The effect of the macronutrient composition of the usual diet on long term weight maintenance remains controversial.373,803 subjects aged 25–70 years were recruited in 10 European countries (1992–2000) in the PANACEA project of the EPIC cohort. Diet was assessed at baseline using country-specific validated questionnaires and weight and height were measured at baseline and self-reported at follow-up in most centers. The association between weight change after 5 years of follow-up and the iso-energetic replacement of 5% of energy from one macronutrient by 5% of energy from another macronutrient was assessed using multivariate linear mixed-models. The risk of becoming overweight or obese after 5 years was investigated using multivariate Poisson regressions stratified according to initial Body Mass Index.A higher proportion of energy from fat at the expense of carbohydrates was not significantly associated with weight change after 5 years. However, a higher proportion of energy from protein at the expense of fat was positively associated with weight gain. A higher proportion of energy from protein at the expense of carbohydrates was also positively associated with weight gain, especially when carbohydrates were rich in fibre. The association between percentage of energy from protein and weight change was slightly stronger in overweight participants, former smokers, participants ≥60 years old, participants underreporting their energy intake and participants with a prudent dietary pattern. Compared to diets with no more than 14% of energy from protein, diets with more than 22% of energy from protein were associated with a 23–24% higher risk of becoming overweight or obese in normal weight and overweight subjects at baseline.Our results show that participants consuming an amount of protein above the protein intake recommended by the American Diabetes Association may experience a higher risk of becoming overweight or obese during adult life. Obesity is a growing epidemic now affecting developing and industrialized countries alike A positive balance between energy intake and energy expenditure is necessary to gain weight. In both the United States Therefore, the consequences of the macronutrient composition of the usual diet in free living subjects still remain controversial, especially as results from the few long term prospective studies The European Prospective Investigation into Cancer and Nutrition – Physical Activity, Nutrition, Alcohol, Cessation of Smoking, Eating Out of Home and Obesity (EPIC-PANACEA) study recruited half a million participants from 10 different European countries with heterogeneous dietary patterns and obesity prevalence. It constitutes a unique opportunity to explore the relation between diet and obesity. The objective of the present study is to investigate the relationship between the macronutrient composition of the usual diet and weight change after 5 years on average, taking into account lifestyle factors.EPIC is a multi-center, prospective cohort study investigating the role of metabolic, dietary, lifestyle, and environmental factors in the development of cancer and other chronic diseases. Briefly, between 1992 and 2000, 521,448 volunteers aged between 25 and 70 years were recruited in 23 centers from 10 European countries . In France, Norway, Utrecht (The Netherlands) and Naples , only women were included. Individuals were selected from the general population except in the French cohort, based on state-school employees and the Utrecht cohort, based on women who underwent breast cancer screening. Furthermore, a large portion of the centers of Spain and Italy were blood donors. Participant eligibility within each center was based essentially on geographic or administrative boundaries. Approval for this study was obtained from the ethical review boards of the International Agency for Research on Cancer and from all local institutions. Details of the recruitment, study design and data collection have been previously published For the present study, we excluded 23,713 individuals with missing dietary and non-dietary questionnaires, missing data on weight and height at baseline, extreme or implausible anthropometric values, pregnant women, and those in the top and bottom 1% of the ratio between energy intake to estimated energy requirement. We further excluded 121,866 subjects with missing weight at follow-up and 2,066 subjects with extreme or implausible weight changes. Thus, 373,803 subjects  were included in the present analysis (more details have been previously published i.e. (weight at follow-up – weight at baseline) ×5/years of follow-up). Recent findings from the EPIC-Potsdam study based on 5 measurements of weight suggest that weight gain can be reasonably well approximated by a straight line over a follow-up period of 8 years on the population-level Two weight measures were available for each participant: one measure at baseline (between 1992 and 2000 depending of the center) and one at the latest follow-up . At baseline, body weight and height were measured in most centers using similar, standardized procedures. The exceptions were France, Norway and the health conscious group of the Oxford center (United Kingdom) where self-reported anthropometric values were collected. At follow-up, self-reported weight were obtained in all centers, except in Norfolk (United Kingdom) and Doetinchem (The Netherlands) where weight was measured. Assessment of self-reported weight was conducted through mailed questionnaires, with several exceptions: Spain and Greece completed the questionnaire on the phone and Varese used a combination of postal survey and telephone interview. The accuracy of all self-reported anthropometric measures was improved with the use of prediction equations derived from subjects with both measured and self-reported measures Usual dietary intake at baseline was measured using country-specific validated questionnaires developed to capture geographical specificity of diet. Most centers adopted a self-administered quantitative dietary questionnaire of 88–266 food items Lifestyle and health factors  were collected by questionnaires at baseline We first examined the distribution of population's main characteristics according to the sex-specific tertiles of the percentage of energy coming from each macronutrient using ANCOVA or chi-square tests as appropriate. Means adjusted for center are presented for continuous variables. The 5y weight gain means were further adjusted for initial BMI.2), follow-up time (years), educational attainment , physical activity index  was investigated using 2-levels  mixed effects linear regression models with each macronutrient on a continuous scale. Sex, age, initial BMI , those likely to misreport energy intake  We explored potential effect modification by age, BMI category at baseline, smoking status, level of education, physical activity and dietary pattern by including interaction terms between each variable and percentage of energy from macronutrient in the models. Dietary patterns were derived from maximum likelihood factor analysis as previously described 2: obese). Subjects morbidly obese at baseline (BMI≥40 kg/m2) were excluded . Relative risks were adjusted for energy from non alcohol sources, energy from alcohol sources, center and confounding factors previously described. The BMI after 5-y was calculated from the 5-y weight change and their baseline height.We studied the association between categories of energy from protein and the risk of becoming overweight, obese, or morbidly obese after 5-y of follow-up. The modified Poisson regression approach of Zou All statistical analyses were performed with SAS 9.2  or STATA 10.0 (College Station TX).Approval for this study was obtained from the ethical review boards of the International Agency for Research on Cancer and from all local institutions where subjects had been recruited for the EPIC study.The average percentages of energy from each macronutrient were 43.8%, 35.4% and 17.0% for carbohydrates, fat and protein respectively. Percentage of energy from carbohydrates was negatively correlated to percentages of energy from fat (r = −0.68) and to a lesser extent to protein (r = −0.27). Percentage of energy from protein was not correlated to percentage of energy from fat (r = −0.04). Characteristics of the population according to sex and percentages of energy from each macronutrient are presented in Adjusted 5-y weight change (g/5y) for the iso-energetic replacement of 5% of energy from one macronutrient by 5% of energy from another macronutrient are presented in We evaluated whether the effect associated with a change in the carbohydrates proportion differed according to the glycemic index of the diet. A significant interaction according to the glycemic index median was observed for the association between weight gain and the substitution of 5% of energy from carbohydrates by 5% of energy from fat in women only . However, associations were not statistically significant in both groups (β (95% CI) = −3 , in women below the median and −41  in women above the median). A significant interaction according to the glycemic index median was also observed in women only for the association between weight gain and the substitution of 5% of energy from carbohydrates by 5% of energy from protein (p for interaction = 0.83 in men and <0.0001 in women). The association was positively significant in both group but stronger in women with low glycemic index (β (95% CI) = 474  vs. 265 ).We also evaluated whether the effect associated with a change in the carbohydrates proportion differed according to the fiber intake of the diet. No interaction was observed for the association between weight gain and the substitution of energy from carbohydrates by energy from fat in men (p for interaction = 0.42). However, a 5% higher proportion of fat at the expense of carbohydrates was negatively associated with weight change in women below (β (95% CI) = −50 ) but not above ) the fiber intake median (p for interaction<0.0001). In addition, a 5% higher proportion of protein at the expense of carbohydrates was associated with a higher weight gain in participants below as well as above the fiber intake median but the association was stronger in participants with high fiber intake (in men: β (95% CI) = 312  vs. 192 , p for interaction = 0.03; in women: 446  vs. 348 , p for interaction = 0.002).The positive association between protein intake and weight change was observed in all age, BMI, smoking status, educational attainment, physical activity, dietary pattern and Goldberg criterion categories although significant interactions were observed according to BMI, smoking status, age, “prudent” dietary pattern and Goldberg criterion categories . The assAdjusted relative risks (95% CI) of the risk of becoming overweight or obese after 5 years according to energy intake from protein and initial BMI are presented in In the present study investigating the relationship between the macronutrient composition of the usual diet and long term weight change in a large European cohort, a 5% higher proportion of protein, at the expense of either fat or carbohydrates, was positively associated with weight gain after 5 years. This association was observed in men and women, in normal weight as well as overweight and obese subjects and in all participating centers. A 5% higher proportion of fat at the expense of carbohydrates was not significantly associated with weight change. Compared to diets with no more than 14% of energy from protein, diets with more than 