This mutationrecapitulates the molecular hallmarks of a disease-causing mutation described inhumans, an m.4290T>C transition affecting also the human mt-Tigene. We could determine that the pathogenic molecular mechanism, induced by both themouse and the human mutations, is a high frequency of abnormal folding of thetRNAIle that cannot be charged with isoleucine. We demonstrate that thecells harboring the mouse or human mutant tRNA have exacerbated mitochondrialbiogenesis triggered by an increase in mitochondrial ROS production as a compensatoryresponse. We propose that both the nature of the pathogenic mechanism combined withthe existence of a compensatory mechanism can explain the penetrance pattern of thismutation. This particular behavior can allow a scenario for the evolution ofmitochondrial tRNAs in which the fixation of two alleles that are individuallydeleterious can proceed in two steps and not require the simultaneous mutation ofboth.About half of the mitochondrial DNA (mtDNA) mutations causing diseases in humansoccur in tRNA genes. Particularly intriguing are those pathogenic tRNA mutations thancan reach homoplasmy and yet show very different penetrance among patients. Thesemutations are scarce and, in addition to their obvious interest for understandinghuman pathology, they can be excellent experimental examples to model evolution andfixation of mitochondrial tRNA mutations. To date, the only source of this type ofmutations is human patients. We report here the generation and characterization ofthe first mitochondrial tRNA pathological mutation in mouse cells, an m.3739G>Atransition in the mitochondrial  Mitochondrial DNA (mtDNA) encodes for 13 proteins that are subunits of the OxidativePhosphorylation system. These proteins are synthesized within the mitochondria by itsown set of ribosomes. In mammals the rRNAs and all the tRNAs required for themitoribosomes to work are also encoded by the mtDNA. Surprisingly, half of mtDNAmutations causing diseases occur in tRNA genes while they represent only 10%of the total sequence. Moreover, these pathological tRNA mutations can occur inhomoplasmic form . It is puzzling that, withthese homoplasmic mutations, some individuals suffer a severe disease while othersare healthy. Recently we have described that common variants of mtDNA in mouse inducedifferences in the performance of the Oxidative Phosphorylation capacity but that thecell is capable to sense these differences and adapt the biogenesis of the OXPHOSsystem to compensate for them. We also showed that this mechanism was triggered bydifferences in the basal level of ROS. It was considered that this adaptationmechanism was of no relevance in pathological mutations. The implication of theROS-mediated mitochondrial biogenesis in modulating the consequences of mildpathogenic mutations and its significance for disease penetrance and mitochondrialtRNA evolution are underscored. Mutations in mitochondrial DNA are knownto be responsible for a wide variety of diseases in humans whose common characteristicis the impairment of the OXPHOS system. Almost half of the ≈250 mutations describedso far are located within tRNA genes Cells carrying pathological mutations in mt-tRNAs usually exhibit impaired respirationand reduced growth rates in medium with galactose instead of glucose. This is due to thefact that mutations in tRNA genes may affect the synthesis of critical subunits ofComplexes I, III and IV and two subunits of complex V. Different mutations produce avariety of defects mt-Tk cause MERRF or MERRF-like syndromes mt-Ti, usually have cardiomyopathy as the main or one of thecardinal features Mutations in mt-tRNAs tend to promote different disease patterns. Thus, while differentmutations in mt-TrIn mice, there is no description of mitochondrial tRNA pathological mutations. However,it has been reported the existence of four different alleles in a highly polymorphicloci at the mt-Ti gene. The mutationis located in the anticodon loop of this tRNA, two bases downstream from the anticodon,and generates a new potential Watson and Crick pair between the first and last base ofthe loop. Interestingly, we previously described an analogous mutation in humans, ahomoplasmic T to C transition two bases upstream the mt-tRNAIle anticodontriplet, responsible for a progressive necrotizing encephalopathy with variablepenetrance Ile that causes a reduction in the effective amountof functional isoleucyl-tRNAIle. As a consequence, mitochondrial proteinsynthesis and the activity of complexes I, III and IV are impaired, causing a mild butsignificant OXPHOS deficiency. We describe also that cells harboring the mutant mtDNAshow a higher ROS production that leads to a compensatory response to this respirationdeficiency by enhancing mitochondrial biogenesis. This response is able to partiallycompensate the deficiency. Therefore we demonstrate the positive implication of theROS-mediated mitochondrial biogenesis also in the expression of mitochondrial tRNApathological mutations found in human patients. These observations highlight thedifferent nature of the mutations affecting protein-coding genes vs. tRNA genes withconsequences to our understanding of pathology and evolution of mitochondrial tRNAs.Thus, this mechanism may generate an epistatic-like effect  by which a partial suppression of deleterious mutations inmitochondrial tRNAs is exerted. This increased mitochondrial biogenesis may allow thesurvival and reproduction of some individuals despite of harboring a deleterious allele,facilitating the appearance of a true compensatory mutation, the bona-fide epistaticmutation.We describe here the first pathological mutation in a mouse mitochondrial tRNA, anm.3739G>A transition in the mitochondrial neopuro . Then, we fully sequenced the mtDNA of these cell linesand we found a unique mutation, consisting in an m.3739G>A transition affectingthe mt-Ti gene (Ile anticodon triplet(m.4290T>C), has been described in humans and it could also promote a newpotential Watson and Crick pair between the first and last base of the anticodon loop in order to generate a proper control for mB77p18 cells.In our laboratory, we systematically induce and isolate mtDNA mutations by randommutagenesis using different mitochondrial backgrounds -Ti gene . This nu-Ti gene , is loca-Ti gene . An idendon loop. The latdon loop. FinallyAs shown in Cells with impaired OXPHOS capacity show difficulties to grow in medium where glucoseis substituted by galactose as carbon source, and this depends very much on theextent of the OXPHOS impairment A hallmark of pathological mt-tRNA mutations is that usually all respiratorycomplexes with mtDNA encoded subunits are affected 50) was significantly smaller for mutant versus wild typetRNAIle cells for complex I, III and IV inhibitors while the onlyrespiratory complex without mtDNA encoded subunits, complex II, was equally affectedby its inhibitor in control and mutant cells  specific activities . Recentlands 2O2  and thatands 2O2 . As a coands 2O2 .in vivo protein synthesisexperiments were performed. Analysis of mitochondrial DNA translation products didnot reveal significant quantitative differences or abnormal migration of anypolypeptide in the mutant cells  analysis  10%polyacrilamide/4M urea gel and electroblotted onto a zeta-probe membrane. Then, theblots were sequentially hybridized with specific probes for different mitochondrialtRNAs. Under these conditions the acylated and deacylated forms of most of the tRNAsmay migrate differently due to the induction of a conformational change in the tRNAupon aminoacylation, while an increase in urea concentration decreases the tRNAfolding and minimizes the migration differences Arg, tRNATrp and tRNALeu, two bands, theslower moving corresponding to aminoacylated tRNA species  or in higher (8 M) urea concentration -3H]-Valine as a control. As shown in inorganello-aminoacylated tRNAs were isolated and electrophoresed underacidic conditions, only one band was observed. These results confirm that the slowermoving extra band present in mutant cell lines was not isoleucyl-tRNAIle,and therefore, that it corresponds to a non-chargeable form of thetRNAIle. As a consequence, the ratio of isoleucyl-tRNAIle/tRNAIle seemed to be abnormally low in the mutant cells.To understand whether the slower-migrating extra band in the mt-tRNAIle as theprimary cause of the protein synthesis defect observed in mutant cells, we alsotested the influence of a drug (pentamidine), that specifically targets mitochondrialtRNAs preventing their proper folding In addition, since we propose the missfolding of the tRNAIle secondarystructure were also present in human cell lines carrying mutations intRNAIle anticodon loop, we took advantage of human transmitochondrialcell lines harboring the m.4290T>C pathological mutation To determine whether the observed changes in the tRNAIlemutant caused reduced levels of respiration when compared to control  showing a variable penetrance that remainsunexplained at the molecular level. In addition, a new mutation (m.4296G>A)corresponding to the one described here, has been found in humans associated with adegenerative encephalopathy .Thirteen mutations affecting Ile, at the same position that the human m.4296G>A, and alsoinduces a similar alternative folding of the tRNA to that promoted by the humanm.4290T>C. This may be possible despite being in a different relative position of thetRNA because both may generate the same new base pairing in the anticodon-loop of thetRNAIleIle is not aminoacylated, representing a defectivetRNA secondary/tertiary structure that cannot participate in protein synthesis and,therefore, reduces the amount of functional tRNA. Together with that, we haveestablished that both mutations, in humans and in mouse cells, cause only a moderatedefect in OXPHOS that is accompanied by accumulation of subcomplexes of the respiratorychain. Therefore, the mouse mutation seems to fully reproduce the molecular hallmarks ofa described human mtDNA mutation affecting a tRNA. We would like to stress that both thehuman and the mouse mutations could reach homoplasmy because the OXPHOS deficiency theypromote is moderate. Therefore, they represent an intriguing set of mitochondrial tRNAmutations with very variable penetrance than can cause no symptoms or, as it is the caseof the tRNAIle mutation, a devastating neurological disease in members of thesame family The mouse mutation described here (m.3739G>A) is also located at the anticodon loopof the tRNAHere we are proposing a model aimed to explain this behavior that we would like to call“functional epistasis model” . The conWe believe that it may be possible to assimilate the homoplasmic tRNA mutations causingdiseases together with their variable penetrance in humans with the rare intermediatevariants required to cross the low fitness- valleys in tRNA evolution as follows :2O2). As has been described for mouse mt-tRNAnon-pathological variants First, a mitochondrial tRNA (mt-tRNA) mutation occurs that destabilizes the functionalstructure of the tRNA making a second but non-functional folding similarly feasible. Ifthe mutation reaches homoplasmy, it substantially reduces the availability of functionaltRNA and can compromise mitochondrial protein synthesis fidelity. As a consequence, anunbalance in the assembly of respiratory complexes induces a compensatory response byincreasing mitochondrial biogenesis through a rise in the basal production of ROS(H0L929neo and ρ0L929puro)were generated by long-term growth of L929 mouse cell line in the presence of highconcentrations of Ethidium Bromide and transfection with the neocassette-containingplasmid pcDNA3.1 (Invitrogen) as previously described 0L929neo cells as described elsewheremt-Ti gene. Balb/cJp1 andmB77p18 cells were generated by TmBalb/cJ or mB77 cytoplasts transference toρ0L929puro cells. Transmitochondrial cell lines wereisolated by growing the cell population in DMEM supplemented with 5% dFBS and10 µg/ml of puromycin (SIGMA). When indicated, cells were cultured in thepresence of 5 mM NAC for a week or 1mM Tiron for 72 hours.All the cell lines were grown in DMEM (GibcoBRL) supplemented with 5% FBS. MtDNA-less mouse cells as previously described To confirm the presence of the mutation, RFLP analysis was achieved. See 4 cells on 12 wells testplates in 2 ml of the appropriate medium , incubating them at 37°C for 5days and performing cell counts at daily intervals.Growth capacity was determined by plating 5*102 consumption determinations in intact or in digitonine-permeabilizedcells were carried out in an oxytherm Clark-type electrode (Hansatech) as previouslydescribed OMitochondria were isolated as described previously Effect of different inhibitors on the viability of control and mutant cells wasevaluated using the MTT reduction assay according to Mosmann et al 3 cells per ml, in the case of human cells, and5*103 cells per ml when analyzing mouse cells. Then, cells werecultured for 3 days to be allowed to reach exponential growth rate before drugaddition. Afterward, cells were cultured in galactose containing medium withinhibitors for 48 hours  and from 7859 to 7927 in human samples (NC_012920)) andSdhA (from position 1026 to 1219 in mouse (AK049441) and fromposition 224 to 295 (AF171018) in human DNA). mtDNA copy number per cell wascalculated using SdhA amplification as a reference for nuclear DNAcontent as previously reported MtDNA quantification was performed by real-time PCR using an ABI PRISM 7000 SequenceDetector System (AB Applied Biosystems) and Platinum SYBR Green qPCR SuperMix-UDG(Invitrogen). Total cellular DNA was used as template and was amplified with specificoligodeoxynucleotides for 2-DA, Fluka).Then, cells were collected and the reaction was stopped in an ice-bath for 5 minutes.After that, cells were disrupted by treatment with Triton X-100 (2%) andcentrifuged at 2,500 g for 20 minutes at 4°C. The supernatant was used to measurefluorescence emission (excitation at 485 nm and emission at 535 nm) in a TECANSpectrafluor plus. The amount of hydrogen peroxide produced was then calculated usinga standard curve of 2,7-DCF in which 1 µM of 2,7-DCF represented 1 µM ofhydrogen peroxide.Production of hydrogen peroxide was measured in cultured cells grown in the absenceor in the presence of NAC for 1 week as previously described 35S]-methionine in intact cells as described elsewhere for 1hour Labeling of mtDNA-encoded proteins was performed with-dATP (Amersham). For the detection ofmt-tRNAIle and mt-tRNAGly, oligodeoxynucleotides specificfor each tRNA were used  from Roche with thespecific oligodeoxynucleotide tIle1: TATCAAAGTAATTCTTTTATC. The mt-tRNAIle fragment wasthen synthesized by PCR using the primers tIle1 and tIle2 (AGTAAATTATAGAGGTTCAAG) and cloned in the TACloning vector (Invitrogen).The 5′ and 3′ ends of the mt-tRNAIle, radiolabeled primer extension was performed. Thus, themt-tRNAIle fragment synthesized as described above was used as templateand the 5′ end 32P-labeled tIle-PE oligodeoxynucleotide was used asa primer: tIle-PE: TTCAAGCCCTCTTATTTCTA. Nucleotide concentrations were: 50 µMdCTP and 500 µM ddATP. For control purposes we used a syntheticmt-tRNAIle (Dharmacon). The radioactive signal was developed using thePersonal Molecular Imager system from BIO-RAD and analyzed with 1-D Analysis softwareQuantity One (BIO-RAD).To confirm the proper 3′-end processing and CCA addition in the mutanttRNAThe mtRNA fraction isolated under acid conditions was electrophoresed at 4°Cthrough a 10% polyacrylamide-0 to 8 M urea gel in 0.1 M sodium acetate(pH = 5) at 100–200 V and then electroblotted and themt-tRNAs charged and uncharged forms were identified by sequential hybridization withspecific probes as described above.  to a concentration of 10 µM each and 75 µCi of a3H-labeled aminoacid (Amersham). Incubation was carried out at 37°Cfor 15 minutes in a rotary shaker (12 rpm) and analysis of aminoacylation wasperformed as described above.For The differences between control and mutant cell lines for the various parametersanalyzed were assessed by analysis of variance (ANOVA). Paired haplotype differenceswere assessed by the post hoc Fisher's protected least significant differencetest (PLSD). All tests and calculations were done with the statistical packageStatView 5.0 for Macintosh .Figure S12O2 production by wild typeand mutant cells . C) Influence of N-acetyl cysteine (NAC) on theH2O2 production and mtDNA copy number in wild type andmutant cells  and n = 3 inall cell lines except in mB77 where n = 4 in mB77 for mtDNAcopy number (right)). D) Influence of NAC on the respiration activity ofpermeabilized cells with different substrates . All values are givenas mean ± SD of the mean. Asterisks indicate significant differencesrespect to each control, tested by ANOVA post-hoc Fisher PLSD (p<0.05).Analysis of the biogenetic response induced by the mutation. A) mtDNA copy numbervariation between wild type and mutant cells  B) H(0.15 MB DOC)Click here for additional data file.Figure S2Ile precursor processing. It has beenreported that some pathogenic mutations in mt-tRNAIle affect steps intRNA maturation including 3′-end processing and CCA addition Ile precursorprocessing, several cDNA clones derived from circularized mt-tRNAIlefrom wild type and mutant cell lines were sequenced Ile overlaps two nucleotides with the 3′ endof the mt-Nd1 gene and three nucleotides with the 5′ end ofthe gene encoding for the tRNAGln. We believe that RNAs derived fromthe processing of tRNAGln and ND1 mRNA explain the finding of thisproportion of circularized products with the lack of 3′ and 5′portions of the tRNAIle. In summary, since the major proportion ofmolecules showed a proper maturation of the 3′ and 5′ and CCAaddition, we conclude that no major defect in the processing of themt-tRNAIle can be attributed to the mutation.m.3739G>A mutation and mt-tRNA(0.20 MB DOC)Click here for additional data file.Table S1Primer sequences.(0.05 MB DOC)Click here for additional data file.Table S2Inhibitor concentration ranges.(0.05 MB DOC)Click here for additional data file.


