Recent studies suggested an important role for vascular factors in migraine etiopathogenesis. Notch4 belongs to a family of transmembrane receptors that play an important role in vascular development and maintenance. The aim of this study was to test the hypothesis that polymorphisms of the NOTCH4 gene would modify the occurrence and the clinical features of migraine.Using a case–control strategy, we genotyped 239 migraine patients and 264 controls for three different non-synonymous polymorphisms  of the NOTCH4 gene and for the (CTG) n-encoding polyleucine polymorphism in exon 1. Although the analyzed polymorphisms resulted not associated with migraine, the clinical characteristics of our patients were significantly influenced by the different NOTCH4 genotypes. Longer duration of disease and severity of neurovegetative symptoms during headache attacks were associated with the R1346P and G835V polymorphisms, respectively. In female patients, worsening of migraine symptoms at menarche was significantly correlated with T320A polymorphism.Our study shows that genetic variations within the NOTCH4 gene significantly modify the clinical characteristics of migraine and may have a role in disease pathogenesis. Migraine is a chronic and disabling neurovascular disorder that affects approximately 15% of the general population in Western Countries .Migraine etiology is complex and multifactorial, involving both genetic and environmental factors that interact in a manner yet to be completely understood. Mutations in CACNA1A, ATP1A2 and SCN1A genes have been associated with familial hemiplegic migraine (FHM), a rare monogenic form of migraine . RecentlAlthough the pathophysiology of migraine is not fully understood, cortical spreading depression (CSD), a depolarization wave that propagates across the brain cortex and activates the trigeminal nerve in animal models, is speculated to cause the neurological symptoms typical of migraine . It has NOTCH4 gene is located at 6p21.3, in the major histocompatibility complex (MHC), a chromosomal region intensively investigated in migraine ,13. The Notch4 belongs to a family that in humans include four receptors (Notch1-4), binding with different ligands, such as Jagged1, Jagged2, Dll1 and Dll4 . MutatioThe aim of the present work was to evaluate the association between migraine and the NOTCH4 gene. We studied several polymorphisms of the gene in a large cohort of Italian migraine patients recruited from a University-based Headache Center and in healthy controls.A group of 239 consecutive unrelated migraine patients , recruited from the Headache Center of the University of Torino , were selected for the study. The diagnosis of migraine was made, according to the ICHD-II criteria ,21.http://www.ncbi.nlm.nih.gov/), are located in regions of putative functional relevance.We examined three non synonymous polymorphisms  and the (CTG) n-encoding polyleucine polymorphism in exon 1 for NOTCH4 gene. All polymorphisms, selected from SNPs database of NCBI (http://www.softgenetics.com).Genomic DNA was extracted from 200 μl of peripheral blood using the QIAamp DNA mini Kit (QIAGEN). PCR reactions were performed with 90 ng of genomic DNA, 0.4 unit of Taq Gold DNA polymerase, 250 nM of each primer, 1.5 mM MgCl2 and 50 mM dNTPs. For non synonymous polymorphisms, PCR conditions used were: an initial denaturation at 95°C 10 min followed by 35 cycles 95°C for 1 min, annealing specific temperature for each couple of primers for 1 minute, then at 72°C for 1 min with a final extension at 72°C for 5 min. PCR products were electrophoresed on a 2% agarose TBE 1X gel and stained with ethidium bromide. All non synonymous polymorphisms were analyzed by enzymatic digestion. Genotyping for T320A, G835V, R1346P polymorphisms was performed by digesting PCR products with BsuRI, XmiI, SmaI restriction enzyme, respectively. Conversely, (CTG)n polymorphism was performed on ABI 3700 sequencer ; PCR product was obtained using the same mixture previously described and using the following primers: forward: 5- CCTGCCTGAAGAGGGACAG-3, reverse: 5-CCCCACTGATCATCCTCCTA-3. The forward primer was fluorescently labeled with FAM. Alleles were scored using GeneMarker v1.5 software package (SoftGenetics LLC – χ2 test and Monte Carlo Markov chain method (MCMC), were used to compare allelic (AF) and genotypic frequencies (GF) between cases and controls. Patients clinical characteristics were analyzed with ANOVA followed by Bonferroni correction for multiple comparisons. Statistical analysis was performed using Statistical Package for Social Sciences – version 20 . The Hardy-Weinberg equilibrium was verified for observed genotype frequency in all tested populations in order to detect deviation from the expected genotype distribution and to detect genotyping errors. The level of statistical significance was taken at p<0.05.Fisher’s exact test, Table Table When we divided the migraine patients into different subgroups , according to IHCD-II criteria, no significant difference in the distribution of the genetic variants was found.Finally, we compared the clinical characteristics  of our migraine patients according to different NOTCH4 genotypes. Interestingly, we found that the carriage of the allele T of the G835V polymorphism was associated with a higher frequency of vomiting , while carrying the allele C of R1346P polymorphism correlated with a longer duration of disease . When we analyzed our patients according to gender, we found that in females there was a significant association between the allele A of T320A and worsening of symptoms at menarche . When patients were divided into migraine with aura and without aura subgroups, no significant difference was found.To the best of our knowledge, this is the first study that investigated the association between migraine and the NOTCH4 gene. We found that the analyzed polymorphisms are not significantly associated with the disease. Genotypic and allelic frequencies of the examined polymorphisms were similarly distributed between cases and controls. When patients were divided into migraine with aura and without aura subgroups, no significant difference was found. It is unlikely that genetic variations within the NOTCH4 gene contribute greatly to migraine susceptibility. In addition, we assessed the relation between different NOTCH4 genotypes and the clinical characteristics of migraine. Intriguingly, we found that several characteristics of the disease, like the presence of neurovegetative symptoms, the duration of the disease and the worsening of symptoms at menarche were influenced by NOTCH4 genotypes. Our data support a role for NOTCH4 as a disease-modifier gene in migraine.There are some limitations in our study that deserve mention and suggest caution in the interpretation of the results. First of all, we performed our study in a sample of patients recruited from an Headache Center that may not be representative of the general migraine population . In addiOur data are in accordance with several studies suggesting a significant involvement of vascular mechanisms in migraine etiopathogenesis. First of all, migraine symptoms are included in the phenotype of several genetic vasculopathies like cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy (CADASIL), retinal vasculopathy with cerebral leukodystrophy (RVCL) and hereditary infantile hemiparesis, retinal arteriolar tortuosity and leukoencephalopahty (HIHRATL) . It is oHow genetic and acquired vascular mechanisms might be involved in migraine pathogenesis is still matter of investigation. A dysfunction in endothelial function has been suggested as a key finding in migraine pathogenesis. The endothelium regulates numerous vascular functions. In response to specific stimuli, endothelial cells secrete local vasoactive mediators, including the vasodilator nitric oxide (NO) and vasoconstrictor endothelin-1 (ET-1) and tissue-type plasminogen activator (t-PA). Several observations suggest that endothelial function is abnormal in migraine patients, including an increased prevalence of anti-endothelial cell antibodies that may induce endothelial damage and raised plasma levels of ET-1 and von Willebrand factor . FinallyIn conclusion, we found no evidence for a strong association between the NOTCH4 gene and migraine and our data does not support the hypothesis that the NOTCH4 gene is a major genetic risk factor for migraine. However, due to the relatively small number of subjects examined, replication studies in larger samples are required in order to confirm our data. Intriguingly, we found that genetic variants within NOTCH4, a gene involved in vascular maintenance and remodeling, significantly influence several clinical features of migraine. Our results further support a role for vascular genetic factor in the disease. Finally, we can hypothesize that the NOTCH4 gene may be particularly involved in some migraine subtypes. Additional studies in families segregating an atypical migraine phenotype are warranted to test this hypothesis.The authors declare that they have no conflicting interest.ER and IR conceived and supervised the project and drafted the manuscript. PDM, MLG, SG, and SG were responsible for migraineurs patient collection. PF and SB carried out the molecular genetic studies. FG and AV performed the statistical analyses. LP edited the manuscript for intellectual content. All authors read and approved the final manuscript.


