However, the results obtained from the transcript expression study and enzymatic activities related to the ascorbate-glutathione pathway did not fully explain the differences in the oxidative damage observed between salinity and the combination of salinity and heat. An exhaustive metabolomics study revealed the differential accumulation of phenolic compounds depending on the type of abiotic stress applied. An analysis at gene and enzyme levels of the phenylpropanoid metabolism concluded that under conditions where flavonols accumulated to a greater degree as compared to hydroxycinnamic acids, the oxidative damage was lower, highlighting the importance of flavonols as powerful antioxidants, and their role in abiotic stress tolerance.Efficient detoxification of reactive oxygen species (ROS) is thought to play a key role in enhancing the tolerance of plants to abiotic stresses. Although multiple pathways, enzymes, and antioxidants are present in plants, their exact roles during different stress responses remain unclear. Here, we report on the characterization of the different antioxidant mechanisms of tomato plants subjected to heat stress, salinity stress, or a combination of both stresses. All the treatments applied induced an increase of oxidative stress, with the salinity treatment being the most aggressive, resulting in plants with the lowest biomass, and the highest levels of H This, together with the expected increase in the world's population, highlight the urgent need for generating crops with increased tolerance to abiotic stress conditions. Traditionally, the effects of abiotic stress on plants have been studied through the application of a single stress, such as salinity, drought or heat, under controlled laboratory conditions  for 2080 indicate a substantial deterioration of current conditions , and hydroxyl radical (HO), are formed in cells by the partial reduction of oxygen. Plants can be damaged by the accumulation of ROS not only on a daily basis, but also on a seasonal basis, as a consequence of their being subjected to long-term abiotic stress conditions, such as drought, salinity, low/high temperatures, or nutrient deprivation , such as the superoxide anion  do not have. The tomato plants in our study were grown under salinity, heat or the combination of both. The results obtained from transcript regulation and enzymatic activities did not fully explain the lower oxidative damage observed under the combination of salinity and heat as compared to salinity. Thus, we hypothesized that additional compounds might be involved in ROS detoxification during the different stresses studied. A metabolomics study performed under these conditions revealed that the type of phenolic compound that accumulated was very different depending on the stress applied, with flavonols accumulating in greater concentrations as compared to hydroxycinanmic acids. Moreover, the higher concentrations of flavonols correlated with lower oxidative damage, highlighting their role in cellular ROS homeostasis, the reduction of oxidative damage, and abiotic stress tolerance.In this work, our main objective was to gain more insight into the changes that occur during ROS metabolism in tomato plants. Tomatoes are an economically-important crop world-wide, have many nutritional and health qualities, and are used as a model plant in many studies due to its many interesting features such as fleshy fruit, a sympodial shoot, and compound leaves, which other model plants  were sown in vermiculite in a growth chamber under control conditions of light (500 μmol m−2 s−1), photoperiod (16/8 h day/night), humidity (60–65%) and temperature (25°C) for 10 days until the appearance of the first two true leaves. A total of 78 plants were transferred to 18 L containers and grown in aerated hydroponic systems containing a modified Hoagland solution . The accession number of the genes assayed for expression and the primer sequence used are listed in Table 2 of the FC was calculated and represented in Figure 5. Relative expression values obtained from qPCR experiments can be found in Table Total RNA was isolated from whole tomato leaves from (as described above) using the TRI Reagent . The RNA was cleaned with an RNAeasy spin column (Qiagen). To eliminate traces of DNA, the RNA was treated with DNase I (Fermentas Life Sciences) according to the manufacturer's protocol. cDNAs were synthesized from three separate RNAs  using the SuperScript VILO cDNA synthesis kit (Invitrogen). From each cDNA, three technical replicates were used, so that every sample was represented by nine replicates in total. Primer3 software was used for primer design based on the tomato unigenes sequences available at Sol Genomics Network , as a degradation product of lipid peroxidation, was determined as Fu and Huang  with theProtein oxidation was assayed as according to Reznick and Packer .g for 20 min at 4°C. The supernatant was precipitated with ammonium sulfate (70% w/v saturation), kept on ice for 1 h, and centrifuged at 16,000 × g for 10 min at 4°C. The resulting precipitate was dissolved in 1 mL of 100 mM sodium phosphate buffer (pH 6.8) containing 8 mM DTT, 40 mM sodium ascorbate, and 1% m/v BSA. After centrifugation, the enzyme extract was purified with ammonium sulfate. All the enzyme extracts were desalted with PD10 columns and then used for enzyme analysis immediately. Enzyme quantifications were performed spectrophotometrically using a temperature-controlled plate reader .The extraction procedures for the enzymatic analyses were performed as in Li et al.  with somPAL activity was assayed in a mixture (250 μL) containing 100 mM Tris-HCl buffer pH 8.0 and enzyme extract. The reaction was initiated by the addition of 150 μL of 200 mg mL−1 L-phenylalanine  and the production of cinnamic acid was measured over 10 min at ΔA290.C4H activity was assayed in a mixture (250 μL) containing 100 mM phosphate buffer (pH 7.5), 1 mM DTT, 1 mM NADPH, and 100 μL enzyme extract. The reaction was initiated by the addition of 10 mM trans-cinnamic acid  and the changes in absorbance at 290 nm was recorded during 10 min.F3H activity was assayed in two independent reactions following the conversion of naringenin to dehydrokaempferol and the conversion of eridictyol to dihydroquercetin. The reaction mixture (250 μL) contained 100 mM Tris-HCl buffer (pH 7.5), 0.5 mM DTT, 0.25 mM 2-oxoglutarate, 0.05 mM ferrous sulfate, 5 mM sodium ascorbate, and 0.5 mM of the substrate (naringenin or eridictyol). The addition of the substrate initiated the reaction and the variation in absorbance at 280 nm was followed for 10 min. Results obtained from each substrate were combined and plotted as F3H activity.CHI activity was assayed as follows: 2′,4,4′,6′-Tetrahydroxychalcone was synthesized from naringenin by treatment with 50% KOH followed by acidification and recrystallization with aqueous ethanol. Enzyme extract (100 μL) was added to 50 mM Tris-HCl pH 7.4 containing BSA  and 50 mM KCN to give a final volume of 250 μL. The reaction was initiated by adding 5 μL of 1 mg mL−1 tetrahydroxychalcone in 2-ethoxyethanol and changes in absorbance at 381 nm was recorded with the cell holder maintained at 30°C. To allow for spontaneous isomerization of the chalcone, the reference cell contained the assay mixture without enzyme. The rate of disappearance of the chalcone (ΔA381), in the presence of enzyme, was used to estimate CHI activity.CHS and UFGT activities were assayed as described by Ghasemzadeh et al.  were also eliminated from our analysis .Six biological replicates of fresh tomato leaves for each treatment  were used for metabolite profiling. The samples were prepared by extracting 1 g of fresh material with methanol:water . Methanol was the preferred solvent used as it was able to successfully extract most of the polar and semi-polar metabolites contained in tomato leaves ; KNApSAcK metabolite database (http://kanaya.naist.jp/KNApSAcK/); KOMOCS ; MassBank (http://www.massbank.jp/); Madison 15 Metabolomics Consortium Database (http://mmcd.nmrfam.wisc.edu/); ARMeC: High Mass resolution annotation database (http://www.armec.org/MetaboliteLibrary/index.html); METLIN (https://metlin.scripps.edu/); Human Metabolome Database (HMBD: http://www.hmdb.ca/) within a mass tolerance of 10 ppm was performed. If no matched metabolites were found, the molecular feature was noted as unidentifiable, and only the matched metabolites were considered as candidates. 2) For each metabolite candidate, the spectrum coverage, the sum of the matched fragments intensities over the total fragment intensities in the spectrum was used to determine the underlying metabolite. If the metabolite could not be identified by spectrum match, we checked whether characteristics fragments existed in the different molecular features to reduce the number of candidates. Isotope ratio (IR) matches were also performed for all of them to ensure a metabolite's identity. Finally, retention times (RTs) from the different identified metabolites were checked again across the different databases, since they are usually relatively stable across experiments. Although similar RTs usually appear across samples, their relationship with a single mass almost always ensures them to be associated with a single metabolite. Finally, a comparison of the results obtained to those found in the literature  A search based on the predicted elemental composition in the available databases  by sonication for 1 h, followed by overnight extraction and another sonication period (1 h). The resulting extract was centrifuged and filtered through a 0.45 um PVDF membrane.Chromatographic separations were carried out on a Phenomenex reverse-phase column . The mobile phase consisted of two solvents: water/acetic acid  and acetonitrile (B), starting with 5% B and using a gradient to obtain 50% at 30 min and 80% at 37 min. The flow rate was 1 mL/min and the injection volume 20 μL. Spectral data from all peaks were accumulated in the range of 200–400 nm, and chromatograms were recorded at 280, 320, and 360 nm. The HPLC/UV-PAD/ESI-MSn analyses were carried out with an Agilent HPLC 1100 series equipped with a Pulsed Amperometric Detector 0.01 < P < 0.05] and a Duncan test when necessary. Relative transcript expression assayed by qPCR was calculated using the 2−ΔCt method. Heat maps for metabolomics results, transcripts expression and enzymatic activities were created using R.Statistical analysis for compound concentration was carried out as described above. Statistical analyses for DW, HTomato plants were grown under control  conditions for 25 days, and then either maintained under control conditions, or subjected to salinity , heat  or a combination of salinity and heat  for an additional 15 d (a whole vegetative-growth cycle). Dry weight of the plants was measured at the end of the experiment  accumulation, peroxidation of membrane lipids  and protein oxidation (measured as protein carbonyl groups). These bioindicators were measured in tomato plants subjected to salinity, heat, and a combination of salinity and heat  and phenolic compounds (43 compounds identified) were the most represented groups of compounds in our tomato plants. Very little is known about how phenolic compounds are regulated during salinity or heat stress, and information is even more scarce for the combination of salinity and heat. Therefore, we decided to deepen our understanding of these compounds and their metabolism under the different stress scenarios investigated.As shown in Table We thus performed a HPLC quantification of the caffeoylquinic acids and flavonols present in our tomato samples under the different stress conditions used Figure  in orderSlC3H-caffeoylquinin acids concentration r = 0.854***; CH3 activity-caffeoylquinic acids concentration r = 0.902***). On the other hand, it was observed that transcript expression of CHS and CHI, the main branch enzymes of flavonol synthesis .To complete the phenylpropanoid pathway study, transcript expression and activities of the main enzymes (PPO and GPX) involved in phenylpropanoid degradation were analyzed under the different conditions used in our experiment. As shown in Figure To date, most molecular studies performed on tomato and other horticultural plants of economic interest have been performed via the application of a single stress condition. However, plants in nature are usually exposed to a combination of two or more stresses at the same time, and laboratory experiments performed on plants subjected to a single stress condition do not appropriately address the metabolic changes that plants suffer under conditions found in the field  while the combination of salinity and heat reduced it by about 66%, compared to control plants Figure  and heat2, leading to ROS production  revealed similar levels to those obtained in previous experiments  and high levels of protein oxidation, with salinity applied as a sole stress being the most devastating in that sense. The combination of salinity and heat also induced high levels of lipid peroxidation and protein oxidation as compared to control plants, but at lower levels than salinity alone.Abiotic stresses cause oxidative damage to plant cells Mittler, . An impa2O2 under the different stresses was due to an overexpression of SISOD-encoding transcripts . The downregulation of the SlGR transcript may be related to the high levels of lipid peroxidation and protein oxidation found in plants grown under salinity and heat combination as compared to control plants, as described by Ursini et al.  occurs  is a key determinant of the flow of carbon into the shikimate pathway , and Seneca Foundation from Region of Murcia, Spain (Grant no.15288/ PI/10).