Whitefly-transmitted geminiviruses (begomoviruses) are a major limiting factor for the production of numerous dicotyledonous crops throughout the world. Begomoviruses differ in the number of components that make up their genomes and association with satellites, and yet they cause strikingly similar phenotypes, such as leaf curling, chlorosis and stunted plant growth. MicroRNAs (miRNAs) are small endogenous RNAs that regulate plant growth and development. The study described here was aimed at investigating the effects of each virus encoded gene on the levels of developmental miRNAs to identify common trends between distinct begomoviruses.African cassava mosaic virus [ACMV], Cabbage leaf curl virus [CbLCuV], Tomato yellow leaf curl virus [TYLCV] and Cotton leaf curl virus/Cotton leaf curl betasatellite [CLCuV/CLCuMB]) were expressed from a Potato virus X (PVX) vector in Nicotiana benthamiana. Changes in the levels of ten miRNAs in response to the virus genes were determined by northern blotting using specific miRNA probes. For the monopartite begomoviruses (TYLCV and CLCuMV) the V2 gene product was identified as the major symptom determinant while for bipartite begomoviruses (ACMV and CbLCuV) more than one gene appears to contribute to symptoms and this is reflected in changes in miRNA levels. The phenotype induced by expression of the βC1 gene of the betasatellite CLCuMB was the most distinct and consisted of leaf curling, vein swelling, thick green veins and enations and the pattern of changes in miRNA levels was the most distinct.All genes encoded by four distinct begomoviruses (Our results have identified symptom determinants encoded by begomoviruses and show that developmental abnormalities caused by transient expression of begomovirus genes correlates with altered levels of developmental miRNAs. Additionally, all begomovirus genes were shown to modulate miRNA levels, the first time this has been shown to be the case. Plant virus infections may result in disease symptoms that can include chlorosis and/or necrosis, leaf curling, altered plant stature and morphology, presumably caused by interference with developmental processes . In recemiRNAs are endogenous, approx. 21nt RNAs that play important regulatory roles in animals and plants by targeting mRNA for cleavage or translational repression . They arGeminiviridae are divided into four genera based on insect vectors, host range and genome organization  the analysis has shown that the other (non-suppressor) proteins can affect miRNA levels, the first time this has been shown to be the case. Nevertheless, at least four of these proteins  have nucleic acid binding activities, of which only one (Rep) is sequence-specific. At the moment, there is no evidence to suggest that they can directly bind small RNAs such as miRNAs. Since these apparently do not have a direct effect on miRNAs, by binding them so as to remove them from the system and thereby up-regulate the genes they control, the implication is that their effects on miRNA levels is indirect, by affecting host factors that in turn affect miRNA levels. Consistent with the idea it has recently been shown that Oilseed rape mosaic virus infection up-regulates transcription from the miR164a promoter  (AJ496461), ACMV-[CM:DO3:98] , CbLCuV-[US:Flo:96]  and TYLCV-Mld[ES:72:97] (AF071228) were amplified with specific primers  at 28°C until the OD600 of the culture was 1. The cells were harvested by centrifugation at 3220 ×g for 8 minutes and resuspended in LB medium containing acetosyringone . For agroinoculation to N. benthamiana, plants at the 4 to 5 leaf stage were selected and were not watered for 24 hours before inoculation. Young leaves were infiltrated by gently appressing a 5 ml syringe to the abaxial surface and depressing the plunger until a water-soaked appearance was achieved.pGR107 and constructs were electroporated into N. benthamiana were grown at 28°C with 16 hours dark period/8 hours light period and 65% humidity in small 5 inch diameter plastic pots containing clay, silt, sand and compost in equal proportions. All plants were watered on daily basis and with Hoagland solution 2.4H2O, 0.5 mM KH2PO4, 1.25 mM KNO3, micronutrients  and Fe-EDTA  once a week if required. Samples were collected 12 days post inoculation (dpi) and photographed with a high resolution camera. Systemic young leaves of inoculated plants were collected, kept in plastic bags labelled with permanent marker, transported on ice and stored at -80°C until utilized.Arabidopsis miRNAs were end-labelled, as described earlier , the coat protein (CP), the (A)V2 protein [(A)V2], the nuclear shuttle protein (NSP) and the movement protein (MP) Additionally the gene encoding the hypothetical protein C5 (C5*) of Cotton leaf curl Kokhran virus and the green fluorescence protein (GFP) were expressed from the PVX. Shown below the northern blots in each case are the rRNA bands of the ethidium bromide-stained agarose gels that were used to normalize the data for loading.click here for filegfpfor cloning in the PVX vectorTable S1: Primers used to PCR amplify begomovirus genes and .click here for file


Recombinant chloroplasts are endowed with an astonishing capacity to accumulate foreign proteins. However, knowledge about the impact on resident proteins of such high levels of recombinant protein accumulation is lacking.Nicotiana tabacum) plastid transformants massively accumulating a p-hydroxyphenyl pyruvate dioxygenase (HPPD) or a green fluorescent protein (GFP). While under the conditions used no obvious modifications in plant phenotype could be observed, these proteins accumulated to even higher levels than ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), the most abundant protein on the planet. This accumulation occurred at the expense of a limited number of leaf proteins including Rubisco. In particular, enzymes involved in CO2 metabolism such as nuclear-encoded plastidial Calvin cycle enzymes and mitochondrial glycine decarboxylase were found to adjust their accumulation level to these novel physiological conditions.Here we used proteomics to characterize tobacco (The results document how protein synthetic capacity is limited in plant cells. They may provide new avenues to evaluate possible bottlenecks in recombinant protein technology and to maintain plant fitness in future studies aiming at producing recombinant proteins of interest through chloroplast transformation. The genetic modification of the plastid genome recently emerged as an alternative to nuclear transformation in fundamental research, for example to understand the interactions between the chloroplast and nuclear genomes, but also in applied research, as a system of transgene expression for high-added-value protein production. Chloroplast transformation Pseudomonas fluorescens p-hydroxyphenyl pyruvate dioxygenase (HPPD) or an Aequorea victoria GFP 2 metabolism, presumably to adapt the CO2 concentration within chloroplasts to the actual Rubisco concentration. The implications of these findings both in terms of biotechnological applications and physiological significance are discussed.Here, we have used proteomics to characterize transplastomic tobacco lines accumulating either a Pseudomonas fluorescens HPPD did not exhibit peculiar phenotypes under the growth conditions used despite the massive accumulation of the recombinant protein (Aequorea victoria GFP (data not shown), in agreement with previous results The transgenic plants over accumulating the  protein . Similarin vivo activities modulated, as the plant physiology is not strongly affected until the content of enzymes such as Rubisco, PGK or PRK reduces to 50% or less of wild type levels Among the 41 varying proteins identified in HPPD transformants photosynthesis proteins (54%) accounted for the major changes  and wereIn contrast to the decreased level in Rubisco subunits, Rubisco activase protein level was up regulated in HPPD transformants . This en2 necessary for Rubisco functioning has been well documented 2 fixation catalyzed by Rubisco Arabidopsis thaliana5,N10-methylene-tetrahydrofolate , a precursor of 5-CH2-THF involved in Met biosynthesis and the C1 metabolism Along with such variations, this proteomic analysis revealed a down-accumulation of three protein spots corresponding to subunits of glycine decarboxylase (GDC)  and S2, The present study showed an increased accumulation of several protein spots belonging to the categories “protein destination and storage” and “disease/defense/detoxification” as various chaperones and peroxidases  and S2. Aequorea victoria GFP  in seed tissues is to simultaneously reduce the accumulation of an abundant resident storage protein Oryza sativa) seeds over accumulating a human therapeutic protein reported a decrease in endogenous storage proteins In conclusion our work strongly suggests the existence of mechanisms limiting protein synthesis in plants. This finding is not without precedent. For example a recent work performed on the green alga Pseudomonas fluorescens p-hydroxyphenyl pyruvate dioxygenase (HPPD) or the Aequorea victoria GFP genes were as described rbcL and accD tobacco plastid genes. The HPPD coding region was placed under the control of the strong tobacco plastid promoter psbA, and the GFP coding region was placed under the control of the corn 16S rDNA plastid promoter. Plastid transformation was carried out according to the procedure described by Svab and Maliga in vitro leaves, measuring 3–5 cm, were bombarded with DNA-coated gold particles using a helium-driven particle influx generator gun −2.s−1).Homoplasmic transgenic tobacco lines (PBD6 cultivar) expressing the Germination assays were carried out on three replicates of 100 seeds and independent experiments for seeds from each recombinant tobacco. Seeds were incubated at 25°C, with 8-h light daily, on three sheets of absorbent paper  and a black membrane filter with a white grid  wetted with 1.3 mL of Millipore water in covered plastic boxes. A seed was regarded as germinated when the radicle protruded through the seed coat.g for 5 min. The final supernatant was recovered for analysis. Protein concentration was determined by the Bradford method Total soluble proteins were extracted from mature fully developed leaves ground in liquid nitrogen using an extraction buffer composed of 10% (w/v) trichloroacetic acid (TCA), 0.07% (v/v) 2-mercaptoethanol (2ME), in cold acetone, supplemented with protease inhibitor cocktail tablets  g, 10 min) at 4°C. The final supernatant corresponded to the total soluble protein extract.Total soluble protein extracts from dry mature seeds (150 mg) ground in liquid nitrogen were extracted at 2°C in 2.2 ml of thiourea/urea lysis buffer Proteins samples were analyzed by 2-DE as described http://www.matrixscience.com) or blasted manually against the current databases as described Protein spots were excised from 2D gels with sterile tips and submitted to in-gel digestion with trypsin . Extracted peptides were analyzed by tandem mass spectrometry on a nanoelectrospray ionization quadrupole time-of-flight hybrid mass spectrometer  coupled with a nano-HPLC . The peptide masses and sequences obtained were either matched automatically to proteins or EST in a non-redundant database  using the Mascot MS/MS Ions Search algorithm using an error tolerant search of all significant protein hits Click here for additional data file.Figure S2Influence of recombinant proteins accumulation on seed proteome and germination.(PDF)Click here for additional data file.Table S1List of the soluble varying proteins from tobacco leaf protein extracts identified by LC-MS/MS.(PDF)Click here for additional data file.Table S2Quantitative data for the normalized volumes of the unique varying spots.(PDF)Click here for additional data file.


