Subclinical low-grade systemic inflammation has been associated with obesity, insulin resistance and metabolic syndrome (MS). Recent studies have highlighted the role of gut microbiota in these disorders. The toll-like receptor 4 (TLR4) plays a key role in the innate immune response activation. We studied two polymorphisms (+3725G/C and 11350G/C) in the 3′ untranslated region (3′UTR) of the TLR4 gene that may alter its expression and their association with metabolic disorders related to systemic inflammation. We cloned the 3′UTR into a luciferase reporter system and compared wild-type 3′UTR (WT) and +3725C variant (MUT) constructs luciferase activities. MUT construct reduced the reporter gene activity by 30% compared to WT (P = 0.0001). To evaluate the association between these polymorphisms with biochemical and clinical overweight related variables, we conducted a population cross-sectional study in 966 men of Argentine general population. Considering smoking as a confounding variable that causes systemic inflammation, we studied these possible effects in both, smokers and nonsmokers. The 11350G/C polymorphism was not detected in our sample whereas the CC genotype of +3725 polymorphism was associated with lean subjects (p = 0.011) and higher Adiponectin levels (p = 0.021). Subjects without any NCEP/ATP III MS component were associated with this genotype as well (p = 0.001). These results were strengthened in nonsmokers, in which CC genotype was associated with lean subjects (p = 0.003) and compared with G carriers showed significantly lower BMI  and waist circumference . None of these associations were found in smokers. These results showed that +3725C variant has a functional effect down-regulating gene expression and it could be considered as a predictive factor against overweight, particularly in nonsmokers. Considering the role of TLR4 in inflammation, these findings would suggest that the presence of +3725C variant could predict a lower prevalence of chronic metabolic disorders. Obesity and Type 2 Diabetes Mellitus (T2DM) prevalence has increased throughout the world, mainly due to rapid lifestyle changes Subclinical low-grade systemic inflammation has been associated with obesity, metabolic syndrome (MS) and T2DM The adipose tissue is an important endocrine and secretory organ, which releases a wide range of signals and specific factors termed adipokines, such as Leptin and Adiponectin On the other hand, as confirmed by numerous population-based studies, a low-grade systemic inflammatory response is also evident in smokers: elevated levels of C-reactive protein (CRP), fibrinogen, and interleukin-6, as well as increased counts of white blood cells (WBC) have been reported 712His) in TLR4 prevents diet-induced obesity and IR In Drosophila, Toll mediates dorso-ventral embryonic polarization and activates immune responses to fungal infection. A human homologue of the fly Additionally, two miss-sense mutations in TLR4 gene (Asp299Gly and Thr399Ile) have been reported to be associated with endotoxin hyporesponsivenes The control of mRNA stability is an important process that allows cells to rapidly adjust protein levels providing a fine tuning mechanism to regulate gene expression. Variations in the 3′-UTR are known to altered gene expression levels through a direct effect on mRNA stability or translation efficiency. In particular, two polymorphisms +3725G/C (rs11536889) and 11350G/C (rs41426344) in the 3′UTR of TLR4 gene were recently found to be associated with alterations in the immune response Taking into account the role of TLR4 as a molecular gate linking inflammation with insulin resistance, diabetes, and obesity In this study we present the analysis of the functional effect of +3725G/C and 11350G/C sequence variants of TLR4 gene on mRNA stability and the association between these polymorphisms and markers of inflammation, MS and metabolic associated traits in non diabetic men, focusing especially on the potential interaction with smoking status.Each subject signed a consent form to participate in this genetic study. The study was carried out in accordance with the Declaration of Helsinki, and approved by the Ethic and Research committees of the “Hospital de Clínicas”, Universidad de Buenos Aires (UBA), Argentina.All 966 male individuals were unrelated subjects from general Argentinian population self-reporting European ancestry, especially from southern European countries , living in Buenos Aires metropolitan area, recruited between April 2006 and April 2010. The clinical characteristics of the sample are shown in www.ba.itb.cnr.it/BIG/UTRScan/) and Targetscan (http://www.targetscan.org/) were used for searching putative UTR functional elements www.bioinfo.rpi.edu/applications/mfold/) Online programs UTRSCAN (GCTAGCAGGAAGCAACATCTATCTGAAGAGGAAAAA-3′5′-GC and TLR4_3′UTR_R: ATCGATTGGTCAGTTATGGTCTGAAAATAGGTGAGT-3′5′-GC. Wild type (WT) and mutant TLR4 +3725C (MUT) constructs were obtained by cloning the PCR products into the pCR2.1 vector by using a TOPO-TA cloning kit (Invitrogen). Plasmid DNA was prepared using the Qiagen plasmid Maxi kit . The primers contain two recognition sites for enzymes NheI and ClaI respectively (underlined above) to enable cloning into the firefly luciferase expression plasmid . Luciferase constructs were verified by sequencing.Genomic DNA from peripheral blood from homozygous individuals for each variant was used as template in a polymerase chain reaction (PCR). The entire TLR4 3′UTR (2908 bp) was amplified using the primers: TLR4_3′UTR_F: E. coli. Twenty four hours after transfection, the cells were harvested and assayed for firefly luciferase and β-gal activities using Luciferase Assay Reagent (Promega) and ortho-Nitrophenyl-β-galactoside (ONPG) respectively. Measurement of luciferase activity was performed on the GloMax®-Multi Detection System Promega. The relative firefly luciferase activities were normalized against the β-gal activities.HeLa Tet-off advanced (Clontech) cells were manteined in DMEM High Glucose medium  supplemented with 10% fetal bovine serum (FBS), 100 units/ml Potassium Penicillin, 100 µg/ml Streptomycin Sulfate, 250 ng/ml Amphotericin B at 37°C in a humidified chamber containing 5% CO2. Cells (200.000 cells/well) were seeded in a 12-well plate 24 h before transfections. Each construct (1 µg) was co-transfected with pCMV β-gal plasmid (100 ng) as an internal control using 3 µl of Polyethylenimine (PEI) 1 µg/ml. The pCMV β-gal plasmid contains the reporter gene β-galactosidase  from Anthropometric measurements , systolic and diastolic blood pressure  were determined by a standardized protocol. After a 12-h overnight fast, blood samples (20 ml) were drawn in every individual, centrifuged and frozen immediately at –20°C. Total cholesterol (TC), triglycerides (TG), and high-density lipoprotein cholesterol (HDL-C) were determined in serum by enzymatic methods using commercial kits  in a Hitachi 727 autoanalyzer. All determinations were performed using standardized procedures. Intra-CV (coefficient of variation) for TG and total cholesterol were 1.3 and 1.1%, respectively. Inter-CVs were 2.4% and 1.5%, respectively. LDL-C (low-density lipoprotein cholesterol) was calculated using the Friedewald formula (LDL-C = TC – HDL-C – [TG/5].Fasting plasma glucose (FPG) was determined by a glucose-oxidase method  in a Hitachi 727 autoanalyzer. Intra- and inter-CVs were 0.9 and 1.8%, respectively. Fasting serum insulin was measured by radioimmunoassay with a commercial kit  by counting on a gamma counter , with a lower detection limit of 2 µU/ml, intra- and inter-CVs being <1 and <7.43%, respectively. Cross-reactivity specified by the suppliers is less than 0.2% to intact human proinsulin. Serum C-reactive protein (sCRP) concentration was determined by Tina-quant CRP (Latex) high-sensitive immunoturbidimetric assay (Roche Diagnostics) in a Hitachi 917 autoanalyzer. Within-run and between-day precisions (coefficient of variation) were 0.4% and 3.4%, respectively. Adiponectin was measured by radioimmunoassay with a commercial kit  by counting on a gamma counter . Intra- and inter-assay CVs were 3.73% and 8.24%, respectively.Cigarette smokers were defined as having smoked at least 1 cigarette per day for 1 year or more. Those who reported smoking at least 1 cigarette per day during preceding year were classified as current smokers.www.dtu.ox.ac.uk/index.php?maindoc=/homa/) Each subject was assessed for the presence of MS using the National Cholesterol Education Program/Adult Treatment Panel III (NCEP/ATP III) criteria TLR4 +3725G/C (rs11536889) polymorphism were scored blindly using allele specific PCR confronting two-pair primers analysis, as previously described C allele, 184 bp for G allele, and 397 bp for a common band. Our lab randomly sampled 10% of the subjects for sequencing to corroborate the initial findings. Using these criteria consistency was 100%. Genotyping of SNP 11350G/C (rs41426344) was performed by KBioscience (http://www.kbioscience.co.uk ) using the KASPar chemistry, which is a competitive allele specific PCR SNP genotyping system using FRET quencher cassette oligos (http://www.kbioscience.co.uk/genotyping/?genotyping-chemistry.htm). Blind duplicates, plate-identifying blank wells, and Hardy-Weinberg equilibrium tests were used as quality-control tests.Genotypes for the 2 test. A two-tailed probability value of 0.05 was considered to be statistically significant. Odds ratios (ORs) are reported with their 95% confidence intervals (CIs). For univariate analysis, differences between groups for qualitative variables were assessed by Fisher’s exact test. One-way ANOVA was conducted for comparison of continuous variables and Levine’s test was performed to study equality of variances. Log-transformed variables were used whenever variables showed non normal distribution. Logistic regression models were used in order to adjust for age the association between variables and single nucleotide polymorphisms on TLR4 gene, for this purpose, logistic regression was used as discriminant factor. Variables that appeared to be associated in the univariate analysis were introduced into the multivariate analysis. Multiple linear regression and logistic regression analyses were used to adjust for possible confounding variables. Subjects were also grouped into tertiles to waist circumference, and differences in events among tertiles were calculated by a contingency Fisher’s exact test. In the in vitro experiments, both cell transfection and luciferase assay were performed in