Photobacterium phosphoreum ANT-2200 has been isolated from the NW Mediterranean Sea at 2200-m depth (in situ temperature of 13°C) close to the ANTARES neutrino telescope. The effects of hydrostatic pressure on its growth and luminescence have been investigated under controlled laboratory conditions, using a specifically developed high-pressure bioluminescence system. The growth rate and the maximum population density of the strain were determined at different temperatures (from 4 to 37°C) and pressures (from 0.1 to 40 MPa), using the logistic model to define these two growth parameters. Indeed, using the growth rate only, no optimal temperature and pressure could be determined. However, when both growth rate and maximum population density were jointly taken into account, a cross coefficient was calculated. By this way, the optimum growth conditions for P. phosphoreum ANT-2200 were found to be 30°C and, 10 MPa defining this strain as mesophile and moderately piezophile. Moreover, the ratio of unsaturated vs. saturated cellular fatty acids was found higher at 22 MPa, in agreement with previously described piezophile strains. P. phosphoreum ANT-2200 also appeared to respond to high pressure by forming cell aggregates. Its maximum population density was 1.2 times higher, with a similar growth rate, than at 0.1 MPa. Strain ANT-2200 grown at 22 MPa produced 3 times more bioluminescence. The proposed approach, mimicking, as close as possible, the in situ conditions, could help studying deep-sea bacterial bioluminescence and validating hypotheses concerning its role into the carbon cycle in the deep ocean.Bacterial bioluminescence is commonly found in the deep sea and depends on environmental conditions.  Carnobacterium, Desulfovibrio, Marinitoga, Shewanella, Photobacterium, Colwellia, Moritella, and Psychromonas within the Gammaproteobacteria subclass reviewed by Bartlett et al.The deep sea, under 1000-m depth, is characterized by a high hydrostatic pressure (≥10 MPa), with, generally, a low temperature and a low organic-matter concentration. Laboratory experiments using pure cultures of piezophilic bacteria have highlighted microbial adaptations to high hydrostatic pressure. The adaptive traits include those related to growth lux genes directly involved in bioluminescence Vibrio, Photobacterium and Shewanella genera within the Gammaproteobacteria subclass Photobacterium phosphoreum is the predominant species found in the Mediterranean Sea Darkness is another major characteristic of this deep-sea environment that can be disturbed by a biological phenomenon named bioluminescence. Bioluminescence is the process by which living micro- or macro-organisms emit light. Amongst the bioluminescent organisms, marine luminous bacteria are ecologically versatile and can be found as free-living forms, epiphytes, saprophytes, parasites, symbionts in the light organs of fishes and squids, and commensals in the gut of various marine organisms Photobacterium profundum SS9  to define temperature and pressure optima for growth and (2) to study pressure effect  on growth and bioluminescence activities of P. phosphoreum ANT-2200 using a new laboratory controlled hyperbaric system dedicated to high-pressure and bioluminescence studies.Those of the most studied micro-organisms are, for piezophily, SS9 e.g. , not knoeum e.g. . Up to dP. phosphoreum ANT-2200 (GenBank accession number EU881910) was isolated from sea water collected in the Northwestern Mediterranean Sea at the ANTARES neutrino-telescope site (42°54′N/06°06′E) at 2200-m depth (13°C)  see . Procedu600 nm) using a spectrophotometer .Triplicates cultures were incubated at pressures of 0.1, 10, 22, 30 and 40 MPa and for temperatures of 4, 13, 20, 30 and 37°C. Syringes were placed into high-pressure bottles (HPBs). In order to avoid decompression-recompression of the samples, each HPB corresponded to one incubation time. Bacterial growth was estimated by measuring the optical density (OD600 nm) according to the Direct counting was also performed using epifluorescence microscopy with DAPI-staining procedure, according to Porter and Feig Traditionally, a linear regression is used to determine the growth rate of a strain during the logarithmic phase. The logistic (or Verhulst) model The birth rate: The mortality rate: 600 nm) that P. phosphoreum ANT-2200 can reach according to the growth conditions  andMeaning that the logistic model is written as:−1) is supposed to be positive (meaning that α>γ).Biologically, the intrinsic growth rate , maximum population density (600 nm) and cross coefficient  was performed according to two different procedures. On the one hand, cells were fixed with 0.2% glutaraldehyde, filtered on 0.2- µm-pore-size nucleopore membranes, washed with filtered and sterilized seawater with 2% osmic acid, and then with MilliQ water. Washed cells were dehydrated and observed using SEM . On the other hand, cells were rapidly frozen in liquid nitrogen and lyophilized for 48 h using a CHRIST beta 2–4 LT+LD lyophilizator, operated at a temperature of −50°C and a pressure of 4 Pa. After complete dehydration, samples were attached onto stubs with double-sided adhesive (carbon type) and sputter coated, in a Baltec MED020 Sputter Coater, with a thin film of platinum to improve electrical conductivity of the sample surface. Samples were subsequently observed using SEM .Transmission Electron Microscopy (TEM) was carried out using cells fixed with 2% glutaraldehyde, buffered with PBS and embedded in 2% agar. Cells were post-fixed in 1% osmium tetroxide, dehydrated in a graded series of ethanol and embedded in Epon. Sections of 70 nm were realized using an ultramicrotome (Leica ultracut S), contrasted with uranyl acetate and lead citrate, and observed under a Philips CM 120 Transmission Electron Microscope at 80 kV.P. phosphoreum ANT-2200 were grown in 300-ml completely-filled polyethylene bottles (188 mL of culture +62 mL of oxygenated Fluorinert™ FC-72), at 13°C and at 0.1 or 22 MPa (into high-pressure bottles). The bottle stoppers were equipped with a septum through which the pressure was applied. Cells in the late logarithmic stage of growth were harvested by centrifugation . Bacterial pellets were immediately frozen at −20°C and lyophilized. Lipids were extracted using the modified method of Bligh and Dyer 2O, 1∶2∶0.8, v/v/v) under sonication. Following the addition of DCM and water to allow phase separation , the lower DCM layer was collected and the upper aqueous phase was further extracted with DCM (x2). The combined lipid extracts were concentrated, dried over anhydrous sulfate and evaporated to dryness (N2 flux) before being trans-esterified  with 2% sulfuric acid in MeOH in the presence of toluene −1) and the oven temperature was programmed from 70 to 130°C at 20°C min−1, and then at 4°C min−1 from 130 to 300°C at which it was hold for 20 min.Cultures of −1) was monitored with a high-pressure bioluminescence system shown in Bioluminescence (photons sec−1) and maximum-population-density (K expressed as OD600 nm) parameters were defined for each temperature  and pressure  conditions  and a low K of 0.167 ± 0.032  was observed at 30°C and 10 MPa (r–K coefficient) in order to define these optima . An extrapolated-contour diagram was drawn for the cross coefficient Cr–K  was found at 30°C and 10 MPa (in situ conditions (13°C) and to compare atmospheric pressure (0.1 MPa) to high pressure (22 MPa). This allowed to checking the piezophilic character of strain ANT-2200 and studying its morphology and its fatty-acid composition, known to be affected by hydrostatic pressure P. phosphoreum ANT-2200 was also characterized.The r and K parameters were used to construct the extrapolated-contour diagram of their temperature-pressure dependence  was not detected in this strain. DeLong et al.P.phosphoreum ANT-2200 may be due to its origin from warmer deep-sea waters , and to its optimal temperature of growth (30°C).The effect of hydrostatic pressure on the cellular fatty-acid composition of  at 13°C . The maiP. phosphoreum ANT-2200, at 0.1 MPa and 22 MPa, 13°C. Our results showed that the higher maximum cell density at 22 MPa than at 0.1 MPa is associated to higher luminescence intensity. Maximum luminescence intensity is reached, in average, at 17.6 h at 22 MPa and at 13.3 h at 0.1 MPa (6 photons sec−1) than at 0.1 MPa (1.2 ± 0.2×106 photons sec−1). When bioluminescence is maximal, the total cell number is 1.40 ± 0.01×108 cells mL−1 at 0.1 MPa and 1.90±0.01×108 cells mL−1 at 22 MPa  and an ideal growth environment for bacteria 8 to 109 cells mL−1) 5 to 106 times more than the surrounding waters Three different ecological niches with high cell density, enhancing quorum sensing and indirectly bioluminescence, have been described in the literature so far. Firstly, light organs of marine squids or fish contain up to 10P. phosphoreum ANT-2200 was isolated from a deep-water sample  close to the ANTARES site in the Mediterranean Sea. It has been shown that, using only growth rate, it was not possible to characterize the strain growth optima. However, using both growth rate and maximum population density of strain ANT-2200, optimal temperature and pressure have been estimated at 30°C and 10 MPa. As observed in other deep-sea strains, the ratio of total unsaturated vs. total saturated fatty acids is higher at elevated pressure. All these points converge to characterize this strain as mesophile and moderately piezophile. The strain ANT-2200 produces higher luminescence intensity at high pressure (22 MPa) than at atmospheric pressure (0.1 MPa). To our knowledge, this is the first time that such phenomenon is described. Genetic determinism and corresponding ecological benefit of this pressure-controlled bioluminescence still have to be determined.The strain Figure S1Representation of growth curves for temperatures of 4, 13, 20, 30 and 37°C and for pressure of 0.1, 10, 22, 30, and 40 MPa. The logistic model (line) improves the r and K parameter estimation on empirical growth data (dots). Dashed lines are levels of confidence for the 0.05 and 0.95 quantile curves and the 0.25 and 0.75 quantile curves. The growth rate  and maximum population density  parameters are indicated.(TIFF)Click here for additional data file.Table S1Phenotypic and enzymatic characterizations of P. phosphoreum strain ANT-2200. The presence of cytochrome oxidase was tested with one- or two-days cultures on SWC medium, using filter papers dropped with 1% N, N-dimethyl-p-phenylene-diamine hydrochloride (Kovacs oxidase test). Catalase activity was determined by looking at bubble production in a 3% (v/v) hydrogen-peroxide solution. Physiological and biochemical characterizations of P. phosphoreum ANT-2200 were performed using the API 20 NE, Biolog GN2 microplates, and APIZYM methods .(DOCX)Click here for additional data file.


