Coordination of cell growth with nutrient availability, in particular amino acids, is a central problem that has been solved by the implementation of complex regulatory cascades. Although the specific regulatory mechanisms differ between kingdoms and species, a common theme is the use of tRNA molecules as sensors and transducers of amino acid starvation. In many bacteria, amino acid starvation leads to high levels of uncharged tRNAs, a signal for the synthesis of the stringent response’s alarmones, halting transcription of stable RNAs and inducing the synthesis of amino acid synthesis pathways  The first modification is found at position 37 of most tRNAs decoding ANN codons, just before the residue that interacts with the first base of the codon. The second modification is found at position 34, the residue that decodes the 3rd base of the codon, of tRNALysUUU, tRNAGluUUC, and tRNAGlnUUG . Both these modifications are found in tRNALysUUU and affect translation efficiency The anticodon-stem-loop (ASL) of tRNAs drives decoding by interacting directly with the mRNA codon . Modifications of the ASL are the most distinct and chemically complex of all RNA modifications 6A and mcm5s2U are both synthesized by elaborate multimeric complexes that were first thought to be transcription factors. The KEOPS complex  in combination with Sua5 is involved in the synthesis of t6A 5s2U Saccharomyces cerevisiae mutants lacking t6A or mcm5s2U display many similar phenotypes. For example, telomere shortening is observed in the absence of either modification 7785s2U deficient strains. Overexpression of tRNALysUUU suppresses the telomere shortening phenotype Schizosaccharomyces pombe, key regulators, including the two subunits Atf1 and Pcr1 of the central regulator of the core environmental stress response 5s2U levels. More generally, proteins involved in translation initiation and elongation are enriched in AAA codons, and reduced in cells lacking mcm5s2U 126A for efficient translation in yeast, but it has been shown in mammals that a decrease in sulfur modified form of t6A (ms2t6A) on tRNALysUUU leads to lower levels of proinsulin t6A or a mcm5s2U deficient S. cerevisiae derivatives is the GCN2 independent activation of GCN4 156A and mcm5s2U.Another phenotype shared by a t5s2U have previously been made: 1) ELP mutants are sensitive to rapamycin and caffeine SIT4 leads to rapamycin hypersensitivity and resistance to zymocin (a tRNase that recognizes mcm5s2U and cleaves tRNALysUUU leading to cell death) et al.5s2U, suggesting that proper tRNA modification affects Gln3 localization and subsequent activation of the nitrogen catabolite repression (NCR) response. Mislocalization of Gln3 occurs both in elp3∆ (removing the mcm5 moiety) and in urm∆ (s2 moiety). A link between t6A synthesis enzymes and TOR has also recently been seen in flies. The Glavic group showed that lowering levels of one of the subunits of the KEOPS complex (Bud32) reduces TOR phosphorylation of S6K (SCH9 in yeast) required for TOR dependent regulation of ribosome biogenesis The TOR kinases regulate the balance between protein synthesis and protein degradation in response to nutrient quality and TOR activity is inhibited by low nitrogen conditions, caffeine or rapamycin 6A and mcm5s2U is still far from understood at the molecular level. Are the Gcn4 activation and TOR repression in strains lacking these modifications due to direct effects caused by poor translation of specific proteins or are they part of general stress responses caused by translation inaccuracy and protein misfolding? The reality might lie in a combination of responses as in addition to the targeted effects described above, low mcm5s2U increases levels of proteins involved in proteasomal degradation 2 synthesis proteins in S. cerevisiae are unstable at high temperature and reduced levels of the modification lead to activation of the heat-shock response regulator (Hsf1) through the synthesis of unfolded proteins  6A synthesis genes Bud32 and Kae1 in flies activates the Unfolded Protein Response (UPR) 2i6A in mouse led to an increase of the Endoplasmic Reticulum (ER) stress response How Gcn4 and TOR signaling depend on t6A and mcm5s2U modifications of tRNALysUUU draw on primary metabolism intermediates 5s2U thiolation pathway; sulfur starvation reduces the levels of the Uba4 thiolation enzyme and hence the levels of mcm5s2U in yeast 5s2U levels trigger an adaptive response: 1) reduced protein expression due to general slow-down of translation of lysine rich proteins that are found predominantly in the ribosomal machinery; 2) increased levels of methionine, cysteine, and lysine synthesis proteins Because the synthesis of the t6A and mcm5s2U a delicate exercise that will require combining of classical genetic and biochemical studies with the genome wide bioinformatics, proteomic and profiling studies that are now available. These studies are all the more critical as derivatives of both modifications have been linked to human diseases as defects in the ms2t6A synthesis enzyme have been linked to type 2 diabetes 5s2U The complexity of the responses with the interplay of central regulators such as GCN4 and TOR , make the dissection of the roles of t


Approximately 30–40% of patients undergo leukemic transformation to acute myeloid leukemia (AML) during the course of their disease.n=5) or to AML (n=36) (5 targeting the hotspots of 31 recurrently (>1%) mutated genes in myeloid malignancies  bone marrow samples from 41 MDS patients before (preprogression) and after progression (postprogression) to a more advanced subtype (L (n=36) . The mutgnancies . Dual-bagnancies . The pro3A total of 99 and 122 mutations across 23 genes were identified in preprogression and postprogression samples, respectively . The numASXL1, TET2, SRSF2, U2AF1, RUNX1 and TP53. ASXL1, encoding an epigenetic regulator, was the top ranking mutated gene with a frequency of 44% in preprogression samples and 46% in postprogression samples . Given that the frequency of ASXL1 mutations in MDS ranges from 11 to 15%, and of SF3B1 mutations from 20 to 28% in unselected studies,6 the mutation data concerning these two genes are clearly strikingly different in our study. It should be noted, however, that the patient cohort used in this present study was highly selected—that is, comprising only patients whose disease had progressed. Our data thus indicate that ASXL1 mutations are strongly associated with MDS cases that show disease progression to AML and conversely that SF3B1 mutations are rarely associated with MDS cases that show disease progression. This finding is consistent both with the status of ASXL1 as a poor prognostic marker in MDS7 and with the strong association of SF3B1 mutation with a good prognosis in MDS and with the low-risk MDS subtype refractory anemia with ring sideroblasts.8The most frequently mutated genes (in >15% of samples) were  samples . In cont9 mutations in splicing factor genes  were mutually exclusive, with the only exception being one case with mutations in both U2AF1 and ZRSR2. Mutations of genes involved in splicing , chromatin modification (EZH2 and ASXL1) and DNA methylation  were present in the preprogression and postprogression samples for almost all cases harboring mutations in these genes, and thus represent early events in the disease course in these cases. Interestingly, age-related clonal hematopoiesis, with the majority of variants occurring in DNMT3A, TET2 and ASXL1, has been shown to be a common condition that is associated with increases in the risk of hematologic cancer.11 Mutations of genes involved in transcriptional regulation  and signal transduction (NRAS and KRAS) were found in the postprogression sample only in the majority of cases, suggesting that these are often late events that may co-operate with early events to drive disease progression . The ASXL1–RUNX1 mutated gene association has been previously shown to be significant in studies on large MDS cohorts.3 We also found that all five preprogression samples in our cohort with ZRSR2 mutations also carried ASXL1 mutations, whereas 13 of 36 preprogression samples without ZRSR2 mutations carried ASXL1 mutations . Co-occurrence of mutations in splicing factor genes and in genes involved in epigenetic regulation has been reported previously;3 however, the ASXL1–ZRSR2 association has not been described. In the postprogression samples, there were five cases with mutations of both NRAS and RUNX1 (compared with two cases in preprogression samples), an association reported as significant in a previous MDS study.3 We have also observed co-occurrence of NRAS and ASXL1 mutations in five postprogression samples (compared with two cases in preprogression samples) in our study. Interestingly, NRas mutation and Asxl1 loss co-operate to drive myeloid proliferation and myeloid leukemia in mice,13 and our data on MDS serial samples support this observation. These co-occurring gene mutations may thus have a role in disease progression in MDS.Emerging data suggest that key differences in disease phenotype can be driven by different combinations of comutated genes in MDS.6 In our study, we found that TP53 mutations were present in all four cases in our cohort with abnormalities of chromosome 5, U2AF1 mutations were present in three of five cases with −20/del(20q) and two of four cases carrying SETBP1 mutations had −7; these association have been previously reported.6Specific associations between mutated genes and chromosomal abnormalities have also been described in MDS.TP53 showing the largest average VAF fold increase (>50%) in postprogression samples compared with preprogression samples among genes mutated in more than five cases, suggesting that this mutation had a major role in driving disease progression in these patients , suggesting that mutations of this gene occurred mainly in a subclone; four of these subclonal mutations expanded with disease progression and two additional TP53 mutations were present in postprogression samples only. Four NRAS mutations identified in preprogression samples were subclonal (VAF range 7–30%); eight NRAS mutations were present in postprogression samples only, suggesting that the emergence of new NRAS mutations during the course of the disease may have a role in disease progression. The VAF of all three subclonal mutations in RUNX1 in preprogression samples (VAF range 12–19%) increased with disease progression; in addition, four RUNX1 mutations were found in postprogression samples only, suggesting that in the case of RUNX1 both the emergence of new mutations and the expansion of existing ones during the disease course may be involved in progression to AML.The large majority of NRAS mutation expanded during disease progression, whereas the allele burden of mutations in ASXL1, RUNX1 and EZH2 remained constant. In another case, within a background of mutations in ASXL1, EZH2 and ZRSR2, a mutation in SETBP1 emerged mid-progression and a mutation in NRAS was found at the AML stage only. This shows that more precise information on the mutational profile and subclone evolution during disease progression can be obtained by the analysis of multiple serial samples.An additional serial sample was sequenced for four cases in our cohort . In one This is the first study to investigate the mutational status of a large group of MDS patients showing disease progression by the study of serial samples using a NGS myeloid gene panel. We have determined the frequency and chronology of myeloid gene mutation acquisition during disease progression in MDS, identifying specific mutations that are associated with disease evolution, and illuminating the role of subclone development in MDS progression. These data suggest that there are several genetic paths for MDS progression.


