PACSIN2 and ITPA, on drug-induced toxicities by applying a multi-analytical approach including logistic regression (LR), classification and regression tree (CART) and generalized multifactor dimensionality reduction (GMDR). In addition to the TPMT genotype, confirmed to be a major determinant of drug related toxicities, we identified the PACSIN2 rs2413739TT genotype as being a significant risk factor for 6-MP-induced toxicity in wild-type TPMT patients. A gene-gene interaction between MTRR (rs1801394) and MTHFR (rs1801133) was detected by GMDR and proved to have an independent effect on the risk of stomatitis, as shown by LR analysis. To our knowledge, this is the first study showing PACSIN2 genotype association with hematological toxicity in ALL patients undergoing maintenance therapy.Adequate maintenance therapy for childhood acute lymphoblastic leukemia (ALL), with 6-mercaptopurine as an essential component, is necessary for retaining durable remission. Interruptions or discontinuations of the therapy due to drug-related toxicities, which can be life threatening, may result in an increased risk of relapse. In this retrospective study including 305 paediatric ALL patients undergoing maintenance therapy, we systematically investigated the individual and combined effects of genetic variants of folate pathway enzymes, as well as of polymorphisms in  Cure rates of approximately 80% can currently be achieved in paediatric acute lymphoblastic leukaemia (ALL)1TPMT can explain the majority, but not all, of the cases exhibiting decreased TPMT activity in individuals. Due to an incomplete genotype-to-phenotype correlation, which can be as low as 50% in heterozygotes in some populations7in vitro on several cancer cell lines9in vivo in a study involving over 1,000 healthy individualsPACSIN2, which has been found to be associated with TPMT activity and with the incidence of 6-MP-related gastrointestinal toxicity6-MP is a prodrug that exerts its cytotoxic effects after several steps of metabolic activation leading to the formation of 6-thioguanine nucleotides (6-TGN). The main inactivating pathways are through xanthine oxidase (XO), which metabolizes 6-MP to thiouric acid, and through thiopurine S-methyltransferase (TPMT) metabolizing 6-MP to 6-methylmercaptopurine (6-MMP). The large variations in responses to 6-MP therapy that have been observed between patients are, to a great extent, attributed to differences in TPMT activity that lead to different accumulations of 6-TGNs in cellsPACSIN2 and ITPA, which have previously been associated with treatment toxicity12Since SAM is synthesized in the methionine cycle, which is closely related to one-carbon metabolism, its intracellular concentration depends on the availability of methionine as well as the folate pool. Based on this knowledge, we hypothesized that polymorphisms in genes coding for enzymes involved in the methionine and folate pathways, such as are presented in In total, 305 paediatric ALL patients with a mean age of 5.9 ± 4.3 years were included in the study. Their clinical characteristics are presented in We analysed 11 SNPs in 8 candidate genes, all of which showed distribution in accordance with the Hardy–Weinberg equilibrium. The list of analysed SNPs with information on variant allele frequencies and p-values of Hardy-Weinberg tests are presented in Hematotoxicity, stomatitis, and recurrent infections, except secondary tumours, were in correlation to each other as well as to 6-MP dose reduction . These observations are expected, since dose was usually reduced as a consequence of experienced toxicity. Furthermore, it was not uncommon for the same patient to experience more than one adverse drug effect, due to overall worse tolerability of treatment.During the period of maintenance therapy, hematotoxicity of grade 3 or 4 was experienced by 15% (46) of patients while 6-MP dose reduction (at least a 10% reduction lasting 3 months or more) was recorded in 20% (63) of patients.In order to assess the effects of the candidate polymorphisms and their interaction in predisposing the patient to 6-MP induced hematotoxicity and dose reduction, we used multivariate logistic regression models along with generalized multifactor dimensionality reductions (GMDR) and classification and regression tree (CART) analyses.GNMT 1298CT or the TT genotype than in those with GNMT 1298CC  and a higher risk in patients with PACSIN2 TT than in those with PACSIN2 CC or the CT genotype . The effect of the MTRR genotype was not statistically significant and neither were the effects of other genotypes, except for TPMT, after Bonferroni correction.As indicated in hematotoxicity revealed a two-locus interaction between TPMT rs1142345/rs1800460 and PACSIN2 rs2413739. The model, adjusted to treatment protocol, age at diagnosis and gender, had a cross-validation consistency of 9/10 and a balanced testing accuracy of 0.6692 (−7), when the model was adjusted to protocol treatment, age at diagnosis and gender. The effect remained significant after Bonferroni correction.The best overall GMDR model for the prediction of f 0.6692 . The resf 0.6692 . The newNext, we conducted a CART analysis during which patients were subdivided successively by the most discriminating polymorphism or haplotype to form a so-called regression tree. The results of this are presented in dose reduction, the logistic regression model with the smallest value of AIC was that including TPMT, GNMT and PACSIN2 genotypes. Besides the influences of treatment protocol and TPMT genotype, described previouslyGNMT genotype on dose reduction, such that a lower frequency of dose reduction was observed in patients with GNMT 1298CT or TT than was the case in those with the GNMT 1298CC genotype . The effect did not remain significant after Bonferroni correction. PACSIN2 genotype contributed to the overall fit of the model, but was not statistically significant  had their dose reduced less often than patients treated according to protocols BFM90, BFM95 or BFM2002. The reason for this is difference in the management of patients, where treating physicians working within the older protocols tended to discontinue 6-MP in the event of adverse events rather than lower the dose. The effect of genotype is, therefore, seen only in the more recent protocols, where the TPMT genotype again emerged as the strongest predictor of dose reduction. In wild-type TPMT patients, an additional influence of PACSIN2 rs2413739 emerged, with patients carrying the TT genotype being at a higher risk of dose reduction relative to patients with CC or CT genotypes . The genDuring the maintenance therapy period, infections of grade 3 or 4 and stomatitis of grade 2 or 3 were experienced by 22% (67) and 6% (19) of patients, respectively. 4% (12) of patients developed secondary tumours during or after the maintenance therapy.stomatitis already including TPMT genotype improved the model, although the effect of this addition was not statistically significant   . None ofstomatitis suggested an interaction between MTRR rs1801394 and MTHFR rs1801133 polymorphisms , for stomatitis (by LR and CART methods) and was associated with the occurrence of dose reductions due to therapy toxicities. An interaction between the TPMT genotype and PACSIN2 rs2413739 was consistently identified by all statistical methods in the case of hematotoxicity; namely, that patients with wild-type TPMT who had the PACSIN2 rs2413739 TT genotype stood a higher risk of experiencing hematotoxicity than patients carrying the PACSIN2 rs2413739 CT or CC genotype. Similarly, the effect of the PACSIN2 genotype in wild-type TPMT patients was associated with dose reductions according to LR and CART analysis , but this association was not shown by GMDR.It has long been recognized that TPMT genotype is the primary risk factor for PACSIN2 is rs2413739, which had previously only been associated with 6-MP-related gastrointestinal toxicity in ALL patients during consolidation therapyPACSIN2 genotype with 6-MP-related toxicity occurs through the modulation of TPMT activity, a hypothesis that was confirmed in in vitro cell models as well as in ALL patients, where individuals with the PACSIN2 rs2413739 CC genotype had higher TPMT activity than patients with the TT genotypePACSIN2 is a member of the ‘protein kinase C and casein kinase substrate in neurons’ family of proteins that are involved in vesicle formation via interactions with the large GTPase dynamin and N-WASP, which forms a critical part of the actin polymerisation machineryIn CART analysis age under 4 years emerged as the risk factor for hematotoxicity forming a split before PACSIN2 genotype in wild-type TPMT patients. However, since in LR model the effect of age was not statistically significant and we found no reference describing the same effect in the published literature, it is likely that this observation has no clinical relevance.Of the genes related to the folate and methionine cycles that were hypothesised as having an effect on 6-MP-related toxicity by modulating SAM levels and, consequently, TPMT activity, only MTHFR was associated with hematotoxicity by two statistical methods (LR and CART), although the effect did not remain statistically significant after Bonferroni correction. This observation is in agreement with previously published results13A major strength of our study is that some gene-gene interactions, including, and in particular that between TPMT and PACSIN2, were consistently identified when analyzing data by different statistical approaches. The analysis was first done by logistic regression (LR), which is considered to be the workhorse of modern epidemiologyThere are some limitations to our findings. Firstly, our study evaluated a limited number of patients and, due to the small number of cases, for example in the analysis of stomatitis and secondary tumours , some risk factors might not have been picked up by the statistical analysis. In addition, patients included in our study were treated according to different treatment protocols over a relatively large period of time. Although the regimens of the maintenance phase of therapy were very similar in all the protocols and all our statistical models were adjusted to this covariable, the genetic effects detected in our study should be evaluated in an independent cohort of patients treated in accordance with the most recent treatment protocols so as to prove their clinical utility. Furthermore, we only investigated a limited number of polymorphisms in our study. Other genes, such as those related to the folate pathway, including TYMS and FOLH1PACSIN2 has previously been identified as influencing gastrointestinal toxicity during consolidation therapy, this is, to our knowledge, the first study showing its association with hematological toxicity in ALL patients undergoing maintenance therapy, a finding consistently detected by different statistical methods. We believe these results warrant further investigation of this and other genetic classifiers (including MTHFR and MTRR genotypes) in larger prospective studies to prove or reject their utility in improving the outcome of thiopurine therapy.In conclusion, although 2) and weekly oral low-dose methotrexate (MTX) (20 mg/m2).ALL patients diagnosed and treated at University Children’s Hospital, University Medical Centre, Ljubljana, Slovenia from 1970 to 2006 were identified through the National Cancer Registry. Of 408 registered patients with childhood ALL, adequate documentation and intact genetic material was obtained from 308 patients. Due to the unsuccessful genotyping of PACSIN2 rs2413739 in 3 patients, these were removed from subsequent analysis, such that the final study group consisted of 305 patients. The following therapy protocols were applied: from 1970 to 1983 the USA Paediatric Oncology Group (POG) protocols were used; from 1983 the therapy was changed to the German Berlin-Frankfurt-Muenster (BFM) protocols . Maintenance therapy was included in all protocols and lasted from 1 to 3 years. The maintenance phase consisted of daily oral 6-MP  which waived the need for written informed consent, since the data was collected retrospectively and was analyzed anonymously. Methods were carried out in accordance with the approved guidelines.We have followed some of the most commonly observed toxic effects during the maintenance therapy, leading to discontinuation of the therapy for longer than one week, a reduction of over 10% of 6-MP dose for a duration greater than 3 months, or hospitalization of the patient and graded them using National Cancer Institute Common Toxicity Criteria (version 2.0), which was valid at the time of medical data collection and classification. Since the criteria for grading leukopenia, stomatitis and secondary tumours in the latest version of toxicity criteria (CTCAE 4.03) have not changed, results would apply also if new toxicity criteria were used. However, we acknowledge the possibly different results when analysing occurrence of infections, since grading differs between the two versions. For the analyses conducted, toxicity grades were used to dichotomize toxicities as “present” versus “absent”. Hematotoxicity corresponding to grade 3 and 4 leukopenia, infections of grade 3 to 4, stomatitis of grade 2 to 3 and secondary tumours of grade 4 were classified as present, while lower grades were considered absent. In addition, associations of polymorphisms and occurrences of dose reductions were investigated. Dose reduction was classified as present when the 6-MP dose was reduced by more than 10% and was of a duration greater than 3 months.Patients were stratified into risk groups according to criteria described previously252627DNA extraction was performed as previously described13The distributions of genotypes and possible deviations from the Hardy-Weinberg equilibrium were assessed using Fisher’s exact test. Odds ratios, 95% confidence intervals (95% CI) and p values, showing the association of the occurrence of adverse effects with studied genotypes, were calculated using logistic regression analysis. All models were adjusted to treatment protocol, age at diagnosis and gender. In addition, for each of the toxicities, models were fit for all possible subsets of covariates, including all of the determined genotypes. The models were then compared using the Akaike Information Criterion (AIC) in order to determine the ‘best’ prognostic model based on the dataIn order to explore the independent and synergistic effects of the studied SNPs on the toxicities, we performed a generalized multifactor dimensionality reduction (GMDR) analysisFor the execution of the CART analysis, polymorphisms as well as haplotypes were used. The maximum tree depth was set to five levels. Attribute selection was based on the information gain (or entropy reduction) criterion. Pruning was set to a maximum depth of 5 levels and splitting was stopped when there were fewer than 10 instances in a node. Leaves with the same majority class were set to recursively merge and post-pruning with an m-estimate (m = 2) was set.Statistical analyses were performed using R (logistic regression), Orange 2.7 (CART)How to cite this article: Smid, A. et al. PACSIN2 polymorphism is associated with thiopurine-induced hematological toxicity in children with acute lymphoblastic leukaemia undergoing maintenance therapy. Sci. Rep.6, 30244; doi: 10.1038/srep30244 (2016).