triplicate and analyzed using the Fisher’s exact test. Statistical significant differences were considered when p<0.05 and results were presented as their means±SD. Statistical analyses were conducted using the program for Statistical Package for the Social Sciences, version 12.0  and Prism 4.0 .Data on continuous variables are shown as means±SD. Categorical variables are reported as proportions (%). Deviation from the Hardy–Weinberg equilibrium was assessed using χConsidering that variations at the 3′UTR may result in differential post-transcriptional regulation or compromised translation, bioinformatic analysis was performed in order to look for functional patterns present in this region.The total length of TLR4 3′UTR determined by the University of California Santa Cruz (UCSC) genome browser was 2840 bp, with the SNP +3725 located in the middle of the 3′UTR sequence. Using the on-line UTRscan program, we found that there are five Au-rich elements (AREs) on the 3′UTR of TLR4. These are located in nucleotide positions 447–451, 1619–1623, 1776–1780, 1860–1864 and 2145–2149. The SNP +3725G/C is located between the first and the second ARE. Additionally, we found other potential regulation areas such as: a Gy-Box, at nucleotide positions 219–225, which may participate in duplex formation with complementary sequences found at 5′ ends of certain miRNAs HeLa cells were transiently transfected with the recombinant luciferase vectors (MUT or WT constructs). Twenty four hours after transfection, the β-gal-normalized luciferase activity of the MUT construct was lower than that of WT construct (p = 0.0001). In addition, the MUT construct reduced the reporter gene activity by 30% compared to that of WT construct .According to the evidence, TLR4 activation induces up-regulation of inflammatory pathways related to the induction of IR, such as IKKß/IκBα/NF-κB and JNK Variations at the 3′UTR could affect several processes such as mRNA transcription efficiency, stability, post-transcriptional modification, degradation and translation. Messenger RNA containing AREs in their 3′untranslated region are particularly labile, allowing a very precise control of their temporal and spatial expression patterns. Between 5–8% of human genes code for ARE-containing mRNAs, mostly related to growth factors, oncoproteins, and cytokines. These sites bind specific proteins that regulate the mRNA stability. The presence of the +3725G/C polymorphism between the first and the second AU-rich element suggested that the SNP might influence mRNA stability, altering gene expression levels and LPS and/or another endogenous ligands response. This differential expression of TLR4 could possibly result in down-regulation of inflammatory pathways related to metabolic disorders induction.The average age of the subjects was 39.67±12.85 years. The clinical characteristics for all participants (966 men) are presented in The prevalence of NCEP/ATP III-MS was 24.6% (n = 237). The frequency of 0, 1, 2, 3, 4 and 5 criteria for MS was 23.9% (n = 231), 27.7% (n = 268), 23.8% (n = 230), 15% (n = 145), 7.2% (n = 69), and 2.4% (n = 23), respectively.The DNA samples from the 966 participants were screened for the presence of two SNPs in the 3′-untranslated region (3′UTR) of the TLR4 gene. The genotype frequency of the TLR4+3725G/C polymorphism (rs11536889) was: 79.8% for G/G, 18.5% for G/C, and 1.7% for C/C, which was in Hardy–Weinberg equilibrium . The minor allele frequency found among our population was 0.12, similar to the reported allele frequency in Caucasians  2 (p = 0.008), OR = 0.27 [CI 95% 0.10 to 0.70] . The association analysis was therefore repeated taking into account different models of inheritance (dominant and recessive). The presence of the CC genotype in the TLR4 +3725G/C polymorphism under a recessive model, was associated with lean subjects, BMI less than 25 kg/mto 0.70] . The assAs hsCRP values were not normally distributed, log transformation was used. The association between log-hsCRP levels and the smoking status was studied. According to ANOVA test, smoking status was significantly associated with log-hsCRP concentration (p = 0.01) When the sample was stratified by cigarette smoking status, the association between rs11536889 and lean status was strengthened. TLR4 +3725 G/C polymorphism was significantly associated with lean status among nonsmokers (p = 0.004), OR = 0.16 [CI 95% 0.05 to 0.56], but not among current smokers (p = 0.65) . The ass2; age adjusted p = 0.023), and WC .Taking into account these results and considering that the waist circumference (WC) is a good marker of intra abdominal adiposity, the association between CC genotype and WC was studied in nonsmokers and smokers. Among nonsmokers frequencies between CC vs. CG+GG genotypes and lower tertile of waist circumference (WC<91 cm) were significant . In this way, CC genotype carriers compared with G carriers showed significantly lower BMI . As shown before, the smoking status behaves as a confounding variable. When introducing it into the multivariate analysis, association between CC genotype and Adiponectin levels remained significant .The relationship between the CC genotype and the presence of MS components was studied. Since the CC genotype showed a potential relationship against development of overweight, we studied the presence of this polymorphism within individuals without any MS component vs. subjects with at least one component. Among individuals carrying the rs11536889 CC genotype, the prevalence of subjects without any MS component was 59% (n = 10) and 41% (n = 7) for subjects with at least one, and among GG and GC genotype 31% (n = 226) and 69% (n = 723), respectively (p = 0.001 age adjusted); OR = 0.166 [95% CI 0.05 to 0.49]; (age and BMI adjusted p = 0.008). These association were significant even after adjustment for confounding variables age, BMI and smoking status (p = 0.020). By studying each component individually, a non significant trend was observed consistent with former results. In particular for each MS component the following ORs were determined: WC >102 cm, OR = 0.60, p = 0.59; fasting TG ≥150 mg/dl, OR = 0.56, p = 0.38; SBP≥130 mmHg, and/or DBP≥85 mmHg, OR = 0.40 p = 0.25; fasting HDL-C <40 mg/dl, OR = 0.65 p = 0.43 and FPG ≥100 mg/dl, OR = 0.56 p = 0.47¨ age, BMI and smoking status adjusted.We performed a functional characterization of TLR4 +3725G/C polymorphism in order to evaluate its impact on gene expression. Our findings show a substantial down regulation in gene expression when comparing luciferase activity in WT or MUT (+3725C) transfected HeLa cells. These results support the hypothesis that +3725G/C polymorphism affects mRNA stability. On the other hand this encourages further functional studies in order to unveil the mRNA regulatory mechanisms involved, and the impact of these variants in the up-regulation of inflammatory pathways related to IR.This is the first study demonstrating significant associations between TLR4+372G/C gene polymorphism (rs11536889) and body mass index, waist circumference, Adiponectin levels and reduced risk for MS components modulated by cigarette smoking status in a general Caucasian male population.Specifically, individuals carrying the CC genotype at the +3725 base-pair position in TLR4 gene showed lower prevalence of overweight, especially nonsmokers (never or former smokers) and higher Adiponectin levels. In addition, CC genotype was associated with lean subjects and waist circumference lower tertile only among nonsmokers. CC genotype carriers, compared with G carriers showed significantly lower BMI and WC among nonsmokers as well. Additionally, we found an association between rs11536889 and subjects without any NCEP/ATP III component for MS.Plasmatic Adiponectin levels are known to be lower in obese than in lean subjects. Particularly a strong negative correlation between body mass index and Adiponectin serum levels has been previously shown. Our results are consistent with these observations, since Adiponectin levels were higher in CC genotype subjects.It has also been suggested that Adiponectin has anti-inflammatory properties and acts as an endogenous modulator of obesity-related diseases through its insulin-sensitizing function. In order to study the inflammatory stage in our population, a well-known marker for systemic inflammation, CRP, was determined. A strong relationship between cigarette smoking and systemic inflammation was confirmed by numerous population-based studies: elevated levels of CRP, fibrinogen, and interleukin-6, as well as increased white blood cells counts have been reported Considering that gene-environment interactions may change the effects of genetic factors by modifying environmental exposures and in particular that a low-grade systemic inflammatory response is evident in smokers, we studied the possible effects of rs11536889 in both, smokers and non smoker subjects. We found that only among non smokers, CC genotype was associated with lean subjects and waist circumference lower tertile. Taking into account that intra-abdominal adiposity correlates with waist circumference In order to explain the loss of association between rs11536889 and body weight and associated metabolic disorders in smokers, we hypothesize that cigarette might act as a confounding variable. On one hand, it is known that smokers display increased pro-inflammatory markers  Another important point to note is that TLR4 genetically deficient mice were reported to be an “ideal body type” when fed on regular chow, having an increased density, size and bone mineral content, as well as decreased body fat. Unlike many laboratory wild-type mice, this strain does not become obese with age. Considering that we studied a young human population (mean age of 39 years old) and that Adiponectin levels are diminished many years before the metabolic disturbances of IR are expressed, our findings are consistent with the known protective effect of Adiponectin on the development of metabolic disorders. The relationship between TLR4 and the amount and distribution of adipose tissue brings the function of TLRs in mammals much closer to that of Toll in the fly, being critical for the establishment of the body plan we have shown for the first time a down-regulating effect of +3725G/C on TLR4 expression and an association with overweight, particularly in nonsmokers. Considering the role of TLR4 in inflammation, these findings would suggest that the presence of +3725C variant would predict a lower prevalence of chronic metabolic disorders.


