-induced alterations in DARPP-32 and CREB activity on altering sensitivity to the locomotor effects of nicotine. More specifically, the basal levels of PFC pDARPP-32 Thr34 are correlated positively with the baseline locomotor activity in rats housed in different housing conditions. Future studies will investigate whether manipulation of prefrontal cortical DARPP-32 phosphorylation attenuates the difference in basal and nicotine-mediated behavior, which will allow us to better understand the neurobiological basis of environmental enrichment in potential resistance to the motivational responses to psychostimulants.


H-cyclopenta[b]thiophene-3-carbonitrile, 5TIO1, is a new 2-aminothiophene derivative with promising pharmacological activities. The aim of this study was to evaluate its antioxidant activity in different areas of mice central nervous system. Male Swiss adult mice were intraperitoneally treated with Tween 80 dissolved in 0.9% saline (control group) and 5TIO1 . Brain homogenates—hippocampus, striatum, frontal cortex, and cerebellum—were obtained after 24 h of observation. Superoxide dismutase and catalase activities, lipid peroxidation and nitrite content were measured using spectrophotometrical methods. To clarify the 5TIO1's mechanism on oxidative stress, western blot analysis of superoxide dismutase and catalase was also performed. 5TIO1 decreased lipid peroxidation and nitrite content in all brain areas and increased the antioxidant enzymatic activities, specially, in cerebellum. The data of Western blot analysis did not demonstrate evidence of the upregulation of these enzymes after the administration of this compound. Our findings strongly support that 5TIO1 can protect the brain against neuronal damages regularly observed during neuropathologies.2-[amino]-5,6-dihydro-4 Human cells are constantly exposed to oxidants . There iOxidative stress has been a common pathogenic mechanism underlying many major psychiatric disorders, such as anxiety, due to the intrinsic oxidative vulnerability of the brain . GrowingAmong the numerous antioxidants available to cells, sulfur compounds  and glutathione and their derivatives have been widely studied due to their antioxidant properties . TherebyH-cyclopenta [b]thiophene-3-carbonitrile), also called 5TIO1 amino]-5,6-dihydro-4ed 5TIO1 , is a neThe aim of this study was to evaluate the antioxidant activity of 5TIO1 in different areas of the central nervous system (CNS) of adult mice by determination of lipid peroxidation level, nitrite content and enzymatic activities of catalase (CAT), and superoxide dismutase (Mn-SOD). To clarify 5TIO1's mechanism on oxidative stress, western blot analysis of Mn-SOD and CAT was also performed in mice brain homogenates 24 h of observation.o 031/12).Male Swiss adult mice (25–30 g) were used. All animals were maintained at a controlled temperature (25 ± 2°C) under 12 h dark/light cycle with free access to water and food. All experiments were performed according to the Guide for the Care and Use of Laboratory Animals, from the US Department of Health and Human Services, Washington DC, 1985. This protocol was previously approved by the Animal's Ethic Committee at Federal University of Piaui (nRf 0.54 (n-Hex./AcOEt. 8.5 : 1.5). It was emulsified with 0.05% Tween 80 , dissolved in 0.9% saline (vehicle), and intraperitoneally administered at doses of 0.1, 1 and, 10 mg kg−1 (n = 7 per group). Control animals received 0.25 mL of 0.05% Tween 80 dissolved in 0.9% saline (n = 7).5TIO1 was from Laboratory of Synthesis and Vectorization of Molecules of the State University of Paraiba. It is a yellow crystal, produced in 89% of yield, melting point 159-160°C, n = 7) were killed by decapitation 24 h after treatment. Their brains were dissected on ice to remove hippocampus, striatum, frontal cortex, and cerebellum for determination of lipid peroxidation levels, nitrite content, and superoxide dismutase and catalase activities.For neurochemical assays, both 5TIO1 and control groups (Drug dosages of 5TIO1 were determined from dose-response studies (data not shown) and from observation of dose currently used in animal's studies –25. The n = 7 per group) and control animals (n = 7) was analyzed by measuring thiobarbituric acid reacting substances (TBARSs) in homogenates, as previously described by Draper and Hadley [−1 μg protein−1. To determine nitrite content of the control mice (n = 7) and 5TIO1 groups (n = 7 per group), 10% (w/v) homogenates was centrifuged . The supernatants were collected, and nitric oxide production was determined based on the Griess reaction [μL supernatant was incubated with 100 μL of the Griess reagent (1% sulfanilamide in 1% H3PO4/0.1% N-(1-naphthyl)ethylenediamine