Body composition, glucose, insulin sensitivity, and lipids were assessed in diet-induced obesity (DIO) mice and Zucker Diabetic Fatty (ZDF) rats after oral administration.KDT501 mediated lipogenesis in 3T3L1 and human subcutaneous adipocytes; however, the gene expression profile of KDT501 differed from that of the full PPARγ agonist rosiglitazone, suggesting that KDT501 has pleiotropic biological activities. In addition, KDT501 showed only modest, partial PPARγ agonist activity and exhibited anti-inflammatory effects in monocytes/macrophages that were not observed with rosiglitazone. In a DIO mouse model, oral administration of KDT501 significantly reduced fed blood glucose, glucose/insulin AUC following an oral glucose bolus, and body fat. In ZDF rats, oral administration of KDT501 significantly reduced fed glucose, fasting plasma glucose, and glucose AUC after an oral glucose bolus. Significant, dose-dependent reductions of plasma hemoglobin A1c, weight gain, total cholesterol, and triglycerides were also observed in animals receiving KDT501.These results indicate that KDT501 produces a unique anti-diabetic profile that is distinct in its spectrum of pharmacological effects and biological mechanism from both metformin and pioglitazone. KDT501 may thus constitute a novel therapeutic agent for the treatment of Type 2 diabetes and associated conditions. The molecular link between inflammation, obesity, and insulin resistance is incompletely understood; however, multiple lines of evidence suggest that inflammation plays a major role in insulin resistance. A number of groups have investigated the link between inflammation, obesity and insulin resistance PPAR-gamma (PPARγ) is the primary target of the drug class of thiazolidinediones (TZDs), which are used to treat diabetes mellitus and other diseases featuring insulin resistance. It has been shown that activation of PPARγ increases adipocyte differentiation, lipid metabolism, insulin sensitivity, and glucose homeostasis In contrast to full PPARγ agonists, partial PPARγ agonists mitigate insulin resistance and hyperglycemia in rodent models of obesity without increasing adiposity and cardiac weight Several studies have reported potential anti-inflammatory effects of PPARγ ligands on monocytes/macrophages. However, it has been demonstrated that PPARγ activation is not essential for PPARγ ligands to exert anti-inflammatory effects in macrophages. For example, in a RAW264.7 cell model Humulus lupulus) have been widely used as flavoring agents in brewing. Previously, it was reported that mixtures of hop extracts show anti-inflammatory activity by inhibiting the NF-κB signaling pathway in vitro and in vivo models of diabetes and insulin sensitivity.Extracts from hops (Lipopolysaccharide (LPS), TNFα and telmisartan were purchased from Sigma .Milliplex MAP human cytokine/chemokine kit and adipogenesis assay kits were purchased from Millipore . 3T3-L1 cells, THP-1 cells and FBS were purchased from ATCC . RPMI-1640 medium was purchased from Hyclone . Penicillin-streptomycin solution was purchased from MediaTech . PPARα, PPARγ and PPARδ assay kits, rosiglitazone, GW590735 and GW0742 were from Indigo Biosciences . DMEM/F12 medium was purchased from Invitrogen . Human subcutaneous primary adipocytes and cell culture reagents were purchased from Lonza . All other reagents were analytical grade.in vitro screens focused on ADME related issues. The protocols and target list are available at .KDT501 was screened over 150  biological targets, binding and enzymatic assays, as well as PPAR assay kits from Indigo Biosciences and supplemented protocols were used in this study. Briefly, the reporter cells were treated in triplicates with various concentrations of KDT501 (25–0.78 µM). Telmisartan (10–0.625 µM), GW590735 (10–0.03 µM), and GW0742 (1–0.01 µM) were used as positive controls for PPARγ, PPARα and PPARδ respectively. Rosiglitazone (1–0.031 µM) was used in all assays to confirm specificity to PPARγ. DMSO (0.1%) was used as a negative control. Cells were incubated for 20 hours as per the protocol and read using a luminometer . Average relative light unit (RLU) values from blank wells were subtracted from all the samples. The final data were compared with DMSO control and statistical analyses were performed using GraphPad Prism.THP-1 cells were maintained in RPMI1640 in the presence of 10% serum according to manufacturer's instructions as described earlier 6 cells in 24-well plates. After confluence, cells were treated with KDT501 , rosiglitazone (10 µM), or DMSO on day 0. Cells were differentiated in differentiation medium consisting of 10% FBS/DMEM, 0.5 mM methylisobutylxanthine, 0.5 µM dexamethasone, and 10 µg/mL insulin. After two days, the medium was changed to progression medium consisting of 10 µg/mL insulin in 10% FBS/DMEM followed by 2 more days in maintenance medium consisting of 10% FBS/DMEM. KDT501 or rosiglitazone were included throughout the maturation phase (day 6/day 7). Human subcutaneous primary adipocytes were maintained as described for 3T3L1 adipocytes and treated with rosiglitazone (1 µM), KDT501 (10 µM), telmisartan (10 µM), DHA (10 µM) or PGJ2 (10 µM) for 10 days.Murine 3T3-L1 preadipocytes were maintained in DMEM supplemented with 10% FBS as described earlier Intracellular lipid was quantified with Oil O-Red staining using an adipogenesis assay kit from . The medium was carefully discarded and washed twice with PBS. 300 µL Of Oil O-Red solution were added and incubated for 15 minutes at room temperature. The cells were washed, and bound dye was extracted and transferred to a 96 well plate. Absorbance was measured at 530 nm using a plate reader (Thermo Electron Corp.). Fold change relative to DMSO control was calculated from four independent wells for each condition. Data represent Mean ± SD.6 cells in 6-well plates with PGM-2 medium (2 mL medium/well) and allowed to grow for 24 hr to confluence. Cells were treated with test compounds KDT501 (25 µM), rosiglitazone (1 µM), telmisartan (10 µM), or DMSO in adipocyte differentiating medium. The differentiation medium consisted of indomethacin, methylisobutylxanthine, dexamethasone, and insulin from PGM-2 single aliquots. After 5 days, the cells were washed in PBS, frozen in liquid nitrogen and shipped to Expression Analysis  in dry ice. Three independent plates were used for each condition.Human subcutaneous primary adipocytes were maintained as described earlier. Cells were seeded at an initial density of approximately 2×10in vitro transcription using T7 RNA Polymerase to produce anti-sense RNA, which was then converted into single-stranded sense cDNA that was fragmented and labeled with biotin prior to hybridization to the GeneChip Arrays. Stained arrays were imaged using a Scanner 3000 (Affymetrix) and probe set analysis results were generated with Expression Console (Affymetrix) using the robust multi-array algorithm.RNA was extracted using the miRNeasy Mini Kit (Qiagen) as described by the manufacturer. RNA was isolated and expression profiling was performed using the Ambion WT Expression Kit (Life Technologies). Labeled target, which was hybridized to GeneChip Human Gene 1.0 ST Arrays (Affymetrix), was prepared essentially as described by the manufacturer. This cDNA was then used as a template for Gene Ontology (GO): Gene expression fold-change was calculated for test compounds ≥ absolute (log2) relative to control and with a p-value <0.05 and PADE value <15. The Bonferroni adjusted p-value (<0.05) was used for ranking and is shown as “E-Value”. The GO terms for KDT501 (Probe Set 24), telmisartan (Probe Set 8) and rosiglitazone (Probe Set 74) denote direct biological pathways.All procedures were in compliance with the U.S. Department of Agriculture's (USDA) Animal Welfare Act  and other appropriate agencies. All animal studies were approved by the Institutional Animal Care and Use Committee of Covance Laboratories .C57Bl6/J male mice were 15 weeks old at the start of the experiment. In general, DIO models are initiated at an early age to evaluate effects on obesity. However, older, overweight mice were used in this study to represent middle-aged human subjects for the assessment of metabolic parameters associated with weight loss/diabetes. The mice were housed individually in cages at 72±4°F with a relative humidity of 30–70% with 12 hour light- dark cycle. High fat diet  TD95217 (Teklad Custom Research Diet) was used in this study. The test compounds were administered orally twice per day at 12 hour intervals (10 mL/kg) in 0.5% methylcellulose and 0.2% Tween 80 (w/v). Mice were randomly allocated on the basis of body weight to seven groups (N = 10/group), with five animals for the oral glucose tolerance test (OGTT) and 5 animals for the insulin tolerance test. KDT501 , pioglitazone (30 mg/kg) and metformin (200 mg/kg) were given twice per day. Body weight and food consumption were determined weekly.After 30 days, the mice were fasted overnight, blood was drawn through the tail vein and baseline blood glucose was measured using a glucose analyzer. One hour later, mice were given an oral gavage of glucose solution in water  and blood samples were taken at 15, 30, 60, and 120 min to determine plasma glucose and insulin levels. Five days before the start of the experiment (t = −5 days) and again at the end of the experiment (t = 28 days), body fat was determined using QNMR. With the exception of the group treated with 200 mg/kg KDT501, 10 animals/group were used in the determinations. Insulin and HbA1c glucose levels were determined on days 1, 15 and 29.Male ZDF rats were obtained from Charles River Laboratories at approximately 7 weeks of age. Rats were maintained ad lib on Purina #5008 diet, delivering approximately 17% of calories from fat. Animals with fasting glucose between 150–350 mg/dL were randomized based on glucose and body weight. Test articles were prepared in 0.5% (w/v) methylcellulose + 0.2% (w/v) Tween 80 and given by oral gavage twice daily. Rats  were administered KDT501 , metformin (200 mg/kg), pioglitazone (30 mg/kg) or vehicle control for up to 32 days. Body weights and food intake were determined weekly.Blood was collected via tail bleed and whole blood glucose was measured on days 1, 8, 15 and 29. Blood was collected in 5 animals on days 15 and 29 for determination of glucose, insulin, lipids, hematocrit, and HbA1c. Animals were fasted overnight and OGTT was conducted on days 31 and 32 for determination of glucose and insulin levels. A second ZDF rat study was conducted and the resulting data on total cholesterol are presented.post hoc determinations were used to compare vehicle treated control. Data were expressed as mean ± SEM.Statistical calculations were performed . One-way ANOVA with Dunnett's test for 50's of 19, 14 and 8.4 µM, respectively. Since we were aware of the pleiotropic nature of hop components, we then embarked on a series of analyses to distinguish KDT501 at a molecular level from traditional glitazone PPARγ agonists.As a first step in our investigation, KDT501 was screened in Cerep's BioPrint Panel to identify its biological targets. Only three binding targets were identified: the ion channel, SCN2a, the type II angiotensin receptor, AGTR2, and PPARγ, with IC50 = 13.4 µM) compared to rosiglitazone (EC50 = 0.42 µM). On the other hand, KDT501 (EC50 = 14.0 µM) exhibited both weak and partial PPARγ activity, reaching only 29±7%  of rosiglitazone's maximum activation.High concentrations of KDT501 increased PPARγ reporter activity, but had no effect on PPARα or PPARδ reporter activity , suggestTo further understand how KDT501 is differentiated from classical PPARγ agonists, we performed studies in monocytic THP-1 cells, an established inflammatory cell line that expresses PPARγ. Changes in cell viability were not observed for any of the compounds at the highest dose used. KDT501 (6.25 to 50 µM) dose-dependently reduced LPS stimulated inflammatory mediators MCP-1, RANTES and IL-6 . Telmisade novo lipogenesis in 3T3L1 adipocytes and human subcutaneous adipocytes. Rosiglitazone (10 µM) increased lipogenesis about 2.8-fold in 3T3L1 adipocytes. KDT501 dose-dependently (3.125 to 25 µM) increased lipogenesis with maximum activation of two-fold  in the high dose KDT501 (200 mg/kg bid) group (data not shown). Significant reduction in fat mass was observed by the end of the study for KDT501 (doses ≥100 mg/kg bid) and metformin , all animals gained weight throughout the trial . Two weeAt the end of the study, 100 mg/kg of KDT501 reduced HbA1c equivalent to levels observed with metformin, while higher doses of KDT501 as well as pioglitazone reduced HbA1c levels to approximately half that of the vehicle-treated control .Administration of KDT501 (150 or 200 mg/kg) prevented the dramatic increase in blood sugar seen in vehicle-treated control animals as they progressed to frank diabetes . KDT501 The reduction seen in non-fasting blood glucose was mirrored when animals were subjected to an OGTT , and allIn another ZDF rat study, we evaluated the effect of KDT501 on total cholesterol and triglyceride levels in the plasma. KDT501 reduced cholesterol, but neither metformin nor pioglitazone had this effect . SimilarIn the present study, we evaluated the effect of KDT501 on several metabolic traits associated with insulin resistance and T2D. Oral administration of KDT501 in DIO mouse and ZDF rat models of diabetes improved glucose metabolism and reduced plasma HbA1c, an important biomarker in diabetes. KDT501 dose-dependently reduced weight gain and total cholesterol in ZDF rats, while rats treated with metformin or pioglitazone gained weight. KDT501 also reduced total cholesterol, unlike metformin and pioglitazone. These results suggest that the anti-diabetic mechanism of KDT501 differs from that of both metformin and pioglitazone and that KDT501 may be a novel therapeutic for the treatment of T2D and related conditions in humans.2 and NO was reduced by KDT501, but not with rosiglitazone (results not shown). These data demonstrate that the anti-inflammatory mechanism of KDT501 differs from that of rosiglitazone and is independent of PPARγ expression, thus serving to highlight substantial differences between KDT501 and the glitazones. Several other natural compounds have been found to exhibit PPARγ-dependent  and independent  anti-inflammatory properties In recent decades, several groups have reported that inflammation plays an important role in insulin resistance. We therefore evaluated KDT501 in several inflammatory models, including monocytic cells, THP-1 cells, RAW2647 macrophages, and human synovial fibroblasts from rheumatoid arthritis subjects (data not shown). KDT501 inhibited several inflammatory mediators  in a dose-dependent manner in LPS-activated THP-1 cells . AlthougThe incidence of diabetes is associated with obesity, a chronic medical condition with increasing worldwide prevalence. Many studies indicate that inflammation plays an important role in diet-induced obesity and diabetes. It has been shown that inflammatory markers such as MCP-1 are involved in the recruitment of inflammatory macrophages to adipose tissue, contributing to the development of insulin resistance and progressing, in many cases, to T2D The pleiotropic effects of many natural compounds  are well documented. KDT501 appears to be involved in the regulation of multiple insulin-sensitizing mechanisms, including the reduction of inflammation and activation of lipogenesis. In Cerep BioPrint assays, we identified only three biological targets, suggesting both an unusual specificity as well as a good safety profile for KDT501. KDT501 activates transcriptional activity of one of these targets, PPARγ, but not of PPARα or PPARδ, indicating ligand-binding specificity. Specificity towards PPARγ suggests reduced side effects associated with nonspecific PPAR agonists, such as PPARα-mediated hepatocarcinoma. Recent reports show that pharmacological activation of a second KDT501 target, AGTR2, in combination with PPARγ activation, ameliorates insulin resistance and reverses β-cell damage in diabetic pancreatic tissue in T2D mice Recently, it has been reported Our studies show that the novel compound KDT501 improves blood glucose levels and insulin resistance in rodent models of T2D. We have also demonstrated that KDT501 moderates inflammation and adipose tissue function through a unique mechanism that is complementary and distinct from current anti-diabetic drugs. Additional research is needed to further delineate this molecule's pleiotropic mechanism and potential as a next-generation therapeutic for the treatment of human metabolic disorders.


Enterococcus faecalis  has become a major leading cause of nosocomial endocarditis. Treatment of such infections remains problematic and new therapeutic options are needed. Nine E. faecalis strains were tested: six obtained from patients presenting endocarditis, one with isolated bacteremia, and two reference strains. Antibiotics included daptomycin, alone or in combination, linezolid, tigecycline, rifampicin, gentamicin, teicoplanin, ceftriaxone and amoxicillin. Time-kill studies included colony counts at 1, 4 and 24 h of incubation. Significant bactericidal activity was defined as a decrease of ≥3log10CFU/ml after 24 h of incubation. Antibiotics were tested at a low (106 CFU/ml) and high (109 CFU/ml) inoculum, against exponential- and stationary-phase bacteria. We also performed time kill studies of chemically growth arrested E. faecalis. Various pH conditions were used during the tests. In exponential growth phase and with a low inoculum, daptomycin alone at 60 µg/ml and the combination amoxicillin-gentamicin both achieved a 4-log10 reduction in one hour on all strains. In exponential growth phase with a high inoculum, daptomycin alone was bactericidal at a concentration of 120 µg/ml. All the combinations tested with this drug were indifferent. In stationary phase with a high inoculum daptomycin remained bactericidal but exhibited a pH dependent activity and slower kill rates. All combinations that did not include daptomycin were not bactericidal in conditions of high inoculum, whatever the growth phase. The results indicate that daptomycin is the only antibiotic that may be able of overcoming the effects of growth phase and high inoculum. Enterococcus faecalis  are infrequent but steadily increasing, especially in elderly or immunocompromised patients, and in the field of health-care-associated infections E. faecalis (90%) Invasive infections with in vitro against enterococci isolates, even those resistant to glycopeptides Staphylococcus aureus (S. aureus) and high inoculum S. aureus and Pseudomonas aeruginosaS. aureus but high antibiotic concentrations could overcome the inhibitory effects of acidity These observations emphasize the need to evaluate new therapeutic options. Daptomycin is a cyclic lipopetide and belongs to a novel class of antimicrobial agents. It has shown its efficacy in vitro activity of daptomycin, alone or in combination, against clinical strains of Enterococcus faecalis isolated from endocarditis. Time-kill studies with daptomycin, alone or in combination with linezolid, tigecycline, rifampicin, gentamicin, teicoplanin and amoxicillin were performed at a standard (106 CFU/ml) and high (109 CFU/ml) inoculum, against exponential and stationary growth-phase bacteria. We also tested nondividing E. faecalis. Various pH conditions were used during the assay and two concentrations for daptomycin: 60 µg/ml, that is close to the peak obtained with a 6 mg/kg dosing regimen in human, and 120 µg/ml, obtained with a higher dosage of 8 mg/kg Therefore, we planed to investigate Nine strains were included in this study. Six strains  isolated from blood culture of patients with confirmed endocarditis according to the Dukes modified criteria Antibiotics as pure titrated powders were supplied by their respective manufacturers . Various combinations were tested at fixed concentrations: daptomycin alone (60 and 120 µg/ml), amoxicillin (5 µg/ml) with ceftriaxonee (20 µg/ml), amoxicillin with gentamicin (15 µg/ml), amoxicillin with linezolid (5 µg/ml), amoxicillin with rifampicin , amoxicillin with teicoplanin (15 µg/ml), amoxicillin with daptomycin (60 µg/ml), daptomycin (60 and 120 µg/ml) with rifampicin , daptomycin (60 µg/ml) with tigecycline (2 µg/ml), daptomycin (60 µg/ml) with linezolid (5 µg/ml).E. faecalis: 1% glucose, 4% Mueller-Hinton broth and calcium 50 mg/l in phosphate buffered saline  Mueller-Hinton broth  was used for all experiments and supplemented with calcium at a final concentration of 50 mg/l (MHBc) due to daptomycin calcium dependency for antimicrobial activity 5 to 106 CFU/ml) according to The European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines 8 to 109 CFU/ml), incubated at 37°C for 24 hours.The Minimal Inhibitory Concentrations (MICs) were determined by Etest  on Mueller-Hinton Agar  inoculated with a standard inoculum  at 4°C. Centrifuged aliquots were then resuspended in MHBc for exponential testing and adjusted to an OD550 of 1. Antibiotics were then added to the bacterial suspension and colony counts performed as detailed.Stationary growth-phase 550 standardized to 0.125 and followed by a 1∶100 dilution to obtain a standard inoculum. Centrifugation (5000×g for 10 minutes) was performed and culture aliquots resuspended in resMHBc and adjusted to an OD550 of 1 to obtain high inoculum suspensions. Antibiotics were then added to the bacterial suspensions and colony counts performed as detailed.Initial stationary growth phase bacteria were obtained as detailed above, ODE. faecalis were prepared as detailed and inoculum adjusted to an OD550 of 0.125. Carbonyl cyanide m-chlorphenylhydrazone (CCCP) was added at a final concentration of 10 µM. Bacterial suspensions were then divided into two portions, incubated 3 hours at 37°C while shaking (250 rpm).OD550 was adjusted to 0.125 and followed by a 1∶100 dilution for the portion used in susceptibility testing, the second being used as control to ensure cell viability.Exponential growth-phase U-test. A p value of ≤0.05 was considered significant. All statistical analyses were performed using GraphPad Prism software 6.0 .Changes in CFU/ml at 24 h in conditions of high inoculum were compared by Mann-Whitney Ethical approval was waived for this study. Patient approval is not necessarily required for the use of bacteria isolated from clinical strains. French legislation exempts the work from requiring it .MICs were determined by Etest for amoxicillin, ceftriaxone, teicoplanin, linezolid, daptomycin and tigecycline . All strDaptomycin at 60 µg/ml and the combination amoxicillin-gentamicin resulted in a 4-log decrease of the initial inoculum on all strains except the strain with high-level aminoglycosides resistance (strain FG) where the 4-log reduction was achieved in 4 to 24 hours . There wAll the combinations tested, but daptomycin, showed lower kill rates when compared to exponential growth phase bacteria . Daptomyp<0,0001) and than the combination amoxicillin-gentamicin   .p<0,0001), than daptomycin at 60 µg/ml and pH 7 , but not