CYP2C19 are very frequent worldwide, particularly in Asia, raising the possibility that reduced metabolism could be advantageous in some circumstances. The evolutionary selective forces acting on this gene have not previously been investigated.Cytochrome P450 CYP2C19 metabolizes a wide range of pharmacologically active substances and a relatively small number of naturally occurring environmental toxins. Poor activity alleles of CYP2C19 genetic markers from 127 Gambians and on 120 chromosomes from Yoruba, Europeans and Asians (Japanese + Han Chinese) in the Hapmap database. Haplotype breakdown was explored using bifurcation plots and relative extended haplotype homozygosity (REHH). Allele frequency differentiation across populations was estimated using the fixation index (FST) and haplotype diversity with coalescent models.We analyzed CYP2C19*2 and *3). REHH was high around CYP2C19*2 in Yoruba  and to a lesser extent in Europeans  and Asians . FST at the CYP2C19 locus was low overall (0.098). CYP2C19*3 was an FST outlier in Asians (0.293), CYP2C19 haplotype diversity < = 0.037, p <0.001.Bifurcation plots suggested conservation of alleles conferring slow metabolism (CYP2C19*2 is subject to positive selective forces worldwide. Similar evidence was also found for CYP2C19*3 which is frequent only in Asia. FST is low at the CYP2C19 locus, suggesting balancing selection overall. The biological factors responsible for these selective pressures are currently unknown. One possible explanation is that early humans were exposed to a ubiquitous novel toxin activated by CYP2C19. The genetic adaptation took place within the last 10,000 years which coincides with the development of systematic agricultural practices.We found some evidence that the slow metabolizing allele  CYP2C19 gene which is situated in the CYP2C gene cluster on chromosome 10 where it is in strong linkage disequilibrium with CYP2C9[CYP2C19 exhibits considerable genetic polymorphism giving rise to profound changes in enzyme activity leading to reduced metabolic capacity [The cytochrome P450 enzymes (hereafter referred to as cytochromes) have a wide range of essential biological functions in humans resulting from oxidation of their substrates. CYP2C19 is a key member of the cytochrome family and is responsible for metabolizing a substantial proportion of pharmacologically important compounds  althoughth CYP2C9. CYP2C19capacity . This ‘pcapacity ,14. HoweCYP2C19 (19154G/A) which creates an aberrant splice site altering the reading frame of the mRNA and introducing a premature stop codon. This allele, designated CYP2C19*2, results in a non-functional protein [CYP2C19*2 is common in Europe , Africa (0.14), China (0.26) and Japan (0.28) [CYP2C19*3 is also common (0.08) [CYP2C19*3 is rare in Europeans and Africans (0–0.01) [CYP2C19*3 is a guanine to adenine mutation at position 636 of exon 4 (636G/A) which creates a premature stop codon downstream resulting in a non-functional protein. The prevalence of these slow metabolizing alleles tends to increase moving east so in some parts of East Asia CYP2C19*2 and *3 allele frequencies rise to 0.71 and 0.13 respectively making the slow metabolizer phenotype predominant [17) , Gambians (0.24), Ethiopians and Swedish (0.18) and is rare in Chinese (0.04) [Early investigations into the genetic basis for the poor metabolizer phenotype revealed a single base pair mutation in exon 5 of YP2C19 194G/A whicn (0.08) . CYP2C19(0–0.01) -20. The dominant . CYP2C19402C > T ) which ie (0.04) .Because of a potential role in metabolizing unknown environmental substances, alleles that cause a change in function of the enzyme would be likely to give an individual a selective advantage or disadvantage in evolutionary terms. For example if the cytochrome is involved in promoting the elimination of a toxic chemical, a gain-of-function allele might confer evolutionary advantage and would thus be positively selected. Conversely if the enzyme catalysed the formation of a toxic compound from an otherwise non-toxic precursor, then alleles conferring low enzyme activity might be selected.New techniques for identifying such selective pressures on genes in specific populations have been developed leading to significant new insights into human genetic diversity . When a In an extension of this idea, relative extended haplotype homozygosity (REHH) is the ratio of the EHH of the core haplotype compared with the EHH of all other haplotypes on the chromosome. The REHH test compares the length of haplotype identity as a function of frequency around each allele compared to an expected distribution . The degRecent studies have highlighted the importance of infectious agents such as malaria ,27 and vCYP2C19 locus for signatures of evolutionary selection. We focussed specifically on the common non-functional CYP2C19 mutations CYP2C19*2 (19154G/A) and *3 (636G/A), measuring their haplotype frequencies and molecular diversity across populations, aiming to identify signals of recent evolutionary selection that might result from exposure to potentially harmful environmental substances.In view of the key role of cytochrome P450 enzymes in metabolism of environmental compounds, we investigated the CYP2C19*2 in the merged genotypic data from Gambians are shown in Figure CYP2C19*2/*3-containing alleles are described in Figure Bifurcation plots for the common loss-of-function allele CYP2C19*2 (AGT) suggests some evidence of positive selection, shown by the predominance of thick branches. This haplotype, present at a frequency of 0.15, had an REHH of 2.7 at −68.3 kb from the core region which is supportive of the suggestion of positive selection for CYP2C19*2. Since our genotyping data did not extend further than −68.3 kb from the core haplotype it was not possible to examine REHH over longer stretches of the genome in the Gambian study group.The bifurcation plot for the haplotype carrying CYP2C19*2 AGT-haplotype was present in the Yoruba at a similar frequency to Gambians (0.14) and displayed extended homozygosity evidenced by conservation on the bifurcation plot and an REHH of 8.3 at 133.3 kb from the core. This implies that the slow metabolism allele can confer an evolutionary advantage and confirms the initial findings in Gambians. The magnitude of the REHH for CYP2C19*2 in Yoruba is similar to that observed for G6PD-202A (glucose-6-phosphate dehydrogenase deficiency) which is thought to confer a 50% reduction in risk of malaria [We therefore examined the HapMap data for the Yoruba to obtain more extensive genomic data from people living in a nearby area of West Africa Figure B. The sa malaria ,33.CYP2C19*2 for positive selection in Europeans showed similar patterns to those observed in Yoruba . The bifurcation plot for the Japanese and Han Chinese showed clear breakdown of haplotype homozygosity arising from development of a new mutation, depicted by thinning of the branch , which also codes for an inactive enzyme only found in Asia shows some evidence of extended homozygosity  was 0.016 implying that only 1.6% of human genetic diversity at CYP2C19*2 is a result of genetic differentiation among the four populations studied. The CYP2C19*3 allele in Japanese and Han Chinese had a high FST implying unopposed positive selection in this population. Pairwise FST values are given in Additional file CYP2C alleles screened in the Gambians and HapMap populations.FCYP2C19  may be subject to selective forces in recent human evolution. Slow metabolizing haplotypes of CYP2C19 showed extended homozygosity which results from recent positive selection. We inferred this initially in the Gambian population for CYP2C19*2 and these findings were confirmed by analysis of genotypic data in Yoruba, from a nearby region of West Africa. We observed a similar pattern of haplotype homozygosity in Europeans, however the CYP2C19*2 EHH is more striking in Yoruba suggesting stronger positive selection in the West African population. Interestingly the fast metabolizing CYP2C19*17 was also under positive selection in Europeans. The REHH statistic observed for CYP2C19*2 in Yoruba (REHH 8.3) is of a similar magnitude to that seen in the same ethnic group for G6PD [We found some evidence that non-functional haplotypes of for G6PD . SelectiCYP2C19*2 accounts for the poor metabolizing phenotype in Europeans and in people of African descent, whilst CYP2C19*3 contributes to the poor metabolizer phenotype only in Asia. In Asian populations we found some evidence that CYP2C19*3 is also positively selected. This suggests that the CYP2C19 poor metabolism phenotype confers an evolutionary advantage in Asia in addition to Africa and Europe even though some alleles responsible for that phenotype differ between the three continents.ST values estimated for the CYP2C19 SNPs are lower than the average FST values for autosomal SNPs in worldwide human populations which is approximately 0.123. The CYP2C19 FST values are consistent with the low FST observed for many of the genes involved in immunity such as the major histocompatibility complex (MHC) and beta-globin gene which are under balancing selection [ST is an indication that balancing or species-wide directional selection has taken place at the CYP2C19 locus, in contrast to the force of geographically-restricted directional selection that leads to high FST values. Thus one would infer from the long extended haplotype and low FST for the slow-metabolizer CYP2C19*2 allele that CYP2C19*2 is still evolving and has not yet reached fixation.All Felection . The lowCYP2C cluster in the past have been relatively limited. Vormfelde and colleagues analysed selection signals in CYP2C9[CYP2C9*3 appeared to be under positive selection. There is a high degree of Linkage Disequilibrium (LD) in the CYP2C cluster so it is possible that the effects we saw on CYP2C19 might reflect selection of alleles of CYP2C9 and CYP2C8[CYP2C19 genomic region. CYP2C9*3 was not screened in this project as the variant has not been found in sub Saharan Africa.Investigations of evolutionary selection of alleles in the in CYP2C9 and shownd CYP2C8 however CYP2C19*2 poor functional variant worldwide is in accordance with the recent findings by