Rhinolophus sinicus are characterized by unusual relative distributions in which R. s. septentrionalis is restricted to a small area within the much wider range of its sister taxon R. s. sinicus. To determine the history of lineage divergence and gene flow between these taxa, we applied phylogenetic, demographic and coalescent analyses to multi-locus datasets. MtDNA gene genealogies and microsatellite-based clustering together revealed three divergent lineages of sinicus, corresponding to Central China, East China and the offshore Hainan Island. However, the central lineage of sinicus showed a closer relationship with septentrionalis than with other lineages of R. s. sinicus, in contrary to morphological data. Paraphyly of sinicus could result from either past asymmetric mtDNA introgression between these two taxa, or could suggest septentrionalis evolved in situ from its more widespread sister subspecies. To test between these hypotheses, we applied coalescent-based phylogenetic reconstruction and Approximate Bayesian Computation (ABC). We found that septentrionalis is likely to be the ancestral taxon and therefore a recent origin of this subspecies can be ruled out. On the other hand, we found a clear signature of asymmetric mtDNA gene flow from septentrionalis into central populations of sinicus yet no nuclear gene flow, thus strongly pointing to historical mtDNA introgression. We suggest that the observed deeply divergent lineages within R. sinicus probably evolved in isolation in separate Pleistocene refugia, although their close phylogeographic correspondence with distinct eco-environmental zones suggests that divergent selection might also have promoted broad patterns of population genetic structure.Closely related taxa living in sympatry provide good opportunities to investigate the origin of barriers to gene flow as well as the extent of reproductive isolation. The only two recognized subspecies of the Chinese rufous horseshoe bat  Closely related taxa that have sympatric or parapatric ranges provide good opportunities to investigate the origins of barriers to gene flow and the extent of reproductive isolation. Although gene flow has a powerful homogenizing effect that can hamper the divergence of populations into new taxa As with many temperate taxa, tropical and subtropical species underwent radical range shifts during Pleistocene glacial-interglacial cycles Rhinolophus sinicus is a common species in East Asia with a wide longitudinal range that encompasses the Qinghai-Tibetan Plateau. Its sister species, R. thomasi and R. rouxii, occur further south and west, respectively, and together with a recently described cryptic species of R. rouxii from South India (R. indorouxii sp. nov) rouxii-group R. sinicus was regarded as a subspecies of R. rouxii based on craniodental features The horseshoe bats (family Rhinolophidae) number over 70 species and show highest diversity in the Old World tropics and subtropics R. sinicus is currently recognized as consisting of just two subspecies . Bats were captured using mist nets at cave exits, and the bats were measured, sampled and subsequently released R. sinicus from 41 localities across the Chinese mainland, including the known range of septentrionalis in Yunnan Province, and the offshore Hainan Island , three autosomal , one X-chromosomal (USP9x) and one Y-chromosomal (Dbx) genes. Information on the samples and primers for these markers are summarized in We amplified and sequenced two mitochondrial genes . Heterozygous sites in the nuclear gene sequences were identified by the presence of clear double overlapping peaks in both the forward and reverse sequence chromatograms. Haplotypes were resolved unambiguously if there was only one heterozygous site, and, for sequences with more than one heterozygous site, we resolved haplotypes probabilistically using PHASE version 2.1 Cytb and concatenated mtDNA sequences (Cytb and ND1). To investigate the relationship between the two recognized subspecies of R. sinicus we also incorporated Cytb data from two outgroups: R. thomasi from the rouxii-group, and R. affinis from a different clade (accession numbers FJ85215 and DQ297582 respectively). To establish phylogenetic consensus we implemented maximum parsimony (MP) and neighbor joining (NJ) in PAUP* 4.0b10 Cytb and HKY+G [G = 0.102] for concatenated mtDNA. For BI, we performed two simultaneous runs of Metropolis-coupled Markov chain Monte Carlo (MCMC) analysis using the chosen molecular evolution model parameters, each comprising four chains and 10 million generations. Trees and parameters were sampled every 100 generations, and the first 25% of the sampled trees were discarded as a burn-in. For NJ and ML, node supports were estimated based on 1000 bootstraps. The best topology was compared with several alternative candidate hypotheses using PAUP and CONSEL Phylogenetic analyses were performed for both Cytb and concatenated mtDNA haplotypes using the maximum parsimony method implemented in TCS version 1.21 At the intraspecific level, bifurcating trees may not be the most appropriate way to represent genealogical relationships among haplotypes Cytb) clade  for each mtDNA . We tested the following three competing scenarios: (1) septentrionalis arose recently via divergence from Central sinicus following a more ancient divergence between the common ancestor of these taxa, and lineages of sinicus from the East and Hainan. We also incorporated a bottleneck early in the history of septentrionalis, because of its small population size. This scenario corresponds approximately to our hypothesis 2 in the Introduction. Scenario (2) is the same as (1) but without the bottleneck. In scenario (3) septentrionalis was considered the ancestor of all R. sinicus with a subsequent divergence between Central sinicus and East/Hainan sinicus  with the thermal profile: initial denaturation step at 95°C for 15 min, followed by 35 cycles of 30 s at 94°C, 30 s at (55–66°C) and 30 s at 72°C and final extension step at 72°C for 20 min. Sample information for each locality is summarized in Nine primer pairs developed for Only individuals genotyped at over seven loci were used in subsequent analyses. Data were checked for possible null alleles and genotyping errors with Micro-checker v2.2.3 6 iterations and with an initial burn-in of 105 iterations per run) and used the admixture model. The most likely number of clusters was inferred based on the method described in 6 iterations and a burn-in of 10%.To assess the relationship among populations, we used STRUCTURE 2.2 Finally, we repeated ABC analyses for microsatellite data, again using DIY ABC v1.0.4.37 septentrionalis and adjacent populations of sinicus, we used the isolation with migration (IM) model Cytb, Chd1 and SWS1, and only nonrecombined regions were used in the subsequent IM analyses.To test for gene flow between Cytb), (ii) combined ncDNA , (iii) combined mtDNA and ncDNA, and (iv) microsatellites. To incorporate differences in the effective population size of the markers, we set inheritance scalars as follows: 0.25 for Cytb, 0.75 for the X-linked locus USP9x and 1 for the three other nuclear genes  and also for the microsatellites. Scaled directional migration rates  were estimated between septentrionalis and adjacent populations of sinicus. Preliminary runs with large parameter intervals were used to determine the starting values of prior distributions for the subsequent runs. Three final runs were conducted with different random seeds and a single chain with a burn-in of 107 steps for 2×108 iterations. For the sequence datasets, each run included five MCMCs with ten multiple chain-swapping attempts, and a geometric heating scheme with the heating terms h1 set to 0.05. For the microsatellites dataset, each run included eight MCMCs with 25 multiple chain-swapping attempts, and a geometric heating scheme with the heating terms h1 and h2 set to 0.05 and 2.IM analyses were performed for four datasets: (i) mtDNA  from 165 individuals contained 56 haplotypes with 66 polymorphic sites, and the alignment of ND1 (957 bp) from 59 individuals contained 28 haplotypes with 88 polymorphic sites. No premature stop codons were observed in either of these genes, suggesting they are functional mitochondrial genes rather than nuclear copies. When concatenated, the mtDNA alignment spanned 1448 bp, and data from 59 individuals contained 40 haplotypes with 124 polymorphic sites.The alignment of R. sinicus is a monophyletic grouping to the exclusion of the closely related congener R. thomasi )]; AU test, P<0.001; SH test, P = 0.03). Consistent with the tree results, network analysis for concatenated mtDNA recovered three networks with a 95% connection limit, corresponding to clade I, II and III+IV, respectively  and for all Central sinicus (clade III) were similar to each other, both estimated to be around 0.3 MYA. This date was more recent than that of the East sinicus (clade I) but older than Hainan (clade II)  from 94 individuals included 5 haplotypes based on 4 polymorphic sites and 11 indels. The alignment of SWS1 sequences (510 bp) from 66 individuals included 33 haplotypes based on 32 polymorphic sites and 20 indels. The alignment of THY (381 bp) from 61 individuals included 12 haplotypes based on 6 polymorphic sites and 10 indels. The alignment of USP9x sequences (591 bp) from 56 individuals included 12 haplotypes based on 12 polymorphic sites. Levels of nucleotide diversity were not consistent across four ncDNA markers  which was deleted in the subsequent analysis. We detected no significant departure from Hardy-Weinberg equilibrium after Bonferroni correction, although significant departure from linkage equilibrium was detected between SF77 and SN30 in several locations of AJ and JF.septentrionalis, and thus private to this subspecies.Genetic diversity decreased broadly from the west  to the east (i.e. East and Hainan) see . Privatesinicus were firstly separated from others at K = 2 , although septentrionalis was separated from the Central sinicus at K = 4  to the Central population of sinicus. In contrast, very restricted or no gene flow was detected between Yunnan and Central in either direction when IM analyses incorporated microsatellite data or ncDNA sequences, either alone or with mtDNA  will be needed to confirm this suggestion in the future. Therefore, of our three original hypotheses proposed to account for the origin and restricted range of septentrionalis nested within sinicus, we are able to reject the first scenario that the current taxonomy is incorrect, and so must consider the alternative explanations.Morphological, echolocation and nuclear DNA sequence data collected from sinicus, corresponding to Central China, East China and the offshore Hainan Island. However, contrary to morphological data, the Central lineage showed an unexpected closer relationship with septentrionalis than with other lineages of sinicus. The paraphyletic arrangement of sinicus with respect to septentrionalis could arise if, as stated in Hypothesis 2, the latter subspecies had recently evolved in situ from a Central lineage of its more widespread sister subspecies. However, phylogenetic discordance can also result from other processes such as incomplete lineage sorting of ancestral polymorphism or past mtDNA introgression septentrionalis.MtDNA gene trees and microsatellite-based clustering together revealed three divergent lineages within the subspecies sinicus mtDNA paraphyly. First, the mtDNA haplotypes causing discordance were not distributed randomly across the range of sinicus as would be expected if this had been due to stochastic incomplete lineage sorting sinicus, then we might expect to see similar patterns in the genealogies of nuclear genes given their larger effective population sizes Chd1 and SWS1 . From the former analysis, we found that contrary to expectations, the estimated species tree showed septentrionalis to be ancestral to sinicus . This latter period coincides with the penultimate glacial period of Pleistocene septentrionalis, and one in the East by both high mtDNA diversity and an ancient TMRCA estimate for bats in this region. Similarly, the Central area harbours high genetic diversity (mtDNA and ncDNA), shows morphological distinctiveness Accepting an ancient origin of inan see . SimilarR. sinicus, the observed strong genetic differentiation among lineages might have also been caused, at least in part, by geological and ecological differences between regions associated with the dramatic uplift of Qinghai-Tibetan Plateau (QTP) in the Quaternary septentrionalis corresponds to the edge of step 1, the Central sinicus population to step 2, and the East sinicus population to step 3. Because each period of uplift of the QTP had different effects on different regions of East Asia Given the distribution of Figure S1Graphic representation of the three scenarios for the history of lineage divergence used in the DIY ABC analysis. Graphs show the logistic regression, showing the posterior probabilities of three tested scenarios. N5 corresponds to a bottleneck lasting a time of db generations.(EPS)Click here for additional data file.Figure S2Maximum parsimony tree and network based on Cytb haplotypes including one sequence of R. thomasi. For the tree, node support is indicated with MP, NJ and ML bootstrap values and Bayesian posterior probabilities . For the network, each circle represents a single haplotype and the circle size is scaled by the haplotype frequency. Filled black circles represent missing or unsampled haplotypes. One unfilled circle represents haplotype of R. thomasi. The numbers in the network represent mutational steps between haplotypes.(EPS)Click here for additional data file.Figure S3The plots of the log-likelihood value L(K) (a) and delta K value (b) based on ten runs for each values of K from 2 to 8.(EPS)Click here for additional data file.Figure S4Geographic distributions of genetic clusters identified by GENELAND at K = 4.(EPS)Click here for additional data file.Figure S5Geographic distributions of genetic clusters identified by TESS at K = 4.(EPS)Click here for additional data file.