dihydrochloride/1% H3PO4/distilled water, 1 : 1: 1 : 1, v/v/v/v) at room temperature for 10 mins. A550 was measured using a microplate reader. Nitrite concentration was determined from a standard nitrite curve generated using NaNO2. The results were expressed in μM.In all experimental procedures, 10% (w/v) homogenates of each brain's area were prepared for both groups. Lipid peroxidation in the 5TIO1 groups (d Hadley . Brieflyreaction . Brieflyn = 7 per group) and control animals (n = 7) was evaluated by using xanthine and xanthine oxidase to generate superoxide radicals [μM), 7.44 mg cytochrome c, 3.0 mL KCN (200 μM), and 3.0 mL EDTA (1 mM) in 18.0 mL 0.05 M sodium phosphate buffer, pH 7.0. The sample aliquot (20 μL) was added to 975 μL of the substrate mixture plus 5 μL xanthine oxidase. After 1 min, the initial absorbance was recorded, and the timer was started. The final absorbance after 6 mins was recorded. The reaction was followed at 550 nm. Purified bovine erythrocyte superoxide dismutase  was used under identical conditions to obtain a calibration curve showing the correlation between the inhibition percentage of formazan dye formation and superoxide dismutase activity and the results expressed as U mg protein−1.Each brain area was ultrasonically homogenized in 1 mL 0.05 mM sodium phosphate buffer, pH 7.0. Protein concentration was measured by the Lowry's method . The 10%radicals . They ren = 7 per group) and control animals (n = 7) was determined by the method that uses H2O2 to generate H2O and O2 [2O2 in 50 mL 0.05 mM sodium phosphate buffer, pH 7.0. The sample aliquot (20 μL) was added to 980 μL substrate mixture. The initial absorbance was recorded after 1 min, and the final absorbance after 6 mins. The reaction was followed at 230 nm. A standard curve was established using purified catalase  under identical conditions. All samples were diluted with 0.1 mmol L−1sodium phosphate buffer (pH 7.0) to provoke a 50% inhibition of the diluent rate . Results are expressed as mmol min−1 μg protein−1 [Catalase activity in the 5TIO1 groups (O and O2 . The actrotein−1 .n = 4 per group), hippocampus, striatum, frontal cortex, and cerebellum homogenates were mixed with protein loading buffer  according to manufacturer's procedure and placed in a heating bath (95°C) for 5 mins. Proteins were separated using SDS-PAGE (gradient gels from 5% to 25%). The protein amount loaded per lane was 10 Ag. After separation, the proteins were stained with Coomassie Brilliant Blue or transferred to nitrocellulose paper, and unspecific protein binding sites were blocked with blocking buffer . The blots were incubated overnight with the primary antibodies against (1) catalase  and (2) Mn-SOD , followed by incubation with horseradish peroxidase-conjugated secondary antibody . Immunoreactivity was visualized using the ECL detection system . The Western blots did not show the B-actin band, since in our results corrections were made for the variability in protein loading in the gels.In the immunoblotting assay . There was no dose-dependent effect of 5TIO1, and its scavenging activity was, statistically, the same in all areas for both assays.Tables −1, resp.) and in cerebellum  in comparison with the control group (P < 0.05). In striatum, groups treated with 5TIO1 0.1 and 1.0 increased catalase levels in 5.37% and 36.72%, respectively; but only 5TIO1 10 group augmented significantly (57.72%). In frontal cortex, the catalase activity increased 32.87% and 51.17% in the 5TIO1 1.0 and 10.0 mg kg−1 groups, respectively, if compared to the control group, 5TIO1 0.1 and 5TIO1 1.0 groups (P < 0.05). Only 5TIO1 at the dose of 0.1 mg kg−1 showed a decrease of 31.54% in catalase activity in frontal cortex compared with control group (P < 0.05).−1 showed an increase in superoxide dismutase activity in hippocampus (58.93%), striatum (8.33%), frontal cortex (14.54%), and cerebellum (22.29%) compared to the control group (P < 0.05). 5TIO1 at the dose of 1 mg kg−1 increased 40.18% and 8.64% SOD activity in hippocampus and frontal cortex, respectively, (P < 0.05). On the other hand, in the dose of 10 mg kg−1 upregulation of this enzyme activity was not observed.