The aim of this study is to quantify the impact of using eGFR instead of measured creatinine clearance (Clcr) on the evaluation of recovery from acute kidney injury (AKI).n = 229), patients on dialysis at ICU discharge (n = 77) and patients for whom Clcr on the last day of ICU was not available (n = 206), 784 patients were included in this analysis. We compared eGFR (MDRD equation) with measured Clcr (based on 24-hour urine collection and corrected for BSA) at ICU discharge for patient groups with different ICU stays. We also evaluated the impact of using the two GFR measurements on the estimation of complete recovery relative to baseline eGFR. Parameters were compared with the paired t test and McNemar's test.From a large RCT's database  we exclu2 (P < 0.0001). eGFR was not significantly different from Clcr in patients with ICU stay <7 days. In patients with ICU stay between 8 and 14 days, eGFR was significantly higher than Clcr  and the difference increased even further in patients with ICU stay over 14 days . The percentage of patients with complete recovery differed significantly when evaluated by eGFR (35.3%) or Clcr (28.7%) (P = 0.007). In patients with ICU stay >14 days, this difference increased to 56.4% by eGFR versus 14.1% by Clcr (P < 0.0001).Amongst the 784 patients with AKI, 456 (58%) reached stage 1, 143 (18%) stage 2 and 185 (24%) stage 3. Mean ± SD Clcr and eGFR at ICU discharge were respectively 54.5 ± 28 and 76 ± 55 ml/minute/1.73 mEstimated GFR at ICU discharge is significantly higher than the measured Clcr in patients with prolonged ICU stay. This difference can be explained by loss of muscle mass with decreased creatinine production and results in an important overestimation of recovery.


Ara h 8 is hypothesized to be the panallergen responsible for oral allergy syndrome between birch pollen (Bet v 1) and peanut. We recently determined the crystal structure of Ara h 8. In this work, we probed microarrays of peptides with peanut allergic and peanut sensitized patient sera for IgE and IgG4 reactivity.15-mer peptides that were offset by 5 amino acids were printed to glass. Patient sera was incubated with the slides. IgE and IgG4 binding was detected with combinations of secondary and fluorescently-labeled tertiary antibodies. The linear epitopes identified were mapped on the 3-D structure and compared with those of birch pollen protein Bet v 1.The majority of the Ara h 8 IgE epitopes mapped in this work align with those identified with Bet v 1. Considerably more IgG4 epitopes than IgE epitopes were found. Peanut allergic sera were more reactive with regard to IgE and IgG4 than peanut sensitized sera.Our results support both the hypothesis that Ara h 8 could be contributing to oral allergy syndrome between birch pollen and peanut.


Intraplexal nerve transfer is defined as nerve transfer of a nerve within the brachial plexus with intact spinal cord connections to a more important injured nerve. For elbow flexion, the most popular one for the upper arm brachial plexus injury is the “Oberlin” nerve transfer. Transferring a part of the ulnar nerve to the branch to the biceps (Oberlin 1) and possibly transferring a part of the median nerve to the branch to the brachialis (Oberlin 2) provides promising results for elbow flexion without any permanent deficit of the donor nerve function.In 1998, we published the paper using the Oberlin 1 technique in 32 cases with upper arm brachial plexus injury. Thirty patients had biceps strength of M4 (flexion power ranged from 0.5 to 7 kg) and 1 had biceps strength of M3. All but 1 patient demonstrated signs of recovery of the biceps muscle. No notable impairment of hand function was observed.For shoulder abduction, most nerve surgeons recommended nerve transfers both to the suprascapular nerve and the axillary nerve. In 2003, we described the nerve transfer to the deltoid muscle using the nerve to the long head of triceps both anatomically and clinically in 7 cases. All patients recovered deltoid power against resistance (M4) at the last follow-up evaluation. Useful functional recovery was achieved in all 7 patients; 5 had excellent recoveries and 2 had good results. The average shoulder abduction was 124 degrees. No notable weakness of elbow extension was observed.In 2006, we published the paper of combined nerve transfers for C5 and C6 brachial plexus avulsion injury in 15 cases. All 15 patients achieved useful functional recovery. Ten patients experienced excellent recoveries and 5 were classified as having good results. The mean shoulder abduction was 115°. The mean shoulder external rotation was 97°. No patients complained of functional deficit from harvesting of the nerve to the long head of the triceps.In our experience, 40 % of our patients with upper arm typed brachial plexus injury demonstrated winged scapula from serratus anterior weakness. In 2009, we described the nerve transfer to serratus anterior muscle using the thoracodorsal nerve in C5 and C6 brachial plexus avulsion injury in 5 patients. All patients recovered serratus anterior muscle function. Two patients had no winged scapula, whereas 3 patients had mild winged scapula after the surgery at the last follow-up evaluation. The result was excellent for 2 patients, good for 2 patients, and fair for 1 patient. The averaged shoulder abduction was increased to 134 degrees and external rotation was 124 degree. No notable weakness of shoulder adduction was observed.For wrist extension in C5, C6 and C7 brachial plexus injury which is difficult to achieve by tendon transfer, we described the nerve transfer using the branch of median nerve to restore the wrist extension. We used Flexor digitorumsuperficialis branch of median nerve transfer to extensor carpi radialis branch of radial nerve. This technique could restore wrist extension in C5, C6 and C7 brachial plexus root avulsion.


Here we report the consensus motif recognized by the DBINO domain identified by SELEX method and demonstrate the specific interaction of INO80 with the consensus motif. We show that INO80 significantly down regulates the reporter gene expression through its binding motif, and the repression is dependent on the presence of INO80 but not YY1 in the cell. The interaction is lost if specific residues within the consensus motif are altered. We identify a large number of potential target sites of INO80 in the human genome through in silico analysis that can grouped into three classes; sites that contain the recognition sequence for INO80 and YY1, only YY1 and only INO80. We demonstrate the binding of INO80 to a representative set of sites in HEK cells and the correlated repressive histone modifications around the binding motif. In the light of the role of INO80 in homeotic gene regulation in Drosophila as an Enhancer of trithorax and polycomb protein (ETP) that can modify the effect of both repressive complexes like polycomb as well as the activating complex like trithorax, it remains to be seen if INO80 can act as a recruiter of chromatin modifying complexes.The presence of a highly conserved DNA binding domain in INO80 subfamily predicted that INO80 directly interacts with DNA and we demonstrated its DNA binding activity  The SNF2 family of chromatin remodelers is known to facilitate different regulatory functions involving chromatin. The INO80, a highly conserved member of the SNF2 family of DNA dependent ATPase shows functional diversity and is implicated in transcription, replication, cell division and DNA repair . The altin vitro DNA binding activity to various degrees, specific targeting and recruitment to nucleosomal DNA, as well as regulation of chromatin remodeling activity is believed to be mediated either by specific interactions between specialized domains of chromatin remodeling complexes with post-translationally modified histones or through the interaction with sequence-specific DNA binding proteins, such as YY1, which recruit different complexes including the INO80 complex [CA][CA][CG] GTCA[GC]CC 3’ with a significant enrichment score of 5.75e-07 . When the T at 6th position is altered to G (M3) there is no interaction, while it is altered to A (M6), the interaction persists at equal molar ratio of 1:1, and is competed out at 1:5, suggesting a decrease in affinity for the mutant oligo. These residues are also among the most conserved residues in the consensus motif. Thus, we confirm the binding of INO80 to its sequence motif and that the binding is specific as seen by competitive EMSA and the INO80 knock-down experiments.To assess the base specificity within the consensus DNA sequence, the consensus motif was altered at various positions and these oligos were used in the competitive EMSA . The mutDrosophila  represents the subset of putative INO80 targets devoid of YY1 binding sites; [INO80(+YY1)] represents the subset of the list having both INO80 and YY1 binding sites in the upstream sequences (2000bp upstream of transcription start site).(TIF)Click here for additional data file.S5 FigQuantitative PCR carried out for expression status of target genes tested for INO80 interaction. The reciprocal of Ct values are plotted. Relative to GAPDH the other genes have low expression.(TIF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 Table(DOC)Click here for additional data file.S4 Table5’GCCATCAT3’ (8mer) and 5’CCGCCATNTT3’ (10mer) (www.genecards.org).The numbers of putative sites of interaction of INO80 and YY1 proteins in the regions upstream of protein coding genes were analyzed. The sequences of YY1 motif we used for analysis are: (DOCX)Click here for additional data file.S5 Table(DOC)Click here for additional data file.


