Since 2000, the Global Alliance for Vaccines and Immunization (GAVI) and WHO have supported the introduction of the Pneumococcal Conjugate Vaccine (PCV) in the immunization programs of developing countries. The highest pneumococcal nasopharyngeal carriage rates have been reported (40-60%) in these countries, and the highest incidence and case fatality rates of pneumococcal infections have been demonstrated in Africa.Studies concerning nasopharyngeal pneumococcal carriage and pneumococcal infection in children less than 5 years old were conducted in Dakar from 2007 to 2008. Serotype, antibiotic susceptibility and minimum inhibitory concentrations were determined. In addition, among 17 overall publications, 6 manuscripts of the Senegalese literature published from 1972 to 2013 were selected for data comparisons.Streptococcus pneumoniae. The five most prevalent serotypes, were 6B (9%), 19 F (9%), 23 F (7.6%), 14 (7.6%) and 6A (6.8%). Fifteen percent of the strains (20/132) showed reduced susceptibility to penicillin and 3% (4/132) showed reduced susceptibility to anti-pneumococcal fluoroquinolones. Among the 196 suspected pneumococcal infections, 62 (31.6%) Streptococcus pneumoniae were isolated. Serogroup 1 was the most prevalent serotype (21.3%), followed by 6B (14.9%), 23 F (14.9%) and 5 (8.5%). Vaccine coverage for PCV-7, PCV-10 and PCV-13, were 36.2% (17/47), 66% (31/47) and 70.2% (33/47) respectively. Reduced susceptibility to penicillin and anti-pneumococcal fluoroquinolones was 6.4% and 4.3%, respectively, and the overall lethality was 42.4% (14/33).Among the 264 children observed, 132 (50%) children generated a nasopharyngeal (NP) positive culture with Streptococcus pneumoniae serotypes contained within the current generation of pneumococcal conjugate vaccines and consistent with reports from other countries in sub-Saharan Africa prior to PCV introduction. Antimicrobial resistance in this small unselected sample confirms a low rate of antibiotic resistance. Case-fatality is high. Introduction of a high valency pneumococcal vaccine should be a priority for health planners with the establishment of an effective surveillance system to monitor post vaccine changes.This study confirms a high rate of carriage and disease caused by The online version of this article (doi:10.1186/s12879-014-0627-8) contains supplementary material, which is available to authorized users. In developed nations, a substantial decrease in the invasive pneumococcal disease (IPD) incidence rate has been achieved through the introduction of the 7-valent- pneumococcal conjugated vaccine (PCV-7) into pediatric immunization programs. PCV-10 and PCV-13 including additional pneumococcus serotypes are now gradually replacing PCV-7 in immunization programs mainly in developed countries  but alsoStreptococcus pneumoniae (S. pneumoniae) serotypes colonize the nasopharyngeal niche as commensal inhabitants and subsequently migrate to cause IPD ..StreptocSince 2000, the pneumococcal infection burden in Africa has been a source of great concern, as this continent has the highest incidence of pneumococcal infections (3627 per 100000) and pneumococcal mortality (399 per 100000), with case fatality rates of 11%, 73% and 58% for pneumonia, meningitis and non-pneumonia non-meningitis diseases, respectively .In West African countries, serotype 1 is the most prevalent serotype in IPD, followed by serogroups 5, 6, 2, 3 and 12 ; howeverThe prevalence of Penicillin Non-Susceptible Pneumococcus (PNSP) has increased worldwide, associated with the spread of specific clones with serotypes 6B, 9 V, 14, 19 F, and 23 F. After the introduction of PCV-7 a reduction in antibiotic-resistant infections has been observed, reflecting a decline in the incidence of IPD caused by PCV-7 serotypes. To go beyond the disease protection capacity of PCV-7, extended valency vaccines have been developed which include 3 and 6 additional capsular polysaccharides . These PCVs designated PCV-10 and PCV-13 are gradually used and licensed in many countries around the world. These implementations provide the opportunity to more deeply investigate the impact these vaccines will have on disease .S. pneumoniae infection and NP carriage in Senegal published from January 1, 1972 to September 30, 2013 and selected pediatric studies for comparison with our current findings. Herein, we report the serotype distribution and antibiotic resistance of S. pneumoniae isolated from NP carriage and IPD patients, and these data were compared with previous data from studies conducted in Senegal. Moreover, the question of the most appropriate PCV for Senegal is discussed regarding the S.pneumoniae serotype distribution in carriage and IPD.In the present study, we performed a PubMed search for studies on This study was approved through the Ethics Committee of the Ministry of Health. Written consent was obtained from parents or legal guardians before inclusion. A detailed description of the study was provided in the native language for illiterate individuals and for both studies different questionnaires were conducted.This study was conducted in Dakar, the capital of Senegal, in 2007 and 2008. All samples were collected from children less than five years of age. Experiments involving pneumococcal carriage were conducted at 2 Health Centers located in Medina and Saint Martin in Dakar between March 2007 and October 2008. These two Health Centers have been chosen for their localization in popular areas and their capacities to draw a large population of children from surrounding areas. Medical activities comprise primary care, vaccinations, medical consultations and hospitalizations.Children consulting for vaccination were asked to provide a nasopharyngeal swab sample . During the 20 month period of the study, in both Health Centers, children were sampled on nine different months over the study period, two days per month, on a total of 18 days. Children were sampled once and very few refusals were recorded. Exclusion criteria were presence of antibiotic consumption within the preceding 2 weeks.S. pneumoniae disease were recruited in the pediatric wards from the Albert Royer/Fann University Hospital. The Albert Royer hospital is the referral pediatric center in Dakar and children attending these pediatric units originate from the city of Dakar and its outskirts, but also from more remote areas outside the capital.In the clinical study, during 21 months, from January 2007 to September 2008, children with suspected S. pneumoniae from a normally sterile body site as blood, CSF or pleural effusion. The diagnosis of meningitis was based on S. pneumoniae identified in CSF through culture. Septicemia was defined as S. pneumoniae cultured in blood with no distinctive clinical syndrome. The diagnosis of empyema was based on cultures positive for S. pneumoniae that were taken from pleural fluids. Repeat samples from sterile sites from the same child were regarded as part of the same episode and one isolate was included if both isolates expressed the same serotype. Specimens were sampled in accordance with the clinical pattern; here are reported results of blood-cultures, CSF, and pleural fluids.An IPD case was defined as culture of S. pneumoniae isolates were then sent to the Pasteur Institute laboratory for susceptibility testing and serotyping. S. pneumoniae was isolated and identified using common bacteriological methods. The suspected samples were streaked onto fresh sheep blood agar plates (5%) containing optochin disks and incubated at 35-37°C with 5% CO2 for 24 h; in cases of negativity, the cultures were incubated up to 48 h. Bacteria showing inhibition zones around the optochin disks were subjected to additional tests, such as gram staining, catalase reaction and latex agglutination . The capsular type was determined for 23 different serogroups through latex agglutination . Then when needed, traditional factor antisera were used to serotyping the PCV-7 serogroups 6, 9, 18, 19 and 23 by the Quellung reaction. The isolates showing no reactivity were defined as “Non Typed” (NT) strains. Beta-lactam (BL) and Fluoroquinolone (FQ) susceptibility was examined using the disk-diffusion method according to the Comité de l’Antibiogramme de la Société Française de Microbiologie . CASFM and EUCAST  guidelines recommend the use of different oxacillin disk screening tests for detection of penicillin resistant pneumococci, a 5-μg oxacillin disk for CASFM and a 1-μg oxacillin disk for EUCAST. As a result, cut-off values of the inhibition zone are 26 mm and 20 mm, respectively. In cases of intermediate or resistant susceptibility, the Minimum Inhibitory Concentration (MIC) was determined for BL and FQ using a standard agar diffusion method (Steer’s multipoint apparatus). The Steer’s device is an inoculum replicator with multipoints delivering simultaneously up to 96 dots of the prepared bacterial suspension on the surface of the agar plates  when relevant. Mean age values and sex ratios were estimated on children from both the carriage and the invasive disease studies.S. pneumoniae cultures. S. pneumoniae NP positive carriers were observed as early as the first month after delivery. The serotype distribution displayed 28 different serotypes. The five most prevalent serotypes were 19 F , 6B , 14 , 23 F , and 6A , accounting for 40.2% of the overall serotypes  carriage study included 264 children less than 24 month of age; 253 (95.8%) children were between 0 and 12 month of age, and the mean age was 5 months, with a sex ratio (M/F) of 1.13. Among the 264 children, 132  children had NP positive S. pneumoniae isolates showed a reduced susceptibility to ciprofloxacin, of which 10 isolates  were completely resistant to this antibiotic (MICs 4 to 8 mg/L), while 3 isolates were levofloxacin resistant  and 1 isolate was moxifloxacin resistant (MIC = 1 mg/L).According to CASFM, among the 132 isolates, 70 showed an inhibition diameter of less than 26 mm on the oxacillin disk (5 μg), 20  of the isolates showed reduced susceptibility to penicillin by MIC determination (MIC ≥ 0.12 mg/L) and 2 of the 20 isolates were resistant to penicillin (>2 mg/L). The first strain, of NT serotype (penicillin MIC = 4 mg/L), was also amoxicillin resistant (MIC = 4 mg/L) and cefotaxime non-susceptible (MIC = 1 mg/L), and the second strain, of 23 F serotype (penicillin MIC = 8 mg/L), was amoxicillin and cefotaxime susceptible. The mean value of penicillin MIC among the 20 PNSP was 0.33 mg/L. Regarding FQ resistance among the 132 isolates, 32 S. pneumoniae infected children out of the 196 suspected children were finally included (31.6%).During the 21 month IPD study, 196 children between 0 and 5 years of age were included; the mean age was 15.5 months, with 54.6% (107/196) of the children less than 1 year old, and the sex ratio (M/F) was 1.3.Two hundred and forty specimens were collected, 144 bloodstreams, 75 CSF and 21 pleural fluid (PF) samples. Among 101 children with blood samples, 74 children with CSF samples and 21 children with PF samples, 12 septicemia cases , 42 meningitis cases  and 8 empyema cases  were observed. Sixty two S. pneumoniae isolates: 29 meningitis cases, 10 septicemia cases and 8 empyema cases. Twenty different serotypes were identified  of the 62 d Figure . SerotypS. pneumoniae meningitis, 16 different serotypes were identified, and serotypes 1 , was the most prevalent followed by 23 F  and 6B   with PCV-7, 66% (31/47) with PCV-10 and 70.2% (33/47) with PCV-13 , amoxicillin (MIC = 32 mg/L) and cefotaxime (MIC = 32 mg/L). Among the 17 isolates with reduced susceptibility to ciprofloxacin , 2 isolates were moxifloxacin (MIC = 4 and 16 mg/L) and levofloxacin resistant (MIC = 32 mg/L). The isolate simultaneously resistant to BL and FQ was of serotype 1, and the second FQ resistant isolate was of serotype 5.S. pneumoniae positive results, showing an overall lethality of 42.4%  (14/33) and a meningitis lethality of 54.5%  (12/22).The outcomes were known for 33 children out of the 62 children (53.2%) and 22 children out of the 42 meningitis cases with Regarding the 6 Senegalese publications, one study concerned both NP carriage and Respiratory Tract Infections (RTI) , one stuThe 50% (132/264) mean NP carriage prevalence rate observed in the present study is consistent with the results obtained in most of the publications concerning young children ; indeed,The high NT prevalence rate observed in our study is partially due to the Pneumotest-kit used for serotype determination in the laboratory, with only 23 different serogroups identified. This corresponds to non-typed strains rather than to non-typable strains. The most prevalent carriage serotypes found in this study were 6B, 19 F, 23 F, 14, 6A, 11 and 19A  to other African countries, such as Ghana (50%) , Kenya , is consistent with the results of the Echave et al. study conducted in a Senegalese rural area in 2003 (13%) . HoweverOur study has limitations due to single hospital site recruitment; however we believe that our surveillance captures most laboratory-confirmed IPD cases and, because pediatric wards in the Albert Royer hospital welcome a large population of children living in the Dakar region, enables comparison with other published data from Senegal or other African countries.The study is also limited by the incomplete serotyping and susceptibility testing of overall cultured isolates. Given these limitations, results have to be discussed cautiously, but in case more isolates had been tested, we are unlikely to see contradictory findings as these data are consistent with previous Senegalese and sub-Saharan Africa published results.The most prevalent serotypes were serotype 1 21.3%), 6B 14.9%), 23 F (14.9%) and 5 (8.5%) in accordance with what is known in Africa [.9%, 23 F%, 6B 14.The vaccine coverage observed in the present study Figure  is consiS. pneumoniae meningitis tested with 28 different antisera, 16 different serotypes were identified, and serotypes 1 , was the most prevalent followed by 23 F , and 6B  . In 2003, Echave et al. reported an 18% PNSP prevalence rate in RTI, with no MIC determination to distinguish penicillin intermediate and resistant isolates in Senegal . Dia et S. pneumoniae positive results, the high overall case fatality rate (CFR)  and meningitis CFR  found are consistent with lethality reported in other African countries, with a mean rate of 73% (18%-94%) in meningitis [Even though outcomes were only known for 53.2% (33/62) of children with ningitis . These Cningitis , and thiSince November 2012, the introduction of PCV-7 has been recommended in Senegal, administered at two doses at 2 and 3 months of life, generating a family cost of 109.2 USD per dose. The invasiveness of individual serotypes is reflected in the case:carrier ratio (CCR) which distinguish the more from the less invasive serotypes . Our stuAlthough cefotaxime- and FQ-resistant isolates have been detected in Senegal, pneumococcal antibiotic resistance is not a major issue. The primary concern is the CFR, and measures should be urgently implemented in Pediatric wards in Dakar to improve children outcomes. An effective pneumococcal surveillance network should be developed to collect representative data on pneumococcal carriage and infection and to detect serotype changes.


