Staphylococcus arlettae through response surface methodology integrated nature inspired genetic algorithm. Response surface model based on central composite design has been developed by considering the individual and interaction effects of fermentation conditions on lipase production through submerged fermentation. The validated input space of response surface model  has been utilized for optimization through genetic algorithm. An optimum lipase yield of 6.5 U/mL has been obtained using binary coded genetic algorithm predicted conditions of 9.39% inoculum with the oil concentration of 10.285% in 2.99 hrs using pH of 7.32 at 38.8°C. This outcome could contribute to introducing this extremophilic lipase  to industrial biotechnology sector and will be a probable choice for different food, detergent, chemical, and pharmaceutical industries. The present work also demonstrated the feasibility of statistical design tools integration with computational tools for optimization of fermentation conditions for maximum lipase production.Microbial enzymes from extremophilic regions such as hot spring serve as an important source of various stable and valuable industrial enzymes. The present paper encompasses the modeling and optimization approach for production of halophilic, solvent, tolerant, and alkaline lipase from  Hydrolases particularly lipases  from extremophilic microorganisms are experiencing a growing demand, due to their versatile catalytic activities (regio- and enantioselectivity) coupled multifold industrial applications , 3.Bacillus and Burkholderia sp. [Staphylococcus xylosus [Geobacillus sp. strain ARM [S. arlettae through SmF for enhanced lipase yield.Lipases from extremophiles are capable of functioning in presence of salts, oxidizing agents, and organic solvents and can withstand the harsh industrial conditions which may permit their use in some specialized industrial applications, such as novel substrates catalysis reactions . Producteria sp. . Geneticeria sp. . It can  xylosus  and Geobrain ARM . Having rain ARM  S. arlet Staphylococcus arlettae JPBW-1, previously isolated from rock salt mine Darang Mandi  and identified as a lipase producer, was maintained on the slants of Luria Agar and subculturing was done on every week to maintain its viability. Staphylococcus arlettae JPBW-1 was cultivated in LB medium at 35°C for 2 days for spore production.The strainp-nitrophenyl palmitate  and LB Broth, Miller  were used for the present study. All other solvents and reagents were either of HPLC grade or AR grade and were obtained from Merck.SmF was carried out by seeding the spore suspension (5 mL) in Erlenmeyer flasks (250 mL) containing 50 mL of the L.B medium, supplemented with soyabean oil (12% v/v). The flasks were incubated at 35°C under agitation (100 rpm) for 3 h. After incubation the fermentation medium was harvested by centrifugation at 6314 ×g for 10 min at 4°C. The supernatant was collected and subjected to estimate the lipase activity.p-nitrophenol produced by hydrolysis of p-nitrophenyl palmitate at 410 nm [p-nitrophenol released per minute under the assay conditions.The lipase activity was evaluated spectrophotometrically by measuring t 410 nm . One uniRSM is a combination of mathematical and statistical techniques for empirical model building and optimization, which examines the relationships between one or more response parameters and a set of experimental input parameters. This model is only an approximation, but it is used extensively because such a model is easy to estimate and apply, even when little is known about the process. RSM had been used not only for optimization of culture parameters in the fermentation process but also for studying the combined effects of medium components .Production of lipase through SmF mainly depends on fermentation process variables, namely, temperature (30–40°C), oil concentration (10–14), inoculum size (8–12%), pH (7–9), and incubation time (2–4 h). In the present study based on the central composite design of RSM, design of experiments (DOE) was planned and performed for developing a polynomial response surface model after considering the above-mentioned fermentation variables at three levels.Based on the one variable at time approach experimental results coupled with literature survey and prior experience in statistical modeling, in the present study, CCD was used by taking five variables at three levels. Nonlinear regression analysis was carried out based on the data collected as per CCD  planningThe coefficient of the nonlinear regression model can be determined using the method of least squares. The effect of the parameters and their interaction terms on the response have been studied by conducting the significance tests and analysis of variance (ANOVA) has been carried out on each response to check the adequacy of the model. The detailed analysis of the effect of parameters and their interactions on the response were also done through the surface plots using MINITAB 14 software.Pc). If the child chromosome is less fit than the parent chromosome, then it will slowly die in the subsequent generation. Mutation was the last operation of GA optimization and used further to perturb the child vector using mutation probability (Pm). It alters the string locally to create a better string and to create a point in the neighborhood of the current point, thereby achieving a local search and maintaining the diversity in the population. The entire process is repeated till some termination criterion is met [S. arlettae JPBW-1 using binary coded GA by utilizing the input space of the developed RSM model of lipase production through SmF. Taking ten bits for one variable, 50 bits (five input variables) were used to represent a GA string. Based on the concept of duality, the maximization problem is converted to minimization problem. This simulation has been executed through C program.Optimization is described as the simulation performed aiming to maximize a certain process objective. The search for the desired optimum is usually done using mathematical algorithms. Generally in case of optimization, the problem of interest must be formulated as a mathematical model which describes the system and its performance. The simulation of the process with a mathematical model facilitates the process optimization against highly expensive experiments, predicting process results for any set of decision variables. The simplicity, robustness, and higher convergence rates in lesser computational time account for their popularity in solving the complex, nonlinear problems. These algorithms differ with traditional and gradient based approaches, in searching a population of points in parallel not just a single point and utilizing the probabilistic transition rules instead of deterministic ones. In this context, the present work aims to optimize the nonlinear RSM model of lipase production using artificial intelligence based GA approach. GA is the most popular evolutionary algorithm (EA) which mimics the principle of natural evolution. In GA, the optimization search proceeds through three operators, namely, reproduction, crossover, and mutation . The repn is met . The mecS. arlettae (Most lipases are inducible enzymes and addition of oils proved to enhance lipase activity . RSM is arlettae  using thLa) of S. arlettae JPBW-1 has been expressed as a nonlinear function of the input process parameters in coded form as follows:X1, X2, X3, X4, and X5 represent temperature, oil concentration, inoculum size, pH, and incubation time, respectively.These experiments were performed in triplicate and lipase activity  as a level of confidence) are considered as significant terms with impact final lipase activity. The P value of the factors X1, X2, X5, and X2X5 is found to be more than the confidence level (0.05) but their square terms P values are found to be less than the confidence level indicating their nonlinear relationship with the response, lipase activity. The significant contribution of linear, square, and interaction terms towards the response, lipase activity, has been revealed through ANOVA results  was seen to be equal to 96.6% which shows the developed model is an adequate predictor of the experimental conditions and confirmed that the selected SmF process variables significantly influence lipase yield [R2 was found to be in reasonable agreement with adjusted R2 (90.5%). The three dimensional response surfaces were plotted to study the interaction among the various factors selected which was found to be its nonlinear relationship with the response, lipase activity , population size (10–350), and maximum generations (25–1000) versus fitness value; from this analysis we have selected the optimum values of Pm, population size, and maximum generation number at which optimum fitness value has been noticed one by one. In the present study, tournament selection of size two, uniform crossover probability (Pc) of 0.5, bitwise mutational probability (Pm) of 0.0015, population size of 210, and maximum number of generations of 815 were employed in search of optimal values of the lipase extraction from the fermented broth for enhanced lipase activity. These optimized parametric parameters  have been utilized for final run of binary coded GA, which results in 50-bit length string that represents the five optimized input variables (10 bits for each variable) of SmF. After decoding these strings to real values through linear mapping, the optimum values of process parameters were seen to be equal to 38.82°C, 10.285%, 9.392%, 7.32, and 2.995 h for