In erythrocytes, the regulation of the redox sensitive Tyr phosphorylation of band 3 and its functions are still partially defined. A role of band 3 oxidation in regulating its own phosphorylation has been previously suggested. The current study provides evidences to support this hypothesis: (i) in intact erythrocytes, at 2 mM concentration of GSH, band 3 oxidation, and phosphorylation, Syk translocation to the membrane and Syk phosphorylation responded to the same micromolar concentrations of oxidants showing identical temporal variations; (ii) the Cys residues located in the band 3 cytoplasmic domain are 20-fold more reactive than GSH; (iii) disulfide linked band 3 cytoplasmic domain docks Syk kinase; (iv) protein Tyr phosphatases are poorly inhibited at oxidant concentrations leading to massive band 3 oxidation and phosphorylation. We also observed that hemichromes binding to band 3 determined its irreversible oxidation and phosphorylation, progressive hemolysis, and serine hyperphosphorylation of different cytoskeleton proteins. Syk inhibitor suppressed the phosphorylation of band 3 also preventing serine phosphorylation changes and hemolysis. Our data suggest that band 3 acts as redox sensor regulating its own phosphorylation and that hemichromes leading to the protracted phosphorylation of band 3 may trigger a cascade of events finally leading to hemolysis. Due to their function in carrying oxygen and their high iron content, red blood cells (RBCs) are constantly exposed to oxidative stress . In addiIt is noteworthy that approximately 7% of world population is affected by those mutations which have been selected by malaria.It is well known that RBCs respond to oxidative stress with a metabolic response finalized to maximize the production of NADPH and to regenerate the stores of GSH and thioredoxin. In parallel, RBCs also respond by activating tyrosine kinases determining the tyrosine (Tyr) phosphorylation of band 3, the most abundant RBC membrane protein and the major linkage between the cytoskeleton and the lipid bilayer –12. In RThe redox regulation of band 3 Tyr phosphorylation apparently involves different components. In a previous report, it has been demonstrated that oxidized band 3 is selectively phosphorylated . Lyn is 2O2 of the inhibitory Cys is 0.005-fold lower than GSH, indicating that, at its normal concentrations, GSH should very effectively protect PTPs from oxidative inhibition [Phosphatases (PTPs) have also been implicated in the phosphorylation of band 3 that follows oxidative stress –29 and ihibition , 31. Addhibition ; Lyn acthibition , 26. Mor2O2 and by hemichromes which cause irreversible oxidation.In the present report we performed a series of experiments to gain more information on the mechanisms that are involved in the Tyr phosphorylation of band 3 following a reversible membrane protein oxidation triggered by diamide and H2HPO4, 1.5 mM KH2PO4, 20 mM HEPES, 1 mM MgCl2, and pH 7.4) in 5 mM glucose (PBS glucose) to obtain packed cells. RBCs were suspended at 20% hematocrit in PBS glucose and incubated at 37°C in 0.5 mM diamide at different incubation times  and then in the presence of different diamide concentrations. Separate experiments were also performed in 5 mM H2O2 or 1 mM phenylhydrazine (PHZ). pH was measured after 180 minutes and adjusted to 7.4 with NaOH. When necessary to avoid tyrosine phosphorylation, RBCs were pretreated with 10 μM of Syk inhibitor II , for 1 hour at 37°C in the dark, before oxidant treatment. For all protocols described, untreated controls were processed identically except that the inhibitor was omitted from the incubation. To prevent further phosphorylation of band 3, after incubation we washed the cells with cold buffer and membranes were immediately prepared.Venous blood was drawn from healthy volunteers following informed consent and pelleted at 1000 g for 10 minutes at room temperature. After removal of the buffy coat, RBCs were again pelleted and washed 3 times with phosphate buffered saline   containing a protease and a phosphatase inhibitor cocktail and then washed up to 4 more times in the same buffer (until membranes became white) in a refrigerated Eppendorf microfuge at 25000 g. The preparations were stored frozen at −80°C until use. Membrane protein content was quantified using the CD Protein Assay (Bio-Rad).Membrane proteins were prepared at 4°C on ice as previously described . Brieflyµg of proteins for analytical gels, 1 µg of proteins for anti-band 3, and 30 µg of proteins for anti-phosphotyrosine, anti-phosphoserine, and anti-Syk antibodies were loaded for western blot analysis and separated on 8% of polyacrylamide gel under reducing and nonreducing conditions. SDS-PAGE analysis was conducted by heating the sample for 5 minutes at 95°C and was run on the Bio-Rad mini-protean 3 setup.To perform one-dimensional electrophoresis, membrane proteins were solubilized in Laemmli Buffer  in a volProteins separated by SDS-PAGE were transferred to nitrocellulose membranes as previously described  and thenµM band 3 concentration. Band 3 concentration was estimated measuring total membrane proteins in packed membranes (approximately 4 mg/mL) considering that band 3 represents approximately 25% of total membrane proteins and a band 3 M.W. of 95.000 Da. Resuspended membranes were incubated for 10 minutes on ice, with 0.1 mM diamide in the presence of increasing concentrations of GSH. The reaction was stopped by washing the solution 3 times with HB. The percentage of oxidized band 3 was evaluated by western blot following nonreducing 8% SDS-PAGE and expressed as percentage of the maximal oxidation measured in the absence of GSH. In the absence of GSH an average of 95.2 ± 4.5% of the band 3 was found present in reducible aggregates with a M.W. >200.000 KDa.RBC membranes were diluted in HB to obtain a 5 μM Syk inhibitor II  was added to prevent further phosphorylation of band 3. The rate of band 3 dephosphorylation was expressed as PTP activity and as a percentage of maximal activity in untreated RBCs.Erythrocyte PTP activity was measured using phosphorylated band 3 as substrate. Phosphorylated band 3 was obtained treating RBCs with 1 mM diamide. Membranes were prepared and incubated for 10 min at 37°C with the cytoplasmic fraction of RBCs treated with different concentrations of diamide. 10 We used a simplified method to measure the relative changes of Hb in RBC cultures supernatant; after discarding RBC membranes by centrifugation, lysis was quantified by measuring hemoglobin absorbance at 405 nm in RBC supernatant and expressed in nmoles/mL .RBCs were solubilized in HB containing 1% Triton X-100, centrifuged at 15.000 g at 4°C. High molecular weight hemichrome aggregates were separated from the supernatant on a Sepharose CL-6B microcolumn. The hemichrome fraction was then diluted and quantified measuring heme absorbance at 560, 577, and 630 nm  and exprRBC membrane proteins were treated in the presence or the absence of 2 mM diamide, solubilized for 10 minutes on ice with 3 volumes of 1% Triton X-100 in HB. After centrifugation in a refrigerated Eppendorf microfuge at 15000 g, supernatants were collected and incubated with anti-mouse anti-band 3 cross-linked to Protein A-Sepharose (1 : 10) via bifunctional coupling reagent dimethyl pimelimidate for 2 hours at 4°C under gentle mixing. Beads were washed three times with 1% Triton X-100 in HB . LaemmliTo obtain the oxidized and nonoxidized cdbd3 fragment, RBCs were incubated with or without diamide (2 mM). Membranes were prepared as described above and cytoskeletal proteins were eliminated incubating the membranes with 0.1 M NaOH at 4°C (stripped membranes). Cdbd3 was then purified from RBC membranes as previously described . The pur4HCO3/ACN (acetonitrile) (50/50 v/v) and successively dried with pure ACN. The gel slices were rehydrated for 45 minutes at 4°C in 20 μL of 5 mM NH4HCO3 digestion buffer containing 10 ng/μL of trypsin. Excess protease solution was removed and the volume was adjusted with 5 mM NH4HCO3 to cover the gel slices. Digestion was allowed to proceed overnight at 37°C.Bands were excised from electrophoresis gels and were destained by doing several washes in 5 mM NHμL of the tryptic digests mixed 1 : 1 with a solution of CHCA  . MS analysis of peptides from 1-DE gel bands was performed with a MALDI-TOF micro MX  according to the tuning procedures suggested by the manufacturer. Peak lists were generated with Proteinlynx Data Preparation using the following parameters: external calibration with lock mass using mass 2465.1989 Da of ACTH fragment 18-39 background was subtracted using the adaptive mode, performing deisotoping with a threshold of 3%. The MS spectra were converted into pkl files using Mass Lynx 4.0. Peak lists containing the 20 most intense peaks of the spectrum were sent to MASCOT PMF search (http://www.matrixscience.com/) using a Swiss-Prot database . Search settings allowed one missed cleavage with the trypsin enzyme to be selected, carboxymethylated cysteine as fixed modification and oxidation of methionine as potential variable modification and a peptide tolerance of 50 ppm. Only protein identifications with significant Mascot scores (p < 0.05) were taken in consideration.Samples were loaded onto MALDI target using 1 2O2) that is physiologically generated from superoxide anion during methaemoglobin formation and by denatured hemoglobin products [Time dependent phosphorylative changes of the RBC membrane proteins have been measured comparing the effects of (i) diamide, a single electron oxidant that induces disulfide formation , 26, . This is plausibly due to the potent scavenging activity of catalase and glutathione peroxidase in RBCs on H2O2.Diamide caused an intense and transient Tyr phosphorylation of band 3 and of proteins 4.1 and 4.2 though to a lesser extent and Ser phosphorylation changes in additional membrane protein Figures . H2O2 inλ-phosphatase, which was used to remove phosphate groups from blotted proteins (data not shown). Control experiments to exclude a direct oxidant effect of PHZ (2 mM) on isolated membranes revealed no effect on band 3 sulfhydryl groups (data not shown). The lack of short term effects of PHZ on membrane protein phosphorylation is coherent with its specific action on hemoglobin and the slow formation of hemichromes [Conversely PHZ caused a slow phosphorylation response measurable only after 60 minutes of incubation Figures . Tyr phoichromes , 39. A lichromes , 18.μM) that, due to the buffering effect of cellular GSH, are not expected to exert an effect on protein thiols. We observed a dose-dependent increase of the phosphorylation signal (p < 0.01) while no significant difference has been observed between the amount of cdbd3 immunoprecipitated from oxidized and nonoxidized samples  in forming band 3 (5 μM) intermolecular disulfide bonds on increasing concentrations of GSH. This experiment showed that at 0.1 mM GSH concentration (20-fold higher than band 3) approximately 40% of band 3 was still oxidized by diamide; at 1 mM GSH concentration (200-fold higher than band 3) approximately 20% of band 3 was still oxidized (μM) no oxidation of GSH was detectable (data not shown).The characteristic accessibility of the two cysteins 201 and 317 located in the cytoplasmic domain of band 3 has been already demonstrated . To obtaoxidized , indicatErythrocyte PTPs have been implicated in promoting the Tyr phosphorylation of band 3 due to an inhibitory Cys residue located in their catalytic domain , 40.To rule out the possibility that diamide treatment, at concentrations that induce band 3 phosphorylation, may also determine a substantial inhibition of PTPs, we compared the levels of band 3 phosphorylation and PTP inhibition at different diamide concentrations. Differently from the effect of diamide that caused reversible changes, after phenylhydrazine treatment, band 3 oxidation, and its phosphorylation, Syk translocation to the membrane and its phosphorylation increased progressively, paralleling the generation of hemichromes . Also inInterestingly, RBCs rapidly react to oxidative stress through very intense Tyr phosphorylation of band 3, their major integral membrane protein. We previously found that the phosphorylation of band 3 affects its interactions with the cytoskeleton inducing membrane destabilization . This phAnyway, the involvement of Syk kinase of band 3 oxidation and of all the major steps of the pathway such as the mechanism of redox sensing, its transduction, the regulation of Syk activation, and docking to band 3 need to be clarified to envisage the physiological role of this intense redox response characteristic of erythrocytes. To address those issues, the present work has been performed to obtain a series of quantitative data to study (i) the temporal and dose effects of different physiological and nonphysiological oxidants in eliciting the minimal band 3 and Syk modifications, (ii) the role of disulfide cross-linked band 3 in docking Syk, (iii) the buffering effect of GSH on the oxidation of band 3 Cys residues to rule out if band 3 could display activity as redox sensor in intact erythrocytes, and (iv) the relative roles of Syk activation and docking versus PTPs inhibition in the phosphorylation of band 3.2O2. The comparative measurement of band 3 phosphorylation and of PTPs inhibition at different concentrations of diamide revealed that intense phosphorylation can occur at concentrations that minimally inhibit erythrocytes PTPs acting on phospho-band 3. This finding is in accordance with the relatively low reactivity of the Cys residue located in the catalytic site of PTPs [The presented results indicate that band 3 possess highly reactive Cys residues capable of being easily oxidized in the presence of physiological concentration of GSH and that disulfide cross-linked band 3 docks Syk and acts as competitive inhibitor of band 3 phosphorylation. Those results support the observed changes in whole RBCs with very low concentration of a specific sulfhydryl reagent (diamide) or following the formation of minute amounts of hemichromes. In both models band 3 phosphorylation exactly parallels its oxidation. On the other hand erythrocytes seem to be fairly protected by H of PTPs  and with2O2 which induces a transient phosphorylation of band 3 does not cause hemolysis, a persistent phosphorylation of band 3 induced by irreversible hemichromes apparently leads to a severe membrane destabilization. It should be anyway noticed that hemichromes formation was also accompanied by serine phosphorylative changes involving some membrane protein; those phosphorylation changes have been usually considered to cause a decrease of the affinity between some components of the RBC membrane junctional complexes [In the present report, we observed that Syk inhibitors are potent inhibitors of the hemolysis that follows to the generation of hemichromes. Considering that treatment with diamide or Homplexes –50 and mConsidering that band 3 phosphorylation may have a function in remodeling the RBC membrane to remove noxious hemichromes , the pre


Prolonged post-operative air leak is a recognised complication in patients receiving lung volume reduction surgery (LVRS). Some patients are transferred to a portable flutter-valve bag to facilitate discharge. TissuePatchTM is a synthetic absorbable self-adhesive film which acts as an adjunct to minimise air leak.Our aim was to see whether the use of TissuePatchTM would reduce post-operative air leak and the subsequent need for a drain in LVRS patients.We retrospectively analysed LVRS cases over a two year period performed by a single surgeon to minimise procedural heterogeneity. Patients were divided into two groups; group 1 received Tissue PatchTM as the staple line adjunct and group 2 did not.There were 26 cases in total ; group 1=12 , group 2=13 . The median length of stay was 15 for both groups (p = 0.40). The median duration of air leak was 13 days for group 1 and 18 days for group 2 (p = 0.95). Only 2/12 (16%) in group 1 did not have full resolution of air leak and drain removal prior to discharge and were placed on a portable flutter-valve bag compared to 5/13 (38%) in group 2 (p = 0.64).We have observed a reduced trend in the number of patients being discharged with persistent air leak following LVRS with the concomitant use of Tissue PatchTM. A larger study is indicated which may demonstrate significant results.