Increasing evidence suggests that the European human settlement is older than 1.2 Ma. However, there is a fierce debate about the continuity or discontinuity of the early human settlement of Europe. In particular, evidence of human presence in the interval 0.7−0.5 Ma is scarce in comparison with evidence for the previous and later periods. Here, we present a case study in which the environmental conditions at Sierra de Atapuerca in the early Middle Pleistocene, a period without evidence of human presence, are compared with the conditions in the previous period, for which a relatively intense human occupation is documented. With this objective in mind, the available resources for a human population and the intensity of competition between secondary consumers during the two periods are compared using a mathematical model. The Gran Dolina site TD8 level, dated to 0.7−0.6 Ma, is taken as representative of the period during which Atapuerca was apparently not occupied by humans. Conditions at TD8 are compared with those of the previous period, represented by the TD6-2 level, which has yielded abundant evidence of intense human occupation. The results show that survival opportunities for a hypothetical human population were lower at TD8 than they were at TD6-2. Increased resource competition between secondary consumers arises as a possible explanation for the absence of human occupation at Atapuerca in the early Middle Pleistocene. Homo. From this perspective, one of the most interesting subjects is the continuity or discontinuity between the hominins of the Early and Middle Pleistocene in Western Europe and the origin of the Neandertal lineage Out of Africa because of the formation and evolution of significant biogeographical barriers between the two continents, such as the Negev desert Megantereon whitei, a hypercarnivorous predator which, presumably, provided hominins with large amounts of scavengable carcasses The current paleoanthropological scenario shows an increasingly complex human population dynamic for the Pleistocene period. Some phenomena, such as migrations, replacements of species and abandonment of territories with unfavourable conditions for human colonization, seem to have played significant role in the evolution of the genus Homo antecessor, described from level TD6 of Gran Dolina, seems to have inhabited this area at least between the Jaramillo Subchron and the Matuyama-Brunhes Boundary H. antecessor and to describe the human scenario at this early time. In the absence of other data, these fossils have been classified as Homo sp. Currently, human settlement during the Early Pleistocene is represented in Western Europe by a short list of sites, such as Fuente Nueva 3 Mosquera et al. Given the shortage of sites and the sparse anthropic evidence from the beginning of the European Middle Pleistocene, three possible scenarios may be considered. In the first scenario, a discontinuity occurred between the Early and Middle Pleistocene human populations, and thus, Europe was almost depopulated at this time. Alternatively, if continuity between the Early and Middle Pleistocene human populations occurred, the scarcity of evidence might be explained by a contraction of the human populations as a response to adverse environmental conditions. In this latter case, the Early Pleistocene hominins would have survived in some refuges where the environment remained more consistent and eventually evolved into the new Middle Pleistocene forms, both anatomical and culturally. A third possible scenario is that the Early Pleistocene human populations survived in some refuges from OIS18 to OIS14 but were eventually substituted by the Acheulean immigrants approximately 0.5 Ma. Alternatively, it could be argued that the scarcity of evidence of human presence is just a consequence of the low completeness of the fossil record. Although this possibility may not be definitively ruled out, the scarcity of lithic record in this period, in comparison to the previous one, is striking, since stone tools are abundantly produced by humans and they have a high probability of being preserved in the archaeological record.Here, we focus on the critical period 0.7−0.5 Ma to test whether the lack of evidence of human presence is related to adverse ecological conditions during this period. Resource availability and competition with carnivores have been repeatedly proposed as key limiting factors for Early Pleistocene European hominin populations The Gran Dolina site in the Sierra de Atapuerca (in northern Spain) provides a unique opportunity to test this hypothesis on a local scale. This site has a long stratigraphic sequence that dates from the Jaramillo Subchron to the end of the Middle Pleistocene. Evidence of human presence at Sierra de Atapuerca before and after the critical period 0.7−0.5 Ma has been registered at different sites and stratigraphic levels. Human settlement in the 1.2−0.7 Ma period is documented at the TE9 level at Sima del Elefante, and the TD3-TD4 and TD6 levels at Gran Dolina, while the GII, GIIIa, GIIIb units of Galería, the TD10-2 and TD10-1 levels of Gran Dolina and the Sima de los Huesos site document human settlement during the period 0.5−0.25 Ma The aim of this study is to compare competition intensity and resource availability at Atapuerca during the period for which evidence of human presence is lacking, represented by the TD8 assemblage, with the conditions at the end of the Early Pleistocene, represented by the TD6-2 assemblage, a period of intense human occupation at Atapuerca. We introduce in this analysis an index of intraguild carnivore competition to compare the changes in the local conditions between these two periods. These data will provide us which information about the main features of the palaeoecosystem and the survival opportunities for a hypothetical human population at the Sierra de Atapuerca during this period.The Sierra de Atapuerca is located 15 km east of Burgos, in the north of the Iberian Peninsula. This Cretaceous limestone massif has various cavities filled with well-stratified Pleistocene sediments, known from south to north as Sima del Elefante (TE), Galería (G) and Gran Dolina (TD). These sites have been excavated since 1981 by Atapuerca Team with permission of the Consejería de Cultura y Turismo de la Junta de Castilla y León. Gran Dolina (TD)  is a cavity approximately 18 m in height that was filled with Lower and Middle Pleistocene sediments. Its stratigraphic sequence was initially divided into 11 stratigraphic levels (TD1-TD11 from bottom to top) and revised in subsequent studies; e.g., The TD8 level was formed by a succession of brecciated flows of red lutites with boulders and gravels Taking into account the species of carnivores recovered at the TD8 level (see below) and the taphonomic characteristics identified in the faunal assemblage , hyena is proposed as the main agent responsible for the ungulate accumulations The faunal list for TD8 was obtained from Rodríguez et al. Bison voigtstedtensis, Cervus elaphus (red deer), Dama vallonetensis, Equus altidens, Eucladoceros giulii, Hippopotamus sp., Macaca sp., Megaloceros solilhacus, Stephanorhinus etruscus, Sus scrofa (wild boar) and Ursus sp. The bear, Ursus sp., is considered to be an omnivore, and in this study, it was included as both a potential prey and a potential predator. Five large predators were identified in the TD8 assemblage: Canis mosbachensis, Crocuta crocuta (spotted hyaena), Hyaena sp. , Lynx sp., Panthera gombaszoegensis and Ursus sp. The small canid Vulpes sp. was not included in the analyses because its diet was presumed to be composed of small mammals, as discussed by Rodríguez-Gómez et al. Hippopotamus sp., Hyaena sp., Macaca sp., Megaloceros solilhacus, Panthera gombaszoegensis and Sus scrofa using allometric equations. Thus, for these species, we used the body masses provided by Rodríguez Hippopotamus sp., for which the body mass for Hippopotamus gr. H. antiquus provided by Mazza and Bertini Thus, we analysed the entire TD8 fossil assemblage and reviewed the body size estimations provided by Rodriguez Homo sp. has been included in the analyses as a hunter-gatherer to estimate competition and resource availability for a hypothetical human population living in Atapuerca at that time. We assume for Homo sp. the same trophic behaviour inferred for Homo antecessor in the TD6-2 assemblage. Hunting has been interpreted as the main food procurement strategy at TD6-2 HomoAlthough we lack evidence of human presence at TD8, Homo sp.Thus, we analysed the distribution of resources between secondary consumers for two different scenarios and examined the effects of these different configurations of the TD8 paleoecommunity: 1) a TD8 assemblage and 2) a TD8 assemblage + Homo antecessorRodríguez Gómez et al. Homo antecessor as a secondary consumer, because it was not included in Homo were tested to represent either a diet with a low  or high  meat component. In addition, we included two scenarios for TD6-2, adding a large felid to its carnivore guild, although none was recorded in the fossil assemblage. Thus, we repeated our analyses, including Homotherium latidens as a member of the TD6-2 predatory guild, to evaluate the effect of its possible presence on our results, as explained in Rodríguez-Gómez To make the results for TD6-2 and TD8 directly comparable we also ran the model for TD6-2, including We investigated the distribution of resources, primary consumer biomass, among secondary consumers in different scenarios. On the one hand, it is necessary to estimate resource availability, i.e., the biomass of primary consumers available to secondary consumers or total available biomass (TAB). On the other hand, the requirements of secondary consumers (TBD) should also be estimated  [78]. A Our model was developed on the basis of the assumption that all of the variations in population size and composition may be taken as oscillations around a mean value that is constant through time-an assumption that is widely accepted in population dynamics studies - The population should be stable .- The population should be stationary .Sus scrofa and Cervus elaphus, the values of these physiological variables were taken directly from the literature. For species without living representatives, the values reported in the literature for closely related living species of similar body mass were used . For species defined to genus level such as Macaca sp., we used values for species of similar size, as Macaca sylvanus. In the cases of Bison voigtstedtensis, Dama vallonetensis, Eucladoceros giulii, Hippopotamus sp., Stephanorhinus etruscus and Ursus sp., which lack closely related living species of similar size, we computed a least square regression equation for each physiological variable with respect to mean body weight using data for species in the same family; see further details in Supplementary Material, Input data are physical and physiological variables  that are species specific. For species with living representatives, such as The population profiles obtained from this model for every primary consumer population provide estimates of the average sustainable biomass output by age classes, which were eventually translated into body size intervals. Biomass output by age interval was obtained from the annual mortality rates obtained from the Leslie Matrix . Each deOnly female individuals are represented in a Leslie Matrix. Thus, we assumed the sex ratio was equal to 1∶1, that the population profile was the same for males and females and that the survival rate was equal for both sexes. A further assumption is that the sub-adult survival rate should be lower than the adult survival rate m is the body mass in grams.The model solutions are not dependent on population size: thus, an estimate of population density is needed to estimate the sustainable biomass output. Mediterranean taxa dominate the TD8 pollen spectrum Combining the mortality profiles