Aeromonas hydrophila. To understand the iron homeostasis mechanism in A. hydrophila, a dimethyl labelling based quantitative proteomics method was used to compare the differential expression of cell envelope proteins under iron starvation.Iron homeostasis is an essential process over the entire lives of both hosts and bacterial pathogens, and also plays roles in many other metabolic functions. Currently, knowledge is limited on the iron scavenging mechanism of the cell envelope in the aquatic pathogen, +-ATP synthase subunits increased in abundance while iron-cluster proteins, electron transport chain and redox proteins were down-regulated. Further q-PCR validation, in vivo addition of exogenous metabolites, and an enzyme inhibition assay revealed that redox, the energy generation process, and ATP synthase elevated the susceptibility of A. hydrophila to iron starvation.A total of 542 cell envelope proteins were identified by LC-MS/MS, with 66 down-regulated and 104 up-regulated proteins. Bioinformatics analysis showed that outer membrane siderophores, heme and iron receptors, periplasmic iron binding proteins, inner membrane ABC transporters and HA. hydrophila may play critical roles in iron acquisition under conditions of iron-stress. An understanding of the iron scavenging mechanism may be helpful for the development of strategies for preventing and treating A. hydrophila infection.Our study demonstrates that the redox and energy generation process, and ATP synthase in The online version of this article (doi:10.1186/s12866-016-0769-5) contains supplementary material, which is available to authorized users. These include photosynthesis, N2 fixation, methanogenesis, respiration, tricarboxylic acid (TCA) cycle, and DNA biosynthesis [Escherichia coli, in which outer membrane proteins (OMPs), such as CirA, FecA, FepA, BtuB, FhuA, FhuE, and YbiL, serve as receptors of siderophores or heme to transport iron into the periplasmic space [Escherichia coli deliver iron compounds to ATP-binding cassette (ABC) transporters in the cytosolic membrane and then into the cytosol [fur gene family which regulates iron homeostasis [Iron is the second most abundant metal in the earth’s crust; however, ferrous iron, the useable form, is scarce, due to an oxidative environment –3. It isynthesis . To prevynthesis . Therefoic space . Some otic space . During ic space . The per cytosol , 9, 10. eostasis .Aeromonas hydrophila, causes serious disease outbreaks in numerous farmed fish populations, and leads to big economic losses in the aquaculture and fishery industry, annually [of A. hydrophila were compared in the presence/absence of the iron chelating medium by a dimethyl labelling based quantitative proteomic method. Bioinformatics analysis found some important biological processes involved. Furthermore, some of the altered proteins were validated by quantitative polymerase chain reaction (q-PCR) analysis and subjected to related functional validation. We provide the first report, to our knowledge, on the iron homeostasis functions of cell envelope proteins in A. hydrophila.It is well known that the aquatic pathogen, annually –14. HoweA. hydrophila, the strain treated with 150 μM 2, 2′-dipyridyl (DIP) grew slower than the control strain, especially after the middle exponential phase  less than 1 % filtered in the total number of 1024 proteins , 323 inner membrane proteins (31 %), 138 periplasmic proteins (13 %), 71 outer membrane proteins (7 %), 55 extracellular proteins (5 %), and 12 fimbrium proteins (1 %) with some overlapping proteins in the predicted locations Fig. .A. hydrophila under iron-limiting conditions, dimethyl labeling based quantitative proteomic technology was used to analyze the differential expression of envelope proteins. In the 837 identified proteins, 170 membrane envelope proteins were found to be differentially expressed. Taking overlapping locations into account, 104 proteins including 37 OMPs, 50 IMPs, 25 periplasmic proteins and three fimbrial proteins were up-regulated, while 66 proteins including seven OMPs, 39 IMPs and 28 periplasmic proteins decreased in abundance in the iron starvation medium.To further investigate the biological behavior of A. hydrophila presented a down-regulated trend, although its biological function is still unknown. Interestingly, besides the outer membrane siderophore receptors, there were more outer membrane proteins altered, including R4VAF7 , A0KK67 , A0KLX3 , R4VCH3 , R4VRX0 , A0KNY2 , R4VNF2 , A0KHF6  and R4VR45 .Of these altered proteins, seven outer membrane proteins, A0KJP9, R4W0J5, R4VC61, A0KJN3, A0KGW8, R4VA05, and K1JLD4, which serve as ferrienterobactin, ferrichrome, hemin, siderophore and heme receptors, and at less five periplasmic ABC transporters  and ExbB family protein (R4W2T6) increased in abundance. However, OmpW (R4VIJ9) of Meanwhile, seven c-type cytochromes  decreased in abundance. These proteins contained iron ion binding domains suggesting they reduce the use of iron and lead to the decrease of the respiratory electron transfer chain under iron starvation. On the contrary, ATP synthase subunits , the final terminal point of cellular respiration, were found to be up-regulated. While the purpose of this opposite phenomenon is unknown, we hypothesize that it provides an ATP source for ferrous iron scavenging.Proteomics analysis also showed that almost all of the altered proteins located in the extracellular space were up-regulated, such as hemolysin (R4VSQ0 and D2XPP9) and fimbrial protein (R4VSB4) could be explained as an attempt by the cell to strengthen the acquisition of scarce iron ions from outside and try to escape from the threatening surroundings. In addition, many proteases such as Q49KA8 (Serine protease), A0KIU6 (Proprotein convertase P-domain), A0KGX4 , R4VU01 (Hg (II) reductase) and A0KFK2 (glycosidase) were found to increase in extracellular space.A. hydrophila. The BLASTP program was run against the Swiss-Prot database, producing 131 (74.0 %) matches from a total of 170 candidates with blast expected values ≤10−3 using BLAST2GO. Figure To further characterize the altered envelope proteins, a biological functional classification was clustered using bioinformatic analysis. The local Blast2GO software was chosen to maximize homologous gene/protein annotations because of the limitation of Gene Ontology (GO) annotations of +-ATPase subunits, seven genes  were related with the transport process, six genes  were involved in the redox process and three genes  were involved in the energy generation process. Results showed that most of the transcription levels of these 21 related genes were consistent with our proteomics conclusions  were HIn the redox process and energy generation process, the down-regulated mRNA levels of R4V7Y6, R4VNP5, R4VF53, A0KL28, and R4VS58 were in agreement with protein levels, while K1JGN0, A0KQV7 only changed slightly. Interestingly, the transcription of R4VNL8 (ammonia-forming cytochrome c nitrite reductase) and R4VH33 (cytochrome c551 peroxidase) increased, contradictory to the proteomic results. In summary, up to 81 % (17/21) of genes displayed the same directional change in both protein and mRNA levels, 19 % (4/21) showed no correlation, while only 10 % (2/21) were dysregulated in the opposite direction.A. hydrophila to iron starvation, especially α-KA and CA, which both depressed bacterial growth significantly at 8 mM , an inhibitor of ATP synthase. When treated with 20 μg/mL DCCD, the bacterial survival rate was significantly decreased by 7.5, 117.2 and 14.9 fold with the increase of DIP concentrations 150 μM, 200 μM and 300 μM, respectively  extraction used in this study was documented to produce the cleanest extraction of envelope proteins up to date [E. coli, using the same method in the mentioned reference, the 57 % of cell envelope proteins enriched in A. hydrophila, should be acceptable [A. hydrophila.In order to systematically study the iron acquisition mechanism of the bacterial cell envelope system under the external pressure of iron limiting conditions, a dimethyl labeling based quantitative proteomic strategy was used to compare the differential expression of cell envelope proteins in  to date . Compareceptable . Thus, iE coli [E.coli [A.hydrophila under iron-limiting conditions.In the present study, according to the final quantitative proteomic analysis, which can be summarized into the following statements: Firstly, the expression of iron source receptors, which are located in the bacterial outer membrane, induced iron scavenging. These are high-affinity iron chelators and competitively bind iron from iron sources. Actually, under iron deprivation, there are many reports about the upregulation of proteins involved in regular channel forming proteins, such as siderophore receptor channels, CirA, FecA, FepA, FhuA, FhuE, and YbiL in E coli , and oth [E.coli . Those rA. hydrophila as well as in other bacteria [E.coli [A. hydrophila as well. Besides, periplasmic iron binding proteins, such as the heme- and vitamin B12- binding proteins, and the associated cytoplasmic membrane ABC permease transporters mediated the transport of ferri-siderophore complexes across the periplasm and cytoplasm similar to the other fish pathogenic bacteria Edwardsiella ictaluri [Secondly, after the ferri-siderophores and other iron sources specifically bind to outer membrane receptors, an energy generation system for transport is required. Because there was no ATP energy supply in the outer membrane, the ExbB–ExbD–TonB system assisted ferri-siderophores translocation from the outer membrane to the periplasm in bacteria –27. Gene [E.coli . Althougictaluri .E.coli in iron-limited medium [Thirdly, besides the siderophore receptors are located in outer membrane, there were many OMPs differentially expressed under iron starvation. In our previous study, we found that the outer membrane protein R4VIJ9 (OmpW) was down-regulated in d medium . Some otd medium , 31. Becd medium , 33. Addd medium . Since Td medium .E. coli [Staphylococcus aureus [Fur-regulated FsrA may repress expression of glutamate synthase (GltAB), and finally down-regulate TCA cycle intermediate α-ketoglutarate, and DctP which imports succinate and fumarate [Fourthly, bacteria have to reduce their intracellular redox reaction rates because many functional related enzymes are iron-cluster proteins. Thus, the energy generation system and electron respiration chain were depressed under iron starvation. We assume that the down-regulation of the energy generation process takes part in the iron homeostasis mechanism rather than simply stimulation from iron depletion. The down-regulation of TCA cycle and its related proteins under iron starvation was reported in other bacteria species, such as FrdA, FrdB, FrdC and FrdC in E. coli , SdhA, Cs aureus . Smaldonfumarate . Thus, t+-ATP synthase was activated in iron starvation and the inhibition of its activity would increase the susceptibility of A. hydrophila to iron starvation. According to the proteomic results of cell envelope proteins involved in iron starvation, the scheme of iron uptake systems in A. hydrophila is represented graphically in the Additional file Finally, the reduction of the electron respiration chain, to some extent, might relieve the danger of bacterial reactive oxygen species (ROS) and help for survival , 38, 39.A. hydrophila. To our knowledge, this is the first report on the iron homeostasis functions of cell envelope proteins in A. hydrophila by using a dimethyl labeling based quantitative proteomic method. The transmembrane transporter activity and ATP synthase increased significantly in abundance to enhance iron transport and maintain cellular iron