FOXG1), Methyl CpG binding protein 2 (MECP2) and Cyclin-dependent kinase-like 5 (CDKL5) have been studied individually, not much is known about their relation to each other with respect to expression levels and regulatory regions. Here we analyzed data from hundreds of mouse and human samples included in the FANTOM5 project, to identify transcript initiation sites, expression levels, expression correlations and regulatory regions of the three genes.Mutations in three functionally diverse genes cause Rett Syndrome. Although the functions of Forkhead box G1  mediated silencing of Foxg1 in cerebellum.Our investigations reveal the predominantly used transcription start sites (TSSs) for each gene including novel transcription start sites for Our analyses provide a comprehensive picture of the regulatory regions of the three genes involved in Rett Syndrome.The online version of this article (doi:10.1186/1471-2164-15-1177) contains supplementary material, which is available to authorized users. MECP2), Forkhead box G1 (FOXG1) or Cyclin-dependent kinase-like 5 (CDKL5) genes [MECP2 differs from the phenotype of patients with FOXG1 or CDKL5 mutations, there are some similarities in the clinical profile that overlap with RTT. Classic RTT patients with MECP2 mutations have a normal period of development followed by regression of acquired skills, deceleration of head circumference, epilepsy, hand stereotypies, breathing abnormalities, inability to walk or talk and intellectual disability while patients with atypical RTT may show some but not all features of classic Rett syndrome [FOXG1 are known to cause the congenital variant of Rett syndrome where the initial normal developmental window is absent [CDKL5 mutations are found in patients with severe epilepsy during early childhood that later show features that resemble atypical RTT syndrome [Rett Syndrome (RTT) is a disorder caused by mutations in Methyl CpG binding protein 2 5) genes  Althoughsyndrome . Mutatio genes  AMECP2 is expressed ubiquitously [MECP2 mutations and copy number variations in humans lead to neurological phenotypes such as classic or atypical Rett syndrome and in rare cases Angelman Syndrome, X-linked mental retardation and Autism  suggestiewed in , 17. Theiewed in  and it iiewed in , but thein cells .FOXG1 protein is a brain specific member of the forkhead transcription factor family with a role in transcriptional repression. Similar to other members of the forkhead family, FOXG1 has a defined binding sequence motif , which bCDKL5 protein is a serine threonine kinase, whose expression is low in embryonic stages, but increases in postnatal stages up to postnatal day 15  CDKL5 mREven though these three genes have different expression patterns, distinct functions and specific regulatory targets, their paths appear to intersect. Both MeCP2 and FOXG1 proteins regulate transcription via DNA binding and association with other transcriptional regulators –1325, 2, 225, 26MECP2 and CDKL5 genes are expressed ubiquitously, their mutations cause a brain specific phenotype suggesting that their expression level, transcription regulation, or function in brain may be distinct from that in other tissues. We tried to resolve these questions through bioinformatics analyses using the FANTOM5 dataset [Although we have some knowledge of their downstream intersecting functions, we are yet unaware of the common genomic features between these three genes, which may provide insights into their regulation. Importantly, although both  dataset .Mecp2 KO mouse model [FOXG1 in mouse and humans. We show that each of these genes use the same TSS in most tissues and provide information on the expression level of the three genes over development in multiple human and mouse samples. Although we did not find a significant correlation between the expression levels of the three genes in the brain, our genome wide analyses uncovered common transcription factors regulating the three genes, suggesting an additional molecular layer in the pathogenesis of Rett Syndrome. The FANTOM5 CAGE dataset also allowed us to locate putative enhancers regulating the three genes in human  of these genes and study their expression profile in hundreds of mouse and human samples using Cap Analysis of Gene Expression method (CAGE) . In conjse model  and stud et al., ) and usiSingle molecule CAGE profiles were generated from RNA obtained from a collection of 573 human primary cell samples (~3 donors for most cell types) covering most mammalian cell steady states. This data set was complemented with profiles of 250 different cancer cell lines, 152 human post-mortem tissues and 456 mouse samples , or provided under informed consent. All non-exempt material is covered under RIKEN Yokohama Ethics applications (H17-34 and H21-14). Mouse tissue samples were collected as per RIKEN Yokohama institutional guidelines. Mouse primary cells were collected as per our collaborators Institutional guidelines and shipped as either purified RNA or as guanidinium isothyocyanate lysates  which were then purified using the miRNeasy kit (QIAGEN). More detailed information for each specific sample is available in Additional file: http://fantom.gsc.riken.jp/5/data/. All CAGE data has been deposited at DDBJ DRA under accession number DRA000991.All the data published by the Fantom5 project and by this study are available through the Fantom5 portal http://fantom.gsc.riken.jp/5/sstar/). Annotation files were built in the context of the FANTOM5 project with respect to Gencode v10 gene model (human), RefSeq (mouse), CpG islands and TATA box in bed format.We used the FANTOM5 database  to idenhttp://www.r-project.org/).We extracted expression information for each TSSs using the FANTOM5 expression dataset for tissues, cell lines and primary cells in human and mouse . All the values for different genes were added together and compared to expression levels of MECP2.We extracted the CAGE defined promoters associated to the genes whose products form the Histone1 transcripts (TSSs identified were expanded by 500 nucleotides on either side (±500bp). ChIP seq data from Human and mouse ENCODE were downloaded as bed files and intersected with our expanded TSS using intersectBed .P < 0.05) with any of the three Rett genes promoters were included.To identify enhancers associated with the human Rett genes, we used the CAGE derived enhancer database from Andersson et al. . In shorWe downloaded the whole-genome alignment of the human genome with 45 other vertebrate genomes, and of the mouse genome with 29 other vertebrate genomes, from the UCSC Genome Browser database . From thFOXG1 and 6 TSSs for mouse Foxg1  and 30% (140) of human and mouse samples respectively, suggesting that the expression of this gene was limited to selected tissues  and chr12:50485112..50485144,+;  suggesting that other than the brain, fibroblast cell lines may be useful for in vitro analysis of Foxg1 in mouse  identified 8 TSSs for human FOXG1 isoform and therefore a single TSS. We found 8 TSSs for human FOXG1 expressed over 5 TPM and the 3 TSSs with the highest expression in human brain were located at chr14:29235961..29236008,+ (p1@FOXG1); chr14:29234581..29234601,+ ; and chr14:29236269..29236285,+ (p3@FOXG1), with distances of 317, 1697 and 9 bases upstream of the RefSeq annotated TSS, respectively , which are known to faithfully recognize active transcription initiation sites [Foxg1 by Polycomb Repressive Complex 2 (PRC2) , however its expression was not entirely discordant with that of Foxg1 (data not shown). Analysis of the ncRNA database and manual annotation of UCSC Genome Browser revealed several ncRNAs within a genomic window of 1.5 MB around Foxg1, but none of the listed ncRNAs were detectable in the FANTOM5 CAGE dataset.Silencing by chromatin remodeling proteins such as PRC2 requires a non-coding RNA to mediate chromatin modification . TherefoMECP2, less than 100 bases upstream of the RefSeq annotated start sites of which p1@MECP2/Mecp2 was expressed predominantly in most tissues and p2@MECP2/Mecp2 displayed a stable low level expression in all tissues (expression less than 10 TPM)  and cell lines   and P30. In kidney, the expression of Mecp2 declined after P20 and in liver the expression of Mecp2 appeared to be induced after birth (P00) but remained unstable up to the age of P30 , pituitary cortex (n = 1 at each age) and visual cortex (n = 4 at P15 and n = 3 at P30 and P60). Our data reveal that p1@Mecp2 expression fluctuates in embryonic cerebellum samples but is clearly induced after postnatal day 9  confirming our earlier observation of similarities in expression of the two Cdkl5 promoters in mouse.To investigate the relationship between the expression of all TSSs of each gene, we conducted intra gene correlations and found a high degree of correlation between p1@FOXG1 and p3@MECP2, FOXG1 and CDKL5 genes result in overlapping neurological phenotypes, we additionally investigated the inter gene expression correlations of the three genes in the brain. We first generated heatmaps from all brain sub-regions and brain related primary cells  and in humans we found negative correlation of −0.1, suggesting slight discordance of expression of the two genes in brain. Thus, our analyses failed to find mathematically significant