22% of energy from protein were associated with a 23-24% higher risk of becoming overweight or obese in normal weight and overweight subjects at baseline.Low carbohydrate/high protein diets have been shown to promote weight loss in obese subjects in short term intervention studies Observational studies follow subjects during a longer time than intervention studies and have found inconsistent results so far The mechanism underlying the positive association between protein intake and weight gain is unclear. We previously shown that meat intake was positively associated with weigh gain In our study, a 5% higher proportion of fat at the expense of carbohydrates was not significantly associated with weight change for participants both below and above the glycemic index median. This is in agreement with a previous 1-y controlled trial in type 2 diabetes patients showing no significant weight change difference between three different diets with various glycemic index, carbohydrate and fat amounts The main strengths of the present study are the very large sample size and the high heterogeneity of macronutrients intake and obesity prevalence in the study population. We also partially corrected for measurement error of diet per se. Third, we used BMI as an indicator of adiposity which is less precise than abdominal obesity measurement such as waist circumference or body composition measured by dual energy x-ray absorptiometry. Fourth, measurement error is likely to have attenuated the observed associations. Using urinary nitrogen excretion as a reference biomarker for protein intake, Kipnis et al have shown that even after calibration using the 24-hour dietary recall , the association between protein intake and disease could still be underestimated by up to 240% However, several limits must be mentioned. First, misreporting may have influenced our results. A previous cross-sectional EPIC study showed that reported energy-adjusted protein intake did not differ across BMI categories whereas nitrogen excretion was significantly higher in obese participants compared to normal weight participants In agreement with other European data


Introduction. Pompe disease (PD), a lysosomal storage disease as well as a neuromuscular disorder, is a rare disease marked by progressive muscle weakness. Enzyme replacement therapy (ERT) in recent years allowed longer survival but brought new problems to the treatment of PD with increasing affection of the musculoskeletal system, particularly with a significantly higher prevalence of scoliosis. The present paper deals with the orthopedic problems in patients with PD and is the first to describe surgical treatment of scoliosis in PD patients. Patients and Methods. The orthopedic problems and treatment of eight patients with PD from orthopedic consultation for neuromuscular disorders are retrospectively presented. We analyzed the problems of scoliosis, hip dysplasia, feet deformities, and contractures and presented the orthopedic treatment options. Results. Six of our eight PD patients had scoliosis and two young patients were treated by operative spine stabilization with benefits for posture and sitting ability. Hip joint surgery, operative contracture release, and feet deformity correction were performed with benefits for independent activity. Conclusion. Orthopedic management gains importance due to extended survival and musculoskeletal involvement under ERT. Surgical treatment is indicated in distinct cases. Further investigation is required to evidence the effect of surgical spine stabilization in PD. Pompe disease (PD), glycogen-storage disease type II or acid maltase deficiency, is a very rare autosomal recessive disorder due to dysfunction of the enzyme acid alpha glucosidase. PD is at once a glycogen and lysosomal storage disorder as well as a neuromuscular disorder, marked by progressive muscle weakness , 2. The PD includes a wide range of symptoms and has a variable classification. The disease is commonly divided into infantile-onset (IOPD) and late-onset (LOPD) PD , 5, 6. TThe late-onset form has symptoms developing anytime after the first year of life and it is subdivided into childhood- 2–13 years), juvenile- (13–20 years), and adult- (>20 years) onset forms , 6. LOPD3 years, A milestone in the treatment of PD was reached in 2006, when enzyme replacement therapy (ERT) was accredited and now offers a first primary therapy of the disease mechanism. The success of ERT was recently shown in different publications –10 with The success of the ERT  brought Against this background, we describe eight patients with PD with a wide range of disease onset and severity from the orthopedic point of view. We specify the conservative and operative orthopedic treatment possibilities, especially the management of scoliosis. Although scoliosis is a common complication of PD  and its Eight patients, aged, 3 to 59  years diagnosed with PD by muscle biopsy were treated during special orthopedic consultations for patients with neuromuscular disorders. There were seven males and one female patient. The patients were interviewed and examined formally during our special muscle-disease consultations. We reviewed the patient data retrospectively from our surgeries and from patient records concerning disease onset and diagnosis, ERT therapy, sitting, standing, and walking abilities, musculoskeletal symptoms, respiratory involvement, and the surgical and conservative orthopedic treatment. Longtime follow-up was very restricted as the patients were partly present only for a particular orthopedic intervention in our neuromuscular disease center with follow-up at their local hospital. Limited and fragmental follow-up data was available for 6 of 8 patients and ranged from 14 months to 10 years.This study is in accordance with the ethical standards of the relevant local Ethics Commission .We analyzed eight PD patients, thereof two patients with adult onset, one patient with juvenile onset, two patients with childhood onset, and three patients with muscular infantile onset of PD. The patient collective is composed of three adult patients with milder disease forms (from 36 to 59 years) and five younger patients aged from 3 to 16 with a wide range of severity of symptoms .Two patients were able to walk independently; another two patients were supplied with walking aids (crutches). Powered wheelchairs were provided in 4 cases, whereof one patient was still able to use an active wheelchair for indoor activities. Three patients were dependent on ventilator support. Patient 4 and 5 were provided with intermittent noninvasive ventilation; patient 7 was ventilated via tracheostomy round the clock .Patient 3 presented with a winged scapula  as one of the first symptoms in childhood which revealed noticeable general weakness and led to the diagnosis of PD. The scapula alata remained regressive with continuous physiotherapy during a surveillance time of 10 years.Patient 2 reported weakness in arms and shoulders at the age of about 40 years before developing weakness in the lower extremities. This patient presented with weakness and atrophy of the shoulder muscles, recurrent pain, and limited range of motion in the right shoulder at the age of 50 due to a rupture of the rotator cuff, tendinosis calcarea, and subacromial impingement. Surgery was carried out to reconstruct the right rotator cuff and for subacromial decompression. The patient had no physical complaint in the shoulder during one-year aftercare. ERT was planned to be started shortly after the last documented control examination.Patient 7 reported recurrent subluxations of the left shoulder with occasional discomfort. Imaging and further examination were refused because of poor general condition of the patient.Patient 1 suffered from progressive walking insecurity and pain in his hip and lumbar region at the age of around 45. Severe degenerative changes in the lumbar vertebral column, a hip joint with progressive dysplastic coxarthrosis, and subluxations were found. The constant pain, especially while walking, led to limited mobility and indicated total endoprosthetic (TEP) surgery of the left hip joint. The joint was secured with a cemented endoprosthesis with a snap cup in order to prevent further luxation. During postoperative care, the patient reported a decrease of pain. The remobilization was prolonged because the disease causes weakness of his hip stabilizing muscles; walking was possible with walking aids (crutches) after 21 days of hospitalization. Longer aftercare data is not available.Patient 2 was diagnosed with PD after noticeable progressive leg weakness and difficulties with climbing stairs. The muscle strength remains sufficient with regular physiotherapy during the 14-month follow-up; further orthopedic treatment was not indicated.Three of five patients with childhood PD form had severe hip dysplasia which led to hip joint surgery in two cases . PatientThe patients with adult and juvenile forms in our collective were not affected by contractures of the lower extremities or foot deformities; however, it was a frequent finding in patients with childhood and muscular infantile PD forms. Two patients presented with distinct hip and knee joint contractures (patients 5 and 6) and four patients had feet deformities  .Surgical correction of hip and knee contractures was performed in the two patients suffering from contractures of the hip and knee flexor muscles (patients 5 and 6). The surgical release of hip and knee flexor muscles was performed on both sides outside of our hospital with consecutive physiotherapy. Preoperative data was not available for patient 5, but the postoperatively achieved correction was found to be deteriorated when the patient was presented 10 years later to our consultation; contractures of 30° of both hip joints and 40° of both knee joints were measured at that time. Patient 6 had preoperative hip contractures of 50° left and 40° right and knee contractures of 25° on both sides which were corrected to 0° after surgery. During follow-up, no mobility limitations were found for the first four years, but contractures increased as of the fifth year to 15° left and 30° right of the hip and 40° left and 10° right of the knee. Night braces were not tolerated by the patient. Both patients were wheelchair bound.Deformities of the feet were mostly flexible and treated conservatively with physiotherapy; additional surgical intervention was indicated only in one patient. Patient 8 underwent frontal achillotenotomy on both sides at the age of 5. Lower leg cast night splints were provided after surgery. Aftercare data is not available as the patient fails to appear to the check-up appointments.Patient 7 has been presented with a swollen thigh after sleeping; a spontaneous right supracondylar femur fracture, which was noticed because of autonomous bearing of body weight, independent sitting, standing, or walking, was never achieved. The fracture occurred without any known history of trauma and has been treated conservatively; clinical symptoms were not presented during 6-year aftercare.Six of our eight patients presented with scoliosis varying parameter values, depending on the severity of the disease. The patients with milder course of the disease (adult and juvenile forms) had a lower grade of scoliosis (less than 30°) which did not affect their mobility or respiratory function (patients 1 and 3). Both patients were provided with physiotherapy. Patient 3 had unaltered scoliosis at the 3-year follow-up. No follow-up data is available for patient 1.Four of five