Finally, we found that exercise increased peroxisome proliferator-activated receptor-γ coactivator 1 (PGC1) mRNA expression in the soleus of diabetic rats. These results indicate that both chronic and acute exercise influence the phosphorylation and expression of components of the AMPK and downstream to PIK3 , and improve GLUT4 trafficking in skeletal muscle. These data help explain the mechanism how exercise regulates glucose homeostasis in diabetic rats.Exercise can increase skeletal muscle sensitivity to insulin, improve insulin resistance and regulate glucose homeostasis in rat models of type 2 diabetes. However, the potential mechanism remains poorly understood. In this study, we established a male Sprague–Dawley rat model of type 2 diabetes, with insulin resistance and β cell dysfunction, which was induced by a high-fat diet and low-dose streptozotocin to replicate the pathogenesis and metabolic characteristics of type 2 diabetes in humans. We also investigated the possible mechanism by which chronic and acute exercise improves metabolism, and the phosphorylation and expression of components of AMP-activated protein kinase (AMPK) and downstream components of phosphatidylinositol 3-kinase (PI3K) signaling pathways in the soleus. As a result, blood glucose, triglyceride, total cholesterol, and free fatty acid were significantly increased, whereas insulin level progressively declined in diabetic rats. Interestingly, chronic and acute exercise reduced blood glucose, increased phosphorylation and expression of AMPKα1/2 and the isoforms AMPKα1 and AMPKα2, and decreased phosphorylation and expression of AMPK substrate, acetyl CoA carboxylase (ACC). Chronic exercise upregulated phosphorylation and expression of AMPK upstream kinase, LKB1. But acute exercise only increased LKB1 expression. In particular, exercise reversed the changes in protein kinase C (PKC)ζ/λ phosphorylation, and PKCζ phosphorylation and expression. Additionally, exercise also increased protein kinase B (PKB)/Akt1, Akt2 and GLUT4 expression, but AS160 protein expression was unchanged. Chronic exercise elevated Akt (Thr Obesity and insulin resistance are markers of type 2 diabetes AMPK is a fuel-sensing enzyme that senses an increase in the intracellular AMP/ATP ratio during exercise or hypoxia Insulin and exercise/muscle contraction promote intracellular glucose transporter 4 (GLUT4) translocation to membrane, increasing skeletal muscle glucose uptake As type 2 diabetes is common in the middle-aged and elderly population, we used 15-month-old Sprague–Dawley rats in this study, and diabetes was established by HFD feeding and low-dose STZ injection. Based on previous studies Male Sprague–Dawley rats, weighing 450–470 g, were provided by the Laboratory Animal Center of Academy of Military Medical Sciences of the Chinese People’s Liberation Army (certification No. SCXK (army) 2007–004). Experimental procedures were performed in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, formulated by the Ministry of Science and Technology of the People’s Republic of China and were approved by the Animal Ethics Committee of China Medical University. All surgeries were performed under anesthesia, and all efforts were made to minimize suffering. The animals were killed under anesthesia  following the recommendations of the US National Institutes of Health.These rats were housed in standard polypropylene cages, with three animals per cage, at a temperature of (22±2)°C, humidity of (50±10)%, and 12-h light/dark cycle. The high-fat diet consisted of 23% soy protein (15% energy), 19.8% pork fat (33% energy), 19.8% corn oil (33% energy), 24.5% sucrose (20% energy), and 5% cellulose, and was supplemented with 1.4% vitamin mixture, 6.7% mineral mix, 0.2% choline bitartrate, and 0.004% dibenzyltoluene. The routine control diet contained 57.3% carbohydrate, 18.1% protein, 18.8% cellulose, and 4.5% fat The rats were randomly assigned to the following groups: control(CON), high-fat diet (HFD), high-fat diet+chronic exercise (HFD +CE), high-fat diet+streptozotocin (HFD+STZ), high-fat diet+streptozotocin+chronic exercise (HFD+STZ+CE), and high-fat diet+streptozotocin+acute exercise( HFD+STZ+AE) (8 rats per group). The CON group of rats received the routine control diet for 20 weeks, with free access to water. The HFD groups were fed with the HFD for 20 weeks, with free access to water. After 8 weeks of diet intervention, the HFD+STZ, HFD+STZ+CE, and HFD+STZ+AE groups received one intraperitoneal injection of low-dose STZ , while other groups were injected once with citrate buffer solution . Four weeks after STZ injection, the animals were deprived of food for 12 h. Diabetes was regarded as successfully induced in rats with fasting blood glucose levels ≥7.8 mmol/L and postprandial blood glucose ≥11.1 mmol/L et alThe rats were trained to swim over 2 d, for 10–20 min per session, to reduce water-induced stress. Two or three rats, as one group, were placed in a plastic pool, 45 cm in diameter, 60 cm deep, and water temperature of 34–35°C. After the initial training, the rats underwent chronic exercise for 1 h/d, 5 d/wk, for 8 wk. The exercise program was conducted essentially as described by Luciano The HFD+STZ+AE rats received acute exercise after 20 weeks of HFD. Acute exercise was conducted in the same conditions as for chronic exercise. Acute exercise consisted of two sessions lasting 1.5 h each, with a 45 min interval between each session. The exercise program was conducted as previously described −1 ). Blood samples were collected from tail veins and centrifuged at 1 100×g for 10 min to separate serum. Serum glucose, triglyceride, total cholesterol, and free fatty acid concentrations were determined using an autoanalyzer . Serum insulin concentrations were determined using a radioimmunoassay, as previously described At 24–36 h after the final session of chronic exercise, or 8–16 h after acute exercise, all rats were anesthetized by an intraperitoneal injection of sodium thiopental (40 mg (kg body weight)Kitt) was calculated using the formula 0.693/biological half life. The plasma glucose half-life (t1/2) was calculated from the slope of least squares analysis of the plasma glucose concentration during the linear phase of the decline Insulin tolerance tests were performed after blood sample collection. Briefly, the rats were fasted for 12 h, anesthetized, and administered with 1.5 IU/kg artificial insulin (Sigma). Blood samples were collected at 0, 5, 10, 15, 20, 25, and 30 min after injection, centrifuged at 1100×g, at 4°C for 15 min, and stored at –20°C to determine glucose concentrations. The rate constant for plasma glucose disappearance  supplemented with 8 mmol/L D-glucose for 20 min. For insulin treatment, muscles were preincubated and then incubated in the presence of 1 µM insulin for 40 min. The buffer was continuously infused with 95% O2/5% CO2 at 37°C After the insulin tolerance test, the animals were intraperitoneally injected with sodium thiopental  and sacrificed. Bisected soleus was used from rat to perform glucose uptake and placed in 6 mL of Krebs–Ringer bicarbonate buffer  and 7 mmol/L D-[14C] mannitol  at 30°C for 10 min g at 4°C for 40 min . Non soluble material was discarded. The protein concentration in the supernatant was quantified using Bradford’s method. Then, 60 µg of tissue extract was mixed with an equal volume of 3× sample buffer solution  at room temperature for 30 min 172), AMPKα1, p-AMPKα2 (Thr172), AMPKα2, p-AMPKα1/2 (Thr172), AMPKα1/2, p-Akt1 (Ser473), Akt1, p-Akt2 (Ser474), Akt2, p-LKB1 (Ser431), LKB1, p-PKCζ/λ (Thr410/403), p-PKCζ (Thr410), PKCζ ; p-AS160 (Thr642), AS160, acetyl CoA carboxylase (ACC), p-ACC (Ser79), p-Akt (Ser473), p-Akt (Thr308), Akt, and GLUT4 . Bands of interest were visualized by enhanced chemiluminescence and absorbance was determined using FluorChem V 2.0 gel imaging analysis software .The frozen muscles were thawed, weighed, roughly cut, placed in protein extraction solution , and ultrasonicated at maximum speed at 4°C for 30 s . The homogenate was centrifuged at 9 000×The frozen muscles were weighed, digested with 1 mol/L NaOH (1∶9 wt/vol) at 80°C for 10 min, neutralized with 1 mol/L HCl, and mixed with 6 mol/L HCl to a final concentration of 2 mol/L HCl. The resulting solution was incubated at 85°C for 2 h and neutralized with 5 mol/L NaOH PGC1α), 5′-CACCGTAAATCTGCGGGATG-3′ and 5′-TATCCATTCTCAAGAGCAGCGAAAG-3′; nuclear respiratory factor 1 (NRF1), 5′-CACTCTGGCTGAAGCCACCTTAC-3′ and 5′-TCACGGCTTTGCTGATGGTC-3′; and β-actin, 5′-CCGTAAAGACCTCTATGCCAACA-3′ and 5′-GCTAGGAGCCAGGGCAGTAATC-3′, respectively.Total RNA was extracted from the frozen muscles using RNAiso Plus , and the RNA concentration was measured at 260/280 nm. RNA samples were reverse-transcribed using a PrimeScript RT reagent kit (Takara). Real-time PCR and subsequent data were analyzed using a 7900HT Fast Real-Time PCR System . β-actin was used as an internal reference, and the mRNA expression of target genes is shown relative to β-actin. The forward and reverse primer sequences were as follows: peroxisome proliferator-activated receptor-γ coactivator .Results are expressed as mean ± SE. Differences between the control and HFD, control and HFD+STZ, HFD and HFD+exercise, and HFD+STZ and HFD+STZ+exercise groups were compared by one-way analysis of variance. Values of Kitt, 3MG uptake and glycogen content were lower in the HFD+STZ group. These results indicate that HFD+STZ induced disorders in glucose and lipid metabolism, common metabolic characteristics of diabetes, and could be used to establish a rat model of type 2 diabetes, mimicking diabetes in humans. Compared with HFD rats, food intake and blood glucose were increased, while body mass, epididymal fat, insulin, and Kitt were reduced in the HFD+STZ rats , but exercise increased PKCζ phosphorylation  and Akt (Ser 473), protein expression of Akt and GLUT4 both in HFD+STZ and HFD rats. Compared with the control group, phosphorylation of Akt1, Akt2, Akt (Thr308), Akt (Ser 473) and AS160 in the muscle was reduced  and AS160 , indicating that acute exercise does not completely compensate for muscle insulin resistance in HFD+STZ rats. Although acute exercise increased glycogen content in the HFD+STZ+AE group compared with the HFD+STZ group, this difference was not statistically significant . NRF1 mRNA expression was also significantly reduced in the HFD and HFD+STZ groups compared with the control group  and Akt (Thr308) phosphorylation to exercise. However, a previous study showed that exercise only restored Akt1 expression in rats with HFD-induced insulin resistance, and Akt2 content was unchanged Akt, AS160, and GLUT4, are all components of the classical insulin pathway. Chronic exercise increased the phosphorylation and protein expression of the Akt1 and 2 isoforms in the HFD+STZ group, consistent with the responses of Akt (Seret alExperiments using mice with muscle-specific LKB1 knockout showed that LKB1 is a major kinase upstream of AMPK involved in the contractile response, and is an important regulator of skeletal muscle glucose transport Acute exercise significantly enhanced insulin’s ability to stimulate glucose absorption in skeletal muscle and counteracted insulin resistance, a typical feature of type 2 diabetes. However, the potential molecular mechanism remains poorly understood PGC1α gene by the AMPK-mediated peroxisome proliferator PGC1α gene expression. The results of this study suggest that by enhancing AMPK activity, chronic and acute exercise reversed the downregulation of gene expression in the HFD+STZ rats. As a transcriptional coactivator, PGC-1 is a major coordinator of mitochondrial biogenesis, and acts by interacting with NRF-1 NRF1 gene expression in this study. We found that acute exercise restored the reduction in NRF1 gene expression induced by HFD+STZ in muscle.Exercise improves skeletal muscle AMPK activity, and enhances mitochondrial biogenesis and function In summary, chronic and acute exercise improved skeletal muscle AMPK signaling and downstream to PIK3 in rats with HFD- plus STZ-induced diabetes. Although the HFD adversely affected the phosphorylation and expression of several components of the AMPK signaling and downstream to PIK3, chronic exercise improved these changes. The present study provides important insights into the mechanisms by which exercise may correct the molecular disorders associated with obesity and type 2 diabetes.