evidence of contrasting expression between FOXG1 and MECP2. The two promoters of CDKL5 were also poorly correlated with the FOXG1 promoter expression in brain, while there was a positive correlation (23-49%) between expression of MECP2 and CDKL5 in both species  was located 7kb upstream of the gene. Many predicted enhancers for MECP2 had an average correlation of 0.37, the closest enhancer (53kb) had an expression correlation of 0.43, while the highest correlated enhancer (r = 0.55) was over 408kb distant. For CDKL5, the only identified active enhancer had a low correlation of 0.2 and was located over 245 kb upstream of the gene  were broad in keeping with the CpG islands in their vicinity , FOXP1 (p = 0.03), and NFY (p = 0.01) transcription factors. NFY was also predicted to regulate MECP2 (p = 0.01) and possibly CDKL5 (p = 0.09). Similar analyses in the mouse genome revealed motifs for 21, 5 and 3 TFs within 500 bp of the Foxg1, Mecp2 and the Cdkl5 promoters respectively  were common to all three genes. Calculating the statistical significance of the estimated number of binding sites revealed that in mouse for all three genes the promoter regions were enriched for motifs associated with transcription factor NFY, as well as Sp1  and humans  are clearly devoid of FOXG1 expression at any developmental stage. We further confirmed our observation of the absence of Foxg1 expression in mouse cerebellum through analyses of chromatin signatures from mouse ENCODE. Our investigation revealed enrichment of H3K27me3 in the Foxg1 genomic region, suggesting PRC2 mediated silencing of Foxg1 in the cerebellum. Although H3K27me3 has also been reported to be present at transcriptionally active or poised loci [Foxg1 promoter region in cerebellum but not in the cortex, strongly suggest specific repression of Foxg1 in the cerebellum. A similar examination in liver also revealed H3K27me3 enrichment at the Foxg1 promoter region , weMECP2, MECP2 mnslation  but we dMECP2 gene gives rise to two mRNA isoforms with same transcription start site [MECP2 in humans and mouse in all tissues, we could not allocate two distinct start sites for the two isoforms of MECP2. Based on our data, we were unable to conclude whether p2@MECP2/Mecp2 represented an independent poorly expressed protein coding isoform, a shorter non-coding regulatory ncRNA transcript arising from the vicinity of the main promoter p1@MECP2/Mecp2 or a tissue specific enhancer RNA (eRNA) [MECP2. Almost 25% of all enhancers are expected to transcribe short bi-directional capped transcripts called e-RNAs [MECP2/Mecp2, do not support its identification as an e-RNA for MECP2. The two TSSs for CDKL5 are highly correlated with each other in mouse as well as humans. Based on their similar expression levels and distinct promoter shapes, we propose that they represent two independently regulated transcripts despite their proximity.The art site , 57 and A (eRNA)  for MECPd e-RNAs . Our obsThe comparison between corresponding promoters in human and mouse samples, including the novel promoter p1@FOXG1 in human and pA@Foxg1 in mouse, revealed remarkably similar shapes, suggesting evolutionary conservation in their regulation. The only exceptions were the human p2@FOXG1 and pB@Foxg1 mouse, which due to their distinctive promoter shapes appear to be regulated in a species-specific manner.The recently released ENCODE Histone ChIP seq data , allowedFOXG1 and MECP2 but not CDKL5 in humans and NFY is likely to regulate all three genes in mouse. Further investigation will be needed to experimentally verify these findings nevertheless, it will be of interest to study the expression level and presence of mutations in the common TFs in mutation negative RTT patients.Almost 20% patients of atypical RTT do not have mutations in the three genes. We conducted genome wide TFBS analyses with the aim to discover the common transcription factors likely to regulate the three genes and thus identify shared pathways upstream. Mutations or functional impairment of such common TFs may affect the expression of the three genes, which may result in disease phenotype. Our data predict that TFs NFY and SP1 are likely to regulate MECP2 in brain or neurons, which could have explained the predominantly neurological phenotype seen in patients with mutations in this ubiquitously expressed gene.Our investigations failed to demonstrate brain specific promoter usage or particularly high levels of expression of Our comprehensive analyses of data from the FANTOM5 project reveal novel insights into the common and distinct genomic features of the three genes, which are related not only by disease phenotype, but also in their regulation in a species-specific manner.Additional file 1: Table S1: List of all tissues, cells and cell lines with the TPM expression of the FANTOM5 defined transcription start sites of the three genes shown per sample in sheets 1 and 2, and averaged TPM expression across replicates shown in sheets 3 and 4. (XLS 24 KB)Additional file 2: Table S2: List of RefSeq and FANTOM5 detected transcription start sites in human and mouse. (XLS 24 KB)Additional file 3: Figure S1: Top 15 samples in expression for each of the three genes. Panels a, c and e represent mouse, while panels b,d and f are human samples showing promoter expression in TPM, on X-axis, in various tissues, as labeled on Y-axis. For each gene, the samples with the highest expression of the main promoter  are shown. The expression of the other key promoters in these samples is also shown . (PDF 21 KB)Additional file 4: Figure S2: Silencing of Foxg1 in mouse. UCSC Browser image of the genomic locus for Foxg1 showing ENCODE tracks for DNAse-I hypersensitive sites, active enhancer specific histone mark (H3K27ac), active promoter specific histone mark (H3K4me3) and PRC2 mediated repressor mark (H3K27me3) in mouse cerebellum, cerebrum, whole brain and liver as labeled. Cerebellum samples lack the DNAse-I hypersensitive sites visible in cerebrum and whole brain samples. Cerebellum samples also lack the active promoter mark H3K4me3 seen in cortex, but contain PRC2 repressive histone mark H3K27me3 not seen in cortex at the locus. (PDF 36 KB)Additional file 5: Figure S3: Expression levels of Mecp2 and Cdkl5 during development in heart kidney and liver. The line plots show the fluctuations in expression for the two promoters for Mecp2 and Cdkl5 in heart, (a and d), kidney (b and e) and liver (c and f) in mouse. (PDF 46 KB)FOXG1 (a), MECP2 (b) and CDKL5 (c) expression in TPM across a set of adult, newborn and fetal brain regions is shown as labeled. FOXG1 shows the highest overall expression as well as having higher expression in fetal than in adult samples as opposed to the expression of MECP2 and CDKL5 in the same samples. (PDF 39 KB)Additional file 6: Figure S5: Developmental profile for the 3 genes in human brain. Human Foxg1, Mecp2 and Cdkl5 during development in mouse cerebellum (panel a), mouse visual cortex (panel b) and mouse pituitary gland (panel c). Refer main text for details. (PDF 41 KB)Additional file 7: Figure S4: Expression profile of the three genes in mouse in developing brain tissues. Line plots showing expression of selected promoters of MECP2. Bar charts showing TPM expression of the key promoter of MECP2 and collective total expression of Histone H1 TSSs in brain related cells (panels a and c) and tissues (panels b and d) in mouse (panels a and b) and humans (panels c and d). Histone expression levels appear to be over 100 fold higher than MECP2 in brain related cells suggesting a massive up-regulation of MeCP2 at the level of protein translation. (PDF 39 KB)Additional file 8: Figure S6: Comparison of mRNA levels of Histone H1 and Additional file 9: Table S3: Comparison of the expression of the Histone H1 genes promoters and MECP2 in both human and mouse. (XLS 134 KB)Additional file 10: Table S4: Pearson and Spearman correlations for all TSSs in human and mouse. (XLS 20 KB)Additional file 11: Figure S7: Intra and inter gene expression correlations between the three genes. Expression correlation plots for all other promoter combinations not present in Figure Additional file 12: Table S5: Location of enhancer and promoter specific Histone marks in relation to TSSs in mouse. (XLS 20 KB)Additional file 13: Table S6: Locations and correlations of human enhancers to the three Rett genes. (XLS 26 KB)Additional file 14: Figure S8: Locations of active enhancers correlated to the three genes in human samples. UCSC snapshot showing the positions of all eRNA producing human enhancers that are correlated to the expression of the three genes: FOXG1 (a), MECP2 (b) and CDKL5 (c). (PDF 58 KB)Additional file 15: Figure S9: Promoter shapes for all the other promoters. The shapes of all the individual promoters in mouse (a-e) and human (f-m) are shown as labeled. The shapes are drawn from the first nucleotide of the first mapped CAGE tag to the first nucleotide of the last mapped CAGE tag, the y-axis shows the counts in TPM for each position. (PDF 41 KB)Additional file 16: Table S7: List of transcription factors with high binding probability of 0.7 and above to the promoters of the three genes in mouse (A) and human (B) genome. Transcription factors common to the three genes are shown in red. (DOC 48 KB)