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Direct evidence for the effects of vegetable intake on weight loss is qualified. The study aimed to assess the effect of higher vegetable consumption on weight loss.2) randomised to two energy deficit healthy diet advice groups differing only by doubling the serving (portion) sizes of vegetables in the comparator group. Data were analysed as intention-to-treat using a linear mixed model. Spearmans rho bivariate was used to explore relationships between percentage energy from vegetables and weight loss.A single blind parallel controlled trial was conducted with 120 overweight adults (mean body mass index=29.98 kg/mP<0.001 time) with no difference between groups (P>0.05 interaction). Both groups increased vegetable intake and lost weight in the first 3 months, and the change in weight was significantly correlated with higher proportions of energy consumed as vegetables . Fasting glucose, insulin and triglyceride levels decreased (P<0.001 time) and high-density lipoprotein cholesterol levels increased (P<0.001 time), with no difference between groups. Weight loss was sustained for 12 months by both groups, but the comparator group reported greater hunger satisfaction (P=0.005).After 12 months, the study sample lost 6.5±5.2 kg (Advice to consume a healthy low-energy diet leads to sustained weight loss, with reductions in cardiovascular disease risk factors regardless of an emphasis on more vegetables. In the short term, consuming a higher proportion of the dietary energy as vegetables may support a greater weight loss and the dietary pattern appears sustainable. The 2013 Australian Dietary Guidelines (ADG) review found suggestive evidence that vegetable consumption is associated with a reduced risk of weight gain4 and the 2010 Dietary Guidelines for Americans found the association was modest, but may be significant in the long term.5 These positions may reflect difficulties with food-based dietary trials.7 Randomised controlled trials provide the appropriate basis for nutrition recommendations,8 but for demonstrating long-term effects there are problematic issues. For example, dietary patterns may end up similar in control and intervention groups, or reduced energy intake may override effects from dietary patterns per se.Weight loss occurs when energy intake is less than energy expenditure,9 In such circumstances, it is difficult to attribute the effects to vegetable consumption alone. Shorter feeding studies under highly controlled conditions can provide ‘proof of concept',8 but in weight management long-term effects are more clinically relevant. This situation raises a number of questions for how to conduct the research itself.9 Some intervention studies have reported beneficial associations between actual vegetable consumption and weight loss for the whole study sample.11In a recent review, we found that randomised controlled trials demonstrated a greater weight loss from high vegetable intake when it occurred as part of a healthy background diet, the control diet constituted ‘usual intake' and behavioural support was provided.12 gender and behavioural factors,13 and background cuisine14 also may be important considerations. Low-fat diets have appeared superior to high vegetable diets,15 but not always.16 Nevertheless, low energy density and increased satiety are plausible reasons for the reduced risk of weight gain with high vegetable intake,17 and emerging research suggests the phytochemical composition may be beneficial.18 Eating more vegetables may help shift cuisine patterns, proving efficacious in the long term. The aim of this study was to demonstrate the effects of a higher vegetable consumption on sustained weight loss in healthy overweight adults.The classification of vegetables ,2. Exclusion factors were major illnesses, diabetes mellitus, thyroid abnormalities, heavy alcohol consumption, recent acute or chronic disease, changing medications affect weight, weight loss >5 kg in last 3 months, fluctuating exercise patterns, strenuous exercise >1 h per day, pregnancy or lactation, dietary limitations, and dislike of vegetables. One hundred subjects were considered sufficient to detect a minimum between-group weight loss difference of 2.7 kg as significant with 80% power and a two-tailed α of 0.05.19 This assumes a 40% post-randomisation dropout rate (20 subjects per group) and a within-group weight loss s.d. of 3.5 kg (using available literature19). A researcher not associated with the clinical interface (MJB) conducted the randomisation using the RALLOC command in STATA V10.0  with the randomisation performed in blocks of 2, 4 or 6 and block sizes randomised within four strata, used to divide the sample by sex and body mass index (25–30 kg/m2 and 30–35 kg/m2). The master list was provided by strata and reference to the block size and order was removed, and consecutive numbers for allocation provided.A single blind parallel randomised controlled trial was conducted between 2010 and 2012 in Wollongong, NSW, Australia. Participants were recruited by advertising in the local media. Inclusion criteria were healthy adults 18–65 years with a body mass index 25–35 kg/m20 that is, vegetables, fruit, grain foods, meat/fish/eggs/cheese, milk/yoghurt and nuts/seeds/spreads/oils, providing ~80% energy requirements for age, weight and sex as per the Mifflin equation.21 The energy intake of the diets was managed by careful dietary modelling of all food groups including vegetables. All participants were requested to consume at least five servings of vegetables each day, but the servings were different between control vs comparator . Doubling portion size has been shown to be effective in increasing vegetable consumption in a disguised way.22 Foods high in saturated fat and added sugars  were discouraged, in keeping with the ADG including the 2013 update.17An accredited practising dietitian provided participants with a personalised diet prescription based on core food groups from the Australian Guide to Healthy Eating,Initial consultations lasted 1 h, with 30-min follow-up at months 1, 2, 3, 6, 9 and 12 by the same dietitian. E-mail messages were sent 2 weeks before clinic visits. Short message service was sent to participants' mobile phones with reminders of appointments and encouragement to maintain study requirements. Booklets outlining the recommended number of servings of food groups per day and a 4-day estimated food record (including one weekend day) were provided. The high vegetable group were given extra support and materials on use of vegetables.The primary outcome of body weight (kg) was measured in an upright position in minimal clothing and without shoes using scales with a bio-electrical impedance component to also estimate body fat (%) . Body weight was assessed at similar times of the day at 0-, 1-, 2-, 3-, 6-, 9- and 12-month time points.Secondary clinical outcome measures were indicators of lifestyle disease risk: fasting insulin, glucose and blood lipids: total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol and triglycerides . Fasting blood samples were sent to quality assured pathology laboratories  for lipids; and Cardinal Bio-Research Pty Ltd, Brisbane, QLD, Australia, for F2 isoprostanes).23Physical activity using the Baecke physical activity questionnaire.25 scale completely coinciding with the 4th day of the 4-day food record kept between appointments).Subjective ratings of food intake behaviour using visual analogue scales  using a scale (100 mm) within the range of 0–10 .General diet acceptability score , and including items related to satisfaction with the diet).Perceptions of health, measured with questions from the Medical Outcomes Study 36-item Short Form Health Survey (SF-36): . During the past 4 weeks, to what extent has your physical health or emotional problems interfered with your normal social activities with family, friends, neighbours or groups? ').Additional information was collected via questionnaire at the 0-, 3-, 6-, 9- and 12-month appointments. The following assessments were also made:26 Participants were asked to report their usual intakes of types and amounts of foods starting with the first meal of the day and indicating variations within a 2- to 4-week period. They were asked specific questions on vegetable intake, including identifying the individual vegetables in dishes. Plasma F2 isoprostanes , were measured, given the potential for the phytochemicals in vegetables to act as anti-oxidants, and the previously observed negative associations with F2 isoprostanes and high vegetable diet patterns.27Dietary intake was assessed by diet history interview conducted by an Accredited Practising Dietitian for each participant at 0, 3, 6, 9 and 12 months.28 As weight loss is predicated on total energy intake, vegetable intake data were presented as a percentage of total energy intake.Data were entered into OpenClinica version beta 3.1.2 clinical trial software  for clinical data management, using the double-entry method by at least two independent researchers for completeness. Dietary data were calculated and analysed using FoodWorks  nutrient analysis software using the AUSNUT 2007 food composition survey database.To examine diet acceptability and monitor changes in appetite, scores were presented so that: for digestive discomfort, the lower the score the less thirst, nausea, diarrhoea or constipation; for satiety, the higher the score the less hungry, less satisfied, lesser sense of fullness, less desire to eat more, and seldom wanting salty, sweet, savoury or fatty foods, respectively; for general diet acceptability, the higher the score the more satisfied, greater ease with preparing food, the greater the effort to adhere to diet plan, more acceptance of core food items and greater ease with continuing on the diet.e or square root transformed before analysis, and values before transformation were reported to assist with interpretations. The analysis was conducted on an intention-to-treat basis on trial completion. Owing to non-normal distributions, Spearman's correlations were used to assess relationships between changes in vegetable intake and isoprostane levels and to assess correlations between