Atmospheric dispersal of bacteria is increasingly acknowledged as an important factor influencing bacterial community biodiversity, biogeography and bacteria–human interactions, including those linked to human health. However, knowledge about patterns in microbial aerobiology is still relatively scarce, and this can be attributed, in part, to a lack of consensus on appropriate sampling and analytical methodology. In this study, three different methods were used to investigate aerial biodiversity over Svalbard: impaction, membrane filtration and drop plates. Sites around Svalbard were selected due to their relatively remote location, low human population, geographical location with respect to air movement and the tradition and history of scientific investigation on the archipelago, ensuring the presence of existing research infrastructure. The aerial bacterial biodiversity found was similar to that described in other aerobiological studies from both polar and non-polar environments, with Proteobacteria, Actinobacteria, and Firmicutes being the predominant groups. Twelve different phyla were detected in the air collected above Svalbard, although the diversity was considerably lower than in urban environments elsewhere. However, only 58 of 196 bacterial genera detected were consistently present, suggesting potentially higher levels of heterogeneity. Viable bacteria were present at all sampling locations, showing that living bacteria are ubiquitous in the air around Svalbard. Sampling location influenced the results obtained, as did sampling method. Specifically, impaction with a Sartorius MD8 produced a significantly higher number of viable colony forming units (CFUs) than drop plates alone. Microbial dispersal in the atmosphere represents a key biological input, directly influencing the gene pool . The disWhilst several studies have focused on the movement of bacteria through the atmosphere, the majority of these studies have failed to consider the viability of these colonists upon arrival in their new environments . Microbi4 to 106 cells per m3 [Atmospheric bacterial abundance generally ranges from 10s per m3 , but, ths per m3 , and cans per m3 . Bacteris per m3 ,20. Bacts per m3 ,22,23, as per m3 . Barberás per m3  collateds per m3 . Howevers per m3 ,22.Patterns of diversity in airborne bacterial communities are central to the emerging field of atmospheric biogeography. Indeed, until relatively recently whether microbial biogeography existed in the atmosphere at all was contentious . HoweverThe Arctic can be defined as the area above the Arctic Circle. The Norwegian Arctic archipelago of Svalbard is one of the northernmost inhabited locations in the world at 79° N. Svalbard is characterised by its remarkably low human population with only 2185 registered Svalbard inhabitants in 2015 . This loOrganisms in the Arctic atmosphere are exposed to extremely low temperatures and hurricane strength winds, seasonal freeze–thaw cycles, extreme exposure to UV and extremely low levels of nutrients. Thus, organisms inhabiting this region are referred to as extremophiles and tend to exploit features such as the ability to form spores, which allow them to survive the harsh conditions. Similar to those microbes inhabiting the Arctic, organisms surviving in the atmosphere also endure extreme temperatures, UV exposure and poor nutrient levels.Sampling techniques for terrestrial and aquatic microbial ecology studies are highly variable but based on common principles, established and used consistently. In contrast, a wide range of techniques are available to aerobiology, despite the low number of studies in the field. In general, sampling methods involve impaction, impingement, membrane filtration or the drop plate mechanism, the results of which are not directly comparable due to strong methodological biases. Furthermore, the strength of the bias is still unknown, due to the lack of studies comparing different methodologies, although recent efforts have been made towards establishing a standard methodology .Analytical techniques can also vary considerably among studies, compromising comparability even further. To date, most aerobiological studies use colony-forming units (CFU) count per unit volume of air sampled to measure the density of cultivable microorganisms in the atmosphere. These studies report density changes over space, time and varying environmental conditions; however, culture based studies only provide a partial picture of the overall microbial diversity . CultureSet against this background, in this study, the influence of different sampling techniques, sampling location and total sample volume on the identification of aerial bacterial communities in the Arctic was explored, based on culture dependent and independent analytical methods, thus presenting a preliminary picture of the microbial community in the air over Svalbard.Airborne microbial samples were collected in July 2015 above Svalbard . SvalbarSamples were collected between 6 and 23 July 2015 above both marine and terrestrial locations using a range of techniques . Marine −1 and humidity averaged 67%  were placed open at Gipsdalen, Mine (Gruve) 7, Deltaneset and Bjørndalen for 15 min; plates were incubated for 10 days at room temperature; following incubation the plates had colony counts and distinct colony counts taken.−1 and 20–80 L m−1 on 13, 15, 16 and 17 July 2015. The gelatine filters collected at all sites were placed directly onto the surface of R2A agar plates . These plates were then incubated at room temperature for 10 days. Total CFU and distinct colony numbers were counted.Additionally, a portable AirPort MD8 , comprising a disposable gelatine filter membrane, was used to compare sampling efficiency and cultivability at two flow rates and different sampling volumes. Sampling sites were chosen to compare with terrestrial plate drop sites but also to assess for the differences at marine sites. Terrestrial samples were collected at Mine (Gruve) 7, Deltaneset, Gipsdalen and central Longyearbyen (UNIS roof) and marine samples at Billefjorden, Sassenfjorden and Adventfjorden, respectively. The sampler was used at respective flow rates and durations ranging 30–50 L m−1 connected to Sartorius filtration unit  containing a 47 mm × 0.2 μm pore size cellulose nitrate membrane filter .As gelatine filters are not amenable to culture independent techniques (due to the presence of gelatine), airborne bacteria from both terrestrial and marine sites were collected via membrane filtration. A Welch WOB-L vacuum pump  was set up at a flow rate of ~20 L mA marine sample was collected at Isfjorden on the 11 July 2015 with a respective sample duration and volume of 8 h and ~9600 L and the terrestrial sample was taken in central Longeyearbyen (UNIS roof) at the following dates, durations and volumes, respectively: 6 July 2015 for 30 (600 L), 60 (1200 L), 120 (2400 L) and 300 (6000 L) min; 19 July 2015 for 30 (600 L), 60 (1200 L), 120 (2400 L) and 300 (6000 L) min; and 21–24 July 2015 for three days  continuously .The cellulose nitrate membrane filters were sent to MrDNA  for extraction and sequencing. DNA was extracted from samples using the MoBio PowerSoil kit  following the manufacturer’s protocol with an additional 1 min of bead beating to account for the filter paper. Extracted samples were then amplified using 16S rRNA universal primers 27Fmod (AGRGTTTGATCMTGGCTCAG) and 519Rmodbio (GWATTACCGCGGCKGCTG) and barcodes were attached at the 5′ end. A 28-cycle PCR using the HotStarTaq Plus Master Mix Kit  was carried out under the following conditions: 94 °C for 3 min, followed by 28 cycles of 94 °C for 30 s, 53 °C for 40 s and 72 °C for 1 min, after which a final elongation step at 72 °C for 5 min was performed. After amplification, PCR products were checked in 2% agarose gel to determine amplification success. Samples were then pooled based on their molecular weight and DNA concentrations, purified and illumina DNA libraries were prepared. Paired end sequencing of the V4 region was then performed on a MiSeq following the manufacturer’s guidelines. The resultant data were analysed using QIIME v1.9.1 . The 776Statistical analyses were performed using PAST  to test p = 0.095, plate fall morphologically distinct CFUs: p = 0.123).Viable bacteria were found in all of the samples. Clear differences were apparent in the mean CFUs from the two culture dependent methods used . A Kruskp < 0.05); no differences in variances, medians or coefficient of variations, but a significant difference in equality of distributions (Kolmogov–Smirnov: p < 0.05). For the morphologically distinct CFUs, however, there were no significant differences for any of the mentioned parameters. When looking at the overall variance and efficiency of both culture dependent methods, only considering the MD8 samples collected at 50 L m−1 for 20 min i.e., 1000 L sampling volume (t-test comparing the two methods showed a significant difference in the mean CFUs from drop plate and MD8 samples (p < 0.001), no significant differences in variances, but with significant differences in coefficients of variation (p < 0.005), medians (Mann–Whitney U p ≤ 0.001) and distributions (p < 0.001). Looking at the statistical analysis of the morphologically distinct CFUs, there was no significant difference in the means from drop plates and the MD8 (p > 0.05), with no significant differences in variances, medians, distributions, or coefficients of variation. These results show that there is a significant difference between the two methods, the MD8 yielding a larger number of CFUs.Comparing the differences in drop plate and MD8 results for the locations where data were available for both methods, the MD8 showed much higher CFU yields, although this difference is not obvious when looking at the number of morphologically distinct CFUs i.e., CFUs of different appearance . Statistg volume , there wp = 0.070). This also held for the morphologically distinct CFUs, there was no significant difference for either of the mentioned parameters between terrestrial and marine sites.Comparing MD8 results for terrestrial and marine samples, the mean CFUs were lower at marine sites than terrestrial sites . StatistTo account for the variable scales across the samples, the normalised coefficient of variation was used, and this showed the highest variation in the CFUs in the drop plate and MD8 samples collected at UNIS. In the MD8 marine sample the variability of morphologically distinct CFUs was the highest. The CFUs at the terrestrial sites varied the least .