In breast cancer, the presence of Foxp3 (Tregs)  within tThe goal of our study was to quantify Foxp3 in breast cancer patients from Qatar and correlate with their survival.Expression of FoxP3 was studied in 132 FFPE samples with known clinico-pathological data by immunohistochemistry technique and quantified by modified H-score system by pathologist. Results were analyzed via SPSS.Analysis was carried for 132 patients. Age at time of diagnosis was 49 ±10.4 years. 76.2% of the patients showed positive expression of FoxP3. FoxP3 expression was not correlated with patient age or hormone receptors. Expression of Foxp3 positively correlate with better patient survival when compared to negative expression .FoxP3 is expressed on lymphocytes that are present in the tumor microenvironment regardless of breast cancer subtypes. Foxp3 is correlated with better survival.


The absence of driver gene mutations in the exomes of three individuals with aUPD14q but no known haematological disorder suggests that aUPD14q may be sufficient to drive clonal haemopoiesis. Analysis of cases with both aUPD14q and JAK2 V617F (n=11) indicated that aUPD14q may be an early event in some cases but a late event in others. We conclude that aUPD14q is a recurrent abnormality that targets an imprinted locus and may promote clonal haemopoiesis either as an initiating event or as a secondary change.Acquired uniparental disomy (aUPD) is a common finding in myeloid malignancies and typically acts to convert a somatically acquired heterozygous mutation to homozygosity. We sought to identify the target of chromosome 14 aUPD (aUPD14), a recurrent abnormality in myeloid neoplasms and population cohorts of elderly individuals. We identified 29 cases with aUPD14q that defined a minimal affected region (MAR) of 11.2 Mb running from 14q32.12 to the telomere. Exome sequencing ( Constitutional UPD is associated with developmental disorders caused by the abnormal expression of imprinted genes, that is, genes that are differentially expressed depending on whether they have been maternally or paternally inherited. By contrast, somatically acquired UPD (aUPD) in cancer is a mechanism by which a pre-existing driver mutation  is converted to homozygosity, thereby providing an additional clonal advantage. aUPD may involve whole chromosomes as a result of non-disjunction or, more commonly, whole chromosome arms or terminal segments as a consequence of mitotic recombination. aUPD cannot be detected by conventional cytogenetics but is readily apparent by the finding of somatically acquired long homozygous tracts without change in copy number by genome-wide single nucleotide polymorphism (SNP) analysis.TET2, EZH2, JAK2, CBL and FLT3, respectively.1 Several other regions of recurrent aUPD have been identified for which the target is unknown, for example, chromosome 14q aUPD (aUPD14q) is seen in myeloid neoplasms2 and is one of the most common abnormalities associated with clonal haemopoiesis in population cohorts of elderly individuals.4 Here we show an unexpected and highly significant parental bias associated with aUPD14q, implicating an imprinted locus at 14q32 as the primary target rather than a specific mutated gene.aUPD is prevalent in myeloid neoplasms: chromosomes 4q, 7q, 9p, 11q and 13q are commonly affected and target mutated 5 and the Prospective Investigation of the Vasculature in Uppsala Seniors (PIVUS).6 The study was approved by the following ethics committees or review boards: the National Research Ethics Service (UK) Committee South West, the Uppsala Regional Ethical Review Board, the Ethics Committee of the Biomedical Research Foundation of the Academy of Athens, Comitato Etico, Azienda Ospedaliero-Universitaria Careggi, Firenze. Informed consent was obtained according to the Declaration of Helsinki.Our study comprised two major groups of individuals: (i) patients diagnosed with a myeloproliferative neoplasm (MPN) or myelodysplastic (MDS)/MPN according to standard morphological, haematologic and laboratory criteria; (ii) population cohorts of elderly individuals from Sweden, specifically the Uppsala Longitudinal Study of Adult Men7 were obtained from peripheral blood or bone marrow leucocyte DNA using the Affymetrix SNP 6.0  or Illumina Human OmniExpressExome v1.2 platforms . Peripheral blood leucocyte profiles for the Swedish population cohorts were obtained using Illumina 2.5M HumanOmni arrays (Illumina) have also been published.8 Exome sequencing for MPN and MDS/MPN cases was performed using the Agilent SureSelect kit  (Human All Exon 50 Mb) and sequenced using an Illumina HiSeq 2000 (Illumina) at the Wellcome Trust Centre for Human Genetics at Oxford, UK. Exome sequencing for the Swedish cases was performed by SciLifeLab, Stockholm, Sweden.Genome wide SNP profiles for MPN and MDS/MPN cases, most of which have been published previously,MEG3 or NHP2L1 with a common FAM labelled reverse primer and analysed with a 3130xl Genetic Analyser  as described.9 Loss of heterozygosity analysis at 14q32 was performed using microsatellites D14S553, D14S267, D14S1006, D14S542, D14S292 and D14S1007, again using a 3130xl Genetic Analyser. All primer sequences are listed in For methylation analysis, DNA was bisulphited  alongside four healthy controls, amplified using forward primers specific for methylated or unmethylated 11 For each SNP the log R Ratio, a measure of normalised total signal intensity, and B Allele Frequency (BAF), a measure of normalised allelic intensity ratio, were determined using the BeadStudio (Illumina) and pennCNV12 software for Illumina and Affymetrix arrays, respectively. Regions of aUPD were identified by BAF segmentation10 which excluded non-informative SNPs (SNPs with BAF >0.9 or BAF <0.1 and SNPs where the absolute difference in BAF between preceding and succeeding SNPs is >0.6), mirrored BAF at 0.5 and used circular binary segmentation to identify regions with similar allelic proportions. For heterozygous SNPs, the BAF is the proportion of the total signal (A+B) accounted for by one allele (B). In a mixed population of cells, the segmented mirrored BAF value will be a combination of values of 1 and 0.5 for cells with and without aUPD, respectively. Regions of aUPD were therefore defined as a region of allelic imbalance (segmented mirrored BAF >0.56) with neutral copy number (log R ratio ~0) that extended to the telomere.12 For samples with known JAK2 V617F levels, determined by pyrosequencing,13 and BAF for both aUPD9p and aUPD14q aUPD, we were able to calculate both the proportion of cells with aUPD14 and the proportion of cells which were homozygous or heterozygous for JAK2 V617F. This allowed us to infer the likely order of acquisition of aUPD14 and JAK2 V617F , variant allele frequencies were used to approximate BAF and were analysed accordingly to confirm regions of aUPD. The minimally affected region identified in patient E5364  was interrogated in all aUPD14q exomes for rare variants that were either novel or had a minor allele frequency of ⩽1% in databases of common variation .Analysis of exome sequencing data was as previously described.20 reads . For varn=21), 1/293 (0.3%) had MDS/MPN, 8/563 (1.6%) had JAK2 V617F negative MPN  and 12/1054 (1.1%) had JAK2 V617F positive MPN, an overall prevalence similar to that identified in other studies of myeloid neoplasms .17 Trisomy chromosome 14 (+14) was seen in 4/293 (1.4%) MDS/MPN cases.2 Of the 1641 individuals ⩾70 years of age in the Swedish population-based cohorts, 4 (0.2%) had aUPD14,8 similar to the frequency in elderly individuals reported by other larger studies of cases recruited for a variety of genome-wide association studies.4 Cases with aUPD14q or other chromosome 14 abnormalities in our study are summarised in We analysed in house array data on cases with MPN or MDS/MPN and identified aUPD14q extending to the 14q telomere in 25 cases. Of the UK cases .3 This region does not include FANCM, a variant of which was associated with aUPD14q in a single case.18The region affected by aUPD14q was variable between individuals and there was no difference between cases diagnosed with a haematological malignancy and those picked up in population-based screens . Since tn=7) or +14 (n=1). We focused on the identification of novel variants in the minimal region of aUPD14q , thus capturing both constitutional and somatic mutations that might provide a selective advantage when reduced to homozygosity. No gene was identified with likely causative variants in more than one individual  or +14  consistent with paternal aUPD . Methylation values and BAF in the region of aUPD14q were strongly correlated  . This is the first time to our knowledge that aUPD has been associated with a specific parent of origin effect, a finding that indicates that aUPD14q targets an imprinted locus.Constitutional maternal UPD14q causes Temple syndrome, whereas paternal UPD14q causes Kagami–Ogata syndrome. Both are developmental conditions resulting from aberrant expression of genes in the imprinted +14 n=6; . MEG3 isosome 14 . Samples=0.0001) , suggestn=5), an abnormality that is also seen recurrently in myeloid malignancies and population cohorts of elderly individuals. Like aUPD14q, the target of aUPD22q has not been identified but there is a candidate imprinted locus (NHP2L1) within the affected region.19 Methylation analysis indicated maternal aUPD22q in two cases and paternal aUPD22q in three cases  that had either been analysed by SNP arrays and were known to be negative for aUPD14q (n=48) or were randomly selected without knowledge of their aUPD14 status (n=48). Most cases had methylation levels that were indistinguishable from healthy controls, but three had a clear gain of methylation ,20 components of which are recurrently inactivated in myeloid neoplasia by mutation of EZH2, SUZ12 or EED.21 It is possible therefore that the consequence of aUPD14q might be functional inactivation of PRC2, in which case we would expect aUPD14q and PRC2 mutations to be mutually exclusive. Partial sequence analysis of EZH2 and SUZ12, the most commonly mutated components of PRC2, revealed causative EZH2 mutations in two out of eight aUPD14q cases  revealed no other obvious driver mutations in three out of four individuals from the population cohorts, suggesting that aUPD14q alone may be sufficient to promote clonal haemopoiesis. The fourth individual (PIVUS 931) was positive for JAK2 V617F as well as a TP53 mutation; he was subsequently diagnosed with polycythemia vera following recruitment into the PIVUS study. In contrast, all three cases with diagnosed myeloid malignancy that were exome sequenced had additional somatic driver mutations in SF3B1, EZH2, JAK2 or TET2  this cluster falls with the 11.2 Mb MAR, an interval that contains 121 known protein-coding genes plus the immunoglobulin region, and (ii) despite extensive searches there are no other confirmed imprinted loci within the MAR, or indeed elsewhere on chromosome 14. Furthermore, expression of DLK1-MEG3 has been reported to be deregulated in diverse neoplasms in the absence of chromosome 14 abnormalities, including acute promyelocytic leukaemia27 and myelofibrosis.29 In our study, we did not have suitably stored material for expression analysis of aUPD14q cases; however, expression analysis alone is unlikely to be informative since by definition aUPD would be expected to distort the normal expression of imprinted genes in the affected region whether they were of pathogenetic relevance or not.Our finding of a highly significant parent of origin effect associated with aUPD14q strongly implicates an imprinted locus as the primary target, and that paternal homozygosity for this target provides a growth advantage over cells that harbour both alleles. Del(20q), another somatic abnormality associated with myeloid neoplasms, has been shown to target an imprinted gene clusterMEG3 methylation in our study, 21 had positive values indicative of paternal aUPD. One case had a negative methylation value. We do not have an explanation for this aberrant case, but potentially other mutations may have been present that promoted clonal expansion. We note that aUPD for other regions has not been associated with a specific mutational target in all cases, for example, we found that only 9 of 12 MDS/MPN cases with aUPD7q had EZH2 mutations, with the target of the remaining 3 cases remaining unclear.2Of the 22 aUPD14q cases analysed for 31 aUPD14q is more prevalent in the elderly. Previous studies found aUPD14q in 21/50 222 (0.04%) individuals, rising to 14/15 101 (0.09%) in those >60 years of age,4 an increase that parallels the increase in myeloid neoplasia seen in the elderly. Analysis of longitudinal data estimated a 10-fold elevated risk of developing a haematological malignancy for individuals with any acquired chromosome anomaly,4 and a similar risk was estimated for individuals with somatic mutations in genes known to be associated with myeloid malignancies.31 The most commonly mutated genes in population cohorts were DNMT3A, TET2 and ASXL1, and the finding that most individuals had only a single abnormality suggested that mutations in these genes are often initiating events for clonal haemopoiesis. Inspection of our exome sequencing data for the aUPD14q cases revealed no mutations in known driver genes in three out of four population cohort cases. By contrast, all three cases with diagnosed myeloid malignancy had additional somatic driver mutations. We suggest therefore that aUPD14q may also initiate clonal haemopoiesis and predispose to overt malignancy. However, when we examined cases that had both aUPD14q and JAK2 V617F, it was clear that aUPD14q may be an early event in some MPN cases but a late event in others. Furthermore, we found that aUPD14q tends to arise before JAK2 V617F in essential thrombocythaemia but after V617F in polycythemia vera. This is reminiscent of the finding that mutated TET2 may precede or follow the acquisition of JAK2 V617F in MPN, with the order of acquisition influencing clinical features and stem cell biology.32Similar to other mutations associated with clonal haemopoiesis in population cohorts,


There is an error in the data in cell Y47 of the Supplementary Table 2. It should read C*03:04. Please see the corrected Table S2 below.Table S2HLA allele information of ELISpot assay responders and its compatibility with previous report. In total, 79 responses among 14 epitopes were identified. HLA allele information of ELISpot assay responders and its compatibility with previous report of responsible HLA alleles listed in Los Alamos database are shown.(XLSX)Click here for additional data file.


Previous studies have shown the C1 spinal nerve has sensory neurons. Direct stimulation of the C1 spinal nerve provokes peri-orbital pain in migraine patients. No data exist which can predict a positive outcome for C1 nerve root block. Tenderness over the greater occipital nerve has been shown to predict outcome of GON block. We propose that tenderness over the GON with periorbital referral on exam predicts periorbital referral on direct C1 stimulation and predicts a positive outcome of block.Predict the outcome of C1 spinal nerve block based on exam findings.Review of 23 patients, 21 of whom had chronic migraine and 2 of whom had chronic cluster headache. All 23 have undergone C1 spinal nerve block.Of the 23 patients, 17 (74%) had GON tenderness on exam with periorbital referred pain and 6 (26%) had only occipital tenderness. Both cluster patients did not have periorbital nor orbital pain on palpation of the GON. All 17 with periorbital referred pain on GON palpation had reproducible periorbital pain intraoperatively on direct stimulation of the C1 spinal nerve with fluoroscopic guidance. Of those, 15/17 (88%) had a positive block. Of the 6/23 (26%) with a negative block, only two had periorbital pain reproduced on exam by GON palpation. Both cluster headaches had negative blocks. Neither had intraoperative periorbital or orbital pain on C1 stimulation.Tenderness over the GON with periorbital pain referral during exam predicts positive outcome of C1 spinal nerve block in patients with migraine.No conflict of interest.