Array findings were validated using qRT-PCR, immunohistochemistry, and extensive in silico analyses of independently performed microarray datasets. To our knowledge, this study provides the first comprehensive genomic analysis of breast cancer in Middle Eastern women in age-specific cohorts and potential markers for cancer progression in young women. Our data demonstrate that cancer appearing in young women contain distinct biological characteristics and deregulated signaling pathways. Moreover, our integrative genomic and cross-species analysis may provide robust biomarkers for the detection of disease progression in young women, and lead to more effective treatment strategies.Breast cancer in young women is more aggressive with a poorer prognosis and overall survival compared to older women diagnosed with the disease. Despite recent research, the underlying biology and molecular alterations that drive the aggressive nature of breast tumors associated with breast cancer in young women have yet to be elucidated. In this study, we performed transcriptomic profile and network analyses of breast tumors arising in Middle Eastern women to identify age-specific gene signatures. Moreover, we studied molecular alterations associated with cancer progression in young women using cross-species comparative genomics approach coupled with copy number alterations (CNA) associated with breast cancers from independent studies. We identified 63 genes specific to tumors in young women that showed alterations distinct from two age cohorts of older women. The network analyses revealed potential critical regulatory roles for Myc, PI3K/Akt, NF-κB, and IL-1 in disease characteristics of breast tumors arising in young women. Cross-species comparative genomics analysis of progression from pre-invasive ductal carcinoma  Breast cancer is the most common type of cancer among women worldwide with an estimated 1,300,000 new cases and 465,000 deaths annually in situ (DCIS), and culminates in invasive ductal carcinoma (IDC) Typically, the most common histologic type of breast cancer initiates as a premalignant lesion known as atypical ductal hyperplasia (ADH), then progresses into the preinvasive stage called ductal carcinoma A cross-species comparative genomics approach represents a powerful strategy to identify target genes that may play a role in tumor initiation and progression to malignancy and thus has great therapeutic potential There are areas of genomic instability reported in many cancers, including breast cancer, and some regions commonly exhibit either deletion or increased gene dosage, leading to changes in DNA copy number (CN) In this study, we analyzed whole-genome mRNA expression profiling from breast tumors and adjacent normal tissues from Middle Eastern women (n = 113 samples) in age-specific cohorts to characterize the underlying biology of aggressive breast cancers appearing in young women. Moreover, we performed an integrative and cross-species comparative genomics approach to identify evolutionarily conserved marker genes for disease progression in young women and validated its prognostic potential.in situ (DCIS). Breast cancer samples were collected from primary tumors of 76 patients who sought treatment and underwent surgery  at the King Faisal Specialist Hospital and Research Center. Signed informed consent was obtained from all patients. On excision of tissues by a surgeon, an anatomic pathologist obtained a sample of the tumor tissue and adjacent normal breast tissue from the same breast having the tumor. 113 samples were collected from patients and fully consented according to institutional review board approved protocols (KFSHRC IRB Protocol). The study was approved by the research ethics board at our institution (RAC# 2031091). Fresh surgical samples including tumors and adjacent disease free tissues were placed in RNAlater™  and stored at −20°C after micro dissection had been performed for pathological confirmation. All normal breast tissues were confirmed by the pathologist to have normal morphology before the results were analyzed. Whenever possible depending on the quantity of the surgical samples, a piece of every sample was also snap frozen in liquid nitrogen and then stored at −80°C for subsequent isolation of DNA and proteins. The majority of samples received no prior chemotherapy; only two had chemotherapy and were excluded from further analysis.In this study, we focused on breast cancer patients diagnosed with infiltrating ductal carcinoma (IDC) and ductal carcinoma Histological assessment of tumors and axillary lymph nodes were done by using formalin-fixed, paraffin-embedded breast cancer samples for HER2, estrogen receptor (ER), and progesterone receptor (PR) status. ER status was determined by immunohistochemistry and measured as a percentage and intensity of positive nuclear staining. The estrogen and progesterone receptors were stained with relevant specific antibodies . For HER2 immunohistochemistry, HercepTest™  was used with scores of 0 and 1+ considered negative and 2+ equivocal and 3+ considered positive.Cancers were categorized as luminal A (ER-positive and/or PR-positive and HER2- and either histologic grade 1 or 2); luminal B ; HER2 (ER-negative and PR-negative and HER2+); and triple negative  as defined previously Total RNA was extracted from tumor and adjacent normal tissue from patients with standard protocols. Sample handling, cDNA synthesis, cRNA labeling and synthesis, hybridization, washing, array  scanning, and all related quality controls were performed according to the manufacturer’s instructions. The Affymetrix GeneChip/GCOS software (Affymetrix Inc.) was used to calculate the raw expression value of each gene from the scanned image. The total RNA quality was assessed by the values of the 3′–5′ ratios for actin and glyceraldehyde- 3-phosphate dehydrogenase (GAPDH). DChip 2 test. The Fisher exact test was used when expected cell counts were less than 5 using the Monte Carlo method as implemented in SAS. A P-value of <0.05 was considered significant. Statistical analyses were performed by using SAS 9.2 , MATLAB (The MathWorks), and PARTEK Genomics Suite softwares. All microarray data reported here are MIAME compliant and have been submitted to the NCBI Gene Expression Omnibus (GEO) database (GSE29044), according to MIAME standards Global expression profiling of samples from tumor, IDC (n = 64) and DCIS (n = 7), and adjacent disease free tissues (n = 33) were probed using Affymetrix’s GeneChip® Human Genome U133 Plus 2.0 Arrays representing over 47,000 transcripts and variants using more than 54,000 probe sets. The open source R/Bioconductor packages,  http://www.ncbi.nlm.nih.gov/geo/). These were used to assess the prognostic potential of our gene signature , and datasets from GSE7390 www.canevolve.org/) were used for independent validation analyses. The Miller et al. For cross-species analysis, the murine markers of disease progression are taken from Kretschmer et al  assigned to that data set is explained by chance alone.Functional pathway, gene ontology and network analyses were executed using Ingenuity Pathways Analysis (IPA) 6.3 , a web-delivered application that enables the discovery, visualization, and exploration of molecular interaction networks in gene expression data. The differentially expressed gene lists were mapped to their corresponding gene objects in the Ingenuity pathway knowledge base. These so-called focus genes were then used as a starting point for generating biological networks. A score was assigned to each network in the dataset to estimate the relevance of the network to the uploaded gene list. This score reflects the negative logarithm of the P that indicates the likelihood of the focus genes in a network being found together due to random chance. Using a 99% confidence level, scores of ESR1, IL1RN, SEPP1, TIAM1, and SCD) were selected and primers designed using Primer3 software. After primer optimization, realtime PCR experiments were performed with 6 µl cDNA using Quantitech SyBr Green Kit (QIAGEN), employing GAPDH as the endogenous control gene. All reactions were conducted in triplicates and the data was analyzed using the delta delta CT method Confirmatory realtime RT-PCR experiments were performed using the ABI 7500 Sequence Detection System (Applied Biosystems). 50 ng total RNA procured from the same microarray study samples were transcribed into cDNA using a Sensicript Kit  under the following conditions: 25°C for 10 min, 42°C for 2 hrs, and 70°C for 15 min in a total volume of 20 µl. Five differentially expressed genes . Reaction was detected either by DAB  or by AEC . The sections were counterstained with Harris hematoxylin (Acros Organics). Staining was visualized using the DAKO Envision kit according to the instructions of the manufacturer .Validation of protein expression was done using immunohistochemistry. Immunohistochemical staining was performed using standard techniques. Monoclonal anti-TGF-α antibody , monoclonal anti PI3 kinase P85 alpha antibody  and polyclonal anti IL1 Receptor I antibody  were run manually. Slides were deparaffinized by routine techniques. Antigen retrieval was done in Tris/EDTA buffer, pH 9 heated at 95°C in a microwave for 25 minutes. After blocking endogenous peroxidase activity with a 3% aqueous HGenome-wide gene expression profiling provides a comprehensive view of the transcriptional changes that occur during the carcinogenic process and enables the understanding of biology beyond what may be apparent from studies assessing only clinicopathologic features. Here, we first analyzed the whole-genome mRNA expression profile from tumors (n = 71) and adjacent disease free tissues (n = 33) and compared tumor with the normal tissue in each age cohort, young women (≤45 years), 45 to 55 years (pre) and ≥55 years (elderly), separately. We identified 2632, 2029 and 2842 significantly dysregulated genes (up- or down-regulated) present in tumors from young, pre and elderly cohorts , respectively . To obtaWe next compared the transcriptomes of tumors across three age cohorts using a multi-factor ANOVA, controlling for ER, PR, HER2, and grade of the tumors (n = 67). The ANOVA identified 567 genes that were significantly modulated among three age groups (unadjusted p<0.01). The unsupervised principal component analysis (PCA) using 567 genes separated samples according to their age group, hence supporting the conclusion that there are distinct gene expression changes associated with tumors that are dependent on the age of the patient . We thenThe gene signature specific to tumors in young women (≤45 years) were obtained by overlapping gene lists. When comparing two groups of samples to identify genes differentially expressed in a given group, we used p-value and the fold change (FC) between two groups as the cut-off criteria. As shown in In Saudi Arabia, almost 50% of all the breast