Preclinical data support further investigation of ascorbic acid in pancreatic cancer. There are currently insufficient safety data in human subjects, particularly when ascorbic acid is combined with chemotherapy.14 subjects with metastatic stage IV pancreatic cancer were recruited to receive an eight week cycle of intravenous ascorbic acid (three infusions per week), using a dose escalation design, along with standard treatment of gemcitabine and erlotinib. Of 14 recruited subjects enrolled, nine completed the study (three in each dosage tier). There were fifteen non-serious adverse events and eight serious adverse events, all likely related to progression of disease or treatment with gemcitabine or erlotinib. Applying RECIST 1.0 criteria, seven of the nine subjects had stable disease while the other two had progressive disease.These initial safety data do not reveal increased toxicity with the addition of ascorbic acid to gemcitabine and erlotinib in pancreatic cancer patients. This, combined with the observed response to treatment, suggests the need for a phase II study of longer duration.NCT00954525Clinicaltrials.gov  Pancreatic cancer survival continues to be amongst the shortest of all cancers, and new therapies are urgently needed. The majority of patients with pancreatic cancer already have metastatic disease at clinical presentation Ascorbic acid  in cancer care has had a labyrinthine history Since then, renewed interest in ascorbic acid and cancer treatment arose serendipitously from clinical pharmacokinetics studies of ascorbic acid in healthy adults Detailed pharmacokinetics studies in humans and animals have confirmed that intravenous ascorbate in pharmacologic doses can produce peak plasma concentrations that are several hundred fold higher than those possible from maximal oral doses There are limited human data on the use of pharmacologic ascorbate, despite surprisingly current wide use by practitioners of complementary and alternative medicine Pancreatic cancer is sensitive to pharmacologic ascorbate both in vitro and in animal models Given what is known about the relative safety of pharmacologic ascorbate and its potential for efficacy, coupled to the pressing need for new treatments, we conducted a phase I trial of intravenous ascorbate added to gemcitabine and erlotinib in patients with stage IV metastatic pancreatic ductal adenocarcinoma with the specific primary aim of assessing safety and the secondary aim of assessing response to treatment. We investigated adverse events, measured peak plasma ascorbic acid concentrations after infusions, and conducted imaging pre- and post-treatment.This research was approved by the Thomas Jefferson University Institutional Review Board. Written informed consent, approved by the Thomas Jefferson University Institutional Review Board, was received from all patients who participated in the study. Clinical Investigation was conducted according to the principles expressed in the Declaration of Helsinki.This was a phase I study of patients with histologically or cytologically confirmed metastatic stage IV pancreatic ductal adenocarcinoma, conducted at the Thomas Jefferson University and Hospital between July, 2009 and July, 2011. The study was an open-label, dose-escalating trial that utilized a 3+3+3 design see . The proThe following inclusion and exclusion criteria were used for subject recruitment into the study. Patients had newly diagnosed, stage IV pancreatic ductal adenocarcinoma, were not eligible for surgical resection, and had not yet been treated. The diagnosis was established by histology or cytology. Patients had an Eastern Cooperative Oncology Group (ECOG) Performance Status of 0–2. Laboratory requirements were an absolute neutrophil count ≥1,500/mm3, hemoglobin >8 g/dL, platelet ≥100,000/mm3, total bilirubin ≤1.5 mg/dL, creatinine ≤2.0 mg/dL, transaminases ≤2.5× upper limit, urine uric acid <1,000 mg/d, urine pH<6, and urine oxalate <60 mg/d. Patients were excluded if they had documented glucose-6-phosphate-dehydrogenase (G6PD) deficiency or a history of oxalate renal calculi, since high levels of ascorbic acid are known to cause hemolysis in G6PD deficient individuals and kidney stones in those patients with a preexisting risk for oxalate stones. Patients were excluded if they were currently receiving chemotherapy or radiation therapy or enrolled in other trials currently or in the preceding 1 month. Patients with co-morbid condition that would affect survival such as end stage congestive heart failure, unstable angina, myocardial infarction within 6 weeks of study, or chronic active hepatitis or cirrhosis also were excluded.2 over 30 minutes, on day 1, once weekly for 7 weeks followed by a 1 week rest. Erlotinib was given orally in a single daily dose of 100 mg per day for eight weeks.All subjects meeting eligibility criteria were enrolled to receive gemcitabine, erlotinib, and intravenous ascorbate as first line treatment. Gemcitabine was administered intravenously at a dose of 1000 mg/mData were obtained regarding laboratory values and adverse events throughout the treatment period. Ascorbic acid concentrations in the serum were measured by HPLC with coulometric electrochemical detection Ascorbic Acid Injection USP was supplied by Bioniche Pharma (Rosemont IL) as sterile solution of ascorbic acid in water for parenteral use. The product was supplied in sterile 50 mL single use glass ampoules. Each mL contained ascorbic acid 500 mg (2.84 mmol), edetate disodium 0.025%, and water for injection with pH (range 5.5 to 7.0) adjusted with sodium bicarbonate, hence providing ∼2.84 mmol sodium and an osmolality of 2.84+2.84 = 5.7 mOsm/mL.To meet the goal of 9 patients completing the trial, 14 patients were recruited from July, 2009 to July, 2011: 4 males and 10 females, mean age of 64.4±10.0 (range 47–81). All patWhen patients received intravenous ascorbic acid, they frequently reported mild lightheadedness or nausea which was expected from the osmotic load and resolved with eating and drinking. Overall, for the total cohort of 14 patients, there were 23 total adverse events with 8 being serious adverse events. The adverse events are shown in Ascorbic acid concentrations were measured immediately after infusion end in the six patients receiving the two upper dosage tiers of either 75 g or 100 g per infusion see . MillimoTo assess treatment response by imaging, all nine patients who completed the protocol underwent pre- and post-treatment CT scans or PET-CT scans. Scans were evaluated, by an expert radiologist blinded to the clinical conditions of the patients, for the change in size of the primary tumor  as well The goals of this phase I trial were to provide an initial safety evaluation of ascorbic acid added to gemcitabine and erlotinib in patients with stage IV pancreatic cancer, to measure whether predicted ascorbic acid concentrations could be achieved, and to preliminarily assess any response to treatment. In the nine patients who completed the study, ascorbic acid concentrations were reached safely and with minimal associated adverse events that could be attributed to ascorbic acid. Overall, the safety data do not reveal adverse events other than what might be expected for progression of pancreatic cancer and/or treatment with gemcitabine and erlotinib. Deaths of three patients who died before completing the study were attributable to underlying and rapidly advancing disease, as affirmed by the Data Safety and Monitoring Board.Peak ascorbic acid concentrations were achieved as high as 30 millimoles/L in the highest dose group. These concentrations are similar to reported concentrations in patients who received ascorbic acid intravenously without concomitant chemotherapy CT images at the beginning and end of 8 weeks of treatment revealed that primary tumor size (target lesion) decreased in 8 of 9 subjects; was stable in the one subject who did not have a decrease; and specifically decreased in the three subjects who received the highest ascorbic acid dose see . ClinicaThe behavior of non-target lesions also was concordant. In the highest ascorbate dose group, non-target lesions were either improved or stable, and 7 of 9 patients who had pre and post treatment CT scan evaluations had stable or improved non target lesions. However, since 3 additional patients died from rapid progression of the disease the overall result would suggest that 7 of 12 patients had stable disease. The data are consistent with observed synergy between gemcitabine and pharmacologic ascorbate in cell and animal experiments increase in target lesions decrease in 8 of 9 subjects may be underestimated. RECIST 1.0 criteria for partial response require that there be at least a 30% decrease in target lesions without definitive increase in non-target lesions. In addition, many efficacy studies of gemcitabine based therapies for pancreatic cancer patients include both metastatic and locally advanced patients It is noted that RECIST 1.0 criteria for stable disease are inclusive of a 19% There were other potential issues with properly capturing the efficacy signal of ascorbic acid plus gemcitabine and erlotinib in this trial design. Ascorbic acid may act differently than classic cytotoxic chemotherapy. In particular, unlike many cancer therapies, ascorbate does not appear to have toxicity on rapidly dividing normal cells such as those in intestine cells, hair follicle cells, and bone marrow. Because of the absence of apparent tissue toxicity, effects of ascorbic acid treatment on human tumors might be expected to be more gradual, and as a corollary to require longer treatment. This possibility is consistent with observations from case reports of patients who received intravenous ascorbic acid as treatment for several types of cancers Given the possibility that longer ascorbic acid treatment is necessary to see disease improvement by RECIST 1.0 criteria, and the somewhat encouraging findings in the nine subjects in this trial, studying a longer treatment period at the 100 gram dosage seems warranted. Although our determination of progression free survival and overall survival were comparable to values previously reported for gemcitabine/erlotinib therapy alone Protocol S1Trial Protocol.(PDF)Click here for additional data file.Checklist S1CONSORT Checklist.(DOC)Click here for additional data file.