Compared to the golden standard glycation index of HbA1c, glycated albumin (GA) has potentials for assessing insulin secretory dysfunction and glycemic fluctuation as well as predicting diabetic vascular complications. However, the reference ranges of GA and a conversion equation need to be clearly defined. We designed this study to determine the reference ranges in patients with normal glucose tolerance (NGT) based on conventional measures of glycemic status and to devise a conversion equation for calculating HbA1c and GA in a Korean population.th to 95th percentile value of GA in subjects with NGT was adopted. The conversion equation between HbA1c and GA was devised using an estimating regression model with unknown break-points method. The reference range for GA was 9.0–14.0% in 2043 subjects. The 95th percentile responding values for FPG, and HbA1c were approximately 5.49 mmol/l, and 5.6%, respectively. The significant glycemic turning points were 5.868% HbA1c and 12.2% GA. The proposed conversion equation for below and above the turning point were GA (%) = 6.960+0.8963 × HbA1c (%) and GA (%) = −9.609+3.720 × HbA1c (%), respectively.In this multicenter, retrospective, cross-sectional study, we recruited antidiabetic drug-naïve patients with available glycemic variables including HbA1c, GA, and fasting plasma glucose regardless of glucose status. For the reference interval of serum GA, 5These results should be helpful in future studies on the clinical implications of high GA relative to HbA1c and the clinical implementation of diabetes management. Currently, HbA1c is the golden standard glycation index for use in clinical practice and research Besides the role of GA as an intermediate glycation index, several previous studies have suggested additional values of GA in reference to HbA1c levels in assessing insulin secretory dysfunction and fluctuating glycemic excursions Based on a previous study, where GA reportedly increased by 2.5–3.2% for every 1% increase in serum HbA1c (range: 6.5–14.0% HbA1c) This study was approved by the independent ethics committee/institutional review board (IRB) at each study site . All enrolled subjects provided written informed consent.2 according to the Modification of Diet in Renal Disease criteria [MDRD]); active thyroid disease; liver cirrhosis; or nephrotic syndrome.Study subjects were recruited from outpatient clinics at 6 major referral centers; a total of 2450 patients who were registered with the health check-up program of Severance Hospital or the Newly Detected Diabetes Registry in Asan Medical Center, Kyunghee Hospital, Kangdong Kyunghee Hospital, Kangbuk Samsung Hospital, Samsung Medical Center, and Severance Hospital were recruited. All patients had their GA, HbA1c, and fasting plasma glucose (FPG) levels measured. All study participantshad fasting C-peptide levels of >0.5 ng/mL. We excluded patients without GA, HbA1c, or FPG data and those with any of the following criteria that might affect GA or HbA1c: hemoglobin (Hb) level <12 g/dL for women and <13 g/dL for men; chronic kidney disease ≥ stage 3  was calculated as weight in kilograms divided by the square of height in meters. All blood samples were obtained in the morning, following an overnight fasting of at least 12 hours. Plasma glucose was measured by the hexokinase method. Lipid parameters, including serum total cholesterol, triglycerides, HDL cholesterol (HDL-C), directly-measured LDL cholesterol (LDL-C) levels, and liver enzymes, including aspartate aminotransferase (AST), alanine aminotransferase (ALT), and gamma-glutamyltransferase (GGT), were measured using an enzymatic colorimetric method. Serum GA was determined by an enzymatic method using an albumin-specific proteinase, ketoamine oxidase and albumin assay reagent , and a Hitachi 7699 Pmodule autoanalyzer . The coefficient of variation (CV) was 1.43%. HbA1c was measured by high-performance liquid chromatography (HPLC) method. The reference intervals of HbA1c were between 4.0% and 6.0%. HOMA of insulin resistance (HOMA-IR) was calculated as the product of fasting serum insulin (µU/ml) and FPG (mmol/l) concentrations, divided by 22.5. HOMA of beta cell function (HOMA-beta) was calculated according to the equation: HOMA-beta (%) = (20×fasting serum insulin)/(FPG-3.5). All enzyme activities were measured at 37°C.In this study, NGT was defined by the previously defined criteria th percentile of patients with NGT). The relationship between glycemic parameters including FPG, GA, and HbA1c were assessed by Pearson correlation analysis. The relevant values of HbA1c and GA corresponding to specific FPG concentrations were calculated using linear regression analysis after assigning HbA1c and GA as dependent variables and FPG as independent variables. R version 2.14.0  was used to analyze the data. All p-values <0.05 were considered statistically significant.Continuous variables are expressed as the mean (± standard deviation [SD]). Categorical variables are expressed as proportions (%). Demographic and biochemical characteristics of the study population between the two arbitrarily defined groups in this study were compared using independent t-test for continuous variables and the chi-squared test for categorical variables. To determine the relationship between serum GA and HbA1c levels according to the deterioration of glucose tolerance, we performed the estimating regression model with unknown break-points as previously described 2. When we fitted estimating regression models with unknown break-points, serum GA levels were drastically increased from HbA1C level of 5.868%  and more obese . The Hb concentration were similar between the two groups . Similar to our previous reports vs. 4.4±0.3 mg/dL in group I and group II, respectively; p<0.001). The percentage of current smoker was similar , but the blood pressure and glucometabolic profiles, including FPG, GA, HOMA-IR, and HOMA-β, were significantly higher in group II. Finally, significantly more patients in group II were taking antihypertensive and antilipid medications.A total of 2043 subjects out of all that were recruited satisfied the inclusion and exclusion criteria of this study. The clinical characteristics of the study participants are shown in f 5.868% . Based oth percentile interval value was approximately 11.0–12.5% for GA, while the 5–95th percentile interval was approximately 9.0–14.0% in patients with NGT. In this study, the 95th percentile values for FPG, GA, and HbA1c were approximately 99 mg/dL, 14.0%, and 5.6%, respectively. The FPG and HbA1c values, according to the individual percentile points, were almost exactly same between sexes; but, the GA values were similar.r = 0.915; p<0.001; r = 0.821; p<0.001, r = 0.817; p<0.001, p<0.001) and 0.912 (p<0.001) for groups I and II, respectively.In Current clinical guidelines for assessment of glycemic control recommend self-monitoring of blood glucose (SMBG) by patient and HbA1c as a part of hospital continuing care th–95.0th percentile reference interval for GA was 9.0% to 14.0% while adopting the cut-off values of impaired glucose tolerance of both FPG levels ≥5.55 mmol/l, and HbA1c levels ≥5.7%. Second, we noted a significant glycemic turning point at 5.868% HbA1c and an apparently linear slope (both below and above this point) on continuous plots for GA and HbA1c in patients with various levels of glucose intolerance . Third, we devised the following conversion equations for GA and HbA1c in groups I (HbA1c<5.868%) and II (HbA1c≥5.868%):GA (%) = 6.960+0.8963 ×HbA1c (%) and GA (%) = −9.609+3.720×HbA1c (%), respectively.The aim of the present study was to develop a simple equation for converting HbA1c to GA in a Korean population. By establishing expected GA levels from HbA1c, we could get additional information on the glycemic fluctuation, insulin secretory dysfunction and pro-atherogenic condition in subjects with T2D in whom the laboratory levels of GA would be above the calculated GA levels. The present study demonstrates 3 main findings. First, the 5.0th percentile) th percentile was 10.8–17.1% GA thpercentile) in this population of Korean patients (n = 2043) did not exactly correspond to other Asian ethnicities, including Japanese and Chinese populations. This could be explained by the different FPG cutoff values that were used to define NGT (<110 mg/dL in Japanese population) Regarding the reference values for GA, it ranged between 11.9–15.8% (mean ±2 SD) in a healthy American population of both white and black patients without a known history of diabetes in North Carolina (n = 201 patients); this range was determined using the 75-g oral glucose tolerance test (OGTT) and demonstrated a significant racial difference in vivo nonenzymatic glycation rate of albumin is approximately 9 times that of human Hb We used 2 steps to develop our conversion equation. In the first step, we identified a significant glycemic turning point  = 6.960+0.8963 × HbA1c (%) and GA (%) = −9.609+3.720 × HbA1c (%), respectively. Using the equation that we proposed, the differences between measured GA level and calculated GA level could be identified. These results should be helpful in future studies on investigating the clinical implication of GA as a glycemic index in specific patients with unexpected high GA levels over the HbA1c and the clinical implementation of diabetes management using GA.