cancer patients were reported to be less than 45 years old. Accordingly, we performed additional analyses within the young women’s subset comparing transcriptomes of women younger than 35 years (very young) to two other age cohorts: 35 to 45 years and >45 years. We identified genes that were specific to tumors in very young women using the same methodology that was described previously. The heat map clearly shows significantly higher or lower expression of these genes in very young women compared to the two older age cohorts . The enrDuctal carcinoma in situ (DCIS) is heterogeneous group of pre-invasive tumors which may progress rapidly or slowly to invasive cancer. Therefore, an ability to identify which DCIS lesions are likely to progress to the potentially life threatening stage of invasive ductal carcinoma (IDC) would greatly help in the treatment plan and prognosis of the disease. To identify the putative genes involved in disease progression in young women, we performed genome-wide gene expression profiles characteristic of the sequential disease stages (DCIS and IDC) of breast cancer and compared them to age-matched normal controls in young women (≤45 years). We defined potential progression genes as genes that are significantly altered in both DCIS and IDC as these likely represent the earliest molecular steps in acquiring the capacity for invasion −5), Mitotic Roles of Polo-Like Kinase , ATM Signaling , Cyclins and cell cycle regulation , and Sonic hedgehog signaling . The network analysis illustrated activated pathways as well as interactions of genes that may potentially play a role in disease progression  that were unique to IDC and DCIS, respectively . Interesignaling . In contd MAP3K5 .https://tcga-data.nci.nih.gov/tcga/). This dataset is composed of samples from invasive breast carcinoma patients (n = 536) and matched normal controls (n = 63). Our cross-species conserved 16-progression gene signature was significantly up-regulated in patients compared to normal controls  and was sufficient to cluster and differentiate samples as tumor versus normal controls (data not shown).To validate our results, we used four independently performed microarray datasets as well as data available in the database developed by Gyorffy et al. ) were of significant clinical relevance in all the datasets tested, especially for identifying a high risk patient group , overall survival , and distant metastasis free survival  in datasets from Gyorffy et al. nt group .As a further validation of our results, we re-analyzed an independently performed microarray dataset from Miller et al The Microarray Literature-based Annotation (MILANO) database ) in very young (≤35 years) and/or young (≤45 years) women compared to older cohorts and validated the expression levels using qRT-PCR. A significant correlation existed between the microarray and realtime RT-PCR results, . In fact, 90% of young patients (n = 10) tested positive, which is in concordance with the microarray result. IL1RN was found to have higher expression in older cohorts compared to young patients in our microarray analysis, which was also validated by qRT-PCR . Indeed,Numerous studies have shown that younger women with breast cancer have a poorer prognosis and disease free survival compared to their older counterparts TGFAErbB2IL-1/IL-1RIL-1) promotes angiogenesis, tumor growth, and metastasis IL-1/IL-1R though autocrine or paracrine mechanisms can lead to a cascade of secondary tumorigenic cytokines, which can subsequently contribute to angiogenesis, tumor-cell proliferation and tumor invasion IL1RN were associated with shortened disease-free and overall survival among Caucasian women with breast cancer IL-1 expression has been shown to be an adverse prognostic factor TIAM1 and VANGL2 in very young women are significantly lower than in their older counterparts. The expression of TIAM1 has been shown to be associated with increased invasiveness and progression of breast carcinomas VANGL2 promotes migration of cells by a metalloproteinase-dependent invasion of extra cellular matrix and therefore influences invasion and perhaps metastasis We performed two different approaches to identify gene signatures for different age cohorts of women with breast cancer. In the first approach, we compared whole-genome mRNA expression profile from tumors and disease free normal tissues in three age cohorts of young women (≤45 years), 45 to 55 years (pre) and ≥55 years (elderly). The network analyses of significantly dysregulated genes revealed the activation of MYC CCNB2, UBE2C, TPX2, KIF4A, BIRC5, NUSAP1, and RRM2, using integrative and cross-species comparative genomics analysis. These genes are related to mitosis, cell cycle, embryonic development, DNA replication, cell division and proliferation. Our findings are consistent with previously performed independent studies of breast cancer progression Previous studies have shown that important driver mutations in various cancers can be identified using comparative genomic approaches et. al.’s study into two clusters that differed significantly in their TP53 mutation status. The cluster which had high expression of these genes comprised nearly of all the p53 mutant tumors. Previous studies have reported that TP53 mutations in breast cancer are associated with poor survival independent of other risk factors Testing our genes on independent microarray datasets using samples from over 3000 breast cancer patients demonstrated that high expression of these genes are significantly associated with poor outcome. Intriguingly, our 16-gene signature separated patients in Miller Differential expression analysis of DCIS and IDC in young women revealed significant down regulation of PI3K, DUSP6, CD22, RB, BCR, MS4A1 , and MAP3K5 as well as alterations in PI3K/Akt, NFkB, Jnk, and ERK pathways. The PI3K/Akt pathway is involved in regulation of cell proliferation and implicated in carcinogenesis In summary, to our knowledge this study provides the first comprehensive transcriptomic analysis of breast tumors that characterizes the underlying biological mechanisms in an age-specific manner in a cohort of Middle Eastern women, and coupled with an integrative cross-species comparative genomics approach has identified genes that could be potential biomarkers for tumor progression in young women. Our global expression profiling resulted in 63 genes that are specific to young women’s breast tumors. The network analyses illustrated the interaction of potential critical genes and the altered pathways associated with breast cancer that specifically appear in young women. The implication from these findings is that these genes may be contributing to the aggressive tumor behavior often present in these patients. Our results confirm previous studies as well as provide additional insights into young age (≤45 years) and very young age (≤35 years) specific oncogenic alterations that may be promoting tumorigenesis. Our cross species data analyses coupled with genomic copy number alterations may provide robust biomarkers for the detection of disease progression in young women and may lead to improved diagnosis and therapeutic options.Figure S1(A) Comparison of each age cohort, young women (≤45 years), 45 to 55 years (pre) and ≥55 years (post), with the age-matched normal controls. We identified 2632, 2029 and 2842 significantly dysregulated genes (up or down) due to tumor in young, pre and old cohorts respectively . (B) Gene interaction networks analysis of differentially expressed genes associated with tumor in each age cohort. Green/red indicates decreased/increased mRNA expression in patients compared to age-matched normal controls. The color intensity is correlated with fold change. Straight lines are for direct gene to gene interactions, dashed lines are for indirect ones (top scoring networks are shown).(DOCX)Click here for additional data file.Figure S2(A) Heatmap of very young-specific tumor genes across all tumor samples. Samples are denoted in columns and genes are denoted in rows. The heatmap clearly shows that those set of genes were significantly up- or down-regulated in tumor samples from very young women. The expression level of each gene across the samples is scaled to  interval. These mapped expression levels are depicted using a color scale as shown at the top of the figure, as such highly expressed genes are indicated in red, intermediate in black, and weakly expressed in green. (B) Validation of microarray data by realtime RT-PCR. Ratio of expression for each gene in Young (age 35 to 45) to very young (< = 35). Red bars represent microarray hybridizations, and, and blue bars represent values from qRT-PCR. (C) Gene interaction networks analysis of genes specific to very young women tumor. Green/red indicates decreased/increased mRNA expression in younger patients compared to older counterparts. The color intensity is correlated with fold change. Straight lines are for direct gene to gene interactions, dashed lines are for indirect ones.(DOCX)Click here for additional data file.Figure S3I. Comparison of the expression profile characteristics of IDC and DCIS. (a) 143 genes have significantly different levels of expression between DCIS compared to IDC. (b) Functional enrichment analysis of genes whose expression altered between DCIS and IDC. (c-d) The network analysis of 143 genes. Green/red indicates decreased/increased mRNA expression in IDC compared to normal controls. II. Network analyses of genes specific to DCIS or IDC in young women (A) Venn diagram illustrating 27 genes and 94 probes (corresponding to 72 genes) that are specific to IDC and DCIS, respectively. (B) Network analyses of genes specific to IDC. Green/red indicates decreased/increased mRNA expression in IDC compared to normal controls. (C) Network analyses of genes specific to DCIS (top two significant networks shown). Green/red indicates decreased/increased mRNA expression in DCIS compared to normal controls. The color intensity is correlated with fold change. Straight lines are for direct gene to gene interactions, dashed lines are for indirect ones. DCIS: ductal carcinoma in situ; IDC: invasive ductal carcinoma.(PDF)Click here for additional data file.Figure S4In Silico Independent Validation Analysis. (A) Re-analyzed dataset from Miller et al et. al.’s study into two clusters which differed significantly with the p53 mutation status. The cluster which had high expression of these genes comprised nearly of all the p53 mutant tumors. (B) GSE7390 BIRC5, TOP2A, NUSAP1, TPX2, and CCNB2 were of significant clinical relevance for identifying patients at high risk patients groups (result for RRM2 has been shown).(DOCX)Click here for additional data file.Table S1Gene signatures specific to malignant stage of invasive ductal carcinoma (IDC) and pre-invasive ductal carcinoma in situ (DCIS) in young women.(DOCX)Click here for additional data file.