homeostasis, while redox processes, precursor metabolites, energy generation processes and other iron-sulfur cluster binding processes decreased significantly in abundance to alleviate the pressure of the iron-limitation, by reducing iron use. In general, this mechanism plays a crucial role in the signal transmission and regulation of the pathogenic bacteria under metal ion limited conditions. A deep understanding of the iron acquisition mechanism of bacteria in the metal ion deficient conditions provided a theoretical support for the invasion mechanism of A. hydrophila disease.The aim of this study was to investigate the iron homeostasis mechanism in the aquatic pathogen, A. hydrophila (ATCC7966) was kindly provided by Prof. Jijuan Cao from Liaoning Entry-Exit Inspection and Quarantine Bureau, Dalian, PR. China. This strain was grown in Luria-Bertani (LB) medium at 30 °C. Iron limitation was conducted by the inclusion of a divalent metal chelator, the ferrous iron binding agent 2, 2′-dipyridyl (DIP)  [In this study, , China) , 45, at A. hydrophila cell envelopes were extracted according to a modified osmotic shock method [2EDTA) was subsequently added to the pellet to extract cell envelope proteins by causing the cells to shrink and release their contents. After incubating on ice for 20 min and additional 20 min in the same volume of ice-cold water, the soluble envelope proteins were separated by centrifugation at 16,000 x g for 30 min at 4 °C. Supernatants were concentrated with Amicon® Ultra-4 centrifugal filter devices with 3 K Da cut offs (Millipore), and then frozen at −80 °C before use.The proteins from the k method . BrieflyA. hydrophila in LB medium with and without 150 μM DIP as the heavy and light isotopes, respectively.The total cell envelope protein concentration was determined using a Bradford assay. Proteins from each sample (100 μg) were reduced with a solution of 200 mM Tris (2-carboxyethyl) phosphine (TCEP) at room temperature for one hour, and alkylated with 25 mM Iodoacetamide (IAA) at room temperature in the dark for 45 min as described previously . TreatedDigested and labeled peptides were analyzed with a Q-Exactive mass spectrometer coupled to an Easy-nLC 1000 HPLC instrument . The peptide mixture was loaded onto an Easy-spray column  and separated with a linear gradient of 3–90 % buffer B (0.1 % Formic acid in acetonitrile) at a flow rate of 250 nL/min over 180 min. Normalized high-energy collision dissociation (HCD) was performed, with the collision energy set at 30 %. The instrument was operated in positive ion mode with an ion spray voltage of 2.3 kV, using a data-dependent top 20 method dynamically choosing the most abundant precursor ions. Isolation of parent precursors was performed with a 300–1800 m/z range and MS/MS was acquired starting at 100 m/z , 50.Aeromonas hydrophila database (download from Uniprot database on Dec.1st. 2014) with the search parameters including alkylated cysteine residue by iodoacetamide, trypsin digestion. Protein quantitation and confidence of the dimethyl labeling quantitative method were performed and evaluated using a highly conservative threshold . The identified proteins with at least two peptides matched and normalized with related peptides in the control were considered for further analysis. Each experiment was performed in at least triplicates and identified in three technical replicate experiments.Fragmentation data were interpreted by Proteome Discoverer version 1.4 against the A. hydrophila database, Blast2GO version 2.7.2 (http://www.blast2go.com/) was used, which is a homology-based approach for gene ontology enrichment analysis [http://www.csbio.sjtu.edu.cn/bioinf/Gneg-multi/#) which combines the gene ontology information and functional domain with the sequential evolution information for Gram-negative bacterial proteins [The Gene Ontology (GO) terms of the differential proteins in this study were analyzed to characterize the related biological functions. To overcome the disadvantage of poor GO annotations in the analysis . The proproteins .GAP-1) was detected as the internal endogenous control, and all samples were normalized to GAP-1 according to a previously described method  according to the manufacturer’s instructions. Isolated RNA samples were eluted in 30 μL RNase-free water and the cDNA was generated from 1 μg total RNA using a PrimeScript ™ RT reagent Kit with gDNA Eraser  after calculations to perform one round of the reverse-transcription reaction. The expression of Glyceraldehyde-3-phosphate dehydrogenase  . The conditions used for q-PCR were 5 s at 95 °C, 34 s at 60 °C, and then 40 cycles of 30 s at 95 °C and melt curve added. Gene expression values were determined by the ∆∆Ct method, which was normalized to the level of l sample . All rea+-ATP synthase activities of A. hydrophila in 0, 150, 200, and 300 μM DIP were measured spectrophotometrically at 340 nm using the H+-ATP synthase assay kit  according to the manufacturer’s protocol, which was based on the oxidation of reduced NADH to NAD+, by the catalysis of lactate dehydrogenase.The HA. hydrophila strain was diluted at 1:100 in 300 μM DIP overnight, with or without 2, 4, and 8 mM oxaloacetic acid (OAA), α-ketoglutarate (α-KA), succinic acid (SA), and citric acid (CA) treatment at 30 °C with shaking at 200 rpm. The cell density was determined via optical density (OD) measurement at 600 nm.The +-ATP synthase inhibition assay, an orthogonal test was further performed. The A. hydrophila strain was diluted at 1:100 in fresh LB medium with a series of DIP overnight from 0, 150, 200 to 300 μM and DCCD from 5, 10, to 20 μg/mL, respectively. After incubation at 30 °C for additional 12 h, cells were diluted appropriately with fresh LB medium for bacterial colony counting, which displayed as color grading according to their survival colony accounting with the control normalized to 100 [For the Hd to 100 .All Statistical analysis was performed by using One-way ANOVA measurement to compare the different treatments with the control sample.2EDTA, TSE buffer; ABC, ATP-binding cassette; BLAST, Basic local alignment search tool; CA, Citric acid; DCCD, N, N’-Dicyclohexylcarbodiimide; DIP, 2, 2′-dipyridyl; FDR, False discovery rate; GAP-1, Glyceraldehyde-3-phosphate dehydrogenase; GO, Gene Ontology; HCD, High-energy collision dissociation; IAA, Iodoacetamide; IMPs, Inner membrane proteins; LB, Luria- Bertani; LC MS/MS, Liquid chromatography tandem mass spectrometry; OAA, Oxaloacetic acid; OD, Optical density; OMPs, Outer membrane proteins; PMF, Proton motive force; q-PCR, Quantitative polymerase chain reaction; ROS, Reactive oxygen species; SA, Succinic acid; TCA, Tricarboxylic acid; TCEP, Tris (2-carboxyethyl) phosphine; TEAB, Triethylammonium bicarbonate; α-KA, α-Ketoglutarate.0.2 M Tris–HCl, pH = 8.0, 0.5 M sucrose, 1 mM Na


Microbacterium terrae KNR 9 was investigated by submerged fermentation. Statistical screening for components belonging to different categories, namely, soluble and raw starches as carbon sources, complex organic and inorganic nitrogen sources, minerals, a buffering agent, and a surfactant, has been carried out for CGTase production using Plackett-Burman factorial design. To screen out k (19), number of variables, k + 1 (20), number of experiments, were performed. Among the fourteen components screened, four components, namely, soluble starch, corn flour, yeast extract, and K2HPO4, were identified as significant with reference to their concentration effect and corresponding p value. Although soluble starch showed highest significance, comparable significance was also observed with corn flour and hence it was selected as a sole carbon source along with yeast extract and K2HPO4 for further media optimization studies. Using screened components, CGTase production was increased to 45% and 87% at shake flask level and laboratory scale fermenter, respectively, as compared to basal media.Cyclodextrin glucanotransferase  production using new alkaliphile Many bacteria can utilize starch as their carbon and energy reserve source. Cyclodextrins (CDs) are produced as a result of intramolecular transglycosylation (cyclization) reaction during degradation of starch by cyclodextrin glucanotransferase  enzyme. Cyclodextrins are cyclic oligosaccharides commonly composed of six, seven, or eight D-glucose units  groups on the outside of the ring molecule with the hydrophobic -CH groups and glycosidic oxygen located inside the cavity of the molecule. Because of this unique property, CDs can form molecular inclusion complexes with range of compounds and hence have found various applications , 3. As aith β-CD . Bacillus, namely, B. circulans 251 [ B. macerans [ B. firmus [ B. cereus [ Bacillus stearothermophilus HR1 [ Bacillus sp. TS1 [ Klebsiella pneumoniae AS-22 [ Paenibacillus sp. F-8 [ Brevibacterium sp. 9605 [ Thermoanaerobacterium thermosulfurigenes EM1 [ Thermoanaerobacter [ Bacillus sp. isolated from a deep sea mud sample for CGTase production. Majority of the CGTases produced by bacteria yield β-cyclodextrin as their major product in a mixture of other CDs in different ratios. Few CGTases have been reported to produce α- and γ-cyclodextrins as their major product along with other CDs [CGTase is an extracellular enzyme, predominantly produced by strains ofmacerans , B. firm. firmus , 9, B. c. cereus , Bacilluilus HR1 , and Bac sp. TS1 . Other kae AS-22 , Paeniba sp. F-8 , and Bresp. 9605 . Thermosenes EM1  and Therrobacter . Georganrobacter  have repther CDs , 15, 19.Production of microbial metabolites can be increased by manipulating nutritional requirements, physical parameters, and genetic make-up of the producing strain . Selectik, where k is the total number of variables). The Plackett-Burman statistical method is a two-level factorial design used to study the effect of k variables in k + 1 experiments [In contrast to this, statistical methods have several merits. They are rapid and reliable for short listings of nutrients, help in understanding the interactions among the nutrients at varying concentrations, and reduce the total number of experiments, resulting in saving of time, glassware, chemicals, and manpower . For opteriments . This meeriments . Microbacterium terrae KNR 9 using Plackett-Burman design.CGTase has been commonly produced by submerged fermentation in media containing various types of starch or other carbohydrates and complex nitrogen sources . Some auβ-Cyclodextrin was purchased from Himedia, Mumbai, India. Soluble starch, yeast extract, and peptone were obtained from Qualigens, India. Phenolphthalein was purchased from Merck India Ltd. Finely grounded corn flour and rice flour were collected from local flour mills and were sieved through fine cheese cloth to get fine uniform raw starch powder. All other chemicals were of analytical grade. Microbacterium terrae KNR 9 by IMTECH, Chandigarh, India (deposited at the same institute as Microbacterium terrae MTCC 8083). It was grown on the basal media containing (g/L) soluble starch, 20; peptone, 5; yeast extract, 5; K2HPO4, 1; MgSO4·7H2O, 0.2; agar, 20; and Na2CO3, 20 (autoclaved separately) at 30°C for 24 h and maintained at 4°C by periodic transfer.The CGTase producing alkaliphilic bacterial culture used in this study was isolated from soil as described by Park et al. . This naThe culture was transferred in a 250 mL flask containing 50 mL inoculum medium (same as above except agar) from a slant culture and incubated at 30°C, 150 rpm for 24 h. Cells were then harvested in sterile centrifuge tubes (15 mL) at 5000 rpm for 10 min. Pellet of cells was resuspended in sterile medium to get an optical density (OD) of 1.0 at 660 nm and used as an inoculum. A 3.0% (v/v) inoculum was transferred to flask containing production medium under aseptic condition.2CO3 in all the media flasks. Inoculated flasks were incubated at 30°C and 150 rpm on a rotary shaker. Media flasks were harvested after 72 h, cells and suspended particles were removed by centrifugation, and the supernatant was used to determine CGTase activity.Fourteen components of different categories were included in the production medium at two levels . 