Pimenoff and colleagues who used a similar technique (EHH) and haplotype structure to describe selection pressure on poor activity variants CYP2C19*2 and CYP2C9*3 in Europeans [CYP2C19*3 to be under selective pressure in Asians and the high activity CYP2C19*17 in Europeans. Poor activity alleles especially are favoured in recent human evolution, being selected in Africa, Europe and Asia. A further analysis of cytochrome gene evolutionary selection in the indigenous American populations would give interesting additional information.Our finding of positive selection for uropeans . Our stuCYP2C19 alleles are likely to have been operating over the last 10,000 years [The population selection forces acting on 00 years ,36. An aCYP2C19. The period of selection would be likely to correspond with the end of the most recent glacial age and beginning of the Mesolithic era (Middle Stone Age), from 8500 BC to 4000 BC. Over this period there is substantial archaeological evidence of developing systematic agricultural practices in Asia and the Middle East in contrast to the previous hunting and gathering lifestyle of early humans [It is interesting to speculate what biological forces are responsible for evolutionary selection of inactive alleles of y humans .Fusarium strains that contaminate cereals and grains [The storage of grain and tubers from one growing season to another, which is essential for effective agriculture, would have exposed early humans to a range of novel toxins derived from fungi growing on stored foodstuffs. Activation of mycotoxins by cytochrome P450 enzymes is well-described ,39 and td grains .In addition to mycotoxins, other classes of harmful environmental compounds may have been encountered for the first time as a result of developing agricultural practices. For example aryl hydrocarbons formed from incomplete combustion of carbon are highly toxic when partially activated by CYP2C19 and might be produced by the use of fire to clear land for crops .CYP2 family occurred nearly 400 million years ago with more than 50 gene duplication events [CYP evolution started much earlier >600 million years ago at the same gene locus referred to as the cytochrome P450 genesis locus from where all CYP clans or families emerged [CYP2C19*2/*3[Earlier speculations by Nebert suggested that rapid evolution of drug metabolizing enzyme (DME) genes and receptor genes occurred as a result of the interaction between animals as they moved on to land and the plants that they encountered there. An explosion of new animal genes in the n events . Nelson  emerged . The DME emerged . Such fa2C19*2/*3-44.CYP2C19*2 rs4244285 with the regulation of blood pressure [The physiological functions of CYP2C19, particularly in the synthesis of steroid hormones, could also potentially be important in increasing survival fitness . Gomes apressure . CYP2C19pressure ,49 and vpressure . These fCYP2C19 seems to be persuasive. The magnitude of the evolutionary pressure appears to be similar to that exerted on the human genome by infectious diseases. These environmental and/or physiological forces that shaped the cytochrome repertoire of modern humans have important consequences for drug metabolism in the present day [Whilst the mechanism for positive selection of individuals with poor CYP2C19 activity remains unknown, the evidence for such selection in The selective forces that we observed are temporally linked to the development of systematic agricultural practices by early humans and CYP2C physiological functions although it is not possible to infer a direct causal association from our data. It would be useful to extend these studies to other populations where different agricultural practices might have exposed humans to a different range of potentially harmful environmental chemical compounds. Similar signatures of selection might be found for other cytochrome P450 enzymes that activate known mycotoxins ,38. In aFurther work could examine the co-evolution of cytochromes with other elements of the metabolic pathways involved in detoxification of xenobiotic substances and metabolism of drugs. Some speculative studies indicate that considerable synergisms may exist between certain isoforms of cytochromes and transporter molecules that regulate influx of their substrates into cells . If thisInclusion of longer stretches of the genome in increased numbers of individuals could identify long range haplotypes in cytochrome gene clusters which have been positively selected. These haplotypes delimited by rapidly decaying LD would identify biologically important combinations of alleles across the gene cluster . Such haCYP2C19 that have low activity in the metabolic transformation of xenobiotic compounds it seems reasonable to suggest that some environmental agent would be responsible for exerting this pressure and shaping the cytochrome profile of modern humans. This agent could be a known substrate for CYP2C19 which has a metabolite with unrecognised toxic effects or alternatively a known toxin which is activated by the enzyme by a novel molecular mechanism. Either of these hypotheses might provide a fruitful starting point for further biochemical and toxicological research. Such studies might cast further light on human evolution and could potentially identify substances not previously known to be toxic.Since we have identified some evidence of global evolutionary selective forces in favour of alleles of Many forces shape the topography of the human genome and strong natural selection resulting from infectious diseases is well–recognised. Here we show that environmental chemicals could also exert a similar effect on human evolution.CYP2C19 locus and hence strongly influenced the drug metabolizing profile of modern humans.We speculate that a ubiquitous environmental compound may be rendered toxic by the activity of CYP2C19 and thus early humans with poor CYP2C19 activity had a survival advantage. This genetic adaptation took place within the last 10,000 years which coincides with the development of systematic agricultural practices. These evolutionary forces, which are of a similar magnitude to those exerted by infectious diseases, could have arisen from exposure to a novel toxin perhaps arising from stored foodstuffs. Selective pressure from this toxin may have driven allelic differentiation at the Eighty-five Gambian blood donors from Sukuta and 42 from Brikama (Western Region), Njaba Kunda and Farafenni (Northern Region) gave informed consent for genetic screening and analysis. The subjects from Sukuta participated in an investigation of nucleotide diversity of the TNF gene region  and the Venous blood was collected in EDTA tubes and deoxyribonucleic acid (DNA) extracted using Nucleon kits BACC3  according to the manufacturer’s protocol. The DNA was quantified using Picogreen (Molecular Probes®) and the NanoDrop 1000™ spectrophotometer (Thermo Scientific) and stored at −20°C until required.CYP2C19 had been previously characterized in fine detail in the adult study group and all common fast and slow metabolizing alleles identified [® drug metabolizing assay mixes for SNP genotyping were used to screen functional polymorphisms and amplification refractory mutation system PCR to genotype promoter and intronic polymorphisms in CYP2C19 and CYP2C9 in all the subjects. The 13 SNPs IDs genotyped were:Metabolic phenotype for CYP2C19: rs7067866, rs11568729, rs4417205, rs4986894, rs3758580 (mRNA990 *2A*2B), rs4986893 (mRNA636 *3), rs17884712 (mRNA431 *9), rs4244285 (mRNA681 *2), rs12248560 (*17) and1. CYP2C9: rs1799853 (*2), rs7900194 (*8), rs2256871 (*9), rs28371686 (*11).2. Genotypic data from the two Gambian study groups were pooled to give a sample size of 127 subjects.Ethical approval was obtained from the Medical Research Council (MRC) Scientific Coordinating Committee and the MRC/Gambia Government Joint Ethics Committee . Consent was written in English and explained in the local language of the subjects by an interpreter and the response was documented in English on the consent form.CYP2C19*2 in the Gambian population using SWEEP [CYP2C19*3 was not present in any Gambian participant. Missing allelic data were filled in using fastPHASE version 2.3 [CYP2C19*2 locus with the core haplotype rs4244285-rs4417205-rs3758580 and for CYP2C19*3 where it was present (rs12778026-rs4986893-rs4304692). We generated REHH and bifurcation plots from rs7067866 (96501916) to rs9332198 (NCBI build35 96731487) for the Yoruba, to rs1934967 (96731416) for CEU and to rs9332198 (96731487) for the Han Chinese. These plots were then examined for signals of selective pressure.First we constructed relative extended haplotype homozygosity (REHH) and haplotype bifurcation plots to look for positive selection of ng SWEEP . CYP2C19sion 2.3 . We thension 2.3 . These dST statistic was calculated to summarise allele frequency differentiation between populations using FSTAT 2.9.3 [The FAT 2.9.3 . HaplotyAT 2.9.3 . The inpCYP2C9: Cytochrome P450 2C9; CYP2C19: Cytochrome P450 2C19; EHH: Extended haplotype homozygosity; LD: Linkage disequilibrium; REHH: Relative extended haplotype homozygosity; SNP: Single nucleotide polymorphism.The authors declare that they have no competing interests.RTW, REJ and AW contributed to study conception and design. REJ and FSJ participated in the acquisition of data. RTW, REJ and AW took part in data analysis. REJ and RTW drafted the manuscript. REJ, AW, SOS, KJL and RTW interpreted the results. All authors have read and approved the final version of the manuscript.ST for CYP2C19 alleles across Gambians, Europeans, Japanese and Han Chinese and Yoruba.FClick here for file