We have previously identified a locus on human chromosome 20q13.1, encompassing related genes of postulated WFDC-type protease inhibitors and semen coagulum proteins. Three of the genes with WFDC motif also coded for the Kunitz-type protease inhibitor motif. In this report, we have reinvestigated the locus for homologous genes encoding Kunitz motif only. The identified genes have been analyzed with respect to structure, expression and function.SPINT3, SPINT4 and SPINT5, and the structure of their transcripts were determined by sequencing of DNA generated by rapid amplification of cDNA ends. Each gene encodes a Kunitz domain preceded by a typical signal peptide sequence, which indicates that the proteins of 7.6, 8.7, and 9.7 kDa are secreted. Analysis of transcripts in 26 tissues showed that the genes predominantly are expressed in the epididymis. The recombinantly produced proteins could not inhibit the amidolytic activity of trypsin, chymotrypsin, plasmin, thrombin, coagulation factor Xa, elastase, urokinase and prostate specific antigen, whereas similarly made bovine pancreatic trypsin inhibitor (BPTI) had the same bioactivity as the protein isolated from bovine pancreas.We identified three novel genes; The similar organization, chromosomal location and site of expression, suggests that the novel genes are homologous with the genes of WFDC-type protease inhibitors and semen coagulum proteins, despite the lack of similarity in primary structure of their protein products. Their restricted expression to the epididymis suggests that they could be important for male reproduction. The recombinantly produced proteins are presumably bioactive, as demonstrated with similarly made BPTI, but may have a narrower spectrum of inhibition, as indicated by the lacking activity against eight proteases with differing specificity. Another possibility is that they have lost the protease inhibiting properties, which is typical of Kunitz domains, in favor of hitherto unknown functions. The mammalian semen coagulum proteins are a heterogeneous collection of proteins secreted at very high concentration by the seminal vesicles. There are two homologous semen coagulum proteins, denoted semenogelin I SEMG1) and semenogelin II (SEMG2), in most primates  and seme. DuplicaThe murine seminal vesicles secrete six proteins, Svs1-Svs6, at high concentration -9. It haThe proposed evolutionary mechanisms suggest that the seminal vesicle-transcribed genes might be related to genes with similar organization that code for secreted, but structurally very different proteins. This hypothesis led to the discovery that the genes of the elastase inhibitors elafin and secretory leukocyte protease inhibitor (SLPI), and the predominating seminal vesicle-secreted proteins are homologous, in spite of lacking similarity between their protein products . Elafin PI3, the gene of elafin, and SLPI are flanking SEMG1 and SEMG2 and an extended analysis of the chromosomal region identified 12 additional, homologous genes encompassing WFDC motif  terminates in a poly-A tail that is preceded by an unconventional poly-adenylation signal, TATAAA, which notably is identical to the canonical TATA-box sequence in eukaryotic genes , formation of disulphide bonds in the cytoplasm would be favored. Studies with recombinant BPTI and BSTI showed that the proteins isolated from the cytoplasm were almost exclusively in a monomeric state, which suggests correct disulphide formation and folding of the Kunitz domain. Furthermore, the recombinant inhibitors were as functionally active as BPTI isolated from bovine pancreas, something that also indicates that they are correctly folded. Expression of SPINT3 yielded an exclusively monomeric protein in a similar way as BPTI and BSTI, which suggests that the Kunitz domain was properly folded. Recombinant expression of SPINT4 yields high molecular mass complexes that probably consist of misfolded peptide chains held together by disulphide bonds in an unorganized way, but also a substantial fraction of monomers that presumably are correctly folded. The structure of SPINT4 differs from that of the consensus Kunitz domain. The latter contains three conserved disulphide bridges, but in SPINT4 there will only be two, as one Cys is missing. The absent disulphide would have linked the C-terminal alpha-helix to one of the beta-strands at the core of the molecule. Earlier experiment has shown that the folding of the Kunitz domain is only minimally affected by removal of this bridge and would not affect the protease-binding loop that is situated at the opposite side of the molecule [Protein domains are building blocks that usually fold to explicit three dimensional structures, which many times have a specific function. The Kunitz domain consists of 50-60 amino acid residues, six of which are Cys that form three disulphide bonds and have characteristic locations in the primary structure. Studies on recombinant Kunitz domains expressed in n bodies ,25. Our molecule . The locDespite several attempts, we did not succeed to isolate recombinant SPINT5 from the cytoplasm of Origami(DE3) cells. It is not clear whether the failure was due to inherent instability of the protein product or whether the problems are due to technical problems with our construct, but future studies will presumably give an answer.The experiments with recombinant BPTI demonstrated, not only that the Kunitz domain can be correctly folded in Origami(DE3) cells, but also that the fusion product with thioredoxin could be an equally efficient protease inhibitor as BPTI isolated from a natural source. It is therefore likely that recombinant SPINT3 and SPINT4 would be functional as well. They were tested by a set of proteolytic enzymes that represent different catalytic specificities, but unfortunately, we did not succeed to identify a target protease. The amino acid residues in BPTI that interact with target proteases have been characterized in detail and homologous residues can be identified in other Kunitz domains by sequence alignment . There aSPINT3 and SPINT4 with high specificity are expressed in the epididymis, which suggests a role in the maturation of spermatozoa. An innate immune function can also inferred, as it has been shown that the Kunitz domain of SPINLW1 inhibits elastase and display antibacterial activity [The WFDC genes at the locus on chromosome 20 could be important during sperm maturation, as several of them are highly expressed in the epididymis. There is this far no report on target proteases in the epididymis. It has also been speculated that the WFDC genes are important but not fully recognized components, of innate immunity; in which some modulate host response by regulating endogenous pro-inflammatory proteases, such as elastase and cathepsin G, whereas others regulate poorly characterized exogenous proteases used for tissue invasion by microbes . The genactivity .We have in this report described three genes that produce small secreted proteins, composed of single Kunitz domains that are relatively specific for the epididymis. The similar organization, chromosomal location and site of expression, suggests that the novel genes encoding Kunitz motifs are homologous with the genes of WFDC-type protease inhibitors and semen coagulum proteins, in spite of lacking similarity in primary structure of their protein products. A likely mechanism, by which the genes could have been formed, include an exon shuffling event, in which the Kunitz motif was taken up by a WFDC gene, and a following purifying process, in which the WFDC domain was lost. This would then be the third mechanism, beside de novo selection of splice site and repeat expansion, by which the regulatory frame of a conserved WFDC/semen coagulum gene could generate a new protein product. The restricted expression of the novel genes to the epididymis suggests that the secreted Kunitz domains are important in male reproduction. The recombinantly produced proteins are presumably bioactive, as demonstrated with similarly made BPTI, but may have a narrower spectrum of inhibition, as indicated by the lacking activity against eight proteases with differing specificity. Another possibility is that they have lost the protease inhibiting property, which is typical of Kunitz domains, in favor of new hitherto unidentified functions.The nucleotide sequence of the WFDC-locus on chromosome 20 was translated in six reading frames and scanned for short sequence motifs as previously described . Kunitz Preparation of RNA samples and conditions for cDNA synthesis has been described earlier . Primers2 and 0.1 mM CaCl2. Following addition of 1 μl of 25 mM EDTA, the enzyme was inactivated by incubation at 65°C for 10 min. The material was then taken to oligo(dT)18-primed cDNA synthesis in a volume of 20 μl using the RevertAid H Minus First Strand cDNA Synthesis kit (Fermentas) for 1 h at 42°C. Finally, the reverse transcriptase was inactivated by heating to 70°C for 5 min, and the samples were diluted with H2O to 200 μl and stored at -20°C until used. Negative controls were produced in the same way, but with the reverse transcriptase omitted.The cDNA for qRT-PCR was made from DNAase-free RNA using reagents and protocol provided by the supplier . Briefly, 3 μg of RNA was treated at 37°C for 30 min with 1 unit of RNase-free DNase in 10 μl of 10 mM Tris-HCl pH 7.5, 2.5 mM MgClCSTB and APRT [The PCR was done in MicroAmp Optical 384-Well Reaction Plates  using the 7900 HT Fast Real-Time PCR System (Applied Biosystems). PCR primers were selected such that the resultant transcript should be less than 150 bp  or Nucleotrap (Clontech) and subjected to DNA sequencing using the Big Dye Terminator Cycle Sequencing Ready Reaction Kit 3.0 and a 3730 DNA Analyzer (Applied Biosystems). The sequences were analyzed with the Sequence Analysis 5.1 program (Applied Biosystems) and bioinformatics tools available at the EBI web server (http://www.ebi.ac.uk/Tools/). Translations of transcripts were screened for signal peptides using the SignalP 3.0 server [AY372172, AY372173, AY372174, and AY542490.Synthesis of RACE ready cDNA and touch down PCR was done with the SMART or the SMARTer RACE cDNA Amplification Kits  and RNA from human epididymis or mouse testis, essentially as described . The pri0 server . SequencEcoRI and NotI  for 30 minutes with 10 s burst separated by intervals of 10 s, using the power setting 3. Cell debris was removed by centrifugation at 16,000 × g for 20 min. The supernatant was dialyzed against 50 mM Na-phosphate pH 8.0, 0.3 M NaCl and then passed through a 0.45 micron filter .Origami(DE3) bacteria containing plasmids designed for prokaryotic expression were grown at 37°C in 500 ml of LB medium containing antibiotics until mid-logarithmic phase. The culture was chilled and the synthesis of recombinant protein was induced by addition of isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM, where after growth continued at 30°C for 5 hours. Bacteria were harvested by centrifugation at 10,000 × The recombinant proteins were purified on a 12 ml column of Ni-NTA superflow (Qiagen) that was run at 2 ml/min in 50 mM Na-phosphate, 0.3 M NaCl and 10 mM imidazole, pH 8.0. Proteins were eluted by increasing the concentration of imidazole to 100 mM or 300 mM.2 instrument  and displayed as the time-dependent increase in absorbance at 405 nm. Inhibitors at various concentrations were incubated with proteases for 15 min at 37°C in the assay buffer. Reactions were initiated by addition of peptide substrate. The following concentrations of proteases  were used: 5 nM of bovine trypsin, 10 and 20 nM of bovine chymotrypsin, 50 nM human plasmin, 10 nM human urokinase, 15 nM bovine thrombin, 15 nM bovine Factor Xa, 10 nm human leukocyte elastase, and 20 nM porcine pancreatic elastase. Analysis was also made with 10 nM human prostate-specific antigen (PSA), purified from human seminal plasma by affinity chromatography using Mab 2E9 [The amidolytic activity of proteases was measured with purified enzymes and synthetic peptide substrate. The assays were run in microtiter plates with enzyme, inhibitor and peptide substrate in wells containing 100 μl of 50 mM Tris-HCl, pH 8.0, 0.15 M NaCl and 0.05% Tween 20. Enzymatic activity was measured at 37°C in a ViktorAC designed and carried out experimental procedures and drafted the manuscript; MP designed and carried out experimental procedures, HL was involved in planning and design of experiments and reviewed and contributed substantive edits of the manuscript, ÅL initiated the study, identified the novel gens, contributed to the experimental design, made the qPCR and wrote the final manuscript. All authors have read and approved the final manuscript.