significantly higher than daptomycin at 120 µg/ml and pH 6   . Daptomy 0,0537) .8 to 1010 bacteria per g of tissue) and mostly nondividing cells encased in a biofilm matrix E. faecalis, daptomycin is the only antibiotic tested that remains efficient. The bactericidal activity at 120 µg/ml seems to be pH dependent. Daptomycin has been authorised by FDA with a maximum dosage of 6 mg/kg that usually leads to a peak concentration of 85 to 95 µg/ml, whereas a dosage of 8 mg/kg can lead to peak concentrations from 110 to 120 µg/ml in vitro studies, animal experiments or clinical trials in the field of endocarditis or bone infections should probably consider higher dosage leading to higher concentrations of daptomycin. This consideration is also supported by the emergence of daptomycin resistance in Staphylococcus sp. and Enterococcus sp. Development of daptomycin resistance in methicillin-resistant S. aureus has been observed following vancomycin-unresponsive S. aureus bacteremia or osteomyelitis 0–24 required to meet the AUC0–24/MIC target, the PK/PD parameter predictive of efficacy. The use of higher dosage, that produce higher peak concentrations and AUC0–24, should be able to reach MPC even in condition of high inoculum, and optimize AUC0–24/MIC in vitro pharmacokinetic/pharmacodynamic model with simulated endocardial vegetations, Ashley D. Hall et al. have shown that vancomycin-resistant E. faecalis developed reduced daptomycin susceptibility with daptomycin at 6, 8 and 10 mg/kg/day but not at 12 mg/kg/day. Additionally, bactericidal activity was sustained over 96 hours with daptomycin at 10 and 12 mg/kg/day regimens but not with the 6 and 8 mg/kg/day regimens Endocarditis and bone infections are specific issues as bacteria are sequestered, with high density  or the Infectious Diseases Society of America (IDSA) still recommend a first line therapy with amoxicillin and gentamicin for 6 weeks that is poorly followed by physicians. The efficiency of daptomycin on enterococcal infections has been proved


Dexamethasone was used as positive control. Among the tested compounds, diosquinone was the only one that caused a NO reduction with statistical importance and without cytotoxicity: an IC25 of 1.09±0.24 µM was found, with 38.25±6.50% (p<0.001) NO reduction at 1.5 µM. In order to elucidate if this NO decrease resulted from the interference of diosquinone with cellular defence mechanisms against LPS or to its conversion into peroxynitrite, by reaction with superoxide radical formed by naphthoquinones redox cycling, 3-nitrotyrosine and superoxide determination was also performed. None of these parameters showed significant changes relative to control. Furthermore, diosquinone caused a decrease in the pro-inflammatory cytokines: tumour necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6). Therefore, according to the results obtained, diosquinone, studied for its anti-inflammatory potential for the first time herein, has beneficial effects in inflammation control. This study enlightens the mechanisms of action of naphthoquinones in inflammatory models, by checking for the first time the contribution of oxidative stress generated by naphthoquinones to NO reduction.The search of new anti-inflammatory drugs has been a current preoccupation, due to the need of effective drugs, with less adverse reactions than those used nowadays. Several naphthoquinones , plus  Diospyros spp., including the species producing the edible fruit persimmon, but also in fungi, lichens and in bacteria Diospyros extracts widely used in African, Chinese and Indian traditional medicine Diospyros chamaethamnus is characterized by the presence of several 7-methyljuglone derivatives, including five dimers , a trimer (xylospyrin) and a tretramer (6-[2-(7-methyljuglonyl)]-isoxylospyrin) Naphthoquinones are secondary metabolites widely distributed in nature. They are present mainly in several families of higher plants, as Antibacterial During inflammation highly reactive species, such as superoxide radical, peroxynitrite, hydrogen peroxide, hypochlorous acid and NO are produced. NO is a small diffusible molecule with important biological functions, including vasodilatation, neurotransmission and inflammation. NO generated by iNOS in activated macrophages is important for host defences. NO modulates the synthesis of prostaglandins, tromboxans and other inflammatory molecules Macrophages are important producers of pro-inflammatory cytokines, such as tumour necrosis factor-alpha (TNF-α), interleukin 1β (IL-1β) and interleukin 6 (IL-6), when they are stimulated by an aggression, like lipopolysaccharide (LPS) exposure Although inflammatory mediators like cytokines are necessary for successful defence against foreign invaders, their production also results in collateral damage to host tissue components, including proteins, lipids/membranes and DNA. A delicate balance between formation and detoxification of reactive species produced in inflammation process allows directing signalling pathways to physiological or pathological conditions. Therefore, inflammation plays a pivotal role in the pathogenesis and development of some diseases, like certain cancers, neurodegenerative lesions and chemical toxicity For the management of inflammation a broad range of immunosuppressive drugs, as calcineurin inhibitors, steroids and anti-inflammatory non-steroids have been used. Nevertheless, these drugs have undesirable side effects like metabolic derangements, development of infections, cancers and gastric toxicity p-hydroquinone and p-benzoquinone (p-hydroquinone and p-benzoquinone); establish possible structure-activity relationships; determine if NO decrease induced by naphthoquinones is due to its consumption in peroxynitrite formation. Pro-inflammatory cytokines , superoxide radical and protein nitration levels were determined for the most promising compound.In the present work, the ability to induce a decrease of NO in LPS-stimulated RAW 264.7 macrophages of several naphthoquinones , oquinone  was evalDiospyros species As far as we know, the contribution of oxidative stress induced by naphthoquinones to NO reduction has not been studied yet. Furthermore, diosquinone, an epoxide dimeric naphthoquinone found in several p-hydroquinone, p-benzoquinone, menadione, sulphanilamide, potassium nitrite, β-nicotinamide adenine dinucleotide reduced disodium salt hydrate (NADH), sodium pyruvate, 3--2,5-diphenyltetrazolium bromide (MTT), methanol, triton, tris-HCl, ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), brilliant blue G, dimethyl sulfoxide (DMSO), DL-dithiothreitol (DTT), phenylmethanesulfonil fluoride (PMSF), nitrotetrazolium blue chloride (NBT), bovine serum albumin, lipopolysaccharide from Salmonella enteric serotype Typhimurium, monoclonal anti-3-nitrotyrosine antibody produced in mouse and GBX fixer and developer of Kodak processing chemicals for autoradiography films were purchased from Sigma-Aldrich . N-(1)-Naphthylethylenediamine, glycerol and ethylenediaminetetraacetic acid (EDTA) were obtained from Merck . Sodium chloride and potassium hydroxide (KOH) were purchased from Vaz Pereira  and ortho-phosphoric acid 85% was obtained from Panreac . Goat antibody to mouse IgG (horseradish peroxidase) (0.8 mg/mL), TNF-α, IL-1β and IL-6 mouse ELISA kits were purchased from Abcam . Hybond – ECL, Hyperfilm™ ECL and the enhanced chemiluminescence (ECL)-Plus reagent Kit were obtained from Amersham™ . Diosquinone was isolated from root barks of D. chamaethamnusDPBS , DMEM (Dulbecco's Modified Eagle Medium)+GlutaMAX™-I, heat inactivated foetal bovine serum and penicillin+streptomycin (Pen Strep) were obtained from Gibco, Invitrogen™ . Plumbagin, naphthazarin, juglone, 1,4-naphthoquinone, Chloroform solution of diosquinone was analysed in a HPLC system (Gilson), using a Spherisorb OSD2 column  . The solvent used was a mixture of acetic acid 5% in water (A) and methanol (B) and the gradient was as follows: 0 min - 75% B, 10 min - 75% B, 25 min - 85% B, 35 min - 100% B and 60 min - 100% B. The HPLC system was equipped with a Gilson diode array detector (DAD). Spectroscopic data of the peak was accumulated in the range of 200–400 nm, and chromatogram was recorded at 255 nm. The data was processed on Unipoint system software .2 at 37°C, in humidified air. Cells were plated at 1.5×105 cells/mL in a plate with 48 wells (1 mL/well). The tested compounds were dissolved in DMSO, at 10 mM, and stored, as small aliquots, at −20°C. The compounds were diluted with supplemented DMEM as needed, before cell exposure. When cells reach confluence, the compounds were added, 1 h before exposition to LPS (1 µg/mL). After addition of LPS, the cells were maintained in culture for 18 h. Dexamethasone at a concentration of 50 µM was used as positive control. The effect of all tested compounds in the absence of LPS was also evaluated, in order to observe if they induced changes in NO basal levels. In negative controls, no LPS was added. The final concentration of DMSO was 0.5% (v/v) and all control groups received the same amount of DMSO.The mouse macrophage-like cell line RAW 264.7 was kindly provided by Prof. Maria S. J. Nascimento . Cells were grown in DMEM+GlutaMAX™ – I supplemented with 10% heat inactivated foetal bovine serum, 100 U/mL penicillin and 100 µg/mL streptomycin, under 5% CO+, at 340 nm. Results are expressed as a percentage of the respective control (with or without LPS).The release of the cytosolic enzyme LDH into culture medium was evaluated as follows: an aliquot of culture medium was taken and mixed with a NADH buffered solution and pyruvate solution Cellular viability was also assessed by the mitochondria dependent reduction of MTT to formazan, according to Sousa and collaborators N-(1)-naphthylethylenediamine in 2% phosphoric acid) In culture, the NO released by the macrophages into the medium is converted to several nitrogen derivatives, from which only nitrite is stable, being easily measured by Griess reagent . The absorbance of reduced NBT, formazan, was measured at 630 nm, in a microplate reader (Multiskan ASCENT Thermo®). Results are expressed as a percentage of the control without LPS.Superoxide radical was measured by the NBT reduction assay Protein quantification was performed by addition of 200 µL of Bradford dye reagent ; phosphoric acid, 10% (v/v) and water) to 40 µL of samples, pre-diluted 100 times. The photometrical measure was performed at 595 nm. Bovine serum albumin was used to generate a standard curve.5 cells/mL, in a plate with 6 wells. After treatment with dexamethasone (50 µM), diosquinone (1.5 µM) and LPS (1 µg/mL), as described above, the cells were washed and subjected to 200 µL triton lysis buffer  with 1 mM DTT and 0.5 mM PMSF, as proteases inhibitors, during 60 minutes. Samples were centrifuged at 12 000 rpm, for 10 minutes, at 4°C, and respective supernatants were collected. Samples solutions were diluted to contain the same protein concentration (0.5 mg/mL) and 100 µL of these solutions were applied in a nitrocellulose membrane, pre-washed with 10% of methanol and pre-hydrated during 5 min. Negative pressure was applied after application of all samples (in duplicate). The membrane was then disassembled from the apparatus and incubated in a TBS-T blocking solution with 5% milk for 5 h, at room temperature. A mouse polyclonal anti-3-nitrotyrosine antibody was used at a dilution of 1∶1000 in TBS-T blocking solution with 5% milk. The membrane and the antibody were incubated overnight, at 4°C, and further incubated at room temperature for 2 h. After washing the