The transfer of patients in extracorporeal membrane oxygenation (ECMO) from a peripheral hospital to a tertiary center represents a high-risk situation of adverse events . The aimWe collected data for the ECMO Regional Reference Centre Careggi Hospital activity from September 2009 to June 2014. In this study, 57 transfers were examined. The ECMO service is activated by a telephone call from a peripheral hospital. The team is represented by an intensivist, a heart surgeon, a cardiologist, a perfusionist and an intensive care nurse, all previously trained in the management of patients with ECMO. Medical personnel and the necessary equipment are transported by an ambulance and a van, specially designed and equipped for the transfer of patients with ECMO.In this study, 57 patients transferred from the peripheral hospital to the ECMO center were examined; in all cases the ECMO system was implanted in the peripheral hospital (54 venovenous ECMO and three venoarterious ECMO). On average, trails were 271 km ± 304 (round trip) (minimum 14 km to maximum 939 km). The activation time from the call to the ambulance departure from our hospital was an average of 2 hours 27 minutes 13 seconds ± 1 hour 25 minutes 35 seconds. Transfer duration (from activation to return to the ECMO center) was an average of 8 hours 25 minutes 6 seconds ± 3 hours 27 minutes 58 seconds (minimum 3 hours to maximum 16 hours 55 minutes). The stop time  was an average of 3 hours 53 minutes 40 seconds ± 1 hour 6 minutes 35 seconds (minimum 2 hours 5 minutes to maximum 7 hours 30 minutes). Major complications related to malfunctions of the devices during transport were not recorded; in some cases it was necessary to manage minor complications .Some studies have found several complications during transfer of patients in ECMO . In our


In Southern Europe Pru p3 is the primary sensitizer of plants fruit and it is responsible of severe reactions. Specific immunotherapy (SIT) brings a new perspective to treat those patients. There is a lack of knowledge regarding cellular responses that include changes in the basophil activation during the IT. We aim to analyse early changes in the basophil response to Pru p 3 and other related allergen (Ara h 9) after the first month of sublingual immunotherapy (SLIT).Forty-six peach allergic patients confirmed by positive specific IgE determined by skin prick test or fresh peach (prick-by-prick), ImmunoCAP IgE and/or a double blind placebo control food challenge with peach. Basophil reactivity was determined by the basophil activation test (BAT) with Pru p 3 and Ara h 9 at two concentrations, 1 and 0.1 µg/ml, before and after 1month of SLIT.Twenty one patients evaluated (45%) performed anaphylaxis and 25 (55%) urticaria and/or angioedema. The 82,6% showed sensitization to other plant foods proteins and 69,5% showed sensitization to pollens. The BAT was done in 25 patients with first month of SLIT completed. The 28% patients have an increase of Pru p 3 reactivity. The 36% patients showed same reactivity after first month and 36% presented a decreased reactivity to Pru p3. Similar results were obtained for Ara h 9 in those patients.Preliminary result disclosed that percentage of patients who underwent changes in BAT reactivity to Pru p3 and Ara h 9 was similar. There have been no differential clinical pattern in the groups studied after one month of SLIT. The BAT shows a good correlation between both Pru p 3 and Ara h 9.


To the Editor: Novel highly pathogenic avian influenza (HPAI) viruses ofsubtypes H5N2, H5N8, and H5N1 have recently caused numerous outbreaks in commercialpoultry farms in the United States and Canada . Of 3,300 samples tested, 1sample tested positive for HPAI virus subtype H5  Figure 1Minnesota and Iowa lie within regions where migrating waterfowl spend their breedingseason, and waterfowl densities on commercial poultry farms are particularly high Figure 2Of particular note, outbreaks in poultry were densely concentrated within Minnesotaand Iowa in a spatial pattern inconsistent with the much more geographicallydispersed spread of infection in wild birds. The magnitude and clustereddistribution of poultry outbreaks are suggestive of local spread, rather thanmultiple introductions from passing migratory waterfowl. Genetic analyses havesimilarly shown evidence for concurrent multiple introductions as well as commonsource exposures, and surveys of affected farms have shown that local spread couldbe facilitated by the sharing of equipment by multiple farms or through animalsentering barns  virus, subtype H5, inNorth America


SLC4A3 has been shown to cause retinal degeneration in a genetically engineered knockout mouse, and in a naturally occurring form of canine progressive retinal atrophy considered to be the equivalent of retinitis pigmentosa in humans (RP). This study was undertaken to investigate if SLC4A3 coding variants were implicated in human retinal degeneration. SLC4A3 exons were amplified and sequenced in 200 patients with autosomal recessive retinal degeneration who had no known molecular diagnosis for their condition, which included 197 unrelated individuals with suspected RP and three individuals with other forms of retinal disease. Three rare variants were identified that were predicted to be potentially pathogenic, however each variant was heterozygous in a single patient and therefore not considered disease-causing in isolation. Of these three variants, SNP-3 was the rarest, with an allele frequency of 7.06x10−5 . In conclusion, no compound heterozygous or homozygous potentially pathogenic variants were identified that would account for recessive RP or retinal degeneration in this cohort, however the possibility remains that the rare variants identified could be acting with as yet undiscovered mutations in introns or regulatory regions. SLC4A3 remains an excellent candidate gene for human retinal degeneration, and with the advent of whole exome and whole genome sequencing of cohorts of molecularly unsolved patients with syndromic and non-syndromic forms of retinal degeneration, SLC4A3 may yet be implicated in human disease.The online version of this article (doi:10.1186/s12952-016-0054-z) contains supplementary material, which is available to authorized users. SLC4A3 , encodes the anion exchanger 3 (AE3) protein, which mediates Cl−/HCO3− exchange across cellular membranes [Slc4a3 identified SLC4A3 as a candidate gene for human vitreoretinal degenerations based on their findings of blindness and retinal degeneration in knockout mice [Slc4a3−/− mice at four months of age had no gross retinal abnormalities; However, ERG analysis revealed an inner retina defect from birth (reduced b-wave and flicker amplitudes), leading to phototransduction failure at four months (reduced a-wave amplitude). At 4–6 months the number of apoptotic nuclei observed by TUNEL labelling increased. By eight months, pathological signs of photoreceptor degeneration were observed including dense astrocytic processes wrapped around inner retina vessels , small diameter major blood vessels, disorganised astrocytic processes at the optic nerve head and rod bipolar cell dendrites aberrantly sprouted into the outer nuclear layer [embranes , and hasSLC4A3 is associated with a form of naturally occurring autosomal recessive (AR) Progressive Retinal Atrophy (PRA) in the Golden Retriever dog breed, known as GR_PRA1 [In addition, we have previously shown that a mutation in  GR_PRA1 .PRA is widely considered to be the veterinary equivalent of Retinitis Pigmentosa (RP) in humans. RP is the name given to a group of inherited retinal degenerations which affects 1 in 3500 to 4500 people . PhotoreRPE65 have been implicated in AR retinal degeneration (LCA) in dogs [RPGR cause XLPRA [At least 17 naturally occurring dog models with retinal degeneration have been described with equivalent human disease (reviewed in ). These  in dogs , 11 and  in dogs , 13, and in dogs –20. Simise XLPRA –23 and ase XLPRA –26. Genese XLPRA . Importase XLPRA .SLC4A3 have been described in humans: a full-length (SLC4A3fl1) isoform comprised of one non-coding (5’UTR) and 22 coding exons and a cardiac (SLC4A3c) isoform with 18 coding exons [SLC4A3fl1 results in a further isoform (SLC4A3fl2) that differs by 81 bp. SLC4A3fl1 (Genbank: NM_201574.2) encodes a 1259 amino acid protein  is the shorter version and encodes a protein of 1232 amino acids. In SLC4A3c exon C1 replaces exons one to six of the full-length transcripts, and encodes a smaller protein product of 1034 amino acids [SLC4A3 gene, Ala867Asp, has been associated with idiopathic generalized epilepsy (IGE) in humans, with carriers exhibiting an increased risk of developing IGE [SLC4A3 has not been implicated in human retinal disease. Evidence from the mouse and canine disease models suggest the SLC4A3 gene is an excellent candidate for human retinal degeneration. We therefore screened SLC4A3 in a cohort of human patients with predominantly recessive retinal degeneration currently lacking a molecular diagnosis in order to determine whether mutations in this gene cause a significant proportion of retinal degeneration in humans.Two main isoforms of ng exons . These a SLC4A3fl isoform  SLC4A3fl isoform no acids . A rare ping IGE , but SLCThe recruitment of all patients was part of a study protocol that adhered to the tenets of the Declaration of Helsinki and had received approval from the Moorfields Eye Hospital Research Ethics Committee. Written, informed consent was obtained from all participants prior to their inclusion in this study with parental written consent provided on behalf of any minors involved.SLC4A3 variants. Of these, 192 probands were affected with progressive retinal degeneration, consistent with a diagnosis of either retinitis pigmentosa or cone-rod dystrophy with presentation in adulthood (during or after the second decade). Eight additional patients with various forms of retinal degeneration were also selected for screening. In each of these 8 cases, autozygosity mapping previously conducted at UCL Institute of Ophthalmology  had identified large regions of homozygosity that included the genomic region containing SLC4A3, amongst other genes  [Two hundred affected unrelated individuals ascertained from the clinics of Moorfields Eye Hospital were evaluated for m (ExAC)  was usedSLC4A3 exons , SLC4A3fl2(GenBank: NM_005070.3) and SLC4A3c [SLC4A3 exons were amplified by PCR using HotStarTaq Plus DNA Polymerase (Qiagen) in genomic DNA. PCR products were purified using PCRμ96 filter plates (Millipore). Amplification products were sequenced using BigDye Terminator v3.1 (Applied Biosystems), and sequence product was purified using the Montage SEQ96 Cleanup Kit (Millipore), then