patients with earlier PD onset suffered from severe scoliosis of 60°–90° and pelvic tilt ranged from 35° to 60° . PatientPatients 5 and 6 were provided with surgical multisegmental dorsal spine stabilization. We used a Stryker XIA-System steel implant and similar operation technique in both cases. Pedicular screws were utilized from L1 to S1, transversal and laminar hook claw construction on Th3 and Th5, and double Luque wiring from Th6 to Th12. The vertebral joints between Th12 and S1 were opened on both sides. To support the stability after fixation, two additional cross connectors were used.The postoperative period for patient 5 was inconspicuous. The thoracic right-convex part of scoliosis was corrected from 60° to 38° and the lumbar left-convex part from 60° to 23°. Pelvic tilt was improved from 35° to 0° . PatientAs already mentioned above, PD is a very rare disease. Our patient collective is a selection of PD patients with orthopedic problems who applied to our neuromuscular disease consultations. The general muscle weakness in the PD course led to lower extremity disturbances including hip dislocations and subluxations, hip and knee flexor contractures, and feet deformities as well as shoulder-girdle involvement with less frequent and milder occurrence than lower limb disorders. Progressive scoliosis was found frequently with severe development during the disease course. These findings are characteristic for PD and in line with other publications , 13, 15.Our small number of patients does not allow a general statement about the prevalence and therapy of orthopedic disorders in PD. Nevertheless, the present paper is the first to describe therapeutic approaches for the orthopedic treatment of scoliosis in PD patients.Weakness in lower extremity and hip joint involvement is common in the progression of the disease  and usuaIn children, the muscle weakness in PD seems to have a strong negative effect on hip joint development . Two of Two of our patients firstly reported weakness in the upper extremities; a winged scapula led to the diagnosis of PD. The clinical manifestation of shoulder-girdle problems mostly appears later than the hip joint disorders, even though some cases with weakness of shoulder-girdle muscles as a first symptom of PD are described . We assuFour patients suffered from feet deformities which are frequently found in PD including pes equinus, talipes equinovarus, and pes planovalgus . AnotherThe spontaneous fracture of the femur as seen in one of our patients (patient 7) seems to be a known problem of PD. Similar cases of spontaneous fractures without known trauma history or independent sitting or walking have been described . FracturSome publications in recent years have explored the effect of low bone mass density probably caused by PD , 18–20. Severe scoliosis was the most frequent and most prominent finding within our patients. It has already been shown elsewhere that scoliosis is more frequent in wheelchair-bound patients and the progress of the disease is faster after loss of ambulation . It has Two patients underwent surgical spine stabilization and showed positive effects after the intervention concerning the radiographical measure of scoliosis as well as posture and sitting stability. However, the diversity of the forms of PD is very large which makes it difficult to draw a general conclusion. Further investigation with a larger number of patients is necessary to evidence the positive effect of spinal surgery on scoliosis and the general status of PD patients. We can conclude from our experience that spinal surgery can be an option for scoliosis treatment in infantile and late-onset PD patients and should be considered as a therapy of choice in higher degree scoliosis. Furthermore, we observed no specific surgical or anesthetic complications during operations or anesthesia . The rigThe course of PD is changed by ERT, leading to skeletal muscle pathology with additional symptoms  and impaThe present paper has some limitations due to small number of patients, the retrospective data collection, and very irregular and nonstandardized follow-up data available. It remains a descriptive case series for a limited number of orthopedic problems. Since publications for the orthopedic surgical treatment for PD patients are not available at present, further analysis of the therapeutic options is required.Orthopedic management gains importance due to extended survival and musculoskeletal involvement under ERT. Continuous care and follow-up is important in order to identify musculoskeletal problems at an early stage and provide a timely and adequate treatment. In order to improve the quality of life, in some cases, surgical treatment as enhancement of the conservative therapy can be indicated. The present paper offers the first description of surgically treated scoliosis and therapy options for continuative orthopedic problems in PD patients. Further evidence for the surgical spine stabilization for PD patients remains to be shown with larger number of patients.


Pygoscelis adeliae) colonies of the southern Ross Sea are among the longest biologic time series in the Antarctic. We present an assessment of recent annual variation and trends in abundance and growth rates of these colonies, adding to the published record not updated for more than two decades. High angle oblique aerial photographic surveys of colonies were acquired and penguins counted for the breeding seasons 1981–2012. In the last four years the numbers of Adélie penguins in the Ross and Beaufort Island colonies (southern Ross Sea metapopulation) reached their highest levels since aerial counts began in 1981. Results indicated that 855,625 pairs of Adélie penguins established breeding territories in the western Ross Sea, with just over a quarter (28%) of those in the southern portion, constituting a semi-isolated metapopulation . The southern population had a negative per capita growth rate of −0.019 during 1981–2000, followed by a positive per capita growth rate of 0.067 for 2001–2012. Colony growth rates for this metapopulation showed striking synchrony through time, indicating that large-scale factors influenced their annual growth. In contrast to the increased colony sizes in the southern population, the patterns of change among colonies of the northern Ross Sea were difficult to characterize. Trends were similar to southern colonies until the mid-1990s, after which the signal was lost owing to significantly reduced frequency of surveys. Both climate factors and recovery of whale populations likely played roles in the trends among southern colonies until 2000, after which depletion of another trophic competitor, the Antarctic toothfish (Dissostichus mawsoni), may explain the sharp increasing trend evident since then.Measurements of the size of Adélie penguin ( Aptenodytes forsteri) and Adélie (Pygoscelis adeliae) penguins, which breed gregariously in large colonies Diomedea spp.) by the local habitat and resources and by changes in conditions and prey stocks.The underlying factors most likely to limit the abundance of breeding seabirds in a region are prey or nesting space availability Population responses of penguins to changing ecosystems can be complex. Spatio-temporal variation in climatic variables resulting from phenomena such as long-term climate change, or shorter-term decadal atmospheric variation, i.e. factors related to the Southern Oscillation and Antarctic Oscillation , are expressed through the physical environment. Examples include changes in sea-ice conditions such as concentration, extent and thickness, air temperatures, winds, sea surface temperatures (SST) and precipitation  and shags  Balaenoptera bonaerensis) in International Whaling Commission Areas V and VI Climate change, however, by no means is the total story. Significant changes in Adélie penguin numbers could warn of changes in the abundance of their prey and/or structure and function of the marine ecosystem owing to other factors Reliable repeated assessments over the long term are therefore crucial for understanding how these and other factors influence breeding population sizes and dynamics for high latitude penguins, and hence for informing management. The Adélie penguin is one of the species monitored by the Commission for the Conservation of Antarctic Marine Living Resources (CCAMLR) as part of its CCAMLR Ecosystem Monitoring Program (CEMP) to potentially detect anthropogenic effects on Antarctic marine ecosystems In the Ross Sea sector of the Southern Ocean, Adélie penguins breed over a latitudinal range of 1200 km, from the Balleny Islands  north of the Ross Sea to Ross Island  in the south  and therHigh angle oblique aerial photographs of Adélie penguin adults occupying nesting territories at colonies along the Victoria Land coast and offshore islands were acquired for the period 1981–2012 (see Taylor and Wilson http://www.landcareresearch.co.nz/resources/data/adelie-census-data).We used photographs that were taken each year as close as possible to 1 December (range of dates: 25 November to 8 December) each season. This is a date on which the colony population, owing to its seasonal dynamics, was represented almost entirely by one member of each penguin pair incubating its eggs, and minimal numbers of non-breeders Prior to 2006, film negatives were developed in a dark room, and the resulting photographs of the colony were printed and manually joined together. Once a mosaic of the colony was constructed, the appropriate photographs were enlarged and each territory occupied by a penguin counted by marking it with a dot to ensure that each was counted only once. This method of counting was slow, and it was difficult to verify counts at a later stage. However, the method had the benefit that a physical record was kept for each census.From 2006, the colony images were captured digitally but the photographs were still processed and penguins counted manually. In 2010, semi-automatic penguin counting software was developed so that colony mapping, counts and verification of counts could occur on the computer screen s) The Cape Bird totals are the sum of counts of 3 partitions of the colony and the Cape Crozier counts are the sum of 2 partitions of that colony, but in 3 different years counts from one of the three Cape Bird partitions were not available. To avoid biasing the total colony count towards zero these missing values for Cape Bird were interpolated by fitting a cubic smoothing spline to the colony partition time series using the “zoo” package in the statistical software “R” e-transformed Ross Island colony counts with first-order correlation (AR(1)) in the error term to account for the inherent dependence of a count in one year on the count last year. Two models were fitted for each colony, one with a linear trend with time across all years (1981–2012), and one with different linear trends in the periods 1981–2000 and 2001–2012, in which the two periods were specified using dummy variables. The best fitting model out of the two was selected using the Akaike Information Criteria corrected for small sample sizes (AICc).Visual inspection of the colony time series indicated a potential change point in the trends in counts of southern colonies around the years 2000–2001. To assess differences in trends 1981–2000 vs 2001–2012, a generalized least squares model was fitted to each of the logt-1 vs t; both loge-transformed) were constructed as a visual means of assessing density dependence n, where n is the length of time series. Spatio-temporal synchrony