Because of relatively small treatment numbers together with low adverse drug reaction (ADR) reporting rates the timely identification of ADRs affecting children and young people is problematic. The primary objective of this study was to assess the utility of unplanned medication discontinuation as a signal for possible ADRs in children and young people.Using orlistat as an exemplar, all orlistat prescriptions issued to patients up to 18 years of age together with patient characteristics, prescription duration, co-prescribed medicines and recorded clinical (Read) codes were identified from the Primary Care Informatics Unit database between 1st Jan 2006-30th Nov 2009. Binary logistic regression was used to assess association between characteristics and discontinuation.During the study period, 79 patients were prescribed orlistat . Unplanned medication discontinuation rates for orlistat were 52% and 77% at 1 and 3-months. Almost 20% of patients were co-prescribed an anti-depressant. One month unplanned medication discontinuation was significantly lower in the least deprived group (SIMD 1–2 compared to SIMD 9–10 OR 0.09 (95% CI0.01 – 0.83)) and those co-prescribed at least one other medication. At 3 months, discontinuation was higher in young people ). Read codes were recorded for digestive, respiratory and urinary symptoms around the time of discontinuation for 24% of patients. Urinary retention was reported for 7.6% of patients.Identification of unplanned medication discontinuation using large primary care datasets may be a useful tool for pharmacovigilance signal generation and detection of potential ADRs in children and young people. Although adverse drug reactions (ADR) represent a major source of morbidity and mortality in adults, the nature and frequency of ADRs affecting children and young people remains poorly defined. Published data suggest that ADRs account for 1.5-2.1% of paediatric hospital admissions and affect 2.6-9.3% of paediatric inpatients and 1.5-11.1% of paediatric outpatients .In the past children and young people have frequently been excluded from clinical trials and where trials are undertaken, they are often insufficiently powered to detect even relatively common ADRs. This means that children and young people are often prescribed medication with limited information about a treatment’s ADR profile, at doses which are not evidence based, and “off–label”; a practice which itself is associated with increased risk of ADRs .Post marketing surveillance is an essential tool in enabling the detection of potentially serious ADRs; however, to detect an ADR with a frequency of 1:10,000, at least 30,000 individuals need to be treated with the drug for at least 1 case to be identified. Therefore, post-marketing surveillance requires the monitoring of large patient cohorts exposed to a medication to detect ADRs. Currently, pharmacovigilance systems rely primarily on spontaneous reporting of ADRs. However under-reporting of ADRs is a significant issue affecting all European ADR monitoring systems. It is widely accepted that only 10% of UK and European healthcare professionals take part in the process of ADR reporting and that only 6-9% of ADRs affecting adults, which should be reported, are actually reported to the regulatory authorities -15. DespA growing area of medical concern and drug treatment is that of obesity ,18. Obes2) adult patients or obese patients with comorbidities. Use in adults is associated with a 30% discontinuation within 3 months due to intolerable ADRs [In the UK, orlistat, is currently the only anti-obesity drug licensed for use in severely obese , as a signal for the identification of possible ADRs, has been previously assessed in the adult population using Primary Care electronic administrative data. This approach is based on the recognition of an early, unplanned discontinuation of the prescription for a medication, possibly accompanied by switching to alternative medication. A close agreement has been previously reported between the UMD rate and ADR rates reported in the literature for a variety of adult medications .The primary objective of this study was to assess the utility of UMD as a signal for possible ADRs in children and young people. Using a large Primary Care electronic administrative dataset and orlistat as an exemplar, we assessed prescribing pattern, medicine use and the potential to generate pharmacovigilance signals for medicines prescribed with low frequency to special populations such as children and young people.The Primary Care Clinical Informatics Unit (PCCIU) research database is a large administrative dataset covering, in 2006, 179 Primary Care practices across Scotland ; represeThe PCCIU database, contains demographic data including gender, date of birth and socioeconomic status based on the practice post code (the Scottish Index of Multiple Deprivation (SIMD) 2006 quintiles) : clinicaThe study population included a closed cohort, all children and young people aged 0–16 years of age registered with a General Practice on the 1st January 2006. This cohort was then followed until they reached their 19th birthday or 30th November 2009.All children and young people newly prescribed orlistat  were identified and tracked through the study period.For this study, a “new prescription” was defined as the first prescription for orlistat during the study period with no previous prescription in the preceding 3 months.The primary outcome of interest was identification of a potential pharmacovigilance signal, defined as the discontinuation of orlistat for at least 3 months without a subsequent prescription. Unplanned medication discontinuations were categorised based on duration of treatment as: <1 month, 1–3 months, 4–6 months or >6 months. Duration was calculated from the date of the first prescription to the date of the last prescription. If prescriptions were ongoing at the end of the study, the child was categorised as continuing treatment.Because the majority of patient records identified in the PCCIU dataset did not contain a specific reason for drug discontinuation, clinical symptoms, diagnoses, investigations and procedures recorded as Read codes around the time of medication discontinuation were examined. All Read codes occurring after starting orlistat were identified. Potential signal associations were limited to codes recorded during the 3 months before and 3 months after the last prescription. Read codes were reported and summarised according to the International Classification of Diseases (ICD)-10 top level categories.All co-prescriptions were extracted from the date of the first orlistat prescription up to 3 months after the last orlistat prescription date and classified into their respective British National Formulary (BNF) therapeutic drug classes . The numAs national guidelines recommend that the BMI be calculated for the purposes of diagnosis, monitoring and initiation of pharmacological treatment, the date of the last recorded weight and height was also noted, and if assessed more than 1 year before start of orlistat was treated as missing.To allow comparison between gender and developmental stages, the BMI standard deviation score (BMI SDS) was calculated based on Cole’s LMS method .Individuals with a BMI SDS between 2.5 – 3.49 were classified as overweight to obese, those with a BMI SDS over 3.5 classified as severely obese and those over 4 as very severely obese.Descriptive statistics , median (IQR)) were used to report the characteristics of the cohort. The incidence rate for orlistat prescription was calculated for 2006.Discontinuations were summarised as percentages and reported graphically.To explore factors that might influence the discontinuation of orlistat treatment, we compared those who discontinued orlistat treatment at one and three months with those continuing treatment. We used binary logistic regression to calculate unadjusted odds ratios (OR) and 95% confidence intervals (CI). The factors considered included: gender, age, socioeconomic status, co-prescribing of antidepressants, and number of co-prescriptions. Analysis was performed using SPSS for windows 19.0 .Data were anonymised and no personally identifiable information was sought. Approval for the study was granted by the Primary Care Clinical Informatics Unit research steering committee in accordance with their research governance process.From the study population of 166,726 children and young people  aged 0–16 years and registered with a participating primary care practice in Jan 2006, a cohort of 79 young people newly prescribed orlistat during the study period were identified. In 2006, at cohort inception, the incidence of orlistat was 0.06/1000/year for children aged 0–16 years.The median age at first orlistat prescription was 17 years (IQR 16 – 18), 81% were female and 55% were from General Practices situated in the most deprived socioeconomic areas (SIMD deciles 7–10) (Table Two thirds (65.8%) of patients were co-prescribed between 1–4 drug classes/items. The medicines most commonly co-prescribed were antibiotics , topical skin treatments (12.4%), analgesics (10.7%), oral contraceptives (9.9%), asthma medications (7.4%) and antidepressants (4.1%). Four (5%) patients prescribed orlistat were also prescribed metformin. For three of these, metformin was initiated immediately following cessation of the orlistat prescription.Fifty-two percent of patients (41) discontinued orlistat medication within 1 month of the index prescription and 77% (61) within 3 months.Discontinuation within 1 month was not affected by age or gender but was significantly less likely if the patient had received any co-prescription (OR 0.12 (95% CI 0.03 – 0.41) or was in the least deprived group (OR 0.09 (95% CI 0.01 – 0.84) (SIMD 1–2) though there was no obvious trend by deprivation category.Gender, co-prescription and socio-economic status, were not significantly related to 3-month discontinuation, however young people over 17 years of age were three times more likely to discontinue their prescription at 3-months when compared with those under 17 years of age  Table .In total, 404 Read codes were recorded from the start of orlistat treatment to 3 months after the last orlistat prescription for 77.2% (61) of the study population. The median number of Read codes per individual was 5; IQR 1–6 (range 0–51), and 20.3% (82) of these Read codes related specifically to symptoms or treatments. Eight (10.1%) patients had a Read Code recorded for: the respiratory, seven (8.9%) for the genitourinary, six (7.6%) for skin, six (7.6%) for endocrine, four (5.1%) for obstetric and gynaecological, four (5.1%) for gastrointestinal, four (5.1%) for musculoskeletal, 3 (3.8%) for mental health and behavioural and 3 (3.8%) for the CNS systems.  