Background. The rs12979860 CC genotype of the interleukin 28B (IL28B) polymorphism is associated with high rates of sustained virological response (SVR) to peginterferon (PegIFN) and ribavirin (Rbv) in hepatitis C virus genotype-1 (HCV-1) patients. The impact of baseline predictors of treatment outcome and their interplay with viral kinetics in HCV-1 CC patients has not been fully evaluated. Aim. To identify baseline and on-therapy predictors of treatment failure in HCV-1 IL28B CC patients. Methods. Treatment-naïve HCV-1 patients, compliant to PegIFN and Rbv who did not discontinue treatment for nonvirological reasons, were analyzed. Results. 109 HCV-1 IL28B CC were studied. Sixty were males, 39 with BMI >25, 69 with >600,000 IU/mL HCV RNA, 15 with HCV1a, and 30 with cirrhosis. Overall, 75 (69%) achieved an SVR; cirrhosis was the only baseline predictor of treatment failure  as SVR rates were 53% in cirrhotics versus 75% in noncirrhotics (P = 0.03). HCV RNA undetectability (<50 IU/mL) at week 4 (RVR) was achieved by 58 patients (53%). The SVR rates were independent of RVR in noncirrhotics, 76% (34/45) RVR (+) and 74% (25/34) RVR (−) (P = 0.9). In cirrhotic patients, SVR rates were significantly higher in RVR (+) compared to RVR (−) (10/13 (77%) versus 6/17 (35%) P = 0.03). Conclusions. In HCV-1 IL28B CC patients, cirrhosis is the only clinical baseline predictor of PegIFN and Rbv treatment failure. However, in IL28B CC cirrhotics, the achievement of RVR identifies those patients who still have high rates of SVR to Peg-IFN/Rbv therapy. Chronic infection with hepatitis C virus (HCV) affects almost 200 million people worldwide, representing a leading cause of cirrhosis and anticipated liver-related death . Until 2HCV-1 naïve patients who received PegIFN plus Rbv therapy in 5 clinical studies –17 conduChronic hepatitis C was defined by at least one year serum positivity for serum HCV-RNA, ALT levels >1.5 times the upper limit of normal, and by a liver biopsy, performed in the year preceding treatment, consistent with chronic hepatitis C. No patient was coinfected with hepatitis B virus (HBV) or human immunodeficiency virus (HIV) or had decompensated liver disease, drug dependence, or >20 g/day alcohol intake. Patients gave their written informed consent to receive therapy and to make their medical records available for this study which was approved by the Institutional Review Board of the Department of Internal Medicine. Genomic DNA was obtained from whole blood or peripheral blood mononuclear cells using the QIAamp DNA Blood Mini Kit . Genotyping of rs12979860 was performed using a 5′nuclease assay with allele-specific TaqMan probes, and assays were run on a 7900HT real time PCR instrument , following manufacturer's instruction or by using the tetra primer ARMS method , 19. Serα2a  180 mcg/week or PegIFNα2b 1.5 mcg/Kg/week  for 48 weeks. PegIFNα2a was associated with Rbv 1000–1200 mg day  while PegIFNα2b was associated with Rbv 800 mg for patients of less than 65 Kg body weight, 1000 mg for 65–85 Kg, and 1200 mg for ≥85 Kg. Therapy was discontinued if quantitative HCV-RNA testing at week 12 dropped by less than 2Logs compared to baseline values and at week 24 if HCV-RNA was still detectable in those patients in whom HCV-RNA dropped >2 Log at week 12. All patients were evaluated for safety and tolerance of treatment every 4 weeks during the treatment period. PegIFNα2a was reduced to 135 mcg and PegIFNα2b to 1.0 mcg/Kg per week in patients with <0.75 × 109/L neutrophils at two consecutive tests, whereas it was withdrawn in patients with <0.50 × 109/L. The same dose reductions were applied if platelets fell under 50,000 cells/mm3 with PegIFN being discontinued when reaching the 25,000 cells/mm3 threshold. In both treatment arms, Rbv dose was tapered by 200 mg/day in patients with haemoglobin <10 g/dL, whereas it was discontinued in patients with <8.5 g/dL haemoglobin. Patients received Rbv  combined with either PegIFNClearance of serum HCV-RNA by RT-PCR was assessed at week 4 , at week 12 , at week 24, and at week 48 of treatment . A sustained virological response (SVR) was undetectable HCV-RNA by RT-PCR at week 24 after treatment. Patients with an ETR who tested HCV-RNA positive during followup were classified as relapsers. Patients who had any other virologic response were considered as nonresponders.U test or Student's t-test for continuous variables and the χ2 or Fisher exact probability test for categorical data. A probability value of P < 0.05 was considered statistically significant. Logistic regression analysis was performed to identify the variables associated with PegIFN/Rbv treatment SVR. All variables with statistical significance at the univariate analysis were included in the final model, and odds ratios (OR) and corresponding 95% confidence interval (95% CI) were computed. Calculations were done with Stata 10.0 statistical package .Comparisons between groups were made by using the Mann-Whitney P = ns). HCV subtype was 1b in 94 (86%) and 1a in 15 patients (14%). 50 (46%) patients were treated with PegIFNα-2a, while 59 (54%) received PegIFNα-2b.Out of 409 HCV-1 patients enrolled in 5 clinical studies at two liver centers and meeting the inclusion criteria, 109 (27%) were IL28B CC and were included in the current analysis. The clinical and epidemiological characteristics of the 109 patients are shown in Overall 75 (69%) patients achieved an SVR. Of the 34 patients with a treatment failure, 3 (9%) were nonresponders while 31 (91%) had a posttreatment relapse. Although SVR rates did not significantly differ in the 5 clinical studies, they ranged from 64% to 76%. P = 0.03). By univariate analysis, male gender, age, BMI > 25, HCV viral load, and HCV-1 subtype did not influence SVR rates, as the only baseline variable associated with treatment failure was the presence of cirrhosis  . Indeed,P = 0.05). Patients with cirrhosis showed numerically lower rates of RVR, EVR, and ETR and higher rates of posttreatment relapse compared to patients without cirrhosis . HoweverP = 0.1). 58 (53%) patients achieved an RVR, 99 (91%) an EVR, and 103 (95%) an ETR. Overall, the achievement of an RVR did not significantly affect the SVR rates, as the SVR rates were 76% in those with an RVR and 61% in those without an RVR (P = 0.9). On the other hand, in patients with cirrhosis, the achievement of an RVR was associated with significantly higher SVR rates compared to those obtained in patients who failed to achieve an RVR, SVR being, respectively, 77% (10/13) in RVR (+) and 35% (6/17) in RVR (−) (P = 0.03), hence identifying RVR as a strong predictor of SVR in these patients   in RVR (+) compared to 74% (25/34) in RVR (−) (.2–31.2) .Our study shows that cirrhosis is the only baseline predictor of treatment failure in HCV-1 IL28B CC patients, with 53% of cirrhotic patients achieving an SVR compared to 75% of noncirrhotics. Our data do not only partially replicate a previous subanalysis of the IDEAL study, showing lower SVR rates in IL28B CC patients with bridging fibrosis and cirrhosis , but addOn the other hand, RVR did play a key role in predicting treatment outcome in IL28B CC patients with cirrhosis, as SVR rates were surprisingly low in those not reaching this virological landmark. Although we cannot provide a definitive explanation for this interplay, it is probable that, in the setting of an altered liver architecture with a deranged microcirculation due to fibrosis deposition , which mWhile this hypothesis further reinforces the dominant role of RVR over pretreatment variables as the most powerful predictor of SVR, our characterization of the interplay between fibrosis staging and on-treatment kinetics has important clinical implications. Since a universal migration to DAA-based regimens in all HCV-1 patients might not be affordable in terms of healthcare resources in countries where a welfare state is in place, correct selection of patients for a DAA-based regimen is mandatory. In Europe, most, local guidelines suggest offering HCV-1 patients with the IL28B CC genotype, PegIFN/Rbv as the first-choice therapy, while offering a DAA-based regimen only to IL28B CC patients with advanced fibrosis/cirrhosis , 25, 26.3/mm3 (range 90–260 × 103/mm3), and had esophageal varices in just 1 case (3%), stressing the concept that our data apply only to well-compensated cirrhotics without clinical stigmas of portal hypertension.We acknowledge that our study has several limitations; first of all it is retrospective, and it includes only patients who were adherent to the optimal treatment schedule and were enrolled in 5 clinical studies where both PegIFNs were administered. Given the different efficacy of the two regimens reported by us and others , 28 and Notwithstanding these limitations, our demonstration of an interplay between cirrhosis and RVR in IL28B CC patients provides evidence for individualized treatment algorithms also in this subgroup of highly responsive patients.