fermentation variables such as temperature, oil concentration, inoculums size, pH, and incubation time, respectively. Moreover, the maximum value of lipase activity for S. arlettae JPBW-1 was found to be equal to 6.456375 U/mL.Genetic algorithm is a stochastic optimization technique that searches for an optimal value of a complex objective function and is used to solve complicated optimization problems by simulation or mimicking a natural evolution process . The appAzohydromonas lata MTCC 2311 [Staphylococcus arlettae JPBW-1 showed stability in presence of salt (up to 30% NaCl) and organic solvents  and has ability to work in extreme conditions of temperature (up to 70°C) and pH (8–12) [To confirm these results, lipase production was conducted with the optimum levels of the significant factors representing the maximum lipase yield 6.45 U/mL. The lipase yield improved about 1.8-fold than the one at a time approach for optimization of lipase production in which the lipase yield was 3.54 U/mL. The significant correlation between predicted and observed values of lipase yield in these experiments justified the validity of the response model and the existence of an optimum point. Among the various artificial intelligence techniques, genetic algorithms, a powerful stochastic search and optimization technique, have received considerable attention. Genetic algorithms can be used to optimize fermentation conditions without the need of statistical designs and empirical models. Implementation of a GA for multiobjective experimental optimization was recently demonstrated  and, thuTCC 2311  and in lTCC 2311 . MoreoveH (8–12) .S. arlettae. A second order polynomial response surface model has been developed successfully and utilized in search of optimal conditions for lipase production through SmF using binary coded GA. The optimum fermentation conditions obtained for the synthesis of lipase from S. arlettae were 38.8°C, oil concentration 10.2%, inoculum volume 9.3%, pH 7.32, and incubation time 3 h for obtaining a maximum lipase activity of 6.45 U/mL. An overall 1.8-fold increase in lipase activity was achieved after fermentation variables optimization, following the statistical approach. The high tolerance of this lipolytic enzyme under extreme conditions will make it an enzyme of choice for many industries and considered to be a good candidate for its viability for commercialization.To summarize, this study presents evaluation of RSM integrated GA in modeling and optimization of lipolytic activity production from


AR) gene cause differences in androgen sensitivity, no straightforward correlation can be observed between the interindividual variation in T levels and different phenotypes. Therefore, we aim to investigate the relationship between genetic variations in the AR, circulating androgens and muscle mass and function in young healthy male siblings.The relationship between serum testosterone (T) levels, muscle mass and muscle force in eugonadal men is incompletely understood. As polymorphisms in the androgen receptor (677 men (25–45 years) were recruited in a cross-sectional, population-based sibling pair study.AR gene , sex steroid levels (by LC-MS/MS), body composition (by DXA), muscle cross-sectional area (CSA) (by pQCT), muscle force  and anthropometrics were studied using linear mixed-effect modelling.Relations between genetic variation in the 2) and free E2 (FE2) concentrations were negatively associated with muscle force. Subjects with longer CAG repeat length had higher circulating TT, FT, and higher E2 and FE2 concentrations. Weak associations with TT and FT were found for the rs5965433 and rs5919392 SNP in the AR, whereas no association between GGN repeat polymorphism and T concentrations were found. Arm span and 2D:4D finger length ratio were inversely associated, whereas muscle mass and force were not associated with the number of CAG repeats.Muscle mass and force were highly heritable and related to age, physical activity, body composition and anthropometrics. Total T (TT) and free T (FT) levels were positively related to muscle CSA, whereas estradiol (EAR are related to sex steroid levels and anthropometrics, we have no evidence that these variations in the AR gene also affect muscle mass or function.Age, physical activity, body composition, sex steroid levels and anthropometrics are determinants of muscle mass and function in young men. Although the number of CAG repeats of the  Skeletal muscle mass and function are highly heritable AR). Polymorphisms in the AR gene have been described to alter this activity. We have previously shown that diminished androgen feedback, and consequently higher serum T concentrations, are associated with the CAG repeat length, and to a lesser extend with the GGN repeat length AR gene, resulting in an altered binding with cofactors, have been linked with the androgen insensitivity syndrome (AIS) However, the interrelationship between T levels, muscle mass and muscle force in eugonadal men is less clear AR, causing differences in androgen sensitivity, contribute to the variation in muscle mass in young healthy men.In order to gain more insight into the between subject variation in muscle mass in young healthy men, we investigated the relationship between androgens and muscle mass and function, as well as the influence of genetic components. We hypothesized that genetic variations in the et al.The study protocol was conducted according to the Helsinki Declaration and was approved by the ethical committee of the Ghent University Hospital. All participants gave their written informed consent and questionnaires about previous illness and medication use were completed. Physical activity was scored using the questionnaire as proposed by Baecke This population-based cross-sectional study is part of a larger study, from which inclusion criteria and study design were described previously Body weight and anthropometrics  were measured in light indoor clothing without shoes. Sternum height was measured using a wall-mounted Harpenden stadiometer . Lean and fat mass of the whole body were measured using dual-energy x-ray absorptiometry (DXA) with a Hologic QDR-4500A device . Isokinetic peak torque of biceps and quadriceps muscles was assessed at the dominant limbs using an isokinetic dynamometer . Grip strength at the dominant hand was measured using an adjustable hand-held standard grip device . Their maximum performance was assumed to best reflect the current status and the history of their musculoskeletal adaptation.A peripheral quantitative computed tomography (pQCT) device  was used to scan the dominant leg (tibia) and forearm (radius). Muscle cross-sectional area (CSA) was estimated using a threshold below water equivalent linear attenuation set at 0.22/cm. This threshold eliminated skin and fat mass with lower linear attenuation in the cross-sectional slice. From the remaining area, bone area was subtracted, revealing the muscle at its maximum CSA.2) levels were determined by liquid chromatography tandem mass spectrometry (LC-MS/MS) . Serum limit of quantification was <0.5 pg/mL (1.9 pmol/L) for E2 and 1.2 ng/dL for T. The interassay coefficients of variation (CV) were 4.0% at 21 pg/mL (77 pmol/L) for E2, and 8.3% at 36.7 ng/dL and 3.1% at 307.8 ng/dL for T 2) concentrations were calculated from serum TT, E2, SHBG and albumin concentrations using a previously validated equation derived from the mass action law Venous blood samples were obtained between 08:00 and 10:00 AM after overnight fasting. All serum samples were stored at −80°C until batch analysis. Serum total testosterone (TT) and estradiol . The CAG and GGN repeats were determined as previously described http://www.hapmap.org) and the data was incorporated into the Haploview program 2 of 0.80 (for their location and the SNPs which they tag). For the SNP analyses, SNPlex Genotyping data for the AR gene for the Caucasian CEPH population was downloaded from the International Haplotype Mapping Project web site  and variables were log-transformed in subsequent linear models. Linear mixed-effects modelling with random intercepts and a simple residual correlation structure was used to study the effect of anthropometrics, sex steroid concentrations and genetic variations in the AR on muscle mass and function, with adjustment for the confounding effect of age, adult height and weight or fat mass and taking into account the interdependence of measurements between brothers. Parameters of fixed effects were estimated via restricted maximum likelihood estimation and reported as estimates of effect size (β) with their respective standard error. A sample size of 677 subjects allowed us a 81% power to detect a minimum effect size of 0.01 at a two-sided significance level of 5%. Validity of the models was assessed by exploring normality of distribution of the residuals. SNPs were considered as a categorical variable, whereas CAG and GGN lengths were analysed as continuous variables for assessing association, and as categorical variable (quartiles) with groups compared by one-way analysis of variance (ANOVA). Associations were considered significant at p-values less than 0.05. Statistical analyses were performed using S-Plus 7.0 . The polygenic program in SOLAR 2.0  was used to estimate heritability, using a variance component model.Descriptives are expressed as mean ± standard deviation or median [12. Body composition and muscle function parameters are given in Six hundred seventy seven subjects with a mean age of 34.5±5.5 years are included in the study. Mean height is 1.79±0.06 m and