change in weight and change in vegetable consumption (% energy). The study was approved by the University of Wollongong Human Research Ethics Committee and registered with Australia New Zealand Clinical Trials Register Network (ACTRN12610000784011).Data were analysed using SPSS  statistical analysis software. Primary and secondary analyses of all continuous variables were conducted using a linear mixed model, which uses all available data regardless of whether the subjects complete the study, the type III fixed effects were used to determine significance. Skewed variables were logn=120 adults were randomised to control and comparator groups and n=93 completed through to 12 months , with no difference between groups in weight loss . Compared with the controls, the comparator group reported consuming a significantly greater proportion of energy intake from vegetables  estimated at about –2000 kJ/day . Vegetable consumption increased substantially in the first 3 months  and increased energy intake from low-energy-dense vegetables  to lower energy vegetables .The sample produced a mean weight loss of 6.5±5.2 kg (range –27.8 to +5 kg) and this was associated with reported change in energy intake  and from baseline to 12 months . There were significant changes in macronutrient intakes: a reduction in energy from total fat by about 5%, an increase in protein energy by about 3% and an increase in carbohydrate energy by about 2% .The change in weight was significantly correlated with the increase in proportion of energy consumed as vegetables at 3 months  with a marginally significant decrease in score by the control group from 6 to 12 months (P=0.077). All trial participants reported being less hungry throughout the trial , they were less inclined to eat more food  and to desire sweet foods . They reported less desire for fatty food .The higher vegetable group reported a greater increase in hunger satisfaction , except ‘ease to continue' item , which was lower score for the higher vegetable group at 6 months  only.All items on general diet acceptability were high (around 7–8 where 10=extremely acceptable), with no differences between groups . The proportion reporting their health as ‘excellent' increased fourfold and the proportion noting ‘very good' doubled compared with the baseline. About half of the trial participants reported their health as being ‘better than a year ago'. The proportion indicating that physical or emotional problems interfered with normal activities decreased three- to fourfold.17 produced substantial and sustained weight loss regardless of differences in advice on vegetables .31 Low-kilojoule/low-fat dietary advice appears to be more sustainable than diets focusing on vegetables intake,32 although providing vegetables in the short term may enable what is known as ‘proof of concept' conditions.8 We need to look beyond the single outcome measure of weight loss. Although we did achieve significant differences between groups in energy consumed as vegetables, this was not enough to have an effect on weight loss , consistent with reports suggesting that vegetables act via controlling for hunger.16 Reviews suggest that encouraging and supporting healthy diets with significant amounts of vegetables are likely to be effective because, although there are limitations on other foods, the central message is positive.33 In terms of the vegetable content of both diets, the literature suggests that establishing long-term habituation through repeat presentation of vegetables at all meals34 and the encouragement of vegetable variety36 may help establish lasting change essential to prevent weight regain,37 and we appeared to be seeing that. It should also be noted that the dietary advice strategy focused on shifting from high- to low-energy-dense vegetables and this was reflected in the reported dietary change for the study sample  results were consistent with observations that a high vegetable intake is associated with a lower level of the markers of oxidative damage.mference , fastingmference . These c7 Dietary change is not just about desirable foods going into the diet; it is also about taking others out. In a recent publication,39 we reported that participants with poor baseline dietary patterns lose substantially more weight than those starting with healthy dietary patterns. Future research might test whether shifting eating patterns from a low to a high vegetable intake may have an impact on achieving weight loss by replacing poor quality foods in the total diet.There were necessary controls on energy intakes and the background diet in this study, which made it difficult to expose effects of a single food group, as acknowledged in the literature.3 Advice to consume a healthy low-energy diet including five servings of vegetables per day can lead to sustained weight loss, with associated reductions in cardiovascular disease risk factors. In the short term, consuming a higher proportion of the dietary energy as vegetables may support a greater weight loss and the dietary pattern appears sustainable.In conclusion, a dietary energy deficit can be achieved in any number of ways.


BRAF-mutant and other cancers. The novel IdyllaTMBRAF Mutation Test has high sensitivity and shorter turnaround times compared to other methods. We used Idylla to detect BRAF V600 mutations in archived formalin-fixed paraffin-embedded (FFPE) tumor samples and compared these results with those obtained using the cobas 4800 BRAF V600 Mutation Test or MiSeq deep sequencing system and with those obtained by a Clinical Laboratory Improvement Amendments (CLIA)-certified laboratory employing polymerase chain reaction–based sequencing, mass spectrometric detection, or next-generation sequencing. In one set of 60 FFPE tumor samples (15 with BRAF mutations per Idylla), the Idylla and cobas results had an agreement of 97%. Idylla detected BRAF V600 mutations in two additional samples. The Idylla and MiSeq results had 100% concordance. In a separate set of 100 FFPE tumor samples (64 with BRAF mutation per Idylla), the Idylla and CLIA-certified laboratory results demonstrated an agreement of 96% even though the tests were not performed simultaneously and different FFPE blocks had to be used for 9 cases. The IdyllaTMBRAF Mutation Test produced results quickly (sample to results time was about 90 minutes with about 2 minutes of hands on time) and the closed nature of the cartridge eliminates the risk of PCR contamination. In conclusion, our observations demonstrate that the Idylla test is rapid and has high concordance with other routinely used but more complex BRAF mutation–detecting tests.Fast and accurate diagnostic systems are needed for further implementation of precision therapy of  BRAF V600 mutation hotspot in melanoma and other malignancies has led to the development of small-molecule kinase inhibitors targeting the BRAF oncogene. These BRAF inhibitors have revolutionized therapy for patients with BRAF V600–mutant advanced melanoma and demonstrated promising results in patients with other BRAF mutation–harboring diseases, including histiocytosis, hairy cell leukemia, non–small cell lung cancer, and biliary cancer. [With the identification of druggable molecular aberrations in cancer, we have increased our understanding of cancer biology and identified novel molecular targets for cancer therapy. –6 In par cancer. , 7–10BRAF inhibitors are contraindicated in the absence of a BRAF mutation. To determine whether a disease harbors a BRAF mutation, and thus whether treatment with BRAF inhibitors is appropriate, various methods to detect BRAF mutations in archived formalin-fixed, paraffin-embedded (FFPE) tissue samples are used. However, the methods that are currently routinely used for this purpose often require several hours to perform owing to time-consuming steps that include incubation, pipetting, and other processes. [ocesses.  In additocesses.  Such delocesses. BRAF mutations in FFPE tumor samples is the novel, fully integrated, real-time polymerase chain reaction (PCR)-based IdyllaTM system. Unlike current routinely used technologies for BRAF mutation detection, the IdyllaTMBRAF Mutation Test does not require manual sample preprocessing steps such as deparaffinization, FFPE tissue digestion, or DNA extraction because all these steps are integrated within a single-use cartridge. A complete FFPE tissue section or macrodissected FFPE material is placed directly into the cartridge and is subsequently processed by the IdyllaTM system, which provides automated sample processing, real-time PCR–based mutation detection, and result reporting. In the present study, we compared the performance of the IdyllaTMBRAF Mutation Test with that of other routinely used diagnostic methods for detecting BRAF V600 mutations.A faster and simplified method for detecting TMBRAF Mutation Test (hereafter referred to as Idylla) in detecting BRAF V600 mutations in FFPE material, we used the test to analyze eight different sections of commercially available FFPE cell-line blends containing wild-type BRAF only, 1% BRAF V600E or 1% BRAF V600K in a wild-type BRAF background and demonstrated 100% agreement among eight independent experiments for each respective mutation  from the BRAF wild-type CHL-1 cell line to identify the extent of cross-reactivity between the BRAF wild-type allele and the BRAF V600E and V600K reactions. Unlike the DNA recovered from FFPE samples, which is degraded by the formalin fixation and only partially amplifiable by PCR, the unfixed, high-quality genomic DNA recovered from the CHL-1 cell line enabled us to maximally challenge the specificity of the assay. In this experiment, the delta Ct between the specific signal of the BRAF V600 wild-type reaction and the cross-reactivity signal in the BRAF V600E and BRAF V600K reactions was greater than 20, demonstrating that the mutation detection reactions are highly specific even in the presence of a high number of wild-type alleles  capacity to detect BRAF V600 mutations using 73 unprocessed  FFPE tumor samples from patients with diverse cancers  obtained from commercial suppliers as outlined in Methods section (Table BRAF V600 mutations in 12 of 30 melanomas (40%), 1 of 23 colorectal cancers (4%), and 2 of 5 papillary thyroid cancers (40%) but did not detect BRAF V600 mutations in breast cancers or NSCLC.We initially tested the IdyllaBRAF and all 15 samples with BRAF V600 mutations as per Idylla and subjected these 60 samples to the cobas BRAF V600 Mutation Test, a U.S. Food and Drug Administration–approved companion diagnostic, according to manufacturer's instructions. The sample size of 60 samples was expected to be adequate to demonstrate concordance of 95%–100% . To help maximize the sensitivity of the cobas test, we used macrodissection to enrich the tumor area of 42 of the 60 tumor samples (70%). Of these 60 FFPE tumor samples, 2 (1 melanoma and 1 breast cancer) could not be analyzed with cobas owing to insufficient DNA concentrations. For 56 of the remaining 58 samples (97%), the Idylla and cobas results were in overall agreement . Compared with Idylla, cobas had a sensitivity of 87% , specificity of 100% , positive predictive value of 100% , and negative predictive value of 96%  contained less than 25% tumor cells. As described below, the Idylla results for both these samples were confirmed using an independent method.From the 73 FFPE tumor samples, we randomly selected 45 samples with wild-type BRAF and 15 with BRAF V600 mutations as per Idylla) to mutation analysis using the MiSeq deep sequencing system, in which the BRAF V600–surrounding region was amplified by PCR and sequenced at high depth (coverage >5000x) to facilitate the identification of mutations present in at least 1% of the DNA. Of these 60 samples, 2 had insufficient coverage and were excluded from further analysis. For the remaining 58 samples, the Idylla and MiSeq results demonstrated an overall agreement of 100% . Compared