The aim of this study was to examine the association between ectopic fat and organ-specific insulin resistance (IR) in insulin-target organs in patients with nonalcoholic fatty liver disease (NAFLD).2H2]glucose). Liver fat, intramyocellular lipid (IMCL), and body composition were measured by liver biopsy, proton magnetic resonance spectroscopy, and bioelectrical impedance analysis, respectively.Organ-specific IR in the liver (hepatic glucose production (HGP)×fasting plasma insulin (FPI) and suppression of HGP by insulin [%HGP]), skeletal muscle , and adipose tissue (suppression of FFA by insulin [%FFA]) was measured in 69 patients with NAFLD using a euglycemic hyperinsulinemic clamp with tracer infusion , %HGP with %FFA , and Rd with %FFA . Liver steatosis score was negatively associated with Rd  as well as with HGP×FPI . Similarly, intrahepatic lipid was negatively associated with Rd . IMCL was not associated with Rd . Fat mass and its percentage were associated with HGP×FPI  and Rd , but not with %FFA .HGP×FPI was significantly correlated with Rd (Unexpectedly, fat accumulation in the skeletal muscle and adipose tissue was not associated with organ-specific IR. Instead, liver fat was associated not only with hepatic IR but also with skeletal muscle IR, suggesting a central role of fatty liver in systemic IR and that a network exists between liver and skeletal muscle. Insulin resistance (IR) is a core pathology of type 2 diabetes mellitus (T2DM), nonalcoholic fatty liver disease (NAFLD), and cardiovascular diseases 1H-MRS) under some conditions Conversely, inter-organ network and organ-derived bioactive hormones such as adiponectin and selenoprotein P may play a role in the development of distant organ IR 1H-MRS.The present study try to address the association of organ-specific IR with ectopic fat among the liver, skeletal muscle, and adipose tissue in Japanese patients with NAFLD, systematically using reliable methods including liver biopsy, assessment of glucose metabolism measured by a euglycemic hyperinsulinemic clamp study with stable-isotope, and The study was approved by the Medical Ethics Committee of Kanazawa University , and written informed consent was obtained from each patient prior to participation. The study was conducted in accordance with the Declaration of Helsinki.We studied 69 patients clinically diagnosed with NAFLD, recruited consecutively between 2010 and 2012 from Kanazawa University Hospital, Japan. The patients were in good general health without evidence of any acute or chronic diseases  as determined by history, physical examination, routine blood chemistries, urinalysis, and electrocardiography. Out of the 69 patients, 37 (54%) had T2DM according to the American Diabetes Association criteria. Of the 37 T2DM patients, antidiabetic agents were administered to 18 patients in monotherapy and 7 patients in combination therapy . None of the patients were taking α-glucosidase inhibitors, rapid-acting insulin secretion agents, sulfonylurea, thiazolidinediones, or long-acting insulin. Participants were excluded if they had a history of alcohol abuse (more than 20 g/day), liver diseases other than NAFLD , type 1 diabetes, or a history of clinically significant renal, pulmonary, or heart diseases.2H2]glucose) infusion; 2) liver biopsy for histology to confirm the diagnosis of NAFLD and score the degree of steatosis, grade, and stage; 3) intrahepatic lipid (IHL) and intramyocellular lipid (IMCL) measured by 1H-MRS, and body composition by a bioelectrical impedance analysis; and 4) 75-g oral glucose tolerance test (OGTT) to evaluate the glucose tolerance according to American Diabetes Association criteria The participants were studied on four separate occasions. Generally, all measurements were performed within 1 month and included: 1) organ-specific IR in the liver, skeletal muscle, and adipose tissue by a euglycemic hyperinsulinemic clamp study with tracer  was started at a rate of 0.05 mg·kg−1·min−1 after a priming dose equivalent. After 100, 110, and 120 min, blood samples were obtained for determination of tracer enrichments. Subsequently, at 0900 h, the euglycemic hyperinsulinemic clamp study was started using an artificial pancreas , as described previously −1·min−1 to attain a plasma insulin concentration of approximately 100 μU/mL. Glucose was infused to maintain a plasma glucose concentration of 100 mg/dL . Simultaneously, glucose infusion was continued at a rate of 0.15 mg·kg−1·min−1. During the last 20 min of the clamp study, blood samples were obtained in 10-min intervals to determine tracer enrichments.After an overnight fast, two intravenous catheters, one for blood sampling and one for infusion of glucose, insulin, and tracers, were inserted in the antecubital vein of each arm. At 0700 h, after obtaining a blood sample for background enrichment of plasma glucose, a continuous infusion of ) and suppression of HGP by insulin during a clamp study (%HGP). The skeletal muscle IR index was calculated as insulin-stimulated glucose disposal (Rd), and the adipose tissue IR index was calculated as suppression of FFA by insulin during a clamp study (%FFA).We calculated and defined organ-specific IR in the liver, skeletal muscle, and adipose tissue as described previously t-test for continuous variables and chi-square test for categorical variables. Data involving more than two groups were assessed by analysis of variance (ANOVA). Statistical significance was considered to be P<0.05.All analyses were performed using SPSS software version 21.0 . All values are expressed as mean ± SEM, unless stated otherwise. The relationship between individual variables was assessed by Pearson’s correlation for parametric variables and by Spearman’s correlation for non-parametric variables. Multiple linear regression analysis was used to assess independent determinants of organ-specific IR. The differences between the two groups were assessed by Student’s −1·min−1 in subjects with normal glucose tolerance (NGT), 2.18±0.10 mg·kg−1·min−1 in subjects with impaired glucose tolerance (IGT) and 2.67±0.12 mg·kg−1·min−1 in subjects with T2DM. Rd was 3.81±0.18 mg·kg−1·min−1 in NGT, 3.27±0.17 mg·kg−1·min−1 in IGT and 3.57±0.14 mg·kg−1·min−1 in T2DM. Basal FFA was 0.47±0.05 mEq/L in NGT, 0.56±0.04 mEq/L in IGT and 0.60±0.04 mEq/L in T2DM. Basal HGP showed a significant positive correlation with fasting plasma glucose levels . Rd showed a significant positive correlation with basal oxygen consumption rate per body weight (VO2) . FFA and HGP were suppressed from baseline by 77.0±1.4% and 69.3±2.8%, respectively. These values are similar to previous data in Japanese The characteristics of the study subjects and their metabolic profiles are shown in r = −0.57, P<0.001), %HGP with %FFA , and Rd with %FFA  suggesting that the IRs in the liver, skeletal muscle, and adipose tissue were significantly associated with each other, although the correlation was not very strong.The relationship between clinical characteristics and organ-specific insulin sensitivity/resistance indices is shown in 1H-MRS .Histological liver steatosis score was strongly correlated with IHL measured by r = −0.47, P<0.001) as well as HGP×FPI   and tended to be correlated with HGP×FPI  (P<0.05) and Rd  after adjusting for age, sex, and BMI. Correlation of liver steatosis score with Rd  was significant after further adjusting for total fat mass  (Liver steatosis score was significantly correlated with Rd (P<0.001) . Similar = 0.09) . In the fat mass . When stctively) .r = −0.16, P = 0.26; r = −0.22, P = 0.08, respectively)  and Rd , but not with %FFA   and adipose tissue were not associated with their own organ-specific IR . IMCL anctively) . Total fctively) .Similar results were obtained when Rd was normalized by steady state plasma insulin (Rd/SSPI) .Because it may be possible that T2DM itself is associated with IR independently with organ steatosis, we analyzed the subjects with or without T2DM. Age, hemoglobin A1c, fasting plasma glucose, 2-h glucose level of 75-g OGTT and basal HGP were significantly higher in T2DM group compared to non-DM group . RegardlWe comprehensively and simultaneously evaluated ectopic fat accumulation and organ-specific IR in insulin-target organs in Japanese people with NAFLD, and found the following: 1) the IRs in the liver, skeletal muscle, and adipose tissue were associated with each other, 2) indices of fat accumulation in the skeletal muscle and adipose tissue were not associated with their own organ-specific IR, and 3) liver fat was associated with skeletal muscle IR as well as hepatic IR, independently of age, sex, BMI and total fat mass .Although the IRs in the liver, skeletal muscle, and adipose tissue were associated with each other, the relation was relatively weak. There are a couple possible explanations for this result. First, the main site and the severity of IR may vary among organs and individuals 2) and subjects may have different physical exercise habits with various intensities. IMCL is increased not only by obesity but also by enhanced physical fitness In the present study, IMCL was not associated with skeletal muscle IR. The participants in this study had a wide BMI range . Fourth, we did not collect arterial or arterialized blood samples to perform the insulin clamp because these were not included in the manufacturer’s protocol of the artificial pancreas model STG-55. Further study should be required to confirm our conclusion by using arterial or arterialized blood samples.In summary, the present study revealed an unexpected lack of an association between fat and local organ-specific IR in the skeletal muscle and adipose tissue. Instead, liver fat is strongly associated with skeletal muscle IR as well as with liver IR, suggesting a central role of fatty liver in the development of IR and that a network exists between liver and skeletal muscle to maintain whole-body energy homeostasis.Figure S1Correlation between ectopic fat and insulin resistance (IR) in the liver, skeletal muscle, and adipose tissue. Liver fat (steatosis score) was associated with skeletal muscle IR index (Rd) as well as with IR in the liver (HGP×FPI). Intramyocellular lipid was not associated with skeletal muscle IR index (Rd). Total fat mass was associated with HGP×FPI and Rd, but not with adipose tissue IR index (%FFA).(PDF)Click here for additional data file.Table S1Multiple regression models predicting HGP×FPI and Rd in subjects without type 2 diabetes (n = 32). HGP, hepatic glucose production; FPI, fasting plasma insulin Model 1, adjusted for, age, sex, and body mass index; Model 2, adjusted for, age, sex, body mass index, and total fat mass.(DOC)Click here for additional data file.Table S2Multiple regression models predicting HGP×FPI and Rd in subjects with type 2 diabetes (n = 37). HGP, hepatic glucose production; FPI, fasting plasma insulin Model 1, adjusted for, age, sex, and body mass index; Model 2, adjusted for, age, sex, body mass index, and total fat mass.(DOC)Click here for additional data file.