−1 flow rate sample, however, had slightly higher CFUs, despite lower volume. There was a clear correlation between CFUs and the sampled volume of air  when this sample was included. For the morphologically distinct CFUs, there was a slight negative correlation (R2 = −0.256) in number of CFUs with increasing volume (−1 showed a considerable negative correlation (R2 = −0.640).At UNIS, where volume and flow rate were varied, a clear trend of increasing CFUs with increasing volume was evident . The 30 g volume B, leavinTargeted amplicon sequencing of the 16S rRNA V4 region resulted in 776,315 total reads across the 10 samples, which were then quality filtered and checked for chimeras leaving 685,583 reads. The range of reads per sample ranged from 32,651 (recorded in the 60 min sample from 6 July) to 145,488 reads (recordedin the 30 min sample collected on 19 July). Samples were then rarefied to 26,190 reads ; rarefied samples averaged 5015 OTUs (range 4143–6402). Rarefaction curves for all of the normalised samples did not reach asymptote suggesting the full extent of the diversity present was not reached for all samples A. The ShThe differences in OTU diversity between the communities was measured using the Bray-Curtis dissimilarity index A and an Twelve phyla in total were detected within the samples: Proteobacteria, Firmicutes and Actinobacteria were present in all of the samples at differing but high relative abundances and were the visibly dominant phyla ; BacteroProteobacteria, Firmicutes and Actinobacteria represented ~99% of the Day 1 sample set in which there were 10 phyla present in total . ProteobPseudomonas, Staphylococcus, Propionibacterium, Delftia and Corynebacterium spp. made up the five most relatively abundant genera. Pseudomonas was the most common and relatively abundant genera representing 18% of the full sample set, however, they were only the most abundant genera in the Day 1, 30 min sample. Pseudomonas, Acinetobacter, Corynebacterium, Staphylococcus, Deltia, Cloacibacterium, Arthrobacter, Sphingomonas, Alcanivorax, Comamonas, Streptomyces and Brevibacterium spp. were all regularly present in the top 10 most abundant genera in each sample  difference in the number of morphologically distinct CFUs produced by the two methods, and none of them showed any particular bias for specific taxonomic groups on R2A. Replicates taken for drop plate samples showed the technique to be robust with no significant difference across the samples. Generally, marine studies tend to present more CFUs than terrestrial samples [All culture dependent samples recorded growth, showing that viable microbes are common in the atmosphere, at both terrestrial and marine locations around Svalbard. The number of viable bacteria measured in the air was considerably lower than the number measured in other environments e.g., surface ice and cryoconite holes, where the number of CFU can be up to tenfold higher using the same media . These r samples . Despite−1 to 30 L m−1 increased the number of cultivable bacteria recovered, possibly due to the decreased impact stress placed on captured bacteria [The number of cultivable bacteria increased with the increase in sample volume when using the MD8. This contradicts previous studies which showed no effect of sample volume on total CFU counts . In addibacteria .Whilst culture dependent studies provide useful information about the proportion of viable bacteria in the atmosphere, it is generally considered that only around 1% of the total bacteria present in the atmosphere are culturable . DormancCulture independent studies using sequencing can provide more information about the diversity and taxonomic composition within an environment. Despite the ability of culture independent studies to generate useful information, they also have major drawbacks, as they contain little information about the viability of the bacteria in the environment. Thus, combining both culture dependent and culture independent methods, provides a better insight into both the structure and viability of bacterial communities.Previous research on bacteria in the atmosphere outside the Arctic has linked temporal and spatial variation to changes in the diversity and abundance . DespiteDelftia, Ralstonia and Pseudomonas. A higher Simpson reciprocal value was seen on the third sampling day (76.61) taken at UNIS, suggesting that sampling for a longer duration increases the diversity of bacteria captured. The marine sample was considerably more diverse than the terrestrial samples when taking into account dominance  are all either polar associated or ubiquitous. Delftia sp. have been described at multiple Arctic locations including Svalbard and Greenland where they are associated with surface ice [Propionibacterium sp. are typically associated with marine sediment in the Arctic Ocean [Pseudomonas sp. are ubiquitous and present in almost all polar studies, however, on Svalbard they are mainly described from fjords [Pseudomonas was recently described from the same region [Corynebacterium sp. have previously been found in soils from the Canadian high Arctic [Staphylococcus sp. were frequently present, but are not routinely described in environmental Arctic studies and could be human or animal associated. Acinetobacter sp. are also commonly found in the top 10 most relatively abundant bacteria in all the locations. Acinetobacter sp. have been found in glacial snow and ice in mountainous locations outside the Artic [Alcanivorax sp. and members of the Oxalobacteraceae family were also common, they appeared on the days dominated by easterly winds and did not appear on the day dominated by westerly winds [Polaribacter sp., a bacterium associated with polar sea ice, was present in the marine sample suggesting that the Arctic Ocean provides a source of bacteria to the atmosphere. Many of the regularly occurring marine psychrotrophs, included in the Pseudomonas, Acinetobacter, Alcanivorax, Psychorbacter genera and members of the Oxalobacteraceae family are associated with the degradation of hydrocarbons in the Arctic [Cytophagales, Lactobacillus, Staphylococcus, Janthinobacterium, Pseudomonas and Polaromonas, which were mentioned but excluded as a chimeric sequence in that study. Bipolar comparisons also give an insight into both long-range transport and biogeography. Thus, Pearce et al. [Acidovorax, Acinetobacter, Cloacibacterium, Pseudomonas and Sphingomonas at Halley station in Antarctica, all of which were present at varying relative abundances in Svalbard air.At the genus level, the patterns were much less distinct. Of the 196 genera, only 58 were present in all samples. The five most relatively abundant genera  present was related to variation in both sampling technique (with the concentration of viable CFUs being higher when using a Sartorius MD8 when compared to drop plates) and sample volume, with increasing volume increasing total viable CFUs. The communities described were fairly homogeneous across sites, suggesting a distinct aerial community above Svalbard. Airborne bacterial abundance was lower than that described from other Arctic environments, such as the soil or the ice surface. The most relatively abundant taxa were polar associated, suggesting that the largest input into the atmosphere on Svalbard was of local origin. The overall diversity of the phyla present in the air above Svalbard was less diverse than in other locations such as urban environments, but was similar to that described previously in the Arctic on WHI. The key phyla remained consistent across studies.Further studies using metatranscriptomics would provide a deeper insight into the ecological role and metabolic activity of airborne bacteria, and potentially their ability to sustain activity, colonize and alter the environment at their final destination. Future studies investigating the biodiversity of the airborne microbes present in the Arctic will provide an insight as to whether an indigenous Arctic community exists.