mean weight 81.4±11.8 kg, with a body mass index of 25.3±3.5 kg/mAs expected, the level of physical activity was associated with muscle mass. Biceps force was positively associated with the level of physical activity during work  but not related to physical activity during sports (p = 0.96), whereas quadriceps force was related to sports  and not to physical activity during work (p = 0.52), independent from age, height and weight.2/y±4; p<0.0001) and tibia , which remained positive after additional adjustment for height, physical activity level and body fat (radius: p<0.0001 and tibia: p = 0.004). With increasing age, lower limb muscle force indices slightly decreased after adjustment for height and weight (p = 0.02). Biceps muscle force and maximal grip strength were unrelated to age.Both fat  and lean mass  increased with age, as well as muscle CSA at the radius  and weight . Also a close relationship between muscle CSA and weight  was found. Moreover, maximal grip strength and muscle force indices at upper (biceps) and lower limb (quadriceps) were all positively related to height  and weight .Whole body lean mass exhibited a strong positive association with muscle CSA and muscle function , whereas whole body fat mass was inversely related to muscle CSA at radius (p<0.0001) and grip strength and muscle force of biceps (p<0.001).The relationship of muscle CSA and muscle force  with height and weight are represented in 2 observed for whole body lean mass.Whole body lean mass and muscle CSA at the radius were positively associated with arm span  as well as with finger (p = 0.0001 to 0.04) and hand length  adjusted for height, weight and age. Fat mass was negatively associated with arm span . Moreover, biceps flexion and hand grip force were related to arm span , even more strongly than to hand length  and finger length . Muscle force and muscle CSA were unrelated to sternum height (data not shown). All associations remained positive after additional adjustment for fat or lean mass.2 and FE2 concentrations were negatively associated with maximal grip strength  and quadriceps force , even after additional adjustment for T. No influence of TT or FT on muscle force was observed (data not shown). The 2D:4D finger length ratio and arm span were unrelated to circulating steroid concentrations (data not shown).TT and FT concentrations were positively related to whole body lean mass  and inversely to fat mass , adjusted for age, weight and height. TT concentrations were positively related to muscle CSA at the tibia , and FT was positively associated with muscle CSA at the radius . EAR on circulating gonadal steroids, body composition and muscle function is shown in 2 and FE2 concentrations. Weak associations were found for the rs5965433 and rs5919392 polymorphisms in the AR. However, only the association between CAG repeat and TT and FT remained significant after Bonferroni correction. No associations between GGN repeat polymorphism and TT or FT concentrations, as determined by LC-MS/MS, were found.The influence of genetic variation in the AR polymorphisms or CAG/GGN repeats were found on either body composition, muscle mass or muscle force  were found to be significantly associated with FT, with the first also borderline significantly associated with TT. However, it is noteworthy that these associations did not remained significant after Bonferroni correction. Two recent genome-wide association studies AR gene was not described in these studies. Considering our relatively limited sample size, we suggest that analysis of our SNPs in those larger study populations may be required to confirm our findings.Based on studies reporting mutations in the AR gene influences circulating androgen levels, but may also affect body composition, muscularity or anthropometrics. Data on the association between CAG repeat length and muscle mass is limited and has been contradictory AR with either body composition or measurements of muscularity. This might indicate that the relation of T with the muscle CSA is not related to genetic factors influencing androgen sensitivity, most likely because lower androgen sensitivity is compensated by elevated T levels.Genetic variation in the 2 levels found in men with longer CAG repeat length, as suggested by Huhtamieni IT et al.2 produced in normal men is formed by aromatization of androgens 2 levels. E2 is considered to be the main sex steroid involved in the development and maintenance of bone mass 2 in men with lower androgen sensitivity, but preserved estrogen action, resulted in earlier termination of longitudinal bone growth during puberty, an event wich is clearly observed in boys with aromatase excess syndrome or familiar hyperestrogenism Interestingly, we found that arm span and the 2D:4D finger length ratio were inversely associated with the number of CAG repeats, but not with the GGN repeat lenght or the analysed SNPs. The 2D:4D finger length ratio has been proposed as a marker of prenatal androgen action and of sensitivity to T, with a lower 2D:4D being associated with high androgen exposure AR CAG number AR and 2D:4D ratios AR and 2D:4D ratios exist.To date, several studies have examined the possible relation of adult sex hormone concentrations 2 levels found in men with a higher CAG repeat number might also play a direct role on muscle force since the negative association between E2 and grip strength and biceps force, and between FE2 and grip strength and biceps force in our study persisted after adjustment for T. Also Auyeung et al.2 levels, though positively related to muscle mass, were negatively related to muscle strength. However, it should be noted that the participants of the latter study were much older, with lower T levels.The higher serum E2 on the regulation of muscle mass and function are still poorly understood. As skeletal muscle myoblasts and mature fibers express functional estrogen receptors (ER), a direct effect of E2 in muscle cells may occur 2 is involved in muscle recovery 2 on the musculature has also been suggested by others. Several studies observed a decrease in muscle mass and force after E2 administration of ovariectomized rats et al.ER knockout mice. However, the exact mechanism by which estrogens regulate muscle mass still has to be elucidated.Possible effects of EWe recognize that our study has some limitations. First, our study may have been limited by the relatively small sample size, by which small but significant associations might have been missed, especially for the genetics analysis. Secondly, observations within brothers are not completely independent from each other. However, all analyses in this study were performed using linear mixed-effects modelling with random intercepts to account for this interdependence. Furthermore, the cross-sectional design of this study does not allow us to draw conclusions on causality.2 serum concentrations. Most other studies used direct immunoassays, which are thought to have a reduced specificity at lower concentrations, especially those for serum E2A major strength of this study is that we have used a highly precise LC-MS/MS method to determine T and EAR gene also contributes to the between subject variation in muscle mass or muscle function in young healthy men.In summary, in this study we showed that age, physical activity, body composition, sex steroid levels and anthropometrics are all determinants of muscle mass and function in young men. Although the number of CAG repeats were related to sex steriod levels and anthropometrics, we have no evidence that variations in the


KCNJ5 gene were implicated in APA, and two germline mutations were associated with familial hyperaldosteronism III.Primary aldosteronism (PA) is the most common endocrine form of secondary hypertension, and one of the most common subtypes of sporadic PA is aldosterone-producing adenoma (APA). Recently, two somatic mutations of the KCNJ5 gene and sporadic PA patients in Xinjiang, China.This case-control study was designed to investigate the relationship between genetic variations in the KCNJ5 gene  were detected in patients with sporadic PA (n = 235) and essential hypertension  by the TaqMan polymerase chain reaction method.Five common single nucleotide polymorphisms (SNPs) of the P<0.05), as well as rs3740835 in male subjects (P<0.05). However, only the association between the rs2604204 genotype and male sporadic PA remained significant after Bonferroni’s correction (P<0.01). Furthermore, logistic regression analysis demonstrated that the CC genotype of rs2604204 was a risk factor for male patients with sporadic PA, after adjusting for age and body mass index .The EH group and the PA group showed significant differences in the distributions of genotypes and alleles of rs4937391 and rs2604204 in total and male subjects (KCNJ5 is associated with sporadic PA in Chinese males, suggesting that KCNJ5 may be involved in the pathogenesis of sporadic PA in these particular patients.The genetic variant rs2604204 of  Primary aldosteronism (PA) is a potentially curable disorder characterized by the autonomous overproduction of aldosterone from the adrenal zona glomerulosa. It is the most common endocrine form of secondary hypertension, with an estimated prevalence as high as 10% in patients with hypertension Aldosterone is known to play a key role in regulating blood pressure and maintaining electrolyte and fluid homeostasis. However, excessive aldosterone secretion can result in hypertension, low plasma renin, and hypokalemia. Aldosterone secretion from the adrenal zona glomerulosa cells is regulated