Obesity was also significantly associated with lower plasma interferon concentrations on days 3, 7, and 29  and lower plasma ribavirin concentrations day 29 (P = 0.025), and lower concentration of interferon in turn was associated with a poorer first phase reduction in HCV RNA (P<0.0001). In multivariate analysis, ribavirin concentrations week 12, interferon concentrations day 29, and baseline HCV RNA levels were independent predictors of achieving SVR among patients treated for 24 weeks (n = 140).Patients with BMI ≥30 kg/mReduced bioavailability of interferon and ribavirin along with higher baseline viral load are dominant risk factors for treatment failure in obese patients with chronic hepatitis C. Hepatitis C virus (HCV) is the foremost cause of parenterally transmitted hepatitis i.e. have a body mass index (BMI) above or equal to 30 kg/m2Thirty percent of American adults are obese, i.e. having received at least 80% of the target dose of peginterferon-α2a as well as at least 80% of the target dose of ribavirin for at least 80% of the target treatment duration) were included. Of 303 eligible patients, a body mass index could be calculated for 300, and these latter patients were included in the present study  24 weeks after completion of therapy, as having relapsed if plasma HCV RNA was undetectable at end-of-treatment but detectable 24 weeks after completion of therapy, and as being non-responders if plasma HCV RNA was detectable at end-of-treatment. Patients were classified as having a rapid virological response (RVR) if HCV RNA was undetectable day 29 and a complete early virological response (cEVR) if HCV RNA was undetectable week 12.Patients were classified as achieving SVR if plasma HCV RNA was undetectable , which quantifies HCV-RNA with a limit of detection of ≤15 IU/mL. HCV-RNA quantification was performed on days 0, 3, 7, 8, 29, week 8, week 12, week 24 (for those receiving 24 weeks of therapy), and 24 weeks after completion of therapy. All samples were frozen (−70°C), and subsequently analyzed at the central laboratory.Baseline fasting glucose (mmol/L) was measured locally whereas fasting serum insulin  was analyzed on frozen samples at the central laboratory. HOMA-IR was calculated using the formula: (Glucose×Insulin)/22.5 i.e. immediately before the second dose of peginterferon-α2a) and 29. All samples were collected using PPT-tubes, frozen (−70°C), and subsequently analyzed at a central laboratory. Quantification was performed using 20 µl plasma (diluted 1∶5) on a solid-phase ELISA using high absorbant plates coated with interferon alfa antibody . The detection limit of the assay was 400 pg/mL.Plasma concentration of interferon-α2a was measured at day 3, 7  followed by UV-detection (wavelength 215 nm).Plasma samples for ribavirin drug concentration measurement were drawn immediately before the morning dose of ribavirin at day 29 (week 4) and at week 12, Liver biopsies were obtained from all patients within 24 months prior to study entry. The evaluation was performed in a blinded fashion by two independent observers according to the Ishak protocol 2) test, and Spearman's rank correlation coefficient (sr) test was utilized to evaluate relationships between variables. After univariate analyses, multivariate analyses were performed on the per-protocol patients treated for 24 weeks with all variables associated with the endpoint (P<0.1) being entered. The variables entered in the multivariate analyses were age, BMI, Ishak fibrosis stage, steatosis grade, HOMA score, peg-interferon concentrations on day 3, 7 and 29, ribavirin concentrations day 29 and week 12, and baseline HCV RNA level. All statistical analyses were performed using SPSS (version19.0) and StatView . All reported p-values are two-sided, and p-values<0.05 were considered significant.Wilcoxon-Mann-Whitney U-test, Kruskal-Wallis test, Chi squared , Regional Committee for Ethics in Medical Research, Bergen, Norway , The Scientific Ethical Committee for the Region of Middle Jylland, Viborg, Denmark (Den Videnskabsetiske Komité for Region Midtjylland), The Scientific Ethical Committee for the Region of South Denmark, Vejle, Denmark (Den Videnskabsetiske Komité for Region Syddanmark), and the Ethics Committee, Department of Medicine for the Hospital District of Helsinki and Uusimaa, Finland (Etiska kommittén för invärtesmedicin)). The study has been registered at the NIH trial registry .s = 0.31, P<0.0001), indicative of insulin resistance and more pronounced steatosis and fibrosis (s) test for correlation between BMI and HOMA-IR was 0.31 (P<0.0001). When grouping according to HCV genotype, the association between steatosis and BMI remained significant for both genotypes 2 and 3, as did the correlation between BMI and HOMA-IR for genotype 3. As expected, patients with BMI ≥30 kg/m2 also had significantly higher systolic and diastolic blood pressure as well as baseline triglycerides levels  as well as HOMA-IR with patients having higher BMI also having higher HOMA-IR , 7 , and 29 ,  and week 12  ; and this association between interferon concentrations and the first phase decline was independent of IL28B genotype. In contrast, ribavirin concentrations were not associated with either the first (rs = 0.018 and P = 0.8 and rs = −0.018 and P = 0.8 for ribavirin concentrations on day 29 and week 12 respectively) or second phase reduction of HCV RNA (rs = 0.018 and P = 0.8 and rs = 0.042 and P = 0.5 for ribavirin concentrations on day 29 and week 12 respectively).Interferon concentrations at all three time-points sampled were significantly associated with the first phase decline in HCV RNA  for boths = 0.12, P = 0.038), and this correlation became more pronounced while on treatment as exemplified by treatment day 3 , day 7 , day 8 , day 29 , week 8 , and week 12 . Similarly a non-significant trend towards a smaller first phase decline in HCV RNA as measured by the reduction from day 0 to 3 (mean 1.66 vs. 1.91 log10 IU/mL BMI ≥30 vs. <30 kg/m2) and second phase as measured by the reduction from day 8 to 29 (mean 0.79 vs. 0.83 log10 IU/mL per week BMI ≥30 vs. <30 kg/m2). However, the association noted between higher BMI and higher HCV RNA levels day 29 was not reflected by the proportion of patients achieving undetectable HCV RNA, i.e. RVR . In contrast, obese patients were less likely to achieve undetectable HCV RNA week 12 . This decrease in SVR was mirrored by increased rates of relapse and non-response for both HCV genotypes 2 and 3. In univariate analysis of the population treated for 24 weeks, both peg-interferon and ribavirin concentration day 29 where significantly associated with SVR (P = 0.01), and in a multivariate analysis, ribavirin concentrations week 12, peg-interferon concentrations day 29, and baseline HCV RNA levels were independent predictors of achieving SVR .In addition to the effect on viral kinetics, BMI profoundly affected SVR especially among patients treated for the standard 24 weeks of treatment duration  with onlving SVR . Among pIn spite of the pending introduction of direct antiviral agents (DAA) in routine clinical practice for HCV genotype 1, few of the currently available DAAs are active against genotype 2 or 3. Thus interferon-α and ribavirin are likely to retain pivotal roles in the management of chronic HCV genotype 2/3 infection, and a fixed dose of 800 mg/day ribavirin in conjunction with fixed 180 µg dosing of peginterferon-α2a et al.  who reported a non-significant trend towards lower interferon concentrations and higher baseline viral load following a single dose of 10 mIU of unpegylated interferon-α2b without the addition of ribavirin among obese patients. Interestingly in contrast to the present study, no difference in the first phase decline in HCV RNA was noted though the sample size was small The lower bioavailability of peginterferon noted in our study corroborates the findings reported by Lam 2 had significantly lower concentrations of ribavirin following fixed dosing of 800 mg ribavirin/day is not surprising, and may substantiate the report by Pattullo et al. that weight based dosing of ribavirin may negate the influence of BMI and weight on outcome et al. noted that among difficult-to-cure  HCV genotype 1 infected patients higher fixed doses of both ribavirin (1600 mg daily) and peginterferon-α2a (270 µg/week) improved viral kinetics and SVR rates as compared to lower doses of ribavirin (1200 mg daily) and peginterferon-α2a (180 µg/week) i.e. body weight ≥85 kg and viral load ≥400,000 IU/mL) HCV genotype 1 infected patients 2, and no subset analysis restricted to patients with BMI ≥30 kg/m2 has yet been reported. Additionally in a per-protocol analysis restricted to patients with at least 80% exposure to the planned ribavirin dose, numerically higher SVR rates were obtained with intensified regimens suggesting that such strategies may be successful if tolerated The finding in the present study that patients with BMI ≥30 kg/m2 may benefit from increased dosing of interferon, which is substantiated by the abovementioned study by Fried et al.Similarly it may be hypothesized that patients with BMI ≥30 kg/m2 would improve outcome is outside the scope of this study. Since replication, assembly and release of HCV occurs in close proximity to intracellular lipid droplets In addition to differences in bioavailability of interferon and ribavirin, we also noted that patients with higher BMI had higher baseline viral load, more pronounced steatosis, in addition to features of the metabolic syndrome as previously reported, e.g. higher systolic and diastolic blood pressure, higher HOMA-IR, and higher baseline triglyceride levels. Although weight reduction among obese HCV patients is known to normalize many aspects of the metabolic syndrome as well as liver histology In conclusion, higher BMI is associated with lower bioavailability of both peginterferon and ribavirin, which subsequently along with higher baseline viral load are dominant risk factors for treatment failure in obese patients with chronic HCV genotypes 2 and 3 infection.