Real life implementation studies performed in different settings have proved that lifestyle interventions in the prevention of type 2 diabetes (DM2) can be effective, although the weight reduction results are typically modest compared to randomized control trials. Our objective was to identify the factors that predict successful weight loss in a less intensive, lower budget, real life setting lifestyle diabetes prevention intervention.n = 175) with increased DM2 risk (Finnish Diabetes Risk Score (FINDRISC) > 14) but no diabetes at baseline received ten group lifestyle counselling sessions, physical activity and motivation sessions during a ten-month intervention. Stepwise regression analysis was used to determine demographic, clinical, and lifestyle predictors of successful weight reduction defined as a reduction of ≥5% of the initial body weight.Study participants (p = 0.03), decreased total fat consumption  and adherence to four-five lifestyle goals  were more often reported among those who managed to lose ≥5% weight versus those who did not.At 12 months following the initiation of the intervention, 23.4% of study participants lost ≥5% weight . Increased physical activity , baseline and medium versus higher education  and a history of increased glucose . A reduction of total fat in the diet was an independent lifestyle predictor, increasing the probability of successful weight loss by 3.8 times .Baseline higher BMI, lower education and a history of increased glucose predicted the successful weight loss among individuals with a high risk for the DM2 following lifestyle intervention in a real life primary health care setting. People who manage to lose weight more often adhere to lifestyle changes, while the reduction of total fat in diet independently predicts successful weight loss. Further studies exploring the predictors of success in implementation studies in DM2 prevention should help health care providers redesign interventions to improve their effectiveness and outcomes.ISRCTN96692060, registered 03.08.2016 retrospectively.ISRCTN, ID  Lifestyle intervention, through dietary and physical change, is very effective in type 2 diabetes prevention and as demonstrated in several studies can reduce DM2 incidence up to 60% –4. WeighThe mean weight loss and the number of people who lost ≥5% of their initial body weight is much higher in randomized control studies (RCTs) than in less intensive and less costly real life implementation studies. Weight loss in RCTs has been shown to be greater for those who were older, had a higher diabetes risk, were engaged in more frequent self monitoring of fat intake, reported lower percentage calories from fat, increased consumption of fiber, and increased physical activity –24. ImpoThe DE-PLAN project , EU initiated and sponsored, based on the principles of the Diabetes Prevention Study  was deveA detailed description of the program including the inclusion criteria, the characteristics of the participants, methods, the intervention, and one-year and follow up results have been published previously , 7, 28.The study was performed in nine independent Primary Health Care General Practitioners’ (GP) practices in Krakow, Poland. The study group consisted of everyday patients, city inhabitants, aged over 25. The inclusion criterion was a high DM2 risk assessed with the Finnish Diabetes Risk Score (FINDRISC > 14) (33% chance of developing diabetes within 10 years). The exclusion criteria was either known diabetes or oral glucose tolerance test (OGTT) screened diabetes as well as known chronic disease which could affect the results of the study.Information about the study and leaflets with the FINDRISC questionnaire were distributed in co-operating practices. Patients with known risk factors were directly approached by nurses and medical staff. Out of 800 FINDRISC questionnaires distributed, 566 were completed and 368 respondents scored FINDRISC > 14. Subsequently, 275 people signed informed consent and agreed to undergo OGTT examination. Of these 262  were invited to participate in the intervention. 184 participants completed the intervention (the number of completed sessions among completers was from eight to eleven), nine participants completed all sessions but not the final examination after one year and were excluded from the final analyses.175 participants with complete baseline and one year data were included in the analyses.The intervention followed the steps of the Diabetes Prevention Study (DPS) modified and adjusted to a local primary health care setting , 20, 28.The initial intensive phase of the intervention (four months) consisted of one individual session followed by ten group sessions (10–14 people), focusing on diet and physical activity changes. During each session, printed educational materials related to the topic of the session were distributed. Social support was emphasized by the group setting and participants were also encouraged to invite their own social environment to the lifestyle changes. A spouse or other family member could also participate in the sessions. From week four of the initiation of the intervention, patients were offered physical activity sessions  twice weekly and free of charge. The ongoing maintenance phase of the intervention (month 4–10) following the intensive phase consisted of six motivational telephone calls and two motivational letters , 20, 28.There was no other post-intervention contact with the participants except for measurements taken at one year.This study followed the Good Clinical Practice guidelines and the guidelines of the Helsinki Declaration. All study participants gave their written informed consent prior to the participation in the study.2). Waist circumference was measured midway between the lowest rib and the iliac crest. Diastolic and systolic blood pressures were taken while sitting following 10 min rest.Patients were examined at baseline and after 12 months of the study. The examination procedure included: standardized questionnaires  and biochemical tests including: fasting and 120’OGTT glucose, serum triglycerides, HDL and total cholesterol. Impaired Fasting Glucose (IFG) was defined as a fasting plasma glucose concentration of 6.1 to 7.0 mmol/l. Impaired Glucose Tolerance (IGT) was defined as a glucose plasma concentration of 7.80 to 11.0 mmol/l after oral administration of 75 g of glucose (OGTT). Diabetes mellitus (DM) was defined as fasting glucose concentration of more than 7.0 mmol/l or a glucose concentration of more than 11.1 mmol/l at two hours of OGTT [Data regarding education, marital status, employment status, history of increased blood glucose, family history of diabetes, diabetes risk score FINDRISC, smoking status, history of hypertension, history of cardiovascular disease (CVD) and history of depression were taken with the use of self-reported questionnaires.Lifestyle changes were also explored with the use of self reported standardized questionnaires regarding the: increase of consumption of vegetables and fruits, decrease of consumption of total fat, decrease consumption of saturated fat, change of saturated fat to unsaturated, decrease of alcohol consumption and increase of physical activity over the past year.Lifestyle goals’ achievement was defined as low if one-three goals were achieved and high if four-five goals were achieved.Participants were categorized into two groups based on the weight reduction achieved at 12 months using a ≥ 5% weight reduction as the cut-off point.Chi-square test for categorical variables and t-test for continuous ones were applied to compare the distribution of the potential predictors in groups of the participants who lost ≥5% of initial body weight compared to those who did not. Stepwise logistic regression models were used to assess the association between the different predictors and the outcome variable. The odds ratios and the respective 95% confidence interval were calculated. Coefficients of contingency were calculated to assess correlations between the lifestyle variables separately for those who achieved loss of ≥5% of initial body weight and those who did not (max. C for tables 2 × 2 = 0.707).p-value of <0.05 was considered statistically significant.The data was analyzed using STATISTICA 12. A p < 0.05) (data not shown). Of the participants, 69% were married or had a partner, 21% were current smokers. Family history of diabetes, history of increased glucose, CVD history, hypertension history and depression history was present in 60%, 60%, 37%, 66% and 16% of participants respectively.175 participants , mean BMI 31.8 (SD = 5.0) completed the core curriculum and participated in the final examination. Men were younger, had a higher BMI and waist circumference, higher serum TG and lower HDL, and had more often IFG than women (p = 0.004).Of the participants, 21% had a higher education and 39% were working. Among the participants with a higher education, 61% were working, while among those with a baseline/medium education, 34% were working  and 23.4% lost ≥5% of their initial body weight (mean weight reduced by 7.9 kg SD = 5.8); 88% of those who managed to loose ≥5% of weight were women.p = 0.02) and less often had a higher education (5% vs 25% p = 0.004)  and decrease in fat consumption  was more often reported among those who achieved ≥5% weight reduction .An increase in physical activity .In participants who managed to lose ≥5% of their weight, an increase of physical activity was correlated with the decrease of saturated fats consumption C = 0.39, decrease of total fat consumption C = 0.31 and increase of fruits and vegetable consumption C = 0.30. The decrease of total fat consumption was strongly correlated with the decrease of saturated fats C = 0.52 and increase of vegetable and fruits consumption C = 0.49. The decrease of saturated fat consumption was also correlated with an increased consumption of vegetables and fruits C = 0.37 , baseline and medium education  and history of increased glucose . Among lifestyle changes, reduction of total fat in diet was an independent predictor, increasing the probability of successful weight loss by 3.8 times . No other baseline and lifestyle factors modified the effect of weight loss  55% of the reduction in incidence of DM2 over 3 years follow- up was explained by a loss of 5 kg –18. TherIt is important to remember that interventions given in RCTs are not easily replicated in translational studies, where lower resources lead to less intensive intervention. Interventions also need to be adapted to local, cultural and health care possibilities. As a consequence, as seen also in our study, the achieved weight reduction usually is lower compared with RCTs , 17, 18.In previous RCTs, weight loss was greater for those who were older , 19, 20,In our study, a higher baseline BMI, which is a marker of increased diabetes risk, predicted the successful loss of weight. This finding is concordant with results of a study by Wadden et al. where the most consistent predictor of absolute weight loss was initial body weight, with heavier individuals losing more weight .In our study older age was not related to weight loss success. This is in contrast to the DPP and DPS results, where older age was a strong predictor of success at meeting the weight loss goal , 20. OurGender was also not related to weight outcomes, as seen in other studies , 19–22. The relation between lower education (baseline/medium vs high) and better weight outcomes, however surprising, might be explained by the almost double higher percent of working people among those with a higher education. This is concordant with other studies showing that lifestyle intervention programs are taken up mostly by non-working people –33. ScheIn our study, the history of increased glucose reported at baseline  was also an independent predictor of successful weight loss. “Medical triggers” like information regarding already existing diabetes or CV risk have been previously reported as factors leading to successful weight loss . suggestOther socioeconomic variables including marital and smoking status were not related to weight loss success which is consistent with some other publications , 20, 23.In the DPS the intervention was found to be most effective among those with a high baseline diabetes risk assessed by the FINDRISC. The Number Needed to Treat (NNT) was 7.7 and 3.6 for the low and high FINDRISC, respectively . In the p = 0.109) and the decrease of fat consumption was the strongest lifestyle predictor of success, increased activity was not meaningless (95% CI 0.9–4.4). The lack of significance in the stepwise analyses model might be explained by the correlation between physical activity increase and decrease of total fat consumption. In the DPS, individuals who increased moderate to vigorous and strenuous, structured leisure time physical activity resulted in 63–65% reductions in diabetes risk, even after adjustment for changes in weight [In our study, among the investigated changes of lifestyle factors, the decrease in dietary fat consumption was a strong independent factor of successful weight loss. In both groups of weight loss we also observed favorable increase of vegetable and fruit consumption and change of saturated to unsaturated fats however the differences between the groups were not significant. In analysis of contingency in both groups there was a correlation between the total fat consumption decrease and change of saturated fats to unsaturated and increase consumption of vegetables and fruit. In the DPS, individuals with low fat and high fiber intake lost more weight than those consuming a high fat, low fiber diet . Similarn weight . Also inSome strength and limitations of our study need to be discussed. This is one of the first studies investigating the predictors of success in the real life, real setting implementation of type 2 diabetes prevention intervention. The participants in our study were volunteers, and like seen in many other studies, as discussed above, the study predominantly attracted women, with only 22% of the total cohort being men and only 12% of men among those who successfully lost weight. Therefore, our results may not be generalizable to both sexes. In addition, the modest weight reduction obtained in our study might be influenced by the female sex domination, whose success in previous diabetes prevention studies was meager when compared to men .In light of the very poor male participation and success, these factors should also be further investigated, separately for both sexes. The sample size was relatively low and may have caused issues in power to detect small effects. The changes in lifestyle were assessed with the use of self- reported questionnaires also used in the DPS, while in some of the RCTs other methods assessing physical activity and diet were used. To deepen our analysis regarding lifestyle changes, we also investigated the correlations between physical activity and dietary patterns. What needs to be added is that in our study we have not examined psychological and behavioural factors influencing weight outcomes , 30, 42.Further studies exploring predictors of success in implementation studies in DM2 prevention should help health care providers redesign interventions with special focus being placed on physical activity to improve their effectiveness and outcomes.Additional insight into participant characteristics, including psychological and behavioral factors that independently predict weight loss, is critical for the further efforts of health care providers in real life diabetes prevention initiatives to identify those who are most likely to succeed and to understand the barriers of those who are not successful.