100 mL μL approximately diluted enzyme was incubated with 1.0 mL of 50 mg soluble starch in sodium phosphate buffer  at 60°C for 30 min. The reaction was stopped by quickly cooling the tubes on ice. Four milliliters of working phenolphthalein solution was added, the tubes were vortexed, and the absorbance of the mixture was immediately measured at 550 nm. The working phenolphthalein solution was made by adding 1 mL of phenolphthalein stock (4 mM in ethanol) to 100 mL of 125 mM Na2CO3 prepared in 4% ethanol. The standard β-cyclodextrin estimation was also carried using the same method. One unit was defined as the amount of enzyme that produced one μmole of β-cyclodextrin per min.CGTase activity was determined by phenolphthalein assay method described by Goel and Nene  with minPlackett-Burman design was used to evaluate the relative importance of various media components including soluble and raw starches, organic and inorganic nitrogen sources, a phosphorous source/buffering agent, minerals, and a surfactant for CGTase production in submerged fermentation. This design was chosen to screen out the important media components with respect to their main effects and not the interaction effects.k + 1, where k is number of variables (media components). For total of nineteen variables (k = 19) including fourteen media components (assigned variables)  is concentration effect of the variable, Mi+ and Mi− are the CGTase production of the trials, where the variable (Xi) estimated is present at its higher and lower concentrations, respectively, and N is the number of trials (20). Experimental error was estimated by calculating the variance of dummy variables as follows:ED is the concentration effect of the dummy variable, and n is the number of dummy variables. The standard error (SE) of the concentration effect was the square root of the variance of dummy variables and the significance level  of each concentration effect was determined using Student's t-test:E(Xi) is the effect of variable Xi.In Plackett-Burman design, the total number of experiments to be carried out is riables)  and fiveriables) . The incStatistical confidence level of each variable was calculated as p = 0.05 corresponds to a statistical confidence level of 95% and hence any component showing a statistical confidence level higher than 95% was considered as significant.A value of 2HPO4 was added as a buffering agent. Three minerals MgSO4·7H2O, CaCl2·2H2O, and FeSO4·7H2O and a surfactant Tween-80 were included in the study as medium components.To formulate economic CGTase production media, fourteen components of different categories were screened using the Plackett-Burman design. Among the carbon sources, soluble starch and raw organic carbon sources like corn flour and rice flour were selected for the study. Organic and inorganic nitrogen sources like peptone, yeast extract, soybean meal, casein hydrolysate, ammonium sulphate, and ammonium nitrate were added in the media to test their significance. KE), standard error (SE), t-value, p value, and statistical confidence level (%) of each component and is represented in CGTase production in 20 experimental runs at 30°C, 150 rpm, after 72 h is represented in 2HPO4, MgSO4·7H2O, CaCl2·2H2O, FeSO4·7H2O, and Tween-80 are having positive concentration effect on CGTase production, whereas peptone, (NH4)2SO4, and (NH4)NO3 are having negative concentration effect. Among the components showing positive concentration effect soluble starch, corn flour, yeast extract, and K2HPO4 have shown confidence level of more than 95% and hence were considered as significant. Rest of the components, namely, rice flour, soybean meal, casein hydrolysate, MgSO4·7H2O, CaCl2·2H2O, FeSO4·7H2O, and Tween-80, were insignificant, having confidence level below 95%.As revealed from  B. firmus during raw tapioca starch degradation. Significance of sago starch as carbon source has been reported using B. stearothermophilus HR1 [ Bacillus sp. TSl-1 [ Mic. terrae KNR 9 was able to utilize various starches and among them corn flour has shown the confidence level of 97.48% which is as good as soluble starch (98.06%). Therefore, soluble starch and corn flour both were included as a sole carbon source in further media optimization studies.Starch is the most important carbon source commonly used for the CGTase production. Goel and Nene  have repilus HR1  and Bacip. TSl-1 . Optimizp. TSl-1 . Howeverp. TSl-1 . In our  Klebsiella pneumoniae AS-22. Yeast extract showed an appreciable effect on CGTase production, presumably due to the presence of some essential nutrients or inducers. Soybean meal and casein hydrolysate showed the positive effect but were found insignificant below 95% confidence level. Peptone was the only organic nitrogen source showing the negative concentration effect. Inorganic nitrogen sources (NH4)2SO4 and (NH4)NO3 were having very high negative concentration effect and can be excluded during further studies. Interestingly no growth of Mic. terrae KNR-9 was observed when inorganic nitrogen salts were supplied as a sole nitrogen source (data not shown). Thatai et al. have reported the repression of CGTase production in presence of ammonium salts [ B. circulans DF 9R [Among the complex organic nitrogen sources tested, yeast extract was found to be most significant component contributing to the synthesis of CGTase at a confidence level of 98.82%. Gawande and Patkar  have repum salts . Howeverns DF 9R .2HPO4 was found significant at 97.87% confidence level, as it is a good buffering agent as well as phosphorus source. Ibrahim et al. have reported the importance of K2HPO4 in the media for CGTase production by Bacillus sp. G1 [Ks sp. G1 .4·7H2O, CaCl2·2H2O, and FeSO4·7H2O were below 95% and hence were considered as insignificant. MgSO4·7H2O has been reported as an essential mineral element for CGTase production [ B. circulans DF 9R [Confidence levels of minerals like MgSOoduction . Importans DF 9R . In our ns DF 9R . A surfans DF 9R .2HPO4, 1; and Na2CO3, 20 (autoclaved separately) at shake flask level (50 mL medium in a 250 mL flask) and laboratory scale fermenter (2.5 lit medium in a 5 lit fermenter vessel). At shake flask level, maximum CGTase production of 3.36 U/mL and 4.87 U/mL was obtained at 30°C, 150 rpm, after 72 h with the basal medium and screened components confirming medium, respectively : corn flour, 20; yeast extract, 10; Kectively . In the ectively . Laborat Eremothecium ashbyii [β-amylase and pullulanase has been reported with Clostridium thermosulfurogenes SV2 in solid state fermentation [ Colletotrichum coccodes [Use of Plackett-Burman design has been reported for screening of media components for production of lactic acid , 33, alp ashbyii . Using tentation . Apart fcoccodes  and desicoccodes . Mic. terrae KNR-9. Application of this design has not only helped in short listing and selecting the best variables such as soluble starch, corn flour, yeast extract, and K2HPO4 for CGTase production but also ensured the better CGTase production. The results of this study have scope for improvement in CGTase production by further optimization of the most significant components.Thus the present study shows successful application of Plackett-Burman design for statistical screening of media components and selection of the significant media components for CGTase production by a newly isolated alkaliphile


FOXP3) methylation of regulatory T cells (Tregs) and relative telomere lengths (RTLs) were investigated in a total and subsamples of 131 patients with panic disorder as compared to 131 age- and sex-matched healthy controls in order to test for a potential dysfunction and premature aging of the immune system in anxiety disorders. Significantly lower TRECs (p = 0.004) as well as significant hypermethylation of the FOXP3 promoter region (p = 0.005) were observed in female  patients with panic disorder as compared to healthy controls. No difference in relative telomere length was discerned between patients and controls, but significantly shorter telomeres in females, smokers and older persons within the patient group. The presently observed reduced TRECs in panic disorder patients and FOXP3 hypermethylation in female patients with panic disorder potentially reflect impaired thymus and immunosuppressive Treg function, which might partly account for the known increased morbidity and mortality of anxiety disorders conferred by e.g. cancer and cardiovascular disorders.Immunological abnormalities associated with pathological conditions, such as higher infection rates, inflammatory diseases, cancer or cardiovascular events are common in patients with panic disorder. In the present study, T cell receptor excision circles (TRECs), Forkhead-Box-Protein P3 gene ( Anxiety disorders are among the most common mental health disorders in Europe in 2010 and confer a high individual and socioeconomic burden , i.e. exPremature immunosenescence and a diminished regulatory T cell (Treg) function are discussed as etiopathological factors driving the immune system towards inflammatory diseases –17: AginMeasurement of telomere lengths helps to estimate the individual replication history of cells. Telomeres are protective caps at the end of chromosomes and shorten with each cell cycle . IndirecFOXP3 promoter and enhancer regions .–37.FOXP3 disease . Anti-CD disease . Develop disease –42.FOXP3 methylation and relative telomere lengths (RTLs) were for the first time concurrently investigated in patients with panic disorder as compared to matched healthy controls in order to test for potential dysfunction and premature aging of the immune system in anxiety disorders. It was hypothesized that panic disorder would be associated with impaired thymus function as reflected by reduced TRECs as well as by FOXP3 hypermethylation resulting in reduced immunosuppressive Treg function and accompanied by lower relative telomere length.In the present study, T cell receptor excision circles (TRECs), One hundred and thirty-one patients with panic disorder and 131 healthy, age- and sex-matched controls, recruited at the Department of Psychiatry and Psychotherapy, University of Muenster, Germany, were included into the study . DiagnosThe study was approved of by the ethics committee of the University of Muenster, Germany, written informed consent was obtained from all participating subjects, and the study was conducted according to the ethical principles of the Helsinki Declaration.DNA was extracted from separated whole EDTA blood using the FlexiGene DNA Kit  according to the manufacturer's instructions. Signal-joint TREC concentrations were determined by PCR as described in detail previously ,44. RecoDetermination of relative telomere length (RTL) was performed by calculating the ratio of a quantitative PCR reaction product from the same sample using specific primers for telomeres and a single copy gene as described previously –47.FOXP3 promoter  and FOXP3 5’ upstream enhancer  (both ensemble releaser 15 February 2014), were designed with the PyroMark Assay Design software . Primers and sequences to analyze are listed in FOXP3 is located on the X-chromosome (Xp11.23), resulting in hemizygosity in male subjects and random inactivation of the second X-chromosome in females, subsequently