obtained from the Leslie Matrix with the mean body size per age class and the estimated population density of the species, the sustainable biomass output can be computed. Because a carcass includes a variable amount of non-edible tissues , this sustainable biomass output cannot be fully used by secondary consumers. The percentage of non-edible TBO is represented in the model by a size-specific “wastage factor” Our model yields several population profiles for each species, corresponding to different mortality rates. We selected extreme values with maximum and minimum pressure on sub-adults  that produce minimum and maximum TAB levels, respectively . We limited the results to those solutions for which sub-adult mortality rates were higher than adult mortality rates because this is the pattern usually observed in natural populations I is the intake rate in kcal per day and m is the body mass in grams. Some adjustments were made for each secondary consumer according to its inferred dietary preferences. Taking the coyote as an analogue, we estimated that large mammal flesh represented only 20% of the energetic requirements of Canis mosbachensis and that it was primarily consumed as carrion Crocuta crocuta, is able to exploit bone marrow thanks to its bone-crashing abilities. We assumed that approximately 2% of the total energetic requirements of the spotted hyaena were satisfied by bone marrow, and consequently, its energetic requirements were reduced by a factor of 0.02 Hyena sp., by analogy with the striped hyaena, carrion of large mammals would represent approximately 75% of its diet; the remainder could be invertebrates, vegetable food and small vertebrates Lynx sp.), was presumed to be similar to that of the recent Iberian Lynx (Lynx pardina) Ursus sp. was likely to have been highly omnivorous, it was assumed that meat represented only 10% of its energetic requirements as in recent European brown bears (Ursus arctos) Carnivore-demanded resources should be estimated as a first step in evaluating resource distribution among secondary consumers. The carnivore intake rate was estimated using the equation reported by Farlow 2 are obtained multiplying the individual annual intake by the population density  or 60% (Hmax) of its energetic requirements  by consuming the meat of large mammals. Population densities and diet compositions observed for recent hunter-gatherers populations are shown for comparison in The requirements of As in the case of TAB, TDB was distributed over the same six body size categories based on the inferred prey size preferences of each predator, on the basis of the behaviour of their living relatives  [57]. Thij) of each species in each body size category. PPPij represents the relative amount of biomass demanded by the jth carnivore species from the ith prey body size category and is calculated as the proportion of the total amount of biomass demanded from a prey body size category by all carnivores that corresponds to the requirements of a single carnivore species. PPPij incorporates intraguild competition in the model because the resources obtained by a carnivore species depends on the number and characteristics of its competitors. See a detailed formal description of resource distribution computation in Rodríguez-Gómez et al. The distribution of TAB between secondary consumers is based on the proportional predation pressure  that relate the estimated and expected :∑Ds is the summation of estimated densities for all species and ∑Dx is the summation of expected densities for all species.Global competition index (GCI):These indices provide information about competition intensity in the ecosystem with regard to an ideal condition in which all species reach optimal densities. We assume that if the densities estimated from our model approach these thresholds densities, all species would fulfil needs with the have plenty of resources available to them. The SCI index shows to what degree a species fulfils its requirements in a given environment. GCI performs similarly but at the scale of the whole guild of secondary consumers guild. In both cases, however, if the estimated densities were closer to the expected densities or optimal ones, we considered that resources were abundant when compared with requirements (i.e. that competition is low). Conversely, if our model results indicate that the secondary consumers were able to coexist only at low densities, this would be so because there was a scarcity of resources in relation to requirements, which implies a high competition.2*year (TAB-m), and the maximum TAB, corresponding to the minimum sub-adult mortality rate, was 643,968 kcal/km2*year (TAB-M). TAB-m is 23% lower than TAB-M. In addition, the distributions of TAB-M and TAB-m by size category are different  at their ecological densities is 693,725 kcal/km2*year . This enHomo sp. In addition, we considered two other factors: TAB (TAB-m or TAB-M) and Homo sp. requirements (Hmin or Hmax). Thus, we had six different scenarios for TD8 (Homo sp. meeting 60% of its energetic requirements from meat (Hmax) and TAB at a minimum (TAB-m). The scenario with less competition between secondary consumers occurs when TAB is at a maximum (TAB-M). As might be expected, the parameter with the greatest effect is TAB  of the species involved, estimated using equations provided by Silva and Downing Panthera gombaszoegensis while Lynx sp. is the least affected by the competition for resources . Moreover, in the TD6-2 assemblage, the maximum available resources (TAB-M) are higher than the maximum consumer requirements  and thus, all species are able to fulfil their requirements. Meat resources were high enough at TD6-2 to sustain the expected densities, even in excess  is insufficient to satisfy the secondary consumers' requirements . Despite that TAB being lower in TD6-2 than in TD8, TD6-2-TAB meets the carnivores' requirements and TD8-TAB does not. This implies a higher degree of competition between secondary consumers in TD8 than in TD6-2. When the global competition indices are compared  or not (Hmin) for TAB-M condition but not for Hmax with TAB-m. In addition, we evaluated two hypothetical scenarios: the TD6-2 assemblage with a large felid such as Homotherium latidensHomo sp. In the hypothetical scenario of the TD6-2 assemblage with Homotherium latidens and TAB-m, TD8-GCI is similar to TD6-2-GCI with Homo antecessor consuming Hmin. When Homo antecessor is considered to be more dependent on meat resources (Hmax), TD6-2-GCI is slightly higher than TD8-GCI. However, if Homo sp. is included in the TD8 assemblage, both for Hmin and Hmax scenarios, competition increases to a level higher than observed in TD6-2 with Homotherium latidens (Homo is included in the TD8 assemblage with high meat consumption requirements (Hmax). Under these conditions, GCI is 0.35 for TAB-m and greater than 0.2 for TAB-M  obtained for TD8 in comparison to TD6-2 suggest an environment with a higher intraguild competition for resources at Atapuerca 0.6 Ma. Consequently, survival opportunities for Homo at Atapuerca would have been higher in the late Early Pleistocene than in the early Middle Pleistocene. Certainly the value of the population density obtained for Homo at TD8 is higher than the minimum viable population size (MVPS) for these species, according to the equation provided by Silva and Downing Evidence of human presence in Europe during the period 0.5–0.7 Ma is very scarce. Although several European sites provide abundant evidence of human presence before and after this period, the European continent was apparently depopulated during the 0.5–0.7 Ma interval. The results presented here suggest that during this period, the environment was more hostile to a hominin population than it was previously. The Atapuerca environment was able to sustain the expected population densities of a diverse carnivore guild, including Homo antecessor and the large red-toothed shrew Dolinasorex glyphodon, and FU5 is considered the local range zone of Microtus ratticepoides and Blanus cinereusCastor fiber and Mammuthus sp. which do not appear in the TD8 assemblage, and Macaca sp., Megaloceros solilhacus and Hippopotamus sp., which do not appear in TD6-2 assemblage. The rest of the species are equal in both assemblages. As Castor fiber and Mammuthus sp. are approximately equivalent in terms of biomass to Macaca sp. and Hippopotamus sp., respectively (because these species belong to the same body mass categories) the main change between both assemblages is the presence of Megaloceros solilhacus. This species provides biomass to the categories of 45 to 90 kg, 180 to 360 kg and 360 to 1,000 kg. As shown in The differences between TD6-2 and TD8 are due to different community structures. Cuenca-Bescós and García Canis mosbachensis, Crocuta crocuta, Lynx sp. and Ursus sp. (Ursus dolinensis to TD6-2 level) are present in both assemblages. The TD8 level marks the last occurrence of Canis mosbachensis and Crocuta crocuta in the Gran Dolina sequence Homo antecessor and the presence of Panthera gombaszoegensis and Hyaena sp. in the younger assemblage. Panthera gombaszoegensis is recorded at Atapuerca in the lower levels of Gran Dolina in TD3-TD4 and TD5 and also in Sima del Elefante in TE9 and TE12, all of which are older than 0.7 Ma Homo antecessor were considered an effective hunter, the main difference between the TD6-2 and TD8 palaeoecosystems would be the presence of the scavenger Hyaena sp. In addition, it may be assumed that Middle Pleistocene human groups also used scavenging as a feeding strategy and were competitors of Hyaena sp., which might also be considered a partial ecological equivalent of Homo. In this interpretation, Homo would be replaced in the TD8 assemblage by two competitors: Hyaena sp. and Panthera gombaszoegensis. Alternatively, the absence of Homo may be interpreted as a key factor in determining the structure of the TD8 carnivore guild, allowing the presence of certain species that would be competitively displaced if a human population, absent for reasons other than competition, were present in the area. In this alternative interpretation, the absence of human settlement at Atapuerca 0.6 Ma would not be the consequence but the cause of the community structure observed in the TD6-2 assemblage.The differences between TD6-2 and TD8 are larger for the secondary consumer group. HomotheriumHomotherium latidens, a large predator that has been recorded at Gran Dolina in level TD5 (Early Pleistocene) and in level TD10-3 (Middle Pleistocene) Homotherium latidens and a proboscidean species were present at Atapuerca 0.9 Ma, biomass output would have been increased in the heavier body mass categories, but little of this additional biomass could have been consumed by predators. The presence of a proboscidean would increase resource availability carrion, which would have reduced competition among scavengers. Conversely, competition between predators would be increased by the presence of a top predator like Homotherium. Thus, the presence of these species at Atapuerca in the early Middle Pleistocene would change the values of the results presented here, but not their meaning nor our interpretation.Although the results presented here suggest more adverse conditions in TD8 than in TD6-2 other possible explanations for the absence of hominins at TD8 should be considered. The Gran Dolina cave and its sedimentation dynamics constituted a favourable environment for the establishment of carnivore dens and shelters 0.6 Ma Homo preferred mosaic landscapes and that homogeneous habitats were ignored by humans throughout the Palaeolithic. Although the Palaeolithic human groups were able to adapt to a wide range of environments Homo for heterogeneous environments could indicate its inability to directly compete against specialist species, like open habitat cursorial predators With respect to the debate about the continuity or discontinuity of the peopling of Europe in the early