Dictyostelium gene showing rapid transcriptional oscillations with the same period as extracellular cAMP signaling waves. Bursting approaches antiphase to cAMP waves, with accelerating transcription cycles during differentiation. Although coupling between signal and transcription oscillations was clear at the population level, single-cell transcriptional bursts retained considerable heterogeneity, indicating that transcription is not governed solely by signaling frequency. Previous studies implied that burst heterogeneity reflects distinct chromatin states [Transcription is highly stochastic, occurring in irregular bursts , 2, 3. Fn states , 5, 6. H •We observe coupled signal and transcriptional oscillations in living cells•Oscillations are collectively robust but heterogeneous at the single-cell level•Response heterogeneity is maintained by a short-term transcriptional persistence•Oscillations can be a mechanism to control gene expression noise in development Corrigan and Chubb, through live imaging of transcriptional bursts, observe coupled oscillations between an extracellular signal and downstream transcription. Oscillations provide a mechanism to reduce transcriptional noise. Transcription occurs in irregular bursts or pulses. Despite this noisy behavior, organisms manage to achieve spatial and temporal precision in their gene expression. A growing body of evidence indicates that the temporal dynamics of signaling can provide specificity in transcription , 11, 12 Dictyostelium csaA gene, which encodes a developmentally induced cell adhesion glycoprotein [csaA bursting data revealed periodic fluctuations in spot intensity  and cell streams (6 hr).lar cAMP , 19, 20.lar cAMP , 25, 26.Cell speed (measured as the two-frame displacement) was used as a proxy for cAMP wave phase . Clear vAutocorrelation of averaged motility G, suggesPeaks of transcription spot intensity were not in synchrony with motility peaks C, with acsaA regulation, wherein a cell has a number of activity states of monotonically increasing initiation rate . In other words, cells with brighter spots showed greater persistence, and in heterogeneous populations (high SD), persistence will appear greater because deviations from the field average (and therefore fluctuation times) are large. The strongest correlation for transcriptional persistence occurred with the response parameters  is generally considered to occur over timescales of a cell cycle or greater , 31, 32,csaA transcripts, we used Dictyostelium AX3 cells with 24 MS2 loops inserted into the 5′ UTR of the csaA coding sequence [4, pH6.2), plated on KK2/2% agar at 2.5 × 106 cells/cm2, and then placed in humidified chambers at 22°C. Prior to imaging, 1 cm squares were excised and inverted onto Bioptechs Delta TPG dishes (0.17 mm). Agar was covered in mineral oil to prevent desiccation. We used a wide-field fluorescence system specifically designed for fast sensitive imaging of photosensitive samples [csaAMS2 cells were mixed 1:4 with AX3 expressing H2B-mCherry from the genomic rps30 gene. csaAMS2 cells developed 2 or 4 hr were mixed with 4 hr H2B-mCherry cells and replated on agar. Agar was inverted after 1 hr, and streams were imaged after a further 90 min, with four xy positions imaged for each time point of the six experimental repeats.To visualize nascent sequence . These c samples . Four xy


Bombyx mori thymosin (BmTHY) protein from a cDNA library of silkworm pupae, which has an open reading frame (ORF) of 399 bp encoding 132 amino acids. It was found by bioinformatics that BmTHY gene consisted of three exons and two introns and BmTHY was highly homologous to thymosin betas (Tβ). BmTHY has a conserved motif LKHTET with only one amino acid difference from LKKTET, which is involved in Tβ binding to actin. A His-tagged BmTHY fusion protein (rBmTHY) with a molecular weight of approximately 18.4 kDa was expressed and purified to homogeneity. The purified fusion protein was used to produce anti-rBmTHY polyclonal antibodies in a New Zealand rabbit. Subcellular localization revealed that BmTHY can be found in both Bm5 cell (a silkworm ovary cell line) nucleus and cytoplasm but is primarily located in the nucleus. Western blotting and real-time RT-PCR showed that during silkworm developmental stages, BmTHY expression levels are highest in moth, followed by instar larvae, and are lowest in pupa and egg. BmTHY mRNA was universally distributed in most of fifth-instar larvae tissues (except testis). However, BmTHY was expressed in the head, ovary and epidermis during the larvae stage. BmTHY formed complexes with actin monomer, inhibited actin polymerization and cross-linked to actin. All the results indicated BmTHY might be an actin-sequestering protein and participate in silkworm development.We identified a novel gene encoding a  Thymosins, one family of thymic hormones, play a crucial role in the development and maintenance of a competent immune system, especially in the differentiation and maturation of T-lymphocytes Drosophila melanogaster, Aedes aegypti, Bombyx mori and other species, and are highly homologous to Tβ. For instance, the homology of the THY related protein from Drosophila to Tβ is 70%. Both of them have a WASP homeodomain However, few studies on thymosin from lepidoptera animal have been reported. There are only 18 related proteins which contain THY conservative structural domain registered in NCBI cDNA; BmTHY) from a pupal cDNA library (GenBank Accession: FJ602790). Using bioinformatics analysis, we have determined the evolutionary relationship and degree of conservation between BmTHY and other thymosin orthologs. We have also used immunohistochemistry, real-time RT-PCR, and Western blotting analyses to determine its subcellular localization in Bm5 cells (a silkworm ovary cell line), expression patterns in different silkworm developmental stages, and tissue distribution in the fifth instar larvae. Actin sequestering properties of BmTHY were evaluated by nondenaturing gel electrophoresis (NPAGE), sedimentation of polymerization and cross-linking of BmTHY to G-actin.The results provide a solid foundation on which to base further research on the developmental and functional roles played by thymosin in silkworm.In this study, we identified a silkworm thymosin complementary DNA (Escherichia coli strains TG1 and BL21(DE3) were grown at 37°C in LB medium, pH 7.5 . Bm5 cells Bombyx mori strain used in this study is Qingsong×Haoyue. Silkworms were reared on mulberry leaves under standard conditions. The head, fatty body, intestine, Malpighian tubules, silk gland, skin, trachea and ovary from the fifth instar larvae were dissected, frozen immediately in liquid nitrogen, and stored at −80°C. Nascent eggs, day-5 fifth instar larvae with mulberry leaves removed from the gut, pupae (3 days after pupation), and moths were also frozen in liquid nitrogen and stored at −80°C. Rabbit skeletal muscle actin (>95% pure) and G-actin buffer were obtained from Cytoskeleton Inc.USA. Tβ4 was purchased from Prospec Ltd. Reagents for polyacrylamide electrophoresis, such as acrylamide, bis-acrylamide, tricine, ammoniumpersulfate, and TEMED were purchased from Sigma (USA).The cDNA sequence encoding the BmTHY protein was obtained from a cDNA library of the silkworm pupa constructed in our laboratory http://www.ncbi.nlm.nih.gov/). The orthologous sequences used for multiple sequence alignments were obtained from NCBI. Multiple sequence alignment was performed with the biosoftware BioEdit . The three-dimensional models for BmTHY protein was built using 3D-JIGSAW software (http://bmm.cancerresearchuk.org/~3djigsaw/).The characteristics of the protein domain and its function were analysed using ExPASy software (http://us.expasy.org/tools).A BmTHY ORF by PCR, with primers complementary to the flanking sequences of the ORF with EcoRI and XhoI recognition sites as follows:We used pHelix-BmTHY constructed by our laboratory 1:GAATTCATGGCCTGCTCC -3′5′- CGC;P2:CTCGAGTCAAGCTGATTTCTCTT -3′5′- CCG;PEcoRI and XhoI, the purified PCR products were subcloned into the expression vector pET-28a  using T4 DNA ligase  and transformed into E. coli TG1 cells (maintained in our laboratory) for screening purposes. A positive colony with BmTHY gene in the plasmid was identified by double digestion of the plasmid, followed by analysis on 1% agarose gel electrophoresis and was subsequently verified by DNA sequencing.The PCR products were purified after electrophoresis on 1% agarose gel using the PCR Rapid Purification Kit .After digestion with E. coli BL21 (DE3) competent cells, which were incubated at 37°C in liquid LB culture media containing 50 µg/mL kanamycin. Expression of the Histag fusion protein was induced at an A600 of 0.6 followed by adding IPTG  to a final concentration of 1 mM before another 5-hour incubation. A 