methylation data was stratified according to sex. Bisulfite conversion of DNA was performed using the EpiTect 96 Bisulfite Kit  according to the manufacturer’s protocol. PCR amplifications were performed on a Tetrad 2 cycler  with an initial denaturation step at 95°C for 5 min, 40 cycles of 95°C for 30 s, primer-specific annealing temperature of 60°C for 30 s, 72°C for 45 s, and a final extension step at 72°C for 10 min. The reaction mixture consisted of 2.5 μl 10x PCR buffer with MgCl2, 0.5 μl 10 mM dNTP mix, 1.25 μl of each forward and reverse primer , 0.2 μl  Taq DNA polymerase , 18.3 μl PCR-grade water, and 1 μl template DNA (75 ng). PCR products were visualized by electrophoresis on 1.5% agarose gel.Assays quantifying the methylation levels of CpGs in the target regions, i.e. Bisulfite pyrosequencing was performed on a PyroMarkTMQ96 MD Pyrosequencing System with the PyroMark Gold Q96 CDT Reagent Kit . Our experimental methodology relied on simultaneous treatment of control and study samples in order to avoid batch effect and technical variability which is estimated around 1–2% of this assay. Bisulfite conversion, PCR amplifications and pyrosequencing of control and study samples were performed together. The sequences analyzed by bisulfite pyrosequencing are listed in Shapiro-Wilk test was used to test for normal distribution, before applying Student’s t-test for normally-distributed and non-parametric Mann-Whitney-U for not normally-distributed independent variables. Correlations between variables were identified by Spearman's rank correlation coefficient. A generalized linear multivariate regression model was generated by stepwise regression to infer the influence of gender, depression, medication with antidepressants and smoking, adjusted for the chronological age  of the patient. Step-down exclusion was performed by excluding variables with the standardized regression coefficient closest to 0 until a significant regression model was generated. Disease duration was excluded from the regression model due to strong correlation with age . A p-val2 = 0.051, p = 0.045) including age (p = 0.086) and gender (p = 0.468) revealed that having panic disorder (p = 0.016) was an independent factor for lower TRECs. Slightly lower TRECs were seen in patients treated with antidepressants compared to those without any medication (p = 0.024). Performing regression analysis  in patients only, age (p = 0.031) was an independent factor for lower TRECs, whereas gender (p = 0.162), smoking (p = 0.727), and comorbid depression (p = 0.925) or antidepressants (p = 0.483) had no significant association.Patients (mean 0.49 ± 0.81) showed significantly lower TRECs/1,000 cells (mean 0.99 ± 1.90) compared to healthy controls (HC) (p = 0.004), which was due to significantly lower TRECs in female patients . In patiFOXP3 promoter methylation across all five CpGs as well as particularly at sequence 1, CpG 1, and sequence 2, CpGs 2 and 3 was discerned in female patients compared to HC  revealed that having panic disorder was an independent factor for higher FOXP3 methylation across all five CpGs (p = 0.020) in female patients. Lower methylation across all five CpGs was seen in patients on antidepressants (mean methylation 75.94 ± 2.63%) compared to those without any medication (77.78 ± 1.98%) (p = 0.014), with high significance at FOXP3 promoter sequence 2, CpG 4 . Using a regression model for methylation across all five CpGs  in female patients, antidepressants (p = 0.004) and age (p = 0.027)  showed the strongest associations, with lower FOXP3 promoter methylation correlating with antidepressant medication and higher age. No significant difference between female patients and female HC could be demonstrated for FOXP3 enhancer methylation  compared to non-smoking male patients (0.78 ± 1.19%) (p = 0.030). In male patients, significantly higher methylation was determined at FOXP3 enhancer, sequence 2, CpG 4 (0.84 ± 0.70%) and 5 (1.16 ± 1.33%) in the case of depression compared to male patients without depression (position 4 (0.51 ± 0.37%) and 5 (0.51 ± 0.33%); p = 0.039 and p = 0.006, respectively). Similarly, higher methylation was seen at FOXP3 enhancer, sequence 2, CpG 5 in male patients on antidepressants (1.15 ± 1.35%) compared to those without antidepressants (0.53 ± 0.31%) (p = 0.013). Methylation at FOXP3 enhancer, sequence 2, CpG 4 highly correlated with methylation at FOXP3 enhancer, sequence 2, position 5 . Multiple regression analysis  showed that methylation at FOXP3 enhancer, sequence 2, CpG 5 was weakly influenced by smoking (p = 0.067) and older age (p = 0.073) but not by depression (p = 0.267) or antidepressants (p = 0.868).In males, s and HC . SmokingFOXP3 promoter, sequence 2, CpGs 2  and 3  correlated with shorter telomeres in female patients.Relative telomere lengths (RTLs) were not different between patients and HC. However, within the patient group, smokers had significantly shorter telomeres (0.91 ± 0.30) compared to non-smokers (1.07 ± 0.37) (p = 0.018) and females (0.96 ± 0.34) had shorter telomeres than males (1.10 ± 0.32) (p = 0.017), although age and distribution of females were not significantly different between smokers and non-smokers. Stratifying for female patients aged ≥35 years, difference in RTLs was significant between smokers (0.84 ± 0.32) and non-smokers (1.12 ± 0.34) (p = 0.010). In patients, regression analysis  identified smoking (p = 0.004), female gender (p = 0.011) and age (p = 0.041) as factors associated with shorter telomeres, whereas depression (p = 0.106) and antidepressants (p = 0.446) were less important. Interestingly, hypermethylation at FOXP3 promoter region in female patients with panic disorder as compared to healthy controls. No difference in relative telomere length was discerned between patients and controls, but significantly shorter telomeres in females, smokers and individuals aged ≥35 years of the patient group.The present study demonstrated significant lower TRECs in both female and male panic disorder patients as well as significant hypermethylation of the Whereas diminished thymic function and dysfunction of immune regulation by FoxP3+ Tregs is well recognized in stress-induced depression in mice ,48, lessFOXP3 promotor region—potentially resulting in reduced immunosuppressive Treg function—in female but not in male patients with panic disorders, corroborates the idea of a prematurely aged immune system in this particular patient subgroup. Although a strong sex bias of autoimmunity, with most autoimmune diseases predominantly affecting females, is well known [FOXP3 promoter [Diminished thymic function has been associated with inflammatory diseases ,14,17 dull known ,52, the promoter ,54 and lpromoter .FOXP3 promoter region. An association between age and FOXP3 hypomethylation with increased immunosuppressive Treg function has been suggested by a recent study in mice [FOXP3 demethylation could constitute a molecular correlate of beneficial effects of antidepressants on the immune system in panic disorder. This notion is supported by first therapy-epigenetic studies showing dynamic methylation changes after successful antidepressant or even psychotherapeutic treatment in depression or anxiety disorders [FOXP3 enhancer region in smokers as well as in patients with comorbid depression compared to non-smoking patients or patients without depression, respectively. Antidepressants were associated with higher methylation at the same CpG position. However, given only minor FOXP3 enhancer methylation in general  contributing to an immune-risk-phenotype ,60, may e models –64 and oe models . Althougphocytes ,65. The y others .FOXP3 enhancer region was found on activated and only transiently FoxP3-expressing T cells with impermanent change of methylation status [FOXP3 promotor regions may contribute to the methylation results. Another important limitation to mention is the fact that bisulfite sequencing cannot discriminate between 5-methylcytosine and 5-hydroxymethylcytosine. Therefore, the output from bisulfite sequencing cannot solely be interpreted as showing only 5-methylcytosines, but it could also include the 5-hydroxymethylcytosines. 5-hydroxymethylcytosine has been postulated to play an important role in the process of demethylation [One limitation of our study is that lymphocytes were not separated into CD25highCD4+ T cells, defining mainly nTregs, and in naive and other T cell subpopulations, as a differentiated methylation pattern at the n status . This mahylation , where 5Regarding the RTL data, it has to be taken into account that several factors could influence the outcome such as current inflammatory state as measurable by C-reactive protein levels, anti-inflammatory medications, paternal age, menopause, exercise, diet or childhood trauma. However, unfortunately, these data were not available and thus, have not been corrected for in the present study. Mechanisms and directions of interaction between anxiety and immune function as well as their relation to an increased risk of mortality and morbidity, also considering early as well as recent life events need to be further evaluated in longitudinal studies.FOXP3 hypermethylation in patients with panic disorder potentially reflecting impaired thymus and immunosuppressive Treg function. From the present results, we expect that female and older patients with panic disorder may show particularly strong effects regarding immunosenescence and its role in development of age-associated diseases, such as cardiovascular events, autoimmune disorders, cancer and infectious rates accounting for the known increased morbidity and mortality of anxiety disorders. Targeted prevention and early treatment of anxiety disorders could therefore aid in mitigating their detrimental effects on the immune system and thereby in lowering the risk of diseases associated with age such as coronary heart disease, cardiovascular death and cancer.In summary, the present study noted reduced TRECs in panic disorder patients compared to controls as well as