Middle Pleistocene, the results presented here show that a discontinuity in hominin occupation existed at Gran Dolina that coincided with an increase in carnivore competition. According to this interpretation, at approximately 0.6 Ma the TD8 palaeoecosystem was not as favourable to maintaining a hominin settlement as it was at 0.9 Ma, and Atapuerca had no conditions that made it favourable as refuge for human populations, thus representing a “bottleneck” Homo were better 0.9 Ma than in later periods, due to higher resource availability and less competition with other carnivores. A faunal turnover occurred in Europe at the beginning of the Middle Pleistocene It is likely, environmental conditions for This dramatic change in the ecological scenario at the beginning of the Middle Pleistocene might be linked to the appearance in Europe of behavioral and technological improvements and innovations that increased the hunting abilities and survival opportunities of hominins. The use of fire for food processing improved the palatability and edibility of foods and increased energy gain 2It is difficult to point towards a unique explanation for the absence of humans from TD8, but what can be said is that survival opportunities for a human population were worse at TD8 than at TD6-2 because intraguild competition was higher. Nevertheless, it is necessary to take into account that the excavation of TD8 affected an area of only 24 mThe structure of the mammalian palaeocommunity and the intensity of competition in the predatory guild likely constrained human ecodynamics in the Pleistocene. This study provides a new avenue for evaluating the relative suitability of a palaeoenvironment for a human hunter-gatherer population, taking into consideration resource availability and competition intensity. Fossil assemblages with and without evidence of human presence may be compared providing a different way to approach the study of variations in human presence in the Palaeolithic. Although this approach does not provide information about the mechanisms that may have produced changes from one community structure to another, it quantifies changes in trophic relationships using the global competition index (GCI). Investigation of more European sites using a similar approach will help to test whether the apparent discontinuity in the peopling of Europe is related to more adverse environmental conditions or decreased survival opportunities at the beginning of the Middle Pleistocene (0.7−0.5 Ma), as suggested by the Atapuerca record, or whether other mechanisms are implicated.Homo in Europe in the 0.5–0.7 Ma interval. Both sites with and without evidence of human presence should be compared to shed light on the fiercely debated question of the continuity vs. discontinuity of human occupation in Europe at the beginning of the Middle Pleistocene. This study demonstrates that mathematical modeling is a helpful tool in addressing this topic.The reconstruction of trophic relationships in palaeocommunities and the comparison of the intensities of intraguild competition, using the global competition index (GCI) presented here make it possible to evaluate the relative suitability of past ecosystems for hominins. Increased intraguild competition made conditions at Atapuerca more adverse for secondary consumers at 0.6 than 0.9 Ma. This increased competition is a possible cause of the apparent depopulation of the Atapuerca area in the early Middle Pleistocene. Nevertheless, more sites of this period should be studied with a similar approach to determine whether increased intraguild competition for resources played a role in changes in the distribution of Table S1Specimen Number.(XLSX)Click here for additional data file.Table S2Sustainable densities of the TD8 secondary consumers for six different scenarios (see text) for maximum and minimum total available biomass (TAB). Estimated density of carnivores , nutritional requirements , total intake , unsatisfied requirements , sustainable density , Species Competition Index (SCI). Total intake is defined as the biomass  consumed by the species after dividing TAB among the secondary consumers, taking into account the distribution of TAB by body size category (category  and the category .(DOCX)Click here for additional data file.Table S3Sustainable densities of TD6-2 secondary consumers for eight different scenarios (see text) for maximum and minimum total available biomass (TAB). Estimated density of carnivores , nutritional requirements , total intake , unsatisfied requirements , sustainable density , Species Competition Index (SCI), Total intake is defined as the biomass  consumed by the species after dividing TAB among the secondary consumers, taking into account the distribution of TAB by body size category (category  and the category .(DOCX)Click here for additional data file.


Emergency departments see an increase in cases during cyclones. The aim of this study is to describe patient presentations to the Emergency Department (ED) of a tertiary level hospital (Townsville) following a tropical cyclone (Yasi). Specific areas of focus include changes in: patient demographics (age and gender), triage categories, and classification of diseases.Data were extracted from the Townsville Hospitals ED information system (EDIS) for three periods in 2009, 2010 and 2011 to coincide with formation of Cyclone Yasi (31 January 2011) to six days after Yasi crossed the coast line (8 February 2012). The analysis explored the changes in ICD10-AM 4-character classification and presented at the Chapter level.There was a marked increase in the number of patients attending the ED during Yasi, particularly those aged over 65 years with a maximum daily attendance of 372 patients on 4 Feb 2011. The most marked increases were in: Triage categories - 4 and 5; and ICD categories - diseases of the skin and subcutaneous tissue (L00-L99), and factors influencing health care status (Z00-Z99). The most common diagnostic presentation across all years was injury (S00-T98).There was an increase in presentations to the ED of TTH, which peaked in the first 24 – 48 hours following the cyclone and returned to normal over a five-day period. The changes in presentations were mostly an amplification of normal attendance patterns with some altered areas of activity. Injury patterns are similar to overseas experience. In Australia over the last 150 years, cyclones (hurricane) are estimated to have caused the deaths of approximately 2000 people, with a financial cost in excess of 6 trillion dollars. making tThe severity of cyclones is rated using a five category system based on wind velocity, with catDuring cyclones hospitals often see a dramatic increase in the number of patients attending the emergency department . As a coThe aim of this study is to describe the patient presentations to the Emergency Department (ED) of a tertiary level hospital (Townsville) following a tropical cyclone (Yasi). Specific areas of focus include changes in: patient demographics (age and gender), triage categories, and classification of diseases.The Townsville Hospital (TTH) is the tertiary referral centre for North Queensland, Australia, which has a population of approximately 700,000 people and an area of 750,000 square km. It also serves an immediate population of approximately 227,000 and is the secondary referral centre for a number of local facilities—the closest of which are all approximately 100 km away [http://www.bom.gov.au/cyclone/history/yasi.shtml#track) [Tropical Cyclone (TC) Yasi was the largest cyclone to cross the Australian coastline in recorded history with a destructive core more than 100 km wide and Category 5 wind speeds . Yasi fol#track) . A littll#track)  Fig 1)..http://wYasi caused considerable disruption to local infrastructure as a result of wind damage and flooding. Part of the population were evacuated, particularly those in low lying areas and 85% of the city lost power for at least 24 hours with approximately 60,000 homes waiting over a week for power to be restored. As a new structure built to cyclone standards, TTH was able to act as a shelter and remain functioning throughout, utilising generator power. The Hospital Mass Casualty plan was activated which included expansion of the ED into the adjacent Women’s and Children clinic area to provide additional space for care of lower acuity patients, all of which are included in the ED attendance data. The timeline of events is shown in Data were extracted from The Townsville Hospital Emergency Department Information Systems (EDIS) for the three periods, 1 January 2009 to 31 March 2009, 1 January 2010 to 31 March 2010, 1 January 2011 to 31 March 2011. The three month period was used to show trend over time.th February 2011. This period includes 2 days before the cyclone crossed the coast (2nd February 2011) to 6 days after (8th February 2011) by which stage patient presentations had returned to normal numbers  study period. A 50% increase was arbitrarily chosen as it was greater than the 40% average change seen in this study and the authors believed that this would be beyond the normal day-to-day variation .Ethics approval was obtained from the Townsville Health Service District Human Research Ethics Committee (HREC) (Number HREC/11/QTHS/77) and James Cook University HREC (number H4230). The data were extracted from the administrative database EDIS by the hospital, the data were anonymized and de-identified prior to being provided to the researchers for analysis. Consent for the patients was not required for this study as the data were de-identified and retrospective and there was no disadvantage to the participant or their relatives or to any collectively involved; also due to the large numbers of patients, it would also be impossible in practice to obtain consent and would prejudice the scientific value of the research.rd- 7th February) and peaked (372 patients) on 4th February. This resulted in mean daily presentations for the study period increasing from 158 (2009) and 162 (2010) to 222 (Yasi) per day.  . When ex2 = .14 p = .933), increase in females presenting the male to female attendance ratio was maintained .There were increased attendances by both males and females, and although there was a slightly larger, but non-significant (Χ2 = .14 p = .003). While the largest group of patients attending was those aged 0–15 years this was consistent across all years and also consistent with the general levels of increased activity. Those aged 16–24 and 25–34 years showed the least increase in attendances and well below that of the daily average, in both comparison years for those aged 16–24 years. The largest proportional increase in attendance was for those aged more than 65 years with levels of importance (based on greater than 50% variation) also reached for the 55–64 year age group. .  increased much less than the general increase and reached levels of significance (Χ= .013). 2 = 52.11 p < .001).  