of all patients prescribed orlistat had a Read code recorded within three months of orlistat discontinuation for the genitourinary system, of whom five were recorded as having urinary catheterisation and one urinary retention. None of these individuals were prescribed any medication or items during the three year study period to suggest neurological, bowel, bladder or malignant disease, which might explain the need for urinary catheterisation.We demonstrate that using UMD as a surrogate for possible adverse events, routinely collected primary care datasets may be used for pharmacovigilance signal generation, although validation of generated signals and confirmation of potential ADRs will require appropriately designed prospective studies. We believe that this approach will be complementary to the spontaneous reporting systems currently in use and enhance the capability of these systems to widen the breadth of ADRs detected while reducing cost and time to ADR detection. Furthermore such an approach permits assessment of prescribing appropriateness together with real life prescribing patterns. In this study, one in every two patients prescribed orlistat discontinued within a month, and more than three quarters discontinued within three months, of starting treatment.For medicines such as orlistat, continued use is likely to reflect patient acceptability and perceived effectiveness. In this context it is likely that early discontinuation reflects the appearance of unwanted poorly tolerated ADRs, while later discontinuation represents a perceived lack of response . The levDiscontinuation at three months was significantly more frequent in the young people aged 17 years and over, possibly reflecting the effects of emerging autonomy and loss of parental influence -39. One In this study orlistat was prescribed predominantly to adolescents females, and those from the most socially deprived areas. Although the relationship between obesity and social deprivation is well recognised, this is the first study to report a link between social deprivation and orlistat prescribing -46.Seventy percent of the cohort prescribed orlistat was co-prescribed medications including antibiotics, topical treatments for skin conditions and analgesia. Five percent of patients were also prescribed metformin, licensed for use in individuals aged 10 years and older for type II diabetes mellitus, but which is being increasingly used for the treatment of obesity and insulin resistance -51.In the present study, one fifth of all patients  prescribed orlistat were also prescribed antidepressants at some point during the study period. In the general adolescent population antidepressants are reportedly prescribed to 1-5/1000 individuals per year -54, therRead code, describing clinical symptoms and treatments, were recorded by a primary care physician for approximately a fifth of patients around the time of orlistat discontinuation. Respiratory, genitourinary, endocrine, skin, gynaecological and gastrointestinal symptoms were reported to occur in 10%, 8.9%, 7.6%, 7.6%, 5.1% and 5.1% of patients respectively. Data from pediatric/young person orlistat trials suggest that adverse effects, primarily diarrhoea, affect 97-100% of individuals, leading to a discontinuation rate of 2% - 30% ,27,57. IAn unexpected finding was the number of individuals for whom urological system Read codes, urinary catheterisation and urinary retention, were recorded. The reasons for this finding are not clear. While urinary retention is frequently associated with neurological disorders and or constipation, assessment of co-prescribed medicines and items for these individuals, identified only one subject prescribed laxatives and none prescribed medicines or items commonly used for the treatment of neurological, bowel, bladder, malignant or infective conditions during the study period. Although orlistat use has been associated with nephrolithiasis and acute renal injury ,59, therCurrent clinical guidelines make it clear that prior to the initiation of orlistat therapy, height and weight should be recorded. In our study, 70% of the study cohort had this information recorded, and of these only a third had measurements recorded within a month of their initial prescription. The failure to undertake appropriate BMI assessment prior to orlistat initiation means that it is not possible to determine for the majority of patients whether orlistat was prescribed appropriately, according to the UK guidelines.We have previously reported that electronic prescribing databases can be used to identify UMD in adult patients, and that such UMD rates can be used as a surrogate for ADR/AE rates ,35. HoweThe use of large primary care electronic datasets enables the linkage of whole population prescription information with recorded clinical symptoms, diagnoses and encounters. As we have demonstrated, this approach permits the assessment of prescribing trends and identification of individuals who discontinue their medication at an early stage before clinical effectiveness might be expected. Assessment of clinical symptoms and diagnosis recorded around the time of orlistat discontinuation identified unexpected findings which might indicate, although not prove, the occurrence of an ADR to the study medication.There were several limitations to our study. It is possible that the small number of patients prescribed orlistat in this study may reduce the generalisability of the orlistat specific findings; however it does not alter the overall conclusion that the linkage of UMD with medical Read codes identifies possible pharmacovigilance signals of interest. Moreover, the PCCIU-R database, like most other prescribing/dispensing databases, only reflects primary care prescribing and does not provide information on secondary care prescribing or over the counter purchase of orlistat. It is therefore possible that patients for whom an initial prescription was issued in secondary care or who purchased their medication OTC may have been missed. However this is unlikely to be a major issue in the Western world, where primary care remains the major source of medication prescriptions. A further limitation is the lack of a specific recorded reason for orlistat discontinuation in the PCCIU-R database, and this is the reason why all Read codes at or about the time of discontinuation were identified and examined.Using orlistat as the exemplar, we have demonstrated that when unplanned medication discontinuation are linked to clinical codes for events occurring at the time of discontinuation, it is possible to generate both predictable and unpredicted pharmacovigilance signals. These findings support the use of this approach together with traditional pharmacovigilance methodologies to identify possible signals and plan more specific and focused pharmacovigilance studies with the aim of validating suspected ADRs.As all data analyzed was fully anonymised and non attributable the approval for the study was granted by the Primary Care Clinical Informatics Unit research steering committee in accordance with their research governance process and individual patient consent was not necessary.ADR: Adverse drug reaction; BMI: Body mass index; BMISDS: Body mass index standard deviation score; BNF: British National Formulary; ICD: International Classification of Diseases; MHRA: Medicines and healthcare products regulatory agency; PCCIUR: Primary care clinical informatics unit research; NICE: National institute for health and clinical excellence; SIMD: Scottish index of multiple deprivation; SIGN: Scottish intercollegiate guidelines network.The authors declare that they have no competing interests.AS: assisted in conceptualization, design and analysis of the study, drafted the initial manuscript, and approved the final manuscript as submitted. BK: assisted in conceptualization, design and analysis of the study and approved the final manuscript as submitted. PH: assisted in conceptualization, design and analysis of the study and approved the final manuscript as submitted. CB: assisted in conceptualization, design and analysis of the study and approved final manuscript as submitted. MB: assisted in conceptualization, design and analysis of the study and approved the final manuscript as submitted. JM: conceptualized and designed the study, assisted with analysis and interpretation of results, and approved the final manuscript as submitted. JM acts as study guarantor. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/2050-6511/15/11/prepub


Miscanthus sinensis, using genotyping-by-sequencing (GBS), identifying all 19 linkage groups for the first time. The result is technically significant since Miscanthus has a very large and highly heterozygous genome, but has no or limited genomics information to date. The composite linkage map containing markers from both parental linkage maps is composed of 3,745 SNP markers spanning 2,396 cM on 19 linkage groups with a 0.64 cM average resolution. Comparative genomics analyses of the M. sinensis composite linkage map to the genomes of sorghum, maize, rice, and Brachypodium distachyon indicate that sorghum has the closest syntenic relationship to Miscanthus compared to other species. The comparative results revealed that each pair of the 19 M. sinensis linkages aligned to one sorghum chromosome, except for LG8, which mapped to two sorghum chromosomes (4 and 7), presumably due to a chromosome fusion event after genome duplication. The data also revealed several other chromosome rearrangements relative to sorghum, including two telomere-centromere inversions of the sorghum syntenic chromosome 7 in LG8 of M. sinensis and two paracentric inversions of sorghum syntenic chromosome 4 in LG7 and LG8 of M. sinensis. The results clearly demonstrate, for the first time, that the diploid M. sinensis is tetraploid origin consisting of two sub-genomes. This complete and high resolution composite linkage map will not only serve as a useful resource for novel QTL discoveries, but also enable informed deployment of the wealth of existing genomics resources of other species to the improvement of Miscanthus as a high biomass energy crop. In addition, it has utility as a reference for genome sequence assembly for the forthcoming whole genome sequencing of the Miscanthus genus.We have created a high-resolution linkage map of  Miscanthus sinensis using next generation sequencing (NGS) of a large number of heterozygous recombinants to open up the genetic mapping of the species and to support breeding studies.The ability to sequence whole genomes has advanced the means of detecting genetic variation genome wide. However, for large genomes that are exceptionally difficult to unambiguously assemble, genetic maps are still needed for genetic studies and plant breeding. Recently, several studies have used genotype-by-sequencing (GBS) to develop genetic maps, but usually on smaller and/or better understood genomes Miscanthus is one of the leading biofuel crops with growing importance Miscanthus Anderss. (Andropogoninae: Poaceae) consists of many rhizomatous perennial species, most of which are endemic to subtropical and tropical regions of southern Asia, with a few species extending to temperate eastern Asia Miscanthus improvement programs. Species showing the greatest potential as dedicated energy crops include M. floridulus, M. lutarioriparium, M. sacchariflorus, M. sinensis and M.×giganteus, among which, M.×giganteus is currently the most cultivated species for biomass production in Europe M.×giganteus is not an ideal candidate for genetic studies and breeding improvement due to its sterility and triploid genome (2n = 3x = 57) resulting from a rare natural cross between diploid M. sinensis (2n = 2x = 38) and allotetraploid M. sacchariflorus (2n = 4x = 76) M. sacchariflorus was inherited from the diploid M. sinensis, M.×giganteus has two genomes with high homology, and a third genome with low homology to M. sinensisM.