Vmax, complexes I, III, and IV activities), Vsucc , and Vtmpd, (complex IV activity), together with mitochondrial coupling (Vmax/V0) were determined in controls conditions and after exposure to 250, 300, and 350 μg/ml Fe3O4 in young and middle-aged rats. In young liver mitochondria, exposure to IONPs did not alter mitochondrial function. In contrast, IONPs dose-dependently impaired all complexes of the mitochondrial respiratory chain in middle-aged rat liver: Vmax , Vsucc , Vtmpd  using Fe3O4 350 µg/ml. Mitochondrial coupling also decreased. Interestingly, 350 μg/ml Fe3O4 in the form of Fe3+ solution did not impair liver mitochondrial function in middle-aged rats. Thus, IONPs showed a specific toxicity in middle-aged rats suggesting caution when using it in old age.We examined the effects of iron oxide nanoparticles (IONPs) on mitochondrial respiratory chain complexes activities and mitochondrial coupling in young (3 months) and middle-aged (18 months) rat liver, organ largely involved in body iron detoxification. Isolated liver mitochondria were extracted using differential centrifugations. Maximal oxidative capacities ( Because of their unique properties, some nanoparticles have been approved for clinical use like iron oxide nanoparticles (IONPs). IONPs hold immense potential in a vast variety of biomedical applications such as magnetic resonance imaging (MRI), targeted delivery of drugs or genes, tissue engineering, targeted destruction of tumor tissue through hyperthermia, magnetic transfections, iron detection, chelation therapy, and tissue engineering –4. ReporHowever, nanoparticles pose a high health risk because of their ability to reach every part of the organs and tissues and their interaction with cellular functions.Concerning NP clearance, it is known that they are primarily phagocytozed by macrophages in the liver (Kupffer cells) . Thus, tMitochondrial involvement in IONPs toxicity remains controversial and either no deleterious effects , 14 or m3O4 nanoparticles, we recently reported a lack of IONPs toxicity in liver mitochondria in young rats [Concerning Feung rats . Neverthung rats . More reung rats  reported3O4 nanoparticles  on young and middle-aged liver mitochondrial respiratory chain complexes activities and on mitochondrial coupling of phosphorylation to oxidation.Since, to date, no study investigated the potential effects of iron oxide nanoparticles on middle-aged mitochondria and since the liver is a key organ in iron and NPs detoxification, we investigated and compared for the first time the effects of three different concentrations of Fe3O4) nanoparticles, an appropriate amount of iron (II) acetate precursor was added to a given volume (125 mL) of DEG to reach nominal iron cations concentration of 0.2 M. The mixture was then refluxed at a rate of +6°C min−1under mechanical stirring up to boiling point and then maintained at this temperature for about 2 h. The powders were washed several times with ethanol and then with acetone under ultrasonication with intermittent centrifugation and then dried in air at 50°C [Iron oxide nanoparticles were acquired from Unit of Research UR11ES30, Faculty of science of Bizerte, Tunisia. They were prepared by the polyol process starting from iron (II) acetate as metal precursor and diethylene glycol (DEG) as solvent purchased from ACROS Organics. All chemicals were used as received without further purification. Deionized water was used in these preparations. For the synthesis of the magnetite  operating at 100 kV equipped with an energy dispersive spectrometer (EDX) .3O4 nanoparticles showed that the powders are constituted by roughly spherical almost nonagglomerated particles. In addition, about 250 particles have been counted for the average particle size and histogram determinations. The calculated average diameter was 9 nm with a SD of 1.6 nm  and middle-aged  Wistar male rats were housed in a thermoneutral environment (22 ± 2°C), on a 12 : 12 h photoperiod, and were provided food and water ad libitum. This investigation was carried out in accordance with the Guide for the Care and Use of Laboratory published by the US National Institute of Health and approved by the institutional animal care committee .Young  in an induction chamber . Anesthesia was maintained with 1.5% isoflurane and 1 L/min oxygen at under spontaneous ventilation. A midline laparotomy was performed and the liver was excised, cleaned, and then immediately used for the study of respiratory parameters. per se.Similarly, three additional Wistar rats aged 18 months were studied, in order to investigate the potential effect of iron oxideAll operations were carried on ice. A piece of tissue was placed into buffer A containing 50 mM tris, 1 mM EGTA, 70 mM sucrose, 210 mM mannitol, and pH 7.40 at +4°C. Tissue was finely minced with scissors, placed in buffer A, and homogenized with a Potter-Elvehjem. Then, the homogenate was centrifuged at 1300 ×g for 3 min and 4°C. The supernatant was centrifuged at 10,000 ×g for 10 min and 4°C to sediment mitochondria. Finally, the mitochondrial pellet was washed twice and then suspended in 50 mM tris, 70 mM sucrose, 210 mM mannitol, and pH 7.4 at +4°C. Protein content was routinely quantified with a Bradford assay using bovine serum albumin as a standard . Mitocho3O4  during 30 min at +25°C.The iron oxide nanoparticles were mixed with a solution of NaCl 9%. The mixture was then stirred vigorously and sonicated for 60 min to break up aggregates. Particle suspensions were vortexed immediately before each use. Before measurement, 3 mL of solution M containing 100 mM KCL, 50 mM mops, 1 mM EGTA, 5 mM Kpi, and 1 mg/mL bovine serum albumin (BSA) was added to the oxygraph chambers for 10 min. Then, 0.50 mg of mitochondrial protein was placed in the oxygraph chambers with 10 mM glutamate and 2.50 mM malate as substrates. The temperature was maintained at +25°C. Isolated liver mitochondria were incubated with different concentrations of Fe3+ solution with concentration of 350 μg/mL Fe3O4. To obtain free Fe3+ ions a precise mass of Fe3O4 nanoparticles was dissolved in a small volume of concentrated HCl under gentle heating [ per animal, and thus number of data was six.To discriminate if the results obtained might be due to the size or to a general response pattern in front of iron oxide exposure, we submitted middle-aged liver mitochondria to a Fe heating . Two expVmax) was measured by adding adenosine diphosphate (ADP). When Vmax was recorded, electron flow went through complexes I, III, and IV. Then complex I was blocked with amytal (0.02 mM) and complex II was stimulated with succinate (25 mM). Mitochondrial respiration in these conditions allowed determining complexes II, III, and IV activities (Vsucc). After that, N, N, N′, N′-tetramethyl-p-phenylenediaminedihydrochloride  and ascorbate (0.50 mM) were added as artificial electron donors to cytochrome c. In these conditions, the activity of cytochrome c oxidase (complex IV) was determined as an isolated step of the respiratory chain (Vtmpd) [Maximal oxidative capacity ((Vtmpd) .Vmax) over basal respiration (without ADP) with glutamate and malate as substrate (V0), as previously reported by Mansour et al. 2012 [Mitochondrial coupling (coupling of phosphorylation to oxidation) was determined by calculating the acceptor control ratio (ACR), ratio between ADP-stimulated respiration .Results are expressed as mean ± SEM. Statistical analyses were performed using one-way ANOVA followed by a Tukey posttest. The unpaired μmol O2/min/g protein, respectively). This holded true for Vsucc (young: 35.0 ± 3.7 versus 33.9 ± 1.7 μmol O2/min/g protein) and for Vtmpd (young: 50.0 ± 6.2 versus 43.0 ± 1.6 μmol O2/min/g protein).As shown in P = NS).Finally, mitochondrial coupling was similar in both young and middle-aged rats  was not modified as compared to the control group  (protein) .Complexes II, III, and IV Activities. Vsucc was unchanged whatever the dose of IONPs  (protein) .Complex IV Activity. Vtmpd, reflecting complex IV activity, was not modified after IONPs treatment as compared with control group  (protein) .3O4 nanoparticles, whatever the doses used, failed to alter any of the liver mitochondrial respiratory chain complexes activities in young rats.Taken together, these data support that FeVmax, Vsucc, and Vtmpd decreased significantly. When the middle-aged liver rats were exposed to different concentrations of IONPs, the Complexes I, III, and IV Activities. The maximal oxidative capacities, Vmax, reflecting I, III, and IV activities significantly decreased whatever the dose of IONPs. At 250 μg/mL, Vmax was decreased as compared to the control group . Vmax at 300 and 350 μg/mL were also decreased as compared to control values .Vmax decrease was greater when using higher IONPs concentration. Thus Vmax was significantly lower after Fe3O4 350 μg/mL as compared to 300 and 250 μg/mL . The statistical significance was reached at 300 μg/mL and 350 μg/mL  and Fe3O4 350 μg/mL  ( < 0.01) . Complex IV Activity. Vtmpd, reflecting complex IV activity, decreased significantly after exposure to Fe3O4 250 μg/mL as compared to the control group . Similarly, Vtmpd, decreased when exposed to 300 μg/mL of Fe3O4  and to 350 μg/mL  (< 0.001) .Vmax/V0), representing the degree of coupling between oxidation and phosphorylation, was not changed after IONPs treatment .In young liver, the acceptor control ratio . Moreover, ACR was also decreased at 300 μg/mL  and at 350 μg/mL   .μg/mL Fe3O4, Vmax (41.90 ± 3.93 versus 38.08 ± 3.77 μmol O2/min/g protein), Vsucc (44.23 ± 3.443 versus 41.42 ± 3.303 μmol O2/min/g protein), Vtmpd (84.90 ± 5.75 versus 70.08 ± 4.71 μmol O2/min/g protein), and ACR (7.70 ± 1.15 versus 8.72 ± 1.06) were not significantly modified . Indeed, liver mitochondrial respiration chain complexes I, II, III, and IV activities are altered whatever the concentration of FeYoung and Middle-Aged Rats Present with Similar Baseline Mitochondrial Respiratory Chain Complexes Activities in the Liver. Several studies indicate that mitochondria are one of the major sources of reactive oxygen species (ROS) and, in turn, are the most adversely affected organelles during aging [ng aging . In factng aging . Oxidating aging , which ang aging , 30.Accordingly, in rat liver, previous reports have found aging-related decrease in electron transfer activity in complex I or complexes I and IV in young (4 months) versus old (30 months) rats –34. On tThis is consistent with our results and might be explained by differences in age range or animals being only middle-aged.Iron Oxide Nanoparticles Impair Liver Mitochondrial Respiratory Chain Complexes Activities in Middle-Aged but Not in Young Rats. Concerning young rats, iron oxide nanoparticles failed to impair any of the liver mitochondrial respiratory chain complexes activities. Thus, mitochondrial oxygen consumption in young liver was not altered by IONPs and, similarly, mitochondrial coupling remained in the normal range after Fe3O4 exposure. This is in agreement with our previous findings [findings .μg/mL significantly decreased Vmax, Vsucc, and Vtmpd corresponding together to complexes I, II, III, and IV activities of the mitochondrial respiratory chain. IONPs exposure also decreased mitochondrial coupling.To the best of our knowledge, no data are available concerning potential related effects of high levels of iron oxide nanoparticles and age on mitochondria. This is particularly interesting since the liver is a major iron storage organ , 37. VerSeveral mechanisms might explain these results like increased fragility of older mitochondria and iron accumulation.First, the fragility of mitochondria seems to increase in function of age. Thus, studies reported that mitochondria isolated from the organs of aged animals are also aged in terms of cytosolic and mitochondrial oxidative stress and losses of enzymatic activities . AccordiSimilarly studies investigated the effects of iron accumulation on mitochondrial integrity and function with age , 41, 42.3O4 NPs with cytochrome c leads to the reduction of the protein. Moreover, [3O4 NPs and at high concentration (1 mg/mL) the cytochrome c protein expression decreased dramatically. Accordingly, [3O4 NPs caused the highest toxic effects on green alga, as compared to other types of NPs.Particularly, in accordance with our data of a NPs-related impairment of complex IV,  demonstroreover,  using hurdingly,  observed3O4 nanoparticles liver toxicity and one might propose that a threshold of toxicity might play a key role. Indeed, as reported by Bakala et al. [Taken together, these data confirm that age likely modulates Fea et al.  and poss3O4 nanoparticles liver toxicity.However, whether the observed effects are specific of iron oxide in its particulate form or is independent of its size is not known. We therefore submitted old liver mitochondria to iron oxide but not in its particulate form and, interestingly, no deleterious effect was observed. Thus, it is likely that age specifically modulates FeIn summary, we demonstrate for the first time that old rats are more susceptible to IONPs nanoparticle exposure in terms of liver mitochondrial respiration and coupling. These age-related changes in liver mitochondrial respiratory chain activity should perhaps be taken into consideration in preclinical and clinical studies of particulate contrast agents.