it was most marked in the lower acuity Category 4 and Category 5. This did not have a significant impact on the distribution of attendances by triage categories. There was a trend toward decreased higher acuity presentations (Cat 1–3) and increased lower acuity presentations (Cat 4–5) (Χ< .001). While numbers increased the actual percentage admission and discharge rate remained consistent, as did rates for those who left without being seen or after treatment started. The only change of importance was a decrease in the proportion of patients being transferred to another hospital. The most common diagnostic presentations were consistent across all years. Injury (S00-T98) was the most common in all three years while the next most common were Z00-Z99  and R00-R99 . Together with respiratory, infections, skin problems and genito-urinary presentations these made up approximately 70% of attendances in all years.There were however, differences amongst ICD-10 codes in the level of variation from the average increase in attendances. Looking only at those diagnostic groupings which accounted for more than 2% of presentations, there were proportionally greater increases in attendances by those with skin problems (L00-L99) and factors influencing health care status (Z00-Z99) while there were proportionally less attendances by those with mental and behavioural disorders (F00-F99), diseases of the genito-urinary system (N00-N99) and abnormal clinical and laboratory findings (R00-R99). This study describes the patterns of attendance to an Australian hospital following a tropical cyclone. There was an increase in presentations to the ED of TTH, which peaked in the first 24–48 hours following the cyclone and returned to normal over a five-day period. The 372 attendances on the 4-Feb-2011 were to our knowledge the highest number of presentations in a single day to any ED in Australia at this time, in 2012–13 the average number of presentations per hospital per day were for Australia were 105.7 . This increase in presentations by those reliant on health services is most likely to represent those with existing, or chronic, disease who are also most likely to be the elderly.Virtually all General Practices were closed on the 2The increasing presentations closely correlate with those of advancing age. Most presentations in this grouping reliant on access to health services, were related to closure of community dialysis facilities, loss of home oxygen supplies or inability of home nursing services to continue to visit. This is similar to the US experience following the closure of 94 dialysis facilities along the Gulf Coast. Older peIn this study there were increased presentations with injury but not out of proportion to the general increase. While this is consistent with other developed countries, it was not seen to the same extent in which minor injuries have been observed to constitute over 80% of presentations following tropical cyclones.–22 ThereInfectious disease outbreaks are not a strong feature of tropical cyclones., 25–27 IVibrio species wound infections, mostly in patients with associated co-morbidities, who had been wading in flood-waters.[Cyclones also carry an increased risk of acute respiratory infection., 27 Whild-waters.There were no deaths directly attributed to Yasi in this study. However this study has not looked at the long-term health impacts of tropical cyclones or examined indirect mortality.The findings of this study have implications for disaster preparedness of not just emergency departments but the health system as a whole. Knowledge of the likely patterns of injury and illness following a disaster helps inform not just surge capacity planning but also measures which can be taken in advance to support vulnerable community members and potentially prevent their need to present to hospital. We would argue that adding General Practice expertise to a hospital’s disaster response, where the disaster shuts all services in an areas would be worthwhile addition to the disasters services.This was a single centre study of a single population and for one cyclone. Further studies are needed to see if these attendance patterns are maintained in other Australian populations and whether these differ with the severity of the cyclone involved and level of community preparedness. While this was a single centre study it should be noted that there is only one public hospital ED in Townsville with no other major hospital for over 300 km, which increases the validity of the data and the importance of the findings. This study also describes only the first week of activity following Yasi. Further studies are needed to address issues such as the impact of Yasi on mental health and psychological well-being as well as the potential for delayed presentations of either injury or illness, particularly infectious disease. Potential preparation time may have also impacted on the presentation as Yasi was the second cyclone to have threatened the region in a very short time period. There was also an annual increase in presentation to the TTH ED from 2009 to 2010, which we have tried to adjust for by have a criteria of a greater than 50% increase in cases.There was an increase in presentations to the ED of TTH, which peaked in the first 24–48 hours following the cyclone and returned to normal over a five-day period. The changes in presentations were mostly an amplification of normal attendance patterns with some altered areas of activity. Injury patterns are similar to overseas experience.This research will help to guide future disaster planning arrangements, particularly in the areas of medical devices; medications and access to other services / stockpiles; injury prevention; staffing and support services.


In this paper we describe a method for the determination of protein concentration using Surface Enhanced Raman Resonance Scattering (SERRS) immunoassays. We use two different Raman active linkers, 4-aminothiophenol and 6-mercaptopurine, to bind to a high sensitivity SERS substrate and investigate the influence of varying concentrations of p53 and EGFR on the Raman spectra. Perturbations in the spectra are due to the influence of protein–antibody binding on Raman linker molecules and are attributed to small changes in localised mechanical stress, which are enhanced by SERRS. These influences are greatest for peaks due to the C-S functional group and the Full Width Half Maximum (FWHM) was found to be inversely proportional to protein concentration. Currently, the most widely used method for analysis of low levels of protein in basic research and clinical diagnostics is the Enzyme-Linked Immunosorbent Assay (ELISA). ELISA detection methodologies commonly employ sensitive fluorescence based immunoassays on protein arrays but require lengthy procedures and are hindered by limited multiplexed detection . In terme peaks) .Numerous applications of SERS in biological assays have been reported ranging from multimodal photodynamic and theranostic probes ,6,7,8, Ilinker) are influenced when bound to a target antigen. The change in frequency was attributed to structural deformations which arise due to the binding event between the antibody and SERS active molecule. These structural deformations were modelled as a form of a nano mechanical stress. In this way, it was found that the concentration of the target analyte could be linked to a red shift of specific frequencies of the linker Raman spectrum. Essentially the linker bound to the substrate acts as an immobiliser for the antibody, and at the same time acts as an efficient Raman probe, the signal then being enhanced by the SERS effect.An alternative to direct sensing of biological analytes is to use an indirect approach. In this case a SERS based immunoassay is followed but now the concentration is measured in terms of frequency shifts in the Raman spectra that are related to mechanical perturbations ,27. In tTwo target analytes, used as important biomarkers in human cancer research are p53 protein and Epidermal Growth Factor Receptor (EGFR). Two target analytes, used as important biomarkers in human cancer research are p53 protein and Epidermal Growth Factor Receptor (EGFR). One of the most widely studied DNA-binding proteins, in relation to cancer, is the p53 protein, mutations of which are found in more than 50% of all tumours. The p53 protein is at the centre of the cellular network that protects organisms against the spread of tumours, most of which are related to alteration of p53 expression. Therefore p53 is regarded as a valuable prognostic marker whose detection at high sensitivity may considerably contribute to early diagnosis of cancers ,29. A nuIn this work, we aim to develop robust methods for the determination of p53 and EGFR concentration using high sensitivity SERS substrates in resonance conditions ,29,30,312. Substrates were obtained in batches of 5, packaged in a nitrogen atmosphere to prevent early oxidation. Wildtype recombinant human p53 protein in aqueous buffered solution  and Purified mouse anti human p53 antibody  were obtained from Becton Dickinson and used according to the manufacturers guidelines. No further purification steps were performed. Active human EGFR protein fragment  and Anti-EGFR antibody  were obtained from Abcam and used according to the manufacturers guidelines. No further purification steps were performed.4-Aminothiophenol (4-ATP), 2-Naphthalene thiol (2NT), phosphate buffered saline, NHS , EDC (N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride, CAS 25952-53-8), Glycine (CAS 56-40-6), Ethanol (spec grade CAS 64-17-5) and 6-MP (6 Mercaptopurine), were obtained from Sigma Aldrich, Ireland and used as received. Ag SERS substrates were obtained from Danish company Silmeco . These sSubstrates were first rinsed gently in 100% ethanol, as an initial cleaning step. These substrates were then incubated in either 4-ATP or 6-MP (1 mM) for one hour. The substrates were then rinsed 5 times in 100% ethanol to remove the excess/unbound linker molecules. One substrate with linker molecule only was set aside at this point. 171 mM and 427.5 mM solutions of EDC and NHS were prepared separately in 0.01 M PBS. 20 µL of Antibody (either anti-p53 or anti-EGFR) solution (0.5 mg/mL) was added to 0.5 mL of EDC and 0.5 mL of NHS. The mixture was placed under vortex for 5 min. The substrates were then covered with the EDC/NHS/Antibody mixture and allowed to incubate for 2 h. The purpose of EDC was to activate the carboxyl terminal on the surface of the antibody structure. NHS greatly improves the coupling efficiency of the reaction. After 2 h the bioconjugated substrates  were rinsed with PBS to remove unbound antibody solution. The reaction was then blocked with EDC/NHS activated Glycine (1 mM Glycine made up in 50:50 by volume mixture of EDC/NHS) and left overnight. The substrates were then rinsed with PBS and at this point an antibody only substrate was set aside from the others. A number of concentrations of protein were prepared by serial dilution and a 20 µL of each protein sample was placed on top of the antibody conjugated substrate and left to incubate for 1 h. After 1 h the substrates were rinsed in PBS buffer to remove the excess unbound protein and Raman spectra were measured immediately. All parts of this protocol were performed at room temperature. A schematic for the substrate functionalisation is given in −1 and spectral resolution was approximately 0.8 cm−1. Typical measurements were performed with a 1 s integration time and for each substrate spectra were taken at multiple positions (25) across the substrate. We expect that the substrate surface contains a mixture of free linker and protein-bound linker molecules. By recording a collection of spectra in the form of a large area scan and averaging these spectra, we obtained an average of the response from free linker molecules and protein bound linker molecules. Cosmic ray reduction (when applicable), background corrections, spectrum averaging and Savitzky-Golay smoothing  were carried out using Project 4 software Witec. Normalisation and graphical analysis was performed using Origin version 9.1. All spectra described are plotted relative to the excitation wavelength.Raman microscope SERS measurements were performed in reflection mode with a Raman microscope (Witec Alpha 500) using 785 nm excitation. The detector grating used was 1200 lines/mm. Spectra were collected using an Andor Ixus cooled charge coupled device (−60 °C). A 100× objective lens (NA 0.9) delivered the laser beam and collected the back-scattered light. Rayleigh scattering was blocked with the appropriate notch filter. The laser spot diameter was ~1 µm with a laser power of 0.05 mW (785 nm) used. The Instrument calibration was verified using the maximum signal from a silicon standard at 520 cm−6 M 2-Naphthalene-thiol (2NT) were found to yield a massive improvement in Raman signal intensities and superior reproducibility over a number of competitor and