run on an ABI 3730 Genetic Analyzer. Sequence traces were assembled, analysed and compared with the human reference sequence (GRCh37) using the Staden Package [Primers for amplification and sequencing of  SLC4A3c  were des SLC4A3c . SLC4A3  Package .The potential pathogenicity of variants identified was assessed with various bioinformatics tools  have previously been identified and have entries in the dbSNP database, and 31 are present in the Exome Aggregation Consortium (ExAC) database. Copy number variants, such as large deletions and insertions within introns, or affecting upstream promoter sequences, were not assessed in this study.Sequencing of all known exons and intron-exon boundaries of SLC4A3 isoforms, while SNPs_2 and _3 affect the full-length and cardiac isoforms  and/or prediction of pathogenicity, three rare variants remained that were predicted to affect exon splicing and/or change the amino acid sequence of the protein Table . SNP_1 aSNPs_1  and _2  are non-synonymous SNPs that result in amino acid changes that are predicted to be pathogenic by at least one of the in silico prediction tools  to be potentially deleterious. Three individuals with AR RP carry one copy of the minor allele at one of these loci  was found in the heterozygous state only at a frequency of 8.66x10−5. The relatively high frequency of SNP_1 in the control exomes and the observation that it was predicted to be potentially pathogenic by only one of the in silico prediction tools suggests the variant is unlikely to play a role in retinal disease. SNP_2 has a relatively high allele frequency and occurs in the homozygous state in the control cohort. This suggests that this rare variant is also unlikely to cause disease in isolation, even though this variant was predicted to be potentially pathogenic by all four in silico prediction tools. Finally, SNP_3 has a very low allele frequency in the control exome data (8.66x10−5), but is predicted to affect exon splicing only. The SNPS predicted to affect exon splicing , human SLC4A3 mRNA is also likely to be undetectable and this was not pursued. In vitro assays could be useful to assess pathogenicity of SNP variants. However, it is difficult to select which variants to test, and the assay to use until more convincing evidence is presented that SLC4A3 is involved in human retinal disease. Such a study would therefore be premature at this stage.All three rare variants were also seen in the control datasets. SNP_1  and SNP_2  were found in the homozygous state in a single control exome each, as well as in the heterozygous state in multiple exomes, at a frequency of 7.89x10SLC4A3 identified in this study are unlikely to cause AR RP in the cohort screened in isolation, we cannot fully exclude these variants or the gene as a candidate for retinal degeneration. Potentially pathogenic rare variants in intronic regions or upstream elements that were not screened for mutations by sequencing could affect exon splicing or regulation of gene expression. In addition, it is possible that disease-causing mutations in this gene are extremely rare and essentially private mutations affecting only one or two affected individuals, as is increasingly the case in consanguineous families with a recessive condition, and that these individuals have yet to be screened. For example, a PRCD gene mutation (p.C2Y) was reported to cause PRA in dogs and RP in one person. However, no other disease-causing mutations were found in PRCD in a further 1240 RP patients screened [PRCD has since been identified in an isolated Muslim Arab village in Northern Israel; This founder mutation was homozygous in all 18 RP-affected individuals, but not in any of the 28 unaffected family members [PRCD [SLC4A3 actually causes a syndromic or non-syndromic form of retinal degeneration in humans that would not be clinically classified as RP. Little is known about the phenotype associated with GR_PRA1 in dogs beyond ophthalmoscopic observations. Clinically the fundus appears identical to other forms of PRA and histological and detailed ERG analysis has not been reported. In addition, the age of onset is difficult to define, although the age at which dogs with GR_PRA1 are diagnosed is typically 6–7 years [SLC4A3 knockout mouse in which a selective inner retina defect is followed by photoreceptor degeneration at eight months [SLC4A3. Alvarez and colleagues concluded that their results in the knockout mouse linked aberrant SLC4A3 to vitreoretinal degeneration [SLC4A3 than AR RP, but other phenotypes should not be excluded from future studies.While it appears that the variants in screened . Disease members . While trs [PRCD , 50. FinSLC4A3 has been shown to cause retinal disease in the mouse and dog, making the gene a strong candidate for human retinal disease. Three rare variants predicted to be potentially pathogenic were identified in the SLC4A3 gene in a AR RP cohort, however all three variants were present in the heterozygous state, and therefore not disease-causing in isolation. Nevertheless, we could not discount the possibility that these variants have some role in the disease that we have yet to decipher. The SLC4A3 gene remains an excellent candidate gene for human retinal degeneration and the variants identified will help to build a picture of its potential contribution.Aberrant The recruitment of all patients was part of a study protocol that adhered to the tenets of the Declaration of Helsinki and had received approval from the Moorfields Eye Hospital Research Ethics Committee. Written, informed consent was obtained from all participants prior to their inclusion in this study with parental written consent provided on behalf of any minors involved.Not applicable.The datasets supporting the conclusions of this article are included within the article and its Additional files


Electronic health records (EHRs) with poor usability present steep learning curves for new resident physicians, who are already overwhelmed in learning a new specialty. This may lead to error-prone use of EHRs in medical practice by new resident physicians.The study goal was to determine learnability gaps between expert and novice primary care resident physician groups by comparing performance measures when using EHRs.We compared performance measures after two rounds of learnability tests . In Rounds 1 and 2, 10 novice and 6 expert physicians, and 8 novice and 4 expert physicians participated, respectively. Laboratory-based learnability tests using video analyses were conducted to analyze learnability gaps between novice and expert physicians. Physicians completed 19 tasks, using a think-aloud strategy, based on an artificial but typical patient visit note. We used quantitative performance measures , a system usability scale (SUS), and qualitative narrative feedback during the participant debriefing session.There was a 6-percentage-point increase in novice physicians’ task success rate  and a 7-percentage-point increase in expert physicians’ task success rate ; a 10% decrease in novice physicians’ time-on-task  and 21% decrease in expert physicians’ time-on-task ; a 20% decrease in novice physicians mouse clicks  and 39% decrease in expert physicians’ mouse clicks ; a 14% increase in novice mouse movements  and 14% decrease in expert physicians’ mouse movements . The SUS measure of overall usability demonstrated only minimal change in the novice group  and no change in the expert group .This study found differences in novice and expert physicians’ performance, demonstrating that physicians’ proficiency increased with EHR experience. Our study may serve as a guideline to improve current EHR training programs. Future directions include identifying usability issues faced by physicians when using EHRs, through a more granular task analysis to recognize subtle usability issues that would otherwise be overlooked. Health information technology’s (HIT) functionality in clinical practice is expanding and physicians are increasingly adopting EHRs as a result of the financial incentives guaranteed by Centers for Medicare & Medicaid Services (CMS) . MeaningAccording to an EHR user satisfaction survey completed in 2012 by 3088 family physicians, approximately 62% of survey respondents were not satisfied with many of the best-known EHR systems, and EHR vendor support and training were the areas with lowest satisfaction ratings . MultiplPrevious studies have shown the importance of usability evaluation in the EHR adoption and implementation process. Current best practices promote the use of cognitive approaches to examine human-computer interactions in EHR systems ,48-50. KEHRs with poor usability present steep learning curves for new resident physicians, who are already overwhelmed in learning a new specialty. This may lead to error-prone use of EHRs in medical practice by new resident physicians. Identifying and addressing early barriers in the learning environment can help improve the overall capacity of new physicians and save costs for organizations. The objective of this study was to determine the difference in learnability by comparing changes in performance measures between expert and novice primary care physicians 3 and 7 months after 2 rounds of learnability tests . We analyzed learnability by addressing 2 specific research questions: (1) Do performance measures of expert and novice physicians improve after 3 and 7 months of EHR experience? and (2) Does the learnability gap between novice and expert physician groups change after 7 months of EHR experience?To determine learnability gaps between expert and novice physicians when using EHRs, data were collected through learnability testing using Morae video analysis software (TechSmith). Twelve family medicine and 4 internal medicine resident physicians performed 19 artificial, scenario-based tasks in a laboratory setting. Four types of quantitative performance measures, a system usability scale (SUS), a survey instrument , and a qThis study took place at the University of Missouri Health System (UMHS), which is a 536-bed, tertiary-care academic medical hospital located in Columbia, Missouri. In 2012, UMHS had approximately 553,300 clinic visits and