The oxidative stress induced by glutamate is reduced after neuroEPO treatment. These results confirm that neuroEPO has a protective effect against neuronal damage induced by excitotoxicity, improving antioxidant activity in the neuron, and protecting it from oxidative stress.Erythropoietin (EPO) is a glycoprotein initially identified as a hormone synthesized and secreted by the kidney that regulates erythropoiesis. EPO, and a group of its derivatives, are being evaluated as possible neuroprotective agents in cerebral ischemia. The objective of this study, using an in vitro model, was to determine how neuroEPO—which is a variant of EPO with a low sialic acid content—protects neurons from the toxic action of glutamate. Primary neuronal cultures were obtained from the forebrains of Wistar rat embryos after 17 days of gestation. Excitotoxicity was induced after nine days of in vitro culture by treatment with a medium containing 100 µM glutamate for 15 min. After this time, a new medium containing 100 ng of neuroEPO/mL was added. Morphological cell change was assessed by phase-contrast microscopy. Oxidative stress was analysed by measuring antioxidant and oxidant activity. After 24 h, the treatment with 100 ng of neuroEPO/mL showed a significant ( Stroke constitutes the third most common cause of death and the most common cause of disability in adults throughout the world . AccordiThe pathophysiology of strokes is complex, and involves mechanisms of excitotoxicity, inflammatory pathways, oxidative damage, ionic imbalances, and apoptosis . Such deStudies that began more than 20 years ago show that erythropoietin (EPO), a 34 kD glycoprotein that regulates erythropoiesis , has neuIn the last few years, non-haematopoietic EPO analogues, which include asialoerythropoietin  , carbamyneuroEPO is a 28 kD recombinant human glycoprotein produced in Chinese hamster ovary (CHO) cells  supplied by the Centre of Molecular Immunology  . It is characterized by its low sialic acid content which means that neuroEPO lacks erythropoietic activity while exhibiting neuroprotective properties . Neuro-EAll experimental procedures were carried out following the guidelines of the Committee for the Care of Research Animals of the University of Barcelona, in accordance with the directive of the Council of the European Community (86/609/EEC) on animal experimentation.2 and the foetuses were decapitated. Dissociated cortical cells were plated in cell culture plates at a density of 105 cells/cm2 in 9.6 cm2 Corning Costar cell culture plates . Previous to its use, each well was coated with a 1 mL poly-l-ornithine solution (0.01%) for 1 h in the incubator at 37 °C, the poly-l-ornithine solution was removed and washed three times with sterile water. Once the water was removed, the wells were dried in the sterile chamber with ultraviolet light. The cells were cultured in conditioned Neurobasal Medium®  containing penicillin/streptomycin, enriched with GlutaMAX® (Gibco) and B27® (containing antioxidants), supplemented with 5% horse serum, 5% foetal bovine serum, and 20 mM glucose. Cultures were incubated at 37 °C in a humidified atmosphere containing 5% CO2/95% air (pH 7.2). To halt the proliferation of non-neuronal cells, three days after plating, half the culture medium was replaced with medium, as previously described, containing cytosine arabinoside . On day 5 after plating, half of the medium was changed with conditioned Neurobasal Medium containing 2 mM glutamine and 2% B27 (AO+), and the neurons were used on day 9.Cerebral cortex neurons in primary culture were used in this study. Cortical cells were chosen because the cortical area is one of the parts of the brain most frequently affected by stroke. Moreover, the neurons in this area contain a large number of glutamate receptors, so it is prone to excitotoxicity, which plays an important role in stroke . Primary2PO4, and 0.33 Na2HPO4·7H2O, and treated with conditioned Neurobasal Medium containing 2 mM glutamine and 2% B27 (AO+) containing different concentrations of glutamate  and cells were further incubated for 15 min at 37 °C, in order to establish the concentration most adequate to cause death by excitotoxicity between 40% and 60% of the cells. After this incubation, the medium containing glutamate was removed, and after two washes with BSS, replaced by the Neurobasal medium containing 2 mM glutamine and 2% B27 (AO+), and the cells were further incubated for 24 h at 37 °C.On the ninth day in culture the medium was removed, neurons were washed once with pre-warmed (37 °C) with basic saline solution (BSS) containing (in mM) 137 NaCl, 3.5 KCL, 0.4 KHNeuronal injury was assessed by examination of cultures with phase-contrast microscopy at 20×, while that lactate dehydrogenase (LDH) assay and the MTT 3--2,5-diphenyltetrazolium (MTT) assay method were used to assess cell death and cell viability, respectively. Cell death was assessed by measuring the LDH in homogenates of the neurons and in the withdrawn culture medium using the microtiter plate assay described by Dringen . The res® STA-360) and Total Oxidant Activity Kit via reactive oxygen and nitrogen species (In Vitro ROS/RNS Assay Kit: Oxiselect® STA-347).Oxidative stress was analysed by measuring antioxidant and oxidant activity in untreated cortical cells, and in those treated with glutamate alone or with glutamate and neuroEPO. Total antioxidant activity was measured using the Total Antioxidant Capacity Assay Kit  testing. Differences were considered significant when In order to define the appropriate glutamate concentration to achieve a viability of 40–60%, cortical cells were treated with one of the different concentrations of glutamate (25–150 µM) or no glutamate (control) for 15 min. Once the glutamate was removed, the neurotoxic effect was analysed after 24 h. The results show that increase in glutamate concentrations resulted in a significant decrease in cell viability . Based oCortical cells were treated with concentrations of neuroEPO to assess the effect of the neuroEPO on the viability of the cells. The results show a slight decrease in viability in the cell culture treated with neuroEPO compared to untreated cells cultures (control). The differences are not significant .To examine the capacity of post-treatment with neuroEPO to protect against glutamate-induced neurotoxicity, primary cortical cells, were exposed to glutamate (100 µM) for 15 min, followed by treatment with different concentrations of neuroEPO (10–100 ng/mL) for 24 h. The results indicate that glutamate exposure induces a significant decrease in cell viability, 40% lower compared to the untreated control cells. This neurotoxic effect caused by the glutamate is significantly reduced by the subsequent treatment with neuroEPO which maintains the cell viability (around 80%) compared to non-treated neurons (control). The protective effect of neuroEPO seems to be similar for the different concentrations of neuroEPO studied here . The microscopic observation using phase-contrast microscopy show that, after nine days of incubation, the culture is characterized by many large cells with thick and abundant cellular processes that are connected to neighbouring cells A. Exposup > 0.001) in antioxidant activity compared with the oxidant activity observed in untreated control cells and in cells exposed to glutamate and that the antioxidant activity increases as a function of a greater amount of neuroEpo in the medium  for 15 min, followed by treatment with concentrations of neuroEPO (50 ng/mL and 100 ng/mL) for 24 h. Oxidative stress was analysed by measuring the antioxidant and oxidant activity in cortical cells that were untreated, treated only with glutamate, or treated with glutamate and neuroEPO. The results show that there are no significant differences in antioxidant activity between cells treated with glutamate and untreated control cells while, in neurons exposed to glutamate, the treatment with neuroEPO induces a significant increase  neuroEPO can substantially attenuate neuronal death that has been induced by the action of glutamate; and (ii) neuroEPO exposure attenuates oxidative stress induced by glutamate. Thus, neuroEPO favours antioxidant activity and decreases pro-oxidant activity, counteracting oxidative stress triggered by the excessive presence of extracellular glutamate. Our results demonstrate that neuroEPO has the ability to induce antioxidant activity in neurons damaged by excitotoxicity.The neurotoxic effect of glutamate on the viability of primary cultured neurons has been extensively studied ,26,27. IIn stroke, the lack of oxygen and glucose causes an excessive release of glutamate leading to excitotoxicity. There is abundant evidence that the presence of a state of oxidative stress induced by excitotoxicity plays a pivotal role in cell death in the brain after stroke ,31. OxidTreatment with neuroEPO, when applied immediately after a glutamate insult, as