in-house developed substrates. Silmeco silver pillar SERS substrates tested using 10−1 peak, corresponding to the CS stretching mode, which has been assigned previously [−1 [−1 peak for ATP in detail, as in The Raman spectrum of 4-ATP from a 25 point acquisition of an area in eviously . This peusly [−1 ,37 was f−1 peak down to the lowest concentration recorded at 0.25 nM. When one expands the region of interest as in −1 peak. Control data is given in We can qualitatively observe changes in the forward edge of the 1080 cm−1 peak in the 6MP spectra, as shown in Correspondingly, one can examine the influence of EGFR on the 860 cm−1 peak in the 4-ATP spectrum. Peak broadening in Raman spectra has been previously linked to samples under stress. This effect has been studied in the materials sciences, in particular relating to silicon, graphene and carbon where induced mechanical stress and strain effects have been linked to frequency shifts in the Raman peaks of interest and a change in the FWHM [etc., we find that a broadening of the 1080 cm−1 peak occurs at lower protein concentrations, indicating that strain in the system is reduced as the protein concentration is lowered. We believe that such changes in strain are due to changes in steric arrangement of the protein-antibody system, and are shown by broadening of the spectra with decreasing protein concentration. Additionally, the peak centre is red shifted with decreasing protein concentration further indicating reduced mechanical stress in the system. If we overlay the data for ATP-EGFR protein we see that the assay is specific to p53; a similar response for FWHM and peak centre for EGFR-ATP is not obtained. Protein interactions and their resulting structural conformations are very complicated processes. Both p53 and EGFR are comprised of electron rich aromatic lateral chains, but p53 is smaller structurally at 53 kDa mol weight compared to 97 kDa for EGFR. This size difference coupled to the difference between the amino and diazonium binding sites for 6MP and 4-ATP respectively influences the efficiency of the linker to antibody to protein binding on the substrate. Based on our data, we believe that the interactions of linker/antibody/protein systems are very specific and only certain combinations work that result in changes in the SERS spectra. The lowest concentration used in this work compares with previous detection limits reported in previous studies using stress based nano sensors [The FWHM  and peakthe FWHM ,39,40. I sensors ,27.In the 6MP-EGFR system, we see that the system stress resulting from protein–antibody binding also results in a broadening of the FWHM  and follAs further validation of the stress sensor model approach, we can correlate peak centre and FWHM, which as shown in etc. In future work, we plan to investigate other linker protein assays and their combinations, assess the limits of detection and compare to ELISA based methods and finally develop a system for multiplexed detection. By combining sensitive detection of SERRS with the rapid readout capability of Raman based sensing, this method may be applied to the development of a point of care clinical biosensors.We have shown that the influence of protein on the properties of specific peaks in Raman spectra can be measured and used as an evaluation of protein concentration. In particular the measurement of FWHM could be correlated with protein concentration in both data sets tested. We propose that measurements of changes to peak FWHM and centre are immune from system based intensity variations, particularly in the low concentration range for biosensing applications where the intensity based signal to noise is typically low. Utilising high sensitivity SERRS substrates is the key approach in this method, enabling detailed measurements at low concentration ranges. We have found that different protein/linker combinations display changes in mechanical-stress responses and this property can be exploited to enable multiplexed detection. This stress based biosensor could be used for any bio fluid that has a cancer biomarker such as oral, head and neck cancers, gastric cancers, bladder cancers,


Glycemic management is central in prevention of small vessel and cardiovascular complications in type 2 diabetes. With the plethora of newer medications and recommendations for a patient centered approach, more information is necessary to match the proper drug to each patient. We showed that BARI 2D, a five-year trial designed to compare two different glycemic treatment strategies, was suitable for assessing different responses according to different phenotypic characteristics. Treatment with insulin sensitizing medications such as thiazolidinediones and metformin was more effective in improving glycemic control, particularly in the more insulin resistant patient, when compared to the insulin provision strategy using insulin and or sulfonylureas. Triglyceride and high density lipoprotein ratio (TG/HDL-cholesterol ratio) was found to be a readily available and practical biomarker that helps to identify the insulin resistant patient. These results support the concept that not all medications for glycemic control work the same in all patients. Thus, tailored therapy can be done using phenotypic characteristics rather than a “one-size-fits-all approach.” Information of long term clinical trials with combination glycemia-lowering therapy in type 2 diabetes mellitus (T2DM) is scarce. Recommendations for initial glycemic control consist of therapeutic lifestyle changes together with monotherapy, mainly metformin . PersonaThe Bypass Angioplasty Revascularization Investigation 2 Diabetes (BARI 2D) clinical trial compared cardiovascular and diabetes treatment strategies with respect to mortality and cardiovascular events in patients with T2DM and stable CAD , 4. It wDetailed descriptions of BARI 2D have been published , 4. WithIn order to define higher degrees of IR we looked for practical clinical markers, a difficult task in an IR-population with type 2 diabetes that was already receiving intensive medical treatment. We determined the ability of the Adult Treatment Panel III (ATP III) criteria  where meA total of 2368 patients from 49 international sites were recruited. HbA1c was determined at baseline and at monthly and quarterly visits during the study follow-up. The duration of follow-up averaged 4.3 years. Of the 2368 patients, 2204 had baseline data to determine IR as defined above and also both a baseline HbA1c measurement and at least one follow-up value during or after the 6-month clinic visit. We identified a subset of 1308 patients whose baseline HbA1c was above the goal of 7.0%; they were designated as “likely candidates for glycemic improvement,” targeting this cohort for detailed analysis. Most participants were taking multiple prescription medications at study entry, with an average of 1.6 drugs for treatment of hyperglycemia, 2.2 drugs for angina, hypertension or heart failure, and 0.9 for lipids. Medications for glycemic control were extensively used with 54% taking metformin, 19% taking TZDs, 53% taking SUOs, and 28% taking insulin. Over 50% were treated with combination therapy.We calculated the mean follow-up HbA1c for each patient as an “area-under-the-curve” (AUC) average, defined as the area under a line connecting the follow-up HbA1c values measured over time, divided by the length of time between the first HbA1c value starting at the 6-month visit and the last HbA1c value in the BARI 2D follow-up among 2204 patients who had HbA1c measurements available both at baseline and at 6 months or later. The multiple HbA1c values were summarized by calculating a single AUC mean per patient. For most patients, greater than >90%, each contributed somewhere between 5 and 25 HbA1c values to these calculations. The HbA1c values used in this analysis included those recorded in the central laboratory as well as those performed at the local sites. We use AUC mean rather than a conventional mean as the latter would be artificially high for patients who underwent repeated HbA1c tests during periods in which they were known or suspected to have poor glycemic control. Focusing on the 1308 patients who entered the study above goal (baseline HbA1c >7.0%), we used linear regression models to compare AUC average between patients randomized to an IP-strategy and patients randomized to an IS-strategy. The use of regression allowed to control for baseline HbA1c and also to do the comparison within groups defined by IR as defined above and also by other variables such as gender, age, and insulin use.Of the 2368 patients enrolled, 2061 had HbA1c measurements obtained at baseline, 2 years, and with three or more visits in between. n = 1308). Using dyslipidemia measured as TG/HDL ratio as a surrogate for IR, the cohort was divided into 509 patients with a lower TG/HDL ratios  and 799 patients with a higher TG/HDL ratios (elevated) and defined as having IR; 22 members of the latter group were treated with fibrates, niacin, or omega 3 polyunsaturates at baseline. The IR patients were more obese, younger, and with shorter duration of diabetes. There were no differences in gender, blood pressure, or blood pressure treatment at study entry. Differences were found among the ethnic/racial groups with a higher TG/HDL ratio more common in the non-Hispanic White and Hispanic populations, particularly those recruited in Mexico City. The non-Hispanic black population, on the other hand, had a lower TG/HDL ratio.Baseline characteristics are summarized in p = 0.05). In the 1308 patients who had HbA1c values of >7.0 at baseline, a greater reduction in HbA1c was observed in those with an elevated TG/HDL ratio . The importance of the TG/HDL ratio as a marker for higher degrees of IR and increased responsiveness to IS medications is more evident in results shown in The degree of reduction in HbA1c in the IS versus the IP group was analyzed as a function of TG/HDL ratio is shown in p = 0.002). The improved response in the IS-strategy arm was more noticeable among non-Hispanic blacks (p = 0.008) and among patients who were not on insulin therapy at baseline (p = 0.0003). There was no difference by gender, blood pressure, waist circumference, and type of revascularization .The TG/HDL ratio and other factors potentially associated with differences in the HbA1c levels between IP-strategies and IS-strategies during follow-up are also illustrated in Glucose control remains a major focus in the management of patients with diabetes, always in the context of a comprehensive cardiovascular risk factor reduction that includes smoking cessation, adoption of healthy lifestyle habits, blood pressure control, lipid management with priority to statin medications, and, when necessary, antiplatelet therapy. Recommendations for glycemic management are based on evidence-based clinical trial results performed in highly selected patient populations. In clinical practice these recommendations do not always address the range of choices available, nor do they address the vital question of which patient might respond better to which therapy and why, knowing patient-to-patient variability , 8.We analyzed the impact of two different strategies targeting different pathogenetic factors with regard to glycemic management. The IP-strategy consisted in providing more insulin by increasing endogenous secretion through SU or exogenous insulin. The IS-strategy utilized metformin and a TZD aimed at reducing the IR that is commonly present in T2DM. The IS-strategy was superior in lowering HbA1c over the length of the trial, suggesting that IR, more than insulin deficiency, was perhaps the main driver of hyperglycemia. We then tested this hypothesis by using TG/HDL ratios as a biomarker of IR in a cohort of patients who were already under intensive medical management. More than half at study entry were treated with combination therapy, mainly metformin and SUOs and one-third were