−1) the total Mn-SOD and catalase activities did not change in comparison with control group. Likewise, 5TIO1 groups did not affect the Mn-SOD and catalase mRNA or protein levels, as tested by immunoblot analyses of hippocampus, striatum, frontal, and cerebellum homogenates of mice treated with 5TIO1  of Mn-SOD for 0.1, 1, and 10 mg kgrebellum .Oxidative stress, which represents a loss of balance in oxidation-reduction reactions, can dramatically alter neuronal function and has been related to anxiety , 10. OurThe brain is believed to be particularly vulnerable to oxidative stress due to neuronal membrane lipids, rich in highly polyunsaturated fatty acids, undergo rapid lipid peroxidation, and –34 with Lipid peroxidation in a tissue is an index of irreversible biological damage of the cell membrane phospholipid, which, in turn, leads to inhibition of most of the sulfhydryl and some nonsulfhydryl enzymes , 36. As The nitrite content is also a fundamental oxidative stress' biomarker, since high levels of nitric oxide and their oxidative derivatives, as peroxynitrite, can be toxic, playing an important role in neurodegenerative diseases –40. In nLipid peroxidation and nitrite content assays suggest that 5TIO1 can be an antioxidant by ROS and RNS scavenging, respectively. These results can be related with 5TIO1's physicochemical properties. Previous research identified sulfur-containing antioxidants as those with the most beneficial therapeutic ratio . 5TIO1 cIn normal conditions, there is a balance between oxidative and nitrosative stress and antioxidant actions. The harmful effect of free radicals to the organism induces several defense mechanisms , 36, 44.2O2 to produce water and molecular oxygen and with H donors  with peroxidase activity. Catalase protects cells against H2O2 generated inside them. Although catalase is not essential to some cell types under normal conditions, it has an important role in the acquisition of tolerance to oxidative and nitrosative stress during the cellular adaptive response [−∙) and thus acts as a first line of defense. In the case of superoxide dismutase deficiency or increased superoxide production, it reacts with nitric oxide to produce peroxynitrite (ONOO−), which is a potent nitrosating agent that can cause direct damage to proteins, lipids, and DNA [Catalase is an enzyme that effectively reacts with Hresponse . Superox and DNA .Our findings suggest higher catalase levels in cerebellum, frontal cortex, striatum, and hippocampus, indicating that this enzyme may exert a protective role in the first two brain regions more specifically over the other areas investigated in animals treated only with vehicle. Alper and coworkers  also obs−1 in all brain areas, specially, in hippocampus and cerebellum. However, no significant changes were observed with 10 mg kg−1. So, a dose-dependent process was not observed, suggesting that in higher doses of 5TIO1, there may be a saturation of this enzymatic activity. Furthermore, it should be noted that the 1 mg kg−1 increased superoxide activity only in hippocampus and frontal cortex, indicating that 5TIO1 exerts its possible antioxidant effect by Mn-SOD modulation in these areas. Like it was previously mentioned, these unpublished results will contribute significantly to fill the gaps of the causes and consequences of oxidative stress modulated by thiophenic compounds.The superoxide dismutase activity was positively modulated by 5TIO1 at the dose of 0.1 mg kgIn our studies, the total Mn-SOD and catalase activities did not change after treatment with 5TIO1. Data from Western blot analysis did not demonstrate evidence for the upregulation of antioxidant enzymes (Mn-SOD and CAT) after treatment. In addition, data confirmed our hypothesis that occurred only an increase in the enzymatic activities studied, since there was no change in protein contents of Mn-SOD and catalase in treated mice  after 24 h of treatment with 5TIO1.Moreover, the increased enzymatic activity observed in our study may be due to its allosteric regulation, since no studies have looked at our Western blot analyses that did not change in gene expression of these enzymes. However, the increased activity of the enzymes studied may also have been induced by covalent modification (phosphorylation). Thus, we suggest that during the establishment of oxidative stress in brains, the phosphorylation of these enzymes may play a central role to control neuronal functions involved in the establishment of anxiety disorders.We clearly showed that 5TIO1 decreased the lipid peroxidation levels and nitrite content and increased the antioxidant enzymatic activities. In our knowledge, these effects of 5TIO1 on oxidative stress observed in brain have not been reported before. Thus, these findings might have important implications for understanding the mechanism of neurodegenerative diseases, promoting new advances in the development of selective, and targeted antiepileptic, antidepressant, and anxiolytic drugs. 5TIO1 can protect the brain against neuronal damages regularly observed during neuropathologies. Further investigation of 5TIO1 effects against necrosis, apoptosis, and/or autophagy observed in brains disorders are in progress to confirm its antioxidant and neuroprotective effects.