×giganteus are available and have been widely cultivated through vegetative propagation However, M. sinensis and M. sacchariflorus, on the other hand, have also been introduced and adapted widely to Europe as potential bioenergy species due to their high biomass potential Miscanthus crops and generating new high performance hybrids M. sinensis has the most widespread geographical distribution, in terms of latitude, longitude and altitude, with correspondingly high adaptability from extensive genetic and phenotypic diversity. It is also generally diploid in nature, demonstrating greater amenability to hybridization. Recent studies have demonstrated the existence of variation in flowering times in M. sinensis that provides extensive options for hybrid generation and subsequent optimization of varieties to different climatic zones Miscanthus genus is focused mainly on field trials, positioning knowledge of its basic biology behind that of current row crops. Little is known about the genetics behind important agronomic traits that must be improved for commercialization. Better understanding of the genetics of biomass yield related traits such as spring emergence date, flowering time control, senescence, nutrient uptake, and abiotic and biotic stress tolerances, is needed to aid genetic improvement of the crop. This in turn can be facilitated by a reference linkage map.Research for the majority of species within the Miscanthus genomes to-date have concentrated on defining phylogenies and taxonomies using limited genetic marker technologies Miscanthus for advanced genome studies M. sinensis is about 2.75 pg/1C or 2,650 Mbp, which is approximately the same size as the maize (Zea mays L.) genome  Miscanthus has caused considerable difficulties in developing genetic markers and linkage maps of the species. At present, there is only one published linkage map for Miscanthus, in M. sinensis, composed of 257 random amplified polymorphic DNA (RAPD) markers spread over 28 linkage fragments with half of the linkage fragments containing 2–4 markers only Sorghum bicolor L.), maize and rice (Oryza sativa L.).The small numbers of molecular studies on Miscanthus linkage map covering all chromosomes is needed for a variety of genetic studies, such as QTL and whole genome association mapping. A complete map would also enable use of QTL accessible from other grass species through alignment based on syntenic relationships. In this work, we report the first complete, high resolution genetic map of M. sinensis and use of comparative genomics to understand its syntenic relationships with other species, particularly sorghum.It is known that a complete M. sinensis in the present work. A significant benefit of GBS is that genome-wide marker representation is ensured, which is critical for coverage and completeness of the linkage map. The linkage maps created from this study include two parental maps  and a composite map, created after confirming that the two parent maps had the same (or similar) structures detected by common markers. Once the M. sinensis linkage maps were aligned to the sorghum genome, two-to-one syntenic matches were observed between the 19 M. sinensis linkage groups and the 10 sorghum chromosomes, except for one fusion, proving a tetraploid nature of the diploid M. sinensis. Therefore, this linkage map not only defines all 19 chromosome linkage groups for the first time, but also provides a bridge for comparative genomics to genetic resources of other crops species for the ultimate improvement of Miscanthus.The linkage map was constructed based on several thousand single nucleotide polymorphism (SNP) markers using a mapping population that is composed of progeny of a “two-way pseudo-testcross”, full-sib family, which is typically used for genetic mapping of highly heterozygous out-crossing plant species FseI, which cleaves gene-rich regions of the genome, was used to construct reduced representation DNA libraries. After DNA sequencing using the Illumina platform, the main steps of processing sequencing data are presented in In order to focus SNP development on gene-rich space, a methylation sensitive restriction enzyme The number of reads varied in libraries from 328,450 to 6,470,626. All reads passing QC steps were mapped onto the sorghum genome using NCBI BLAST+. Only the best BLASTN match, having at least 90% identity and being at least 55 nucleotides long, was considered . The coverage of reads varied significantly for different restriction sites. Most of the restriction sites were covered by a very small number of reads, whereas a few restriction sites generated a very large number of reads. The number of reads per restriction site correlated between the libraries indicating that restriction sites had different accessibilities  .We considered a nucleotide position to be homozygous in a plant if there was coverage of at least five reads in the corresponding library, having a nucleotide at this position with a quality score ≥15 and at least 90% of these nucleotides were the same. A position was considered to be heterozygous in a plant if there were at least five reads having a nucleotide at this position with a quality score ≥15 and there were two different alleles with frequencies between 0.4 and 0.6.According to these two definitions, in an average plant, 97.8% of the sampled nucleotide positions with five or more reads were homozygous, 0.7% positions were heterozygous, and 1.5% positions were undetermined. We identified a total of 49,007 SNPs using this set of data.Reads which did not match the sorghum genome were clustered using CLC Bio software into 20,169 contigs. For plant Mb111 as an example, this resulted in 1,432,240 additional positions with coverage of at least five reads. An additional 13,897 SNPs were identified from this dataset using the same criteria as the reads mapped to sorghum. To get better coverage of the discovered SNPs by as many plants as possible, we used sequences previously excluded at the QC step to increase the number of reads in defined SNP positions. We considered all discovered SNPs with at least four reads and defined the plant as homozygous if the frequency of a certain nucleotide in this SNP was ≥0.9; heterozygous if there were two different nucleotide variants with frequencies between 0.2 and 0.8. Special codes were then assigned to those SNPs having nucleotide frequencies between 0.8 and 0.9.de novo SNPs the most frequent allele variations were complementary pairs of mutations A/G and C/T, which occurred about twice more than the next allele pair, C/G.In further analyses we considered only SNPs with major allele frequency in the population less than 0.8. As a result, we obtained 4,437 SNPs aligned and 2,669 SNPs not aligned to the sorghum genome, making a total 7,106 SNPs, with determined alleles in at least 120 plants . Eleven The SNP markers were then coded for JoinMap and 5,600 out of the 7,106 SNPs were selected for segregation and mapping analyses. More heterozygous markers were identified in the female than in the male. Of the 5,600 markers, 2,574 (46%) were heterozygous only in the female; 1,468 (26%) only in the male; and 1,558 (28%) were heterozygous in both parents. Chi-square tests indicated that 54%  of the female markers and 49%  of the male markers showed segregation distortion , as did 69%  of the female and male common markers . Thus, markers that were heterozygous in both parents tended to be more distorted in allele transmission than when heterozygous in only one of the parents.The coded data were first used to create parental maps using two JoinMap populations, one using markers segregating in the female, and the other in the male For linkage mapping, framework maps of both parents were created first with no (p<0.05) segregation distorted markers, and used for homologous identification and structural comparisons between the two parental maps. One-to-one homologous relation was confirmed using markers common between the 19 linkage groups of the female and the 19 linkage groups of the male. In addition, good collinearities were observed and no obvious structural heterogeneities were seen between homologous framework maps of the two parents.Then, segregation distorted markers were added to the framework maps by attempting different LOD statistics and/or different numbers of markers. The final maps selected were usually the ones having the best collinearities with the framework maps. Since the female had more heterozygous markers than the male, the number of markers mapped in each female linkage was generally more than that in the corresponding linkage of the male .Since there were no obvious structural heterogeneities between the female and male maps, a composite map containing both female and male markers was created to facilitate future QTL mapping of the population. The composite mapping was done with an independent JoinMap mapping population by simultaneous analysis of all polymorphic markers from both parents. Again, 19 linkage groups were formed and framework maps without segregation distorted markers were created first. Comparisons between the composite and the two parental framework linkages showed perfectly collinear marker order agreements for all 19 linkage groups, indicating good integration of the two parental linkages by the simultaneous mapping analysis.The composite framework maps were then extended by mapping segregation distorted markers using the same mapping criteria and strategy as the parental maps. Ultimately, a high density genetic linkage map representing all chromosomes was established . The linThe composite map comprised 3,745 SNP markers spanning 2396 cM on 19 linkages. The numbers of markers mapped varied among the 19 linkage groups from 131 markers in LG10 to 289 in LG8. The linkage sizes in cM were also very different, from 91 cM of LG17 to 159 of LG8. The marker density of the composite map was highly variable from region to region and chromosome to chromosome, with an average resolution of 0.64 cM .http://www.atgc.org/XLinkage/), was used to validate the linkage map  were poor because the ratios of BLAST alignments were very low in comparison to the sorghum alignment. Moreover, there was a two-to-one syntenic match between the 19 Miscanthus linkages and the 10 sorghum chromosomes, indicating complete, genome-wide chromosome duplication, with one exception: one of the syntenic copies of sorghum chromosomes 4 and 7 was fused into a single linkage group, LG8, in Miscanthus, thus making 19 basic chromosomes of the Miscanthus genus  occurred in sorghum, not in M. sinensis, because the same paracentric inversion was also observed when sorghum was compared to switchgrass, maize and rice, and also was partially seen when compared to B. distachyon  that were syntenic to sorghum chromosomes 1, 2, 3 and 4 generally contained more markers and also longer linkages (in cM) than other linkage groups  syntenic to sorghum chromosome 5 contain the least markers and the lowest percentages (<30%) of markers that map to sorghum, however markers which mapped to other linkages exhibit much higher percentages (60% on average) of sorghum syntenic alignments , sorghum has the closest phylogenetic relationship to Miscanthus. As predicted, the Miscanthus DNA reads better matched the sorghum genome than the maize genome, which in turn was a better match than the rice and B. distachyon genomes. Similar findings were reported recently using a survey sequence of M.