Here, experiments were performed to test the hypothesis that the PNS of insulin-resistant mice displays altered insulin signal transduction in vivo. For these studies, nondiabetic control and type 2 diabetic ob/ob mice were challenged with an intrathecal injection of insulin or insulin-like growth factor 1 (IGF-1) and downstream signaling was evaluated in the DRG and sciatic nerve using Western blot analysis.A reduction in peripheral nervous system (PNS) insulin signaling is a proposed mechanism that may contribute to sensory neuron dysfunction and diabetic neuropathy. Neuronal insulin resistance is associated with several neurological disorders and recent evidence has indicated that dorsal root ganglion (DRG) neurons in primary culture display altered insulin signaling, yet ob/ob mice are also present in the PNS. A robust increase in Akt activation was observed with insulin and IGF-1 stimulation in nondiabetic mice in both the sciatic nerve and DRG; however this response was blunted in both tissues from ob/ob mice. The results also suggest that upregulated JNK activation and reduced insulin receptor expression could be contributory mechanisms of PNS insulin resistance within sensory neurons.The results indicate that insulin signaling abnormalities documented in other “insulin sensitive” tissues  of These findings contribute to the growing body of evidence that alterations in insulin signaling occur in the PNS and may be a key factor in the pathogenesis of diabetic neuropathy. Diabetes and metabolic syndrome are risk factors for several neurological diseases, and emerging evidence has indicated that neuronal insulin resistance may be involved in disease pathogenesis . While ain vivo nerve regeneration [Although neurons do not appear to rely on insulin for glucose uptake , insulinneration ,10,11, ineration ,13, mainneration ,15, suppneration ,17, and neration ,19. WhilInsulin and insulin-like growth factor 1 (IGF-1) signaling is propagated by phosphorylation events that begin with the intrinsic tyrosine kinase activity of the insulin or IGF receptor  and In an insulin-resistant state, the cellular effects of insulin are blunted due to improper signal propagation resulting from several different mechanisms, including 1) degradation of the insulin receptor -25, 2) rin vivo evidence of PNS insulin resistance has been presented, and the cellular mechanisms associated with PNS insulin resistance have not been thoroughly investigated. Here, we demonstrate that the DRG and sciatic nerve of ob/ob mice display reduced insulin signaling in response to an intrathecal injection of insulin. Furthermore, the PNS of ob/ob mice has alterations in cellular mechanisms of insulin resistance, including decreased DRG insulin receptor expression and upregulation of JNK activity in the sciatic nerve.Growing evidence suggests that neurons may become insulin resistant similar to other tissues. However, no ob/ob mice, nondiabetic and diabetic ob/ob mice underwent an IPGTT at 9 weeks of age  was also significantly elevated for ob/ob mice  compared to nondiabetic mice (8.2 ±0.5 mmol/L)  compared to nondiabetic mice Akt/total Akt) in both the DRG and sciatic nerve , whereas ob/ob mice, a dose of IGF-1 equimolar to 0.1U insulin was administered via an intrathecal injection. Blood glucose levels in both nondiabetic control and ob/ob mice once again appeared to increase with intrathecal PBS injection, 7.7 ± 0.3 mmol/L at baseline as compared to 9.7 ± 0.8 mmol/L (p = 0.056) after 30 minutes for nondiabetic mice and 12.1 ± 1.9 mmol/L at baseline to 23.7 ± 3.1 mmol/L after 30 minutes for ob/ob mice. IT IGF-1 did not significantly alter blood glucose levels in nondiabetic mice, 9.3 ± 0.4 mmol/L versus 8.1 ± 0.3 mmol/L. Ob/ob mice that received IT IGF-1 had similar blood glucose profiles to ob/ob mice that received IT PBS, with a significant increase in blood glucose after 30 minutes, 16.3 ± 2.7 mmol/L as compared to 27.7 ± 1.2 mmol/L. Akt was significantly activated in the DRG from both nondiabetic (13.3 fold) and ob/ob diabetic mice (6.0 fold). However, Akt activation was significantly lower in the DRG from ob/ob mice compared to nondiabetic mice , p70S6K (protein synthesis), AS160 (glucose uptake), and GSK3β (glycogen synthesis). At the insulin dose (0.1U) and time point (30 minute stimulation) that were investigated, no statistical differences (p > 0.05) were observed in the activation of these proteins even in control mice . Interestingly, there was significant activation of the stress kinase JNK (p(Thr183/Tyr185)JNK/total JNK) in the sciatic nerve of ob/ob mice compared to nondiabetic mice   Figure C.ob/ob mice, nor did insulin stimulation appear to alter its expression levels . This some time ,14,43,50Future studies will focus on mechanisms through which insulin supports proper PNS function, as revealing these pathways may provide insight into how decreased insulin support contributes to the pathogenesis of DN. Furthermore, delineating the details of PNS insulin signaling may open new avenues for therapeutic intervention in patients with DN.ob/ob leptin null mutant and age-matched control mice (ob/+) were purchased from Jackson Laboratories  at 8 weeks of age. Mice were given access to food and water ad libitum and housed on a 12-hour light/dark cycle. Weekly blood glucose , serum insulin  and weights were monitored and mice were sacrificed at 11 weeks of age.All experiments were approved by the University of Kansas Medical Center Institutional Animal Care and Use Committee. Male At 9 weeks of age, an intraperitoneal glucose tolerance test (IPGTT) was used to assess the response of mice to a glucose challenge. After a 6-hour fast, mice were given an intraperitoneal injection of glucose at 1g of glucose per kg body weight. Blood glucose levels were measured via tail clip immediately prior to the glucose bolus and then at 15, 30, 60, and 120 minutes after injection.At 10 weeks of age, mice underwent an insulin tolerance test (ITT). Mice were fasted for 6 hours and then administered IP insulin  at a dosage of 1.5 U per kg body weight. Blood glucose levels were monitored immediately prior to insulin injection and then at 15, 30, 60, and 120 minutes thereafter.Fasting insulin and fasting glucose levels were used to calculate the homeostatic model assessment of insulin resistance (HOMA-IR). Scores were calculated with the following equation: (blood glucose (mg/dl) X (serum insulin (uU/mL))/405) .Mechanical behavioral responses to Semmes Weinstein-von Frey monofilaments (0.07 to 5.0 g) were assessed at 8, 9, 10, and 11 weeks of age. Mice underwent acclimation 2 days prior to the first day of behavioral testing. Mice were placed in individual clear plastic cages (11×5×3.5 cm) on a wire mesh grid 55 cm above the table and were acclimated for 30 minutes prior to behavioral analysis. The filaments were applied perpendicularly to the plantar surface of the hindpaw until the filament bent. Testing began with the 0.7 g filament, and in the presence of a response, the next smaller filament was applied. If no response was observed, the next larger filament was used. Filaments were applied until there was a change in response, followed by an additional 4 more applications. The withdrawal threshold was calculated using the formula from the up-down method previously described .ob/ob type 2 diabetic mice via a one-time intrathecal injection. Previously, intrathecal 0.1U insulin and equimolar IGF-1 have been shown to have beneficial effects on the symptoms of DN [ob/ob mice. The doses administered and stimulation time frames used were confirmed to be sufficient for Akt activation in the PNS with dose curve and time course studies .Sterile PBS (vehicle), 0.1U (~0.7 nmol) Humulin R insulin, or recombinant IGF-1 equimolar to 0.1U insulin was directly administered to both nondiabetic and ms of DN . All inj3VO4, and 200 mM NaF. Following sonication, protein was extracted on ice for 60 minutes and vortexed every 10 minutes. After centrifugation, protein concentration of the supernatant was measured with a Bradford assay . Samples were then boiled with Lane Marker Reducing Sample Buffer  for 3 minutes. Equal amounts of protein (30 μg) were loaded per lane and samples were separated on a 4-15% gradient tris-glycine gel (Bio-Rad), and then transferred to a nitrocellulose membrane. Membranes were probed with the following primary antibodies and all antibodies were purchased from Cell Signaling  unless otherwise noted: total Akt (1:2000), p-(Ser473)Akt (1:500), total p70S6K (1:500), p-(Thr389)p70S6K (1:500), total GSK3β (1:1500), p-(Ser9)GSK3β (1:1000), total JNK (1:1000), p-(Thr183/Tyr185)JNK (1:500), total mTor (1:500), p-(Ser2448)mTor (1:500), Insulin-like growth factor 1 receptor β subunit (1:500), PTP1B (1:500) , total AS160 (1:1000) , p-(Thr642)AS160 (1:500) (Millipore), Insulin Receptor β subunit (1:500) , and α-tubulin (1:5000) (Abcam). Bands were visualized with either anti-mouse or anti-rabbit HRP-conjugated secondary antibodies (Santa Cruz) and ECL with X-ray film. Densitometry with ImageJ (NIH) was then used to analyze each lane. All samples from each tissue were run simultaneously across multiple gels and each group was equally represented on each gel . Data is presented as the ratio of integrated density of the phosopho-protein normalized to the integrated density of the total protein. The normalized ratio was averaged for each group and the mean ± SEM is represented in the corresponding figures. Representative immunoblots are shown.After a 30 minute insulin stimulation period, the right and left lumbar DRG and sciatic nerves were harvested for each sample from 11 week old mice and frozen at −80°C. Tissues were sonicated in Cell Extraction Buffer  containing 55.55 μl/ml protease inhibitor cocktail, 200 mM Nat-test. Blood glucose changes at 30 minutes in response to insulin or IGF-1 were analyzed with a paired Student’s t-test. Western blot results were analyzed with 2-way ANOVA and Bonferroni’s post hoc analysis when appropriate. Outliers greater than or less than 2 standard deviations from the mean were not included in the analysis. All statistical tests were performed using SigmaPlot software and a P value <0.05 was considered significant.All data is expressed as means ± standard error of the mean. IPGTT, ITT, and behavior data were analyzed with a repeated measures analysis of variance (RM-ANOVA). In addition, the area under the curve (AUC) for IPGTT and ITT was analyzed using a Student’s CNS: Central nervous system; PNS: Peripheral nervous system; IRS: Insulin receptor substrate; mTor: Mammalian target of rapamycin; GSK3β: Glycogen synthase kinase 3 beta; MAPK: Mitogen-activated protein kinase; PTP1B: Protein tyrosine phosphatase 1B; JNK: c-Jun N-terminal kinase; DN: Diabetic neuropathyThe authors have no competing interests to report.CG carried out the Western blot analysis, participated in metabolic characterization of mice, performed the statistical analysis and drafted the manuscript. AG carried out the behavior analysis of mechanical sensitivity. JR performed intrathecal injections, tissue dissections, and participated in metabolic characterization of mice. PG helped conceive the study and participated in the design of the study. EF participated in data interpretation and experimental design and helped draft the manuscript. DW conceived the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.