by serum potassium. Genetic deletion of TWIK-related acid sensitive K+ channels (known as TASK1) not only leads to low-renin hypertension, attributed to the autonomous overproduction of aldosterone, but also a marked depolarization of the adrenal zona glomerulosa cell membrane potential KCNJ5 gene encodes a potassium inwardly rectifying channel , which is a member of the G protein-sensitive inwardly rectifying potassium (KG) channel family. The subunit composition of the KG channel family members varies among different cells and tissues, corresponding to their diverse functional roles + current (IKACh) The KCNJ5 gene mutations, rs6590357 and rs7118833, associated with this cardiac event KCNJ5 gene variants have found that two somatic mutations (G151R and L168R) and two germline mutations (T158A and G151E) are associated with APA and FH-III, respectively KCNJ5 polymorphisms and sporadic PA are less well studied. In order to better understand the role of KCNJ5 genomic variation in sporadic PA, five common KCNJ5 polymorphisms  were detected in sporadic PA patients from Xinjiang, China.Studies of the underlying genetic factors of atrial fibrillation have identified two The study was approved by the Ethics Committee of the People’s Hospital of Xinjiang Uygur Autonomous Region. All procedures were conducted according to the standards of the Declaration of Helsinki. All study participants provided signed informed consent.A total of 1356 consecutive hypertensive patients were recruited from the inpatient population at the Hypertension Center of the People’s Hospital of Xinjiang Uygur Autonomous Region from January 2010 to December 2010. Patients were excluded from the study if they were diagnosed with FH, adrenocortical carcinoma or any other secondary forms of hypertension, or if they had hypertension combined with congestive heart failure or renal function insufficieny. The final study number was 1148, which including 235 PA and 913 EH patients.PA diagnosis was made upon demonstration of inappropriate autonomous hypersecretion of aldosterone, as determined by a salt loading assay All PA patients underwent an adrenal computed tomography scan. In addition, 120 PA patients underwent adrenal vein sampling to assess the presence of unilateral or bilateral adrenal disease in the absence of ACTH stimulation. A cortisol-corrected aldosterone ratio from the high side to the low side of more than 2∶1 was considered to indicate unilateral aldosterone excess, as suggested by the Endocrine Society Guidelines Hypertension diagnosis was made according to the criteria published by the JNC 7. In particular, patients were required to have one of the following features: at least three occasions of systolic blood pressure (SBP) ≥140 mmHg and/or diastolic blood pressure (DBP) ≥90 mmHg, or previous history of hypertension and/or current treatment with antihypertensive medication despite blood pressure readings <140/90 mmHg. After excluding other forms of secondary hypertension through systemic examination, all patients who did not meet the criteria for PA were diagnosed as EH.Following an overnight fast, blood samples were drawn from an indwelling catheter positioned in an antecubital vein after 30 min of rest in the sitting position between the hours of 8∶00 AM and 10∶00 AM. Serum or plasma from each blood sample were separated within 1 h after blood collection, and all samples were stored at −20°C until use. In addition, isolated blood cells were stored at −80°C until subsequent genotyping.125I] angiotensin I radioimmunoassay kit , and the intra- and inter-assay coefficients of variation were 10% and 15%, respectively. Serum cortisol was measured by the iodine [125I] cortisol radioimmunoassay kit , and the intra- and inter-assay coefficients of variation were 10% and 15%, respectively. Serum sodium, potassium, and creatinine levels were measured on a C800 automated biochemistry analyzer .Serum aldosterone was measured by radioimmunoassay using a commercially available kit , and the intra- and inter-assay coefficients of variation were 4.5% and 9.8%, respectively. PRA was measured by an iodine [http://www.ncbi.nlm.nih.gov and http://www.hapmap.org, respectively). Two SNPs (rs6590357 and rs3740835) were TagSNPs. The other three SNPs  were associated with metabolism syndrome Taq amplification method in the 7900 HT Fast Real-Time PCR System  TaqMan SNP genotyping assay primers and probes were chosen based upon specific sequences for the genes of interest available on the Applied Biosystems Inc. website (http://myscience.appliedbiosystems.com).Five SNPs  were selected from the National Center for Biotechnology Information (NCBI) dbSNP and the International HapMap Project databases  in the second exon; rs2604204 (6262A/C) in the 3′ untranslated region (3′UTR); rs11221497 (−20559C/G) in the promoter region; and rs3740835 (−10811A/C) and rs4937391 (5126A/G) in the first and second intron region. In addition, three mutations in the second exon  were genotyped. To ensure and verify genotyping quality, the case and control subjects were distributed randomly across the plates. Genotyping was carried out by investigators who were blinded to the phenotypic information, with controls of the known genotype included in each genotyping run. The success rate for genotyping was >99%, and the minor allele frequencies for all subjects was >5%.The five following SNPs in the Based on the genotype data for the genetic variations, linkage disequlibrium (LD) analysis was performed along with haplotype-based case-control analysis and haplotype construction. The pairwise LD analysis of the five SNPs was carried out with |D′ values|>0.5 used to assign SNP locations within a single haplotype block. In the haplotype-based case–control analysis, haplotypes with a frequency <0.01 were excluded.t-test, Mann-Whitney U test, or Chi-squared (χ2) test. The case-control-based haplotype analysis and Hardy-Weinberg equilibrium test were carried out by the SNPAlyze software, version 2.1 . Inter-group differences of SNP genotype or allele frequency were analyzed by the Chi-squared test. Logistic regression analysis was used to calculate odds ratios (ORs) with 95% confidence intervals (CIs). P-values <0.05 were considered to indicate statistical significance.All statistical analyses were performed with SPSS statistical software, version 16.0 (USA). The data are expressed as mean ± SD or median (interquartile range). Inter-group comparisons were made with the unpaired Baseline characteristics of the 235 PA patients and 913 EH patients are summarized in KCNJ5 gene  were successfully genotyped and in Hardy–Weinberg equilibrium (P>0.05). In contrast, the G151R, L168R, and T158A mutations were not detected in any of the study cases or controls. The EH group and the PA group showed significant differences in the distributions of genotypes and alleles of rs4937391 and rs2604204 overall and in male subjects , as well as the rs3740835 in male subjects . The association between the genotype of rs2604204 and male patients with PA remained significant after Bonferroni’s correction . No significant differences were observed between the PA cases and the EH controls for all SNPs tested in the female subjects. In addition, the genotype and allele frequencies between 77 bilateral cases and 39 unilateral cases also were analyzed, but no significant differences were observed (data not shown).KCNJ5 gene polymorphisms on PA patients, logistic regression analysis was performed. After adjusting for age, smoking, drinking, and BMI, the CC genotype of rs2604204 was identified as a risk factor of male patients with PA .To further investigate the effect of the KCNJ5 gene are shown in The patterns of pairwise LD between the five common polymorphisms in the P<0.05).Haplotype construction for the five common SNPs yielded a total of 21 haplotypes. Among these 21 haplotypes, the frequencies of six were >1% , while tThere was no significant difference in serum potassium, serum sodium, plasma renin activity, serum aldosterone, or aldosterone/renin activity ratio among the three genotypes of rs2604204 in male subjects with PA .KCNJ5 polymorphisms  and sporadic PA were investigated in a patient population from Xinjiang, China. The preliminary results showed that three of the SNPs, rs4937391, rs3740835 and rs2604204, were significantly associated with in male patients with sporadic PA in our study group.In this study, the relationship between five common However, only the association between rs2604204 genotype and male patients with PA remained significant after Bonferroni’s correction. Further logistic regression analysis demonstrated that the CC genotype of rs2604204 was a risk factor for the male patients’ sporadic PA, after adjusting for age, sex, and BMI.e.g. KCNJ5) and specific traits (e.g. sporadic PA) KCNJ5 was found to be significantly associated with sporadic PA. Furthermore, distributions of the five polymorphisms were found to be in Hardy-Weinberg equilibrium, suggesting that the results of this study are unlikely to be biased by population stratification. Therefore, our results indicate that the rs2604204 SNP may be associated with male patients with sporadic PA in the Chinese.Haplotype analysis is a relatively recent approach for elucidating the relationships between candidate genes  associated with APA and a germline mutation (i.e. T158A) associated with FH-III in an American family. More recently, an additional KCNJ5 variant, G151E, was identified in European families and associated with FH-III KCNJ5 mutations (G151R and L168R) may be associated