in the present study, protects cultured neurons from oxidative stress induced by excitotoxicity. neuroEPO increases cellular antioxidant defences and reduces the oxidant activity in neurons exposed to glutamate. This protective effect seems to be associated with the capacity of neuroEPO to increase antioxidant activity, which allows the neuron to keep its redox balance. Together with averting the loss of the antioxidant capacity of neurons, neuroEPO inhibits the increase of the oxidative capacity induced by glutamate in neurons. Thus, oxidative stress is inhibited by neuroEPO. It has been reported in rat primary cortical neurons that, under conditions of excitotoxicity, EPO reduces the concentration of free intracellular calcium . ExcessiIn the present study, we show that glutamate can induce morphological changes, oxidative stress, and neuronal death; and that these changes are attenuated by treatment with neuroEPO. Previous studies demonstrated that the neuroprotective effects of EPO require the addition of EPO to the culture several hours prior to glutamate exposure, which suggested that both mRNA and protein synthesis are important . HoweverIt is well known that glycosylation plays a critical role in the biological and pharmacological function of many proteins, affecting their solubility, cellular processing, secretion, and metabolism . NeuroEPAlthough results obtained in cell cultures must be extrapolated to animal models with caution, our new findings we report here, coupled with those obtained in animal experiments ,21, supp


The renal microvasculature is targeted during aging, sometimes producing chronic kidney disease (CKD). Overdiagnosis of CKD in older persons is concerning. To prevent it, a new concept of “healthy aging” is arising from a healthy renal donor study. We investigated the renal microcirculatory changes of three older persons and compared them with that of one patient with nephrosclerosis using a three-dimensional (3D) reconstruction technique that we previously developed. This method uses a virtual slide system and paraffin-embedded serial sections of surgical material that was double-immunostained by anti-CD34 and anti-α smooth muscle actin (SMA) antibodies for detecting endothelial cells and medial smooth muscle cells, respectively. In all cases, the 3D images proved that arteriosclerotic changes in large proximal interlobular arteries did not directly induce distal arterial change or glomerulosclerosis. The nephrosclerotic patient showed severe hyalinosis with luminal narrowing of small arteries directly inducing glomerulosclerosis. We also visualized an atubular glomerulus and intraglomerular dilatation of an afferent arteriole during healthy aging on the 3D image and showed that microcirculatory changes were responsible for them. Thus, we successfully visualized healthy aged kidneys on 3D images and confirmed the underlying pathology. This method has the ability to investigate renal microcirculatory damage during healthy aging. The microcirculation of the kidney is essential for controlling glomerular filtration and tubular reabsorption and for regulating the medullary concentration gradient . DysfuncAging is an inevitable biological process that results in structural and functional changes. The kidney is one of the most susceptible organs to aging. One-half of adults over the age of 70 years have a low estimated glomerular filtration rate and are diagnosed as having CKD . A biopsIn this study, however, many older donors were systematically evaluated as “healthy” or at least “healthy for age” before biopsy. It is concerning that too many older patients could be unnecessarily labeled as “diseased” and treated without any proven clinical benefit . ElderlyIn a previous article , we propThe clinical features and pathological characteristics of four patients  are shown in In the original 2D images, glomeruli were seen as tufts of capillaries positive for CD34 (brown) A,B. ArteIn case 1, the interlobular arteries showed no distortion and a regular distribution of green smooth muscle cells as seen by inspection C–F. The Compared with case 1, the interlobular artery of case 2 showed irregular distribution of the smooth muscle cells and was mildly distorted in the 3D image as seen by inspection A–E.The diameter of the proximal interlobular artery (102 μm) was larger than that of case 1 (92 μm), and the distal interlobular artery was much smaller than the proximal one. In the corresponding 2D image G, the inp < 0.05)  and medial atrophy but with preserved lumens in the 2D image arrow, A,G. The  < 0.05) . The cor < 0.05) B–F showe < 0.05) G.We examined 3D images of the renal microvasculature, especially glomeruli and their connecting vessels, in older persons with preserved renal function or mild renal dysfunction. We used the images to investigate the morphological changes seen during healthy aging.The renal morphological changes in kidneys due to aging have been investigated using both 2D ,14,15,16The abovementioned changes were found in each of our four cases in different degrees and morphology. Three had normal or nearly normal renal function for their age and had no complications. Compared with case 1, cases 2 and 3 showed more prominent sclerotic changes in the proximal interlobular arteries and increased sclerotic glomeruli and interstitial damage. Three-dimensional imaging, however, proved that the glomeruli and distal arteries and arterioles supplied by the damaged proximal interlobular artery had no severe morphological changes, although the increased number of sclerotic glomeruli had increased in the distal ends. Case 4, with severe intimal changes in the distal interlobular artery and many sclerotic glomeruli, exhibited only mildly impaired renal function. In the 3D images, the distal arteries and arterioles and glomeruli showed marked pathological changes. Our results revealed that the morphological changes in the large proximal interlobular artery or arcuate arteries did not directly induce severe renal damage, whereas small arterial changes were more critical to the development of glomerulosclerosis and interstitial damage. Nimomiya et al.  presenteWe also visualized glomerular modifications in a case of healthy aging, in which an atubular glomerulus and cystic dilatation of the afferent arteriole were apparent. The glomerulus showed long, atrophic, proximal tubules—part of the definition of an atubular glomerulus . Proxima−/− rat kidney [Recent technical advances have enabled us to reconstruct high-resolution 3D images of the renal microvasculature, which have proved useful for investigating renal damage caused by an altered microvasculature ,23,24,25t kidney . Confocat kidney  and the t kidney ,23 in a All 3D imaging of the renal microvasculature requires special tools. In addition, the techniques are rarely applied to humans and do not provide the underlying histology. Our technique is superior to those methods because it uses conventional paraffin-embedded sections and enables detection of the underlying histology. The disadvantages of our method are that it requires an expensive virtual slide system and human labor for reconstruction. In addition, the reconstructions are of limited size. Clearer figures of 3D reconstructions of the renal microcirculation may be obtained by X-ray micro-computed tomography , or scanConventional surgical, formalin-fixed, paraffin-embedded tissue blocks from non-neoplastic renal parenchyma of four Japanese patients diagnosed with renal cell carcinoma were used in this study . The tumConventional stains—e.g., hematoxylin-eosin, periodic acid Schiff (PAS), Masson trichrome, elastic-van Gieson—were used for morphological analysis and determination of suitable areas for tissue preparation. Details of tissue preparation are described elsewhere ,9,10,11.Double immunohistochemical staining with mouse anti-CD34  and anti-SMA (Dako) was performed to identify endothelial cells and smooth muscle cells of the arterial media A. The PAImage registration, segmentation of tissue components, and geometric reconstruction were performed using two software packages, Image-J  and TRI/3D-SRF  as described previously ,10. Hist®; Mitani Corp., Tokyo, Japan)  . Using st-tests and one-way ANOVA, respectively. A value of p < 0.05 was considered to indicate statistical significance.Results are expressed as means ± standard error of the mean (SEM). Comparisons between two and among cases were performed using paired Student’s In conclusion, our 3D imaging of the renal microvasculature has the ability to prove a direct relation not only between the arteries and glomeruli but also between arteries and arterioles and between glomeruli and tubular structures. We successfully visualized 3D images of healthy aging microvascular changes. Our 3D imaging was of great help for clarifying the mechanism of microvascular renal injury in older persons and others with chronic renal disease.