Human CRC HCT-116 cells were implanted in BALB/c nude mice; mice with xenografted tumors were randomly administrated vehicle (control), 20, 40, or 80 mg/mL VB, or 1 mg/mL fluorouracil (5-FU). HIPK2, p53, Bax, and Bcl-2 expression in these tumors were determined by IHC. IP < 0.05). In vivo, the inhibition rates of 20, 40, and 80 mg/mL VB on CRC xenograft tumor weight were 42.79%, 53.90%, and 60.99%, respectively, and were accompanied by increased expression of HIPK2, p53, and Bax, and decreased Bcl-2 expression in treated tumors. In vitro, VB significantly inhibited proliferation of CRC cell lines HCT-116, HT-29, LoVo, and SW620, in a time- and dose-dependent manner. The apoptosis rates of 25, 50, and 100 μM VB on HCT-116 cells were 10.83 ± 1.28, 11.25 ± 1.54, and 20.19 ± 2.87%, and on HT-29 cells were 18.92 ± 6.12, 21.57 ± 4.05, and 25.14 ± 6.73%, respectively. In summary, VB treatment significantly enhanced the protein expression of pro-apoptotic HIPK2, p53, p-p53, Bax, and decreased anti-apoptotic Bcl-2 expression in CRC cells.IHC analysis for 100 human CRC tumor samples and 20 normal intestinal tissues, showed HIPK2 expression to inversely correlate with Dukes stage and depth of invasion in CRC (HIPK2 protein modulates the phosphorylation status of p53, and levels of Bax and Bcl-2 in CRC. We also found that VB effectively activated the HIPK2–p53 signaling pathway, resulting in increased CRC cell apoptosis. Colorectal cancer (CRC) is one of the most common malignancies in the world. With economic development and lifestyle changes, the incidence of CRC has been increasing yearly, with a significant rising rate. According to Global Cancer Statistics 2011, the incidence of CRC ranked third among male cancer patients and second among female cancer patients. In 2011, people who died from CRC accounted for 8% of all cancer deaths. It is the fourth most common cause of cancer death .Various factors contribute to CRC development, including intestinal mucosa losing normal growth control at the genetic level, leading to cell hyperproliferation. Most recent investigations of CRC tumorigenesis have therefore focused on functional abnormalities of relevant genes and their products.Homeodomain Interacting Protein Kinase 2 (HIPK2) is a member of the serine/threonineproteinkinase family, located inside the cell nucleus. It is a transcription mediator that interacts with homobox plastein. Reportedly, HIPK2 is associated with late embryogenesis, and neural, retinal, and muscle tissue development, and also participates in various aspects of tumorigenesis, including oncogene expression , apoptosHIPK2 knockdown in colon cancer cells led to the loss of target gene activation of wild-type p53 × 100%; where OD0 was the absorbance value of the blank group, ODC the control group, and ODN groups with different doses of VB. The IC50 of VB was calculated using three independent experiments.Human CRC HCT-116, LoVo, HT-29, and SW620 cells in logarithmic growth phases were plated at 5 × 10Rapid growing HCT-116 and HT-29 cells were treated with VBat different concentrations  for 48 h. Cells were then stained with 2 μL Annexin-V and 2 μL PI in 50 μL of apoptosis reaction solution at 4°C for 30 min. FACScan flow cytometry was used to detect apoptotic cells. Cell debris in different quadrants was calculated statistically. Cells in the upper right quadrant represented early apoptosis; cells in the lower right quadrant represented late apoptosis.6/mouse) were injected subcutaneously into the right axilla of nude mice. Ten to 14 days later, when tumors formed, the nude mice with good growth state and unbroken tumors were used as tumor supply mice, and were then sacrificed. Tumors were dissected out under aseptic conditions, with necrotic and fibrous tissues removed. Fresh parts on the edge of tumors were cut into 1-mm3tumor blocks, which were implanted under the axillar skin of the right front legs of nude mice. With this method, three generations of mice were produced. The third-generation mice with unbroken transplanted tumor and sound growth state were sacrificed, and using the above-described method, the tumors were re-implanted and when they reached a size of 50–100 mm3, the tumor-bearing mice were randomly divided into five groups (six mice for each group): the control group , the low-, medium-, and high-dose VB groups  and the fluorouracil (5-FU) group (1 mg/kg/day). VBand 5-FU were administered by tail vein injection. At days 1, 4, 7, 10, and 14, the long diameter (a) and the short diameter (b) of each tumor was measured, and tumor volume was calculated as [(a × b2)/2]. After 14 days of treatment, mice were sacrificed and their tumors were dissected and connective tissues were removed. The tumors were weighed. We then calculated the tumor volume inhibition rates [ × 100%]; and the tumor weight inhibition rates [ × 100%].HCT-116 cells  phosphorylation is critical to transcription of apoptosis-related genes. HIPK2 overexpression stabilizes and activatesp53 and promotes its binding to form the HIPK2–p53 complex, leading to Ser46 phosphorylation and increased apoptosis [As a newly found auxiliary transcription inhibition factor, HIPK2 has been suggested to affect many aspects of cancer. Studies showed that HIPK2 participates in a variety of signal transduction pathways, including p53 , Wnt/β-cpoptosis .We conducted a retrospective analysis on 100 primary CRC tumor samples, and found that the average age of CRC diagnosis was 67.25 ± 11.91 years, which did not significantly vary by sex. Common symptoms of CRC include changes in bowel habits, hemafecia/melena, and abdominal pain or discomfort. Among them, hemafecia is the most common symptom, seen in 93.75% of patients with CRC. As for the clinicopathological features, the average tumor diameter was 5.31 ± 2.21 cm, with glandular cancer as the most common histology (91%), and ulcerative type as the major morphological type (37%). IHC analyses showed HIPK2 expression in normal colorectal mucosal tissues to be higher than in CRC samples. These data are consistent with previous reports showing a similar pattern for HIPK2 expressions in breast cancer and thyroid cancer –34. Corrin vivo mouse model of human CRC, and found VB to significantly inhibit xenograft tumor growth. IHC analyses showed heightened levels of pro-apoptotic proteins HIPK2, p53, Bax, and decreasedBcl-2 in VB-treated tumors. These results imply that VB promotes cancer cellapoptosis throughHIPK-2- and p53-related signaling. To study the mechanisms of this anti-cancer effect, we used VB to treat human CRC cell lines. As with the in vivo studies, VB had a remarkable anti-proliferative and apoptosis-promoting effect in HCT-116, HT-29, LoVo, and SW620 cells, in a time- and dose-dependent manner. In addition, this nicely correlates with the previous finding that VB induces genotoxic stress [We next tested VB’s anti-tumor activity in an c stress .Reportedly, theHIPK2–p53 apoptotic pathway is downregulated in different human cancer cells –42. In iin vivo, and represses CRC cell proliferation, and promotes apoptosis, by modulating the HIPK2–p53 signaling pathway.In summary, we found that HIPK2 expression inversely correlates with primary CRC, Dukes staging, and infiltration degrees. We also found that VB significantly inhibits CRC growth


Melatonin, a hormone that functions as an internal signal of darkness for the organism, has opposite effects, aggregating the melanin granules. Because light functions as an important synchronizing signal for circadian rhythms, we further investigated the effects of both hormones on genes related to the circadian system, namely, Per1 (one of the clock genes) and the melanopsins, Opn4x and Opn4m (photopigments). Per1 showed temporal oscillations, regardless of the presence of melatonin or α-MSH, which slightly inhibited its expression. Melatonin effects on melanopsins depend on the time of application: if applied in the photophase it dramatically decreased Opn4x and Opn4m expressions, and abolished their temporal oscillations, opposite to α-MSH, which increased the melanopsins' expressions. Our results demonstrate that unlike what has been reported for other peripheral clocks and cultured cells, medium changes or hormones do not play a major role in synchronizing the Xenopus melanophore population. This difference is probably due to the fact that X. laevis melanophores possess functional photopigments (melanopsins) that enable these cells to primarily respond to light, which triggers melanin dispersion and modulates gene expression. Color change is an important strategy for animals' camouflages, which allows them to match their body color with the environment . In ectoα-Melanocyte stimulating hormone (α-MSH), which is produced in the pars intermedia of the pituitary gland, is a darkening agent in all vertebrates. It induces a very rapid dispersion in dermal melanophores of ectothermic vertebrates [α-MSH is produced in sites other than the pituitary, such as the skin itself and its systemic action promotes melanin production by the human skin in response to UV light [α-MSH is produced in the mammalian hypothalamus affecting behaviors such as food intake and lordosis [tebrates –13, and tebrates , 15, in tebrates , and immtebrates , 18 of eUV light . In addilordosis , 21.α-MSH, melatonin aggregates melanin granules acting as a lightening hormone [Melatonin (5-methoxy-N-acetyl tryptamine) is an indoleamine synthesized by the pineal gland and released at night. Contrary to  hormone , 22. Man hormone , 24. Mel hormone . This ho hormone . In addi hormone , some of hormone . Xenopus laevis melanophores led to the discovery of the photopigment melanopsin, which is present in the retina of all vertebrates, including humans [ Opn4 genes, encoding melanopsins in nonmammalian vertebrates. The second melanopsin, Opn4m, presents a much higher homology with the mammalian than with the X. laevis melanopsin, which was then named Opn4x. Despite the presence of melanopsins in nonmammalian retinas, their function as a photoentrainment pigment has yet to be defined.Studies on the photoresponses ofg humans , 30. In g humans , which cg humans , 33, theg humans , 35. In g humans  demonstr Per, Cry,α, Reverb- and Ror which after being translated inhibit CLOCK/BMAL1 effect [The molecular basis for endogenous biological rhythm is a loop of transcriptional/translation of clock proteins. In general, CLOCK and BMAL1 dimers acting through E-box elements increase the transcription of1 effect . Additio1 effect –40. Tyro1 effect .