Clinical trials document that as low as 0.5-1mg tasquinimod/day is therapeutic against castrate resistant metastatic prostate cancer. Tasquinimod is metabolized via cytochrome P4503A4, but ketoconazole at a dose which completely inhibits CYP3A metabolism does not affect tasquinimod's ability to inhibit endothelial “sprouting” d < 35 μM) to the IIA subdomain of albumin (Sudlow's site I). As blood vessels within the compromised cancer microenvironment are characterized by a higher degree of leakiness than those in normal tissues, this results in an enhanced uptake of tasquinimod bound to albumin in cancer tissue via a tumor specific process known as the “enhanced permeability and retention”  effect. Thus, despite plasma levels of < 1 μM, the EPR effect results in intracellular drug concentrations of 2-3 μM, levels several-fold higher than needed for inhibition of endothelial sprouting (IC50 ~ 0.5 μM) or for inhibition of HDAC4 and S100A9 mediated tumor growth.Tasquinimod's potency is facilitated by its reversible binding (K The total plasma membrane associated drug concentration (PMt) is determined by subtracting the ICt from the CAt value.A rapid filtration method was developed and validated to measures the kinetics of tasquinimod cell uptake on a series of human cell lines. To do this, exponentially growing cells in a tissue culture flask are trypsinized, and the number of single cells counted and their average diameter determined electronically using Cellometer AutoT4 . Then multiple replicates (N=3-6) of 107 cells were incubated in 1ml of tissue culture media containing 10% fetal bovine serum (FBS) plus 1μM 14C-labeled tasquinimod for varying times before the cells were centrifuged rapidly  to remove the drug containing media. The cells were rapidly resuspended in phosphate buffered saline and exposed to pressurized  N2 in a nitrogen cavitation devise  for 5 min before the plasma membranes were lysed by the rapid release of the pressure. This detergent free procedure lyses >98% of the cells without disrupting either mitochondria or lysosomes. The lysate was then centrifuged at 800Gx 2 minutes to restrictively pellet only cell nuclei. The radioactivity of this pellet was counted and used to calculate the total amount of tasquinimod associated with 107 cell nuclei. To determine the nuclear drug concentration, this total nuclear drug amount was driving by the total volume of 107 cell nuclei determined from the average diameter of the nuclei determined electronically using Cellometer AutoT4.To specifically determine the concentration of tasquinimod within the cell nucleus, 10in vitro angiogenic sprouting and tube formation assay was used as described previously [The 3-dimensional (3D) eviously . This 3DAll of the values reported are presented as means ± SE of representative data generated from 1 of a minimum of 3 independent experiments in which there were a minimum of 5 replicates per data point. Statistical analysis was conducted by a 1-way ANOVA with the Newman- Keuls test for multiple comparisons with significance being p<0.05.


The seed dormancy gene is expressed specifically in the embryo. The AlaAT isoenzymes encoded by the long and short dormancy alleles differ in a single amino acid residue. The reduced dormancy allele Qsd1 evolved from barleys that were first domesticated in the southern Levant and had the long dormancy qsd1 allele that can be traced back to wild barleys. The reduced dormancy mutation likely contributed to the enhanced performance of barley in industrial applications such as beer and whisky production, which involve controlled germination. In contrast, the long dormancy allele might be used to control pre-harvest sprouting in higher rainfall areas to enhance global adaptation of barley.Dormancy allows wild barley grains to survive dry summers in the Near East. After domestication, barley was selected for shorter dormancy periods. Here we isolate the major seed dormancy gene  Seed dormancy allows wild barley grains to survive dry summers in the Near East but has been selected against for industrial applications such as beer and whisky production that require quicker germination. Here Sato et al. show that Qsd1 is a major seed dormancy gene in barley and encodes an alanine aminotransferase. Hordeum vulgare) was domesticated ca. 10,000 years ago from its wild progenitor H. vulgare subsp. spontaneum in the Fertile Crescent1Barley (Qsd1) and another in the telomeric region of the long arm (Qsd2) have been identifiedQsd2 was isolated and proved to encode a mitogen-activated protein kinase kinase 3  on chromosome 5H, one near the centromere  and Qsd1/Qsd1 (short dormancy) in a Mendelian fashion , a protein of 495 amino acid residues  with the pANDA vectorQsd1 RNAi knockdown on seed germination. Of the two events, #7 T2 transgenic homozygous positive plants showed <5% germination after 21 days at 21 oC  and all of them were located in the coding region of AK372829 , indicatresidues . For proat 21 oC , while haAT gene .qsd1 allele of wild barley H602   . Transcr1 plants . These tQsd1 alleles were compared with the parents used for Qsd1 mappingQsd1/qsd1 alleles. This SNP is associated with a change from a leucine (L) residue at amino acid position 214 in wild barley H602 to a phenylalanine (F) residue in cultivar Haruna Nijo  was much lower than haplotypes having allele C (n=307). Most of the barleys having the allele G have pedigrees that track malting and brewing applicationsTo further confirm that the SNP alleles were correlated with dormancy in barley grain more generally, the germination scores of 353 cultivated and 14 wild barley accessions were analysed. The germination (low-dormancy) scores were compared among haplotypes of non-synonymous SNPs in AK372829 . Average72829 n=4 was muchQsd1 mutation that resulted in the reduced dormancy time, the genomic regions of Qsd1 in 27 wild and 210 cultivated barley accessions were sequenced  for AlaATs in other plant species and for four barley sequences  of Haruna Nijo  of Qsd1, corresponding to E9, was located on motif 11  was further analysed by RNA in situ hybridization 19 days after flowering. Transcripts were detected in the embryo but not in the endosperm  and mutant Qsd1 (F) alleles , but also from Lebanon (W1585) and Jordan (W1215). The result indicates that the Qsd1 allele is in the lineage from wild barleys of the southern Levant, where the first barley domestication also occurred1The haplotype analyses  showed tnd Korea . The C35Jordan W115. The rqsd1 gene is located almost exclusively in the embryo  rather than the cumulative increase in expression that causes different levels of dormancy.As shown in  Promise  causes aactivity . Howevermination . In thesBrassica napus also controls dormancy and has been implicated in alanine metabolism2O2) to break dormancy in barley seedsAs mentioned above, dormancy and germination are complex processes and dormancy has been linked with redox signalling by reactive oxygen species32qsd1 gene for long dormancy appears to be a mutable gene that might be used for adaptation to different environments and this is consistent with the large variation in qsd1 sequence that exists in nature. For example, the long dormancy qsd1 gene could be used to control pre-harvest sprouting in higher rainfall areas to enhance global adaptation of barley. On the other hand, certain haplotypes which carry the Qsd1 mutation are associated with barley lines that have been developed for industrial uses, such as in the malting and brewing industries. The Qsd1 mutation that was discovered soon after barley domestication has proved to be essential for the transition of barley utilization from food to beverage in human diets and contributes further to the debate as to what extent the development of ancient agrarian societies was driven by the human appetite for flour and bread, or for beer and alcoholDespite these interpretative constraints, it is clear from the genetic data presented here that AlaAT is centrally important in the release of dormancy in barley grain. In practical terms, our data suggest that the Spike samples are harvested at physiological maturity (when the colour of first internode turns to yellow). Spikes are dried 48 h by the dehumidifier at the condition of 30 °C and 10% RH, and then stored at −20 °C until use. Fifty seeds of each sample were exposed to 25 °C for dormancy reduction treatment and germinated for 4 days on moistened filter paper in Petri dishes at 25 °C. Seed non-dormancy was scored as the percentage of germinated seeds. Each germination test was replicated twice.Qsd1 was initially mapped with the 93 doubled haploid phenotype data on a map derived from the cross between the cultivar Haruna Nijo and the wild barley line H602 consisting of 2,890 EST markersQsd1 mapped at the centromeric region of the long arm of chromosome 5H. The method of population development is described in Sato et al.3F1 recombinant chromosome substitution lines (RCSLs) derived from the cross between H602 (donor parent) and Haruna Nijo (recurrent parent) were genotyped and plants heterozygous for a chromosome segment with Qsd1 were selected. A RSCL carrying the long dormancy allele qsd1 and short dormancy alleles for other three QTLs was identified132 plants and 4,792 F3 plants derived from the heterozygous F2 plants were produced to genetically map Qsd1  that showed high sequence co-linearity with the other 10 barley genes (EST2 to EST11). By scoring dormancy . A co-segregated marker with an estimated genotype of Qsd1 was identified in two plants from the F3 population. From each of these two plants, 18 F4 plants were used to confirm the respective genotype of F3 by segregation in the progeny.sd1 ref. . The segsd1 ref. . The dirreatment . Becausereatment , the pladormancy , Qsd1 waQsd1 was used to select BAC clones of the less-dormant parent Haruna NijoHindIII cloning site. The average insert size was 180 kb and the total clone number was 172,800 (6 ×). From the BAC library of Haruna NijoEST4R marker and a contig sequence of 121,395 bp was obtained from another BAC clone (GenBank ID: LC054175). Each shotgun library from the selected clone was sequenced using a 3730xl sequencer (Applied Biosystems). Reads were assembled by phred/phrap software into contigs. The EST sequence was mapped on contig sequences to confirm the target sequence on the BAC clone. The Rice Genome Automated Annotation System (http://ricegaas.dna.affrc.go.jp/) was used for gene prediction on the contig sequence. Sequence polymorphisms between Haruna Nijo and H602 were detected by the alignment of contig sequences by CLUSTALX .The primer set of co-segregated markers with AK372829 was amplified using the forward primer 5′-caccaaagctaggagatgg-3′ and reverse primer 5′-gaacacactgccccaaaagt-3′and cloned into the pENTR/D topo vector (Invitrogen). The construct was recombined (according to the manufacturer's instructions) into the binary vector pANDA modified to contain a GatewayTM cassette down-stream of the maize Ubi-1 promoterAgrobacterium strain EHA101. Immature embryos of cv. Golden Promise were used for Agrobacterium-mediated transformation. Transformation was performed by the protocol of Hensel et al.0 plants which had single copy inserts, as shown by Southern analysis, were examined further. We obtained several independent transgenic lines, and two (ids. #6 and #7) were used for further analysis. Genotypes of T1 plants were determined by the inserts of segregating T2 progeny and each T2 plant from the T1 plants were further genotyped by segregation in the T3 progeny.A 509-bp cDNA region from qsd1/qsd1 allele in the genetic background of cv. Golden Promise. Twenty-three B1F1 plants were genotyped by the Golden Gate Assay (Illumina Co.), using selected 384 genome-wide SNP markers from barley OPA1 (ref. 1F1 plants with the substituted segment were self-pollinated to generate homozygous qsd1/qsd1 substitution lines. Agrobacterium-mediated transformation of the substituted line (id. B1H602GP20-7), using a vector construct carrying a cloned Haruna Nijo Qsd1 genomic DNA  and sequenced. The open reading frame region was digested by SacI and HindIII and ligated into binary vector pBUH3 (ref. 0 transgenic plants. Segregating T1 plants (ids #1 and #4) were produced from single T0 plants and further genotyped for the Qsd1 transgene in the T2 progeny.The recombinant chromosome substitution line was used as a donor of dormant PA1 ref.  to identPA1 ref. . Selecteomic DNA , was conTo measure dormancy levels, transgenic barley lines were grown in a growth chamber with 12 h day length and at 15 °C. Expression levels were measured with the same method described below. Dormancy levels were measured at the time when the difference of long dormancy and short dormancy samples were maximized.de novo using I-TASSERde novo predictions as templates. Modeller DOPE functions, ProSAThe barley AlaAT amino acid sequences were aligned to the barley AlaAT crystal structureA total of 365 barley accessions preserved at Okayama University were evaluated for seed dormancy for two growing seasons. Nucleotide polymorphisms were detected by sequencing PCR amplicons from target regions. Primer sequences are shown in Qsd1 in 27 wild and 210 cultivated barley accessions are sequenced. Non-redundant haplotypes were collected and aligned according to their similarities with the haplotypes of H602  on a Step One thermal cycler (Applied Biosystems Co.) with initial conditions of 95 °C, 60 s, 40 cycles of denaturing of 95 °C, 60 s and extension at 60 °C for 60 s. The qsd1 gene segment comprising part of the 3′-UTR (300 bp) was amplified from cDNA isolated from immature barley spike using specific primers  at 0, 1, 2, 3, 4 and 5 weeks after flowering were measured for  primers . The PCRLC054174: cv. Haruna Nijo BAC clone HNB550I12 and LC054175: wild barley H602 BAC clone HSP216G11. The data that support the findings of this study are available from the corresponding author upon request.BAC clone sequences described in this paper have been deposited in the DDBJ nucleotide database with accession codes How to cite this article: Sato, K. et al. Alanine aminotransferase controls seed dormancy in barley. Nat. Commun. 7:11625 doi: 10.1038/ncomms11625 (2016).Supplementary Figures 1 - 10 and Supplementary Tables 1 - 8Origin and name of accessions of 27 wild and 210 cultivated barleys.