Gestational diabetes mellitus (GDM) and vitamin D deficiency have been associated with increased risk of adverse perinatal outcomes but the consequences of both conditions simultaneously present in pregnancy have not yet been evaluated. Our objective was to study the influence of vitamin D deficiency in neonatal outcomes of pregnancies with GDM.184 pregnant women with GDM referred to specialized prenatal monitoring were included in this cohort and had blood sampled for 25-hydroxyvitamin D measurement. Vitamin D was measured by chemiluminescence and deficiency was defined as < 20 ng/mL. Participants were followed until puerperium and adverse neonatal outcomes were evaluated.Newborns of women with vitamin D deficiency had higher incidences of hospitalization in intensive care units (ICU) , of hypoglycemia , and were more frequently small for gestational age (SGA) . After adjustment, relative risk (RR) for hypoglycemia requiring ICU was 3.63 (95%CI 1.09–12.11) and for SGA was 4.32 (95%CI 1.75–10.66). The incidence of prematurity, jaundice and shoulder dystocia was no statistically different between groups.In this cohort of pregnant women with GDM, vitamin D deficiency was associated with a major increase in the incidence of adverse neonatal outcomes such as SGA newborns and neonatal hypoglycemia. Several physiologic adaptations of the calcium and vitamin D metabolism are necessary during pregnancy in order to provide sufficient nutrients to the fetus and the neonate. FetusesStudies have demonstrated that lower maternal levels of 25(OH)D result in reduction of birth weight and that vitamin D deficiency is associated with a higher risk of neonates small for gestational age (SGA)–11. ReduGestational diabetes (GDM) is another common condition associated with adverse perinatal outcomes, such as macrosomia, neonatal hypoglycemia and shoulder dystocia. MoreoveTherefore, our objective was to investigate the neonatal complications of vitamin D deficiency in the presence of GDM.The study protocol and the informed consent form were approved by the Ethics Committee of Hospital de Clínicas de Porto Alegre. Participants received information about the study in order to enable the voluntary decision to participate or not as research subjects; all of the pregnant adult women included in the study provided written informed consent; we did not included minors.th week of gestation. This cohort was prospectively followed until the puerperium period and the newborns of participants were evaluated.The multidisciplinary team specialized in the care and research of diabetes during pregnancy at Hospital de Clínicas de Porto Alegre assists pregnant women with hyperglycemia referred to high risk prenatal monitoring. Between November 2009 and May 2012, we invited all women with a diagnosis of GDM to participate in the study. They answered a questionnaire requesting information on socio-demographic variables, medical history and pregnancy. Physical examination was performed at all prenatal visits. Blood was sampled after 8 hours fasting in the third trimester of pregnancy since the diagnosis of GDM is usually made after the 24nd Meeting of The Diabetes and Pregnancy Task Force[The diagnosis of GDM at our institution follows the recommendations of the 2ask Force: a 75g oask Force, we also2 (kg/m2). Routine prenatal laboratory tests and HbA1c were evaluated. Data about delivery route, medical emergencies, neonatal intensive care unit (ICU) hospitalization, neonatal weight immediately after birth, sex and health status were extracted from medical records.Several clinical variables were evaluated at the maternal interview: maternal age, self-reported skin color (white or dark skin tone), socioeconomic status  , neonatGestational age was based on an ultrasound early in pregnancy. Adequacy of birth weight for gestational age was based on the traditional curve of Alexander et al, but we ®, MN, USA). This assay measures between 4.0 and 150 ng/mL, and inter-assay precision had a coefficient of variation of < 4% and < 6% for 15 and 50 ng/mL values, respectively. Vitamin D deficiency was defined as 25(OH)D below 20 ng/mL, in agreement with current recommendations of the Institute of Medicine[Glucose was measured by the colorimetric method  and HbA1c by high performance liquid chromatography . Serum 25(OH)D was measured by chemiluminescent immunoassay D measurement was prepared by centrifugation and was stored at -80°C until analysis. After delivery, 25(OH)D was measured and women were classified according to the presence of vitamin D deficiency (risk group) or not (control). Blood was sampled at all seasons. Season was categorized by winter (June 21 –September 22), spring (September 23 –December 20), summer (December 21 –March 20), and autumn (March 21 –June 20). Porto Alegre city is located at latitude 30°S.Descriptive statistics were used to present the data about clinical and demographic variables of mother and neonate in each vitamin D category (below or above 20 ng/mL). Number, percentage, mean ± standard deviation, median and interquartile range were used.Comparison between risk and control groups was performed. Categorical variables were analyzed using the chi-square test, and continuous variables were evaluated with the t-test when parametric and with the Mann-Whitney test when non-parametric. A general mixed model (Poisson regression with robust standard errors) was used to study the association of SGA, neonatal hypoglycemia, and ICU hospitalization with serum vitamin D status. Adjustment models took into consideration potential biological confounders such as maternal glucose at OGTT, HbA1c, maternal BMI, skin pigmentation, birth weight, gestational age, smoking, nulliparity, hypertensive disorders of pregnancy , medication requirement for diabetes treatment, maternal age, low socioeconomic status, years of study and season. Confounding factors were considered not to influence the main result and were removed from the model if their inclusion did not change relative risk (RR) by at least 5%. Correlations were evaluated with Pearson´s or Spearman´s test for parametric and non-parametric variables, respectively. Analyses were also performed between ethnic subgroups.The required number of newborns to be included in the cohort was 158, based on a 7% prevalence of SGA and on aThe reporting of this study is in accordance with the Strobe Statement.Two hundred and thirty women with GDM were referred to high risk prenatal monitoring and 184 of them were included in this cohort . Their bThe incidence of SGA newborns was higher in the group of vitamin D deficient women . The anaPrematurity rates were not statistically different between the group with or without vitamin D deficiency  and seruAnalysis of data considering maternal skin pigmentation differences, showed an incidence of SGA of 9.6% in white and 18.8% in dark-skinned toned women (P = 0.095). In white women, the vitamin D deficient group had a higher incidence of SGA . The RR for SGA in white women with 25(OH)D < 20 ng/mL was 6.04 (CI95% 1.90–19.23) after adjustment for maternal glucose at OGTT, maternal body mass index, smoking and season. However, in dark-skinned women, there was no difference in the rates of SGA according to vitamin D status (19.4% in the deficient group vs 17.6% in the group with 25(OH)D ≥ 20 ng/mL, P = 0.885).th week of pregnancy had neonates with lower birth weight than patient with vitamin D levels above 30 ng/mL [Maternal vitamin D deficiency was associated with increased rates of neonatal hypoglycemia requiring ICU hospitalization and of SGA newborns in our cohort of pregnant women with GDM. Many previous studies have reported an increased risk of SGA newborns in general women with deficiency of vitamin D–9,11. Thectively). In agreSince vitamin D deficiency is more prevalent in dark-skinned women, it could be one of the possible mechanisms to explain ethnic differences in neonatal outcomes. However, available data are not conclusive. In our cohort, vitamin D influenced the SGA incidence in white women, but it did not change the risk in non-white women. Previous studies also did not find any relationship between serum 25(OH)D and SGA risk in black women or descrThe biological mechanism of vitamin D influence on fetal growth is still not fully understood. One of the plausible explanations is that maternal vitamin D deficiency may result in abnormal maternal calcium metabolism, and lower calcium levels in cord blood are associated with lower birth length. AnotherIt is also important to highlight that intrauterine growth restriction is a relevant neonatal outcome, since it results in increased fetal and perinatal morbidity and mortality . MoreoveTransient asymptomatic hypoglycemia may be part of the transition to extrauterine environment, but severe or persistent neonatal hypoglycemia could result in ICU hospitalization, higher cost and neurologic damage. This is the first study to show the association of vitamin D deficiency and increased rates of neonatal hypoglycemia. The association is significant for any hypoglycemia but, more importantly, we also show an association of vitamin D deficiency with severe hypoglycemia requiring ICU hospitalization. This is a relevant outcome in an at risk population because they are newborns of women with GDM. Further studies are necessary to reinforce this association.The incidence of prematurity was higher in the pregnancies with vitamin D deficiency and serum vitamin D was lower in the group with preterm newborns, although there was no statistically significant difference in our study. Some other studies did not find a lower gestation age at birth in pregnancies complicated by vitamin D deficiency,33 whileBased on observational study findings, vitamin D supplementation has been recommended during pregnancy. Two trials from the 1980s reported a lower incidence of SGA neonates and a beThis cohort study adds novel data about vitamin D deficiency in a group of women with GDM. This population has a high incidence of vitamin D deficiency and it is associated with increased rates of adverse neonatal outcomes. The strengths of our study are the prospective design and novel data in a previously not studied group of DMG women. Moreover, we used both a standard international chart for birthweight analysis and the recently built national chart. Relative risk for SGA was very similar between the charts, which reinforced our findings. However, this study also has some limitations. First, we have not assessed calcium intake or serum level. It is relevant since calcium levels in cord blood have been reported to be associated with birth length. Second,In conclusion, vitamin D deficiency was associated with an increased incidence of adverse neonatal outcomes, such as neonatal hypoglycemia requiring ICU and SGA newborns, in our cohort of high-risk pregnant women with GDM. We consider it is imperative to confirm our findings in other independent large cohorts. Vitamin D is a modifiable risk factor in pregnancy with important public health implications and we believe that national authorities should implement routine vitamin D measurement during pregnancy.