employed more than 70 primary care physicians. The Department of Family and Community Medicine (FCM) runs 6 clinics, while the Department of Internal Medicine (IM) oversees 2 primary care clinics . The HeaThere is presently no evidence-based approach to measure a user’s EHR experience; therefore, novice and expert physicians were distinguished based on clinical training level and number of years using the EHR. This decision was based on a discussion with an experienced physician champion (JLB). This study will examine and confirm if after 1 year of EHR use, resident physicians have gained sufficient skills to be considered an expert . Thus, 1In Round 1, 10 novice physicians and 6 expert physicians participated in the study. Out of the 10 novice physicians in Round 1, 7 were from family medicine and 3 from internal medicine. Of the 10 novice physicians, 6 (60%) were male, 8 (80%) identified their race as white, 1 (10%) identified as Asian, and 1 (10%) identified as both Asian and white. The age of novice physicians ranged from 27 to 31 and the mean age was 28 years. In Round 1, 4 (40%) novice physicians had no experience with an EHR other than the one at UMHS, 2 (20%) had less than 3 months of experience, 1 (10%) had 7 months to 1 year of experience, and 3 (30%) had over 2 years of experience with an EHR other than the one at UMHS. In this study, 5 family medicine and 1 internal medicine expert physicians participated in the study. Of the 6 expert physicians, 5 (83%) were female and all (100%) identified their race as white. In this study, 2 did not provide information on their date of birth and EHR experience and were not included in the calculation of age range, mean age, and EHR experience. The age of expert physicians ranged from 30 to 33 and the mean age was 31 years. In this study, 1 (17%) expert physician had no experience with an EHR other than the one at UMHS, 1 (17%) had 7 months to 1 year of experience, and 2 (33%) had over 2 years of experience with an EHR other than the one at UMHS.Of the 8 novice physicians and 4 expert physicians who participated in Round 1 also participated in Round 2 of the study. A total of 2 novice and 2 expert physicians who participated in Round 1 declined participation in Round 2. Conducting 2 rounds of data collection was a major strength of this study, because it allowed us to measure valid learnability. Out of the 8 novice physicians in Round 2, 5 were from family medicine and 3 from internal medicine. Of the 8 novice physicians, 5 (63%) were male, 8 (75%) identified their race as white, 1 (13%) identified as Asian, and 1 (13%) identified as both Asian and white. The age of novice physicians ranged from 27 to 30 and the mean age was 28 years. In Round 2, 3 (38%) novice physicians had no experience with an EHR other than the one at UMHS, 2 (25%) had less than 3 months of experience, 1 (13%) had 7 months to 1 year of experience, and 2 (25%) had over 2 years of experience with an EHR other than the one at UMHS. Four family medicine expert physicians participated in the study. All 4 (100%) were female and all (100%) identified their race as white. The age of expert physicians ranged from 30 to 33 and the mean age was 31 years. In this study, 1 (25%) expert physician had no experience with an EHR other than the one at UMHS, 1 (25%) had 7 months to 1 year of experience, and 2 (50%) had over 2 years of experience with an EHR other than the one at UMHS. Because of the small sample size, we did not attempt to control for age or gender.Two sets of artificial but realistic scenario-based tasks were used in the study. The tasks were created based on discussion with an experienced physician champion (JLB) and 2 chief resident physicians from both participating departments . When completing Round 1 of the learnability test, resident physicians were given a scenario for a “scheduled follow-up visit after a hospitalization for pneumonia.” When completing Round 2 of the learnability test, resident physicians were given a scenario for a “scheduled follow-up visit after a hospitalization for heart failure.” While different, these 2 scenarios were equivalent in difficulty, workflow, and functionalities used. These scenarios were employed to assess physicians’ use of the EHR with realistic inpatient and outpatient information. We included 19 tasks that are generally completed by both novice and expert primary care physicians. These tasks also met 2014 EHR certification criteria 45 CFR 170.314 for meaningful use (MU) Stage 2 [1. Start a new note (§170.314[e][2]).2. Include visit information (§170.314[e][2]).3. Include chief complaint (§170.314[e][2]).4. Include history of present illness (§170.314[e][2]).5. Review current medications contained in the note (§170.314[a][6]).6. Review problem list contained in the note (§170.314[a][5]).7. Document new medication allergy (§170.314[a][7]).8. Include review of systems (§170.314[e][2]).9. Include family history (§170.314[a][13]).10. Include physical exam (§170.314[a][4] and §170.314[e][2]).11. Include last comprehensive metabolic panel (CMP) (§170.314[b][5]).12. Save the note.13. Include diagnosis (§170.314[a][5]).14. Place order for chest X-ray (§170.314[a][1] and §170.314[e][2]).15. Place order for basic metabolic panel (BMP)  to supplement the performance measures. The SUS is a 10-item survey measured on a Likert scale that provides fairly robust measures of subjective usability and is a widely used, validated instrument in HIT evaluation ,55,66. TTwo rounds of data collection were scheduled to measure learnability by comparing whether participants’ performance measures  improved and if participants experienced fewer usability issues with longer exposure to the system. Learnability pertains to the amount of time and effort needed for a user to develop proficiency with a system over time and after multiple use . The 2 gbetween comparison of 2 within comparisons. Therefore, we measured the difference between the novice and expert resident physician groups and the difference within novice and expert physician groups, 3 and 7 months after EHR training. Comparisons of learnability between the 2 groups were between comparisons. Time-on-task, mouse clicks, and mouse movements were measured while users interacted with the EHR system and performance measures were calculated automatically by the Morae Manager usability analysis software program. Percent task success was calculated by creating subtasks out of each task and then identifying each subtask the physician completed successfully. For example, for Task 8 (Include review of systems) the subtasks created to calculate the task success rate were the following: (1) go to review of systems, (2) add “no chills,” (3) add “no fever,” (4) add “fatigue,” (5) add “decreased activity,” (6) add “dry mouth,” (7) add “no dyspnea,” and (8) add “no edema.”We confirmed there were no EHR interface changes between data collection in Rounds 1 and 2 that may have influenced the study and tasks. The recorded sessions were examined using Morae Manager, a video analysis software program that was used to calculate performance measures using markers to identify difficulties and errors the participants encountered. Video analysis took approximately 1.5 hours for each 20-minute recorded session. The first step in the analysis was to review the recorded sessions and label any tasks that were unmarked during data collection. The second step was to divide each of the 19 tasks into smaller tasks to determine the task success rate and identify subtle usability challenges that we may have otherwise failed to notice. Geometric means were calculated for the performance measures with confidence intervals at 95% . PerformGeometric mean values of percent task success rates were compared between the 2 physician groups across 2 rounds  69]. Th. Th69]. In Round 1, the novice physician group achieved a higher success rate than expert physicians for 7 tasks , the same success rate for 7 tasks , and a lower success rate for 5 tasks . In Round 2, the novice physician group achieved a higher success rate for 3 tasks , the same success rate for 15 tasks , and a lower success rate for Task 15.Both novice (6%) and expert physician groups (2%) had equally low task success for Task 7 (Add a medication to your favorites list) in Round 1. However, in Round 2 all physicians in both groups successfully completed Task 7 (100%).Geometric mean values of time-on-task (TOT) were compared between the 2 physician groups across the 2 rounds . There wIn Round 1, the novice physician group spent less time than expert physicians completing 4 out of 19 tasks , the same amount of time completing Task 17, and more time completing 14 tasks . In Round 2, the novice physician group spent less time completing 4 out of 19 tasks , the same time completing Task 18, and more time completing 14 tasks .In Round 1, both physician groups had the longest time spent on Task 7 . However, in Round 2, time on Task 7 decreased by 52% for the expert physician group (87s to 50s) and 29% for the novice physician group (133s to 95s).Geometric mean values of mouse clicks were compared between the 2 physician groups across the 2 rounds . There wIn Round 1, the novice physician group achieved lower mouse clicks than the expert physician group for 7 tasks , higher mouse clicks for 9 tasks , and a comparable number of clicks for 3 tasks . In Round 2, novice physicians used less mouse clicks when completing 6 tasks , the same number of clicks when completing 5 tasks , and more clicks completing 8 tasks .In Round 1, both novice and expert physicians had the highest number of mouse clicks out of all tasks when completing Task 7 (Add a medication to your favorites list). However, in Round 2, the task with the highest number of mouse clicks by expert physicians changed from Task 7 to Task 15 (Place order for basic metabolic panel [BMP]) and novice physicians had the highest mouse clicks when completing Task 14 (Place order for chest X-ray) in Round 2, compared to Task 7 in Round 1.Geometric mean values of mouse movements (the length of the navigation path to complete a given task) were compared between the 2 physician groups across the 2 