membrane with TBS-T, during five minutes, three times, a goat anti-mouse secondary antibody conjugated with horseradish peroxidase (1∶2000) was applied to the membrane for 2 h, at room temperature. All incubations were made with mixing. Finally, the membrane was washed three times with TBS-T buffer and one time with TBS. Blots were revealed using the ECL system kit and 3-nitrotyrosine were visualized on high performance chemiluminescence film, after 15 min of exposition of film to membrane. Three assays were performed. This method is in accordance to Moreira-Gonçalves and collaborators with some modifications Image J 1.44 (http://rsbweb.nih.gov/ij/download.html).RAW 264.7 macrophages were plated with 4 mL/well at a density of 1.5×105 cells/mL, in a 6 wells plate. Supernatants from dexamethasone (50 µM), diosquinone (1.5 µM) exposed macrophages, either without or with LPS (1 µg/mL) stimulation, were collected at 18 h and centrifuged at 4000 rpm for 3 min, to remove any cells. Cell free culture media were stored at −70°C until use. TNF-α, IL-1β and IL-6 secreted cytokines were measured in duplicate, using purified biotinylated antibodies in ELISA sets according to the protocol provided by the supplier . The ELISA plates were read using a microplate reader (Multiskan ASCENT Thermo®).RAW 264.7 macrophages were plated with 4 mL/well at a density of 1.5×10P values of 0.05 or less were considered statistically significant. Statistical analysis was made using: PAWS Statistic 18 Software  and Graphpad Prism 5 Software .OneWay ANOVA and Dunnett test, as post-hoc test, were used to determine the statistical significance in comparison to control. Data are expressed as the mean ± standard error of the mean (SEM) of at least four independent experiments, performed in duplicate or triplicate, as described above. p<0.001) (p<0.001) of cell death, as evaluated by MTT assay. However, for the same concentration of dexamethasone and in the presence of LPS, cell viability of RAW 264.7 macrophages was higher than 100% relative to control (p<0.01). These differences in cell viability were not verified in the LDH assay (The exposure to LPS (1 µg/mL) for 18 h induced a decrease in cellular viability , with statistical importance (p<0.001) . DexametDH assay . TherefoDH assay  and 3, t25 = 0.73±0.07 µM), followed by plumbagin (LC25 = 1.26±0.28 µM) (p<0.001) of cell death, by MTT assay, while in the LDH assay cell viability was higher than 100% (p<0.01) (Naphthazarin was the most toxic compound (LC0.28 µM) . For plu(p<0.01) . In ordep<0.001) and diosquinone (p<0.01) showed some toxicity at concentrations of 5 µM and 2.5 µM, respectively (about 35% of cell death relative to control). Juglone caused nearly 20% of cell death at 10 µM (p<0.001). Menadione (0.5–10 µM), p-hydroquinone and p-benzoquinone (0.5–5 µM) revealed no cytotoxicity under the concentration range used .25 of 1.72±0.54 µM. With 50 µM of dexamethasone, the amount of NO was similar to basal levels  relative to control at 1.5 µM, with an IC25 of 1.09±0.24 µM . Menadione did not induce NO reduction with statistical significance. Juglone, naphthazarin and plumbagin did not cause a decrease of NO at non cytotoxic concentrations  did not reveal significant differences . HoweverDiosquinone (1.5 µM) alone did not increase protein nitration, relative to control. On the other hand, dexamethasone (50 µM) by itself led to protein nitration, which was potentiated by LPS .Diosquinone (1.5 µM) was able to reduce TNF-α by 51.38±9.72% and IL-6 by 39.42±19.79% relative to LPS exposed cells only. With respect to IL-1β, macrophages stimulation with LPS did not cause a measurable increase of this cytokine under the assay conditions. So, no reduction in this interleukin could be observed in cells exposed to diosquinone and LPS.D. chamaethamnusSeveral compounds, the majority of them being naphthoquinones , were scMacrophages, which express high levels of inducible NO synthase, play a central role in host's defence against bacterial infection, being the major cellular targets for LPS action Among inflammatory modulators produced in response to LPS, NO is an important cytotoxic mediator p<0.01), while the treatment only with dexamethasone gave the opposite result. This could be explained by the mechanism of action of dexamethasone, which inhibits NF-κB transcription factor The results obtained after incubation with dexamethasone in MTT assay depended on the presence of LPS : when cep<0.01) in the LDH assay  of cell death relative to control  of NO reduction relative to control at 1.5 µM (p-hydroquinone caused 75.92±5.53% (p<0.001) of NO reduction at 5 µM, NO reduction for 3.20±0.16 µM corresponded to 40%. Thus, diosquinone was more active at lower concentrations than p-hydroquinone. Diosquinone had an IC25 of 1.09±0.24 µM, lower than that of dexamethasone (1.72±0.54 µM). However, dexamethasone caused a decrease in NO for all used concentrations, reaching basal levels at 50 µM  did not induce more nitration than control , indicatThe potential of diosquinone to reduce LPS induced inflammation was confirmed by its ability to reduce the pro-inflammatory cytokines TNF-α and IL-6, which have a central role in inflammation. The decrease in TNF-α was the most expressive and since this cytokine has a fundamental role in the activation of macrophages themselves, being the first mediator in the inflammatory cascade In conclusion, among the several compounds screened for anti-inflammatory activity, diosquinone, the only dimeric naphthoquinone, was the most promising naphthoquinone, causing a reduction of NO, without cytotoxicity. The NO decrease induced by this compound was probably due to its interference with the mechanism of action of LPS and not to NO consumption in the reaction with superoxide. This hypothesis was confirmed by the reduction of the inflammatory mediators TNF-α and IL-6 in macrophages stimulated with LPS. Thus, according to these results, diosquinone brings beneficial effects to cells by inhibiting the inflammatory response. This study contributes to the knowledge of naphthoquinones properties, mainly anti-inflammatory activity, and, as far as we are aware, constitutes the first work concerning diosquinone anti-inflammatory potential. Furthermore, it is the first study assessing whether the oxidative stress induced by naphthoquinones contributes to NO reduction, in this model.


Acacia confusa. Under strict sterile conditions the strain could successfully nodulate Acacia confusa, A. crassicarpa and A. mangium, with nitrogenase activity ranging from 18.90 to 19.86 nmol·g−1·min−1. In the phylogenetic tree based on a complete 16S rRNA gene sequence, the sequence of strain D5 shared 99% homology with that of four species of genus Pseudomonas. The 685 bp nodA fragment amplified from strain D5 shared 95% homology with the nodA sequence of 9 species of genus Bradyrhizobium, with a genetic distance of 0.01682. The 740 bp nifH gene fragment was amplified from strain D5. This strain D5 nifH gene and Bradyrhizobium spp. formed a branch, showing 98% homology and a genetic distance of 0. The homology between this branch and the Bradyrhizobium spp. DG in another branch was 99%, with a genetic distance of 0.007906. These results indicate that this strain D5 is a new type of nitrogen-fixing bacterium.We isolated a novel strain D5 from nodules of  Rhizobium, Sinorhizobium, Mesorhizobium, Bradyrhizobium, Azorhizobium and Agrobacterium, but also some symbiotic nitrogen-fixing bacteria that have previously been categorized as non-symbiotic: some species in Phyllobacterium, Ochrobacterum, and Methylobacterium can all grow symbiotically with legumes and fix nitrogen In the recently years, many new genera and species of symbiotic nitrogen-fixing microorganisms of legumes have been discovered using emerging new technology Acacia is a generic name for all the arbor plants in subfamily Mimosoideae, genus Acacia. So far, Rhizobia strains have been successfully isolated and purified from the root nodules of species including A. melanoxylon, A. impdexa, A. dealbata, A. mearnsii, A. auriculiformis, A. mangium, A. crassccarp, A. mangium×A. auriculiformis, A. confusa, and A. cincinnata, and their physiological, biochemical properties and stress resistance have been studied A. spp.Acacia in Senegal of the Sudan and found that they belonged to Sinorhizobium and Mesorhizobium, respectively. Zhang et al. A. senegal and found that at a mean genetic distance of 0.725 (which seperates Rhizobium and Bradyrhizobium) these rhizobia could be divided into 19 strains. Trinick Acacia are of fast-growing type; some strains were close to fast-growing Rhizobium meliloti. Lv et al. nifA gene fragment of Rhizobia strain HJ06 from A. crassicarpa; they showed that in the phylogenetic tree constructed with the 16S rDNA sequence of strain HJ06, strain HJ06 belonged to a Rhizobium branch, and the cloned nifA gene fragment shared 99.3% homology with Klebsiella pneumoniae. Huang et al. A. cincinnata, and found that this strain was phylogenetically located on the Mesorhizobium branch. Wang et al. Acacia using partial sequences of two housekeeping genes atpD and glnII, and compared the result with the phylogenetic tree constructed with 16S rDNA gene sequence; the results showed that Rhizobia isolated from A. mangium in Fujian and Guangdong belonged to Mesorhizobium, whereas strain isolated from A. confusa in Guangdong belonged to Bradyrhizobium.Lajudie A. spp. may exchange information including genetic information with local species in addition to adapting to the local environment when cultivated in Guangxi. In particular, gene exchange is extremely likely to happen among strains in species such as Acacia rhizobia that show relatively large phylogenetic variations. Aside from transitional Acacia rhizobia, are there endophytic bacteria that are “non-traditional Rhizobia” but are symbiotic with Acacia plant and can nodulate to fix nitrogen? In this study, a “non-traditional” rhizobia, strain D5 isolated from A. confusa was investigated in depth. Inoculation and cross inoculation of this strain were performed under strict sterile conditions, and the nitrogenase activities of its nodules were measured; the complete 16S rRNA sequence of strain D5 and its nodA and nifH gene sequences were examined for symbiotic nodulation genes and nitrogen-fixing genes, and the phylogenetic position of this strain and the homology of its nodA and nifH genes were analyzed. Our findings are of great significance in accurately revealing the diversity and the phylogenetic position of Acacia rhizobia and in searching for potential lateral gene transfer and gene recombination, thus provides a scientific basis for researches that truly uncover phylogenetic relationships in biological systems.Guangxi is located in the north tropical and Subtropical area, the geological condition of which supports life greatly. Thus biodiversity in this region is massive, including that of the microorganisms in the soil. As alien species, A. confusa (A. crassccarp (A. mangium (Glycine max (Linn.) Merr. , P. sp. DK4 (EU158318), P. sp. AUTH 28 (FR725962) and P. sp. 12C (AY689083). The homology was 99%. Strain D5 also had a very close distance to P. stutzeri (AY364327), P. stutzeri GAPP4 (GU396288), P. stutzeri AT11 (JQ621964), and P. stutzeri (AB682251), while traditional rhizobia were located in another branch, far from genus of Pseudomonas , B. elkanii (HQ259488), B. elkanii (HQ259471), B. sp. (AJ430728), B. elkanii (AJ300251), B. elkanii (AB610460), B. elkanii (AM117554), B. elkanii (U04609) and B. elkanii (AB610459); the sequence homology between the nodA sequence of strain D5 and that of these bacteria was 95%, and the genetic distance was 0.01682  formed a branch; the homology between them was 98%, and the genetic distance was 0. The homology between this branch and another B. sp. DG (HM063961.1) was 99%, and the genetic distance was 0.007906.Rhizobium, Sinorhizobium, Allorhizobium, Mesorhizobium, Bradyrhizobium, and AzorhizobiumA. confusa, yet it does not belong to any of these six genera. Based on the Blast result of its complete 16S rRNA sequence and the phylogenetic tree constructed with the 16S rRNA sequence, strain D5 belongs to Pseudomonas. This study shows that under strict sterile conditions, strain D5 can successfully nodulate A. confusa, A. crassicarpa and A. mangium, and the nodules all have nitrogenase activity; nodA nodulation gene and nifH nitrogen-fixing gene are amplified from strain D5, suggesting that there is a diversity of endophytic nitrogen-fixing bacteria symbiotic with A. spp., and demonstrating there are strains in Pseudomonas that contain symbiotic nodulation gene and nitrogen-fixing gene. This has not been reported previously.Currently there are six genera of bacteria that can nodulate legumes, including P. stutzeri A1501 was isolated from the root soil of Chinese southern rice; whole genome “shotgun” method was used to complete the whole genome sequencing of this bacterium, and the genome structure and function were analyzed P. stutzeri A1501 have been cloned In the 1980’s, Bradyrhizobium that shares 95% homology with the nodA gene and 98% homology with the nifH gene of strain D5 belonging to Pseudomonas is a slow-growing genus of rhizobia. A relatively high homology between certain specific gene of two bacterial strains that belong to different genera may be due to horizontal gene transfer (HGT) between bacteria. HGT can occur through transformation (bacteria absorbing free DNA), conjugation  and transduction (between bacteria and virus). Rhizobia plasmids carry genes related to functions including nodulation and symbiotic nitrogen fixing, and are transferrable P. aeruginosaAgrobacterium tumefaciensnodA and nifH genes of strain D5 and those of Bradyrhizobium? One possible explanation could be that the symbiotic nodulation gene nodA and the nitrogen-fixing gene nifH carried by rhizobia plasmids are transferred between bacteria through conjugation, and that these genes are replicated and expressed in strain D5, equipping strain D5 with the ability to nodulate plants and fix nitrogen. To test this hypothesis further investigations on Bradyrhizobium plasmids and the mechanism of its conjugation with strain D5 are needed. This study sets foundation for future research, and provides scientific basis for investigations on HGT and gene recombination.A. confusa. The sampling location was at Chongzuo, Guangxi (21°36′ to 23°22′ N and 106°33′ to 108°6′ E). There is a wide stratigraphic distribution of limestone in this region. The sample was collected from a shallow and dry soil horizon.Strain D5 was isolated from nodules of The stored strain was cultured in a shaking flask containing YMB medium for 5∼7 d  to be activated A. confusa, A. crassicarpa, A. mangium and Glycine max (Linn.) Merr. To avoid infections caused by microorganisms in the soil, Acacia seeds were directly collected from the trees; they were then subject to surface sterilization twice as follows: soaked in 75% ethanol for 1–2 min, rinsed by sterile water for 4 times, soaked in 0.1% acidic mercuric chloride solution for 7–8 min, rinsed by sterile water for 6 times. Inoculation of strain D5 and seedling cultivation was carried out in fully enclosed settings. In each plant 2 ml logarithmic-phase culture of strain D5 was used for inoculation, and 10 plants for each species were used. Plants that were not inoculated or inoculated with other Pseudomonas strains were used as the control The inoculation experiments were performed under strict sterile conditions. The inoculated plants were A. confusa are relatively small in size. In order to avoid loss of nitrogenase activity when removing the nodules from the plants, the intact root system containing the nodules was placed in a 10 ml small bottle, and acetylene reduction method was used to measure nitrogenase activity. Each measurement was repeated twice. Un-inoculated root systems were used as controls. The nitrogenase activity was calculated using the following formula The nodules of 2H4·g−1 biomass·min−1) = K×ethylene peak height for sample (µV) × volume of reaction bulb (µL)/ethylene peak height for standard (µV) × loading volume of sample ethyne (µL) × biomass (g) × reaction time (min), where, K is a constant.E .The primers used were as follows: forward primer Pf (5′-AGA GTT TGA TCA TGG CTC AG-3′), and reverse primer Pr , and the reverse primer was nodA-2(5′-GGN CCG TCR TCR AAW GTC ARG TA-3′).Forward primer for nodA-1 and reverse primer nodA-2 1 µl each, 10×PCR buffer 2.5 µl, 2.5 mmol/L dNTP 2 µl, Taq polymerase 1U; dd H2O was added to make a total volume of 25 µl.The totals volume of PCR mixture was 25 µl, including: template DNA 2 µl, 10 µmol/L forward primer Reaction conditions: denaturation: 94°C 5 min, denaturation: 94°C 30 s, annealing: 50°C 45 s, extension 72°C 60 s, 34 cycles, extension: 72°C 5 min.nifH gene was nifH-f (5′-AAA GGY GGW ATC GGY AAR TCC ACC AC-3′), and the reverse primer was nifH-r (5′-TTG TTS GCS GCR TAC ATS GCC ATC AT-3′).Forward primer for nifH-f and reverse primer nifH-r 1 µl each, 10×PCR buffer 2.5 µl, 2.5 mmol/L dNTP 2 µl, Taq polymerase 1U; dd H2O was added to make a total volume of 25 µl.The totals volume of PCR mixture was 25 µl, including: template DNA 2 µl, 10 µmol/L forward primer Reaction conditions: denaturation: 94°C 3 min, denaturation: 94°C 45 s, renaturation: 65°C 45 s, extension 72°C 2 min, 33 cycles, extension: 72°C 7 min.After PCR reaction was over, 2.5 µl amplification sample was loaded to 1.2% agarose gel for electrophoresis at 100 V. Amplification samples showing bright, pure, specific electrophoretic bands were sent to Beijing Sainuo Biotech Service Inc. for sequencing.nodA and nifH gene sequences. We then used CLUSTALX for multiple sequence comparison, and MEGA4.0 software to construct the phylogenetic trees based on 16S rRNA, nodA or nifH gene sequence. Reference strains and their accession numbers used for sequence similarity and homology comparisons were all from GenBank.We logged into NCBI to access GenBank for sequence similarity analysis and homology comparison of the obtained 16S rRNA,


Sleep-wake cycling is controlled by the complex interplay between two brain systems, one which controls vigilance state, regulating the transition between sleep and wake, and the other circadian, which communicates time-of-day. Together, they align sleep appropriately with energetic need and the day-night cycle. Neural circuits connect brain stem sites that regulate vigilance state with the suprachiasmatic nucleus (SCN), the master circadian clock, but the function of these connections has been unknown. Coupling discrete stimulation of pontine nuclei controlling vigilance state with analytical chemical measurements of intra-SCN microdialysates in mouse, we found significant neurotransmitter release at the SCN and, concomitantly, resetting of behavioral circadian rhythms. Depending upon stimulus conditions and time-of-day, SCN acetylcholine and/or glutamate levels were augmented and generated shifts of behavioral rhythms. These results establish modes of neurochemical communication from brain regions controlling vigilance state to the central circadian clock, with behavioral consequences. They suggest a basis for dynamic integration across brain systems that regulate vigilance states, and a potential vulnerability to altered communication in sleep disorders. All individuals possess an internal circadian timing system, which regulates the appropriate timing of behavior within the 24 hour light-dark cycle. In mammals, the master circadian pacemaker is located within the suprachiasmastic nucleus (SCN) in vitroin vivoAcetylcholine (ACh) was initially thought to provide the signal of light to the SCN. Early studies found that carbachol (a cholinergic agonist) injected into the lateral ventricles produced delays in the onset of activity, similar to those seen in response to light Some insight may be gained from looking at the location of potential cholinergic inputs. Neuroantomical tracing studies have demonstrated cholinergic projections to the SCN arising both in the basal forebrain from the nucleus basalis magnocellularis (NBM) and from the brainstem from the pedunculopontine tegmental nucleus (PPTg) and laterodorsal tegmental nucleus (LDTg) The laterodorsal tegmental (LDTg) and pedunculopontine tegmental (PPTg) nuclei are two regions of a cell group of established importance for regulating vigilance state transitions Outputs from the SCN transmit time-of-day information that generates circadian oscillations in the functional state and physiology of brain stem regions that control sleep and waking in vivoWe examined the hypothesis that signals from brain regions that control vigilance state engage the circadian system. Despite anatomical evidence for neural projections connecting LDTg and PPTg neurons of the brain stem with the SCN To investigate neurochemical communication between sleep/wake and circadian regulatory centers, we stimulated the LDTg or PPTg of freely-behaving mice on a 12hr:12hr light-dark schedule, while collecting microdialysate from the SCN for ACh analysis. Both LDTg and PPTg stimulation  results in a significant increase in ACh release  = 15.3, p<0.001; time F  = 9.98, p = 0.002; treatment × time interaction F = 15.8, p<0.001, PPTg: treatment F = 30.56, p<0.001, time F = 10.74, p<0.001, treatment × time interaction F = 10.92, p<0.001), when compared to baseline measurements . Sham coTo determine whether the magnitude of ACh release depends on time-of-day, the data presented above were analyzed based on the time of stimulus application. Animals received the stimulus at 6, 15, 20 or 24/0 h after lights-on, Zeitgeber Time (ZT) 6, 15, 20 or 24/0. LDTg stimulation at each time-point significantly increases ACh release at the SCN, but differences among these time-points are not significant  = 17.96, p<0.001, ZT F = 1.60, p = 0.196, treatment × ZT F = 0.64, p = 0.88 vs. PPTg results in differential neurotransmitter release, using the same settings we stimulated a second set of animals at ZT 15, this time co-analyzing releasate from each sample for both ACh and glutamate. Results indicate that following LDTg stimulation both ACh and glutamate levels increase significantly at the SCN  = 6.544, p<0.001, releasate F = 1.057, p = 0.306, time × releasate interaction F = 1.16, p = 0.329, To evaluate whether stimulation of the LDTg Three stimulus conditions were used for the PPTg; parameters are detailed in the methods section. PPTg stimulation with Condition 1 significantly increases ACh (188±39%) but not glutamate (136±14%) release at the SCN  = 4.45, p<0.001, releasate F = 10.75, p = 0.001, time × releasate interaction F = 1.36, p = 0.228, To evaluate whether neurotransmitter release at the SCN is specific to the brain region stimulated, we evaluated the response to stimulating the serotonergic median raphe nucleus (MRN), which does not synthesize ACh or glutamate. We analyzed SCN dialysate for serotonin (5-HT), as well as for ACh and glutamate. Stimulation of the median raphe significantly increases 5-HT at the SCN  = 1.74, p = 0.11, releasate F = 