Streptomyces coelicolor A3 (2) strains: the wild type M145 and its derivative mutant M1146, in which gene clusters encoding the four main antibiotic biosynthetic pathways were deleted. Metabolic Flux Analysis and 13C-labeling allowed us to reconstruct a flux map under steady-state conditions for both strains. The mutant strain M1146 showed a higher growth rate, a higher flux through the pentose phosphate pathway and a higher flux through the anaplerotic phosphoenolpyruvate carboxylase. In that strain, glucose uptake and the flux through the Krebs cycle were lower than in M145. The enhanced flux through the pentose phosphate pathway in M1146 is thought to generate NADPH enough to face higher needs for biomass biosynthesis and other processes. In both strains, the production of NADPH was higher than NADPH needs, suggesting a key role for nicotinamide nucleotide transhydrogenase for redox homeostasis. ATP production is also likely to exceed metabolic ATP needs, indicating that ATP consumption for maintenance is substantial.Our results further suggest a possible competition between actinorhodin and triacylglycerol biosynthetic pathways for their common precursor, acetyl-CoA. These findings may be instrumental in developing new strategies exploiting S. coelicolor as a platform for the production of bio-based products of industrial interest.Metabolic Flux Analysis is now viewed as essential to elucidate the metabolic pattern of cells and to design appropriate genetic engineering strategies to improve strain performance and production processes. Here, we investigated carbon flux distribution in two  Since the late 1960s, considerable effort has been devoted to discover new antibiotics. However, the discovery of biologically active molecules has proved progressively more difficult due to biochemically and technically long and costly processes. Furthermore, the prevalence of multi-resistant bacteria has now increased quite substantially Streptomyces, are significant sources of new bio-active molecules Streptomyces species Streptomyces species, the genome comprises 20 to 40 gene clusters presumably involved in the production of many secondary metabolites, while three to five only are currently produced under ordinary laboratory culture conditions.In this regard, the soil-inhabiting, Gram-positive bacteria, belonging to the genus The genetic organization of genes encoding proteins involved in secondary metabolite production has been studied extensively. Several gene clusters encoding antibiotic biosynthetic pathways, as well as specific or pleiotropic regulators, have now been described Streptomyces strains in order to define conditions under which the accumulation of precursors, preferential expression of the above-mentioned gene clusters, or antibiotic synthesis are optimal.However, although primary metabolism provides precursors, reductive power and energy used for biomass and secondary metabolites biosyntheses, an integrated picture of relationships between primary and secondary metabolism is still missing. A metabolic flux analysis describing carbon flux distribution within the metabolic network, that would complete previous transcriptomic Streptomyces coelicolor A3 (2) M145 is a model strain often used to investigate the regulation of antibiotic production and its genome was the first one to be sequenced and annotated amongst Streptomyces species S. coelicolor M145, which presumably indicates an efficient synthesis of the polyketide precursor acetyl-CoA, S. coelicolor M145 has been chosen as a ‘genetic platform’ to create a super-host for the heterologous expression of gene clusters associated with secondary metabolism from other Streptomyces species as well as organisms from other genera S. coelicolor M145 were deleted, yielding strain M1146 Considering the production yield of S. coelicolor M145, production of the Calcium-Dependent Antibiotic (CDA) coincides with growth In S. coelicolor metabolic fluxes under steady-state conditions. N-limitation was chosen as a typical condition promoting actinorhodin production by S. coelicolor M145, as previously described μ∼0.04 h−1), M145 producing only actinorhodin during its growth phase.In the present paper, we developed a N-limited minimal medium to study S. coelicolor using a combination of stoichiometric inventory described by Holms 13C isotopic methods . Data from genome We took advantage of metabolic steady conditions to compute a carbon flux distribution in S. coelicolor M145 and M1146 strains and compare them so as to gain insights on effects of the impairment of secondary metabolism on metabolic homeostasis.We elucidate the flux distribution in primary carbon metabolism in both S. coelicolor A3 (2) strain M145 and S. coelicolor A3 (2) strain M1146 . S. coelicolor strain A3 (2) M1146 cannot produce actinorhodin (ACT), undecylprodigiosin (RED) and calcium dependent antibiotics (CDA) and no cryptic type I polyketide synthase (Cpk) was expressed. Both strains were a generous gift from M.J. Bibb and J.P. Gomez-Escribano from the John Innes Centre .The present study was carried out with −1) or HCl (0.5 mol L−1). CO2 and O2 concentrations were monitored with a Bluesens BCpreFerm gas analyzer. Aeration was adjusted to 0.4 volume air (volume culture)−1 min−1 (vvm) and agitation was 800 rpm so as to maintain dissolved oxygen near 70% saturation (aerobic conditions).Batch cultures were grown in a bioreactor (Infors Labfors 3) with a 2 litre working volume, 2 Rushton-type impellers and 3 baffles. The bioreactor was equipped with a cooled condenser to avoid medium evaporation. Temperature and pH were controlled at 28°C and 7.0, respectively, with automatic addition of NaOH  L−1 and to grow for more than 5 generations in the exponential growth phase. Note that exponential growth during at least 5 generations was a prerequisite for steady-state isotope labeling in metabolites 13C] glucose (CortecNet). For fermentation experiments, 100 mL of medium no. 1 , 3.0 g L−1 NaCl, 1.0 g L−1 CaCO3, pH 7.2) in a baffled polycarbonate 500-mL Erlenmeyer flask were inoculated with 109 spores from a 30% glycerol stock. The culture was incubated at 28°C and agitated with a magnet  for 48 h. Eight mL were then used to inoculate 100 mL of medium no. 2 , pH 7.2). The second culture was incubated for 24 h. Cells from 40 mL were collected by centrifugation , suspended in 20 mL of TES solution (270.5 mmol L−1), and immediately used to inoculate the bioreactor. An additional sample of 10 mL was used for cell dry weight quantification, after filtration through a cellulose acetate filter (0.22 µm) and distilled water washing before drying at 95°C for 24 h.A defined minimal medium  was deve2 Exhaust Rate (CER) and the O2 Uptake Rate (OUR) could be calculated at each time with online measurements: In bioreactors, liquid and gas phases were well mixed and gas diffusion was rapid such that both the CO2 and N2 in the liquid phase was negligible. Flows (ginf) as well as mole fractions  were measured with dehydrated air to avoid any mole fraction imbalance due to water vapour. CER and OUR were plotted versus time and summed in order to quantify total CO2 evolution and O2 consumption. The latter values are used in section Materials and methods 12.1. to determine CO2/SY and O2/SY respectively. All the abbreviations and symbols used in this study are listed in In these equations, it is assumed that the build-up of dissolved COet al. et al.et al.et al. S. coelicolor was determined with an elemental analyser  on cells harvested during the exponential phase of growth. Elemental analyses were carried out at the Institut de Chimie des Substances Naturelles, UPR 2301, CNRS, Gif sur Yvette, France.Nucleic acids were quantified after Norris and Ribbons 4 35% (0.1 mL (mL supernatant) −1) and then centrifuged 2 min at 6,000 g at room temperature. Supernatants were neutralized with cold KOH (7 mol L−1), centrifuged 2 min at 6,000 g at room temperature and filtered through a 0.22 µm polyether sulfone membrane.Supernatants were de-proteinated by acid precipitation with HClO2SO4 (5 mmol L−1) as the eluent (0.5 mL min−1). Metabolites were detected either with an Agilent SPD UV detector (210 nm) or an Agilent RID detector (40°C).Glucose, α-ketoglutarate (2-oxoglutarate), pyruvate, L-lactate, acetate and ethanol were analysed from de-proteinated supernatants on an Agilent 1200 HPLC instrument with an Aminex HPX-87H column at 35°C using H−1) pH 8.0, NADH (0.2 mmol L−1), ADP (0.6 mmol L−1), α-ketoglutarate (10 mmol L−1), and 5 units of glutamate dehydrogenase from bovine liver (Sigma #G2626). The A340 was monitored until the end of the reaction (∼8 min).The ammonium content was measured enzymatically in 20 µL of de-proteinated samples with glutamate dehydrogenase in 1 mL of an assay mixture made of Tris/HCl . The −1) buffer pH 7.8, MgCl2 (10 mmol L−1), 10% glycerol. RNase A (1 mg mL−1) and DNase I (1 mg mL−1) were added when needed. The sample was sonicated 8 times with a Branson Sonifier 250 sonicator set at a power level  =  2, during 20 seconds at 30 seconds intervals. Cell debris was removed by centrifuging at 10,000 g for 15 min at 4°C. The supernatant was used as the crude extract. The protein concentrations were measured by the Bradford method using BSA as a standard et al. et al. 40 mL of culture were harvested and bacterial cells were suspended in 10 mL Tris-Tricarballylic acid  trifluoroacetamide and injected on a GC-MS . Peak integration was performed using LECO Pegasus software. Because automated peak integrations have been occasionally unreliable, manual controls and/or corrections were systematically performed for each metabolite.GC-MS analyses of amino acids to determine the μ, Sq, O2q, CO2γ, X/SY, ACT/SY, CO2/SY and O2/SY values were constant and at their maximal values. MID values for Glu, Thr, Asp, Ala and Val were identical, with less than 1% RDS. We therefore conclude that both metabolic and the isotopic steady-states were ensured in our experiment.Five independent samples were collected along a 1.5-generation period and analysed in triplicate. During the sampling period, S. coelicolor was deduced primarily from genome annotation The central metabolic network of S. coelicolor were well described Glucose entry and phosphorylation through the Glk-GlcP permease complex in S. coelicolorAll genes and enzymes involved in the Embden-Meyerhof-Parnas pathway (EMP), the tricarboxylic acid cycle (TCA), and the pentose phosphate pathway (PPP) were detected in Streptomyces species (i) there was little growth with two carbon substrates and (ii) both isocitrate lyase and malate synthase were not detected in the S. coelicolor proteome The Entner-Doudoroff pathway (ED) has been shown to be non-functional in several CO2γ values, and analysis of 13C-labeling in valine, alanine, glutamate, glutamine and proline provided further evidence of these assumptions for S. coelicolor under our experimental conditions (see 3.4.).In the present paper, comparison of the experimental and calculated Streptomyces species PEP carboxylase was shown to be the sole anaplerotic enzyme in Streptomyces species are available S. coelicolor could be branched, with a type I NADH dehydrogenase excreting 4 protons per NADH, an