with MiSeq, Idylla had a sensitivity of 100% , specificity of 100% , positive predictive value of 100% , and negative predictive value of 100% -certified Molecular Diagnostics Laboratory from routine clinical biopsies and resections. The CLIA laboratory detected BRAF V600 mutations in 66 of the 100 specimens (66%), and Idylla detected BRAF V600 mutations in 63 of the specimens (63%). The results of Idylla and the CLIA laboratory had overall agreement in 96 cases . Compared with the CLIA laboratory, Idylla had a sensitivity of 95% , specificity of 97% , positive predictive value of 98% , and negative predictive value of 92%  resulting in an overall agreement of 89% for this subset  of four patients with discrepant BRAF V600 mutation status between the CLIA laboratory and Idylla received a MEK inhibitor with disease progression and one patient  received a combination of BRAF inhibitor with chemotherapy and responded for 3 months. In addition, a patient with prostate carcinoma  and colorectal cancer  never received BRAF or MEK inhibitors.We then used Idylla in a retrospective study to test FFPE tumor samples from 100 patients with advanced cancers  in two samples that had relatively low tumor cellularity and that cobas identified as having wild-type BRAF, plausibly due to the test's higher detection limit (at least 5% of V600E mutant DNA) and because cobas is not validated for the detection of V600R. Furthermore, the Idylla results were confirmed using the MiSeq next-generation sequencing platform with 100% concordance. In an independent study, we found an overall agreement of 96% between Idylla and a CLIA-certified laboratory on 100 FFPE samples despite the fact that the testing was not performed simultaneously and that a different block had to be used for Idylla testing in 9% of cases. Although intratumoral heterogeneity for the BRAF V600 mutation might confound the concordance analysis, this effect appears to have a limited impact in our study, since the overall agreement level between both tests reached 96%. [First, we found an overall agreement of 97% between Idylla and cobas on 60 FFPE tumor samples. Idylla detected hed 96%. BRAF mutation–detecting methods used in the present study differ considerably. Unlike the FFPE sample processing steps in methods such as cobas, the FFPE sample processing steps in Idylla are completely integrated within one cartridge, which eliminates the need for manually performing time-consuming procedures such as deparaffinization, tissue digestion, and DNA extraction. Both the cobas test and MiSeq platform require a separate upfront DNA extraction step, which can take up to 3 hours, including about 1 hour for manual pipetting and incubation; subsequent PCR testing requires an additional 2 hours, including 30 minutes for manual procedures, when cobas is used. While these procedures are mostly performed in a sample batching mode, Idylla allows random access analysis of individual samples, leading to a turnaround time of hours instead of up to several weeks. The MiSeq workflow is even more complex and time-consuming than that of cobas owing to the different steps needed for preparing the sequencing library and performing the sequencing itself. In addition, for both cobas and MiSeq, the use of separate pre-PCR and post-PCR rooms to prevent sample contamination is strongly recommended. In contrast, the IdyllaTMBRAF Mutation Test produces results in about 90 minutes with about 2 minutes of hands on time and the closed nature of the cartridge eliminates the risk of PCR contamination. Furthermore, given its simple workflow and quick turnaround time, the IdyllaTM system can be used at nearly any facility, including those that would not be able to implement technologies with more complex workflows such as cobas or MiSeq.The workflow complexities and turnaround times of the different BRAF V600E, V600K, V600R, V600M, and V600D mutations, it does not distinguish between BRAF mutations occurring on the same nucleotide, i.e., the V600E/D or V600K/R/M mutations. However, this is clinically acceptable, because patients carrying either one of these mutations have been reported to benefit from treatment with a BRAF inhibitor.[BRAF V600E down to at least 5% of mutant allele and BRAF V600K down to at least 30% of mutant allele. In the present study, 70% of the samples analyzed with the cobas test were macrodissected to maximize the test's sensitivity; in contrast, no samples analyzed with the Idylla test were macrodissected, although the test is compatible with micro- or macrodissected tissue. In addition, the Idylla test detected a BRAF mutation in one unprocessed FFPE sample that MiSeq analysis revealed to have less than 5% BRAF mutation , whereas the cobas test did not detect this mutation even in tissue that had been macrodissected. These findings suggest that Idylla can detect all clinically relevant BRAF V600 mutations and that, compared with the cobas test, Idylla has a higher sensitivity for detecting BRAF V600 mutations and thus could be used to identify a greater number of patients who may benefit from treatment with a BRAF inhibitor.Although Idylla detects nhibitor. Whereas TMBRAF Mutation Test, which offers an integrated and sensitive “sample-to-result” approach to detecting BRAF V600 mutations in FFPE samples, has a high concordance with routinely used methods for detecting BRAF V600 mutations in such samples.In conclusion, the IdyllaBRAF V600 mutations in MD Anderson's CLIA-accredited Molecular Diagnostics Laboratory. Patients had to have enough archived tissue to allow the study and patient accrual was enriched to ensure that at least 60% of patients in the study had BRAF V600 mutations. Patient registration in the database, tumor pathology assessment, and tumor mutation analysis were performed at MD Anderson. The study was conducted in accordance with MD Anderson's Institutional Review Board guidelines. Additional FFPE tumor tissue samples from cancer patients who had signed an Institutional Review Board– or Ethical Committee–approved informed consent form were acquired through commercial suppliers .From May 2012 until April 2014, patients with advanced cancers referred to the Department of Investigational Cancer Therapeutics at MD Anderson Cancer Center were enrolled in the study. FFPE tumor tissue samples from these patients were tested for TM system  is a random-access molecular diagnostic system that provides quantitative allele-specific real-time PCR–based sample-to-result functionality using a disposable cartridge that can detect and quantify up to 30 molecular biomarker groups from a variety of solid and liquid samples, including plasma and FFPE tissue. The instrument is composed of a sample preparation module integrated with a combined PCR thermocycling and fluorescence detection module. For FFPE specimens, the sample preparation module uses high intensity focused ultrasound technology to emulsify the paraffin and simultaneously rehydrate the tissue sample in an aqueous solution, thereby liberating DNA. Nucleic acids are then transported via microfluidic channels in the cartridge into 5 separate PCR chambers that contain pre-deposited dried PCR reagents . Each PCR chamber allows for the identification of up to 6 different biomarker groups , each of which can be composed of multiple individual biomarkers.The IdyllaBRAF Mutation Test  is a single-use cartridge-based test designed to detect the nucleotide G1798 > A and T1799 > A changes in the BRAF gene with a sensitivity limit of 1% BRAF mutant DNA in wild-type background. The G1798 > A change is present in patients with V600K, V600R, and V600M mutations, whereas the T1799 > A change is present in patients with V600E, V600K, V600E2, and V600D mutations. The test requires an analytic time of about 90 minutes and a hands-on time of about 2 minutes. Once the sample is inserted into the cartridge and the lid is closed, the cartridge is sealed, thereby eliminating the possibility of cross-contamination between different samples. The test does not require that FFPE samples be manually deparaffinized or preprocessed; all reagents required for sample preparation  and real-time PCR detection are included in the IdyllaTM cartridge. Although the test is compatible with macrodissected FFPE material, unprocessed FFPE sections were used in this study. For the clinical concordance analyses in the present study, a single 10-μm section per FFPE sample was processed according to the manufacturer's instructions by a molecular biology-trained operator who was blinded for the results of the reference methods.The Idylla BRAF V600 mutation test  was performed according to the manufacturer's instructions at the College of American Pathologists– and CLIA-accredited molecular laboratory of HistoGeneX . Briefly, 5-μm sections were stained with hematoxylin and eosin and examined by a certified pathologist who delineated the tumor areas and determined the tumor cell content. For optimal sensitivity, macrodissection was used to enrich the tumor area. DNA from two 5-μm sections was isolated using the cobas DNA Sample Preparation Kit , diluted to 5 ng/μl, and tested on a cobas 4800 System v2.0 (Roche Molecular Systems) according to the manufacturer's instructions by a molecular biology-trained operator who was blinded for the IdyllaTM and MiSeq results.The cobas 4800 BRAF V600 mutation detection with the MiSeq deep sequencing system , DNA was first extracted from 10-μm FFPE sections using a QIAamp FFPE tissue DNA extraction kit  according to the manufacturer's instructions and eluted in 50 μl of elution buffer. Different samples' DNA was normalized to 10 ng/μl based on measurement by Nanodrop , and an amplicon surrounding the BRAF V600 codon was generated using the forward primer 5′-CTACTGTTTTCCTTTACTTACTACACCTCAGA-3′ and the reverse primer 5′-ATCCAGACAACTGTTCAA ACTGATG-3′. The DNA samples were combined with a PCR reaction mixture consisting of 10 mM Tris, 50 mM KCl, 500 μM each PCR primer, 3 mM MgCl2, 0.2 mM dNTPs, 2 U FastStart Taq DNA polymerase , and 2 μl DNA input. The mixture was then subjected to PCR thermocycling for 10 minutes at 95°C followed by 50 cycles of denaturation at 95°C for 10 seconds, annealing at 62°C for 15 seconds, an extension step of 1 minute at 72°C, and a final extension step of 7 minutes at 72°C. The PCR products were visualized on Experion gel to confirm the presence of a single band of the correct length. Subsequent steps, including PCR product purification, sample barcoding, preparation of the MiSeq sequencing library, MiSeq sequencing, and bioinformatics data analysis, were performed by the Nucleomics Core at the Vlaams Instituut voor Biotechnologie . At least 5000x coverage of the target region in each sample was required for MiSeq to have sufficient sensitivity. During the data analysis, the mutation threshold percentage for variant reporting was set at 1% mutant allele in a wild-type background to identify all mutations occurring in at least 1% of the sample DNA.For BRAF V600 mutations utilizing primers designed by the Molecular Diagnostics Laboratory. In January 2011, the assay was changed to mass spectrometric detection , and in March 2012, the assay was changed to next-generation sequencing . The lower limit of detection is approximately 5–10%.Archived tumor tissues obtained from enrolled patients' primary or metastatic sites using routine diagnostic and/or therapeutic procedures were subjected to mutation testing in the CLIA–certified Molecular Diagnostics Laboratory in the Division of Pathology and Laboratory Medicine at MD Anderson. All histologies were centrally reviewed at MD Anderson. DNA was extracted from microdissected paraffin-embedded tumor sections and analyzed using a PCR-based DNA sequencing method for Concordance among mutation analyses was assessed using the kappa coefficient, sensitivity, specificity, and positive and negative predictive values, which were calculated using the GraphPad software program .