22.48, p<0.001, time × releasate interaction F = 2.83, p = 0.001), as anticipated, however does not affect ACh or glutamate levels (p = 1.000) .To determine whether these signals from the brainstem engage the circadian timekeeping mechanism in the SCN, we evaluated effects on phasing of wheel-running activity. Mice received LDTg stimulation  in constant dark environments at circadian time (CT) 6, 14, 15, 17, 20, 22, or 24/0. Stimulation in early subjective night (15) causes a significant delay in locomotor rhythms  = 3.03, p = 0.01, treatment F  = 0.273, p = 0.603, CT × treatment interaction F = 1.854, p = 0.1, As with LDTg, stimulating the PPTg at CT 15 produces significant delays in circadian activity rhythms  when compared to sham stimulation  = 1.99, p = 0.081, treatment F = 7.18, p = 0.009, CT × treatment interaction F = 1.75, p = 0.124 To determine whether the SCN undergoes circadian changes in sensitivity to these neurotransmitters, we applied the differential stimuli at points around the circadian cycle to animals under constant conditions. Animals received PPTg stimulation using the three conditions described above at CT 6, 15, 20 or 24/0. In early subjective night, at CT 15, significant delays are seen following Conditions 1 and 2 (p = 0.01) when compared to CT 6 or CT 0  = 4.66, p = 0.004, treatment F = 1.80, p = 0.15, t-test), whereas injection with either 90 ng/100 nl glutamate or aCSF has no effect on circadian rhythms  at the University of Illinois at Urbana-Champaign . All animal care and surgical procedures were conducted in full compliance with the recommendations outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Survival surgeries were performed under sodium pentobarbital anesthesia with pre- and post-operative analgesia to minimize pain. Every effort was made in experimental planning to reduce the number of animals required for this study.Male C57BL/6J mice were obtained at 5 weeks of age from Jackson Laboratories . Animals were housed and cared for as described in animal protocols in full compliance with NIH guidelines for the humane care and treatment of animals, approved by IACUC and supervised by the Division of Animal Resources at the University of Illinois at Urbana-Champaign.bregma were: SCN, -0.1 mm AP, -0.1 mm ML, -4.0 mm DV; LDTg, -5.2 mm AP, -0.5 mm ML, -3.3 mm DV; PPTg, -4.3 mm AP, -1.8 mm ML, -3.7 mm DV; MRN, -3.8 mm AP, 0.0 ML, -4.8 DV. DV measurements were made from the dural surface, and coordinates were determined experimentally with the aid of a mouse brain atlas Surgeries on mice (18–23 g) were performed ≥1 week after animals arrived. Mice were anesthetized with sodium pentobarbital (80 mg/kg i.p.). A microdialysis guide  was implanted in the SCN, and a bipolar stimulating electrode  was implanted into the LDTg, PPTg, or MRN using a stereotaxic apparatus . Coordinates based on Mice were housed individually in cages (30×17×22 cm) equipped with running wheels (14.5 cm dia.) that were placed inside ventilated light-tight wooden boxes (171×41×34 cm). Wheels were locked on the first day to minimize novel wheel-induced phase shifts, which can increase the time necessary for entrainment. For light-dark cycles, lights were kept at 100 lux for 12 h, followed by 12 h of darkness, regulated by computer. Up to 6 mice could be housed in each box. For experimental procedures, animals were placed in light-tight circadian activity monitoring systems (CAMS) and maintained in darkness under constant conditions Three stimulus conditions were used for the PPTg: 1) 150 μA, 10 Hz, 2-msec pulse duration applied continuously for 20 min, as in previous experiments; 2) 40 μA, 0.2 Hz, 0.2-msec pulse duration continuously for 20 min; and 3) 400 μA, 60 Hz, 0.2-msec pulse duration applied as a 1-sec train every minute for 20 min. Conditions 2 and 3 have been shown previously to favor glutamate or ACh release, respectively, from these neurons For microdialysis procedures, animals were gently restrained, a microdialysis probe  was inserted into the dialysis guide aimed at the SCN and was attached to the animal using a head-block tether system . Connecting cables for stimulation  also were attached at this time. Animals were placed into a CAMS designated for stimulation experiments, where they were attached to fluid  and electronic swivels . Placing animals in CAMS for the duration of the experiment allowed experiments to be performed on a freely-behaving, undisturbed animal, under controlled environmental conditions.2PO4 (anhydrous), 2 mM Na2HPO4, 1 mM dextrose, 2 mM MgCl2, 1.7 mM CaCl2) containing 500 nm neostigmine, a cholinesterase inhibitor. Animals were allowed to equilibrate for at least 1 h before samples were collected. For experiments performed during the animal's dark period, the microdialyis probe was attached during the light period and animals were perfused with aCSF until 2 h before the first collection, at which point the perfusion fluid was switched to aCSF containing 500 nM neostigmine. This allowed all probes to be attached during the light period, while standardizing the length of time each animal was exposed to neostigmine. After ∼1 h for equilibration, baseline samples were collected every 20 min for 1 h. Animals were then stimulated, and microdialysis sample collection continued every 20 min for an additional 100 min. Samples were split for analysis of either ACh or glutamate content, and kept at −80°C until analysis. Samples collected for analysis of ACh, glutamate, and 5-HT content were split 3 ways. Following collection of the last sample, mice were sacrificed and brains harvested for histological verification of electrode and guide-cannula placement.Before each experiment, the microdialysis probe was placed in a standard solution containing ACh (20 nM) and choline (20 nM) for 20 min to determine the recovery rate of the probe. When inserted into the SCN, the probe was continuously perfused at a rate of 1 μl/min with artificial cerebral spinal fluid  and 0.5% Kathon (BAS P/N CF-2150) at a rate of 140 μl/min. Samples first passed through an ion-exchange microbore analytical column  followed by a microbore ACh/choline immobilized enzyme reactor (IMER) containing acetylcholinesterase (AChE) and choline oxidase . This series of columns separated ACh from choline, and converted it to H2O2. The concentration of H2O2 was measured using a horseradish peroxidase-coated 3 mm glass-carbon working electrode held at a 100 mV potential vs. a Ag/AgCl working electrode. ACh concentrations were determined by comparing peak heights of samples to peak heights of standard solutions and were then corrected based on the recovery values determined for the probe used for each experiment Microdialysis samples were analyzed for ACh content using HPLC in combination with an electrochemical detector . Sample (5 μl) was injected using a low-dispersion injection valve with a 5 μl polyetheretherketone loop (Rheodyne model 9125-087). Samples were run through a mobile phase containing 50 mM Na−6 fluorescein, 2 μl of 10 mM naphthalene-2,3-dicarboxaldehyde (NDA), 1 μl KCN, and 1 μl of 50 mM borate buffer (pH 9.4). NDA forms a stable fluorescent product with glutamate that can be measured by capillary electrophoresis/laser-induced fluorescence (CE-LIF) Several CE-LIF systems with different characteristics were used to quantify the glutamate and 5-HT samples A distinct automated CE-LIF system was used for the glutamate measurements from the SCN after stimulation to the median raphe (as shown in 5-HT and its metabolites were identified and quantified using native fluorescence detection + nm). The 50-μm I.D./360-μm O.D. uncoated fused silica capillary  was 70 cm long. The inlet of the capillary was grounded and -30 kV  was applied to the waste reservoir. The background electrolyte for all analyses was 40 mM borate buffer, pH 8.8, and the sheath flow solution was 25 mM citric acid, pH 2.4.Fluorescence is collected orthogonal to the excitation by a 15x all-reflective microscope objective . The collimated beam then impinges upon two custom coated dichroic beamsplitters , with transitions at 310 nm and 400 nm, respectively. The signal is directed, based on wavelength, into one of three photomultiplier tubes , referred to as “blue” (250–310 nm), “green” (310–400 nm), and “red” . Electropherograms were normalized to laser intensity and smoothed using 6-point boxcar averaging. Quantitation was performed using equations derived from calibration curves in the appropriate concentration ranges for each analyte of interest.For analyzing circadian state in mice, animals were individually housed in cages equipped with a running wheel. A magnet  was attached to each wheel, and each revolution of the wheel moved it past a hermetically sealed reed switch , closing a circuit Because of the need to include neostigmine in the perfusion fluid for microdialysis experiments, which could interfere with the circadian behavior of the animal, microdialysis experiments were performed separately from behavioral experiments. Animals were entrained to a 12-h L:12-h D cycle for at least 10 days following surgery, and then were released to constant darkness (DD). Following at least 10 days in DD, treatment times were calculated as described previously Tau (τ) was calculated before and after each treatment, with no significant difference seen following any of the treatment conditions . Onset of activity was determined using Clocklab's template matching algorithm, and was confirmed visually. The magnitude of phase resetting was measured as the difference between regression lines drawn through the 5 days immediately before treatment and 5 days after reestablishment of a stable circadian rhythm after treatment. nditions .Following experiments, animals were sacrificed and brains were frozen on dry ice. Brains then were sliced into 40 μm sections using a cryostat, and stained with cresyl violet to determine the placement of the microdialysis probe within the SCN, or with cholinesterase staining to verify the placement of the stimulating electrodes within the LDTg, PPTg, and MRN using standard procedures post hoc comparison, or for the PPTg injection experiments, where all animals received all treatment, by paired Student's t-test, using SigmaPlot analysis software . The level of significance was p<0.05.Data were analyzed with either two-way analysis of variance (ANOVA) with Holm-Sidak Figure S1Stimulating the MRN increases 5-HT release at the SCN, but not ACh or glutamate. Stimulation of the MRN at ZT 15 with Condition 1  or Condition 2 , does not significantly increase ACh, glutamate (Glu), or 5-HT levels at the SCN. However, stimulating the MRN with Condition 3  significantly increases 5-HT release at the SCN, without affecting ACh or Glu levels. MRN, median raphe nucleus; 5-HT, serotonin. * indicates p<0.05 by Two-Way ANOVA with Holm-Sidak post-hoc test.(TIF)Click here for additional data file.Figure S2Coordinates and anatomy of electrode and cannula placements in mouse brains. Pictured on the left of each panel are the relevant coronal planes of sterotaxic coordinates A is stained with cresyl violet, while B–E are stained for cholinesterase. All coordinates are based on distance from bregma. (A) SCN . (B) Left: MRN electrode , Right: PPTg electrode . (C) LDTg . (D) PPTg injection probe .(TIF)Click here for additional data file.Table S1Average τ values for mice before (pre-) and after (post-) treatment. Analysis of the data with paired t-test revealed no significant difference in τ before vs. after treatment for any of the groups.(TIF)Click here for additional data file.