5 mL Ni2+-SephadexTM G-25 Superfine column (Amersham) was used to purify the expressed recombinant BmTHY (rBmTHY) protein, as instructed by the manufacturer. The concentrated rBmTHY was further purified by reversed-phase FPLC with acetonitrile linear gradient from 5 to 95% in 20 min with the flow rate of 1.7 ml/min and analyzed by the Q Trap LC/MS/MS System  to determine its molecular weight. The presence and purity of rBmTHY were evaluated by 12% SDS-PAGE and quantified by the Bradford method The recombinant plasmid pET-28a- BmTHY was transformed into Polyclonal antibody was prepared by immunizing New Zealand White rabbits using purified rBmTHY as antigen Eggs, the fifth instar larvae, pupae, moths and tissues isolated from the fifth instar larvae were collected and ground to powder in liquid nitrogen followed by suspending in buffer M ), then incubating for 30 minutes on ice. The homogenates were centrifuged at 12 000×g for 15 minutes at 4°C. Protein concentrations were quantitated by the method of Bradford, in which BSA was used as the protein standard 2O, and scanned using the Odyssey Infrared Imaging System  at 700 nm.Protein samples were equalized and electrophoresed by 10% SDS-PAGE and electrotransfered to polyvinylidene difluoride (PVDF) membranes by semi-dry method with constant current of 2 mA/cm2 for 90 min. The membranes were blocked with 5% skim milk in TBS (pH 7.5). Following incubation with purified anti-BmTHY IgG, membranes were washed and incubated with HRP-labeled anti-rabbit IgG . Membranes were washed three times with TBS, then washed three times with ddHTotal RNA was extracted from different developmental stages of silkworm and tissues of the fifth instar larvae using Trizol reagent (Invitrogen) according to the manufacturer's instructions. The purity of extracted RNA was determined by UV spectrophotometer. Ratios of UV 260/280 were between 1.8 and 2.1 for all RNA samples analyzed. The concentration of total RNA was determined by measuring the absorbance at 260 nm using SPECTRA max PLUS384 (Molecular Devices). RT-PCR primers were designed using the Primer Select program of the DNA STAR software.A pair of primers was also designed to amplify 18S ribosomal RNA (rRNA), which was used as an internal control. The primer pairs were as follows:5′-TGACACTCCCTCCCTGAAAGA-3′, Reverse 5′-CTCCTGGGTCTTCTCAGTGGC-3′;-(BmTHY) Forward primer, 5′-CGATCCGCCGACGTTACTACA-3′, Reverse 5′-GTCCGGGCCTGGTGAGATTT-3′.-(18S rRNA) Forward primer,−ΔΔCT, where ΔCT = CT(target gene)−CT(18S rRNA), ΔΔCT = ΔCT(target gene)−ΔCT(maximum)SuperScript III Platinum SYBR Green One-Step RT-PCR Kit with ROX (Invitrogen) was used. 15 µL reaction mixtures contained 7.5 µL 2× SYBR-Green Reaction mixed with Rox, 0.3 µL SuperScript III RT/platinumTaq mix, 1.2 µL 1 µM forward and reverse primers, respectively, 1 µL total RNA and 3.8 µL DEPC-water. RT-PCR was performed by ABI Prism 7300 Sequence Detection System (Applied Bio systems) under the following conditions: an initial cycle at 50°C for 3 minutes, one cycle at 95°C for 5 minutes, followed by 40 cycles of 95°C for 15 seconds, 59°C for 15 seconds, and 72°C for 30 seconds. Each reaction was performed in triplicate in 96-well plates, with the endogenous 18S rRNA control. Dissociation curves were performed to check for the presence of nonspecific dsDNA SYBR Green hybrids, such as primer dimers. Data analysis was performed using ABI Prism 7300 SDS Software V1.3.1 . Expression levels of the target genes were normalized against the expression level of the 18S rRNA gene. The relative expression level was calculated using 2Bm5 cells were seeded in dishes for confocal microscopy, and cultured overnight, washed for 10 min three times in PBS, and fixed  at room temperature for 15 min. The fixed cells were blocked with 3% BSA at room temperature for 2 h followed by three 10-min washes in PBST (0.05% Tween-20 in PBS). Cells were then incubated with purified anti-rBmTHY polyclonal antibody (diluted 1∶200 in blocking buffer) at 4°C overnight; cells were simultaneously incubated with negative serum as a control. The negative serum was obtained from the rabbit before immunizing with antigen. After three 10-min washes in PBST, cells were incubated with Cy3-labeled goat anti-rabbit IgG  at 37°C for 2 h and were then washed twice for 10 min in PBST. Cells were then incubated with 4′-6-diamidino-2-phenylindole (1 g/ml in PBS) at room temperature for 10 min. Cells were washed three times with PBST (10 min each), and analyzed with a Nikon ECLIPSE TE2000-E Confocal Microscope  with image analysis software EZ-C1.Nondenaturing polyacrylamide gel electrophoresis (NPAGE) was performed in gels containing 25 mM Tris, 194 mM glycine, 0.2 mM ATP, 0.1% Triton X-100, 7.5% acrylamide, 0.2% N,N′-methylenebisacrylamide, 0.03% N,N,N′JV′-tetramethylethylenediamine, and 0.1% ammonium persulfate. The electrode buffer contained 25 mM Tris, 194 mM glycine, and 0.2 mM ATP. Gels were prerun at 20–25 V/cm for 1 h in a 4°C coldbox; the electrode buffer was poured off and replaced, and samples were then loaded and run at 20–25 V/cm at 4°C 0.5 mM β-mercaptoethanol,0.2 mM KCl, 10 mM Tris-HC1, pH 7.5. After a 10-min incubation at room temperature, 3 µL of glycerol with a trace of bromphenol blue was added, and the samples were electrophoresed at 4°C at 20–25 V/cm until the bromphenol blue dye front ran out.The proteins were stained with Coomassie Blue to get electrophoresis pattern.0.6–1.2 µg of Tβ4 or BmTHY were mixed with 2.0 µg of rabbit muscle actin monomers in 12 µLof G-buffer containing 0.5 mM ATP, 2,0.2 mM dithiothreitol, 0.2 mM NaN3, 3 mM Tris-HCl, pH 7.6); Tβ4 or BmTHY were added as 10 µM solutions in KCl buffer . For each assay, 100 µL of actin was mixed with the specified volume of peptide solution, and the final volume was brought to 200 µL with KCl buffer. After mixing, the actin was allowed to polymerize for 15 min at 25°C before centrifuging at 100,000× g for 30 min. The supernatants were pipetted off, and each pellet was solubilized in 50 µL of SDS sample buffer. Equal aliquots were analyzed by SDS-PAGE on a 10% gel.Sedimentation Assays for Actin Polymerization was performed using a Beckman Airfuge with fixed-angle rotor as previously described 2, 0.2 mM ATP, 0.2 mM NaN3, pH 7.5, was incubated at 4°C for 45 min with either BmTHY or Tβ4 at different molar ratio. Aliquots of 10 µL were then mixed with 12.2 µL of 5.4 mM l-ethyl-3-(3-dimethylaminopropyl) carbodimidie in 0.1 M MES, pH 6.5, and incubated for 2 h at 25°C. Equal aliquots were taken up into SDS sample buffer and analyzed by SDS-PAGE on a 10% gel under reducing conditions.Cross-linking of BmTHY or Tβ4 to G-actin was analyzed according to the method described by Safer BmTHY cDNA sequence (GenBank accession number FJ602790) was identified from a cDNA library of the silkworm pupa constructed in our laboratory BmTHY, contains two introns and three exons ≥2.1 was positive; 1.5≤P/N<2.1 was suspicious expression; P/N<1.5 was negative. From this, the titer of the antibodies was greater than 1∶25600 at a concentration of 10 µg/mL, which had met requirement for next experiments . The speextracts . These rWe performed Real-Time RT-PCR analysis on mRNA to determine BmTHY transcription levels at four different silkworm developmental stages like eggs, the fifth instar larvae, pupae, and moths. From the melting curves (data not shown), we established, there were no overt primer dimers, indicating good primer specificity. The amplification curves (data not shown) showed good reproducibility. The PCR results indicated an obvious difference in BmTHY transcription levels between the four silkworm developmental stages. BmTHY mRNA was highest in moth, lower in instar larva, pupae, and lowest in egg .In order to determine the protein expression levels of BmTHY at silkworm various developmental stages, we extracted total protein from the egg, fifth instar larvae, pupa, and moth. Western blotting analyses were performed on protein extracts to determine BmTHY expression levels. The highest amount of expressed BmTHY was in moth, decreasing in the fifth instar larvae;no BmTHY was expressed in egg or pupa .In order to determine transcription levels of BmTHY in the head, Malpighian tubule,epidermis, spiracle, silk gland, testis,ovary, gut, and fatty body of the fifth-instar larvae, total RNA was isolated from these tissues for RT-PCR. From the amplification curves and melting curves(data not shown), we determined that there was excellent reproducibility and no overt primer dimers formed during amplification, indicating good specificity for the primer pair. BmTHY mRNA was universally distributed in the examined tissues, with the transcription level of BmTHY highest in the head and ovary,and lower in the gut, spiracle, Malpighian tubule, epidermis, fatty body, silk gland and testis .To