with a more typical PA phenotype KCNJ5 variants might play an important role in the molecular pathogenesis of APA KCNJ5 in peripheral blood cells of our study population, either in cases or controls. This finding is consistent with those reported from previous studies In 2011, Choi KCNJ5 gene may result in loss of channel selectivity. In addition, the newly reported two somatic variants, delI157 and E145Q, may also result in loss of channel selectivity 2+ channels and elevated intracellular Ca2+ levels. Ultimately, these changes could promote aldosterone secretion and cellular proliferation in vivo. Collectively, however, these findings indicate that different mechanisms exist for different mutations of KCNJ5 in PA.It has been reported that the two somatic variants, G151R and L168R, and the two germline mutations, T158A and G151E, of the et al. demonstrated that 3′UTRs can also influence gene transcription KCNJ5 in the pathogenesis of PA in male patients.The mechanisms by which the rs2604204 SNP might contribute to sporadic PA in the male patients are unknown. The rs2604204 SNP is located in the 3′UTR and plays an important role in the translation, transcription, localization, and stability of mRNA. Several studies have shown that 3′UTRs mediate gene regulation mainly at the translation level, and mRNA stability by providing docking sites for microRNAs and RNA binding proteins et al. reported that KCNJ5 mutation carriers are more likely to be females KCNJ5 was associated with male PA patients. The inconsistent results may be attributed to the difference in the rate at which these mutations occur in female or male patients with PA. Of course, this result requires further validation with a larger sample size.Boulkroun KCNJ5 (rs2604204) was associated with sporadic PA in Chinese males. This finding suggests that common genetic variations of KCNJ5 may be involved in the pathogenesis of sporadic PA in Chinese males. However, further functional analyses of the rs2604204 mutation are necessary to clarify the functional defects caused by the genetic findings.In summary, we found that a common SNP of


The purpose of this study was to describe an approach that aims to provide fundamental information for the application of natural cuttlefish bone. Before applying cuttlefish bone as a bone defect filling material, we evaluated proliferation, adhesion, and cell viability of human mesenchymal stem cells (hMSCs) cultured on cuttlefish bone. Cuttlefish bone was separated into two parts  and each part was used. Cell proliferation and viability were assessed using the MTS assay and live/dead fluorescence staining method. The morphology was observed using scanning electron microscopy (SEM). hMSCs were stimulated with osteogenic medium and osteoblast differentiation was evaluated. The fluorescence images showed that the seeded cells grew well and that cell distribution was in accordance with the surface morphology of the cuttlefish bone. Compared with the dorsal shield, cells penetrated deeper into the three-dimensional inner space of the lamellar part. Furthermore, under osteogenic differentiation conditions, alkaline phosphatase activity increased and the mRNA expression of ALP, runt-related transcription factor 2, and collagen type I α1 was increased in hMSCs cultured on both the dorsal shield and lamellar block. These results indicate the potential of cuttlefish bone as an ideal scaffold for bone regenerative materials. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2012. Numerous conditions, for example, regeneration of damaged bone resulting from disease, trauma, and infection, require bone graft replacement. Despite recent results obtained in bone regeneration and bone graft transplants, there are still crucial issues to be resolved. Although autologous bone grafts have been used as a gold standard for surgical procedures, this approach has some disadvantages, for example, the need for a second surgery at the donor site and limited amounts and shape of bone.3In the bone tissue engineering area, there are several biomaterials that can be used as scaffolds, such as coral, fibrin, and demineralized bone.710[PO4]6[OH]2). Therefore, most reports have focused on the conversion of calcium carbonate materials to hydroxyapatite.Cuttlefish bone is the hard tissue in cuttlefish, which acts as a floating tank in the animal. Cuttlefish bone consists of two regions. One is a thick external wall  and the other is an internal lamellar matrix. The dorsal shield is a thick, hard cover that overlays the lamellar matrix. The internal lamellar matrix consists of a parallel sheet structure of calcium carbonate and the spaces between the sheets (200–600 μm) result in highly porous properties.Therefore, the aim of this work was to demonstrate the biocompatibility and osteoblast differentiation of hMSCs cultured on natural cuttlefish bone. In this study, we used bone marrow-derived hMSCs, which we seeded and then subsequently evaluated for proliferation and attachment. In addition, cell osteoblast differentiation was observed using particular markers of osteoblastic differentiation during culture on cuttlefish bone.Sepia esculenta, from the Korean western Sea), gently washed with distilled water, and dried. First, the dorsal shield and lamellar regions were separated from the cuttlefish bone. The samples were cut into blocks of 4 × 4 × 1 mm3 and 10 × 10 × 2 mm3. For cell culture experiments, these blocks were sterilized with ethylene oxide (EO) gas and transferred to tissue culture dishes.Cuttlefish bone was removed from cuttlefish  were loaded on sterilized dorsal shield and lamellar cuttlefish bone blocks. After predetermined time intervals, 25 μL of MTS reagent was added to each well and incubated for 4 h. Absorbance was measured at 490 nm using an ELISA reader .Proliferation of cells attached and grown on cuttlefish bone blocks was measured using CellTiter96® Aqueous One solution . Briefly, hMSCs . Briefly, hMSCs were seeded on cuttlefish bone blocks and cultured for 3 days. To remove phenol red and serum, cells cultured on cuttlefish bone blocks were washed with phosphate-buffered saline (PBS) for 30 min. The samples were then incubated in a Live/Dead® Viability/Cytotoxcity solution for 30 min. Calcein acetoxymethyl -stained healthy cells appeared green and ethidium homodimer-1 -stained nuclei of dead cells appeared red. After staining, the samples were observed under a fluorescence microscope .M β-glycerophosphate , 50 μg/mL ascorbic acid (Sigma), and 0.1 mM dexamethasone (Sigma). The OS-containing media was changed every 2 days during osteogenic induction.To induce osteoblast differentiation of hMSCs, cells were cultured in media supplemented with an osteogenic stimulant (OS), that is, 10 m3 cells/scaffold. SEM was performed on 3-day culture samples. The cuttlefish bone blocks seeded with hMSCs were washed with PBS buffer and fixed with 2.5% glutaraldehyde solution at 4°C for 16 h. The samples were postfixed with 0.1% osmium tetroxide solution and dehydrated in a series of ethanol . The dehydrated samples were sputter coated with platinum and viewed using SEM .To observe the morphology of cuttlefish bone and behavior of hMSCs on cuttlefish bone blocks, cells were seeded at 1 × 105 cells/scaffold. After 5 days of culture with OS-containing medium, total mRNA was isolated using a RNA purification kit  and cDNA was transcribed with reverse transcriptase  and oligo (dT) primers. cDNA was amplified using the TaqMan Universal PCR Master mix (Applied Biosystem) and primers and TaqMan probe sets for alkaline phosphatase , collagen type I α1 , runt-related transcription factor 2 , and 18S (Hs99999901_s1) (Applied Biosciences). All TaqMan PCRs were performed using the StepOne Plus real-time PCR system . The 18S rRNA gene was coamplified as an internal standard.To assess the gene expression of osteogenic markers, hMSCs were seeded at 1 × 10M glycine NaOH buffer (pH 10.4) and 100 μL of 15 mMp-nitrophenyl phosphate (p-NPP) (Sigma) and incubated for 30 min at 37°C. The reaction was terminated by adding 0.1N NaOH. The degree of p-NPP hydrolysis was determined using an ELISA reader (Spectra MAX M3) at 410 nm; p-nitrophenol  was used as a standard. Protein concentrations were measured using a BCA protein assay reagent kit  according to the manufacturer's instruction. The experiment was repeated at least three times.The degree of osteoblastic differentiation of the cells was evaluated by determining ALP activity. After 5 days of culture using osteogenic induction medium, the adherent cells that were removed from the bone blocks were homogenized in PBS with 1% Triton X-100. A 100-μL aliquot of the suspension was then mixed with 200 μL of 0.1t-test and one-way ANOVA. p < 0.05 was considered to indicate statistical significance.All values were expressed as the mean ± standard deviation of the mean (SD). Statistical analysis was performed using Student's Cuttlefish bone was prepared and cut into small blocks . To obseThe representative graph for cell proliferation shows that both lamellar and dorsal shield blocks exhibit a similar cell growth pattern during cultivation. At day 1, the cell density was not significantly different between lamellar and dorsal shield blocks. From day 10 of cultivation, the cell density on the lamellar block was higher than that on the dorsal shield block. Furthermore, at day 15, proliferation of the cells cultured on the lamellar block was significantly higher than on the dorsal shield block .To evaluate the cytotoxic effects of natural cuttlefish bone, cells were seeded