between annual colony growth rates (rt = loge(Nt+1) – loge(Nt)) was assessed by calculating cross-correlation coefficients  between all pairs of the Ross and Beaufort island colonies. The mean correlation coefficient was calculated to quantify spatial synchrony across colonies in the southern metapopulation. Because of autocorrelation in the data, the confidence intervals of the mean correlation coefficient were estimated using the bootstrap algorithm of Bjørnstad et al. Phase plots of current colony counts against lagged counts  in annual colony size variations across colonies in this population was similar  indicating a lack of cycles in the data During our 30-year survey period 855,625 pairs of Adélie penguins on average held nesting territories just prior to hatching at colonies in the western Ross Sea . Around A regression model with two separate trend lines fitted the colony count data best for the Beaufort Island, Cape Bird and Cape Crozier colonies, but not for Cape Royds, where a single trend line was selected . Our modChanges in abundance of Adélie penguins at seven colonies of northern Victoria Land for which we had data from six or more surveys over the 30 years were variable. There appeared to be initial stability or perhaps decrease through 1991, but thereafter, with sparse counts, no clear trend was evident ; Fig. 3.e-transformed colony sizes showed no evidence of cycles in the time series, with significant lags occurring only at 1 year or both years 1 and 2, though the length of the time series is relatively short for such detection. The slow decay of the ACF to negative values at high lags is indicative of a trend in the data (as discussed in the previous section). The partial rate correlation functions (PRCF) for the rates of change versus loge-transformed colony size all showed a significant negative correlation at lag 1 indicative of direct density dependence in the number of pairs returning to the colony each year. This indicates that high colony counts in one year tend to result in lower growth rates to the following year, and vice versa: low colony counts in one year tend to result in higher growth rates to the following year (The auto-correlation functions (ACF) for loging year . There wr) for the southern population showed a striking level of synchrony through time (R = 0.35–0.59). Synchrony between the other three colonies  was higher, with correlation coefficients of 0.68–0.77.Annual colony growth rates , as well as since all counts began in 1959 cf. . Even soThe most current assemblage of counts indicates about 38% of the circum-Antarctic Adélie penguin population breeds in the Ross Sea The presence of unoccupied sub-colony mounds with ample supplies of pebbles for nest-building indicates that the colonies on Ross Island are not space-limited. However, recent recession of ice fields on Beaufort Island have resulted in more nesting habitat, meaning that recruits within this metapopulation no longer have to emigrate to the Ross Island colonies to find nesting space The similar low level of amplitude associated with colony size variation across the southern metapopulation indicates that colonies could be responding in concert to a single or multiple common drivers . This is supported by the high level of synchrony and correlation in annual growth . The relAmong the possible common drivers is sea-ice variation, with the mega-icebergs showing this quite well at the local, mesoscale. The depression in abundance of Adélie penguins in the southern Ross Sea population in 2001 was a common low point coinciding with the arrival of the giant icebergs B-15A and C-16 in January 2001, and which remained in place until winter 2005. These icebergs, as well as B-15B, resulted in a one-season reduction in primary production in the Ross Sea polynya, 2000/01 Many Adélie penguin adults failed or abandoned breeding attempts early in the breeding season in the initial year of the B-15A and C-16 iceberg groundings (2001/02). In the case of Cape Royds, conditions were especially daunting with 70 km of fast ice remaining in place in McMurdo Sound for most of the period 2001–2005; besides nest desertions, many adults eventually emigrated to Cape Bird Leptonychotes weddelli) numbers, but which recovered entirely once the icebergs departed and annual fast ice returned The effects of ice bergs and the altered sea-ice regime were not confined just to the southern Adélie penguins. B-15A and C-16 presence resulted in an initial decrease in Beaufort Island and Cape Crozier emperor penguins, followed by slow recoveries still ongoing Sea-ice cover at the larger scale can also affect Adélie penguins in a number of ways. A previous study analysing trends up to 1998 demonstrated a correlation between declines in the southern colonies 4–5 years after anomalously extensive sea ice in the Ross Sea sector of the Southern Ocean The more favorable polynya behaviour also likely played a complex role in trends through the 1990s Dissostichus mawsoni) Pleuragramma antarcticum). Over the southern shelf, Antarctic silverfish are a major prey of both Adélie penguins and Antarctic toothfish , could well apply.It is unfortunate that colony assessment became so sparse for northern Victoria Land after 2000. Before then, trends appear to be approximately similar to the southern colonies. Since then, it is impossible to judge trends, and it appears that the slow movement of the B-15, C-16 and C-19 icebergs along the Victoria Land coast negatively affected the northern colonies, coincidently in most of the years when counts were done. The mega-iceberg C-19 delayed sea-ice breakout offshore of most of the northern Victoria Land colonies as it made its way north in Spring 2002. This would help to explain the low counts that year, with the ice having retarded or discouraged penguin arrival in time for egg laying (and counting). Similarly, B-15J  and C-16 appear to have blocked the exit of sea ice offshore of northern colonies as they made their way north in 2006 (another year of low counts). It would have been interesting to have had colony counts from Possession, Sven Foyn and Cape Adare colonies in 2005, as the largest part of B-15 spent several summer months offshore of those colonies in late 2005 As a sea-ice obligate, the Adélie penguin generally exists in areas of intermediate sea-ice concentration (∼15% is ideal


Microarray single-nucleotide polymorphism genotyping, combined with imputation of untyped variants, has been widely adopted as an efficient means to interrogate variation across the human genome. “Genomic coverage” is the total proportion of genomic variation captured by an array, either by direct observation or through an indirect means such as linkage disequilibrium or imputation. We have performed imputation-based genomic coverage assessments of eight current genotyping arrays that assay from ~0.3 to ~5 million variants. Coverage was determined separately in each of the four continental ancestry groups in the 1000 Genomes Project phase 1 release. We used the subset of 1000 Genomes variants present on each array to impute the remaining variants and assessed coverage based on correlation between imputed and observed allelic dosages. More than 75% of common variants  are covered by all arrays in all groups except for African ancestry, and up to ~90% in all ancestries for the highest density arrays. In contrast, less than 40% of less common variants  are covered by low density arrays in all ancestries and 50–80% in high density arrays, depending on ancestry. We also calculated genome-wide power to detect variant-trait association in a case-control design, across varying sample sizes, effect sizes, and minor allele frequency ranges, and compare these array-based power estimates with a hypothetical array that would type all variants in 1000 Genomes. These imputation-based genomic coverage and power analyses are intended as a practical guide to researchers planning genetic studies. Microarray genotyping has been widely adopted as an efficient means to interrogate variation across the human genome. Such arrays are the technological foundation of genome-wide association studies (GWAS), which have uncovered numerous genetic associations for a wide variety of traits and diseases .2r) between discrete allelic dosages at the untyped variant and any of the genotyped variants. Thus one way to describe genomic coverage is as the fraction of variants in a reference set having a maximum 2r with array variants above a given threshold . Genomic coverage for the first generation of SNP microarrays typically was estimated using this pairwise LD paradigm and with the HapMap Project  allelic dosage at a genomic variant and the imputed (continuous) allelic dosage, over a defined set of samples. For a variant with two alleles A and a, the imputed dosage is calculated as 2P(AA) + P(Aa), where P(AA) and P(Aa) are posterior genotype probabilities from imputation. Under this imputation paradigm, genomic coverage can be reported as the fraction of variants in a reference set with imputation 2r above a given threshold   .2r coverage metrics is to estimate the power to detect an association when the causal locus is not observed directly. 2r for observed genotypes at a marker locus, where 2r is the squared correlation between the discrete allele dosages at the causal and marker loci. This relationship has been used to estimate the genome-wide power to detect an association with various SNP arrays, using the maximum pairwise 2r for each variant in a genome-wide reference set such as the HapMap , assuming a linear relationship. We also provide genome-wide power estimates with imputation 2r values, estimated for each ancestry group in a 1000 Genomes Project reference set.An important application of 2 values . Here we2r. Updated coverage assessments are provided here using new arrays, a denser and more robust data release from the 1000 Genomes Project, and imputation-based 2r. New arrays have been developed in response to both the changing needs of the genetic research community and technological advances in genotyping. While the design of the first generation of arrays was informed primarily by the HapMap Project  Infinium HumanCore and the Affymetrix (www.affymetrix.com) Axiom Biobank. Although vendors frequently provide genomic coverage estimates in product documentation, the methods used are often different and/or not well-described, making it difficult to objectively compare across arrays. Here we have used a consistent, imputation-based approach to evaluate genomic coverage and power across eight different genotyping arrays. These analyses are intended to function as a practical guide to researchers who are planning genetic studies. The results may be used to compare the cost efficiency of commercially available arrays, to assess the power to detect trait associations using imputed allelic dosages, and to design custom arrays. They also provide an evaluation of the accuracy of imputation for different categories of minor allele frequency (MAF).Despite a growing focus on rare variants, the tagging, LD-based variant selection scheme is still of prime importance in array design. Many arrays still include a core, or “backbone,” of GWAS markers selected using the same principles as the first generation of arrays. Each of the major commercial vendors has recently released  productsftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/release/20110521/), comprising 1,092 samples from 14 populations  the concordance between observed and most likely imputed genotype, the “genotype concordance”; and (3) the concordance between observed and most likely imputed genotype, when at least one of those two genotypes contains one or two copies of the minor allele, which we call “minor allele (MA) concordance.” Array (observed) variants are included in these metrics summaries and are given imputation 2r, genotype concordance, and MA concordance values of 1. A more detailed account of data preparation, pre-phasing, imputation, and metrics calculation is available in Supporting Information, File S1.To assess imputation accuracy and by extension genomic coverage, we compared imputed results at all the nonarray variants to observed genotypes from the initial VCF files. These comparisons were performed separately in the four different ancestry groups  and restricted to variants with at least two copies of the minor allele. For each imputed variant we calculated three metrics: (1) the squared correlation between observed and imputed allelic dosage, which we call “imputation 2r as the primary coverage metric mainly due to its simple relationship to power, in addition to the following advantages: (1) precedent in the literature for evaluating both imputation accuracy  low MAF . OverallA is the risk allele and a is the alternate allele: genotype relative risk  from 1.1 to 1.4 in increments of 0.1; disease prevalence (K) of 0.05; frequency of the risk allele (p) assumed in each case to be the minor allele; the sample size (N cases and N controls) from N = 1000 to 10,000 in increments of 50; and a significance level of 5 × 10−8 (appropriate for genome-wide testing). The assumption of an additive model is based on how imputed dosages are used in association tests, and the range of GRR is based on a summary of effect estimates from genome-wide association studies (2 with 1 degree of freedom) using the non-centrality parameter in the Appendix. Sample size was N for observed (array) variants and N/(imputation 2r) for imputed variants. A genome-wide power estimate was obtained for each array and parameter set by averaging over all variants in a reference set, as indicated by 2r values) and averaged over all variants in the set. In practice, to reduce computation, we binned all autosomal variants by a combination of MAF and 2r values, calculated power for the mean values of MAF and 2r within each bin, and took the mean over bins weighted by the fraction of variants in each bin, as in 2r was from 0 to 1 in increments of 0.05.The power to detect a variant-trait association was calculated for a case-control study under the assumption of an additive genetic model, using the following parameters, where i.e., at least two copies of the minor allele observed in any one of the four ancestry groups). As may be expected, the overlap with 1000 Genomes for arrays with a high proportion of rare and exome variants is less than for arrays with less content devoted to rare and exome variants. The possible impact of the differential 1000 Genomes overlap across arrays on the estimated genomic coverage appears to be small (see File S1).We have assessed imputation-based genomic coverage for eight commercially available genotyping arrays using the four ancestry groups in the 1000 Genomes Project phase 1 release. The number of assays and unique genomic positions assayed by each array are summarized in 2r threshold of 0.8, by MAF bin and ancestry group, whereas 2r and genotype concordance and the counts of variants in each MAF bin, as this count differs by ancestry group. Line plots of mean imputation 2r and mean genotype concordance are available in Figure S1 and Figure S2. Plots organized by ancestry panel rather than by metric are available for AFR in Figure S3, AMR in Figure S4, ASN in Figure S5, and EUR in Figure S6.2r ≥ 0.8 is greater than 75% in these three ancestry groups, and up to ~90% in all four ancestry groups for the highest density arrays. The most dramatic increase in coverage occurs for common variants in the AFR group: the fraction of common variants passing the imputation 2r threshold of 0.8 increases by almost 40% from the least to most dense array. This finding is likely explained by the genetic diversity and lower LD characteristic of present day African ancestry populations compared to the other ancestry groups  are well-covered by all the arrays in the AMR, ASN, and EUR ancestry groups. The percentage of common variants with imputation However, coverage at common variants eventually levels off with increasing array density, a phenomenon of diminishing returns previously observed by 2r ≥ 0.8 is substantially lower than for MAF greater than 0.05: less than 40% for low density arrays in all ancestries and 50–80% in high density arrays, depending on ancestry. In all ancestry groups there is some improvement in coverage of less common variants when moving from the Omni2.5M to the Omni5M array, most notably in EUR . Rare (MAF < 0.01) variants are generally not well imputed by either low or high density arrays. Imputation accuracy for rare variants is greatest in the AMR ancestry group, which might be a consequence of the lower genetic diversity and greater LD in Native Americans than in other ancestry groups, due to a recent population bottleneck associated with human migration into the Americas , which is equivalent to direct observation of all 1000 Genomes Project variants passing the ancestry-specific MAF filters  and thus does not involve uncertainty introduced by imputation. Power is generally low for the range of parameters considered here (even for “1000 Genomes” array), except at the upper end of genetic effect and sample sizes. This is particularly true for the less common variants, where power is generally < 50% even at the high end of the parameter space for the actual arrays and up to ~60% for the “1000 Genomes” array.Genome-wide power estimates for GRR values of 1.2, 1.3 and 1.4 are displayed in vs. 8900). Generally, the array differences required to achieve a set power are notably larger in regions where the power curves plateau. One array is more cost-efficient than another when the ratio of cost per sample is less than the ratio of sample sizes required to achieve equal power. Of course, this evaluation assumes that the extra samples required are available and that the main interest of the study is to detect effects characterized by the parameters used in the power calculation. All of the data used to generate the power plots are available for download at http://jhir.library.jhu.edu/handle/1774.2/36508, so that users can select parameter sets appropriate to their project goals. We also provide the number of variants per MAF and 2r combinations within each ancestry group so that users can readily calculate power using parameters beyond the set provided here.The cost efficiency of achieving equal power can be compared among array choices using these power estimates. For example, for the variant set with MAF > 0.05 and GRR = 1.3, achieving 50% power requires N = 3000 for the Omni2.5M array and N = 4000 for the HumanCore array in the African ancestry group. A power of 80% requires a larger sample size difference .In addition to planning experiments with existing arrays, our genomic coverage results can be used to design new genotyping arrays. For example, one might wish to design an array focused on specific candidate variants or regions, while also providing a backbone for genome-wide imputation, such as the existing HumanCore or Biobank variant set. In that case, one might begin with the backbone and then add candidate variants that are not already well imputed by the backbone variants in the target ancestry group(s). For this purpose, we also provide the individual imputation i.e., which arrays are currently in use). Our analysis has the advantage of consistently applying the same approach to evaluate several different arrays from different manufacturers. There are, however, some limitations to this study design that may result in either over- or underestimated genomic coverage. First, the genomic coverage reported here might be over estimated by imputing subsets of 1000 Genomes samples using the remaining samples as reference, as the haplotypes are likely to match better than in real world applications where the samples to be imputed are likely from different source populations than the reference. This advantage may be offset to some degree by using a smaller reference panel than is available for independent study populations, although this effect is likely to be small because the reference panel used here is approximately 90% of the full 1000 Genomes phase 1 reference panel. Second, coverage may be underestimated by having pre-phased each batch separately, as small sample sizes may negatively impact phasing accuracy . Finally, some of the arrays assay a substantial number of variants that are not in the 1000 Genomes phase 1 release (see File S1), the 1000 Genomes data set is clearly the best resource available for evaluating complete genomic coverage in multiple ancestry groups. We expect the results presented here to be useful in planning studies with the current generation of human genotyping arrays.In conclusion, these imputation-based genomic coverage and power analyses are intended as a practical guide to researchers planning genetic studies. Deciding among available arrays is often a delicate balance between scientific aims, monetary resources, and genotyping timeframe (accuracy . An increase see . These o


Sarcomas are a heterogeneous group of mesenchymal malignancies that very often lead to death. Nowadays, chemotherapy is the only available treatment for most sarcomas but there are few active drugs and clinical results still remain very poor. Thus, there is an imperious need to find new therapeutic alternatives in order to improve sarcoma patient's outcome. During the last years, there have been described a number of new molecular pathways that have allowed us to know more about cancer biology and tumorigenesis. Sarcomas are one of the tumors in which more advances have been made. Identification of specific chromosomal translocations, some important pathways characterization such as mTOR pathway or the insulin-like growth factor pathway, the stunning development in angiogenesis knowledge, and brand new agents like viruses have lead to the development of new therapeutic options with promising results. This paper makes an exhaustive review of preclinical and clinical evidence of the most recent targeted therapies in sarcomas and provides a future view of treatments that may lead to improve prognosis of patients affected with this disease. Sarcomas are a rare and heterogeneous group of malignant tumors of mesenchymal origin. They can occur through all the lifespan and affect patients of all ages, although certain specific subtypes are more frequent in children and young adults. Almost every part of the body may be involved, bone and soft-tissue being the most typical place of arising. Sarcomas are associated with bad prognosis and approximately 50% of all patients develop metastases even if they are diagnosed at early stages. Lungs are the most frequent site of dissemination and metastases are the final cause of death in almost all these patients . These hFor most sarcomas, chemotherapy is currently the only available treatment. Unfortunately, a very limited number of useful drugs are active against this disease and responses used to be poor and short. In fact, advanced-stage patients treated with the most active drugs in this disease (anthracyclines and ifosfamide) achieve only a median survival of around 1 year . Thus, iIn the last years, great advances have been made in the understanding of sarcomas' molecular biology . Consequ In the last years, angiogenesis has been one of the most studied processes in tumor biology with interesting results. Patients with several malignancies such as renal cancer or colorectal cancer are currently being treated with angiogenic inhibitors alone or in combination with conventional chemotherapy. These patients achieve significant improvement in overall survival (OS) and/or progression-free survival (PFS) . Sarcoma One of the key effectors in angiogenesis is the vascular endothelial growth factor (VEGF). It is well known that the interaction between VEGF and its receptor 2 (VEGF-R2) is the most crucial step in angiogenesis –7 and thα (HIF-1α) is another important player in angiogenesis since it is a transcription factor that acts as an upstream regulator of VEGF. In a 2006 paper by Shintani et al. HIF-1α expression was determined by immunohistochemistry in a group of 49 specimens of STS. The analysis showed that patients with a strong or moderate expression of HIF-1α had poorer OS than those with a weak or negative expression [ Hypoxia-inducible factor1pression .