α-MSH and melatonin have opposite effects on pigment translocation, both mediate light responses, that is, α-MSH disperses melanin granules in the same manner as light does, whereas melatonin is the messenger of the darkness. α-MSH and light have the same effect on pigment translocation within X. laevis melanophores; however, the intracellular signaling pathways are different [α-MSH acts through MC1R receptor coupled to a Gs-protein, triggering the activation of the cAMP pathway [ X. laevis melanin aggregation through the activation of Mel 1c receptor, which is coupled to a Gi-protein, thus leading to a decrease in cAMP [Although ifferent , 43, whi pathway , whereas pathway . On the  in cAMP .X. laevis melanophores incorporate all components of a circadian system, as they express clock genes and photopigments. In addition, Per1 expression increases in response to a blue light stimulus, indicating the mediation of a blue sensitive pigment [α-MSH and melatonin, signal external environmental light conditions to the organisms in an opposite manner, and that light is an important zeitgeber in X. laevis melanophores, we hypothesized that these hormones may differentially affect the melanopsins Opn4x and Opn4m (blue sensitive photo-pigments) as well as Per1 expression. pigment . The fac X. laevis melanophores  were kept in 60% L-15 medium, supplemented with 5 mg/mL insulin/transferrin/selenium, 2X hypoxanthine/thymidine, 1% antibiotic/fungicide solution , and 10% non-inactivated fetal calf serum  at pH 7.5 and 25°C [Immortalized cultures of embryonicand 25°C . The cul6 per 25 cm2 flask, the serum was reduced to 2%, and 2 × 10−7 M retinaldehyde was added. Cells were manipulated in the dark using Konex illuminator with a 7 W red light (~5 lux) and a Safe-Light GBX-2 filter . Each experiment was repeated 2 to 3 times with duplicate flasks of cells (n number presented in the graphs).For the experiments, the cells were plated 2 × 10 Xenopus melanophores aggregate the melanin granules in DD, or in response to melatonin, and disperses pigment granules in response to light or to α-MSH. Therefore, α-MSH was employed to mimic light signal in aggregated cells, that is, in DD, whereas melatonin was applied on already dispersed cells; there is LD.The protocols described below were based on the fact that−9 M α-MSH . Twelve hours later, both groups received a new medium change without hormone. At 8:00 h of the sixth day (24 hours after the beginning of the treatment) total RNA was extracted every 4 hours .Cells were kept under constant darkness for 5 days. At 8:00 h of the fifth day, the flasks were divided into two groups: (1) cells were subject to a medium change without hormone (control); (2) cells were subject to a medium change containing 10−9 M melatonin  in one of the following conditions: (a) beginning of the light phase of the 6th day and (b) beginning of the dark phase of the 6th day. Control cells were kept in the absence of the hormone but were subject to similar light conditions and medium changes. Twenty-four hours after beginning of the treatment, total RNA was extracted every 4 hours . During the light phase, the flasks received irradiance of 87.85 µw/cm2 .Cells were kept in 12 : 12 LD for 5 days and then treated for 6 hours with 10turbo-DNA-free), according to the manufacturer's instructions . RNA concentration was determined with a spectrophotometer  and 1 μg was used in reverse transcription reaction with SuperScript III and random primers .Total RNA was extracted with TRIzol reagent; the RNA pellet was then resuspended in DEPC water and treated with DNase  based on Genbank sequences (http://www.ncbi.nlm.nih.gov/PubMed/) and synthesized by IDT  , probes , Platinum Supermix  supplemented to final concentrations of 6 mM MgCl2, 0.4 mM dNTPS, 0.1 U/µL Platinum Taq DNA polymerase.Quantitative PCR reactions were carried out with primers and probes specific for the genes of interest or ribosomal 18S, designed withIA, USA) . MultiplTo normalize the results, 18S ribosomal RNA was used, as recommended for mammalian and nonmammalian species –51. The −ΔΔCT). The log values  were averaged and graphed as mean ± SEM relative to the minimal value expression for each protocol. The levels of significance of differences among time points were determined comparing the log data by one-way ANOVA, followed by Tukey; between time points in different protocols, as well as control versus hormone-stimulated groups, the log data were compared by two-way ANOVA followed by Bonferroni's test (significant at P < 0.05).The results were analyzed using the ΔΔCT method . The thrThe vertebrate circadian system consists of an element that detects environmental light, an internal oscillator, and one or more output signals. The internal oscillator is comprised by a central clock and several peripheral clocks which, when in synchrony, display a time-accurate output . NotablyPer1 expression in X. laevis melanophores kept in constant dark (DD) did not statistically vary throughout ZTs after medium changes , and at ZT8 when was compared to ZT12 and ZT20 (P < 0.01). Per1 mRNA has also been reported to increase in undisturbed X. laevis melanophores in LD 14 : 10 [ X. laevis eye [ changes . Several changes , 61. How 14 : 10  cycles. evis eye .α-MSH on melanin granule dispersion, we evaluated the action of this hormone on one of the clock genes, Per1, as well as the effects of melatonin, an internal dark messenger for the organism. Melatonin was applied at the beginning of the photophase for 6 hours, whereas α-MSH was applied for 12 hours at the beginning of the subjective photophase in DD, thus mimicking the presence of light in causing melanin dispersion. One-way ANOVA analyses showed that there was no temporal variation in either 10−9 M α-MSH treated cells or in cells subject to medium changes in DD. On the other hand, two-way ANOVA showed that the hormone treatment considerably affected the results  = 10.19, P = 0.0026, Figures Because of the similar effects of light and of  Per1 expression seen in control cells persisted after melatonin treatment. Higher levels of Per1 mRNA were seen at ZT4 when compared to ZT0, ZT12, ZT16, and ZT20  = 20.06, P < 0.0001 and F = 19.74, P < 0.0001, resp.). Bonferroni's post-test analysis of each time point indicated that Per1 mRNA levels were 2 times lower at ZT4 (P < 0.001) and ZT12 (P < 0.01) after 10−9 M melatonin treatment in the photophase  [ Per1 expression [ X. laevis melanophores. Thus, we hypothesized that melatonin decreasing effects on the amplitude of Per1 expression found here could be due to melatonin-induced reduction of the photopigment expression. In fact, 10−9 M melatonin treatment in the photophase dramatically decreased Opn4x and Opn4m expression during the light period, as compared to cells only subject to medium changes  , also lepression . These r Figures  as descr Opn4x  = 16.49, P < 0.0001 and F = 2.84, P = 0.0214 resp.) and Opn4m  = 21.48, P < 0.0001 and F = 3.64, P = 0.0053 resp.) expressions. In addition, the temporal oscillation of Opn4x and Opn4m seen after medium changes in the photophase  and ZT4 (P < 0.001). Therefore, melatonin is affecting the sensitivity of X. laevis melanophores to an important light-wavelength (460–480 nm), which we believe is the entrainment factor in this model. Melatonin per se does not seem to participate in the entrainment mechanism of the biological clock, as demonstrated by its lack of effect on Per1 expression when applied either in the photophase  and at ZT4 as compared to ZT12, ZT16, and ZT20 (P < 0.01). Opn4m expression was higher at ZT0 (P < 0.05) and at ZT4 (P < 0.01) than at ZT12, 16 and 20. When medium changes were performed in the scotophase, the highest levels of Opn4x and Opn4m mRNA were found at ZT8 (P < 0.01 and P < 0.05 resp.), that is, at the end of the light period . So, Opn4m requires LD cycles plus medium changes to display a temporal oscillation as shown here  = 60.7, P < 0.0001 and F = 10.96, P < 0.0001 resp.), which implied an altered temporal profile of both melanopsins. Opn4x mRNA levels decreased at ZT0 (P < 0.05) and increased at ZT4 , and Opn4m mRNA levels were higher at ZT8 compared to ZT0, ZT4, and ZT16 (P < 0.05). After melatonin treatment, both melanopsin expressions gradually increased during the photophase.Melatonin applied in the scotophase strongly affected Figures . In cont X. laevis melanophores in DD [ X. laevis melanophores, therefore, is an interesting model to study melatonin regulation of the circadian peripheral systems.Melatonin administration during the scotophase mimics pineal melatonin secretion in physiological conditions, so one would expect a more relevant effect of melatonin. However, this hormone was incapable of significantly altering melanopsin temporal oscillations. Both diurnal and nocturnal animals release pineal gland melatonin at night, indicating the duration of the dark phase of the day, thus melatonin is a crucial compound that sets circadian temporal system . During es in DD . X. laevα-MSH exerts opposite effect on Opn4x expression when compared to melatonin, as it does on pigment translocation. After 10−9 M α-MSH treatment, Opn4x expression increased 3-4 times at ZT0 (P < 0.05) and ZT16 (P < 0.05). The stimulatory effect of α-MSH on the overall expression was considered very significant  = 7.7, P = 0.0077)  and increased at ZT12 (P < 0.001) as compared to the control group   and lower at ZT8 as compared to ZT16   ; but hig to ZT16 ; after α < 0.01) . Therefoα-MSH and melatonin seem to be conserved, because they have previously been described in other groups of animals [ X. laevis melanophores: α-MSH increases the nucleotide concentration [In fact, the opposite effects of  animals , 66. Thintration , whereasntration .α-MSH (Figures  Opn4x and Opn4m expression; however, unlike the hormone, light was able to induce temporal variation in both melanopsin expressions. This may be explained by the fact that the signaling pathway leading to light-stimulated responses of gene expression and pigment dispersion is distinct from the one triggering melanin dispersion by α-MSH. In fact, light stimulates PKC pathway leading to an increase of cytosolic Ca2+ and cGMP [α-MSH promotes an increase of cAMP [2+ rise inhibits adenylyl cyclase [ Per1 promoter is known to possess cAMP responsive element [ X. laevis Per1 promoter. In Danio rerio embryonic cells, we and others have demonstrated that the light-stimulated pathway increases both Per1 and Per2 expressions, what is blocked by MAP kinase inhibitors [ Per1 promoter in vertebrate peripheral clocks.We would expect similar effects of  Figures  and ligh Figures  on Opn4xand cGMP , whereas of cAMP . This re cyclase . Althoug element , little hibitors –71, wherhibitors , suggest Per1 is sensitive to light-dark cycles, regardless of the presence of melatonin or α-MSH, which slightly inhibited its expression. It is worth to mention, however, that the hormones may be affecting the expression of other clock genes, such as Per2 or Bmal1, or even exerting a posttranslational effect. Melatonin effects on melanopsins depend on the time of application. Melatonin applied in the photophase dramatically decreases Opn4x and Opn4m expressions and abolishes its temporal oscillation, opposite to α-MSH which increases the melanopsin expressions. Our results demonstrate that unlike what has been reported for other peripheral clocks and cultured cells, medium changes or hormones do not play a major role in synchronizing Xenopus melanophore cell population. This difference is most probably due to the fact that X. laevis melanophores possess functional photopigments, the melanopsins, enabling these cells to respond primarily to light, which triggers melanin dispersion and modulation of gene expression.In summary,