There is an error in the sixth sentence of the Abstract. The correct sentence is: Reduced GM volumes in three brain areas including right hOC3v in the collateral sulcus of visual cortex (hOC3vR), left cerebellar vermis lobule 10 (vermisL10) and right cerebellar vermis lobule 10 (vermisR10) were found in patients with schizophrenia.There is an error in the first sentence of the Reduced GM volume in patients with schizophrenia subsection of the Results. The correct sentence is: By using SPM Anatomy Toolbox, we found that GM volumes in three brain areas including left hOC3vR in the collateral sulcus of visual cortex (hOC3vL), left cerebellar vermis lobule 10 (vermisL10) and right cerebellar vermis lobule 10 (vermisR10) were significantly reduced in patients with schizophrenia .The term hOC3vL appears incorrectly throughout the manuscript. The correct term is hOC3vR.S2 Fig(TIF)Click here for additional data file.


P. falciparum may provide false negative results in some parts of South America due to the emergence of P. falciparum parasites that lack the pfhrp2 gene, and thus produce no PfHRP2 antigen. Pfhrp2 and pfhrp3 genes were amplified in parasite isolates collected from Guyana and Suriname to determine if there were circulating isolates with deletions in these genes. Pfhrp3 deletions were monitored because some monoclonal antibodies utilized in PfHRP2-based RDTs cross-react with the PfHRP3 protein. We found that all 97 isolates from Guyana that met the inclusion criteria were both pfhrp2- and pfhrp3-positive. In Suriname (N = 78), 14% of the samples tested were pfhrp2-negative while 4% were pfhrp3-negative. Furthermore, analysis of the genomic region proximal to pfhrp2 and pfhrp3 revealed that genomic deletions extended to the flanking genes. We also investigated the population substructure of the isolates collected to determine if the parasites that had deletions of pfhrp2 and pfhrp3 belonged to any genetic subtypes. Cluster analysis revealed that there was no predominant P. falciparum population substructure among the isolates from either country, an indication of genetic admixture among the parasite populations. Furthermore, the pfhrp2-deleted parasites from Suriname did not appear to share a single, unique genetic background.Guyana and Suriname have made important progress in reducing the burden of malaria. While both countries use microscopy as the primary tool for clinical diagnosis, malaria rapid diagnostic tests (RDTs) are useful in remote areas of the interior where laboratory support may be limited or unavailable. Recent reports indicate that histidine-rich protein 2 (PfHRP2)-based diagnostic tests specific for detection of  Plasmodium falciparum malaria cases compared to other countries in the Caribbean region .P. falciparum-infected samples received from Guyana, thirteen were collected from female patients while the rest were from males. All 100 patients reported travelling within the two weeks prior to seeking medical attention for clinical symptoms; the majority had travelled to Cuyuni-Mazaruni (55%) and Potaro-Siparuni (34%) (18S rRNA and msp2 genes.Of the 100 ni (34%) . Three o18S rRNA/msp2-positive samples from Guyana were positive for both pfhrp2 and pfhrp3 genes (pfhrp2 (PF3D7_0831900) and one sample was negative for the 3’ flanking gene PF3D7_0831700  and Brokopondo (19%) districts. Travel information could not be retrieved for 30 samples. Twenty-five samples were removed from final analysis because they failed to meet the inclusion criterion requiring amplification of both 18S rRNA/msp2-positive samples from Suriname were pfhrp2-negative. Two of the eleven patients from whom the pfhrp2-deleted isolates were collected, reported recent travel to Brokopondo district and nine patients had been to Sipaliwini district. All eleven of these pfhrp2-negative isolates were collected in 2011; in eight of these samples, exon 2 of pfhrp2 was intact; three samples had deletions in both exon 1 and 2 of pfhrp2. Seventeen of the 78 isolates (22%) had deleted the 5’ pfhrp2 flanking gene, PF3D7_0831900, while three (4%) had deleted the 3’ flanking gene PF3D7_0831700 (Eleven (14%) of the 78 _0831700 .pfhrp2. Although the majority of samples (67%) were positive for pfhrp2 and its flanking genes, fourteen isolates (18%) had only deleted the 5’ flanking gene, PF3D7_0831900 (pfhrp2-negative isolates with both flanking genes intact (8%); two PF3D7_083900/Pfhrp2 double-negative isolates (4%); and one Pfhrp2/PF3D7_0831700-double negative isolate (3%) (We further examined the parasite isolates for gene deletion patterns around _0831900 . Other date (3%) .pfhrp3-negative  was intact in all 78 samples while one sample (1%) was negative for the gene found downstream of pfhrp3, PF3D7_1372400 (Only three of the 78 isolates (4%) were negative . Two of _1372400 .pfhrp3. We found that 96% of the 78 isolates from Suriname had intact pfhrp3 and flanking genes (pfhrp3-negative samples with intact neighboring genes and one (1%) isolate that was pfhrp3/PF3D7_1372400-negative but PF3D7_1372100-positive .Neutral microsatellite genotyping and cluster analysis were performed to investigate a possible relationship among parasite isolates with deletions in Median joining network diagrams were created using allele length data at seven neutral microsatellite loci in order to evaluate the genetic relationships among the parasite isolates. No distinct clustering of Suriname isolates separately from those collected in Guyana was observed, indicating that the parasites from the two countries may be very similar genetically . This oupfhrp2-negative isolates, which were collected in Suriname in 2011, did not cluster together as may have been expected, indicating that their genetic backgrounds differed from each other and that they likely did not all originate from a single clonal type  district in Suriname, consists of tropical rainforest and borders French Guiana to the east, Guyana to the west and Brazil to the south. The Suriname-French Guiana border region along the Marowijne river is a relatively high malaria transmission region [P. falciparum  parasite transmission. Further studies will be required to determine if the pfhrp2 deleted parasites found in Suriname are genetically related to those found in other South American countries.Taken together, these data indicate that the occurrence of h as 40% ,17. Givest areas . It is tpfhrp2 gene deletion was not found among P. falciparum isolates collected in Guyana, it is intriguing that 41% of the parasites had deleted the 5’ flanking gene, PF3D7_0831900  had to be excluded from our analyses because of poor quality DNA; these samples did not meet our inclusion criteria of being able to amplify pfhrp3-negative isolates in Guyana and only a limited number of pfhrp3 deletions in Suriname. In Peru, a larger proportion of parasites was pfhrp3-negative compared to those that had deleted pfhrp2 [pfhrp2 is located on chromosome 8 while pfhrp3 is located on chromosome 13), and that the biological significance of these gene deletions is not known, the implications for the apparent differences in proportions of pfhrp2 versus pfhrp3 deletions in Suriname compared to Peru is unclear.It was surprising to find no d pfhrp2 . Howeverpfhrp2 or pfhrp3 gene deletions occurred in P. falciparum isolates collected in Guyana. On the other hand, pfhrp2 gene deletions also did not occur in isolates collected between 2009 and 2010 in Suriname, but were detected in samples collected in 2011. However, it should be noted that a very small number of specimens were collected in 2009 and 2010. The outcome from Suriname illustrates the importance of regular monitoring for pfhrp2 and pfhrp3 deletions if PfHRP2-based RDTs continue to be used in the region. Use of non-PfHRP2-based RDTs that target P. falciparum-specific parasite lactate dehydrogenase (pLDH) with either pan species or P. vivax-specific pLDH can also be considered as an alternative test for use in Suriname. Furthermore, given that Suriname, Guyana and French Guiana experience influxes of migrant workers, it is likely that pfhrp2 gene deletions may spread through this mobile population. Therefore, a current sampling of P. falciparum isolates to update the findings reported here should lead to careful consideration given in choosing appropriate RDTs for use in this region.In summary, no S1 Supporting InformationWritten permission for the use and modification of the maps in (PDF)Click here for additional data file.