uncoupled type II NADH dehydrogenase and two terminal oxidases, the bd complex that excretes 2 protons per NADH and the 1/aa3bc supercomplex that excretes 6 protons per NADH Escherichia coli1/aa3bc was shown to have a lower affinity for oxygen and to be the predominant complex in aerobic conditions 1/aa3bc supercomplex were identified in S. coelicolor proteome 1F0-ATP synthase. Under the assumption that the synthesis of one ATP by F1F0-ATP synthase requires three to four protons, NADH re-oxidation via the bc1/aa3 branch yields 1.50–3.33 ATP. FADH re-oxidation was assumed to be due to succinate dehydrogenase Few experimental data on the respiratory chain efficiency in 2 consumed) of 2.0 could be reasonably considered for S. coelicolor. Nevertheless, our results on carbon flux distribution are not sensitive to the P/O value used.Considering all these data, a P/O value (moles of ATP produced per 0.5 mole of OS. coelicolor (SCO7622 and SCO7623) putatively encode a PntAB-type nicotinamide nucleotide transhydrogenase that interconverts NADP + NADH to NADPH + NAD in E. coliTwo genes of S. coelicolor appeared to be identical to those present in other bacteria Amino acids, nucleic acids, small molecules, peptidoglycan, triacylglycerol and lipids biosynthetic pathways in S. coelicolor were reconstructed using S. coelicolor genome and published data The biosynthetic pathways for the 3 known antibiotics produced by S. coelicolor cells had to be determined. The respective amounts of the main polymers involved in the S. coelicolor biomass composition, namely proteins, DNA, RNAs, triacylglycerol, lipids and peptidoglycan were taken from Borodina et al. To calculate carbon fluxes according to the stoichiometric approach of Holms E. coliS. coelicolor was calculated. We also calculated specific precursor, energy and reducing power requirements to synthesize 1 mole of antibiotics.Using the biomass composition, reactions associated with building-block biosynthesis (primary metabolism) and reactions for secondary metabolism, we calculated precursor, cofactor and energy requirements to synthesise 1 g of dry cells. On the basis of Ingraham's calculations for ln(X(t)) versus t, X(t) versus S(t), ACT(t) versus S(t), RED(t) versus S(t), KET(t) versus S(t), PYR(t) versus S(t), 2(t)CO versus S(t), 2(t)O versus S(t), where X denotes biomass and S denotes source glucose. During exponential growth, each curve was linear, and the slope allowed us to determine growth rate constant μ (h−1) and the following yields: X/SY, ACT/SY, RED/SY , CDA/SY, KET/SY, PYR/SY, CO2/SY, O2/SY. For example, X/SY is the yield of biomass production with respect to glucose, etc. Note that yields are expressed in carbon equivalents, X/Se.g. Y is in moles of biomass carbon per mole of glucose carbon. Using these values, the glucose consumption and productivity of metabolites (μmol (g cell dry weight)−1 h−1) were computed as :For each experiment, we calculated the following graphs: Carbon balance (in %) was calculated as follows:enolpyruvate, pyruvate, acetyl-CoA, α-ketoglutarate and oxaloacetate are the 11 precursors from central metabolism. These precursors are sufficient to synthesize any macromolecule in the bacteria. That is, carbon flux to anabolic reactions can be represented by 11 individual fluxes from precursors to biomass and/or secreted metabolites . Known molar requirements of biomass or antibiotic for precursors −1 and μmol (μmol antibiotic)−1, respectively) and productivity  allow one to calculate anabolic requirement fluxes iΦ and the sum iv for any precursor i, as follows: Glucose-6-phosphate, fructose-6-phosphate, ribose-5-phosphate, erythrose-4-phosphate, glyceraldehyde-3-phosphate, 3-phosphoglycerate, phosphoiΦ were calculated as described in Materials and Methods 12.2, catabolic fluxes in central metabolism calculations remained to be determined to obtain a full carbon-flux distribution. For this purpose, metabolic reactions were represented as a stoichiometric matrix (S) of size {m, n}. Each row of this matrix represented one unique compound amongst the m = 48 compounds and each column represented one reaction amongst the n = 46 lumped metabolic reactions. Note that the 46 lumped reactions correspond to 265 enzymatic reactions. S was a sparse matrix, with 0 as the most represented value. The flux through all reactions in the metabolic network was represented by the vector v (dimension n). The set of concentrations in metabolites was represented by the vector x (length m). The system dynamics is described by dx/dt = Sv. In the steady state, dx/dt = 0, that is, Sv = 0. In the present study, this equation is true as metabolism was studied at the steady state, that is, in the exponential growth phase in which turn-over fluxes are constant and therefore there is no build-up in intracellular metabolite concentration. Glucose specific consumption (Sq) and the 11 anabolic fluxes (iΦ) are used as external constraints. However, the system still has one degree of freedom. Data from labeling experiments were used to solve the system. O2 consumption, CO2 evolution and the respiratory quotient (QR) were not used as constraint here and were rather compared to the computed O2 and CO2 fluxes to check the reliability of our system, as discussed in section Results 2.Although anabolic fluxes 13C-labeling data. The EMU framework used here for the simulation is provided in 13C-1] glucose was used here as the labeling substrate and allowed us to determine the flux-ratio between the pentose phosphate pathway and glycolysis (PPP/Glyc ratio). In fact, when [13C-1] glucose was metabolized by the PPP, the 13C label was lost by decarboxylation, while the 13C label was incorporated into amino acids when it enters glycolysis. We recognize that reversible triose phosphate isomerase and aldolase activities could lead to a 13C-labeling in the C-6 atom position in fructose-1, 6-bisphosphate and then possibly in hexose phosphates 13C-labeling in tryptophan and histidine should provide evidence of the labeling in C-6 because their -COOH group comes from this very atom position. While tryptophan was absent from the spectra since it is degraded to a considerable extent by acid hydrolysis 13C-labeling pattern in histidine  framework istidine  showed tMID data from mass-spectrometer were normalized to total mass-signals as follows, giving relative isotopic abundance:Sq, the 11 anabolic fluxes iΦ and the normalized MID in glutamate (m0/z = 348), threonine (m0/z = 320), aspartic acid (m0/z = 334), alanine (m0/z = 218) and valine (m0/z = 246) were combined to provide a unique solution for all carbon fluxes, thereby solving vector v.Normalized MID were also corrected for natural isotope  abundance during flux calculations. The glucose specific consumption et al. For sensitivity analyses, carbon fluxes were estimated by minimizing the variance-weighted sum of squared residuals (SSR) between the simulated and model-predicted MIDs using non-linear least-squares régression −1 h−1) or relative fluxes with respect to glucose uptake (μmol (μmol glucose) −1). Relative fluxes allowed the comparison between strains, as M145 and M1146 did have different glucose uptake rates.Metabolic fluxes were expressed as absolute (μmol (g cell dry weight)2, CO2). The total CO2 evolution rate was computed from catabolic CO2 production  and anabolic production as: With the formula used for precursors, the stoichiometry of reactions, including those for antibiotics syntheses , and the2 production rate to verify the validity of the carbon-flux distribution (see section Results 4). In other words, experimental CO2 evolution was an independent data point not used as a constraint to calculate carbon-flux distribution. We assumed that NADPH requirement and production should have been equal since the metabolism was in steady state. If the production of NADPH was more important than the need, excess NADPH might have been converted to NADP+, thereby reducing NAD+ into NADH, by nicotinamide nucleotide transhydrogenase.Then, this value was compared with our experimental CO2 consumption, the anabolic demand was completed by the extra consumption required for excess NADH re-oxidation with the following stoichiometry: 1 mole of re-oxidized NADH consumed 0.5 mole of O2. For ATP as well, anabolic and catabolic fluxes were completed by the production of ATP associated with excess NADH re-oxidation. At this stage, knowledge of the actual P/O value (moles of ATP generated per mole of oxidized NADH) would be ideal. As stated in section Materials and Methods 11.1, we assumed that under 70% dissolved O2, the 1/aa3bc cytochrome pathway probably prevailed, and so a P/O value of 2 could be used to estimate ATP production from oxidative phosphorylation. Under this assumption, as for NADH, ATP production would be higher than ATP consumption. Excess ATP production would be likely compensated for by the consumption required for cell maintenance. Such a consumption is hereafter denoted as mATP, that is, energy requirements for homeostasis and turn-over of structural molecules as well as others processes that could not be quantified precisely (e.g. energy needed to excrete antibiotic).Similarly, the metabolic requirement for NADH should match NADH production; but here and quite generally, NADH production was larger than NADH oxidation. In order to satisfy our steady state condition, we assumed that excess NADH was re-oxidized by the respiratory chain. For Oet al. −1) in cell wall composition As mentioned in section Materials and Methods 11.4, we mainly used here metabolite contents reported by Borodina The difference in phospholipid content was considered to be acceptable  since it was previously shown that such a low difference in biomass composition had a negligible impact on carbon flux determination −1). As the mass of the S. coelicolor genome is 1.86-fold that of E. coli, which has a well-accepted DNA content of 3.1% S. coelicolorThe experimental DNA content obtained here was 6.0% (0.06 g (g cell dry weight)2O excess 1.62 O0.45 N0.25 , with 5.7% of ash. This is close to those found in other microorganisms The chemical elemental formula calculated from the data in −1 for M145 and 0.03 g (g cell dry weight)−1 for M1146 .A difference in the triacylglycerol content was found in the biomass composition of M145 and M1146, with a triacylglycerol content of 0.02 g (g cell dry weight)i) the biomass composition (ii) the stoichiometry of reactions describing the biosynthesis of metabolites (iii) the energy requirements re-calculated for S. coelicolor on the basis of published energy requirements for the polymerisation of macromolecules in E. coliUsing −1 and no detectable activity with NAD. Furthermore, we found that 6-phosphogluconate dehydrogenase was strictly NADP-dependent, with a specific activity of 236.4±6.4 µmol min−1 (mg protein)−1 and no detectable activity with NAD. We also verified that isocitrate dehydrogenase was strictly NADP-dependent, with a specific activity of 132.7±4.3 µmol min−1 (mg protein)−1 and no activity at all with NAD.In S. coelicolor genome contains two genes (SCO7622 and SCO7623) encoding a PntAB-like nicotinamide nucleotide transhydrogenase, but no UdhA-encoding gene. In E. coli, PntAB reduces