treated with insulin. From a mean HbA1c of 7.8%, the individuals treated according to the IS-strategy experienced better glycemic control, a difference that was unintended as the study attempted to attenuate this variance in order to compare these two arms on cardiovascular outcomes at similar HbA1c levels.Preventing progression of ischemic heart disease was a major goal in BARI 2D and none of the two strategies had a definitive impact. Rosiglitazone was the most commonly used TZD drug, and despite findings suggesting that rosiglitazone increases the risk of cardiac ischemic events , this waMetformin remains the optimal drug of choice for monotherapy due to the low cost, proven safety record, weight neutrality, and possible benefits on cardiovascular outcomes. Little information is available regarding long term responses to the different medications and or identifying those who are better responders or no responders. Since monotherapy fails to achieve glycemic control in the majority, combination therapy appears to be necessary , but lonβ-cell function to the fasting hyperglycemia ; however, the low precision of the estimates from the model limits its use particularly in a diverse treated population with T2DM. Similarly, the oral minimal model remains cumbersome and will provide different values in different populations and be managed by different treatments.Defining IR clinically remains a challenge, as insulin mediated glucose disposal is distributed continuously throughout the general population without absolute criteria with which to classify individuals as being IR. There is no optimal method to measure insulin resistance or insulin secretion in large clinical studies or in clinical practice. The convenient methods that have been proposed as suitable for large clinical studies have limitations, particularly when studying individuals with diabetes, with different etiologies of glucose dysregulation and or populations of diverse racial and ethnic backgrounds. Comparison of a patient's fasting values with the model's predictions allows a quantitative assessment of the contributions of insulin resistance and deficient Lipids and lipoprotein abnormalities are closely intertwined metabolically with insulin resistance and hyperglycemia. The combination of elevated TG, low HDL cholesterol, and relatively normal LDL cholesterol carried in small, dense, cholesterol-poor LDL particles has been known as diabetic dyslipidemia. People with obesity and insulin resistance have a characteristic dyslipidemia with an overproduction of very low density lipoproteins (VLDL) and hypertriglyceridemia –13. The Having an easy-to-perform practical method of measuring insulin sensitivity can facilitate research, and ultimately one would hope it might help clinicians to better target personalized medicine for patients. Since the gold standard of IR are clamp studies which are not clinically practical, we used TG/HDL ratio as a readily available and useful biomarker of IR. A fixed threshold of TG/HDL ratio of 3.75 in men and 3.00 in women has been previously validated as a marker of IR in nondiabetic individuals , and theβ-cell dysfunction that characterizes T2DM, insulin may be necessary even when the individual appears to be IR [We found the IR-population in this study to be younger, with shorter disease duration and a higher HbA1c, as previously described . These oto be IR . The IP-Preventing progression of ischemic heart disease was a major goal in BARI 2D and rosiglitazone was the most commonly used TZD drug in the study (donated by GlaxoSmithKline). Due to restrictions on rosiglitazone use by the Food and Drug Administration requiring submission of a Risk Evaluation and Mitigation Strategy (REMS), pioglitazone remains now the only TZD prescribed and found to be beneficial when added to existing treatment , 26. WhiEffective treatment of T2DM requires multiple drugs used in combination to correct the multiple pathophysiological defects, should be started early and based on pathogenic abnormalities and not simply on the reduction in HbA1c . HoweverStrengths and Limitations. The major strength of this study is that it was randomized, prospective, and in a large population. It provided information of the phenotypic characteristics of a selective population with T2DM and stable CAD. It also offered information on the effect of glycemic control using two different strategies of combination therapy in a specific population. While we were successful in studying a diverse ethnic/racial populations, the non-Hispanic black population was singular in that the criteria used for IR based on dyslipidemia while being effective were less reliable. Using lipid parameters to predict IR in overweight individuals of other racial/ethnicities is a limitation in this post hoc analysis. Moreover, metabolic abnormalities may differ among racial groups. A further limitation is that the study was performed using “older” agents and many of the newer medications that have different glycemic impact were not studied.The results of our study confirm that BARI 2D was suitable for assessing different phenotypic characteristics and dissimilar outcomes according to different interventions. The use of TG/HDL-cholesterol ratios represents a simple clinical tool that may help identify the IR patient and could help tailor diabetes therapies. Treatment with IS-strategies in the IR-population appears to be more beneficial in reducing glycemia. It is imperative that properly planned randomized long term control studies using combination therapy are performed in order to establish best practices in treating the ever expanding IR-population with T2DM. Coronary artery disease is common in type 2 diabetes population with the dyslipidemia of diabetes. Dyslipidemia of diabetes characterized by elevated triglycerides and low high-density lipoprotein-cholesterol is commonly associated with insulin resistance. Glycemic control is suboptimal in patients with type 2 diabetes, particularly those with insulin resistance. It provides phenotypic characteristics of patients with type 2 diabetes and coronary artery disease and shows a high prevalence of the metabolic syndrome risk factors. Insulin sensitizing therapy is more effective than insulin providing therapy in achieving glycemic control in patients with type 2 diabetes and coronary artery disease. The use of triglycerides/high-density lipoprotein cholesterol ratio is a simple surrogate to help identify individuals that achieve better glycemic control with use of insulin sensitizers. Response to medications for glycemic control differs among patients and they can be chosen according to pathophysiology or phenotypic characteristics rather than using a “one size fits all approach.”


Primary care high-risk prescribing causes significant harm, but it is unclear if it is largely driven by individuals (a ‘bad apple’ problem) or by practices having higher or lower risk prescribing cultures (a ‘spoiled barrel’ problem). The study aimed to examine the extent of variation in high-risk prescribing between individual prescribers and between the practices they work in.Multilevel logistic regression modelling of routine cross-sectional data from 38 Scottish general practices for 181 010 encounters between 398 general practitioners (GPs) and 26 539 patients particularly vulnerable to adverse drug events (ADEs) of non-steroidal anti-inflammatory drugs (NSAIDs) due to age, comorbidity or co-prescribing.Initiation of a new NSAID prescription in an encounter between GPs and eligible patients.A new high-risk NSAID was initiated in 1953 encounters . Older patients, those with more vulnerabilities to NSAID ADEs and those with polypharmacy were less likely to have a high-risk NSAID initiated, consistent with GPs generally recognising the risk of NSAIDs in eligible patients. Male GPs were more likely to initiate a high-risk NSAID than female GPs . After accounting for patient characteristics, 4.2% (95% CI 2.1 to 8.3) of the variation in high-risk NSAID prescribing was attributable to variation between practices, and 14.2% (95% CI 11.4 to 17.3) to variation between GPs. Three practices had statistically higher than average high-risk prescribing, but only 15.7% of GPs with higher than average high-risk prescribing and 18.5% of patients receiving such a prescription were in these practices.There was much more variation in high-risk prescribing between GPs than between practices, and only targeting practices with higher than average rates will miss most high-risk NSAID prescribing. Primary care prescribing safety improvement should ideally target all practices, but encourage practices to consider and act on variation between prescribers in the practice. The study used data routinely recorded in general practitioner (GP) electronic medical records making it possible to examine actual practice on a large scale, and the data used came from practices participating in a national morbidity data set which had all received training and financial support to maintain particularly high quality data.We additionally examined data quality carefully, and restricted analysis to a topic  prescribing) and type of prescription (initiation of NSAID prescribing) where we could demonstrate that data quality was high.All routine data studies share limitations, notably the type and quality of the data recorded, although our choice of practices, topic and type of prescription makes us confident in the findings of this analysis. However, clinical IT systems contain minimal data on GP characteristics and data about practices are restricted to structural characteristics rather than internal organisation, which limits the extent to which we can explain the variation observed.Prescribed drugs are the single most commonly used medical intervention. They deliver significant individual and population benefit, but are also a common source of harm. Approximately 6.5% of emergency hospital admissions in the UK are caused by adverse drug events (ADEs),4Many healthcare safety failures are largely attributable to shortcomings in the environment that individuals work in.There have been a small number of studies of primary care prescribing which have used robust statistical techniques such as multilevel modelling to examine variation,13We have previously shown that high-risk prescribing is common in UK primary care, with an approximately fourfold variation between practices after accounting for casemix.Data were extracted from 38 Scottish general practices via the University of Aberdeen Primary Care Clinical Informatics Unit. We deliberately restricted analysis to these 38 practices because we considered that high-quality data recording was required for an analysis at GP level, and all these practices contributed to an NHS Scotland national morbidity recording data set and had therefore received training in, and financial support for, high-quality data recording. Like almost all UK general practices, these practices all used an electronic medical record which included data on morbidity , demography and prescribing. Patients are only allowed to be registered with one general practice at a time, and that practice has responsibility for all community prescribing including that recommended by hospital specialists who only ever directly prescribe a small number of drugs for community use such as some cytotoxic agents.We chose to focus on oral NSAID prescribing because it is a common cause of harm, and because the decision to initiate NSAIDs is almost completely attributable to the prescribing GP. This is not the case for many other high-risk drugs prescribed such as oral methotrexate in rheumatoid arthritis or antipsychotics in older people with dementia, where specialists frequently recommend initiation. Although the GP who acts on that recommendation takes legal responsibility for the prescription, attribution of the decision to the GP alone is clearly not straightforward. During initial data exploration, we