DRB1 gene second exon and susceptibility or resistance to hydatidosis in three sheep breeds of Sinkiang. The MHC-DRB1 second exon was amplified by polymerase chain reaction (PCR) from DNA samples of healthy sheep and sheep with hydatidosis. PCR products were characterized by the restriction fragment length polymorphism (RFLP) technique. Five restriction enzymes, MvaI, HaeIII, SacI, SacII, Hin1I, were used, yielding 14 alleles and 31 restriction patterns. Frequencies of patterns MvaIbc, Hin1Iab, SacIIab, HaeIIIde, HaeIIIdf, HaeIIIdd (P < 0.01) in Kazakh sheep, SacIab (P < 0.05) in Duolang sheep, and HaeIIIab, HaeIIIce, HaeIIIde, HaeIIIee (P < 0.01) in Chinese Merino (Sinkiang Junken type) sheep, were significantly higher in healthy sheep compared with infected sheep. These results indicated a strong association between these patterns and hydatidosis resistance. In contrast, the frequencies of MvaIbb, SacIIaa, Hin1Ibb, HaeIIIef (P < 0.01) and HaeIIIab (P < 0.05) in Kazakh sheep, SacIbb, HaeIIIae, Hin1Iab (P < 0.05), HaeIIIaa, HaeIIIbe, HaeIIIef (P < 0.01) in Duolang sheep, SacIIaa (P < 0.05) and HaeIIIbd, Hin1Ibb, HaeIIIcf, HaeIIIef (P < 0.01) in Chinese Merino sheep (Sinkiang Junken type) were significantly lower in healthy sheep compared with infected sheep. This indicated a strong association between these patterns and hydatidosis susceptibility. In addition, sheep with the pattern of HaeIIIef demonstrated a high hydatidosis susceptibility (P < 0.01) in all three breeds, while sheep with the pattern HaeIIIde demonstrated significant hydatidosis resistance (P < 0.01) in Kazakh and Chinese Merino sheep (Sinkiang Junken type). These results suggest that the Ovar-DRB1 gene plays a role in resistance to hydatidosis infection in the three sheep breeds.The present study aimed at detecting the association of ovine major histocompatibility complex class II (Ovar II)  Echinococcus granulosus) is recognized as one of the world major zoonoses, and is found all over the world  confers the strongest resistance to the herpes virus responsible for Marek’s disease . EquallyDRB1 was chosen as the immune response gene in this study because it is highly polymorphic, transcribed, and there are over 100 different DRB1 alleles reported in Genbank based upon either restriction fragment length polymorphisms (RFLP) or the deduced amino acid sequence for the β1 domain encoded by exon 2 .MHC Class II Ovar-DRB1 exon 2 was detected by PCR-RFLP analysis in three sheep breeds. We characterized the relationship between the Ovar-DRB1 exon 2 polymorphism and hydatidosis resistance, and screened the genotypes associated with hydatidosis resistance and susceptibility in each sheep breed. Results of the present study may play an important role in developing new sheep breeds that are resistant to hydatidosis.In the present study, the polymorphism of the class II Ovar-Blood samples of Chinese Merino sheep  were donated by agricultural construction division 9 in Sinkiang. Blood samples of Duolang sheep  were donated by agricultural construction division 3. Blood samples of Kazakh sheep  were donated by agricultural construction division 4. The sheep with hydatidosis were distinguished from healthy sheep by a commercially available enzyme-linked immunosorbent assay (ELISA) kit . Genomic DNA were obtained from whole blood by phenol-chloroform method (DRB1 was amplified by PCR in two rounds. The first round of PCR was performed with primers OLA-ERB1 (5’-CCG GAA TTC CCG TCT CTG CAG CAC ATTTCT T-3’) and HL031 (5’-TTT AAA TTC GCG CTC ACCTCG CCG CT-3’) . Reactions were performed in a thermocycler  under the following conditions: a single cycle of 5 min at 94 °C, followed by 15 cycles of 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 60 s, with a final extension at 72 °C for 10 min. We used 3 μl of the resulting mixture and primers OLA-ERB1and OLA-XRBI (5’-AGC TCG AGC GCT GCA CAG TGAAAC TC-3’)  in a total volume of 20 μl. The products of enzyme digestion were analyzed by a 2.5% or 3% agarose gel electrophoresis.PCR products (10 μl) from the second round were digested for 4 h at 37 °C with 5 U of DRB1 genotypes was analyzed by χ2 test. The distribution of genotypic frequency in healthy sheep and sheep with hydatidosis within a breed was analyzed by χ2 test. SPSS version 13.0 was used for statistical analysis.Hardy-Weinberg equilibrium of Ovar-DRB1 exon 2 was amplified by PCR with primers OLA-ERB1, OLA-HL031, and OLA-XRBI. A 296-bp band corresponding to the expected size of exon 2 was observed by 1.5% agarose gel electrophoresis  and HaeIII); fragments the genotypic restriction map is shown in DRB1 exon 2 of Chinese Merino, Duolang and Kazakh sheep was analyzed by PCR-RFLP using restriction enzymes SacII , MvaI , SacI , Hin1I , and HaeIII . Polymorphisms were detected at base pairs 229, 225, 208, 210, 178, 173, 159, 87.Restriction enzyme analysis with DRB1 exon 2 alleles of three breeds were analyzed by χ2 test to determine whether they were consistent with the Hardy-Weinberg distribution, using data shown in Table 3. The χ2 value of the patterns in Kazakh sheep were 173.85 , 9.24 , 0.33 , and 5.84 . These results indicated that patterns produced with restriction enzymes SacII and Hin1I were in Hardy-Weinberg equilibrium, while patterns produced by restriction enzymes MvaI and SacI were not.The Ovar-2 value of patterns in Duolang sheep were 1.25 , 13.63 , 7.44 , and 16.11 . These results suggested that patterns produced by MvaI were in Hardy-Weinberg equilibrium, while patterns produced by SacI, SacII and Hin1I were not.The χ2 value of patterns in Chinese Merino sheep were 0.03 , 1.62 , 0.38 , and 2.14 . These results suggested that patterns produced by MvaI, SacI, SacII, and Hin1I were in Hardy-Weinberg equilibrium.The χMvaIbc, Hin1Iab, SacIIab, HaeIIIde, HaeIIIdf, and HaeIIIdd (P < 0.01) in Kazakh sheep, SacIab (P < 0.05) in Duolang sheep, and HaeIIIab, HaeIIIce, HaeIIIde, and HaeIIIee (P < 0.01) in Chinese Merino sheep (Sinkiang Junken type) in healthy sheep compared with infected sheep, indicating a strong association between these patterns and hydatidosis resistance. Frequencies of patterns MvaIbb, SacIIaa, Hin1Ibb, HaeIIIef (P < 0.01) and HaeIIIab (P < 0.05) in Kazakh sheep, SacIbb, HaeIIIae, Hin1Iab (P < 0.05) and HaeIIIaa, HaeIIIbe, HaeIIIef (P < 0.01) in Duolang sheep, SacIIaa (P < 0.05) and HaeIIIbd, Hin1Ibb, HaeIIIcf, HaeIIIef (P < 0.01) in Chinese Merino sheep (Sinkiang Junken type) were lower in healthy sheep compared with infected sheep, indicating a strong association between these patterns and hydatidosis susceptibility.Comparison of genotypes in sheep with hydatidosis and healthy controls is shown in DRB3 polymorphism in sheep and goats. DRB1 exon 2 polymorphisms of 52 Suffolk sheep by PCR-RFLP with restriction enzymes SacI , SacII , Hin1I , and HaeIII , which was consistent with our results. Peng et al. (2007) determined Ovar-DRB1 exon 2 polymorphisms of 211 Chinese Merino sheep (Sinkiang Junken type) by PCR-RFLP with restriction enzymes SacI  and Hin1I , which was also consistent with our results. However, six alleles and 15 patterns were found using the restriction enzyme HaeIII in their study, while six alleles and 19 patterns were found using this enzyme in the present study. This difference may be due to different numbers and species of sheep. Last but not the least, the genotypes of HaeIII bc and bf weren’t appearance in the population which we chosen. Perhaps they will be found in a larger population.At present, both domestic and international studies have indicated that MHC genes show extensive polymorphism in humans, mice, cattle  and Kazakh sheep with the pattern HaeIIIde had strong hydatidosis resistance; in Duolang sheep, the HaeIIIde appeared to confer some hydatidosis resistance, but the association was not statistically significant. In addition, restriction analysis using enzyme HaeIII produced 19 patterns in Chinese Merino sheep (Sinkiang Junken type) in the present study; however, four of these patterns  were not detected in Kazakh sheep, and three of these patterns  were not detected in Duolang sheep. Kazakh sheep are the local sheep of Sinkiang Province. The Duolang sheep is a cross between Feitun sheep from Afghanistan and local sheep from Kashi City of Sinkiang . The Chinese Merino sheep (Sinkiang Junken type) is a cross between a Merino ram from Australia and Sinkiang Junken sheep. The difference among the three breeds in HaeIII patterns of the Ovar-DRB1 second exon may be due to different breeding histories.Analysis of the restriction patterns revealed that Chinese Merino sheep (Sinkiang Junken type), Duolang sheep, and Kazakh sheep with the pattern In the present research, we screened restriction patterns associated with hydatidosis resistance and susceptibility in three sheep breeds by PCR-RFLP. Additional research is needed to determine whether these patterns could serve as genetic markers for hydatidosis.


Lonicera japonica extracts and irradiation in CH27 cells. Rottlerin, a PKCδ-selective inhibitor, reversed the photoactivated Lonicera japonica extract-induced decrease in PKCδ protein expression and change in cell morphology in this study. In addition, rottlerin inhibited the photoactivated Lonicera japonica-induced decrease in protein expression of Ras, ERK, p38, PKCα, and PKCε, which are the kinases of prosurvival signaling pathway. We also demonstrated that pretreatment with rottlerin prevented actin microfilaments and microtubules from damage during the photoactivated Lonicera japonica-induced CH27 cell death. Furthermore, the promotion of the cytoskeleton-related signaling cascade following rottlerin by upregulation of cytoskeleton-related mediators  and molecules of downstream of F-actin (mitochondria-mediated apoptosis pathway) reduces CH27 cell death, indicating that cytoskeleton is the potential target in the photoactivated Lonicera japonicaextract-induced photokilling of CH27 cells.This study demonstrated that many apoptotic signaling pathways, such as Rho family, PKC family, MAP kinase family, and mitochondria-mediated apoptotic pathway, were triggered by  Lonicera japonica extracts exhibited significant photocytotoxicity in human lung squamous carcinoma CH27 cells at a concentration range of 50–150 μg/mL, with 0.4–1.2 J/cm2 light dose -5,7-dihydroxy-2,2-dimethyl-2H-1-benzopyran-8-yl]-3-phenyl-2-propen-1-one), Tris, and TRITC (tetramethylrhodamine isothiocyanate)-conjugated phalloidin were purchased from Sigma Chemical Company . FAK, β-actin, and HSP27 antibodies were from Sigma Chemical Company. Ras, Rho, Cdc42, ERK, JNK, p38, PKCα, PKCδ, and PKCε antibodies were purchased from BD Biosciences . Tubulin and paxillin antibodies were purchased from Calbiochem .The voucher specimens (Lonicera japonica (200 g) were soaked three times with 1 L of 95% ethanol at room temperature for 3 days. The extracts were filtered. The filtrates were collected and then concentrated under reduced pressure at 40°C. The yield of dry extract of Lonicera japonica was about 11%.The air-dried plants of μg/mL streptomycin (Gibco BRL), and 2 mM glutamine  at 37°C in a humidified atmosphere comprised of 95% air and 5% CO2. When CH27 cells were treated with Lonicera japonica extracts, the culture medium containing 1% FBS was used.CH27 cells were grown in monolayer culture in Dulbecco's modified Eagle's medium  containing 10% FBS , 100 U/mL penicillin and 100 2). The cells were irradiated at 40 W for 30 min that correspond to 0.8 J/cm2 light dose.The irradiation source was a set of fluorescent lamp  located at a made-to-measure box. The wavelength of fluorescence lamp is in the range of 400–700 nm. The intensity of light was measured as Lux, a system international illumination measure. Lux was inverted to light dose . Cell pellets were resuspended in lysis buffer  for 30 min. Lysates were clarified by centrifugation at 13,000 rpm for 30 min at 4°C and the resulting supernatant was collected, aliquoted, and stored at −80°C until assay. The protein concentrations were estimated with the Bradford method.Cells were seeded at a density of 1.7 × 10α, Ras, Rho, Cdc42, ERK, JNK, p38, tubulin, and paxillin) or antirabbit IgG . The enhanced chemiluminescent  detection system was used for immunoblot protein detection.Samples were separated by various indicated concentrations of sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The SDS-separated proteins were equilibrated in transfer buffer  and electrotransferred to Immobilon-P Transfer Membranes . The blot was blocked with a solution containing 5% nonfat dry milk in Tris-buffered