α in sarcomas. Microvascular density (MVD) is one of them. Thus, in a work by Comandone et al., patients with high MVD had worse OS and DFS than patients with lower MVD [ There are many other angiogenesis markers but not all of them are so clearly related with prognosis and survival as VEGF and HIF-1ower MVD . In contower MVD –16.These previous data provided a rationale for the development of preclinical and clinical studies with angiogenic inhibitors in sarcomas. An increasing number of these drugs have been developed in the last years and the effects of many of them have been assessed .Sunitinib is a multitargeted tyrosine kinase inhibitor (TKI) active against VEGF-R1, 2, 3, PDGFR and KIT, among others. In 2009, Stacchiotti et al. reported 3 responses and 1 stabilization in a cohort of 5 patients affected with advanced alveolar STS treated with sunitinib . The sam Sorafenib is another TKI recently added to the group of drugs with activity in sarcomas. This TKI targets VEGF-R 2 and 3, PDGFR, Raf, and KIT, and several preclinical studies have demonstrated efficacy in different soft-tissue and bone sarcoma cell lines –25. Thesin vitro, which can lead to further development of clinical trials [Pazopanib is a new TKI that also has effects on angiogenesis by blocking VEGF-R 1, 2, and 3, PDGFR, and KIT. A phase II trial performed by the European Organisation for Research and Treatment of Cancer (EORTC) in patients with relapsed or refractory advanced STS treated with pazopanib achieved promising results . 142 patl trials –37.in vitro and in vivo [Bevacizumab is the only monoclonal anti-VEGF antibody to date that has been proved to have activity in sarcomas. A set of preclinical studies demonstrated activity in sarcoma models  in vivo –42. The  in vivo . There w in vivo . After a The insulin-like growth factor (IGF) system is a well-known complex network that regulates growth and development in superior organisms . The IGFin vitro and in vivo [The background described previously has led to the development of several studies that test different strategies to assess the inhibition of the IGF pathway in sarcoma models  in vivo –67. The Among the different strategies developed to inhibit the IGF pathway, the most promising results have been achieved with monoclonal antibodies. There are several phase I studies that assess the safety and efficacy of inhibiting the IGF system in sarcomas with these drugs. Thus, Tolcher et al. published in 2009 an early clinical study with AMG 479, a fully human monoclonal antibody to IGFR-1. 15 out of 53 patients enrolled were sarcoma patients . Interestingly, 1 durable CR and 1 PR were achieved in 2 patients with Ewing's sarcoma . In anotSeveral phase II trials with anti-IGFR-1 antibodies are currently being conducted. Preliminary data of treatment with IMC-A12 (cixutumumab) in patients with advanced or metastatic STS and Ewing's sarcoma have been reported in the 2011 ASCO Annual Meeting. The best results were observed in the adipocytic sarcoma arm, with clinical benefit being achieved in 22 out of 37 patients . Moreover, the PFS at 12 weeks in this group of patients was 50% . With suA number of small molecules that inhibit IGFR-1 by binding to the tyrosine kinase intracellular portion of the receptor are currently in clinical development. No results of efficacy and safety are available yet but there is a body of preclinical data that support this therapeutic approach –78. Repo The mammalian target of rapamycin (mTOR) is a serine/threonine kinase integrated in the phosphatidyl-inositol 3-kinase (PI3K) complex network of signaling. It forms part of 2 multiprotein complexes named mTOR complex 1 and mTOR complex 2 (mTORC1 and mTORC2) and plays a key role in cell growth, proliferation, angiogenesis, and survival . Due to To date, 4 compounds with anti-mTOR activity have reached the clinical setting. All of them belong to a single family of drugs and are derived from an initial molecule called sirolimus (rapamycin). Thus, temsirolimus, everolimus, ridaforolimus, and the already mentioned sirolimus are nowadays under investigation in sarcomas and other tumors. Sirolimus inhibits mTOR kinase activity by binding to FK506 binding protein (FKBP12), one of the proteins that form mTORC1. This leads to cell cycle arresting in G1 phase and the subsequent inhibition in proliferation and cell growth. Preclinical data suggest activity of sirolimus in some paediatric malignancies including Ewing's sarcoma, rhabdomyosarcoma, and osteosarcoma , 90. ButA very rare type of mesenchymal malignant tumor named Perivascular Epithelioid Cell Tumor (PEComa) has been specially related to the mTOR pathway. Dysfunction in tumor suppressors TSC1 and 2 and the subsequent upregulation of mTORC1 seems to be a crucial step in the development of this disease. Thus, responses with sirolimus and temosirolimus have been reported in at least 2 studies , 102. HoAll 4 rapalogs described previously belong to a first generation of mTOR inhibitors able to inhibit mTORC1 but not mTORC2. mTORC2 seems to be responsible for feedback phosphorylation of Akt in the PI3K/Akt pathway, which could be a possible mechanism of resistance in sarcomas. So, a new generation of mTOR inhibitors with activity against mTORC1 and mTORC2 is under early development in an attempt to block this escape route. About 1/3 of sarcomas are associated with specific chromosomal translocations. These translocations are an early step in carcinogenesis, promoting some of the processes that finally lead to the appearance of sarcomas . Thus, tClear cell sarcoma is also associated with a specific chromosomal translocation, t in most of the cases. One of the consequences of this chromosomal rearrangement is the activation of the hepatocyte growth factor receptor (MET). This activation is involved in invasion and angiogenesis. A response in a clear cell sarcoma patient with the MET inhibitor ARQ197 has recently been reported in a phase II trial, which is especially relevant in this treatment-resistant disease .But probably the most studied translocation-related sarcoma is Ewing's sarcoma. This disease characteristically has a t translocation that leads to expression of the oncogenic fusion protein EWS/FLI1. This chimerical protein is involved in Ewing's sarcoma development since it acts as an oncogenic transcription factor but needs binding to other proteins such as RNA helicase A for its oncogenic function. Recently described YK-4-279, a new compound that blocks RNA helicase A binding to EWS/FLI1, induces apoptosis in Ewing's sarcoma cell lines and reduces tumor growth in orthotopic xenografts . A cliniin vitro and significant tumor growth suppression in a human osteosarcoma murine xenograft model when treated with a telomerase-specific oncolytic adenovirus [One of the most innovative approaches in cancer treatment developed in the last years is oncolytic virotherapy. The general basis of this strategy is that the therapeutic virus is capable to recognize specifically tumor cells, replicate into them, and lead to their death without damaging normal cells. A paper recently published in Nature by Breitbach et al. assessed the safety and efficacy of an intravenous delivery of an oncolytic poxvirus in humans. Treatment was generally well tolerated and the only sarcoma patient enrolled  achieved SD by RECIST criteria for >16 weeks . This reenovirus . More in Although clinical experience with targeted therapies in sarcomas as single treatment is short, there are some published studies with combination of 2 of these drugs. The most studied double-inhibition-targeted therapies are those related with IGFR-1 pathway, with preclinical evidences of activity in sarcoma models –113. Bas An increasing number of new targets in treatment of sarcomas are being identified in the last years. The finding of new important key effectors in sarcomas biology has resulted in a growing development of inhibitor drugs that need to be tested. For instance, increased activity in Hedgehog pathway has recently been reported in certain sarcomas such as rhabdomyosarcoma, osteosarcoma, chondrosarcoma, and Ewing's sarcoma –121. Oth Anaplastic lymphoma kinase (ALK) is another protein recently related to sarcomas that is upregulated in approximately 50% of cases of inflammatory myofibroblastic tumor. Interestingly, in a phase I trial with the ALK inhibitor crizotinib, a patient with ALK overexpression related to inflammatory myofibroblastic tumor experienced a durable PR .Histone deacetylase (HDAC) inhibitors have also shown signs of efficacy in preclinical models of sarcomas –128 and in vitro and in vivo [PI3K/mTOR dual-inhibitor NVP-BEZ235 that induces G1 cell cycle arrest in sarcomas  in vivo  has been Other molecules such as MDM2 and protein families BCL-2 and CDKs have been described lately as associated with sarcomas –132. Fut Despite the low incidence of sarcomas in general population regarding other types of cancer, the finding of new active treatments is essential since it is a rarely curable disease. Hence, a rapidly increasing number of targeted therapies have been developed in the last years with different results. In general, most of these treatments do not achieve significant tumor shrinkage and SD is usually the best response reported. In addition, OS has not been dramatically increased in the majority of these patients, showing the necessity of keep working. In an attempt to improve response rates, one of the strategies that are currently ongoing is the combination treatment with targeted therapies and conventional cytotoxic drugs. Toxicity is an important issue when using this approach and more clinical trials are needed to assess the safety of this therapeutic option. Combinations of more than one targeted agent are another reasonable choice. The strategy of inhibiting a signalling pathway and simultaneously others that could be possible ways of escape is an attractive alternative still under early development. Phase I trials reported to date with 2 targeted therapies show favourable toxicity profiles, making this strategy a feasible and promising issue to be explored. Different approaches like those, future identification of new pathways and their correspondent inhibitors and the arise of innovative agents such as oncolytic viruses, make the final endpoint of cure sarcsomas a goal not so far to be reached.