Peripherin/retinal degeneration slow (rds) is an integral membrane protein specifically localized to the light-sensing organelle of the photoreceptor cell, the outer segment. Within the outer segment, peripherin is found at the edges of photoreceptor discs, where it plays a critical role in disc morphogenesis and maintenance. Peripherin loss or mutations are often associated with severe forms of visual impairments. Like all other resident outer segment proteins, peripherin is synthesized in the photoreceptor cell body and subsequently transported to the outer segment. In an effort to further examine peripherin’s delivery to outer segments, we undertook a careful examination of its targeting sequence. Using a fluorescently labeled reporter expressed in the rods of transgenic tadpoles, we narrowed peripherin’s targeting sequence to ten amino acids within its C-terminal tail. This small stretch of amino acid residues is both necessary and sufficient for outer segment targeting. We also conducted alanine scanning of all residues within this sequence and found that only a single residue, valine at position 332, is essential for outer segment targeting. This valine is conserved in all species and its mutation is sufficient to completely abrogate the targeting of full-length peripherin in mouse rods. Vision is initiated in the retina where light is captured by the outer segment organelle of photoreceptor cells. The outer segment is a modified primary cilium that contains large quantities of proteins involved in visual signal transduction. Similar to all cilia, the outer segment lacks the machinery required to synthesize proteins and therefore relies on the import of proteins produced in the cell body of photoreceptor cells. The importance of accurate protein targeting to the outer segment is highlighted by observations that defects in protein targeting result in retinal degenerative diseases Membrane proteins destined for the outer segment are synthesized in the endoplasmic reticulum, transported through the Golgi, and then sorted at the trans-Golgi network into transport vesicles specifically directed to the outer segment. The fidelity of sorting is guided by targeting signals, which are short stretches of amino acid residues encoding protein localization information rds mice, in which the peripherin gene is severely truncated, essentially making them a peripherin knockout http://www.retina-international.org/files/sci-news/rdsmut.htm).The second known targeting sequence resides within the C-terminus of peripherin/retinal degeneration slow  Xenopus rod outer segments Unlike rhodopsin, it is unclear how peripherin is delivered to the outer segment. One of the first studies to examine this question showed that peripherin accumulates in intracellular vesicles while rhodopsin accumulates in the plasma membrane of photoreceptors in detached cat retinas The essential requirement for peripherin in outer segment morphogenesis prompted us to further characterize its outer segment targeting. Targeting signals are often 4–7 amino acids long, with only 2–3 residues being critical to the specific targeting of the protein Xenopus rhodopsin C‐terminus lacking the VXPX targeting motif (YFP‐xRhoCTΔ5). This construct retains two palmitoylated cysteine residues critical for membrane attachment, but lacks specific outer segment targeting information. When expressed in Xenopus under the rhodopsin promoter, the majority of the reporter localizes to the outer segment, but a distinct portion spills into the photoreceptor plasma membrane domain  resulted in loss of exclusive targeting to outer segments Previous study of peripherin’s C-terminus found that deletions affecting a twenty amino acid stretch  approved by the Institutional Animal Care and Use Committees of Duke University. Adult Xenopus YFP fusion constructs and their point mutants, overhang extension PCR primers were used (primer sequences available upon request). The FLAG tag was appended to full length mouse peripherin’s N-terminus by introducing the sequence into the forward primer. To make the V332A point mutation in mouse peripherin, we utilized the QuikChange II XL kit (Stratagene). All forward and reverse primers were designed to introduce 5′-AgeI and 3′-NotI sites respectively. PCR products were subcloned into either the XOP5.5 vector Xenopus transgenesis or pRho2.2 in vivo electroportation. DNA templates were obtained as follows: IMAGE clones corresponding to Xenopus peripherin (xRDS38), Xenopus rhodopsin, and mouse peripherin were purchased from American Type Culture Collection; YFP was cloned from pDV-NTAP-NYFP obtained from the The Dicty Stock Center; HTR1A was obtained from a mouse brain cDNA library (Stratagene). All constructs were confirmed by direct sequencing.DNA constructs were generated using standard PCR-based subcloning methods. To generate all Xenopus tadpoles were generated using the restriction enzyme-mediated integration method Xenopus sperm nuclei in the presence of the restriction enzyme XhoI to promote integration. Transgenic sperm nuclei were then injected into Xenopus oocytes, and the resulting embryos were allowed to develop until stage 45–54 when tadpoles were sacrificed by immersion in 0.2% tricaine. A minimum of four positive transgenic animals were analyzed for each DNA construct.Transgenic 2 inhalation followed by decapitation. A minimum of three positively expressing mice retinas were analyzed for each DNA construct.Retinal transfection of neonatal mice was performed as described by Matsuda and Cepko µm cross-sections through the central retina. Sections were collected in 24-well plates, blocked in 5% goat serum in the presence of 0.5% Triton X-100, incubated overnight with monoclonal mouse anti-FLAG M2 (Sigma-Aldrich) diluted in blocker, and stained with goat anti-mouse Alexa Fluor 488 secondary antibody (Invitrogen) and 10 µg/ml Hoechst 33342 (Invitrogen) to label nuclei. To examine the eyes of transgenic tadpoles, the entire animal was fixed in 4% paraformaldehyde, cryoprotected in 30% sucrose, and frozen in 100% tissue-freezing medium. 12µm cross-sections through the eye were collected directly onto glass slides and stained with 2 µg/ml Hoechst to label the nuclei.Posterior eyecups from mouse eyes were obtained by microdissection, fixed in 4% paraformaldehyde for 1 hour, rinsed three times in PBS, and embedded in 4.0% agarose (Invitrogen). A vibratome (Leica VT1200S) was used to collect 100All samples were mounted with Fluoromount (Electron Microscopy Sciences) under glass coverslips and visualized using a Nikon Eclipse 90i microscope and a C1 confocal scanner controlled by EZ-C1 v 3.10 software (Nikon). Manipulation of images was limited to adjusting the brightness level, image size, and cropping using either EZ-C1 v 3.10 Viewer or Photoshop (Adobe).


EPAS1 and EGLN1, were identified in multiple studies. By applying the same analytic methods to genome-wide SNP information used in our previous study of a Tibetan population (n = 31) from the township of Maduo, located in the northeastern corner of the Qinghai-Tibetan Plateau (4200 m), we have identified common targets of natural selection in a second geographically and linguistically distinct Tibetan population (n = 46) in the Tuo Tuo River township (4500 m). Our analyses provide evidence for natural selection based on iHS and XP-EHH signals in both populations at the p<0.02 significance level for EPAS1, EGLN1, HMOX2, and CYP17A1 and for PKLR, HFE, and HBB and HBG2, which have also been reported in other studies. We highlight differences  in the two distinct Tibetan groups examined here and report selection candidate genes common to both groups. These findings should be considered in the prioritization of selection candidate genes in future genetic studies in Tibet.Recent studies have used a variety of analytical methods to identify genes targeted by selection in high-altitude populations located throughout the Tibetan Plateau. Despite differences in analytic strategies and sample location, hypoxia-related genes, including  EPAS1 gene, which encodes the hypoxia inducible factor (HIF)-2α subunit, and the EGLN1gene, which encodes a proline hydroxylase, PHD2, that regulates hypoxia-induced factors in an oxygen-dependent manner. Variation in the EPAS1 region is associated with hemoglobin concentration ([Hb]) in Tibetan populations examined in two separate studies EGLN1 and PPARA  are associated with [Hb] in Tibetans from the Maduo township in the northeast section of the Qinghai-Tibetan Plateau EPAS1 and PPARA haplotypes in Tibetans from the Tuo Tuo River examined here are associated with elevated serum lactate and free fatty acid concentrations, respectively, providing further support for important biological roles Native highlanders have specific traits that enable their survival despite the stress imposed by decreased oxygen availability at altitude. Several regions of the genome, including those that harbor hypoxia-sensing and -regulated genes, were recently identified as putative targets for high-altitude adaptation in Tibetans Tibetans inhabit a vast area of the Qinghai-Tibetan Plateau, which spans approximately 1.5 million square km (0.96 million square miles). At least three major Tibetan dialects are spoken among these geographically distinct groups  compared to other Asian populations , which are commonly used for genetic comparisons  in a high-altitude population from India and are associated with EGLN1 expression and high-altitude pulmonary edema (HAPE) in this population 2 = 1.0) with the putatively adaptive selected haplotypes identified in both of the Tibetan populations examined here, providing additional evidence for an adaptive role of EGLN1 across the Qinghai-Tibetan Plateau. Interestingly, EGLN1 was identified as a target of selection in Andean highlanders, although it is unclear whether the putatively adaptive variants are the same as those reported in Tibetan highlanders EGLN1Highly differentiated SNPs in the EPAS1EGLN1PPARAHMOX2 gene, a heme oxygenase involved in HIF-independent hypoxia sensing, was a top selection candidate in our Maduo and Tuo Tuo River populations and was also reported as a selection candidate in a pooled sample of 50 Tibetan samples collected from various regions of the Qinghai-Tibetan Plateau PKLR and HBB/HBG2, respectively identified in the Tuo Tuo River and Maduo Tibetan populations at the p<0.02 level, were also reported as top selection candidates in an independent examination of genetic variation in 50 Tibetan exomes CYP17A1 gene, contained within a top selection candidate region identified in both Maduo and Tuo Tuo River populations, are associated with hypertension in European and Asian populations While PPARA gene, highlighting regulatory variation as a potential factor underlying adaptive advantages within Tibetan genomes.Considering the lack of differentiation detected through analysis of the protein-coding regions of