elucidate the expression of BmTHY protein in the head, Malpighian tubule, epidermis, spiracle, silk gland, ovary, testis, gut, and fatty body of fifth-instar larvae, we extracted protein from these tissues, and performed Western blotting analysis to determine the amount of BmTHY in each. The result of immunoblots on protein extracts revealed that anti-BmTHY serum reacted with a 18.4-kDa protein in extracts isolated from head, ovary and epidermis, but no signal was detected in extracts isolated from silk gland, spiracle, gut, Malpighian tubule, testis and fatty body .The treated cells were examined under a Nikon ECLIPSE TE2000-E Confocal Microscope,and images were analyzed using EZ-C1software. Cy3-labeled goat anti-rabbit IgG emitted red fluorescence when stimulated with light having a wavelength of 550 nm, and DAPI-stained nuclei emitted red fluorescence when stimulated with light having a wavelength of 353 nm. The results indicated that BmTHY was mainly located in nucleus, although it was rarely present in the cytoplasm .et al.Binding of BmTHY to Actin in Nondenaturing Gels was firstly analyzed. BmTHY and Tβ4 both formed complexes with skeletal muscle G-actin. Incubation of BmTHY with G-actin inhibited salt-induced polymerization by an equivalent amount as assayed by sedimentation. Tβ protein family has a relatively conserved motif ‘LKKTET’ involving Tβ binding to actin E. coli and was mostly in the supernatant of E.coli Rosetta/pET-28a-BmTHY induced by IPTG after supersonic treatment. Recombinant BmTHY protein was also purified easily by affinity chromatography. The molecular weight of the purified fusion protein was confirmed by mass spectrometry, which indicated a molecular weight of 18.4 kD  extracted from the fifth-instar larvae. BmTHY transcription level in the tested tissues from high to low in turn is orderly: head>ovary>midgut>spiracle>Malpighian tubule>epidermises>fatty body>silk gland>testis. The result of Western blotting showed that BmTHY was expressed in the head, ovary and epidermis. Why was BmTHY expressed in ovary but testis? We presume that BmTHY might be involved in sex differentiation in the developmental stage of silkworm. Why was BmTHY expressed in head and epidermis? We speculate that BmTHY might participate in cell differentiation and migration because both head and epidermis are important parts of morphological changes in silkworm.Because both Real-Time RT-PCR and Western blotting analysis testified BmTHY expressed in the fifth instar larva, we extracted total RNA and proteins from nine fifth-instar larva tissues to further examine transcription and translation of the We carried out subcellular localization of BmTHY in Bm5 cell by immunofluorescence. BmTHY was found mostly in the nucleus but was also observed in the cytoplasm. Some actin-binding proteins have been previously reported to localize intracellularly to or shuttle into the nucleus In a word, the presented data indicated that BmTHY might participate in the regulation of morphological changes and physiological function in silkworm development by means of binding to actin.


Axinyssa djiferi, collected on mangrove tree roots in Senegal, was investigated for glycolipids. A mixture containing new glycosphingolipids, named axidjiferoside-A, -B and -C, accounted for 0.07% of sponge biomass (dry weight) and for 2.16% of total lipids. It showed a significant antimalarial activity, with a 50% inhibitory concentration (IC50) of 0.53 ± 0.2 μM against a chloroquine-resistant strain of Plasmodium falciparum. They were identified as homologous β-galactopyranosylceramides composed of 2-amino-(6E)-octadec-6-en-1,3,4-triol, and the major one, axidjiferoside-A (around 60%), contained 2-hydroxytetracosanoic acid. Cytotoxicity was studied in vitro on human cancer cell lines . Results of this investigation showed that axidjiferosides are of interest, because they proved a good antiplasmodial activity, with only a low cytotoxicity against various human cell lines and no significant antitrypanosomal and antileishmanial activity. Thus, it seems that galactosylceramides with a β anomeric configuration may be suitable in searching for new antimalarial drugs.The marine sponge,  COSYhomonuclear correlation spectroscopyFAMEfatty acid methyl estersLCBlong chain baseGC-MSgas chromatography-mass spectrometryGLglycolipid(s)GSLglycosphingolipid(s)amuatomic mass unitHMBCheteronuclear multiple bond coherenceHSQCheteronuclear single quantum coherence50IC50% inhibitory concentrationESI-MSelectrospray ionization mass spectrometryTLCthin layer chromatographydwdry weightMECminimum effective concentrationPlasmodium, is one of the leading causes of mortality and morbidity in more than 100 tropical and subtropical countries of the world, P. falciparum remaining the most dangerous species and causing the most lethal form of malaria ; Rt = 37.1 min , similar to that of an authentic sample.An aliquot (3 mg) of axidjiferoside mixture was heated with 1 mL of MeOH/Het al. [3H]hypoxanthine was then added to each well, and parasites were maintained for another 24 h. Growth inhibition was determined by comparison of the radioactivity incorporated into the treated culture with that in the control culture maintained on the same plate. Concentrations causing 50% inhibition of parasite growth (IC50) were calculated from the drug concentration-response curves. Each experiment was performed in triplicate.According to Labaied et al. , extractdonovani (MHOM/ET/67/HU3) line, called LV9. Promastigote forms were grown in M-199 medium supplemented with 10% inactivated fetal calf serum, 40 mM HEPES, 100 μM adenosine and 0.5 mg·L−1 hemin in the presence of 50 μg·mL−1 gentamicin at 26 °C in 5% CO2. Peritoneal macrophages were harvested from female CD1 mice three days after an intraperitoneal injection of 1.5 mL of sodium thioglycolate (Biomérieux) and were dispensed into eight-well chamber slides  at a density of 5 × 104 cells per well (400 μL per well) in RPMI 1640 medium supplemented with 10% FCS, 25 mM HEPES and 2 mM glutamine. Four hours after, wells were washed to eliminate fibroblasts. After a 24 h incubation period, the macrophages were infected with promastigotes at a stationary phase in a ratio of 10 parasites per macrophage. After 18 h, free promastigotes were eliminated, and intramacrophagic amastigotes were treated with various concentrations of compounds. Pentamidine and amphotericin B were used as reference compounds. The culture medium was renewed 48 h later, and a new culture medium containing the drug was added. The experiment was stopped after five days, and the percentages of infected macrophages were evaluated microscopically after Giemsa staining. IC50 values were determined by linear regression analysis. Each experiment was performed in triplicate.Antileishmanial activity was evaluated on the Leishmania T. brucei were purified by centrifugation from the blood of an infected mouse and were maintained in vitro for 24 h at 37 °C in a 5% CO2 atmosphere in a minimum essential medium supplemented with 25 mM HEPES, Earle’s salts, 2 mM glutamine, 1 g·L−1 of glucose, minimum essential medium non-essential amino acids, 0.2 mM 2-mercaptoethanol, 2 mM sodium pyruvate, 0.1 mM hypoxanthine, 0.016 mM thymidine, 15% heat-inactivated horse serum and 50 μg·mL−1 of gentamycin. Drug evaluation was carried out in 96-well plates in a final volume of 200 μL containing 2 × 105 trypomastigotes and the compounds to be tested. Pentamidine was used as the reference compound. The minimum effective concentration (MEC) was defined as the minimum concentration at which no viable parasite was observed microscopically. This value was confirmed by injecting intraperitoneally the culture from the well corresponding to the MEC into a mouse to confirm that the non-motile parasites were really killed and not able to divide within mice.Antitrypanosomal evaluation was performed as follows: the bloodstream forms of 50 values were calculated from the dose-response curves.The antiproliferative activity of axidjiferosides was investigated upon the cell lines, NSCLC-N6,  , A549 (oAxinyssa djiferi, which lives in the particular biotope of the African mangrove, produced a series of glycosphingolipids, including the three new axidjiferosides isolated in this work. They contained the unusual Δ6-phytosphingosine and a not-so-common galactopyranose. Interestingly, axidjiferosides showed a promising antiplasmodial activity, with only a low cytotoxicity against various human cell lines and no significant antitrypanosomal and antileishmanial activity. If α-galactosylceramide, as a NKT cell ligand, showed a rapid and strong antimalarial activity, it seems nevertheless that a galactosylceramide, such as axidjiferoside with a β anomeric configuration, may be suitable in searching for new antimalarial drugs.The sponge