and cultured on lamellar and dorsal shield blocks. After 3 days of cultivation in normal growth media, live/dead staining images were taken. The results showed that hMSCs were viable and very few dead cells were detected on both lamellar and dorsal shield blocks from cuttlefish bone. In addition, cells cultured on these blocks maintained a fibroblast-like morphology during cultivation . These rTo assess the distribution of hMSCs throughout the blocks, the blocks were sectioned and stained with Calcein AM. The sections used were from the central part of the blocks and images were taken by using fluorescence microscopy. The fluorescence images clearly demonstrated a difference in cell distribution between lamellar and dorsal shield blocks. The seeded cells proliferated in a multilayer fashion and most of the cells were observed on the surface of the dorsal shield block, whereas on the lamellar block, the cells proliferated inside the pore channels after a period of 10 days .Cellular morphology and cell interaction of hMSCs seeded on lamellar and dorsal shield blocks were visualized by using SEM after 3 days of culture. On both the dorsal shield block and lamellar block, hMSCs grew following the surface morphology and maintained their fibroblast-like shape [To determine whether hMSCs differentiated into osteoblasts during OS treatment and when cultured on natural cuttlefish bone, ALP activity was measured and qRT-PCRs were performed. The ALP activity of hMSCs was quantified after 5 days of culture . The ALPin vivo, such as an immune response. In this study, we used natural cuttlefish bone prepared with minimal processing and focused on ensuring that, by providing an appropriate environment, hMSCs attach, proliferate, and differentiate into osteoblasts.The development of porous materials, used as scaffolds for cell transplantation therapy, is of particular interest in tissue engineering. The biological responses of a material require the use of cell cultures adapted to tissue engineering. MSCs are multipotent stem cells that can differentiate into osteoblasts. Therefore, the behavior of MSCs in the presence of a scaffold material is an important indicator in the evaluation of the material's biocompatibility. The aim of this study was to evaluate natural cuttlefish bone as potential candidate for tissue-engineered bones. Natural cuttlefish bone is composed of calcium carbonate and contains small amounts of heavy metals and other organic components. These ancillary components have the potential to cause adverse reactions Sepia officinalis L.) consists of two regions: a thick external wall and an internal matrix. The dorsal shield is a hard cover that overlays the lamellar matrix. This layer has a nonporous structure. The lamellae are separated by pillars and form chambers, that is, areas with spaces between 200 and 600 μm.Sepia esculenta obtained from the Korean Sea, the lamellar pore size and structural patterns were not significantly different from previously observed results.8First, we separated the cuttlefish bone into two structural parts  and confirmed the surface morphology of the lamellar and dorsal shield parts of natural cuttlefish bone. Birchall et al. have reported that cuttlefish bone  contains small amounts of heavy metals such as mercury (0.05 ppm), copper (0.52 ppm), zinc (2.42 ppm), lead (0.39 ppm), and cadmium (0.07 ppm).18Cell culture level tests are a starting point for establishing whether materials are noncytotoxic, and the tetrazolium-based colorimetric MTS assay is a quantitative method for assessing the biological response of cells to a material, whereas the direct contact test permits qualitative assessment of the cell's response after culture. According to the report by Cho et al., cuttlefish bone (The SEM study of hMSCs seeded on cuttlefish bone showed that hMSCs attached to both the lamellar and dorsal shield surface of cuttlefish bone blocks . These rALP activity is one of the most commonly used markers of osteogenesis and reflects the proportion of osteogenic differentiation. ColIα1 is known to play an important role during osteoblast differentiation and cell adhesion.In this study, we have demonstrated that natural cuttlefish bone supports the adhesion, proliferation, and differentiation of hMSCs. In particular, hMSCs grew only on the outer surface of dorsal shield blocks, whereas they penetrated deeper into the lamellar blocks. These findings suggest that the lamellar and dorsal shield parts of cuttlefish bone could be potentially used as bone substitute and as a barrier to inhibit fibroblast infiltration near the bone defect during bone regeneration, respectively. However, one of the major disadvantages of the lamellar matrix of cuttlefish bone as bone regenerative scaffold is its fragility. Therefore, further study will be needed to improve its mechanical strength and stability.


TEM coupled with EDX confirmed that distinct nanoparticles were composed of silver. Yeast extract and mannitol were key components of YEM medium responsible for the formation of nanoparticles. PXRD analysis indicated crystalline geometry and Ag/Ag2O phases in nanoparticles generated with YEM medium, yeast extract and mannitol. Our investigations also revealed that both mannitol and yeast extract possessed potential to convert ∼80% of silver ions in 0.5 mM AgNO3 to nanoparticles, on autoclaving for 30 min at 121°C under a pressure of 1.06 kg/cm2. Addition of filter sterilized AgNO3 under ambient conditions to pre-autoclaved YEM medium and yeast extract brought about color change due to the formation of silver nanoparticles, but required prolonged duration. In general, even after 72 h intensity of color was significantly less than that recorded following autoclaving. Silver nanoparticles formed at room temperature were more heterogeneous compared to that obtained upon autoclaving. In summary, our findings demonstrated that (i) YEM medium and its constituents promote synthesis of silver nanoparticles; and (ii) autoclaving enhances rapid synthesis of silver nanoparticles by YEM medium, yeast extract and mannitol.We discovered that Yeast Extract Mannitol (YEM) medium possessed immense potential to generate silver nanoparticles from AgNO Nanotechnology is one of the most advancing areas of research in modern material science. Nanoparticles exhibit completely new or improved physical  and chemical properties. Silver (Ag) nanoparticles possess strong antimicrobial effects, making them suitable for use in medical devices such as catheters, wound dressings, skin donation, prostheses, contraceptives, in daily commodities such as shampoos, soaps, toys, paints, textile, shoe insoles, washing machines etc. Various physical, chemical and physico-chemical approaches such as the use of laser ablation, mechanical milling, inert gas condensation, thermal irradiation, laser irradiation, chemical reduction, photochemical reduction and electrochemical techniques have been demonstrated to generate metal nanoparticles Rhizobium sp. to synthesize silver nanoparticles [using silver nitrate (AgNO3)], we noted the change in color of yeast extract mannitol (YEM) medium supplemented with AgNO3 from pale yellow to brown up on autoclaving. Such a change in color of medium supplemented with AgNO3 is often associated with the formation of silver nanoparticles. This prompted us to carry out investigations for identifying the component(s) of YEM medium responsible for generation of silver nanoparticles.During an attempt to assess the potential of a symbiotic bacterium + under aqueous conditions. Our investigations revealed that autoclaving promotes rapid synthesis of stable, well dispersed silver nanoparticles in aqueous phase and the same has been compared with the production of silver nanoparticles under ambient conditions.In this communication, we report for the first time that the YEM medium and two of its components namely mannitol and yeast extract have potential to synthesize silver nanoparticles from Ag2HPO4, MgSO4.7H2O and NaCl were of Himedia (India) make. AgNO3 was obtained from Merck. All the chemicals and reagents used were of Analytical grade, and double distilled water was used for all the investigations. YEM broth composed of 53 mM Mannitol, 2.8 mM K2HPO4, 0.81 mM MgSO4.7H2O, 1.7 mM NaCl and 1 g/L yeast extract + with YEM medium, the later was supplemented with 0, 0.1, 0.25 and 0.5 mM AgNO3. In order to establish the component of YEM medium that resulted in the synthesis of silver nanoparticles upon autoclaving, individual components namely, Mannitol, Yeast Extract, K2HPO4, MgSO4.7H2O and NaCl were used (as per the concentrations in YEM medium) with 0.25 mM AgNO3. The pH in all cases was adjusted to 6.8. The media were dispensed into test tubes and placed in ∼75°C pre-heated vertical autoclave  and autoclaved for 30 min at 121°C under a pressure of 1.06 kg/cm2 (∼10 min was required for attaining a temperature of 121°C). About 20–30 min was taken for bringing down the autoclave to atmospheric pressure and temperature. After initial screening, the potential of mannitol and yeast extract to generate silver nanoparticles was evaluated with 0, 0.1, 0.25 and 0.5 mM AgNO3.Mannitol, Yeast Extract, K3 were added under sterile conditions and incubated for different time intervals under ambient conditions.In order to test, if silver nanoparticles can be generated at room temperature, YEM medium, yeast extract and mannitol were autoclaved and after cooling different levels of filter sterilized AgNOThe presence of silver nanoparticles in the resultant colloidal solution was established