Climate oscillations in the Cenozoic reduced species richness and genetic diversity of terrestrial and aquatic animals and plants in central and northern Europe. The most abundant arthropods in temperate soils are Collembola that live in almost any soil-related habitat. Extant species show little morphological variation to Eocene fossils, suggesting persistence of species in stable habitats for millions of years. Collembola are able to evade adverse climatic conditions by moving into deeper soil layers and are tolerant to frost and draught. If these adaptations sufficed for surviving glacial periods remains open and needs to be investigated in a phylogeographic context, i.e. investigating spatial structure on molecular level. We investigated the molecular variation of three common species of Collembola at a pan-European scale to identify glacial refuges and post-glacial colonization patterns with three genetic markers.All genes revealed remarkable genetic structure between but not within populations, suggesting density dependent processes for establishment of populations , which is common for European animals and plants. In contrast to the post-glacial recolonization patterns of many aboveground organisms, divergence times of most geographic lineages indicate preservation of genetic structure since the Miocene.Collembola survived severe climatic changes including those during Quatenary glaciation and kept high genetic variance across Europe. Likely the buffering of temperature oscilliations in soil and the ability to evade adverse climatic conditions due to cold-tolerance and horizontal migration enabled Collembola to evade strong selective pressure of abiotic forces.The online version of this article (doi:10.1186/s12862-016-0719-8) contains supplementary material, which is available to authorized users. Fossind./m2; ) and sigind./m2; , 10.The above-and belowground food web is intimately linked  but alsoCollembola form part of the decomposer system and predominantly feed on dead organic matter and associated microorganisms , 22, resSteganacarus magnus [S. magnus survived Quaternary glaciations in central Europe in cryptic refugia and indicates that phylogeographic patterns of aboveground animals cannot easily be transferred to those of the belowground system. Collembola resemble oribatid mites in various respects but typically are faster reproducing, more mobile and less frost tolerant [Few studies investigated the genetic structure of Collembola in Europe and the existing studies focused on large surface-dwelling rather than soil-dwelling species –28. Genes magnus . In agretolerant , 29–33.Ceratophysella denticulata  (Hypogasturidae) and Folsomia quadrioculata  (Isotomidae) are large species (up to 2.5 mm) that live in the uppermost soil layer (hemiedaphic) and reproduce sexually. In contrast, the third species, Isotomiella minor  (Isotomidae), is small (up to 1.3 mm), lives deeper in soil (euedaphic) and reproduces via parthenogenesis. The investigated species are generally described as palearctic or holarctic in their distribution ranges, only I. minor is considered cosmopolitan, but its taxonomic status needs further attention [Ceratophysella denticulata feeds on bacteria and fungi but presumably also on other soil animals such as nematodes [C. denticulata functions as secondary decomposer, F. quadrioculata is a typical primary decomposer feeding predominantly on litter [I. minor presumably mainly feeds on bacteria [I. minor likely recovers more quickly from disturbances and colonizes new habitats faster than sexual species [The ability to survive Quaternary glaciations and the recolonization potential from glacial refuges likely differs between Collembola and oribatid mites. We investigated the genetic structure of three species of Collembola that are widely distributed and common in the northern hemisphere across thirteen European countries, including potential refuge areas south of the Alps. ttention –36. In fttention . Ceratopematodes , 38. Whin litter . In contbacteria , 40. Due species . Species up to 2. mm that  species  suggestiTo explore divergences of lineages at a wide timespan, we analyzed one mitochondrial and two nuclear markers with different substitution rates. The mitochondrial COI gene is widely used for barcoding but previous studies demonstrated high intraspecific variance in Collembola , 44. In Assuming that soil buffered Quaternary temperature extremes, Collembola presumably survived in local patches in central Europe and expanded from these patches. Accordingly, endemic haplotypes are present in central and northern Europe. Further, we expected different genetic lineages to coexist locally due to expansions from isolated central European refuge populations. Due to more extensive survival during Quaternary glaciations of Collembola in southern Europe, the local molecular variance and genetic distances within populations from southern Europe should be higher than those within central and northern European populations.Leaf litter including humus layers from about two square meters of deciduous and coniferous forests were collected in 19 locations in 13 countries in Europe, including northwest and middle-west Russia, and the Pleistocene refuge areas of the Balkan , Italy and Spain, covering a geographic sampling range of 4,500 km on the east–west and ~4,000 km on the north–south axis, extending other studies on European Collembola by including countries from the Mediterranean to Scandinavia and western Eurasia. Animals were extracted by heat , collectC. denticulata (n = 54), F. quadrioculata (n = 56) and I. minor (n = 55) using the DNeasy® Blood and Tissue Kit  following the manufacturer’s protocol for animal tissue. Purified DNA was eluted in 30 μl buffer AE and stored at−20 °C until further preparation. PCRs of the nuclear Histone 3 and 28S rDNA (D3-D5 region) and the mitochondrial COI gene were performed in 25 μl volumes containing 12.5 μl SuperHot Taq Mastermix  with 1.5 μl of each primer (10 pM), 4.5 μl H2O, 2 μl MgCl2 (25 mM) and 3 μl template DNA. Primers and PCR programs are given in Additional file Genomic DNA was extracted from single individuals of Sequences were edited and corrected with Sequencher 4.10 , coding nucleotide sequences (H3 and COI) were translated into protein sequences using the standard and invertebrate mitochondrial codes implemented in Sequencher. For each species, nucleotide and protein sequences, were aligned separately and combined (as concatenated nucleotide matrix of three genes) with Clustal W  implemenThe best fit model of sequence evolution for each alignment  was inferred with to the hLRT in TOPALi v2.5  using thHypogastrura (five species), Xenylla grisea and Gomphiocephalus hodgsoni for Hypogastruridae and Folsomia (three species), Parisotoma notabilis, Anurophorus septentrionalis and Isotoma (three species) for Isotomidae  with a burnin of 25 % was confirmed using Tracer v1.4 [C. denticulata, F. quadrioculata and I. minor, we generated a phylogenetic tree of 10 families with 23 genera and eight outgroup taxa including the taxa studied by D’Haese [Median-joining haplotype networks for the nucleotide datasets of COI, H3, 28S and the concatenated dataset were generated for all three species using the program Network 4.6 . Molecular variance (AMOVA) within and between populations and isolation by distance (Mantel test) of all three genes (uncorrected p-distances) were analyzed separately in ARLEQUIN  with 20, D’Haese , using 2C. denticulata the nucleotide alignments of COI, H3 and 28S had 240, 75 and 45 variable positions, respectively. Respective values for F. quadrioculata were 234, 65 and 2 and for I. minor 253, 50 and 15.Intraspecific variance of the three genes was similar in the three species. For C. denticulata), 87–88 % (F. quadrioculata) and 92–99 % (I. minor) of the genetic variance of COI, H3 and the concatenated dataset . The only exceptions were one population of C. denticulata , two populations of F. quadrioculata  and two populations of I. minor  in which distinct COI and H3 lineages co-occurred. Genetic distances between and within regions (COI and H3) were highest in southern Europe and decreased towards the north, but were lowest within central Europe , from Montenegro and Italy , and from Norway and Spain .Isolation by distance was rejected as not being significant for all datasets and all species; however, genetic variance among populations was high, explaining 96–97 %  or almost no variation between populations (0.2 % for F. quadrioculata). Accordingly, the network analysis separated only haplotypes of C. denticulata into distinct geographic clusters  had the lowest genetic variance with little  were monophyletic in the single gene phylogenetic trees and carried identical 28S sequences. In I. minor, individuals from Norway and Spain had distinct mitochondrial haplotypes, but nearly identical H3 and 28S alleles. Coexistence of genetic lineages did not occur in each of the three species except for one population from Croatia of F. quadrioculata. Different to the other species, C. denticulata had a central-eastern phylogeographic clade (black lineage) but the geographic range and spatial coherence of this lineage remains open due to the limited number of sampling locations.Phylogenetic trees based on the combined matrix gave the best resolution and statistical support for internal and terminal nodes Figs. , 2 and 3Haplotype diversity of the mitochondrial gene was generally high in the three investigated species (Table 6) but most haplotypes differed in only a few positions. Haplotype networks of the COI and H3 gene were complex in each of the species as the majority of populations were separated by >10 mutation steps , Greece (F. quadrioculata), Croatia (I.minor) and Montenegro (C. denticulata and I. minor) differed from the trees calculated with the combined matrices. Divergence estimates of major lineages fell into the Miocene (23.03-5.3 mya) in each of the three species, considerably predating Quaternary glaciations (1.8 my to present). Results were consistent among the three datasets, demonstrating robustness of the molecular clock analyses  divergence estimates of larger phylogeographic clades were younger in dataset (1) (combined COI and 28S) compared to datasets (2)–(3) (COI only), with predominantly Middle Miocene origin (16–11.5 mya). However, this probably is an artefact of the 28S gene which is strongly conserved among genera of Folsomia  and probably other Isotomidae. In general, divergences of clades and populations were more recent in F. quadrioculata than in the other two species but still concentrated in the Miocene, independent of the dataset analyzed.Three major radiation events can be extrapolated from the datasets of the three species  were dated to be of Late Miocene origin. The lower than average genetic distance in these clades suggests that a single or a few lineages expanded into these areas, possibly due to fragmentation of forest habitats into open woodlands, causing local extinctions or adaptations. In contrast, lineages of the parthenogenetic species I. minor were less separated into geographic regions, but rather split continuously from a ubiquitous and widespread ancestral lineage. This is also reflected by the equal distances of haplotypes in the network of the nuclear marker (28S), indicating that no region is more ancestral to others and suggesting separation of these lineages from a common ancestor. Accordingly, the two closely related (28S and H3) but geographically separated populations of Norway and eastern Spain likely represent relict populations that separated from an ancestral lineage about 12 mya and survived in isolated patches. Related lineages either went extinct or were not sampled. Further, divergences into geographic lineages mainly occurred during the Early and Middle Miocene and Pleistocene in I. minor, with only one divergence within the central European clade during the Late Miocene. In contrast, divergences of central European lineages of C. denticulata and F. quadrioculata predominantly took place several million years later in the Late Miocene and Early Pliocene.The Miocene was warm and humid resembling subtropical climate with central Europe separating later into marine and continental climatic zones along west–east gradients with seasonality increasing to the east . For litCollembola are fast colonizers of newly formed and young habitats such as glacier forelands , but theLepidocyrtus in the Mediterranean basin [S. magnus [S. magnus was more common than in the studied Collembola. This suggests that different factors regulate the establishment of populations among belowground invertebrates and that competition and early habitat colonization is more important for springtails than for oribatid mites [Southern European populations were dominated by isolated lineages, supporting the existence of south European refuge areas but also suggesting that the Alps function as major dispersal barrier. Accordingly, regional extinctions in central Europe and rapid recolonization of empty habitats from the surrounding, rather than from southern refugia, likely resulted in the strong phylogeographic structure of populations north of the Alps. Strong genetic differentiation, little or no mixing between populations and ancient diversifications resemble the pattern reported for epigeic springtail species of the genus an basin  and the . magnus . This inid mites , 74, 75.. Divergence dates and geographic structure suggest that soil microarthropods are less affected by global and long-term climatic changes than aboveground animals and plants. Buffering of adverse climatic conditions, the presence of organic matter colonized by bacteria and fungi in soil, large population sizes and the ability to tolerate freezing conditions likely contributed to the lower responsiveness of soil microarthropods. This suggests that in soil evolutionary processes are slowed down compared to the aboveground system, rendering soil-living detritivore microarthropods “living fossils” with communities in central Europe reflecting Miocene rather than Quaternary diversification events.Similar to distribution patterns of European springtail species , we show