TRIM5 were inversely correlated with disease. Conversely, SNPs around one retroviral locus, HERV-Fc1, showed a highly significant association with disease. The latter association was limited to a narrow region that contains no other known genes. We conclude that HERV-Fc1 and TRIM5 play a role in the etiology of multiple sclerosis. If these results are confirmed, they point to new modes of treatment for multiple sclerosis.We have investigated the role of human endogenous retroviruses in multiple sclerosis by analyzing the DNA of patients and controls in 4 cohorts for associations between multiple sclerosis and polymorphisms near viral restriction genes or near endogenous retroviral loci with one or more intact or almost-intact genes. We found that SNPs in the gene  Two views have dominated the discussions on the etiology of multiple sclerosis (MS[MIM 126200]) in recent decades: It could be a genetic disease, or it could be a disease caused by an infectious agent. The genetic view is most clearly expounded by studies of twins, showing that concordance among monozygotic twins is approximately 25 percent, while concordance among dizygotic twins is only 2–3 percent pol genes have shown that HERV-Fc is closely related to HERV-H, more distantly related to HERV-W and only remotely related to HERV-K The endogenous human retroviruses exist in multiple types, generally named after the amino acid of the tRNA that serves as primer for first strand synthesis. Some endogenous virus types (−H and −W) are old and universal in a large range of species, whereas certain of the −K type integrations are recent and still segregate in the human population. HERV-Fc endogenous viruses can be traced as far back as the simian forefather, however present copies in our genome are more recent acquisitions and specifically the HERV-Fc1 locus on the X-chromosome, with which this paper is concerned, is shared by man, chimpanzee, and gorilla, but not orangutan. This put the time of integration at about 10 to 15 million years ago In this study, we have attempted to evaluate the role of retroviruses in the etiology of Multiple Sclerosis, both indirectly by studying genes that restrict retroviruses, and directly by searching for an endogenous retrovirus associated with occurrence of disease.Fv-1TRIM5 appears to be the closest functional relative of Fv-1TRIM5 and disease. Initially, we investigated a small cohort consisting of 28 verified cases TRIM5 intron 1. The nearby exon 2 encodes the RING domain in the N-terminus of the protein. This domain plays a major role in recognition of retroviral cores. The SNPs and their primers are listed in Inspired by the classical genetic studies of the mouse restriction gene for retroviruses, We were similarly inspired by the classical Mendelian studies of the highly active endogenous ecotropic viruses of the AKR mouse Initially, we investigated 193 SNPs in the DNA from Cohort 2. There were clear-cut clusters of significant SNPs in a few chromosomal locations. These locations were substantiated by analyzing additional nearby SNPs. A total of 220 SNPs were tested. The most significant results are indicated in −6 (2-sided) for association with disease, when calculating on the basis of C-allele carriers . Here, 3 close-lying SNPs gave p-values less than 0.001. The SNP rs391745 was lowest with a p-value of 4*10We retested the association of rs391745 and multiple sclerosis in Cohort 3. This cohort consisted of 542 cases and 1165 controls with 67 percent female cases and 65 percent female controls. rs391745 was again associated with MS (p = 0.01) . In thisTo substantiate the association of HERV-Fc1 with multiple sclerosis, we performed a scan of the region surrounding the HERV-Fc1 provirus in Cohort 2. TRIM5 we depicted the odds ratio for disease as a function of the SNPs rs391745 and rs3802981, representing the two genes. The results are shown in Looking further for an interaction or additive effect between HERV-Fc1 and pol gene. The reading frames of the gag and env genes appear to be complete (NCBI build 37.1). We cannot exclude the contribution of other endogenous viruses to putative infectivity through pseudotyping or recombination. Perhaps the relevant determinant of infectivity in the organism is not the individual locus, but the repertoire of proviral sequences TRIM5.By treating the endogenous retroviruses as inheritable loci, we have brought the formidable knowledge of human genetics to bear on their role in disease, and avoided the difficulties of establishing the causality of a ubiquitous agent by conventional means. It is not clear if HERV-Fc1 is infectious, but analyses of the proviral DNA suggest the presence of two stop-codons and a frame-shift in the TRIM5 encodes an ubiquitin ligase that restricts the replication of many retroviruses to varying degrees Fv-1. The association of TRIM5 with multiple sclerosis is independent confirmation that a retrovirus plays a role in the etiology of MS.The gene Our results should be seen in combination with previous findings in genomic scans that demonstrate that the major histocompatibility complex on chromosome 6 is important for this disease Our suggestive evidence that a gene for multiple sclerosis is located on chromosome X could provide a natural explanation for the old observation that more women than men suffer from this disease. This is the normal pattern for X-dominant diseases. For Cohort 3 we obtained information about the clinical subtypes of the patients, but the cohort was too small for rigorous analyses of the subtypes. However, there were no signs of association with rs391745 among the patients with a primary progressive disease, and the association of the remaining (relapsing-remitting and secondarily progressing) was slightly stronger (p = 0.008) than that of the total cohort. This may indicate a different etiology of primary progressive sclerosis and is in accordance with the observation that the primary progressive form of multiple sclerosis has a sex ratio around 1.In general, the finding that a disease is caused by an infectious agent is an encouraging one. These are the diseases, which we know best how to treat. If our results are confirmed, they point to new modes of treatment of Multiple Sclerosis.2, and 1 mM of each deoxynucleotide. The primers were added to a final concentration of 500 nM each. The total reaction volume was 4 µl. PCR reactions with primers of each plex were performed in 384 well plates with V-shape by performing a 15 min preheat to 94 C followed by cycling the samples 45 times, 94 C for 20 sec, 56 C for 30 sec and 72 C for 1 min, followed by 3 min at 72 C. The plates were stored at −20 C. Treatment with Shrimp Alkaline Phosphatase and extension with molecular weight-modified nucleotides was performed according to the manufacturer's recommendations. The reactions were cleaned with resin and spotted on SpectroChip arrays according to the manufacturer's recommendations. The reactions were analyzed by MALDITOF mass spectrometry on the Sequenom equipment, and the results were analyzed using the MassARRAY Typer 4.0 (Sequenom). Significance was tested by a χ2 test .The studies were approved by the Science Ethical Committee of Mid-Jutland and were performed with written and oral consent from all persons. Four cohorts of ethnically Danish patients, with verified multiple sclerosis Figure S1A cartoon of the human genome with locations of the relevant endogenous viruses and indication of significant markers nearby.(TIF)Click here for additional data file.Table S1TRIM5 and their primers.SNPs used for the analysis of (DOC)Click here for additional data file.Table S2TRIM5 and their association with MS.SNPs used for the analysis of (DOC)Click here for additional data file.Table S3SNPs used for the analysis of selected endogenous retroviruses and the primers used.(DOC)Click here for additional data file.Table S4SNPs used for the analysis of selected endogenous retroviruses and their association with MS.(DOC)Click here for additional data file.Table S5Association of the alleles of rs391745 with MS in the sex-stratified cohorts.(DOC)Click here for additional data file.