Phalacrocorax carbo L.) is on the rise. These birds, characterized by rapid metabolism, can deposit large quantities of feces, and because they breed on the land but forage on water, both terrestrial and aquatic environments can be simultaneously affected by their activities. The contribution of great cormorants in the dispersal of bacterial and viral pathogens has been immensely studied; whereas, the occurrence of eukaryotic parasites such as protozoans and microsporidians in these birds is little known. The present study investigated the presence of dispersive stages of potentially zoonotic protozoans belonging to the genera Blastocystis, Giardia and Cryptosporidium, and Microsporidia spores in feces collected from birds inhabiting the breeding colony established at one lake island in Poland, Europe. The feces were examined by coprological techniques , and with immunofluorescence antibody MERIFLUOR Cryptosporidium/Giardia assay. As found, the Cryptosporidium oocysts were identified rarely in 8% of samples  and no cysts of Giardia and Blastocystis were detected. Microsporidian spores were detected in 4% of samples (1/25) but at very high frequency (4.3 × 104/g). No dispersive stages of parasites were identified in water samples collected from the littoral area near the colony. Despite the profuse defecation of cormorants, their role in the dispersion of the investigated parasites may not be as high as hypothesized.The global population of great cormorants ( Phalacrocorax carbo L.) is on the systematic rise, particularly in some parts of Europe where the number of breeding pairs is estimated to exceed 400,000 . To the best of our knowledge, this is the first study not only to survey such number of these birds in this regard but also to highlight that the role of great cormorants in dispersion of human intestinal protozoan and microsporidian parasites may not be as significant as expected.The present study aimed to investigate the presence of o 36′ 57″ N and 16o13′ 23″ E, respectively. An island has an area of 0.9 ha and slight elevation, up to 40 cm above the lake level. The counting of birds was performed by two independent observers during dawn-dusk prior to collection of fecal samples. During the investigated period (June 2013), 170 breeding pairs  were recorded. The foraging area of cormorants during this period is in the radius of 50 km from the colony, but outside the breeding season, adult birds and younglings may spread across the Europe  which is in the state of moderate eutrophy (Klimaszyk Samples of cormorant feces were collected from the colony area in June 2013 using 10 trays (60 × 60 cm) located directly beneath bird nests at various points within the island Fig. .i) modified Weber’s chromotrope 2R–based trichrome stain for Microsporidia spores  Ziehl-Neelsen stain for Cryptosporidium oocysts, or (iii) iron hematoxylin stain for cysts of Giardia and Blastocystis . The remaining three smears were stained with either:  test kit, MERIFLUOR Cryptosporidium/Giardia , was used according to the manufacturer’s instructions.Additionally, to confirm identification of Cryptosporidium oocysts), modified Weber’s chromotrope 2R-based trichrome stain (Microsporidia spores) and iron hematoxylin (Giardia and Blastocystis cysts) methods, and immunofluorescent assay (IFA).All water samples were examined using modified U.S. Environmental Protection Agency Method 1623  of pooled fecal samples of great cormorant  and width (±SD) of identified oocysts was 5.0 (±0.0) and 5.4 (±0.5) μm, respectively. Spores of Microsporidia  and width (±SD) of these spores was 1.8 (±0.4) and 1.2 (±0.2) μm, respectively. None of the investigated pooled fecal samples contained detectable cysts of Giardia and Blastocystis.ant Fig. . All samdia Fig.  were ideCryptosporidium, Blastocystis and Giardia.None of investigated water sample was identified to contain microsporidian spores and dispersive stages of The birds represent an important factor harboring and dispersing the microorganisms, including pathogens  were also detected in feces collected from ten P. carbo individuals in the Netherlands inhabiting areas nearby man-made human reservoirs. The prevalence amounted to 20% and mean concentration in positive samples was estimated at 64 oocysts per gram—high enough to significantly contribute to the contamination of water reservoir  was found in the Slovakian pilot study examining 40 samples of great cormorant feces at a relatively high prevalence of 42.5% ; no dispersive stages of Microsporidia were identified in lake water near the colony. It should be highlighted that however the most widely used staining method to detect spores (chromotrope 2R modified trichome) was employed in the present study, it does not allow to distinguish particular species or genotypes of microsporidia. This is important if one considers that only some species (at least 15 from over 1200 identified so far) are known to be pathogenic for humans (Ramanan and Pritt The infection of the gastrointestinal tract by a Didier . As showThe present study was limited only to one great cormorant colony situated at the lake of low human pressure; therefore, the results should be treated cautiously upon any extrapolation. Sewage discharge can lead to increased contamination of water with dispersive stages of parasites and their presence in biota including waterfowls (Słodkowicz-Kowalska et al. Blastocystis, Cryptosporidium and Giardia, and Microsporidia spores in feces of great cormorant. It was hypothesized that due to specific behavior and metabolism, these birds may represent an important vector for these parasites. Contrary to this, the prevalence of Cryptosporidium oocysts and microsporidian spores was very low, and no cysts of Giardia and Blastocystis were identified. The study indicates that this species may not play, at least at certain locations, a profound role in the dissemination of investigated parasites. Further research employing immunological and molecular methods is necessary to elucidate exact species of Microsporidia, and evaluate whether cormorants may disseminate those associated with human infection.The present study investigated the presence of dispersive stages of potentially zoonotic protozoans belonging to the genera


KRAS, BRAF, NRAS and PIK3CA mutations in a large collection of CRC patients from genetically-homogeneous Sardinian population.Activation of oncogenes downstream the EGFR gene contributes to colorectal tumorigenesis and determines the sensitivity to anti-EGFR treatments. The aim of this study was to evaluate the prognostic value of RAS and BRAF genes, using pyrosequencing assays, and in PIK3CA gene, using automated DNA sequencing assays.A total of 1284 Sardinian patients with histologically-proven diagnosis of colorectal carcinoma (CRC) and presenting with metastatic disease were included into the study. Genomic DNA was isolated from formalin-fixed, paraffin-embedded primary tumour tissue samples of CRC patients and screened for mutations in KRAS, 4.1 % for NRAS, and 2.1 % for BRAF. Among available DNA samples, 114/796 (14.3 %) primary CRCs were found to carry a mutation in the PIK3CA gene. In this subset of patients analysed in all four genes, a pathogenetic mutation of at least one gene was discovered in about half  of CRC cases. A mutated BRAF gene was found to steadily act as a negative prognostic factor for either time to progression as metastatic disease  or partial survival  or overall survival (p < 0.001). No significant impact on prognosis was observed for mutated KRAS, NRAS, and PIK3CA genes or combined RAS mutations .Overall, mutation rates were 35.6 % for Our study defines both prevalence and prognostic role of main activated oncogenes in a population-based large collection of CRC patients.The online version of this article (doi:10.1186/s12967-016-1053-z) contains supplementary material, which is available to authorized users. Colorectal cancer (CRC) is the third most incident malignancy in both sexes, after lung and prostate cancer in men and breast and cervix cancer in women; it also represents the fourth most frequent neoplastic cause of death after lung, stomach, and liver cancer . The disFrom the pathogenetic point of view, CRC is a complex and heterogeneous neoplastic disease, exhibiting multiple genetic and epigenetic alterations. The accumulation of acquired molecular aberrations is involved in colorectal tumorigenesis, able to transform normal colonic epithelieum into adenocarcinomas. Starting from the model proposed by Fearon and Vogelstein in 1990, not only tubular and tubulovillous adenoma may progress to invasive adenocarcinoma, but it is now recognized that the serrated adenomas (SSA) and traditional serrated adenomas (TSA), originally excluded, may also undergo malignant transformation , 4.APC gene and/or the loss of chromosome 5q where it is located, mutations in the KRAS oncogene, the loss of chromosomal arm 18q, and deletion of chromosome 17p where is mapped the TP53 tumour suppressor gene  or overall survival    or PIK3CA mutated status were instead observed in our series  , the occurrence of BRAF mutation presented strongly significant poorer partial [p < 0.001] and overall [p < 0.001] survivals in comparison with those carrying a BRAF wild-type gene   Fig. .Fig. 3KaNo correlation between occurrence of gene mutations and response to therapies (EGFR inhibitors or others) has been inferred into the present study .Colorectal cancer is one of the most prevalent malignancies worldwide , 2, 28. KRAS mutations have been demonstrated to reduce the effectiveness of the anti-neoplastic agents mentioned, and they are currently a validated predictive marker of negative pharmacological response to anti-EGFR therapies. Mutations on codons 12 and 13 in exon 2 have been initially established as biomarkers of resistance to anti-EGFR antibodies; soon after, mutations on codons 61 and 146, as well as mutations on exons 3 and 4, have been discovered to produce the same effect [NRAS oncogene as well as in the exon 20 of the PIK3CA gene, which are rarer in CRC patients, have also been demonstrated to be predictors of reduced response to anti-EGFR therapies [e effect –38. As we effect –18. Mutaherapies –38.KRAS, NRAS, BRAF, and PIK3CA mutations were observed in 47.5 % of the CRC cases examined, mostly confirming the mutation prevalence observed in our previous study  [KRAS exons 2-4, NRAS exons 2 and 3, BRAF exon 15, and PIK3CA exons 9 and 20 [KRAS, NRAS, BRAF, and PIK3CA, respectively (about 12 % of cases carried both KRAS and PIK3CA mutations) [KRAS (35.6 %) and BRAF (2.1 %), slightly lower for PIK3CA (14.3 %) and slightly higher for NRAS (4.1 %). Focusing on KRAS mutations only, Sardinian CRC patients were found to carry a mutated KRAS gene in 30 to 36 % of the cases [In our series, C cases) . In ItalC cases) . Using t9 and 20 . Converstations) . In the he cases .RAS genes [The distribution of the mutations within the territory of the island was confirmed to be heterogeneous, with the northern populations presenting higher rates of mutations, especially in the AS genes . This paAS genes , 41–43. AS genes , 44.BRAF mutations have been more frequently observed in right-sided, poorly differentiated mucinous tumors [In our study, we did not evidence any relation between the mutation rates and the clinical and/or pathological parameters examined . Most of the lesions were located in the left colon, but most of the mutations involved the rectum, even if the differences were not statistically significant. Some authors advocate that the anatomical location, histology and grading of the primary lesions has a relevant prognostic role, especially because s tumors , 45–47.BRAF have been widely demonstrated, indeed, to negatively impact the prognosis of patients with CRC, independently of any clinical parameter studied [RAS and BRAF genes—presented a worst OS. This finding is in line with those of other authors who found that the all-wild type malignancies  had globally a better prognosis in comparison to the mutated ones [PIK3CA is less clear; it is known that it often coexists with KRAS mutations and predicts resistance to anti-EGFR therapies, but its exact prognostic role is poorly understood [PIK3CA is associated with poor prognosis among patients with curatively resected colon cancer, but such an association was not confirmed neither in our series, nor in other studies in early or advanced stage patients [Somatic mutations in the exon 15 of  studied , 45–47.  