Peste des petits ruminants (PPR) is an acute viral disease of small ruminants. The disease was first reported in Tanzania in 2008 when it was confined to the Northern Zone districts bordering Kenya. The present study was carried out to confirm the presence of PPR virus (PPRV) in Tanzania and to establish their phylogenetic relationships. Samples  were obtained from live goats with clinical presentation suggestive of PPR and goats that died naturally in Ngorongoro (Northern Tanzania) and Mvomero (Eastern Tanzania) districts. The clinical signs observed in goats suspected with PPR included fever, dullness, diarrhea, lacrimation, matting of eye lids, purulent oculonasal discharges, cutaneous nodules, erosions on the soft palate and gums and labored breathing. Post mortem findings included pneumonia, congestion of the intestines, and hemorrhages in lymph nodes associated with the respiratory and gastrointestinal systems. PPRV was detected in 21 out of 71 tested animals using primers targeting the nucleoprotein (N) gene. Phylogenetic analysis, based on the N gene, indicated that PPRV obtained from Northern and Eastern Tanzania clustered with PPRV strains of Lineage III, together with PPRV from Sudan and Ethiopia. The findings of this study indicate that there are active PPRV infections in Northern and Eastern Tanzania, suggesting risks for potential spread of PPR in the rest of Tanzania. Morbillivirus of the family Paramyxoviridae. The PPR virus (PPRV) is highly contagious and easily transmitted by direct contact through the secretions and/or excretions of infected animals to nearby healthy animals (Peste des petits ruminants (PPR) is a highly contagious viral disease of wild and domestic small ruminants and camels characterized by oculonasal discharge, stomatitis, diarrhea and pneumonia . It is a animals . ContactThe disease was first reported in West Africa in the early 1940s and later recognized as endemic in both West and Central Africa . CurrentThe present study was conducted in Ngorongoro district in Arusha region located in Northern Tanzania and Mvomero district in Morogoro region located in Eastern Tanzania. The Ngorongoro district was selected owing to occurrence of several PPR outbreaks since 2008 even in the areas where vaccination was practiced. The District is considered to be a risk area because it borders Kenya, from which it is believed that the disease was introduced into Northern Tanzania  and it fg for 30 min at 4°C. The upper layer was discarded while the opaque interface containing the buffy coat was carefully collected. Nasal and ocular swabs were collected and placed in universal viral transport medium (BD Biosciences) followed by vortexing to dislodge cells from the swabs and centrifugation at 8000 g for 5 min at room temperature. Portions of lungs, intestines and lymph nodes from the same animal were pooled before they were homogenized in F-12 basal cell culture medium  to obtain a 10% tissue suspension. The tissue supernatant was obtained by centrifugation of tissue suspensions at 8000 g for 5 min at room temperature. All samples and the buffy coat were stored at −80°C until RNA extraction was performed.Nasal and ocular swabs, and whole blood samples were obtained from randomly selected live goats with clinical presentation suggestive of PPR. Whole blood was collected from the jugular vein using EDTA-containing vacutainer tubes . In addition, lungs, intestines, lymph nodes, whole blood and, ocular and nasal swabs were obtained from five goats in Mvomero district and four goats in Ngorongoro district that died naturally. Buffy coat from whole blood was obtained by centrifuging 500 μl of histopaque  layered with 1 ml of whole blood at 400 Viral RNA was recovered from buffy coat, swabs and homogenized tissue samples using a commercial QIAamp viral RNA kit  according to the manufacturer's instructions. PPRV nucleoprotein (N) gene was amplified in a 25 μl reaction using AgPath-ID One-Step RT-PCR kit  using PPRV-specific primers NP3 (5′-TCTCGGAAATCGCCTCACAGACTG-3′) and NP4 (5′-CCTCCTCCTGGTCCTCCAGAATCT-3′) as previously described by mega 5  and clustering pattern was determined by neighbor-joining method using the Kimura-2-parameter option implemented within mega 5 .The main clinical signs observed in goats suspected with PPR included fever, dullness, diarrhea, lacrimation, matting of eye lids, purulent oculonasal discharges, cutaneous nodules, erosions on the soft palate and gums and laboured breathing. In some animals, raised cutaneous nodules of up to 3 cm in size were observed especially on the neck region Fig. . The recn = 45) and 26.9% (n = 26) were positive for PPRV N gene in Mvomero and Ngorongoro districts, respectively. The rate of detection of PPRV was higher in tissues than in buffy coat, and ocular and nasal swabs . BLASTN analysis of both Dakawa and Ngorongoro N gene nucleotide sequences of PPRV showed 96% identity to PPRV from Sudan and Ethiopia. A phylogenetic tree based on N gene nucleotide sequences was constructed using the PPRV N gene sequences obtained in this study and other viruses representing the four PPRV lineages. The N gene sequences of PPRV from Mvomero and Ngorongoro districts clustered into Lineage III together with other PPRV previously reported from Ethiopia, Sudan, Oman and United Arab Emirates  currently being used as vaccine in the region.


Objective. To study the prevalence of osteoporosis and vitamin D deficiency in healthy men and to explore the influence of various life style factors on bone mineral density (BMD) and also to look at number of subjects warranting treatment. Methods. Ambulatory south Indian men aged above 50 were recruited by cluster random sampling. The physical activity, risk factors in the FRAX tool, BMD, vitamin D, and PTH were assessed. The number of people needing treatment was calculated, which included subjects with osteoporosis and osteopenia with 10-year probability of major osteoporotic fracture >20 percent and hip fracture >3 percent in FRAX India. Results. A total of 252 men with a mean age of 58 years were studied. The prevalence of osteoporosis and osteopenia at any one site was 20% (50/252) and 58%, respectively. Vitamin D deficiency (<20 ng/dL) was seen in 53%. On multiple logistic regression, BMI  and physical activity  had protective effect on BMD. Twenty-five percent warranted treatment. Conclusions. A significantly large proportion of south Indian men had osteoporosis and vitamin D deficiency. Further interventional studies are needed to look at reduction in end points like fractures in these subjects. Osteoporosis in men is now recognized as a major underestimated public health problem . With thIn addition to genetic determinants, several life-style related factors like physical activity, calcium intake, smoking, alcohol consumption, and vitamin D status may influence the bone mass in men . HoweverScreening for osteoporosis in men is usually recommended above the age of 70 years , 5. HoweThere is also a paucity of data with regards to the risk factors that have been mentioned and their influence on bone health in an Indian context. Many studies have shown that a low Bone Mineral Density (BMD) may not be the sole factor which determines the risk of fracture . This leIn this study, we have attempted to look at the prevalence of osteoporosis and vitamin D status in healthy South Indian men and to study the influence of various life style factors on bone mineral density. The number of subjects warranting treatment in these healthy subjects was also computed.The study was a cross sectional one conducted over a duration period of 1 year. This study was approved by Institutional Review Board.We conducted a survey of the total number of houses in an urban region of south India. Men above 50 years of age in that locality were recruited by cluster random sampling after obtaining a written informed consent. Men with a history of chronic liver or renal disease, hyperthyroidism, hyperparathyroidism, hypogonadism, and malabsorption and those on medications such as anticonvulsants, antiretrovirals, and antituberculous therapy which will affect the bone health were excluded.The risk factors which were mentioned in the FRAX tool assessment included age, sex, height, weight, a past history of fragility fracture, parental history of hip fracture, history of smoking or alcohol intake (3 or more units/day), the presence of rheumatoid arthritis, and any other history suggestive of secondary osteoporosis . PhysicaHitachi Roche Modular P 800 model). The intra-assay and interassay coefficients of variation for these analytes were 1 to 5%.An overnight fasting blood sample was obtained for estimation of serum calcium (8.6–10.2 mg/dL), phosphate (2.5–5 mg/dL), albumin (3.5–5.0 gm/dL), alkaline phosphatase (40–125 iu/L), creatinine (0.6–1.2 mg/dL), 25-hydroxy vitamin D (30–75 ng/mL) hereafter referred to as vitamin D, intact parathyroid hormone (iPTH) (8–50 pg/mL), and testosterone (300–1000 ng/dL). These reference ranges are as provided by the local laboratory. Vitamin D deficiency was defined as a vitamin D level of less than 20 ng/mL and a level less than 10 ng/mL was considered to indicate severe vitamin D deficiency . The vit T score ≤−2.5, osteopenia, or low bone mass −1 to −2.5 and normal as >−1.BMD was assessed using the Hologic DXA QDR 4500 Discovery A machine at the lumbar spine and femoral neck by the same technician. Precision was 2 percent at both themeasured sites (spine and neck of femur). The WHO classification was used for categorization of BMD . OsteopoIn subjects who were found to have osteopenia, data regarding all risk factors were incorporated into a web based software—Fracture Risk Assessment—FRAX India for calculating the 10-year probability of major osteoporotic fracture and hip fracture. The number of people needing treatment was calculated, which included the total number of subjects with osteoporosis at any site and osteopenia with a 10-year probability of major osteoporotic fracture more than 20 and hip fracture more than 3 as calculated by FRAX India. This was as per the Endocrine Society clinical practice guidelines . T-test was used to compare the means of two continuous variables if they were normally distributed and nonparametric tests were used if their distribution were not normal. Statistical analysis was done using the SPSSv.16.0 software .The prevalence of male osteoporosis above the age of 50 years in published literature was about 6% . Total nTwo hundred and fifty-two men above the age of 50 years were included in the study. The demographic parameters, biochemistry including vitamin D status, and BMD of the study subjects are summarized in 2. Forty percent (n = 100) of men were above the age of 60 years. In relation to physical activity, 77 percent were sedentary with the rest being