through UV-Vis spectroscopy, transmission electron microscopy (TEM) and powder X-ray diffraction (PXRD) analysis.The production of silver nanoparticles was initially confirmed by the UV-Vis spectrum of the resultant colored colloidal solutions. The spectra of the media were recorded using Analytikjena Specord 200 at a resolution of 10 nm and a scan rate of 20 nm/sec, to ascertain the presence of peaks arising due to surface plasmon resonance specific to silver nanoparticles.3 in combination with YEM broth, yeast extract or mannitol were centrifuged at 15,000×g for 30 min at 25°C. The resultant pellet formed was suspended in desired amount of water and used for TEM and PXRD studies.The colored colloidal solutions obtained on autoclaving AgNOFor TEM investigations the suspended pellet was subjected to sonication for 30 min. 10 µl of the sonicated solution was drop coated onto a 200 mesh copper TEM grid with an ultrathin continuous carbon film and allowed to dry in a desiccator. The grids were viewed in the transmission electron microscope (Technai G2 T30 U-TWIN) at a voltage of 300 kV. The hardware associated with the machine also allowed (i) energy dispersive X-ray (EDX) to measure the elemental composition and (ii) selected area electron diffraction (SAED) pattern to reveal the crystalline nature of nanoparticles.For PXRD studies the pellet suspended in water, was drop coated on the silica surface, and dried in the desiccator. The PXRD pattern was collected using RIGAKU ROTAFLEX RAD-B using Cu target CuK(α) 1 radiation with tube voltage 40 kV and 60 mA in the 2θ (degree) range of 20–80°.g in Sigma 3K30 centrifuge to obtain supernatant and the pellet. Silver left in the supernatant was determined with GBC Scientific Equipment SensAA atomic absorption spectrophotometer (AAS) at 328.1 nm.For estimating the product yield, resultant colloidal solutions were centrifuged at 57438×3 changed from pale yellow to brown on autoclaving , (211), (220), (221) and (222).The SAED pattern revealed the crystalline nature of silver nanoparticles inset , which w3 in combination with 53 mM (∼0.9%) mannitol or 0.1% yeast extract are shown in TEM images of silver nanoparticles formed on autoclaving AgNOThe PXRD pattern showed Bragg reflections that can be assigned to (111), (200), (220), (311) planes, and matched with JCPDS No. 04–0783. These results confirmed that silver nanoparticles synthesized with mannitol as well as yeast extract were crystalline with fcc structure. In a manner similar to YEM medium, the PXRD patterns of silver nanoparticles formed with mannitol and yeast extract  also sho3 autoclaved independently with yeast extract and mannitol did not show the presence of any silver ions in the supernatant, as they might have been converted into nanoparticles that pelleted down following centrifugation at 57438×g. However, 0.5 mM AgNO3 autoclaved independently with yeast extract and mannitol retained about 20% silver in ionic state.The AAS studies reveal that yeast extract as well as mannitol have equal potential to generate silver nanoparticles. Lower concentrations (viz. 0.1 and 0.25 mM) of AgNOThe silver nanoparticles formed with mannitol as well as yeast extract were well dispersed and stable, and showed no signs of agglomeration even on storing for a period of over a month under ambient conditions .3 from pale yellow to brown just upon autoclaving even without the inoculation of any microorganism. It has been established that colorless AgNO3 solution turns yellow to brown due to the formation of silver nanoparticles 3 and accordingly this medium showed absorption peak at ∼410 nm, which is known to arise due to surface plasmon resonance in the silver nanoparticles During present investigations, we observed alteration in color of YEM medium supplemented with AgNO4, K2HPO4 and NaCl 3 autoclaved independently with different components of YEM medium clearly indicated that two of its constituents namely, mannitol and yeast extract possessed potential to generate silver nanoparticles. As expected, silver nanoparticles generated with mannitol and yeast extract also showed absorption peaks at ∼410 nm due to their specific surface plasmon resonance. However, the absorption peak obtained with yeast extract was broader when compared to mannitol, indicating that the nanoparticles formed with the former were heterogeneous. The heterogeneity of silver nanoparticles formed by yeast extract was also evident from TEM images which not only showed variation in shape but also exhibited significant variation in size. Such a heterogeneous population of nanoparticles might have resulted due to the complex composition of yeast extract YEM is a complex medium constituted of yeast extract, mannitol, MgSO3 upto 0.25 mM. However, about 80% of silver ions were converted into nanoparticles with 0.5 mM AgNO3 within a duration of 1 h on autoclaving. The retention of 20% of silver ions in 0.5 mM AgNO3 indicated that the level of yeast extract and mannitol used was not sufficient for reducing 100% silver into nanoparticles. We believe that it is important to increase the level of mannitol or yeast extract depending on the concentration of AgNO3 used for obtaining optimal yield of silver nanoparticles, i.e. for converting cent percent of silver ions into nanoparticles.Both yeast extract and mannitol possessed potential to convert cent percent of silver ions into nanoparticles when autoclaved with at concentrations of AgNO3 to cooled, pre-autoclaved YEM medium, yeast extract and mannitol. This revealed that the former two possessed potential to alter the color and generate silver nanoparticles, but this alteration in color and the formation of silver nanoparticles under ambient conditions required prolonged duration. Infact, the intensity of the color of YEM medium as well as yeast extract incubated with AgNO3 under ambient conditions for a duration of 72 h was significantly less than that noted in a duration of 1 h through autoclaving. Further, TEM investigations revealed that silver nanoparticles formed under ambient conditions were more heterogenous when compared to those formed through autoclaving.During the course of present studies, experiments were also carried to assess, if silver nanoparticles can be generated under ambient conditions by adding and incubating filter sterilized AgNO3 to pre-autoclaved cooled medium required prolonged duration for the generation of silver nanoparticles under ambient conditions and reasonable quantity of silver nanoparticles could be generated after ∼72 h. In contrast, autoclaving AgNO3 independently with YEM medium, yeast extract and mannitol promoted generation of large quantities of silver nanoparticles within a duration of 1 h. Further, the nanoparticles generated under ambient conditions were more heterogeneous when compared to those obtained through autoclaving.Addition of AgNOThe stability of nanoparticles depends on the medium, inter-particle forces and chemical reactivity, which affect aggregation, size and shape of the nanoparticles For apt application in medicine and pharmaceutical industry it is important to generate silver nanoparticles under sterile conditions. We believe that autoclaving is a suitable approach for generation of metal nanoparticles under sterile conditions, as autoclaving is widely used for general sterilization purposes.+ and formation and stabilization of silver nanoparticles. Similarly, vitamins such as riboflavin present in yeast extract could have also played an important role in generation of silver nanoparticles as has been reported earlier Yeast extract being autolysate of yeast cells is a cocktail of proteins/polypetides, amino acids, carbohydrates, vitamins etc. + to Ag0 following Fetizon oxidation mechanism which involves oxidation of a primary alcohol to an aldehyde group followed by further oxidation to form lactone/ketone 0 can further nucleate to generate silver nanoparticles.The nanoparticles formed with mannitol were relatively uniform in shape and size. In all likelihood mannitol might have generated silver nanoparticles through reduction of AgFor effective commercial endeavors, it is important to generate nanoparticles of uniform size and shape. It is equally important to ensure that the molecules/agent used for generating nanoparticles are completely safe and compatible with living systems and must be eco-friendly. Unlike yeast extract, which is a complex mixture of several biomolecules, mannitol has been well established to be a compatible solute, which has potential to protect living systems against various stresses The generation of stable nanoparticles with good dispersion potential under aqueous conditions is one of the key concerns associated with practical applications of nanoparticles in medicine and industry. Researchers across the globe have been looking for appropriate green protocols for improving the dispersion stability of nanoparticles for wider application and performance + in YEM medium prior to autoclaving lead to generation of silver nanoparticles and therefore, it is not appropriate to include silver salt in the culture medium prior to autoclaving for evaluating the impact of Ag+ on microorganisms; (ii) YEM medium and two of its constituents namely, yeast extract and mannitol promote rapid generation of nanoparticles by autoclaving ; and (iii) the silver nanoparticles generated on autoclaving AgNO3 with yeast extract and mannitol remain stable in aqueous phase under ambient conditions for long durations.In summary, our results demonstrated for the first time that (i) inclusion of Ag