Domestic cats ranging freely in natural areas are a conservation concern due to competition, predation, disease transmission or hybridization with wildcats. In order to improve our ability to design effective control policies, we investigate the factors affecting their numbers and space use in natural areas of continental Europe.We describe the patterns of cat presence, abundance and space use and analyse the associated environmental and human constraints in a well-preserved Mediterranean natural area with small scattered local farms. We failed in detecting cats in areas away from human settlements (trapping effort above 4000 trap-nights), while we captured 30 individuals near inhabited farms. We identified 130 cats, all of them in farms still in use by people (30% of 128 farms). All cats were free-ranging and very wary of people. The main factor explaining the presence of cats was the presence of people, while the number of cats per farm was mostly affected by the occasional food provisioning with human refuse and the presence of people. The home ranges of eight radio tagged cats were centred at inhabited farms. Males went furthest away from the farms during the mating season , using inhabited farms as stepping-stones in their mating displacements (2.2 km of maximum inter-farm distance moved). In their daily movements, cats notably avoided entering in areas with high fox density.The presence, abundance and space use of cats were heavily dependent on human settlements. Any strategy aiming at reducing their impact in areas of conservation concern should aim at the presence of settlements and their spatial spread and avoid any access to human refuse. The movements of domestic cats would be limited in areas with large patches of natural vegetation providing good conditions for other carnivore mammals such as red foxes. Panthera leo population, caused a mortality of 30% of the individuals Lycaon picus, in the Serengeti due to the same disease Felis catus) is no exception and is currently considered a major conservation problem. It is the carnivore species with a wider distribution range, being present in all continents and in many islands, including several subantartic islands The presence of domestic species in the wild often represents a conservation problem due to competition with and predation of wild species and due to the potential hybridization with the wild ancestor types The effects of domestic cats on wildlife depend on where they are found and on the factors controlling their numbers and space use 2), as in low humanized natural-rural areas, ranges are large and the rate of intra-specific encounters is low Felis silvestris). In many island ecosystems domestic cats are dominant predators that cause a very relevant impact on breeding seabird colonies and endemic species Lynx pardinus), the most endangered feline of the world, the transmission of Feline Immunodeficiency Virus (FIV) and Feline Leukemia Virus (FeLV) by domestic cats may become a serious threat to their populations However, cats whose needs are not satisfied by people  pose the main conservation threats. Domestic cats may live and reproduce with little (in the case of stray cats) or no human intervention  and survive by scavenging or hunting. The almost unlimited food supplies that cities provide allow for the presence of large numbers of feral cats; for example, about 30 million cats are estimated to live in the streets of the United States Quercus rotundifolia), patches of sclerophylous scrubland and rocky areas and boulders along the main rivers and streams. Elevation ranges between 200 and 400 m. There are no villages within the Natura 2000 site and human settlements are reduced to isolated traditional farms. The climate is characterized by dry warm summers and cold winters. Human activity is spatially limited, and consists in cattle rising, traditional agriculture and game hunting. This agro-silvo-pastoral system is characterized by a heterogeneous combination of patches with open tree cover for cattle grazing (montado or dehesa) and shrubby forest patches. Moura-Barrancos Natura 2000 study site belongs to two municipalities: Barrancos, with a single village occupied by about 1,800 people  and Moura that encompasses five small villages close to the study area (17.1 inhabitants/km2) The study was carried out in Moura-Barrancos Nature 2000 site  and part of a Bird Special Protection Area, encompassing the agroforestry areas around the village of Barrancos in the Southeast Portuguese-Spanish border . The inhabited farms selected for the captures of radio tagged domestic cats represent well preserved game areas : Noudar −1) and ketamine hydrochloride  in a 2∶1 proportion. After handling, we reversed the anaesthesia with 0.08 mg/kg of Antisedan. We estimated the age class (young/adult) from body weight and dentition. We considered adults those animals with full adult dentition and a weight above 2.5 kg and 3.5 kg for females and males respectively The animals captured were weighted after being anaesthetized with an intra-muscular injection of medetomidine hydrochloride  from March to May 2006, while in the second area we used 11 cameras from May to August 2006 .We identified all farms (inhabited and abandoned farms) in the whole study area and evaluated the presence and number of cats during survey visits. The aim of these surveys was to establish the pattern of distribution of cats around farms in the study area, and to describe its relation with farm location and human use. We complemented this information with interviews to the owners/workers to obtain information on their use of the farm, the number and type of cats and on their relationship with cats (including food provisioning).We selected variables aiming at describing the impact of human-related variables associated with the commensality of cats around people and, additionally, those associated with the plasticity of cats when using more natural areas, including vegetation types. We used a ground-validated 1∶5000 orthophoto (year 2005) to build the digital land cover cartography. We considered three land cover classes: human settlements, corresponding to the building areas in farms and other human settlements; natural vegetation, including sclerophylous vegetation, coniferous forests and riparian vegetation, and agro-forestry areas, which consisted in the oak montado-dehesas without understory and olive groves . Additios>0.7, selecting the one with a higher correlation with the dependent variable and/or the variable with most clear biological interpretation We analysed the environmental and human-related factors affecting the presence of cats at farms using generalised linear models with a binomial error distribution and a logit link function (glm function in R Software version 9.1). We considered that two independent variables were strongly correlated when rkernelUD function, adehabitat package in R software) We located the radio marked cats by triangulation using two bearings taken at less than 10 minutes apart to minimize the errors caused by animal movements. Only bearings between 60° and 120° were accepted We investigated cat habitat use using five covariates, which expand in habitat categories . We compR software v.2.9.1 We analysed the environmental and human-related factors affecting the length of the movements of the radio-tracked cats. These variables represent the most important environmental and human features related with the behaviour and ecology of domestic cats Genetta genetta), 7 red foxes (Vulpes vulpes), 3 Egyptian mongooses (Herpestes ichneumon), 3 badgers (Meles meles) and 2 stone martens (Martes foina). At the same site, we obtained 498 photos with an effort of 612 trap-nights. Considering only one capture per day and camera we photo-captured 15 wildboars (Sus scrofa), 13 red foxes, 8 badgers, 8 common genets, 7 Egyptian mongooses and 4 stone martens. Similarly, at the Southern site, with an effort of 1117 trap-nights, we live captured 12 red foxes, 5 Egyptian mongooses, 3 badgers, 2 common genets, and 2 stone martens. Photo-trapping provided 480 photos with an effort of 814 trap-nights, including 22 red deer (Cervus elaphus), 8 wildboars, 9 red foxes, 8 common genets, 7 Egyptian mongooses, 5 badgers, and 2 stone martens.In spite of our efforts, we were unable to detect any domestic cat in the two trapping sites selected in the natural area away from farms . At the On the contrary, at the ten farms where we carried out a trapping effort of 297 trap nights we captured 30 different individuals: 12 males (of which 8 were adults) and 18 females (12 adults). After these results we concentrated our work in and around farm buildings and in the adjacent scrubland areas.−2. Cats were present in 39 of them (92.5%), with a total of 130 individuals (3.3±1.85 cats per farm) and a density of 0.26 cats km−2 . The sex ratio of 88 individuals  was 1M∶2F but for the remaining 41 individuals sex was unknown. The average nearest distance between farms with cats was 2.7±12.7 km. Farm owners or users considered all cats to be free ranging i.e., they were no kept as pets. In none of the farms the cats received veterinary support. Food provisioning was never provided on a regular basis, with 33 farms feeding cats only sporadically with human refuse (84.6% of the occupied houses). Except for one cat out of the 130, farm owners defined them as very wary .We identified 128 farms within our study area, the majority of which (67.2%) had no resident people. Many of them were abandoned or even in ruins. There were no cats in the abandoned houses. The average distance to the nearest house was 1.1±5.51 km (±SD). The 42 farms in use give a density of 0.09 farms kmThe best model describing the presence of domestic cats in farms included the presence of people and the length of roads and rivers around the farm (explaining 30.5% of the deviance); the next model included only the first two variables . The maximum distances that males travelled are associated to the distance to the nearest farm with cats (r2 = 0.67) only during the mating season (r2 = 0 outside the season). Males used farms as stepping stones in their mating displacements; the maximum inter-farm distance moved was 2.2 km while the male that never made any excursion to other farm was 3.4 km away from the nearest occupied farm. None of the females moved between farms.We marked eight cats  with radio-collars. On average, we tracked them for 10±0.7 (±SD) months, obtaining an average of 176±14.8 fixes per animal. The area covered by all the radiolocations of the marked cats was 10,416 ha . All individual home ranges included the farm where each cat was captured . The aveP = 0.008). The compositional analysis showed that there was a clear order of preference headed by human settlements (House), followed by areas at less than 200 m from roads and with a smaller slope . Considering the management of problematic of cats in natural areas, the control of populations should be more effective when trapping near human settlements (e.g. our farms). The presence and number of cats was dependent on the presence of people and the resources they provide. In fact, the area around farms was the preferred land use type for cats, as confirmed by the highest value of selection ratios.Outside islands and Australian mainland domestic cats can become feral not only in rural environments, but also in semi natural environments that are settled by people, as shown in the literature, with no evidence for cats living on their own in natural areas away from people. For example, in Scotland, the putative wildcats were in contact with farm cats Nevertheless, we can still find domestic cats roaming in natural areas far away from any human settlement. In our study area, their home ranges are centred on farms, but males can make long displacements in search of females during the mating season Canis familiaris have the same behaviour in natural areas with a healthy large carnivore comunity, where their movements were restricted to the vicinity of human dwellings In summary, in natural areas cats may live strictly depending on only wild resources, as in some deserted islands, but the general pattern is that they do not. Feral cats have the capacity to move long distances away from households, but still restricting their movements to the vicinity of human settlements. The free ranging domestic dogs Like in islands, cats living in farms do not compete with other carnivores, but unlike the confined environment of islands, in many mainland areas they have to share space with other predators when moving away from houses. In fact, mesopredator species, like the domestic cat, appear to be ecologically released by increased urbanization not only because they can adapt well to those environments, but also because such sites may provide refuge from top predators Tyto alba) that also leverages on buildings within human settlements.If we cannot manage the presence of people living in the field, in order to maintain low cat numbers, food provisioning should be banned while the access of cats to human refuse must be controlled. In the worst case scenario in which people is living in a network of well-connected settlements and provisioning cats with food, males should be neuter to reduce the distances they move away from houses. Moreover, the movements of domestic cats would be limited in areas with large patches of natural vegetation, promoting the presence of other carnivors such as foxes. Finally, because private landowners are the ultimate controllers of their land, providing them with information is essential to increase the awareness of people before the implementation of any measure. The presence and tolerance of domestic cats in human settlements in rural areas is associated to the ancestral role of cats in controlling rodent populations, but this function can be performed by the barn owl (