NADH using NADP as a reductant in vivo while UdhA catalyses the reverse reaction −1 (mg protein)−1, and no activity at all with thio-NADP and NADH. Since no evident NADPH phosphatase-encoding gene was found in the S. coelicolor genome, transhydrogenase was then introduced in our model as the enzyme that balanced NADPH overproduction.The −1 and 9 g (cell dry weight) L−1, respectively. The stationary growth phase began when the medium turned to be nitrogen-depleted, glucose and phosphate being limiting approximately 9 h after nitrogen depletion for both strains.Batch-cultivation of the strains M145 and M1146 yielded a bacterial density of 8 g (cell dry weight) Lμ) of M1146 was 13±0.1% higher compared to M145 while the glucose specific uptake rate was nearly 13±0.5% lower (2 consumption and CO2 production) was 20±2% lower in M1146 compared to M145 and furthermore, strain M145 produced 2.03±0.08 µmol (g cell dry weight)−1 h−1 actinorhodin (up to 400 mg L−1 at the end of the culture) while no antibiotic production was detected at all in M1146.During the exponential phase in bioreactors, the growth rate constant  and slightly lower than 100%, suggesting that a certain carbon amount was not recovered. HPLC, LC-MS analyses and bioassays on culture supernatants did not allow the identification of extracellular metabolites that could account for this apparent loss of carbon. Nevertheless, these carbon balances are similar to the 94±9% value found by Borodina −1 h−1 in M145 and 162 µmol±6 (g cell dry weight)−1 h−1 in M1146) and pyruvate (21 µmol±1 (g cell dry weight)−1 h−1 in M145 and 167 µmol±11 (g cell dry weight)−1 h−1 in M1146). There was, therefore, a clear difference in carbon allocation between the two strains, with M1146 liberating more pyruvate and α-ketoglutarate in the medium, likely as a result of carbon re-direction in the absence of growth and antibiotic production, as discussed in section 4.4.During the stationary phase, both strains produced α-ketoglutarate (82 µmol±4 (g cell dry weight)13C-labeling ([13C-1] glucose) and 13C Metabolic Flux Analysis in the isotopic steady-state to estimate the relative commitment to PPP as opposed to glycolysis. Isotopic patterns obtained by GC-MS in amino acids from protein hydrolysates are shown in 13C-labeling  in the strain M1146, clearly showing a lower commitment of the 13C-label to glycolysis and the TCA (from which those amino acids are synthesized) and thus a larger commitment to PPP.The use of stoichiometric coefficients and biomass composition to infer metabolic fluxes  could not be carried out properly since an uncertain flux value remained at the PPP/glycolysis branching-point. We thus used 13C-label patterns in alanine and valine clearly showed the absence of 13C label in the C-1 atom position in both amino acids  with a 95% confidence level in all cases. According to Wiechert et al. (1997), our flux maps are representative descriptions of the metabolic processes in S. coelicolorWe analyzed the statistical significance and confidence of estimated fluxes in In M145 and M1146, the flux through the PPP was found to be 22±0.3% and 38±0.2% of available glucose-6-phosphate, respectively.−1 h−1 or normalized to glucose uptake (μmol (μmol glucose)−1). Consistently, the committed step of PPP (glucose-6-phosphate dehydrogenase) was found to be associated with a flux of 0.372±0.002 µmol (μmol glucose)−1 in M1146, which is 1.7-fold higher than in M145 (0.214±0.003 µmol (μmol glucose)−1). In contrast, the commitment to the TCA was 1.019±0.02 µmol (μmol glucose)−1 in M145, that is, 1.4-fold higher than in M1146 (0.730±0.018 µmol (μmol glucose)−1). Precursor abstraction from primary carbon metabolism to biomass synthesis was larger in M1146 −1, giving 60±1% (molC mol−1C) of carbon uptake allocated to biosyntheses) compared to M145 (0.813±0.02 µmol (μmol glucose)−1, 47±1%). There was a much larger commitment of phosphoenolpyruvate to carboxylation by the PEP carboxylase in M1146 and therefore, a larger anaplerotic oxaloacetate production to sustain the TCA.Our results from Metabolic Flux Analysis and isotopic labeling were combined to draw a carbon flux map in M145 and M1146 during the exponential phase of growth , Fig. 2.2 gave the total calculated CO2 evolution rate . Experimental and computed CO2 and O2 exchange rates gave respiratory quotients (CO2γ/O2q) of 0.982±0.002 for both strains, as would be expected during aerobic growth.Summing anabolic and catabolic fluxes for CONADPH production was similar in both strains , allowing to quantify reliably the 13C-commitment to glycolysis and the TCA versus the PPP.Nevertheless, Metabolic Flux Analysis is usually viewed as unlikely to give a completely faithful representation of the actual flux distribution, and consideration of areas in which thecarbon flux-balance model diverges from known metabolic behaviour may be potentially more informative. For example, the oxidative reactions of the oxidative pentose phosphate pathway typically carry no flux in flux-balance solutions of heterotrophic metabolism in plants S. coelicolor M145  was near 0.04 h−1Using this approach, we determined a contribution of PPP to glucose catabolism in 22±0.3%,  close toμ near 0.04 h−1) in S. lividans (35%) during actinorhodin production Streptomyces noursei (33%) during nystatin production Streptomyces C5 (30%) during ε-rhodomycinone production In M1146, our estimate of the PPP contribution is 38±0.2% , similarS. coelicolorWe postulate that the higher flux through the PPP in M1146 is the consequence of a higher NADPH demand to sustain both a higher growth rate .−1) would cover ATP needs for anabolism (3.927±0.052 µmol ATP (μmol glucose)−1). To the contrary, an apparent ATP deficit (1.118±0.013 µmol ATP (μmol glucose)−1) would exist in strain M1146.In strain M145, ATP generated by catabolism (4.000±0.043 µmol ATP (μmol glucose)−1 and 4.453±0.089 µmol NADH (μmol glucose)−1 were generated in excess by metabolism and from putative NADPH transhydrogenation, respectively.For M145 and M1146, 5.643±0.113 µmol NADH (μmol glucose)As the carbon balance do not close, we are aware that the calculation of ATP needs for anabolism may be affected if ATP is required for biosynthesis of the unidentified metabolites. This should not be the case for NADH, as NADH needs for anabolism are negligible .The amount of ATP generated by NADH reoxidation by the respiratory chain obviously depends upon the P/O ratio. Here, even a P/O ratio as low as 0.25 would have been sufficient to cover ATP needs in M1146.etc.) would have produced an apparent ATP excess in both strains.If P/O ratios larger than 0.25 were taken into account, both the oxidation of total NADH to NAD by the respiratory chain and intrinsic metabolic phosphorylation −1 h−1) of those found in various bacteria S. coelicolorHere, taking into account a P/O value of 2, mATP values (on a biomass basis) would be 7.545±0.3 and 4.482±0.2 mmol ATP (g cell dry weight)−1 h−1) as a function of the dilution rate D (h−1) in a chemostat y intercept being the maintenance energy value Sq values in Streptomyces and a value of 0.425±0.05 mmol glucose (g cell dry weight)−1 h−1 is found. Using our own experimental data, maintenance energy should correspond to 0.498±0.019 mmol glucose (g cell dry weight)−1 h−1 in M145 and 0.341±0.015 mmol glucose (g cell dry weight)−1 h−1 in M1146. Even though such values agree reasonably well with the average value from literature, an experimental determination of energy maintenance under our conditions remains to be determined.A standard way to estimate maintenance energy is to plot specific glucose uptake rate (mmol glucose (g cell dry weight)Since M145 produced solely actinorhodin from acetyl-CoA during its growth under our conditions, the genetic deletion of the cluster for actinorhodin biosynthesis in M1146 expectedly resulted in visible metabolic changes , Fig. 2.−1 h−1) than in M145 (21±1 µmol (g cell dry weight)−1 h−1) during the stationary phase. In the absence of growth, acetyl-CoA could not be redirected to biomass in M1146 and accumulation was simply avoided by excreting its direct, freely diffusible precursor pyruvate.Other evidences for the absence of acetyl-CoA accumulation came from the excretion of larger amounts of pyruvate in M1146 (167±11 µmol (g cell dry weight)−1 h−1 are dedicated to biomass production and 32.5±1 µmol acetyl-CoA (g cell dry weight)−1 h−1 to actinorhodin production. In M1146, with no other modification in glucose uptake and carbon flux distribution, a complete redirection of 32.5±1 µmol acetyl-CoA to biomass would result in a 40% increase of the specific growth rate (0.0552 h−1), a 40% increase of the anaplerotic flux and a decrease of O2q −1 h−1) and CO2γ −1 h−1) values in M1146.During the exponential growth of M145, 80.4±1 µmol acetyl-CoA (g cell dry weight)O2q −1 h−1) and CO2γ −1 h−1) values were also lower.However, the specific growth rate of M1146 was only 13±0.1% higher than that of M145 and the rate of glucose consumption was 13±0.5% lower. In M1146, there was therefore a slightly lower flux to acetyl-CoA synthesis due to the lower glucose consumption rate and as a result, the flux through the TCA and ATP production were lower . AccordiS. coelicolor could grow with μ values up to 0.16 h−1, a high Sq value (2 mmol (g cell dry weight)−1 h−1) and a low actinorhodin production rate (ACTγ = 6 nmol (g cell dry weight)−1 h−1) The reasons of the limited increase in M1146 growth rate are not straightforward since Streptomyces peuceticus var. caesius and S. coelicolor, a transcriptional regulation of glucose uptake rate has been demonstrated First, in et al. S. coelicolor. This regulon encodes proteins involved in the transport or the turn-over of small redox-active toxic compounds E. coli by Fridovitch Second, the absence of actinorhodin synthesis in M1146 might also cause growth limitation as a side effect. Indeed, quite recently, Shin μ) and antibiotic synthesis by accumulating neutral lipidic compounds . In a similar molecular context, that is, in the bldA mutant of S. coelicolor unable to produce actinorhodin and undecylprodigiosin, Plaskitt and Chater (i) a higher actinorhodin production has been observed in the sco0958 mutant impaired in triacylglycerol synthesis (ii) the simultaneous deletion of the gene clusters encoding 5-polyketide synthase and addition of a plasmid comprising the gene cluster encoding actinorhodin biosynthesis, resulted in a dramatic increase of actinorhodin production in S. coelicolorThird, the carbon metabolism in M1146 partly compensated for the reduced growth rate , noviose (derived from glucose-1-phosphate) and 3- or 5-methyl-pyrrole-2-carboxylic acids. These pyrrole groups originate from threonine  or proline (precursor α-ketoglutarate). Flinspach et al. In the case of clorobiocin and coumermycin A1, precursors are, in that order of incorporation, prephenate Click here for additional data file.Table S2Additional GC-MS analyses.(DOC)Click here for additional data file.File S1Supplementary Material and Methods.(DOC)Click here for additional data file.File S2Abbreviations and symbols.(DOC)Click here for additional data file.File S3S. coelicolor.The metabolic network of (DOC)Click here for additional data file.