studied , 33. Furted ones , 39. Thipatients , 49.KRAS mutations as a global prognostic factor of disease progression and survival in CRC patients is controversial. Several studies have reported a statistically significant reduction in disease-free survival (DFS) and OS in the presence of KRAS mutations [KRAS and BRAF mutations on the prognosis of 649 patients with IV stage CRC [KRAS mutations on prognosis, but the authors provided evidence that CIMP high appears to be an independent predictor of a low CRC specific mortality, while BRAF mutations are associated with a high CRC specific mortality [KRAS gene alterations, BRAFV600E status, and MSI, Zlobec et al. found that specific KRAS mutations act as informative prognostic factors in both sporadic and hereditary CRC [KRAS codon 13 mutations, compared to those with wild-type genes, further suggesting that different types of KRAS mutations may be differently associated with OS in patients with CRC [KRAS codon 13 mutations were associated with poorer OS but not DFS [KRAS-mutated cases [KRAS mutations were associated with worse prognosis, 15 failed to demonstrate any statistically significant association between such mutations and prognosis, and, finally, one data set identified an improved survival rate for patents with CRC and KRAS mutations [KRAS mutations were not associated with CRC prognosis, either before or after adjustment for the effect of publication bias [The prognostic role of utations –52. Ogintage CRC . The stuortality . By conttary CRC . In anotwith CRC . Finally not DFS . Finallyed cases . Among tutations . Globallion bias .KRAS mutations; we hypothesized that such a finding may be associated with the specific types of KRAS mutations observed in males and females as consequence of exposure to different lifestyle factors [NRAS negatively affects partial survival, even if it did not show any impact on OS or TTPM. Global cases with RAS mutations had a prognostic value on PS and TTPM close to be significant, and this further enhances the above-mentioned observation that colorectal malignancies with wild-type status in all these candidate genes generally present a better prognosis.In a previous study on Sardinian patients, we observed an improved TTPM in male patients with CRC harbouring  factors . NeverthRAS and PIK3CA mutations in CRC may be caused by some confounding factors. The most frequent are: (1) heterogeneity of the study populations; (2) discrepancies in study designs and methodologies; (3) strategies for mutational detection; (4) presence of underlying predisposing conditions (i.e. ulcerative colitis); (5) variability of the staging systems ; and (6) differences in clinical management and treatment. The advantage of our study is to avoid several bias through a prospective collection of data on a consistent high number of patients from a homogeneous population, processed and analysed in a single institution. To date, we can consider as validated the prognostic role of BRAF mutations as well as that of the wild-type status in all main candidate genes. Further studies are probably necessary to better assess the prognostic role of the single RAS and PIK3CA mutations.The discrepancy of results among the mentioned studies on the prognostic role of KRAS, NRAS, BRAF, and PIK3CA genes. The KRAS mutations are the most frequent, followed by those in NRAS, and BRAF, as observed in other studies, and they are strongly confirmed to be mutually exclusive. On the other hand, PIK3CA mutations often coexist with the RAS mutations. With the exclusion of mutations in any of the other genes, harbouring a mutated BRAF was demonstrated to negatively impact the prognosis—regardless the type of survival taken into consideration—in CRC patients from Sardinian population. This makes the role of the latter mutations clear; as for the others, further investigations are necessary to better comprehend their prognostic impact.Our findings evidence that approximately a half of the Sardinian patients with CRC present one or more mutations in one or more of the


A nomogram based on both western and eastern populations to estimate the Disease Specific Survival (DSS) of resectable gastric cancer (RGC) has not been established. In current study, we retrospectively analyzed 4,379 RGC patients who underwent curative resection from the Surveillance, Epidemiology, and End Results (SEER) database. Patients diagnosed between 1998 and 2009 were assigned as training set , and the rest were selected as SEER validation set . An external validation was performed by a set of independent 1,358 RGC patients after D2 resection from Sun Yat–sen University Cancer Center (SYSUCC) in China. The nomogram was constructed based on the training set. The multivariate analysis identified that patient's age at diagnosis, race, tumor location, grade, depth of invasion, metastatic lymph node stage (mLNS) and total number of examined lymph node (TLN) were associated with patient's DSS. The discrimination of this nomogram was superior to that of the 7th edition of AJCC staging system in SEER validation set and SYSUCC validation set . Calibration plots of the nomogram showed that the probability of DSS corresponded to actual observation closely. In conclusion, our nomogram resulted in more–reliable prognostic prediction for RGC patients in general population. Although the incidence has declined recently, gastric cancer is still the second leading cause of cancer related–death and the 5–year survival was less than 30% . RadicalCurrently, the American Joint Committee on Cancer (AJCC) classifies gastric cancer into nine groups in the 7th edition staging system . This syNomogram, a simple statistical predictive tool, has been constructed in gastric cancer previously and proved to be useful and effective –17. By cIn the present study, we aim to develop and validate a nomogram for RGC based on a multi–institution and multi–population data from SEER database which contains both western and eastern patients with RGC. Additionally, we used a separate cohort from Asia for external validation.4,379 gastric cancer patients from the SEER database between January, 2004 and December, 2012 were eligible for the present analysis . The clinical pathological characteristics of validation sets were listed in Table Next, considering that the longest follow–up of SEER validation set was 35 months, the 5–year calibration cannot be executed in SEER validation set. Therefore, the calibration plots were separately performed by the primary cohort and SYSUCC validation set. As shown in Figure In this study, we developed and validated a novel nomogram of RGC patients underwent curative resection to predict DSS based on general population. A total of 4,379 gastric patients from SEER database and 1,385 RGC patients from Asia were analyzed. Our nomogram showed better predictive accuracy than the 7th edition of AJCC staging system in DSS prediction for the RGC patients .Several nomograms have been constructed in RGC patients, and show more accurate survival prediction than the conventional staging system in different populations. In 2003, Kattan et al developed a nomogram to predict 5–year DSS for gastric cancer patients based on 1,136 patients from Memorial Sloan–Kettering Cancer Center (MSKCC), and Han et al developed and validated a nomogram in a cohort of 10,454 gastric cancer patients who underwent curative resection form Seoul National University Hospital  and Cancer Institute Ariake Hospital  in 2012 , 20. BotCompared with previous MSKCC nomogram and SNUH nomogram, our nomogram was developed and validated based both on western and eastern population. In this study, the race of patients was categorized as API and nonAPI. Interestingly, on the multivariate analysis, we found the nonAPI patients had a worse prognosis than API patients in the SEER data , which was consistent with previous studies , 7, 9. IImproving the accuracy of the survival estimation is exceedingly important for clinical decision. There are several advantages by using nomogram. Firstly, the accurate prediction would be favor for designing postoperative treatment. For example, in 2010, a phase III trial confirmed that adjuvant chemotherapy with S1  was an effective treatment for advanced gastric cancer patients who underwent D2 gastrectomy . HoweverThere are some limitations should be acknowledged. Firstly, only the patients who had complete information were included in present study, there may be a selection bias. Secondly, as this nomogram was based on SEER database, analysis was limited to the prognosis factors in the database. Several predictors such as Lauren classification, genetic differences, protein expression differences and postoperative treatments had not been included –25.In summary, we first develop and validate a prognostic nomogram based on a multi–institution and multi–population database predicting short–term and long–term DSS for RGC patients. Compared with the 7th edition of AJCC staging system, the proposed nomogram represents better prognostic discrimination and predictive accuracy for DSS. It can be used to calculate individualized survival prediction and provide better treatment allocation after curative resection.The SEER program is a national collaboration program by the National Cancer Institute. It collects and publishes approximately 3 million cases from a variety of geographic regions and covers 26% American population's cancer incidence and survival data. A retrospective review of all gastric cancer patients underwent gastrectomy from SEER database between 1998 and 2012 was performed. A total of 31,988 cases from SEER 18 registries were initially screened. Patients were excluded if they had incomplete information on depth of invasion, tumor size, positive lymph node (PLN), TLN or status of distant metastasis. Given that the 7th edition of AJCC staging system bases mLNS definition on the absolute PLN and suggests that “at least 16 regional lymph node be assessed pathologically”, the patients  with TLN less than 16 were excluded from present study [The proposed nomogram was also externally validated by SYSUCC validation set. Of the 2,205 RGC patients who underwent D2 resection in SYSUCC between 2000 and 2011, 1,385 patients met the following inclusion criteria: no history of receiving anti–cancer therapy before surgery; no history of other malignancies; no distant metastasis; complete resection of cancer (R0 resection) with D2 lymphadenectomy; number of examined lymph more than 15; without one or more missing characteristics. The median follow-up was 36.8 months in training set, 14.0 months in SEER validation set and 36.7 months in SYSUCC validation set.The data of patients' clinicopathological characteristics such as age at diagnosis, sex, race, surgery, tumor location, size, histology, grade, depth of invasion, PLN and TLN were collected. The pathological tumor stage, depth of invasion and mLNS were restaged according to the 7th edition of AJCC staging system . The priBased on clinical findings, categorical variables were grouped before modeling. Restricted cubic splines were used to evaluate the linear relationship between continuous variables and DSS . Continuhttp://www.r-project.org/).Nomogram was established based on the training set data. Based on the results of Cox PH regression, a nomogram combining all the independent prognostic factors was constructed for 1–year, 3–year and 5–year DSS predicting by using the package of rms in R software version 3.1.3  curve.http://www.r-project.org/) and the software statistical package for social sciences version 19.0 .p<0.05 will be considered as statistically significant. All statistics analysis were performed by the R software version 3.13 (