rounds. There was a 14% increase in novice physicians’ mouse movements between Round 1  and Round 2 . There was also a 14% decrease in expert physicians’ mouse movements between Round 1  and Round 2 . When mouse movements were compared between the physician groups, the novice physician group showed slightly longer mouse movements than expert physicians did across the 19 tasks in both rounds.In Round 1, the novice physicians showed longer mouse movements for 15 of 19 tasks , and shorter mouse movements for 4 tasks . In Round 2, novice physicians used shorter mouse movements in completing 8 out of 19 tasks  and used longer movements completing 11 tasks .In Round 1, novice physicians had the longest mouse movements out of all tasks when completing Task 7 (Add a medication to your favorites list) and expert physicians had the longest mouse movements when completing Task 13 (Include diagnosis). In Round 2, the task with the longest mouse movements by novice physicians was Task 14 (Place order for chest X-ray) compared to Task 7 in Round 1 and expert physicians had the longest mouse movements when completing Task 15 (Place order for basic metabolic panel [BMP]).In Round 1, 5 out of 6 expert physicians and all 10 novice physicians completed the SUS. In Round 2, all 4 expert physicians and all 9 novice physicians completed the SUS. The SUS illustrated that novice physicians ranked the system’s usability at a mean of 69  in Round 1 compared to 68  in Round 2. Experts rated the system’s usability at a mean of 74 (acceptable) in both rounds. A novice physician and 2 expert physicians had a score of 50 (not acceptable) or below. These results may indicate that expert users who have achieved a certain level of proficiency may be more confident using the EHR than novice users. A debriefing session confirmed the overall learnability test experience but did not reveal specific learnability issues. After analyzing the recording, however, it was clear that physicians encountered some difficulties when completing the tasks.Because of space limitations, a second manuscript is in preparation with a full review of the usability themes. Sub-task analysis was instrumental in identifying multiple usability concerns. We identified 31 common and 4 unique usability issues between the 2 physician groups across 2 rounds. Themes were created by analyzing and combining usability issues to form an overarching theme . Five thOur findings show that there were mixed changes in performance measures and expert physicians were more proficient than novice physicians on all four performance measures.P=.01). No statistically significant difference between novice and expert nurses was found when considering only completely solved tasks (P=.08). Our study did not report P values due to the small sample size; however, we observed overall improvement in performance measures for both novice and expert physician groups across 2 rounds. The contradictory results from this study and the study by Kjeldskov, Skov, and Stage, suggest that further research is necessary to draw more definite conclusions about task success between novice and expert physicians.In our study, differences were found between expert and novice physicians’ performance measures across Round 1 and Round 2. A study by Kjeldskov, Skov, and Stage  identifyP<.00) [Alternatively, a study by Lewis et al measured performance of novice health sciences students and a predictive model of skilled human performance when performing EHR tasks using a touchscreen. Novice participants were adults with no prior experience using an EHR touchscreen interface using CogTool. CogTool is an open-source user-interface prototyping tool that uses a human performance model to automatically evaluate how efficiently a skilled user can complete a task. Participants completed 31 tasks commonly performed by nurses and patient registration clerks in an Anti-Retroviral Therapy clinic. The mean novice performance time for all tasks was significantly slower than predictions of skilled use (P<.00) . AlthougPhysicians’ perceptions of the usability of a system may have relations to learnability; that is, physicians may find the system more user-friendly (usability) if the amount of time and effort needed to develop proficiency with the system is shorter (learnability). In our study, the SUS, which measures overall usability, illustrated that there was only a slight change in novice  and expert  physicians’ rankings of the system’s usability. In a study by Haarbrandt et al, primary care providers gave a SUS rating of 70.7  when asked about their perception of a health information exchange system, which was similar to the physicians’ scores in our study. Expert and novice participants found the graphical user interface easy to use; however, they only rated the system as acceptable  measuredThis study had several limitations in terms of the methodology. First, it involved a small sample of physicians; therefore, the sample size may not have been sufficient to obtain statistical significance when reporting quantitative results of learnability. However, the sample size was sufficient when identifying usability issues experienced by participants when interacting with the EHR system. This study was conducted at a health care institution where only 1 EHR system was used and may not be representative of all primary care practice. As such, the study’s findings may have limited generalizability to other ambulatory clinic settings, due to different types of EHR applications and physician practice settings. However, the EHR platform employed in this study is one of the top commercial products with significant market share. Based on data from Office of the National Coordinator for Health Information Technology, Cerner was reported as the primary EHR Vendor by 20% of hospitals participating in the CMS EHR incentive programs, making it the second most implemented EHR in hospitals . Second,Overall, this study identified varying degrees of learnability gaps between expert and novice physician groups that may impede the use of EHRs. Our results suggest that longer experience with an EHR may not be equivalent to being an expert or proficient in its use. The physicians’ interactions with the EHR can be communicated to EHR vendors, to assist in improving the user interface for effective use by physicians. This study may also assist in the design of EHR education and training programs by highlighting the areas  of difficulty that resident physicians face. Resident physicians in primary care are offered extensive EHR training by their institutions. However, it is a great challenge for busy physicians to find time for training. Furthermore, it is an arduous task attempting to meet the needs of users and provide hands-on, on-site support , and evi


CDK4 amplification in liposarcomas is associated with favorable prognosis. We aimed to identify the factors associated with tumor recurrence in patients with well-differentiated (WD) and dedifferentiated (DD) liposarcomas.The absence of MDM2 and CDK4 gene amplification were analyzed by quantitative real-time polymerase chain reaction (Q-PCR).From 2000 to 2010, surgical resections for 101 WD and DD liposarcomas were performed. Cases in which complete surgical resections with curative intent were carried out were selected. P<0.05). Q-PCR analysis of the liposarcomas revealed the presence of CDK4 amplification in 44 cases (91.7%) and MDM2 amplification in 46 cases (95.8%). WD liposarcomas with recurrence after surgical resection had significantly higher levels of CDK4 amplification compared to those without recurrence (P = 0.041). High level of CDK4 amplification (cases with CDK4 amplification higher than the median 7.54) was associated with poor recurrence-free survival compared to low CDK4 amplification in both univariate (P = 0.012) and multivariate analyses (P = 0.020).There were 31 WD and 17 DD liposarcomas. Locoregional recurrence was observed in 11 WD and 3 DD liposarcomas. WD liposarcomas showed better patient survival compared to DD liposarcomas (CDK4 amplification determined by Q-PCR was associated with the recurrence of WD liposarcomas after surgical resection.Level of  MDM2 and CDK4, which are known to play crucial roles in the control of the cell cycle progression CDK4 amplification has been observed in several malignancies including glioma, breast cancer, lymphoma, melanoma, and sarcoma CDK4 amplification in WD and DD liposarcomas is associated with lower rate of recurrence and favorable prognosis Liposarcomas are the most common type of soft tissue sarcoma accounting for approximately 20% of all soft tissue sarcomas MDM2 and CDK4 amplification in a homogeneous population of patients with WD and DD liposarcomas of the abdomen undergoing complete surgical resection.In this study, we sought to identify factors associated with tumor recurrence and patient survival including the levels of From December 2000 to December 2010, 139 patients underwent surgical resection for liposarcoma at Samsung Medical Center, Seoul, Korea. Among these patients, 101 cases were diagnosed as WD or DD liposarcomas. Retrospective review was performed for consideration of inclusion in the study. Cases referred to our institute from other centers for management of recurrent tumors were excluded from the analysis. Cases were selected for this analysis when complete surgical resections with curative intent were carried out for WD or DD liposarcomas of the retroperitoneum and peritoneal cavity. Complete surgical resection of the tumor was achieved when all of the following three criteria were met: no gross residual tumor in the surgical field as observed by the surgeon (pathologic R0 or R1 status), histologic confirmation of negative surgical margins and no radiologic signs of residual tumor in the first postoperative follow-up abdomen computed tomography (CT) scan, typically done between postoperative 1 to 4 weeks.Common practice for surgical resection of abdominal liposarcomas was wide excision of the mass with combined resection of adjacent viscera when the organ is suspected to have direct tumor invasion by preoperative radiologic evaluation and inspection in the