moderately to strenuously active.The mean age (SD) of the study subjects was 58 (11.8) years and the mean (SD) BMI was 23.3 (4.5) kg/mP = 0.01). Osteoporosis at the spine was also more commonly seen among subjects aged above the age of 60 years; however, it was not statistically significant.The prevalence of osteoporosis and osteopenia at spine and femoral neck is shown in n = 133) and 7% had severe vitamin D deficiency (<10 ng/dL). Eight percent of subjects had biochemical hypogonadism (<300 ng/dL).Overall the prevalence of osteoporosis and osteopenia at any one site was 20% (50/252) and 58% (146/252), respectively. However, in men aged above 60 years, the prevalence of osteoporosis was 45%. Vitamin D deficiency (<20 ng/dL) was seen in 53% . Multiple logistic regression analysis was performed to assess the impact of various factors that influence bone mineral density at femoral neck in healthy male subjects. These included body mass index, hypogonadism, age, vitamin D deficiency, smoking, alcohol use, and physical activity. We found that body mass index  and physical activity  had a statistically significant protective effect on bone mineral density.Among the risk factors studied, BMD at the femoral neck was significantly higher in subjects who were physically more active when compared to men who had a sedentary lifestyle (0.811 versus 0.733 gm/cmn = 146) 13 men had a greater than 3% probable risk of hip fracture; however, none of these subjects had more than a 20 percent risk of major osteoporotic fracture calculated by FRAX India.Amongst the subjects who were having osteopenia at any one site . However, there is no significant correlation between vitamin D and BMD at any site and also no significant correlation was found between PTH and BMD.Therefore, out of the total 256 subjects, 63 warranted treatment (50 had osteoporosis plus 13 osteopenics with a more than 3% probable risk of hip fracture). The mean (SD) age of subjects warranting therapy in our study was 67.5 (11.65) years. There was a significant negative correlation between vitamin D and PTH levels (Male osteoporosis is an underreported public health problem. In our study, we attempted to look at the prevalence of osteoporosis and the various risk factors in South Indian healthy men above the age of 50 years.In this present study, about one-fifth of them had osteoporosis at any one site. Vitamin D deficiency was found in over half of the study population. Men who were physically active or having higher body mass index had a better BMD at femoral neck. As per the FRAX score, about 10 percent of the osteopenic subjects had more than a 3%, ten-year probability of sustaining a hip fracture. Mean age of warranting treatment to prevent fractures was close to the recommendations published in the international guidelines . TreatmeIn previously published literature, a 9 percent prevalence of osteoporosis has been reported in Northern India  and in oBMD begins to decline after the third decade and is influenced by genetic and environmental factors. Prepubertal BMD is similar in both sexes. However, the pubertal increase in BMD is more in men when compared to women due to a greater cross sectional area in view of increased periosteal apposition under the influence of androgens . Bone reThe most dreaded complication of osteoporosis is hip fracture, which has been reported to be more in men when compared to women . OsteopoThe economic burden of osteoporotic fractures is not only borne by the patient but by the country as a whole , 23. TheA high prevalence of vitamin D deficiency has been reported in various cohorts of Indian population . VitaminA positive impact of physical activity on bone mass or BMD demonstrated in our study is probably due to a skeletal response to mechanical strain by stimulating bone formation . Also inAn increase in body mass index had a significant impact on BMD. Several studies have shown the positive correlation between BMI and BMD. This is explained by a higher gravitational load on the femur neck, increased peak bone mass, and higher circulating estradiol level .The socioeconomic status, nutritional status, prevalent vertebral fractures, and bone turnover markers in these individuals were not assessed. Also this data has been collected from an urban society in southern India and cannot be generalized to the entire Indian population.A significantly larger proportion of otherwise normal healthy men in our community had osteoporosis and vitamin D deficiency compared to previously published studies. Men with a higher BMI were physically active and had a better BMD. Large scale prospective studies with interventions are needed to look at the reduction in the end points like number of incident fractures and morbidity associated with them.


A 15 nm SiO2 layer was deposited by plasma enhanced chemical vapour deposition (PECVD), onto a resorbable PLGA membrane. Samples were characterized by X-ray photoelectron spectroscopy, atomic force microscopy, scanning electron microscopy, and infrared spectroscopy (FT-IR). HOB cells were seeded on sterilized test surfaces where cell morphology, spreading, actin cytoskeletal organization, and focal adhesion expression were assessed. As proved by the FT-IR analysis of samples, the deposition by PECVD of the SiO2 onto the PLGA membrane did not alter the composition and other characteristics of the organic membrane. A temporal and spatial reorganization of cytoskeleton and focal adhesions and morphological changes in response to SiO2 nanolayer were identified in our model. The novedous SiO2 deposition method is compatible with the standard sterilization protocols and reveals as a valuable tool to increase bioactivity of resorbable PLGA membranes.The possibility of tailoring membrane surfaces with osteoconductive potential, in particular in biodegradable devices, to create modified biomaterials that stimulate osteoblast response should make them more suitable for clinical use, hopefully enhancing bone regeneration. Bioactive inorganic materials, such as silica, have been suggested to improve the bioactivity of synthetic biopolymers. An in vitro study on HOB human osteoblasts was performed to assess biocompatibility and bioactivity of SiO Cell-biomaterial interaction is a major challenge for tissue engineering, since not only the physic-mechanic-chemical features of the natural extracellular matrix must be mimicked, but also a successful cell to biomaterial adhesion should be achieved in the shortest time. Cell adhesion is critical in the first stages of the tissue engineering methodology where adhesion is the basis and the key process that allows other important cell activities such as migration and growth. In the fields of tissue engineering and guided bone regeneration (GBR), an appropriate cell culture scaffold is required to enhance cell attachment, proliferation, and differentiation in vitro, as cells interact with native or topographically and chemically tailored structures in many ways –5.The GBR therapeutic approach is based on the concept that regeneration of osseous defects is attainable via the application of occlusive membranes, resorbable or nonresorbable, which mechanically exclude nonosteogenic cell populations from the surrounding soft tissues. Nowadays GBR is subject of growing interest due to the increasing needs for permanent, temporary, or biodegradable orthopaedic devices designed for bone repair and regeneration –11.A variety of polymeric bioresorbable GBR membranes that permit single-step procedures, thus reducing patient discomfort and costs, and potential surgical complications are currently available. Polymeric coatings for biomedical devices are especially interesting due to the diversity of their chemical and physical properties and natural biopolymers appear promising as biomimetic coatings, as a result of their similarity to human tissues. Although these membranes are biocompatible, nontoxic, or immunogenic and permit mechanical support during bone formation, they do not actually possess bioactive properties to induce osteogenesis , 12–14.Poly(lactide-co-glycolide) (PLGA) is among the few synthetic polymers approved for human clinical use due to its biocompatibility, controllable degradability, and relatively good processability and has been used in the design of scaffolds for bone tissue engineering and for drug delivery purposes , 16. HowThe physicochemical properties of the material at the cell-material interface are decisive for the cell-material interaction, due to its interfacial role between the material and the host tissue, and are able to trigger a wide variety of processes, from the initial inflammatory reaction to ultimate tissue remodelling , 17. AltBioactive inorganic materials, such as silica granules, have been suggested to improve the bioactivity of synthetic biopolymers in promoting the osteogenic performance of osteoblast-like cells. It has been strongly suggested that silicon/silica is functionally active during bone formation with a close relationship between silicate and calcium deposition. Silica is a low cost and biocompatible inorganic material having excellent chemical stability with great commercial value and ideal for tailoring surface topographies –24.2 functionalized PLGA membranes intended to be used as barrier membranes for bone guided regeneration.We have designed an in vitro model in order to assess normal human osteoblasts, HOB, response to a series of SiO2 on the surface of the PLGA membranes (SiO2/PLGA) [For the synthesis of the PLGA foils we have followed classical synthesis procedures based on the solution/evaporation method –27. A prO2/PLGA) , in orde2 until the experiments were started. HOB cells did not exceed ten population doublings. Test surfaces were sterilized under ultraviolet (u.v.) light for 20 min each side, in a laminar flow chamber prior to cell seeding. HOB osteoblasts were seeded at a density of 5000 cells/cm2 on test surfaces, that is, bare PLGA or SiO2/PLGA membranes, and immunolabelled after 24, 48, and 72 hours. HOB osteoblasts, media, and sera were obtained from Promocell .HOB human osteoblasts were incubated in Osteoblast Growing Medium with 10% foetal calf serum at 37° and 5% CO2. The thickness of the membranes was of the order of 50 microns.The PLGA membranes used as substrates were prepared from a 1.5 wt% PLGA dichloromethane solution by evaporation of the solvent on a teflon plate. The size of the membranes was 15 × 15 mm2 was deposited onto the PLGA membranes by PECVD in a plasma reactor with a remote configuration. The deposition system consisted of a quartz tube with a funnel shape termination attached to a stainless-steel chamber where the samples were placed. The plasma source is a surfatron (Sairem) launcher that was supplied with a microwave power of 60 W. These conditions provide a typical plasma electron density of about 1 · 1011 cm−3. Distance from substrate to the quartz tube was 5 cm. The plasma gas and the precursor used were pure Ar (7.5 sccm) and hexamethyldisiloxane , respectively. Synthesis of the films was carried out at room temperature, working at a pressure of 0.3 Torr. The gas flow was controlled by mass flow controllers (MKS). Both the stainless steel receptacle which contained the precursor and the dosing line were heated at 323 K to prevent any condensation in the tube walls. Before depositing SiO2, the polymeric substrate was exposed to plasma of pure argon for 2 min, a pretreatment that favours the adhesion of the oxide layer onto the polymer surface. A thin layer of SiO2 of 15 nm, determined by measuring with an adjacent quartz crystal monitor, was deposited on the polymeric substrate. These measurements were calibrated with the data obtained for thicker layer of SiO2 deposited on a silicon substrate that were cleaved and examined by scanning electron microscopy. Before the treatments, Ar or the mixture Ar + HDMSO was kept flowing for at least 30 min to ensure the purity of the plasma gas in the reactor [2 functionalized membranes will be designated as PLGA and SiO2/PLGA, respectively. The samples were characterized by X-ray photoelectron spectroscopy (XPS) to determine their surface chemical state and composition, atomic force microscopy (AFM) to ascertain their roughness, scanning electron microscopy (SEM) to assess the film microstructure, infrared spectroscopy (FT-IR) to determine the bonding structure, and water contact angle measurements to check the wetting characteristics of their surfaces. XPS spectra were recorded with a SPECS PHOIBOS-100 