AbstractDreissena polymorpha) and the quagga mussel (Dreissena rostriformis bugensis) are considered as the most competitive invaders in freshwaters of Europe and North America. Although shell characteristics exist to differentiate both species, phenotypic plasticity in the genus Dreissena does not always allow a clear identification. Therefore, the need to find an accurate identification method is essential. DNA barcoding has been proven to be an adequate procedure to discriminate species. The cytochrome c oxidase subunit I mitochondrial gene (COI) is considered as the standard barcode for animals. We tested the use of this gene as an efficient DNA barcode and found that it allow rapid and accurate identification of adult Dreissena individuals.The zebra mussel ( PageBreakspecies are defined as “alien species whose introduction does, or is likely to, cause economic or environmental harm or harm to human health”  and the quagga mussel  are invasive freshwater bivalves in Europe and North America  and Dreissena blanci Westerlund, 1890) begin to expand in the area and may be potentially invasive in Europe.The zebra mussel  mitochondrial gene, the most-widely utilized gene for animal DNA barcoding  could be used as an inexpensive method to distinguish between zebra and quagga mussel.Although features , this tareissena , b. For reissena . Here wearcoding  and we tDreissena samples were collected in the Meuse River  according to manufacturer guidelines. To minimize cost, DNA extraction with the CTAB (hexadecyltrimethylamoniumbromide) protocol proposed by Total genomic DNA was extracted from 241 Dreissena species in the Meuse River we tested three barcoding methods: (i) the “Operational Taxonomic Units” (OTU) (Sequences were collapsed into unique haplotypes using DnaSP . In ordes” (OTU) , (ii) ths” (OTU) , and (iis” (OTU) . The K/θs” (OTU) . Neighbos” (OTU) . Sequencs” (OTU)  were uses” (OTU) .restriction map application (http://www.bioinformatics.org/sms2/rest_map.html), we selected two endonucleases to differentially cut the COI gene of Dreissena species: Hinf I and Nla III. We also tested two other enzymes used in previous studies: Nla IV . For each haplotype, the RFLP was performed in 31 μl total volume including 10 µl of PCR reaction mixture, 18 µl of distilled water, 2 µl of buffer (supplied by the manufacturer with the enzyme), and 1 µl of enzyme. Digests were incubated at 37 °C for 3 hours and then loaded on 2% agarose gels.PageBreakDreissena individuals. The OTU method revealed, by a NJ tree, two clusters separated by a genetic distance of 18.5%  cluster with Dreissena polymorpha. This, together with the three barcoding methods which each identified two clusters, shows that both Dreissena polymorpha and Dreissena rostriformis bugensis species occur in the Meuse River.Our network  revealedPageBreakPageBreakPageBreakDreissena species. Digestion with Nla IV produced four fragments in quagga mussels (Q haplotype) of approximately 70, 79, 211, and 294 bp and three distinct patterns for the zebra mussel (Z haplotype): haplotype Z1 and Z2 , haplotype Z3 and Z4 , and haplotype Z5 . We suggest here that the 200 bp fragment of the haplotype Z5 is an artefact, as confirmed by the restriction map, since the summed fragment lengths do not add up to the expected 654 bp. We infer that haplotype Z5 has the same pattern as haplotype Z3 and Z4. Digestion with Hinf I produced two fragments in quagga mussels of approximately 73 and 581 bp and five fragments in zebra mussels of approximately 31, 101, 114, 195, and 213 bp. The small fragments can not be distinguished on the gel but the difference between quagga and zebra is clear. Digestion with Nla III produced two fragments in quagga mussels of approximately 193 and 461 bp and three fragments in zebra mussels of approximately 193, 319, and 335 bp. Digestion with Scr FI produced five fragments in quagga mussels of approximately 42, 53, 120, 171, and 268 bp and three fragments in zebra mussels of approximately 95, 152, and 407 bp. The digestion pattern for the quagga mussel using the endonuclease Scr FI is not clearly defined (smear) since the five fragments are very short.Digestion profiles for each haplotype are illustrated in Dreissena species may become invasive and should be detected promptly.On September 9 2013, the European Commission has published a proposal for a Regulation on the prevention and management of the introduction and spread of invasive alien species. This proposal highlights three types of interventions: prevention, early warning and rapid response, and then management of invasive species . In thisDreissena polymorpha and Dreissena rostriformis bugensis. Moreover, it is possible to conduct a RFLP analysis on this gene to obtain results without sequencing cost. This method could also easily be applied to Dreissena presbensis and Dreissena blanci since the COIgene have already been sequenced by EF414478–EF414492, EF414496, HM209829–HM210081). We showed that the endonuclease Nla IV, previously used by Dreissena blanci and Dreissena presbensis different from those observed in the zebra and quagga mussel.In order to help managers and national agencies, we propose here the use of the COI mitochondrial gene as a barcode to discriminate This study is the first step of an extensive phylogeographical analysis on the invasion of Western Europe by the dreissenids. Further experiments will be needed to assess potential risks of both zebra and quagga mussels on native biodiversity in Western European rivers, e.g. predation on phytoplankton, infestation on native bivalves and alteration of macro-invertebrate communities.


Biomarkers of selenium are necessary for assessing selenium status in humans, since soil variation hinders estimation of selenium intake from foods. In this study, we measured the concentration of plasma selenium, selenoprotein P (SEPP1), and glutathione peroxidase (GPX3) activity and their interindividual differences in 383 low-income blacks and whites selected from a stratified random sample of adults aged 40–79 years, who were participating in a long-term cohort study in the southeastern United States (US). We assessed the utility of these biomarkers to determine differences in selenium status and their association with demographic, socio-economic, dietary, and other indicators. Dietary selenium intake was assessed using a validated food frequency questionnaire designed for the cohort, matched with region-specific food selenium content, and compared with the US Recommended Dietary Allowances (RDA) set at 55 µg/day. We found that SEPP1, a sensitive biomarker of selenium nutritional status, was significantly lower among blacks than whites , with blacks less than half as likely to have highest vs. lowest quartile SEPP1 concentration (Odds Ratio (OR) 0.4, 95% Confidence Interval (CI) 0.2–0.8). The trend in a similar direction was observed for plasma selenium among blacks and whites, , while GPX3 activity did not differ between blacks and whites . Levels of the three biomarkers were not correlated with estimated dietary selenium intake, except for SEPP1 among 10% of participants with the lowest selenium intake (≤57 µg/day). The findings suggest that SEPP1 may be an effective biomarker of selenium status and disease risk in adults and that low selenium status may disproportionately affect black and white cohort participants. Over the past eight decades, there has been a great increase in the scientific literature on the health effects of selenium – from its first discovery as a toxic agent in large animals Selenium has been implicated as a possible inhibitor of a variety of cancers, key among them being prostate Selenium is widely distributed in foods, but its availability depends on the concentration of selenium in the soil. Where there are low selenium soils, selenium deficiency has been demonstrated, especially in China It is thought that levels of selenium needed to protect against cancer may be higher than those needed to correct nutrition deficiency The purposes of this study were: (1) to measure differences in plasma biomarkers of selenium status  among low-income blacks and whites selected from a stratified random sample of adults aged 40–79 years, who were participating in a long-term cohort study in the southeastern US, and (2), to determine the utility of plasma biomarkers in assessing selenium status and related demographic, socioeconomic, dietary, and other indicators.Study protocols were approved by Institutional Review Boards at Meharry Medical College and Vanderbilt University and participants provided written informed consent administered by trained interviewers.The SCCS is a prospective cohort study conducted in the southeastern US with a major goal to understand the causes of cancer and other key health disparities among blacks and whites. Details of the methods have been described elsewhere , inAmong the biomarkers, SEPP1 showed the strongest racial differences, with blacks having mean SEPP1 significantly lower than whites (p = 0.006) . We alsoth percentile crude r = 0.41, p<0.05; adjusted r = 0.56, p<0.005)  . Our finThe primary purpose of this study was to determine whether there may be variation in selenium status and to provide clues to differentials in indications of selenium insufficiency among black and white participants in the SCCS. This study demonstrated the feasibility of assessing selenium status using three plasma selenium biomarkers in a representative sample of low income black and white adults. SEPP1 values in this group were slightly below those in more affluent strata