surgical field. A tissue expander was left in the space previously occupied by the tumor when adjuvant radiotherapy was planned.Patients were followed at our outpatient clinic with abdominal CT scans and chest plain x-rays every 3 to 6 months. When local recurrence was suspected, abdominal CT scans were repeated after 1 month or positron emission tomography-CT (PET-CT) was done to confirm the presence of locoregional recurrence or distant metastases. When possible, surgical resection of the recurred tumor mass was attempted. Unresectable tumors or distant metastases to multiple sites were managed with systemic chemotherapy.This research has been approved by the institutional review board at Samsung Medical Center . All data collection and analysis were done anonymously, and written or verbal consent were not provided by the participants of this work. The lack of consent for this study was also approved.MDM2 and CDK4 amplification was analyzed by quantitative real-time polymerase chain reaction (Q-PCR) performed on a PRISM 7500HT Fast Realtime PCR system  by using a HotStart-IT SYBR Green qPCR Master mix . Ten nanograms of target DNA was dispensed into each sample with a final reaction volume of 10 µl. Each sample was amplified in triplicate. The PCR was carried out as follows: preheating at 50°C for 2 min and then at 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and 60°C for 1 min.  located at 4q11-q13, and were normalized to normal tissue genomic DNA as a calibrator. The level of amplification for MDM2 or CDK4 was calculated as follows: copy number of the target gene /copy number of the reference gene (ALB). Each gene was considered to have positive amplification when the copy number was greater than 2 times that of the reference gene.Immunohistochemical (IHC) staining for CDK4 was performed on representative paraffin-embedded tumor material from the biopsy cut into 4 µm-thick sections and placed onto glass slides. For IHC staining, the slides were deparaffinized in xylene. Staining for CDK4 was performed automatically using the Leica Bond Max immunostainer with Bond Polymer Refine Detection Kits and heat-induced epitope retrieval pH 6.0 (Bond max ER1 (EDTA) solution, Australia) for 15 min. The CDK4 expression was analyzed using mouse monoclonal antibody specific for CDK4 protein  P-values were less than 0.05.Chi-square tests  were used to compare categorical variables. Pearson’s correlation was used to measure the correlation between variables. The Cox proportional hazard model was used for survival and risk factor analysis. All analyses were done using IBM SPSS Statistics version 19 program. Results were considered to be significant when One hundred thirty-nine patients were surgically treated for liposarcoma at our center during the study period. Pathologic examination of the resected tumors of 101 patients showed either WD or DD histologic subtype. Ninety-two tumors were found in the retroperitoneum or peritoneal cavity. Fifty-four patients had their first surgical treatment at our center and 48 had complete surgical resection of the tumor. The patient selection process for inclusion in this study is outlined in The 48 patients who had complete tumor resection  were included in this analysis. The median age of patients was 57 years (range 37∼78 years). There were 31 WD and 17 DD liposarcomas. Tumors were located in the retroperitoneum in 43 cases, the mesentery in 4 cases and the pelvis in 1 case. Tumor size ranged from 6.8 cm to 60 cm (median 21 cm). The characteristics of the 48 patients and clinicopathological features of the liposarcomas according to histologic type are outlined in p = 0.021). Microscopic examination of the surgical margin was negative in 11 (35.5%) WD and 3 (17.6%) DD liposarcomas. Tumor recurrence after surgical resection with curative intent was observed in 20 patients ; 11 were WD and 9 were DD liposarcomas . Recurrence was locoregionally limited in all 11 WD cases while 6 of the 9 DD liposarcomas recurred as distant metastases. A significant difference in recurrence pattern was observed between the 2 histologic subtypes (p = 0.001).p<0.05).Locoregional recurrence was observed in 11 WD and 3 DD liposarcoma cases. The median time interval between surgical resection and the occurrence of the first recurrence was 13.5 months and 11.8 months for WD and DD liposarcomas, respectively. Primary WD liposarcomas expressed variable histologic types in their recurrence lesions; pathologic analyses of the recurrent tumor specimen were WD in 6 cases, DD in 2 cases and pleomorphic in 1 case. All recurrences of primary DD liposarcomas were of the DD histologic subtype. Surgical resection of locoregional recurrence was done in 9 WD and 2 DD cases. Patients underwent an average of 2.0 surgical resections for recurrences .Six cases had distant metastases during their follow-up period . Of thesSurgical resection of the metastatic tumor was carried out in 2 cases and systemic chemotherapy was done in 1 case. Metastatic liposarcoma was the cause of death in 4 patients. Patient survival after metastasis was 22.2% at 24 months.CDK4 amplification in 44 cases (91.7%) and MDM2 amplification in 46 cases (95.8%). The levels of CDK4 amplification were not different between the two histologic subtypes , and also in each histological subtypes .CDK4 immunohistochemistry results were interpretable in all cases. Positive immunoreactivity for CDK4 was observed in 32 out of 48 cases 66.7%, . HoweverCDK4 amplification (cases with CDK4 amplification higher than the median 7.54) was associated with poor recurrence-free survival compared to low CDK4 amplification in both univariate (P = 0.012) and multivariate analyses (P = 0.020). Disease-specific survival and recurrence-free survival curves of WD liposarcoma patients are shown in CDK4 amplification levels greater than 7.54 (CDK4 high) had significantly inferior recurrence-free survival compared to cases with CDK4 amplification less than 7.54 (CDK4 low). None of the analyzed variables were found to significantly affect survival of DD liposarcoma patients.Predictors of recurrence-free survival of WD liposarcoma are shown in MDM2 and CDK4, belong to distinct amplicons and no consistency exists in the amplified sequences between MDM2 and CDK4. It has been well documented that these two genes are involved in the oncogenesis and progression of WD and DD liposarcomas MDM2 amplification and overexpression is present in most WD and DD liposarcomas, CDK4 amplification is absent in a small proportion of cases CDK4 amplification represent a distinct clinical subgroup with a lower recurrence rate and are more likely to be peripherally located et al. was the fact that the better prognosis observed in liposarcomas without CDK4 amplification may have been confounded by these tumors more often being located in the extremities. Liposarcomas of the extremities are more likely to be resected with sufficient free surgical margin compared to those in the retroperitoneum. The liposarcomas included in the current study were limited to tumors arising in the retroperitoneum and peritoneal cavity. Q-PCR of the tumor specimens revealed varying levels of MDM2 and CDK4 amplification, with 44 cases (91.7%) and 46 cases (95.8%) showing positive amplification of CDK4 and MDM2 genes, respectively. Moreover, WD liposarcomas with recurrence after surgical resection had significantly higher levels of CDK4 amplification compared to those without recurrence and the degree of CDK4 amplification was an independent risk factor for recurrence of WD liposarcoma after complete excision. To the best of our knowledge, this study is the first to describe the correlation between CDK4 amplification and WD liposarcoma recurrence in a quantitative manner. In accordance with the study by Italiano et al., four cases in our series that were negative for CDK4 amplification are free of disease recurrence at median 25 months (range 16∼39 months) after surgical resection.Amplification of chromosome 12q13-15 is a typical feature of WD and DD liposarcomas CDK4 to DD liposarcoma progression was presented in a study by Barretina et al., in which shRNA was used to knockdown CDK4 in two DD liposarcoma cell lines. Sustained knockdown of CDK4 led to inhibition of proliferation of the cell lines. In addition, pharmacological inhibition of CDK4 with a selective CDK4 inhibitor (PD0332991) induced G1 arrest in the same two cell lines CDK4 gene. Our study adds further rationale to the current body of evidence that the use of CDK4 inhibitors in DD liposarcoma may prove to be beneficial.In vitro data on the contribution of CDK4 amplification by Q-PCR. These results suggest that translational or post-translational events function in fine tuning the final outcome of amplication and protein expression can be induced by other mechanism rather than amplication. Discrepancies between CDK4 IHC and Q-PCR findings were also observed in a study by Sirvent et al., in which negative staining by IHC for CDK4 was observed despite the presence of amplification by Q-PCR in approximately 40% of cases of WD and DD liposarcomas MDM2 was almost ubiquitously overexpressed in WD and DD liposarcomas and therefore did not affect tumor biology MDM2 Q-PCR analysis results also showed MDM2 amplification in 46 out of 48 cases in our liposarcoma specimens.Immunohistochemical analysis of CDK4 has been shown to be helpful in the differential diagnosis of liposarcomas and benign lipomatous tumors Our group previously analyzed 94 cases of liposarcomas in both the trunk and the extremities and concluded that high grade histologic subtype and positive margin status (microscopic and macroscopic) were independent risk factors for poor survival CDK4 amplification determined by Q-PCR was associated with the recurrence of WD liposarcomas of the retroperitoneum and peritoneal cavity. Well-differentiated liposarcomas with higher level of amplification of CDK4 (≥7.54) were more likely to recur after surgical resection. Utilization of Q-PCR for analysis of CDK4 amplification may aid clinicians in the postoperative surveillance and management of patients with abdominal WD and DD liposarcomas.In conclusion, the level of