spectrometer working in the constant pass energy mode fixed at a value of 20 eV. The Mg Ka radiation was used as excitation source. For calibration of  the binding energy scale, a value of 284.6 eV was considered for the C1s component attributed to C–H and C–C bonds. Surface composition was estimated by calculating the area behind the C1s, Si2p, and O1s peaks and by correcting the obtained values with the sensitivity factors of these elements. AFM images were taken with a Cervantes AFM microscope driven with a Dulcinea control system  working in tapping mode and using high frequency cantilevers. The images were processed with Nanotec WSxM software. SEM images were acquired in a Hitachi S4800 microscope. FT-IR spectra were collected in attenuated total reflectance (ATR) mode in a JASCO FT/IR6200 spectrometer, performing 64 scans with a resolution of 0.96 cm−1 and a spot size of 1 cm of diameter. Static contact angles of water were determined by the Young method in a SCA20 instrument , with an estimated error of 3%.SiO reactor . The nonHOB osteoblasts seeded on test surfaces were daily examined with a DM ILLED Leica phase contrast microscope in order to evaluate cell morphology and spreading, cell alignment, and initial adhesion phase to surfaces, prior to fluorescence and CLSM examination.At the designed experimental time, cells in each group were washed with prewarmed phosphate buffered saline (PBS) and fixed with 3.7% paraformaldehyde at room temperature, permeabilized with 0.1% Triton x-100, preincubated in 1% bovine serum albumin in PBS, and then immunolabelled for actin cytoskeleton with rhodamine-phalloidin and for focal adhesion identification with monoclonal antivinculin FITC conjugate. After 20 min samples were twice PBS rinsed prior to mounting with Vectashield . All chemicals were obtained from Sigma-Aldrich  unless noted.Samples were visualized in a Leica TCS-SL confocal microscope. At least five samples were analysed for each experimental group to assess surface influence on cytoskeletal organization, focal adhesion number and development, and cell morphology. Samples were exposed to the lowest laser power that was able to produce a fluorescent signal, with a pinhole of 1 airy. Images were acquired at a resolution of 512 × 512, mean voxel size of 209.20 nm.http://rsb.info.nih.gov/ij/).To analyze the differences in focal adhesion number between different sample groups, images were collected and processed. Area, perimeter, roundness, circularity, solidity, and axial ratio were analysed as shape variables. Sample images were collected as frames obtained at 40x magnification and processed using Leica imaging software and Image J software , the PLGA surface was totally covered by SiO2, as can be directly retrieved from the XPS spectra , 1185, 1080–980 (C–O stretch), and 754 (CH-bend) cm−1 [μm) and the SiO2 films (15 nm) and the typical thickness proved by ATR analysis (a few tens of microns).The C1s spectrum of the PLGA depicts three components at 284.6, 286.4, and 288.6 eV, attributed to C–C/C–H, C–O, and >C=O/COOH species , 23. Thi in SiO2 , 39. It reported  for the iO2 film . The othnd) cm−1 . No sign2/PLGA membranes and the values obtained were 99.3° and 93.8°, respectively, indicating no significant differences in wetting between these two surfaces.Since wetting behavior and surface tension are important parameters for cell development of cells on bioactive materials , 42 the 2/PLGA membranes extracted from the PBS and gentled dried in air did not reveal significant changes suggesting any significant degradation. membrane degradation  could only be observed after 20 weeks of immersion in liquid media, although clear signs of degradation  could be observed after 20 weeks of immersion in liquid media. The exposure of PLGA and SiO2/PLGA membranes to u.v. irradiation under similar conditions to those used in the sterilization protocol prior to cell seeding did not produce any detectable change.During the experimental time, no clear signs of degradation were observed in the membranes, During the experimental time, no clear signs of degradation were observed in the membranes. FT-IR, AFM, and XPS analysis of the surface of the PLGA and SiOLiving HOB  cells examination revealed a successful cell spreading on silica treated membranes, with cell elongation and marked filopodial emission, and the establishment of clear cell contacts and attachment to the functionalized surface in the first 24 hours. Cells clustered and elongated during the next hours, and after 48 hours in culture, cell spreading evolved to a clear cell packaging, with osteoblasts well adhered to the silica treated surface and to the neighboring cells Figures . Cells g2 functionalized PLGA membranes showed defined stress fibers formation, with a clear polarization of actin cytoskeleton towards strongly evident vinculin positive sites. Cell polarization was clearly determined by SiO2 functionalized surfaces, with osteoblasts showing extensive lamellipodial emission reinforced with vinculin positive focal adhesion in the leading edge and supported by a highly defined actin network. Focal adhesions appeared to be significantly more numerous and well developed in those cells grown on the SiO2 functionalized membranes HOB grown on bare PLGA randomly orientated and showed a reduced stress fibers and focal adhesion development at the same experimental time. Although some evenly distributed filopodia were observed, cells did not show a well-organized actin network or polarization, and vinculin positive sites appeared randomly distributed in the cell periphery  indicates that silicon, time, or both influence focal adhesion number. Using the two-way ANOVA, the analysis of the effects of each factor and their interaction with the ranks of the variable [ silicon is a significant factor (P = 0.000), while time factor is not (P = 0.929). Furthermore, silicon treated groups  and untreated groups (P = 0.873) can be considered homogeneous at any time, and both groups are different from each other (P = 0.05)  , Table 1 silicon or time. Then, an exploratory factor analysis was performed, in the attempt to reduce the number of morphometric indexes (latent factors). The correlation matrix and the factor loadings matrix were obtained first by the method of principal components and later by varimax rotation, which quantify the relationship between the original variables and the indexes achieved. The first index positively related to circularity and solidity and negatively related to perimeter. This first index represents the property of solid roundness (SR) showing higher values in those cells that are circular, without voids, and with a medium perimeter. The second index contrasts roundness and AR, thus representing stretch (ST) or lengthening, with the highest values for the more elongated cells, with high AR, and negative values more circular cells, with AR close to 1. The third index relates to area and perimeter and can, therefore, be interpreted as the cell size, with higher values for larger cells.The descriptive analysis of the shape variables in the osteoblasts grown in the four experimental groups did not establish a clear relationship with the factors silicon significantly increased (P = 0.000) the mean score for SR index, estimating this increase by CI95% = , and also caused a decrease in ST index (P = 0.016), estimated by CI95% = . Moreover, the factor time caused a decrease in the average score for the ST index (P = 0.012), estimated by CI95% = , while a significant interaction (P = 0.029) was obtained for the index size, as cell size decreases in cells grown on bare PLGA surfaces, CI95% = , and increases in silicon treated ones, CI95% = . These results indicate that silica treatment, independently of time, favors the presence of cells that were circular and solid (without voids). On the other hand, and independently of silica treatment, the experimental factor time favors a lower ST, or roundness, and increases the size in cells grown on silica treated membranes.Growing cells were scored for the morphometric indices according to the values obtained in each one by each individual cell. The two-way ANOVA contrast performed for these indices showed that the experimental factor2 functionalization methodology employed by our group enhances HOB cells response to nonresorbable PET devices and stands for standard u.v. sterilization [2 functionalized PLGA membranes has been assessed.There are several ways to modify the surface of a material in order to enhance its attractiveness for cell colonization and osteogenic cell differentiation. For biological applications, there is an additional need for versatile deposition methods in the case of devices with potential clinical applications, which should also stand, without alterations, the sterilization processes , 16, 18.lization . We now Trace amounts of silicon have been described to substantially contribute to strengthening of bones with promotion of bone formation especially during ageing. Well-dispersed nanosized inorganic particles have been described to enhance the mechanical properties of organic materials . A recen2, as active layer, by PECVD. A key feature of this method is that it preserved the integrity of the substrate, which remained practically unaltered after the SiO2 as revealed by FT-IR analysis (2/PLGA substrate showing that the surface roughness is not significantly altered after the deposition of SiO2. Stability of these membranes after 72 hours of immersion in a PBS liquid and after UV irradiation sustains that the in vitro cell growth analysis basically refers to the “as prepared” samples without any significant alteration during its manipulation or during their immersion in the culture medium.Our PLGA membranes were fabricated by using a conventional resorbable PLGA polymer as substrate and then functionalized by depositing a very thin layer of SiOanalysis . In comp princeps issue in cell adhesion is the development of focal adhesions which, furthermore, are established sites for signal transduction, acting as conveyors of mechanical forces directly to the nucleus via the cytoskeletal network by the complex process of mechanotransduction, where vinculin is required for a strong network formation [Cell adhesion onto a biomaterial surface is a major contributing factor of its cytocompatibility and the host response when implanted, ultimately determining device biocompatibility. Without adhesion, osteoblasts cannot spread or differentiate. Aormation , 4, 44. ormation , 20, 45.2 layer identified in our model, parameters that have been established as important mediators of the mechanotransductive processes, parameters that have been established as important mediators of the mechanotransductive processes [The relation between focal adhesion number, functionality, cell movement, and growth of skeletal cells has been described, by our group and others , and therocesses , 47, 48.2 functionalized PLGA membranes actively probe the physical properties of the ECM, with their contractile machinery clearly facilitating the formation of polarized lamellipodia and evenly distributed filopodia, which gather spatial, topographical, and chemical information from the surface. Initial cell tethering and filopodia exploration are followed by lamellipodia ruffling membrane activity and cellular spreading. The results, obtained after a carefully designed in vitro study, are reinforced with a powerful statistical treatment and reveal that in vitro HOB cell morphologies are significantly different and vary greatly depending on the substrata and are consistent with an increased cytoskeletal tension related to focal adhesion increase in SiO2 treated membranes.Adherent cells require extracellular matrix anchorage to proliferate and undergo differential function. As shown by our data, HOB cells grown on SiO2 treated PLGA both the morphological changes and the focal adhesion development identified in our model are significantly related to SiO2 treated PLGA. The novedous SiO2 deposition method reveals as a valuable tool to increase bioactivity of PLGA membranes by combining cell nanotopography cues with the incorporation of bioactive factors, that in addition, stands the standard sterilization protocols.The methodology described provides a biocompatible and durable functionalization on our PLGA barrier membranes that elicits a significant osteoblast response. Both the morphological changes and the focal adhesion development identified in our model significantly related to SiO