×giganteusThe marker sequences were mapped to genome sequences of several grass species. Among grasses with sequenced genomes  consisting of 230 genotypes was produced between two parental lines, female Mb111 (synonyms EMI11 and MS-88-110) and male Mb121 (synonym H0023). Parent Mb121 is one of the progenies derived from a cross between siblings (F1.1 and F1.7) that originated from another cross between MS-90-2 and Mb111 FseI enzyme (New England Biolabs (NEB®), Hitchin, UK) at 37°C for 3 h; subsequently the enzyme was heat-inactivated at 65°C for 20 min.A total of 192 (out of the 230) genotypes of the mapping population, including parents, were selected for genotyping using the GBS platform FseI digested DNA sample was ligated to one of the P1 adapters in a 20 µl reaction mixture containing 1 µl of 10 nM adapter, 1 µl rATP , 1 µl NEB buffer 4, and 0.5 µl T4 ligase  by incubating for 4 h at 16°C. The reaction was stopped by incubating at 65°C for 20 min. Every 12 samples, each with a unique barcode, were pooled and randomly sheared by Bioruptor UCD-300 (Diagenode) to an average size of 450 bp. The barcoded samples were electrophoresed on 1.5% agarose gels, and fragments of 300–500 bp were recovered and purified. The fragments were treated with T4 DNA polymerase, T4 polynucleotide kinase, and Klenow DNA polymerase (NEB) for end repairing, followed by treatment with Klenow exō (NEB) and dATP (Invitrogen) to generate 3′ adenine (A) overhangs Twelve unique P1 adapters were designed, each with a different 6 bp barcode to enable a 12× sample multiplex per Illumina flow cell lane. Each A common P2 adapter containing thymine (T) overhangs (Illumina) was ligated to sheared, size-selected, P1-ligated and pooled DNA templates with 1 µl of 100 nM adapter and T4 ligase for 4 h at 16°C FseI. DNA sequences were analyzed using the NCBI BLAST+ program for mapping reads into the sorghum genome sequence and CLC BIO software for de novo assembly of reads having no match with sorghum. An internally developed proprietary Oracle-based program was used for quality control, marker discovery, allele calling and data storage. All Miscanthus DNA sequences from this study were submitted to the National Center for Biotechnology Information (NCBI) Short Read Archive (study SRA050103).The DNA sequences were produced using two Illumina flow cells, consisting of eight lanes per cell and a multiplex of 12 samples per lane. DNA sequence reads were 76 nucleotides, with the first six nucleotides barcode sequence specific for given samples within the lane and the next six nucleotides partial restriction sites for The SNP markers were coded following the coding scheme of CP population type of JoinMap 4 x2>100 of goodness-of-fit tests; and (D) contained identical recombinant information. Eventually 5,600 SNPs were selected and imported into JoinMap for linkage mapping analyses.To ensure linkage map quality, the SNP markers used for final mapping were selected by removing markers that (A) showed differences of allele frequencies between the first 96 plants and the second 96 plants due to a potential library preparation bias between the two 96-well gDNA plates or a sequencing quality bias between the two flow cells; (B) had more than 60 missing values if they were heterozygous in only one of the parents (segregation type lmxll or nnxnp), or more than 30 missing values if they were heterozygous in both parents (hkxhk); (C) showed segregation distortion with Marker data were analyzed with JoinMap 4 using the CP population type Markers were assigned to correct linkage groups using two-point grouping analyses in three steps. First, only markers that showed un-distorted segregation  were assigned into linkage groups at the minimum independence test LOD score of 12. Second, ungrouped, including segregation distorted, markers were assigned to existing linkage groups based on “strongest cross link (SCL)” with a minimum LOD threshold of 10. Third, markers from different groups that showed strongest cross link were combined until no strongest cross link was seen between markers from different groups. After these three steps, the expected 19 linkage groups were formed for all three linkage analyses including the female, the male, and the composite mapping.Markers within each group were mapped using the regression mapping algorithm with the minimum LOD score of 1.0 to 5.0 and maximum recombination frequency of 0.35 or 0.40 depending on the linkage group http://www.atgc.org/XLinkage/). Any markers that were detected as having weak linkage positions were remapped by adjusting JoinMap parameters, or removed from final mapping.Linkage maps were all visually inspected by graphic genotyping in the JoinMap program. Due to potential sequencing errors and uncertainties caused by missing values and sequence duplication, the qualities of the final maps were carefully addressed and validated by an independent program CheckMatrix using PyMap BIT and REC scores as described by Kozik (Brachypodium distachyon (L.). Only BLASTN matches longer than or equal to 55 nucleotides with identity greater than or equal to 90% were used. The alignment to sorghum was performed using raw sequence reads of M. sinensis as described in the sequence analysis and SNP calling section; however, the alignment to other species used only sequences that produced SNP markers. The syntenic genome relationships of M. sinensis to other species were visualized using an internally developed visualization and comparative genomics software Persephone . In addition, a dot matrix plot was generated with Persephone using linkage map positions of M. sinensis markers against aligned sequence positions of the sorghum genome, allowing a whole genome comparison view of the two species.Using NCBI BLAST+ the DNA sequences containing the SNPs of interest were aligned to the genomes of sorghum, rice, maize and Table S1Composite map and genotype data of the mapping population.(XLS)Click here for additional data file.


The results of the study suggest that the four cardiac hormones lead to significant reductions in hypothalamic leptin levels, which may be an important mechanism for alleviating leptin-induced hypertension in obesity.Circulating levels of leptin are increased in obesity and have been proposed to contribute to the development of hypertension in obese individuals. Four cardiac hormones, specifically, vessel dilator, long-acting natriuretic peptide (LANP), kaliuretic peptide and atrial natriuretic peptide (ANP), have blood pressure-lowering properties and correlate with the presence of hypertension in obesity. The objective of this study was to determine whether one or more of these cardiac hormones was able to decrease the levels of leptin in the hypothalamus, an area of the brain that has been demonstrated to synthesize more than 40% of leptin in the circulation. The effects of these four cardiac hormones on leptin were examined using dose-response curves in the rat hypothalamus, which synthesizes leptin. Vessel dilator, LANP, kaliuretic peptide and ANP maximally decreased the levels of leptin in hypothalamic cells by 79, 76, 80 and 62%, respectively (P<0.0001 for each). The cardiac hormones decreased leptin levels over a concentration range of 100 pM to 10  Leptin is a 16 kDa-peptide (167 amino acids) that is synthesized and secreted predominantly by white adipose tissue –4. HowevAlthough leptin has been suggested to reduce appetite, obese individuals generally exhibit high circulating leptin levels  and, as The heart and adipose tissue are endocrine organs and studies have increasingly suggested that cross-talk exists between them, although the precise mechanism is poorly defined –19. The The cardiac hormones  were obtained from Phoenix Pharmaceuticals, Inc., Belmont, CA, USA.Hypothalamic cells  were obtained from the American Type Culture Collection (ATCC) Manassas, VA, USA. The ATCC authenticated this cell line.6 cells/ml. Cells were dispensed into new flasks with subculturing every 6 days. The growth medium was changed every 3 days.Propagation of the hypothalamic cells was performed in Dulbecco’s Modified Eagle’s medium with an addition of 10% heat-inactivated fetal bovine serum  and 1% penicillin, streptomycin and fungizone at a temperature of 37°C, as recommended by the ATCC. The number of cells in culture was 1.44×10® leptin immunoassay ELISA used to measure leptin levels was obtained from R&D Systems . This 3.5 h solid phase ELISA contained E. coli-expressed recombinant leptin and antibodies raised against the recombinant leptin. This quantitative sandwich enzyme immunoassay utilized a monoclonal antibody specific for leptin. The immunoassay has been shown to quantitate recombinant leptin accurately. Results obtained by measuring natural leptin revealed that the dose-response curves obtained with the recombinant Quantikine® assay paralleled the curves with natural leptin. The assay had a 98% recovery of leptin in the previously mentioned cell culture media. The minimal detectable concentration of leptin in this assay was 7.8 pg/ml. The levels of leptin measured are the amount in the cells plus the amount of leptin secreted into the media.The Quantikine6 cells/ml) were subcultured for 24 h, prior to 50 μl cell culture supernatant being added to 96-well plates with 50 μl media, containing 100 pM, 1 nM, 10 nM, 100 nM, 1 μM and 10 μM of each of the four cardiac hormones, separately (n=9 for each concentration). The hypothalamic cells were subsequently evaluated using the leptin ELISA from R&D Systems. Following this, the mean of the nine measurements at each concentration of the respective peptide hormones was then calculated and this was compared with the mean of the leptin concentrations in the control hypothalami, which had not been exposed to any of the cardiac hormones. The leptin data are shown in the figures as the decrease in the leptin level  compared with the level of leptin in the untreated hypothalami. The standards from R&D Systems were added to the blank wells to serve as reference points for known leptin concentrations. In this assay, absorbance was recorded at a 540 nm wavelength using a 96-well BioTek Gen 5, Synergy Mx microplate reader . There were 48 hypothalamic controls in these experiments.The hypothalamic cells (1.44×10Data are expressed as the mean ± standard error of the mean (SEM). Statistical analysis of the data were performed by one way analysis of variance (ANOVA) with a repeated measures design for within-group comparisons, using a statistical module of Excel software . A value of P<0.05 was considered to indicate a statistically significant difference.μM was used  from the control value of 85±4 pg/ml. The maximal reduction was obtained when the highest concentration of vessel dilator, i.e., 10 was used . At the μM  in the hypothalami, with the maximal reduction occurring at a LANP concentration of 1 μM . LANP, sectively .μM and its smallest reduction  at a concentration of 100 pM .ANP, like LANP, caused its maximal reduction  in hypothalamic leptin levels at a concentration of 1 f 100 pM . There wμM . Thus, with respect to the maximal reduction in leptin levels, the effects of vessel dilator, LANP and kaliuretic peptide were approximately equal and each of these cardiac hormones had a stronger ability than ANP to decrease leptin levels.Kaliuretic peptide decreased leptin levels by up to 80% (P<0.0001), with a maximal reduction occurring at the highest concentration of kaliuretic peptide, i.e., 10 μM . KaliureVessel dilator, LANP, kaliuretic peptide and ANP each significantly decreased leptin levels in the hypothalamus, an area of the brain that synthesizes leptin . The braIn the present study, the cardiac hormones were demonstrated to directly decrease leptin levels in the hypothalamus. It may be expected that the cardiac hormones also have the ability to decrease leptin levels in other leptin-synthesizing tissues, as leptin promotes angiogenesis by increasing vascular endothelial growth factor (VEGF) levels  and the Hormone-sensitive lipase breaks down triglycerides into non-essential fatty acids and glycerol . This hy


The British Council Connect initiative was set up to support strategic alliances and partnerships, promoting student learning overseas. With suitable training, risk analysis and agreements in place, this gave us the potential to explore off shore practice based learning, clinical attachments.A successful grant application was made to the British Council as part of the Prime Minister's Initiative for International Education (PMI2). A remote clinical educators network was identified, and training delivered. Five, third year students spent a month ‘hands on’ placement in five different hospitals in Singapore. Qualitative and quantitative data was collected from educators and students.16 applicants competed for five places. All students passed their clinical attachment without incident, Overall mean rating for the placement; 5 out of 5 (1= poor and 5 = excellent). Mean student score for the value of ‘clinical activities’; 4.8. Overall mean student score for all 14 criteria; 4.51. Accommodation, induction and preparation for the placement; although satisfactory, were identified as areas for improvement.This is the first UK podiatry initiative providing undergraduate students with hands on credit bearing clinical attachments overseas. Repeat funding is being sought. Students benefited greatly from high quality clinical education and the cultural experience.