The aim of our study was to determine whether some bacterial components as well as some proinflammatory cytokines can affect surface mast cell levels. By the use of flow cytometry technique, we documented that freshly isolated mature rat peritoneal mast cells do express surface TLR2 and TLR4 protein, but not CD14 molecules, and respond to stimulation with TLR2 and TLR4 ligands by cysteinyl leukotriene generation. The level of TLR2 protein is modulated by PGN and CCL5 treatment, but not by LPS, LAM, TNF, or IL-6. Surface mast cell TLR4 expression is affected by LPS, LAM, IL-6, and CCL5. Considering that TLR-mediated activation conditions not only engaged these cells in antibacterial defense and development of inflammation but also might influence allergic processes, our observations that surface TLR2 and TLR4 expression can be regulated both bacterial components and proinflammatory cytokines seem to be very intriguing and importance. Mast cell-derived mediators can exert diverse proinflammatory, anti-inflammatory, and/or immunoregulatory effects [Mast cells are found throughout connective tissues of the body and are particularly prevalent beneath the subepithelial surface of the skin, in the respiratory system, in the gastrointestinal and genitourinary tracts, and adjacent to blood vessels and nerves , 2. Upon effects . That is effects  but also effects , 6 and s effects , 7, 8. M effects , 10, inc effects , 12. Mor effects –15. ConsPseudomonas aeruginosa, lipoarabinomannan (LAM) from Mycobacterium smegmatis, and peptydoglican (PGN) from Staphylococcus aureus as well as some proinflammatory cytokines, that is, interleukin (IL)-6, tumor necrosis factor (TNF), and chemokine CCL5 (RANTES), could influence surface TLR2 and TLR4 levels on mast cells. It is without any doubt that toll-like receptors (TLRs), a class of pattern recognition molecules, play a crucial role in early host defense against invading microbes . Several2, CaCl2, glucose, calcium ionophore A23187, sodium azide, and LPS from P. aeruginosa were obtained from Sigma-Aldrich Co. . PGN from S. aureus and LAM from M. smegmatis were obtained from InvivoGen . Nuclear factor (NF)-κB inhibitor—MG-132 was purchased from Biomol . rr (rat recombinant) CCL-5, rrIL-6 and rrTNF were obtained from R&D Systems  and CysLT Immunoassay kit was purchased from Cayman Chemical .Dulbecco's Modified Eagle's Medium (DMEM), Hanks' balanced salt solution (HBSS), sodium bicarbonate, fetal calf serum (FCS), paraformaldehyde, gentamicin, and glutamine were obtained from GIBCO, Life Technologies . Percoll, toluidine blue, trypan blue, bovine serum albumine (BSA) N-2-hydroxyethylpiperazine-N′-ethanesulphonic acid (HEPES), NaCl, KCl, MgClRabbit anti-rat TLR2 IgG antibodies, rabbit IgG isotype control, phycoerythrin (PE)-conjugated donkey anti-rabbit IgG F(ab′)2 fragments, PE-conjugated mouse anti-rat TLR4 IgG antibodies, and PE-conjugates mouse IgG isotype control were obtained from Abcam Inc. . Goat anti-rat CD14 IgG antibodies, goat IgG isotype control, fluorescein isothiocyanate (FITC)-conjugated donkey anti-goat IgG F(ab′)2 fragments, goat IgG antibodies blocking TLR2 and goat IgG antibodies blocking TLR4 were purchased from Santa Cruz Biotechnology . μg/mL gentamicin, and 2 mM glutamine . To prepare purified mast cells, the peritoneal cells were resuspended in 72.5% isotonic Percoll solution and centrifuged . Next, isolated mast cells were washed twice in cDMEM by centrifugation. Purified mast cells were counted and resuspended in an appropriate volume of cDMEM or medium for rat mast cells containing 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM HEPES buffer, 5.6 mM glucose, and 1 mg/mL BSA (pH of the medium was adjusted to 6.9). The mast cells were prepared with over 98% purity, as determined by metachromatic staining with toluidine blue. Mast cells were collected from peritoneal cavities of female albino Wistar rats weighing 200–250 g by lavage with 50 mL of 1% HBSS supplemented with 0.015% sodium bicarbonate. After abdominal massage (90 s), the cell suspension was removed from the peritoneal cavity, centrifuged , and washed twice in complete (c) DMEM containing DMEM supplemented with 10% FCS, 10 6 cells/mL and incubated for 60 min at 37°C with LPS at final concentration of 102 ng/mL, LAM or PGN at final concentrations of 102 μg/mL, calcium ionophore A23187 at final concentration of 5 μg/mL (positive control) or buffer alone (spontaneous cysLT generation). The supernatants were collected by centrifugation  and analyzed by an ELISA commercial kit that detected LTC4 and its degradation products LTD4 and LTE4. The sensitivity of this assay was <13 pg/mL. Purified mast cells were resuspended in appropriate volume of medium for rat mast cells to obtain mast cell concentration of 1.5 × 10μg/mL), with NF-κB inhibitor MG-132  or medium alone for 15 min in a humidified atmosphere with 5% CO2 at 37°C. The cells were then washed twice, resuspended in medium and incubated with bacterial antigens, as described above. To determine the specificity of bacterial antigen action on cysLT generation in some experiments, mast cells were preincubated with anti-TLR2 or anti-TLR4 antibodies . Subsequently, 10 μL of LPS at final concentrations of 100 ng/mL and 102 ng/mL, PGN and LAM at final concentrations of 100 μg/mL and 102 μg/mL, CCL5, IL-6 or TNF , or cDMEM (control TLR2 and TLR4 expression on unstimulated mast cells) was added. Mast cells were incubated for 1, 3,  6,  12, and 24 h at 37°C in a humidified incubator with 5% CO2. After each period of incubation the cells were washed twice in 1% PBS  and resuspended in 90 μL of PBS. Additionally, after each period of incubation mast cell viability was examined by trypan blue exclusion assay. Purified mast cells resuspended in cDMEM were divided into 90 μL of 1% PBS and incubated with: (1) rabbit anti-rat TLR2 IgG antibodies or rabbit IgG isotype control and (2) goat anti-rat CD14 IgG antibodies or goat anti-rat IgG isotype control, respectively, at final concentrations of 1 μg/106 mast cells at 4°C for 45 min. After incubation the cells were washed in 1% PBS  and incubated with PE-conjugated donkey anti-rabbit IgG F(ab′)2 fragments (TLR2 detection) or FITC-conjugated donkey anti-goat IgG F(ab′)2 fragments (CD14 detection) at final concentrations of 1 μg/106 mast cells at 4°C for 45 min in dark. For direct TLR4 staining, mast cells were suspended in ice cold 1% PBS supplemented with 1% FCS and 0.05% sodium azide and incubated with PE-conjugated mouse anti-rat TLR4 IgG antibodies or PE-conjugated mouse IgG isotype control at final concentrations of 1 μg/106 mast cells at 4°C for 60 min in dark. After incubation with antibodies/isotype control, the cells were washed twice with 1% PBS  and finally resuspended in 300 μL of 1% paraformaldehyde. Stained mast cell fluorescence was measured with a FACSCalibur flow cytometer with CellQuest software. The stimulated mast cell TLR2, TLR4, and CD14 expression was presented as percentage of TLR2, TLR4, and CD14 MFI (mean fluorescence intensity) measured in unstimulated mast cells (referred to as 100%).For indirect TLR2 and CD14 staining, mast cells were fixed with 1% paraformaldehyde for 15 min, washed twice with 1% PBS , resuspended in 90 t test for “small groups”. Values of P < .05 were considered statistically significant. Statistical analysis included mean value, standard error of the mean (SEM), and Student's We first examined the presence of TLR2, TLR4, and CD14 proteins on freshly isolated rat peritoneal mast cells by flow cytometry analysis. We stated that rat mast cells expressed both TLR2 and TLR4 molecules, and expression of surface TLR4 was higher than TLR2 level . We alsoP < .01) inhibited cysLT generation  decrease in LPS-, LAM-, and PGN-induced cysLT release  increase in TLR2 expression level following 1 and 3 h incubation, as compared with the control unstimulated cells  downregulated the expression of TLR2 after 24 h incubation  for 1,  3,  6,  12, or 24 h and TLR2 level was estimated by flow cytometry analysis. We found that stimulation of mast cells with LPS used at concentrations of 10 Figures . Treatme Figures . We also Figures ; however Figures . P < .01) downregulation of TLR4 level following 3 h incubation, while the exposure of mast cells to 102 ng/mL of LPS resulted in a statistically significant (P < .01) increased expression of TLR4 after prolonged 12 and 24 h incubation  increase in TLR4 expression level following 3 h incubation, as compared with the control unstimulated cells (Figures The exposure of mast cells to TNF did not cause essential alteration in surface TLR4 level, at any time point examined, as shown in  Figures , while s Figures . It is well documented that mast cells express mRNA for TLR2 and TLR4 proteins. TLR2 and TLR4 mRNA expression was detected in both immature murine –25 and hConsidering the significance of TLR molecules in the development of both innate and adaptive immunity , 38 it sMycobacterium, that is, TLR2 and TLR4 ligands, respectively, on TLR2 and TLR4 expression on mast cells. Taking into account that mast cells play a crucial role in inflammatory processes [γ, and Yang et al. [The impact of humoral factors and bacterial components on mast cell TLR expression is, as yet, not well recognized. Therefore, we decided to determine the influence of some important bacterial antigens, such as PGN—a main cell wall component of Gram-positive bacteria, LPS—an important component of Gram-negative bacteria, and LAM—a major virulence factor in the bacteria genus rocesses , 6, we arocesses  examinedrocesses , 49. We rocesses  found thg et al.  stated tg et al.  and cathg et al. . de novo synthesis and release various cytokines [The changes in TLR2 and TLR4 level on mast cells induced by bacterial components may be caused by a direct effect of ligands, that is, PGN or LPS, on its own receptors TLR2 and TLR4, respectively. However, observed modification of TLR expression could be mediated by an indirect mechanism involving cytokine production. It has been established that prolonged mast cell stimulation with bacterial cell wall components, such as LPS and PGN, resulted in ytokines , 54, 55.Considering that TLR-mediated activation of mast cells conditions not only engaged these cells in antibacterial defense and development of inflammation but also might influence allergic processes , 57, our


Nutrient intake is critical to a bodybuilder in terms of improving the overall muscular appearance of their physique. Total energy intake and the proportion of the kilocalories derived from carbohydrates, protein, and fats are often precisely planned and implemented to maximize skeletal muscle hypertrophy and reduce body fat. The purpose of this study is to describe the self-reported energy intakes of male and female bodybuilders and to determine if differences exist between the genders in regards to total energy intake and macronutrient composition.A comprehensive literature review was performed using the PubMed database. Every effort was made to generate all relative articles pertaining to male and female bodybuilders’ self-reported energy intakes. The search yielded a total of 13 articles, 8 male bodybuilder studies and 5 female bodybuilder studies. The studies summarized contained professional, collegiate, and international bodybuilders during the offseason or non-competitive/non-dieting phase. In 12 of the 13 studies included, energy intakes were derived from food records ranging from 3 days to 7 days. The other study used a food frequency questionnaire. Total kilocalories, kilocalories/kg of body mass, kilocalories/kg of fat-free mass (FFM) and macronutrient composition were recorded and analyzed. Differences between male and female bodybuilders were analyzed via an independent samples t-test using IBM SPSS Statistics (v20).All data are reported as means ± standard deviations. Total kilocalories were 4,049 ± 892 and 2,067 ± 525 for male and female bodybuilders, respectively. The males ingested significantly more total kilocalories than the females (p = 0.001). When kilocalories were expressed per kilogram of body weight, male bodybuilders ingested 47.4 ± 10 and females ingested 35.8 ± 9. No significant differences existed between male and female bodybuilders (p = .064). When kilocalories were expressed per kilogram of FFM, male bodybuilders ingested 54.3 ± 12 and female bodybuilders ingested 41.6 ± 11. There were no significance differences in the amount of kilocalories per kilogram of FFM (p = .126). Total % of carbohydrate ingested was 48 ± 6% and 54 ± 3% for males and females, respectively. No significant differences were demonstrated between the genders (p = .070). The total % of protein ingested for males were 21 ± 2% and females was 24 ± 6%. No significant differences were demonstrated (p = .245). The total % of fat ingested for males were 31 ± 4% and females was 25 ± 8%. Although males reported a higher percentage of total fat ingested, no significant differences existed (p = .060).Based on the data, male bodybuilders reported ingesting significantly more total kilocalories than female bodybuilders. However, when adjusted for body mass and fat free mass, no significant differences exist between the genders. In relation to macronutrient composition , no significant differences exist between male and female bodybuilders.