saline (10 mM Tris-HCl and 150 mM NaCl) with 0.05% Tween 20 (TBST) for 1 h, washed, and incubated with various indicated antibodies. Secondary antibody consisted of a 1 : 20,000 dilution of horseradish peroxidase (HRP)-conjugated goat antimouse IgG -2,5-diphenyltetrazolium bromide (MTT). At each end point, the treatment medium was replaced with fresh serum-free medium containing 2.4 × 10−4 M MTT at pH 7.4. Cells were incubated with MTT medium for 1 h at 37°C. After solubilization in dimethylsulfoxide, absorbance was measured at 550 nm.Cells were seeded at a density of 1 × 105 cells per well onto 12-well plate 48 h before being treated with drugs. The cells were incubated with 0.1% ethanol or 100 μg/mL Lonicera japonica extracts for 4 h and then irradiated with 0.8 J/cm2 fluence dose. After irradiation, the cells were photographed immediately with an Olympus IX 70 phase-contrast microscopy (300X). A field was chosen in the center of each well at approximately the same location for photography.Cells were seeded at a density of 1 × 105 cells per well 48 h. CH27 cells were incubated with vehicle alone or 100 μg/mL Lonicera japonica extracts for 4 h and then irradiated with 0.8 J/cm2 fluence dose. To visualize F-actin, cells were fixed in 3.7% formaldehyde for 15 min, permeabilized with 1% Triton X-100 for 10 min, and incubated with 1.9 × 10−7 M TRITC-phalloidin for 40 min. To detect tubulin, cells were incubated for 30 min with 250 nM Tubulin Tracker Green reagent. After three washings, the cells were observed by Olympus IX 70 fluorescence microscopy (300X).Qualitative F-actin and tubulin staining was obtained by plating CH27 cells at 1 × 106) were incubated with vehicle alone or 100 μg/mL of Lonicera japonica extracts for 4 h and then irradiated with 0.8 J/cm2 light dose. After irradiation, cells were stained with 2 μM JC-1 for 30 min, washed with PBS, and analyzed on a flow cytometer  using 488 nm excitation with 530 and 590 nm bandpass emission filters.Mitochondrial membrane potential (MMP) was measured with JC-1 . CH27 cells  and analyzed using ModFit LT 3.0 Software .Cells were seeded at a density of 6 × 10t-test and regression analysis were used. The results are expressed as percentage ±S.D. of control.Standard statistical methods based on Student's Lonicera japonica extracts, this study detected the protein expression of PKC, Rho, and MAP kinase family members by Western blot analysis. After treatment of CH27 cells with 100 μg/mL Lonicera japonica extracts and 0.8 J/cm2 light dose, the expression of PKCα, PKCδ, PKCε, Ras, Rho, Cdc42, ERK, JNK, and p38 proteins significantly decreased in photoactivated Lonicera japonica extract-treated cells within 6 h  did not exhibit significant cell toxicity following MTT assay (data not shown). Light alone did not affect cell survival in this study  significantly inhibited the photoactivated Lonicera japonica extract-induced changes in the organization and distribution of tubulin , paxillin, and heat shock protein (HSP27). As shown in μg/mL Lonicera japonica and irradiation with 0.8 J/cm2 light dose. The photoactivated Lonicera japonica-induced decrease in tubulin, FAK, paxillin, and HSP27 protein levels was partly reversed by pretreatment with 10 μM rottlerin  family, Rho family, and mitogen-activated protein kinase (MAP kinase) family have been demonstrated to be involved in apoptosis. PKC family, which functions through serine/threonine kinase activity, is involved in signal transduction pathways necessary for cell proliferation, differentiation, and apoptosis , 13. PKCc agents , 15, whe effects , 17. A f effects , 19. Thr effects , 20. Fur effects , 22. Mhativation . Ras, a tivation , 25. Thetivation , 27. To Lonicera japonica extracts and light. The results of PKC protein expression are consistent with those of other studies reporting that the overexpression and activation of PKCα and PKCε have been mainly associated with inhibition of apoptosis [δ by caspase-3 at the V3 (hinge) domain of the enzyme releases a catalytically active fragment of approximately 40 kDa. It is generally believed that proteolytic activation of PKCδ is responsible for apoptotic execution [δ catalytic fragment after Lonicera japonica extracts with irradiation. In this study, PKCε is clearly identified as a doublet with molecular masses about 90 kDa. PKCε has often been detected as a doublet, but this has been rarely commented on [ε purified from rat brain migrates as two distinct bands with molecular masses of 93 kDa and 96 kDa; on phosphatase treatment, these are reduced to a single 90 kDa species [Lonicera japonica extract-induced change in PKC protein expression. Based on the above data, it seemed reasonable to surmise that prevention of CH27 cells from the photoactivated Lonicera japonica extract-induced cell death of rottlerin is associated with the activity of PKC family. In this study, there was a significant decrease in protein expression of PKC, Rho, and MAP kinase family members after treatment with poptosis , 17. Proxecution , 15. Howented on , 29. It  species . RottlerLonicera japonica extract-induced cell death. It is noteworthy that Western blot analysis of the protein expression of Ras exhibits two bands (21 and 22 kDa) in this study. The amount of 21 kDa of Ras significantly decreased after treatment of CH27 cells with Lonicera japonica extracts and light, whereas the protein expression of 22 kDa of Ras was increased. It is wellknown that Ras prenylation in which a lipid anchor is added to the C-terminus of proteins to allow membrane association plays a central role in the development of cancer. When the prenylation of Ras was blocked, a shift to a higher mobility band was seen on SDS-PAGE [Lonicera japonica extracts induced CH27 cell death through blocking the prenylation of Ras. In this study, it was also found that rottlerin reversed the protein expression of many kinases of prosurvival signaling pathway, such as Ras, ERK, and p38, during the photoactivated SDS-PAGE , 32. TheLonicera japonica extract-induced apoptosis has been suggested by our previous study [Lonicera japonica extract-induced disruption of actin microfilaments was prevented by pretreatment of CH27 cells with rottlerin. In addition to actin microfilaments, the microtubule also serves as key components of the cytoskeleton. This study demonstrated that rottlerin prevented the photoactivated Lonicera japonica extract-induced changes in the protein amount and distribution of tubulin. These data suggest that rottlerin can prevent the photoactivated Lonicera japonica-induced rearrangements of the actin microfilaments or microtubules and support the possibility that rottlerin, directly or indirectly, prevents the aggregation of cytoskeleton and stabilizes the CH27 cell cytoskeleton. HSP27 is a component of the large and heterogeneous group of chaperone proteins, and its main functions are prevention of aggregation of actin filament and inhibition of apoptosis [Lonicera japonica-induced decrease in p38 and HSP27 protein expression was significantly reversed by rottlerin in this study. This result suggested that the protein expression of p38 and HSP27, which may stabilize the actin cytoskeleton, may be one of the mechanisms for the protective effects of rottlerin against the photoactivated Lonicera japonica-induced CH27 cell death. In this study, the change in the protein expression of cytoskeleton-related proteins, such as tubulin, FAK, paxillin, and HSP27 was significantly observed in photosensitized Lonicera japonica extract-induced rapid cell death. Many intracellular events, including the assembly of focal adhesion, actin microfilament reorganization, and recruitment of many signaling molecules to the focal adhesions were involved in the change in cell shape [Lonicera japonica extracts induced a marked morphological change, where normally flat cells became round in shape and detached from the extracellular matrix. This finding suggested that the photoactivated Lonicera japonica extract-induced cell death and changes in cell morphology are mediated in part through its effect on cytoskeleton in CH27 cells. The present study also demonstrated that the photoactivated Lonicera japonica extract-induced CH27 cell death and changes in cell morphology were blocked by pretreatment with 10 μM rottlerin.Several reports have indicated that cytoskeleton may play a primary role in the initiation phase of apoptosis in certain circumstances , 5. Micrus study . In thispoptosis . It has poptosis . The phoc or apoptosis-inducing factor, to the cytosol to initiate apoptosis [c or apoptosis-inducing factor into the cytoplasm [Lonicera japonica extracts and light resulted in a significant induce of the opening of MPT pores accompanied by a disruption of mitochondrial membrane potential in CH27 cells. Therefore, we hypothesize that the photoactivated Lonicera japonica extract-mediated apoptosis occurs through the disruption of mitochondrial function. We also demonstrated that the photoactivated Lonicera japonica extract-induced disruption of mitochondrial membrane potential was significantly blocked by pretreatment with rottlerin. Rottlerin had a slight effect on the photoactivated Lonicera japonica extract-induced opening of MPT pores. Based on the above data, the opening of MPT pore may be involved in photoactivated Lonicera japonica extract-induced CH27 cell apoptosis, but not a major factor. However, pretreatment of CH27 cells with rottlerin could prevent the photoactivated Lonicera japonica extract-induced disruption of mitochondrial membrane potential, providing further support for the role of PKCδ as a mediating factor of mitochondria during the photoactivated Lonicera japonica extract-induced cell apoptosis. Many evidences indicated the potential role of actin cytoskeleton in facilitating the mitochondrial recruitment of various proapoptotic proteins, such as cytochrome poptosis –7. The mpoptosis . Therefoytoplasm , 37. ThiLonicera japonica extracts and irradiation in CH27 cells. Our findings suggested that promotion of the cytoskeleton-related signaling cascade following rottlerin by upregulation of cytoskeleton-related mediators  and molecules of downstream of F-actin (mitochondria-mediated apoptosis pathway) reduce CH27 cell death, indicating that cytoskeleton is the potential target in the photoactivated Lonicera japonica extract-induced CH27 cell apoptosis.This study indicated that many apoptotic signaling pathways, such as Rho family, PKC family, MAP kinase family, and mitochondria-mediated apoptotic pathway, were triggered by


Brace treatment in idiopathic scoliosis is the only efficacious method of non operative treatment. The effectiveness of dynamic SpineCor brace with corrective movement principle has been shown, but differences between rigid braces and SpineCor brace is still unclear.The aim of this study is to evaluate differences between rigid brace and SpineCor brace in terms of curve progression, spinal height increase, and SRS-22 questionnaire during treatment.A total of 76 consecutive adolescent idiopathic scoliosis patients who were treated with brace were included in this study. 45 patients were treated with SpineCor brace, 31 patients were treated with rigid braces. After detailing braces and their costs, choice was made by patients’ family. Patient’s height, T1-Coccyx distance, gibbosity, and Cobb angles were documented at the beginning of the treatment and last control. At last visit SRS-22 questionnaire applied to the patients to evaluate clinical effect of braces.Average age of SpineCor group was 12.8±1.5 and average follow up period was 25±10.6 months. In rigid brace, the group average age was 12.2±1.3 and average follow up period was 23±6.7months. There were no differences between groups according to age, gender, height, T1-Cx distance, Cobb angles, gibbosity before brace treatment initiated. In both groups, height and T1-Cx distance increased and there were no difference. Cobb angle decreased 1.5° in SpineCor group and increased 1.1 ° in rigid brace group . Gibbosity decreased 0.6° in SpineCor group and increased 0.3° in rigid brace group . According to SRS-22 questionnaire, SpineCor brace patients’ pain, self image and activity/function scores were statistically better than rigid brace patients’ scores, while mental health and satisfaction from treatment scores were similar.Although SpineCor brace and rigid braces have similar effects on curve correction, height and spinal height, the real benefits of SpineCor brace is less pain, less anxiety about self image and more activity, and function.