CCN2 acts as an anabolic growth factor to regulate osteoblast differentiation and function. CCN2 is induced by TGF-β1 and acts as a mediator of TGF-β1 induced matrix production in osteoblasts and Src is required for CCN2 induction by TGF-β1; however, the molecular mechanisms that control CCN2 induction in osteoblasts are poorly understood. AFAP1 binds activated forms of Src and can direct the activation of Src in certain cell types, however a role for AFAP1 downstream of TGF-β1 or in osteoblats is undefined. In this study, we investigated the role of AFAP1 for CCN2 induction by TGF-β1 in primary osteoblasts.We demonstrated that AFAP1 expression in osteoblasts occurs in a biphasic pattern with maximal expression levels occurring during osteoblast proliferation (~day 3), reduced expression during matrix production/maturation (~day 14–21), an a further increase in expression during mineralization (~day 21). AFAP1 expression is induced by TGF-β1 treatment in osteoblasts during days 7, 14 and 21. In osteoblasts, AFAP1 binds to Src and is required for Src activation by TGF-β1 and CCN2 promoter activity and protein induction by TGF-β1 treatment was impaired using AFAP1 siRNA, indicating the requirement of AFAP1 for CCN2 induction by TGF-β1. We also demonstrated that TGF-β1 induction of extracellular matrix protein collagen XIIa occurs in an AFAP1 dependent fashion.This study demonstrates that AFAP1 is an essential downstream signaling component of TGF-β1 for Src activation, CCN2 induction and collagen XIIa in osteoblasts. CCN2lization , 5 and alization , 6–11. Clization . One of lization –16, howelization . The siglization .Src is the founding molecule of a family of non-receptor tyrosine kinases that, when activated, are involved in numerous physiological and pathological processes including cell proliferation, survival angiogenesis and matrix secretion –20. Src -/- null mice we were the first to demonstrate a novel physiological role for AFAP1 in lactation [Actin filament-associated protein 1  is the prototypical member of a family of three structurally related proteins: AFAP1, AFAP1 like 1 (AFAP1L1), and AFAP1 like 2 . AFAP1 was discovered over two decades ago as a binding partner for oncogenic Src . AFAP1 iactation . These sactation . AlthougConsidering that AFAP1 is an important regulator of Src activity and that Src activity plays a central role in relaying TGF-β1 signaling to induce CCN2 expression and controls osteoblast functions including ECM production, we hypothesized that AFAP1 plays a role in the TGF-β1 signaling pathway and the regulation of Src activity in osteoblasts. Thus, this study characterizes the role of AFAP1 in regulating Src activation and CCN2 induction downstream of the TGF-β1 receptor in osteoblasts.The three stages of osteoblast differentiation in primary osteoblast cultures have been well-characterized and include an initial period of cell proliferation until the cells reach confluency (~day 7), followed by a phase of matrix production and maturation (~day 7–14), and ending with a stage of mineralization in which mineral deposition accrues in the matrix (~day 14–21). We sought to assess the temporal pattern of AFAP1 expression in differentiating primary osteoblast culture and to determine if TGF-β1 was capable of inducing AFAP1 expression at different time points within the spectrum of osteoblast differentiation. Western Blot analysis revealed that AFAP1 protein expression occurred in a biphasic pattern with maximal expression levels occurring during osteoblast proliferation (~day 3), reduced expression during matrix production and maturation (~day 14–21), an a further increase in expression during mineralization (~day 21) . This exWe previously showed that in osteoblasts TGF-β1 activates Src and TGF-β1 induction of CCN2 requires Src . Since AWe have previously demonstrated that blocking Src expression/activity impairs CCN2 promoter activation and protein expression in response to TGF-β1 treatment in osteoblasts , 42. To We have previously demonstrated that CCN2 is an essential downstream mediator for the TGF-β1-induced, extracellular matrix (ECM) protein collagen type I in osteoblasts . AdditioAFAP1 is considered as an adaptor protein that directs the activity and the location of cSrc and as an effector protein that crosslinks actin filaments. The function of AFAP1 has been studied mainly in prostate cancer and breast cancer where AFAP1 contributes to the progression of cancer by regulating the adhesion of cancer cells , 44. We CCN2 is an important factor in the induction and control of osteogenesis. CCN2 is highly expressed in active osteoblasts during osteogenesis , during We found that reducing the expression of AFAP1 using AFAP1 siRNA impaired Src activation after TGF-β1 treatment in osteoblasts. This finding is consistent with other previous reports concerning the involvement of AFAP1 in binding to activated forms of Src , 54. In levels of CCN2 were inhibited by the activation of PKC, but stimulated by the inhibition of PKC and tyrosine kinase [Interestingly, PKC isoforms have been previously shown to be involved in TGF-β1 induction of CCN2 in mesangial cells , hepatoce kinase . Future TGF-β1 is known to influence bone metabolism through effects in both osteoblasts and osteoclasts. Specifically, in osteoblasts, TGF-β1 recruits and induces the proliferation of osteoblast precursors and inhibits their apoptosis and can modulate expression of factors that control the formation and activation of osteoclasts . In geneA major function of TGF-β1 during osteoblast differentiation is to stimulate production of the extracellular matrix components that compose osteoid , 73. In In conclusion, this study demonstrates that a normal physiological role of AFAP1 is to mediate the downstream signaling pathway of TGF-β1 to induce CCN2 induction and extracellular matrix protein production, the prominent anabolic pathway in osteoblast. This study further supports the role of AFAP1 as an adaptor for Src to direct the activity of the kinase upon receiving a specific input signal, TGF- β1.No human subjects were used in this study. This study was approved by the IACUC board of The Commonwealth Medical College, Scranton PA. . All animals were handled according to national and international guidelines following the principles in the NIH Guide for the Care and Use of Laboratory Animals  and in accordance with principles established in the Weatherall report.Transforming Growth Factor-β1 (TGF-β1) was purchased from Calbiochem  and reconstituted as 1μg/ml in 4mM HCl with 0.1% bovine serum albumin. Anti-actin antibody was purchased from Sigma . Anti-CTGF, anti-collagen I and anti-collagen XII antibodies were purchased from Santa Cruz . Anti-Src antibodies (clone GD11) were from BD Bioscience and antibodies specific for active form of Src (clone pY416) were from cell signaling Technologies . CCN2 promoter reporter was constructed as previously described . The genOsteoblasts were transfected with 100 pmol of siRNA specific for AFAP1  or 100 pmol control Luciferase siRNA  using Lipofectamine and Plus reagent  according to the manufacturer’s instructions.The generation GFP AFAP1 fusion protein construct was previously reported . GFP exp2 inhalation followed by cervical dislocation. Neonate rats were euthanized by decapitation.Primary osteoblasts were derived from bone  of neonatal Sprague Dawley rats purchased from Charles River . All animals were handled at the AAALAC approved animal facility in The Commonwealth Medical College , according to the principles in the NIH Guide for the Care and Use of Laboratory Animals  and guidelines established by the IACUC of The Commonwealth Medical College . Adult animals were euthanized by COrd–5th digestions of the calvarial pieces. These cells were plated in 100mm dishes  at 5 × 105 cells/plate in osteogenic media consisting of Earle’s Minimal Essential Medium  supplemented with 10% fetal calf serum , 50μg/ml ascorbic acid (Sigma) and 10mM βcglycerophosphate (Sigma). The cells were incubated at 37°C with 5% CO2 with a change of media every three days until they reached 80% confluence. Cells were sub-cultured under identical conditions for utilization in experiments following the third passage. We have previously shown that these culture conditions result in an enhanced (>90%) population of cells committed to the osteoblast lineage using specific markers of osteoblast differentiation  [Primary osteoblast cultures were obtained using neonatal rats as previously described , 10, 11.Osterix) , 11.Lysate preparation, Immunoprecipitation, Western Blotting and trasfection were preformed as described . Blots w4 cells/well), transfected with CCN2 promoter reporter vector and co-transfected with Renilla  luciferase vector. Following transfection, the cells were serum starved overnight and treated with TGF-nd co-transfected with Renilla . Relative luciferase activity was expressed as a ratio of firefly/renilla luminescence values. All samples were normalized to an untreated (cells only) or mock treated (empty vector or diluent only) control reaction.Luciferase activity was determined using the Dual-Glo luciferase assay  according to the manufacturer’s instructions as described . BrieflyFor all quantitative data, analysis of variance (ANOVA) was employed to evaluate the effect of one variable on two or more independent groups. In the event of a significant group effect, individual pairs of means were compared using the Bonferroni post-hoc test. Data were calculated as mean + SEM, and in some cases, converted to percent of control. A value of p<0.05 was used to determine whether differences were statistically significant.


Ole e 1 is one of the major allergens from olive tree pollen. Up to date there are no specific studies that evaluate in depth the in vitro responses to this purified allergen. The goal of the study was to thoroughly evaluate the celullar responses to nOle e 1 in allergic rhinitis (AR) and local allergic rhinitis (LAR) patients with sensitization to olive tree pollen (OL) demonstrated by nasal allergen provocation test (NAPT).Twelve subjects with AR  to OL), 12 subjects with LAR , and 12 subjects as control group (CG)  were selected. Basophil activation tests (BAT) with OL and nOle e 1, along with dendritic cell (DC) maturation/proliferation studies in response to nOle e 1 stimulation, were carried out in all subjects. Local ethical committee approved the study.All AR subjects had positive BAT responses to OL and 10/12 to nOle e1 (83%); 8/12 LAR (66.6%) had a positive BAT with OL and 4/12 (33%) to nOle e1, with only one subject of the control group with a positive BAT to both OL and nOle e1 (8%). DC proliferation and maturation were increased in SAR>LAR>CG but with no significant differences .BAT with OL and nOle e1 in LAR group showed sensitivity between 66.6 and 33%, demonstrating specific basophil activation with pollens in patients with LAR. DC proliferation and maturation were demonstrated in SAR and LAR subjects although with no significant differences with CG.


After multiple discrete introductions of influenza A(H1N1)pdm09 virus into Sri Lanka, the virus was transmitted among humans, then swine. The spread of virus between geographically distant swine farms is consistent with virus dispersal associated with a vehicle used for swine transportation, although this remains unproven. The first known transmission of influenza A(H1N1)pdm09 virus to humans from swine was in 2009. As the virus spread among humans worldwide, it was transmitted from humans to swine repeatedly 1, chaTo understand the molecular epidemiology and spatial and temporal dynamics of spillover events, we compared our data with full-genome sequences of H1N1pdm available in public databases as of August 28, 2013. These include all available full genome sequences from swine H1N1pdm viruses (n = 82), all human H1N1pdm viruses from outside of the USA (n = 957), and 100 randomly selected full genome sequences from 1,500 human H1N1pdm sequences from the United States. Reassorted swine or human viruses containing H1N1pdm virus genes were excluded from this analysis. Our final dataset included the full genomes of 35 human and 26 swine samples from Sri Lanka and a global sample of 1,057 human and 82 swine virus sequences.The single breakpoint recombination and genetic algorithm for recombination detection methods 3 exclIn Sri Lanka, swine H1N1pdm clusters sw1 2009/10) and sw2 (2011) were genetically distinct from each other and from other swine viruses isolated globally, indicating 2 separate introductions to local swine that circulated among swine for 11 and 4 months, respectively, for each cluster. The sw1 and sw2 lineages did not appear to establish long-term sustained transmission within pigs. However, the reduced surveillance of farms during the period 2012�?"2013 Table 1 009/10 anTo clarify transmission patterns between affected swine farms in Sri Lanka, we obtained contact patterns by interviewing pig farmers using a structured questionnaire  with apOf the 15 farms on which the common truck was used, swine on 3 (20%) were infected by a sw2 clade virus; on 2 farms on which the common truck was not used, no swine were infected. This association was not statistically significant (p = 1.0), however, given the small numbers of farms investigated. Our findings are consistent with dispersal of sw2 clade viruses in association with the truck to infect multiple farms that were geographically distant, but this remains unproven. It was previously documented that influenza viruses can remain viable for prolonged periods of time in water at a temperature of �%^28A�C  10 andDespite widespread inter-farm transmission of sw1 and sw2, our results show that only animals on farm C were infected in both spillover events. Farms A and G, on which swine were infected by sw2 in 2011, appeared not to have had infected swine during 2009�?"2010, as shown by both virus isolation and serologic testing Table 1.This study demonstrates natural independent spillover events of H1N1pdm influenza viruses from humans to swine. H1N1pdm viruses appear to be spread by multiple, discrete introductions to swine, after which clonal expansion occurs within the swine. The spread of such virus lineages across multiple farms is consistent with virus dispersal by breaches of external biosecurity measures, including the manner of swine transportation, although this remains unproven given the small sample size. Unlike classical swine influenza, North American triple reassortant, and European avian swine viruses that have persistently circulated among swine for several decades in other countries 15, H1Overview of the swine industry in Sri Lanka and surveillance of human and swine pandemic influenza A(H5N1) viruses.