established that the electronic medical record did not record the name of the doctor printed on ‘repeat’ prescriptions (those authorised for reissue by receptionists). We therefore focused on examining initiation of high-risk oral NSAID prescriptions, since initial analysis showed that clinician identifiers were reliably recorded in this context.and the 84 days after the NSAID prescription. NSAID prescribing for these individuals was clearly stated as risky and to be avoided in the March 2005 edition of the British National Formulary which is the most commonly used source of drug advice by UK prescribers. We therefore assumed that the potential risk of NSAID use in such patients was widely known in the time period examined.17The outcome examined was the issuing of a one-off oral NSAID prescription to an individual particularly vulnerable to NSAID ADEs who had not had an oral NSAID prescription in the 12 months before each encounter examined. Oral NSAIDs were defined as all oral preparations of drugs listed in British National Formulary (BNF) chapter 10.1.1 (which does not include aspirin).and the patient was particularly vulnerable to NSAID ADEs at the time of the encounter and had not had an NSAID prescribed in the year before the encounter. We chose to use 2006 because that year had the most practices available who were eligible for the study as described above. Encounters could be in a range of different contexts, including face to face in normal surgery, on home visits or on the telephone. An individual patient could therefore only have high-risk NSAID initiation once during the 1 year study period, and their encounters ceased to be eligible for inclusion after that NSAID initiation.An encounter between a patient and a GP was eligible for analysis if it occurred between 1 January 2006 and 31 December 2006 at a time when the patient was permanently registered with the practice Explanatory variables were at the encounter, GP or patient level. Available data at the encounter level were the encounter type , the number of risk factors for NSAID ADEs that a patient had at the time of the encounter, whether the patient had a ‘relevant diagnosis’ recorded in the encounter ). We additionally fitted variables which were characteristics of the patient having the encounter including: sex, age, socioeconomic status  and the number of encounters each GP had with patients at risk during the year . At practice level, data on structural characteristics were available, namely the number of registered patients , practice remoteness , and remote (≤60 min)), whether or not the practice was accredited for postgraduate training of GPs, whether or not the practice was a dispensing practice, and whether or not the practice holds a General Medical Services contract  or a locally specified contract.In the UK, patients are only allowed to be permanently registered with one practice at a time, but since registration is with the practice rather than an individual GP, they can and do see multiple GPs. The outcome being examined happens in encounters between GPs and patients, but patients typically encounter several GPs and GPs encounter many patients. This means that there is no neat hierarchical clustering of patients with GPs within practices. We explored using several models of varying complexity, including models where encounters were cross-classified between GPs and patients , but these more complex models would not converge. Consequently, the analysis presented here is a three-level hierarchical model of encounters clustered within GPs clustered within practices, which is an approximation to the reality but a useful model for this purpose. Since the outcome is binary (a high-risk NSAID is either initiated in the encounter or not), multilevel logistic regression models with random slopes at GP and practice level were fitted. Assumptions about the normality of higher level residuals were checked graphically. The intraclass correlation (ICC) coefficient at GP and practice level was calculated in the empty model with no explanatory variables to estimate the proportion of variation in outcome that was attributable to variation between GPs and variation between practices. Encounter level explanatory variables were fitted first and multilevel univariate and adjusted ORs of associations between the explanatory variables and high-risk NSAID initiation calculated.GP level can be interpreted as the median difference in the odds of high-risk NSAID initiation if the same patient was to randomly encounter two different GPs in the same practice, and the median OR at practice level as the median difference in the odds of high-risk NSAID initiation if the patient was to randomly encounter two different GPs from different practices. Finally, associations between GP and practice characteristics and high-risk prescribing were examined.Variation between GPs and between practices was then re-examined after adjusting for differences in encounter/patient characteristics. GPs and practices with statistically significantly higher or lower high-risk NSAID prescribing were identified using the GP and practice level residuals. Median ORs at GP and practice level were calculated to provide an estimate of variation on the same scale as the fixed effects ORs.Initial data management and analysis was carried out in SPPS V.21, and multilevel modelling in Stata IC V.11. The NHS National Research Ethics Service had previously approved the anonymous use of these data for research purposes; therefore, this study did not need individual ethics approval.The data set for analysis consisted of 181 010 encounters in the calendar year 2006 between 26 539 eligible patients and 398 GPs in 38 practices. Each patient had a mean of 6.8 (95% CI 6.7 to 6.9) eligible encounters, and each GP a mean of 455 (95% CI 414 to 496) encounters with eligible patients. The median age of included patients was 76 years (IQR 67–82), and 14 062 (53.0%) were women. At the start of the year, the median number of risk factors per patient for NSAID ADEs was 1  and the median number of repeat drugs taken was 4 . A total of 239 (60.1%) GPs were male.During the year of observation, a high-risk NSAID was initiated in 1953 of the 181 010 eligible encounters . Put another way, 1953 of 26 539 patients particularly vulnerable to NSAID ADEs had a high-risk NSAID initiated . At practice level, the high-risk prescribing rate varied from 0.37% to 3.50% of encounters , and at GP level the rate varied from 0 to 20.0% of encounters . The practice level, ICC in the empty model was 0.055 (95% CI 0.029 to 0.102) compared to 0.042 (95% CI 0.021 to 0.083) in the model adjusted for patient/encounter level variables . The GP The median OR at GP level was 2.22 (95% CI 2.00 to 2.50) in the empty model and accounting for patient and encounter characteristics reduced it slightly to 2.06 (95% CI 1.87 to 2.30), while the median OR at practice level was 2.52 (95% CI 2.15 to 3.09) in the empty model and accounting for patient and encounter characteristics reduced it to 2.28 (95% CI 1.98 to 2.76). On average, the likelihood of high-risk NSAID initiation therefore varied approximately twofold if a patient was to randomly encounter two different GPs in the same practice, and only slightly more if a patient was to randomly encounter two GPs working in different practices, again indicating that the greatest variation is between GPs.After adjustment for patient and encounter characteristics, 3 of the 38 (7.9%) practices had statistically significantly higher than average rates of high-risk NSAID initiation, and 2 (5.3%) had statistically significantly lower rates. At GP level, 51 (12.8%) of 398 GPs had statistically significantly higher than average high-risk NSAID initiation, and 10 (2.5%) had statistically significantly lower rates. High-risk NSAID initiation in patients at particularly high risk of NSAID related ADEs occurred in 1.1% of eligible encounters in 2006. In the full multilevel model, high-risk NSAID prescribing was more likely to occur in normal surgery encounters and less likely to occur in encounters with patients with more risk factors for NSAID adverse drug effects, in encounters with older patients, and in encounters with patients prescribed more repeat drugs. These findings suggest that GPs generally perceived the NSAID prescribing examined to be risky because they were on average less likely to prescribe in people at higher risk of NSAID related ADEs. At GP level, male prescribers were more likely to initiate a high-risk NSAID than female GPs (OR 1.73) even after adjustment for casemix. None of the practice structural characteristics examined were associated with high-risk prescribing.After accounting for encounter and patient characteristics, 4.2% of the variation in high-risk NSAID prescribing was attributable to variation between practices . The study is therefore better at quantifying the extent of variation (which was the primary objective of our study) than explaining the variation observed. For example, we do not know whether some GPs were not aware of the risks  or had higher risk tolerance .et alStudies of variation at multiple levels of healthcare systems are relatively rare, with only 12 identified by a recent systematic review of 39 studies using multilevel modelling or other appropriate techniques to examine variation.Key implications are that measurement of high-risk prescribing at GP level is not routinely feasible using electronic data in the UK because electronic data do not reliably record who signed the paper prescription, which is a particular issue for ‘repeat’ prescriptions authorised for regular reissue. For new prescriptions, electronic data are reasonably reliable, but attribution may still be problematic because, although the GP who signs the prescription takes legal responsibility for it, many drugs are initiated on specialist recommendation. Measurement at practice level can identify practices with higher than average prescribing, but most (84.3%) GPs with significantly raised high-risk prescribing and most (81.5%) patients initiated on a high-risk prescription are not in these practices. Although some practices with particularly high rates of high-risk prescribing may need individual support, primary care prescribing safety improvement will therefore most likely have to target all practices. This could take the form of regular feedback of practice rates with facilitation of review of patients receiving high-risk prescribing and examination by the practice of internal variation between GPs to inform an appropriate practice response.Finally, although this study has quantified variation in high-risk prescribing between practices and between GPs, the data available do not allow much exploration of factors explaining such variation. The only GP or practice characteristic associated with high-risk prescribing was GP sex, with male GPs being more likely to initiate a high-risk NSAID than female GPs, but whether this relates to a different casemix beyond what could be controlled for in this study, or greater knowledge gaps about the risk and benefit of drugs, or greater tolerance of risk by men is uncertain. Research is needed to better understand such factors and how these influence prescribing decisions. For example, although high-risk NSAID initiation was less common in people with multiple risk factors for NSAID ADEs implying that GPs were generally aware of increasing risk, actually understanding how risk and benefit are balanced in decision-making using appropriate qualitative  or quantitative  methods would be very useful. Similarly, it is unclear whether practices or GPs have a general tendency to be high-risk prescribers across multiple measures or whether most practices have one or more areas where they are different from average. Finally, there are relatively few studies of interventions to reduce primary care high-risk prescribing, with the best evidence to date for pharmacist-led interventions,