Han Chinese and Tibetan samples Differences among studies published to date could also result from variation in the genetic background of Tibetan groups. Such factors could influence the extent to which selection signals are captured (the early stages of a selection sweep versus those that are fixed or nearly fixed in the population) or the potential for selection events to occur in different groups. In order to better understand differences in Tibetan adaptation to altitude, it will be necessary to fully characterize population histories in various Tibetan groups, determine the precise functional variants and timing of selection events, and evaluate additional phenotypic differences related to adaptive functional variants.Selection and association signals identified in Tibetan populations may be influenced by the demographic history of these groups and by the choice of analytic methods. Despite genetic heterogeneity between Tibetan groups as shown here, several putatively adaptive genetic variants are common to Maduo and Tuo Tuo River Tibetans. Further genetic and phenotypic characterizations of physiological traits  are required to determine if the remaining selection targets highlighted here are of biological significance, whether they are related to or independent of [Hb], and how combinations of such factors influence the overall physiology of native highlanders living in the Tibetan Plateau.We extracted DNA from whole blood samples obtained from 85 highland natives  residing in the Tuo Tuo River township in Qinghai Province .Participants provided written agreement as indicated by a signature or mark on a sheet of paper that described the study in their native language. Consent was obtained for all participants, and this study was approved by the Institutional Review Board at the High Altitude Medical Research Institute .We employed the Affymetrix 6.0 SNP Array technology  to genotype 70 DNA samples at Capital Bio Corporation . Default parameters for the Birdseed algorithm (version 2) were used to determine genotypes for all samples . Genotypic data were analyzed using the Affymetrix Genotyping Console 3.1 (Affymetrix) and included all autosomes but excluded the X and Y chromosomes and the mitochondrial genome.http://jorde-lab.genetics.utah.edu/ under published data.We attempted to exclude all first-degree relatives who visited the clinic from our study. We used the ERSA software package We performed principal components analysis on genetic distances as previously reported We had previously generated a list of genes likely related to high-altitude adaptation based on Gene Ontology and Panther Pathway categories as described in ADMIXTURE2 = 1.0), we first filtered out SNPs that had r2>0.2 within 100 kb using PLINK ADMIXTURE.A model-based algorithm implemented in We performed the iHS We focused on the selection candidate genes contained within 200 kb regions significant at the p<0.02 level in either test. We further excluded regions where the iHS test was significant at this level in neighboring populations as previously described Hemoglobin concentration, hematocrit, and percent oxygen saturation were determined from venous blood samples using the Mindray Hematology Analyzer  and the Pulse Oximeter , respectively.We identified the putatively advantageous haplotypes as previously described Figure S1The relationships between EPAS1, EGLN1, and PPARA haplotypes and [Hb] for Tuo Tuo River Tibetans (shown in open circles) and Maduo Tibetans (closed circles). The number of PPARA haplotype copies, previously associated with [Hb] in Maduo Tibetans , is associated with [Hb] when data from both populations are combined (p<0.02).(TIF)Click here for additional data file.Figure S2Statistical power to detect an association of [Hb] with a haplotype. Simulated data sets were constructed with varying sample size (n = 30–500), assuming that the putatively selected haplotype at one locus decreases [Hb] by e g/dl when present in two copies and e/2 if present in one copy . [Hb] was simulated as a normally-distributed variable with mean 19.6 and standard deviation of 1.6 g/dl, as observed in the Tuo Tuo River sample, and the effect of adaptive haplotype copies was added to that variate for each individual. The frequency f of the adaptive haplotype was set at 0.65, 0.75 or 0.85, per the legend. To mirror the actual tests performed, haplotypes with no effect on [Hb] were simulated for two additional loci (haplotype frequency of 0.65 for both). Genotypes were assigned in Hardy-Weinberg equilibrium. Ages were assigned from a normal distribution, mean 37 years and standard deviation 11.5, then truncated to the range of 18–68, mirroring the observed distribution. Sex was assigned randomly with a 50/50 ratio. Multiple stepwise linear regression was performed using the five simulated predictors: age, sex and haplotype copies at three loci . Considering our modest sample size, it will be necessary to collect more data from the Tuo Tuo River population to achieve greater power to detect genotype-phenotype associations.(TIF)Click here for additional data file.Table S1ST for Tibetans and neighboring Asian populations examined.F(DOCX)Click here for additional data file.Table S2Intersection of selection candidate regions and hypoxia-related miRNAs.(DOCX)Click here for additional data file.Table S3Multiple stepwise linear regression including age, sex, and three haplotypes previously identified as selection candidates in Tibetans  in Tuo Tuo River Tibetans. Gender was the only significant (p<0.01) predictor in this analysis .(DOCX)Click here for additional data file.


Because of the scarceness and absence of hectic painful radiculopathic feature of L1-L2 disc, surgeons don’t usually pay enough clinical attention to them. Their main manifestation is severe axial pain or myeloradiculopathy failure attributed to conus or high cauda equine syndrome.During 2006 to 2011, a total of 11 patients with L1 L2 dysfunctions underwent a surgery. Their mean age was 56 years old and sex ratio of 6 male to 7 female. 4 of them had florid neurological deficit as paraparesis and sphincter dysfunction. In other patients, radiculopathies and axial pain were dominant clinical manifestations.Considering the conus position and the extent of canal compromise and disc consistency  either anterior or posterior surgical approach followed by segmental fusions was used.3 patients underwent thoracoabdominal and fusion surgery, while ten underwent a posterior transfacet pedicle sparing surgery combined with transforaminal lumbar interbody fusion (TLIF) surgery. In all patients, satisfactory outcomes were achieved; all radiculopathy and signs of conus dysfunction were improved except one case that showed sphincter dysfunction persistent post operation. Axial pain was resolved and in follow-up assessments no kyphosis or axial pain were observed. Due to the biomechanical traits of L1-L2 disc and its adjacency to thoracolumbar region as well as high degree of osteodiscal degeneration on its manifestation we propose TLIF and Posterior spinal fusion (PSF) surgical approaches after canal decompression to alleviate axial pain and also prevent later kyphosis. Furthermore, because of nearby conus and the risk of thecal sac retraction, the same strategy as thoracic disc was performed in this region considering reverence for conus if good results are to be achieved.Surgical strategy, L1-L2 disc, Thoracoabdominal, Transforaminal lumbar interbody fusion


Diseases have traditionally been defined predominantly by clinical criteria, sometimes with the assistance of “conventional” laboratory data. Recent advances in genomic technology have begun to find previously undiscovered sub-divisions within what were thought to be single diagnoses, sometimes with significant clinical implications. This talk will discuss the potential for such technologies to redefine the classification and treatment of autoimmune diseases, using two recent examples from Cambridge.PRTN3) was found to be a central etiological feature of PR3-ANCA associated vasculitis. Thus PR3- and MPO-ANCA-associated vasculitis must be considered as distinct autoimmune syndromes [The first is the use of a Genome Wide Association Study to determine the genetic underpinning of ANCA-associated Vasculitis (AAV). This study has confirmed a genetic component to AAV pathogenesis, and demonstrated that the AAV clinical syndromes granulomatosis with polyangiitis (GPA: formerly Wegener’s) and microscopic polyangiitis (MPA) are genetically distinct diseases better defined by ANCA specificity for proteinase-3 (PR3) or myeloperoxidase (MPO) rather than clinical syndrome. In addition the response against the autoantigen PR3 . These otherwise “invisible” groups have markedly different long term outcomes ,3. This


Photoreceptor degeneration is the most critical cause of visual impairment in age-related macular degeneration (AMD). In neovascular form of AMD, severe photoreceptor loss develops with subretinal hemorrhage due to choroidal neovascularization (CNV), growth of abnormal blood vessels from choroidal circulation. However, the detailed mechanisms of this process remain elusive. Here we demonstrate that neovascular AMD with subretinal hemorrhage accompanies a significant increase in extracellular ATP, and that extracellular ATP initiates neurodegenerative processes through specific ligation of Purinergic receptor P2X, ligand-gated ion channel, 7 . Increased extracellular ATP levels were found in the vitreous samples of AMD patients with subretinal hemorrhage compared to control vitreous samples. Extravascular blood induced a massive release of ATP and photoreceptor cell apoptosis in co-culture with primary retinal cells. Photoreceptor cell apoptosis accompanied mitochondrial apoptotic pathways, namely activation of caspase-9 and translocation of apoptosis-inducing factor (AIF) from mitochondria to nuclei, as well as TUNEL-detectable DNA fragmentation. These hallmarks of photoreceptor cell apoptosis were prevented by brilliant blue G (BBG), a selective P2RX7 antagonist, which is an approved adjuvant in ocular surgery. Finally, in a mouse model of subretinal hemorrhage, photoreceptor cells degenerated through BBG-inhibitable apoptosis, suggesting that ligation of P2RX7 by extracellular ATP may accelerate photoreceptor cell apoptosis in AMD with subretinal hemorrhage. Our results indicate a novel mechanism that could involve neuronal cell death not only in AMD but also in hemorrhagic disorders in the CNS and encourage the potential application of BBG as a neuroprotective therapy. Age-related macular degeneration (AMD) is the leading cause of irreversible vision loss in the elderly in the developed world The most crucial stage of severe visual impairment is photoreceptor loss due to development of the CNV and related events such as subretinal hemorrhage or exudative retinal detachment in neovascular AMD c that triggers the Apaf-1 (apoptotic protease activating factor 1) apoptosome activation. MOMP also results in the mitochondrial release of apoptosis-inducing factor (AIF), which translocates to the nucleus and participates in the caspase-independent peripheral chromatin condensation and large-scale DNA fragmentation Photoreceptor degeneration involves the activation of several signaling pathways of regulated cell death that can constitute potential therapeutic targets. Accordingly, attempts have been made to inhibit caspases, which play major roles in the apoptotic machinery An alternative strategy to inhibit the mitochondrial apoptotic pathway is to intercept the initiating upstream proapoptotic signals. Recently, adenosine-5′-triphosphate (ATP) has been discovered as a major extracellular messenger that can contribute to lethal signaling Hence, we hypothesized that ATP acting on P2RX7 is involved in the pathogenesis of photoreceptor loss in subretinal hemorrhage. To examine the clinical relevance of this hypothesis, we compared extracellular ATP concentrations in human vitreous samples from patients with retinal diseases, including AMD. Our results showed that increased extracellular ATP substantially contributes to photoreceptor loss and that BBG provides substantial neuroprotective effects in cases of subretinal hemorrhage.Procedures using human samples were conducted in accordance with the Declaration of Helsinki and approved by the Kyushu University Institutional Review Board for Clinical Research. We obtained written informed consent from all the participants. All procedures using mice were performed in strict accordance with the guidelines for Association for Research in Vision and Ophthalmology (ARVO) and Proper Conduct of Animal Experiments (Science Council of Japan). The experimental protocol was approved by the Animal Care and Use Committee of Kyushu University. All efforts were made to minimize the number of animals used and their suffering.−/− mice, the latter kindly provided by Pfizer Inc. , were used in this study. Recently, the rd8  mutation was reported to be widely inherited across strains supplied by common commercial vendors and ES cell lines −/− mice do not carry the rd8 mutation by DNA sequencing (Adult (8 weeks of age) male C57BL6JJcl mice  and isogenic P2rx7quencing . All pron = 10), epiretinal membrane , or age-related macular degeneration (AMD) with vitreous hemorrhage (VH) (n = 15). The clinical characteristics of the patients are summarized in Procedures using human samples were conducted in accordance with the Declaration of Helsinki and approved by the Kyushu University Institutional Review Board for Clinical Research. We obtained written informed consent from all the participants. Vitreous samples (1.0–1.2 ml) were collected from patients who underwent pars plana vitrectomy at the Vitreoretinal Center in Kyushu University Hospital and Kagoshima University Hospital, for the treatment of macular hole  were immediately determined by using luciferin-luciferase reaction buffer  and a Flex Station 3 Multi-Mode Microplate Reader . The ATP levels detected by the luminometer were expressed in relative light units (RLU).Mice were anesthetized with an intraperitoneal injection of pentobarbital and exsanguinated via cardiac puncture into collection tubes containing heparin. For the isolation of plasma, collected whole blood was immediately centrifuged at 18000 g for 60 seconds at 4°C to isolate plasma as previously described Adult primary retinal cell cultures were prepared as previously described with minor modifications + or CMTMRos+ photoreceptor cells were counted in 10 random fields by blinded observers using ImageJ software. Values are given as the means ± SDs of 10 replicate wells.To assess the viability of primary retinal cells, we used calcein AM (2 µM) or MitoTracker Orange CMTMRos , added for 30 min to primary retinal cell cultures. Then, cultured cells were fixed with 4% paraformaldehyde and recoverin-labeled calceinExperimental retinal detachment was performed as previously described TUNEL analysis and quantification of TUNEL-positive cells were performed as previously described using the ApopTag Fluorescein In Situ Apoptosis Detection Kit (Millipore) The posterior segments of enucleated mouse eyes were fixed in PBS containing 1% glutaraldehyde and 1% paraformaldehyde, postfixed in veronal acetate buffer osmium tetroxide (2%), dehydrated in ethanol and water, and embedded in Epon. Ultrathin sections were cut from blocks and mounted on copper grids. The specimens were observed with an H-7770 transmission electron microscope .Immunofluorescence was performed with slight modifications to a previous method P<0.05 (*) and P<0.01 (**). All values were expressed as the means ± SDs.Statistical differences between the two groups were analyzed by means of the Mann–Whitney U-test. Multiple group comparison was performed by ANOVA followed by Tukey–Kramer adjustments. The sex ratio in each group was analyzed by Fisher’s exact test. Differences were considered significant at + photoreceptor cells were detected in the outer nuclear layer (ONL) 24 h after the injections of autologous blood, while quite a small number of apoptotic photoreceptor cells were detected in control retinal detachment within 24 h . There is minimal breakdown of the blood retinal barrier (BRB)–i.e., exudation or bleeding–in MH or ERM. In contrast, vitreous hemorrhage–that is, diffusion of a large amount of blood into the vitreous cavity from the subretinal hemorrhage–often occurs in AMD, and in some of these cases vitrectomy is performed for removal of the vitreous opacity To identify potential neurotoxic factors released from the extravascular blood clots, we prepared mouse primary retinal cells in double chamber slides. Primary mouse retinal cell cultures were prepared as previously described + or CMTMRos+ photoreceptor cells, while BBG reversed the decline in live photoreceptor cells . Subretinal injections without ATP induced only limited TUNEL-positive apoptotic events in the ONL after 24 h of injections  mice  mice , indicatin vitro and in vivo experiments led us to investigate whether the pharmacological P2RX7 antagonist would have a beneficial effect on retinal damage in a more pathological setting–namely, an animal model of subretinal hemorrhage. Autologous blood was injected into the subretinal space alone or with a mixture of BBG (50 µM). Twenty-four hours after experimental subretinal hemorrhage, abundant TUNEL+ photoreceptor cells were detected in the ONL with AIF translocation to the nucleus and caspase-9 cleavage  and subsequent DNA sequencing demonstrated that the genotype of P2rx7−/− mice is the wild type allele (n = 6). Ocular fundus examination also confirmed that P2rx7−/− mice have no phenotype of retinal degeneration, namely drusen-like lesions, which are found in mice carrying the rd8 mutation Genotyping of the rd8 mutation by DNA sequencing in P2rx7(PDF)Click here for additional data file.Supporting Information S2Characteristics and clinical data in patients with vitreous hemorrhage due to AMD. About 2,000 AMD patients are referred to our Vitreoretinal Center every year. Patients undergo ophthalmologic examinations, including visual acuity testing with standardized refraction using decimal charts and slit-lamp biomicroscopy. Decimal fractions of visual acuity were converted to the logarithm of the minimal angle of resolution (LogMAR) according to previous reports (TIF)Click here for additional data file.Supporting Information S3The height of retinal detachment by injections of sodium hyaluronate, sodium hyaluronate with a mixture of ATP, and autologous blood. The height of retinal detachment was measured as the length between the photoreceptor outer segment and RPE in cryosections. The mean lengths in the three groups were not significantly different.(TIF)Click here for additional data file.


We investigated the prevalence of dengue in patients with suspected viral meningitis/meningoencephalitis in a dengue-endemic area. Cerebrospinal fluid analysis showed positive results and a 6.74× greater likelihood of identifying positive fluid in patients who died. Our findings support testing patients with neurologic manifestations for the virus in dengue-endemic areas. Dengue is the most prevalent arboviral infection in humans .We enrolled 183 patients with suspected viral meningitis/meningoencephalitis admitted to São José Hospital of Infectious Disease and 26 deceased patients with suspected fatal meningitis who had been sent to the city of Fortaleza Coroner’s Office. Cerebrospinal fluid (CSF) was collected from all 209 patients. Study inclusion criteria were suspicion of viral meningitis/meningoencephalitis, a CSF cell count <500 cells/mmDengue meningitis was suspected when a patient had fever and symptoms of irritation of the meninges, such as headache and neck stiffness; a diagnosis of dengue meningoencephalitis was established when the patient showed signs of focal involvement of the central nervous system (CNS). A diagnosis of dengue was confirmed with a DENV-positive CSF result by reverse transcription PCR (RT-PCR), nonstructural protein (NS) 1, or IgM against DENV  was used to detect the dengue NS1 in 209 CSF specimens in accordance with the manufacturers’ instructions  showed positive results in s Office . RevieweDENV as a causal agent for meningitis has been rarely reported, although some cases have been described in the literature. In Jamaica, a study of 401 patients with suspected cases of viral infection of the CNS showed that 54 (13.5%) were positive for dengue; 18 (33.3%) of those patients showed clinical signs of meningitis (An investigation of dengue patients with suspected CNS infection conducted in Vietnam found 4.2% (16/378) of persons positive for DENV; 1 (0.3%) patient had meningitis  has been associated with virus replication and viremia with the risk for development of DHF . DetectiThe prevalence of CNS involvement in patients with dengue infection seems to vary with severity of dengue cases . The relThe high risk for death among patients with dengue meningitis/meningoencephalitis in this study supports the need for increased surveillance. Dengue should be suspected in patients with neurologic manifestations in dengue-endemic areas, and appropriate treatment should be given to prevent death.


Circulating endothelial cells and microparticles have prognostic value in cancer, and might be predictors of response to chemotherapy and antiangiogenic treatments. We have investigated the prognostic value of circulating endothelial cells and microparticles in patients treated for advanced non-small cell lung cancer.Peripheral blood samples were obtained from 60 patients before first line, platinum-based chemotherapy +/− bevacizumab, and after the third cycle of treatment. Blood samples from 60 healthy volunteers were also obtained as controls. Circulating endothelial cells were measured by an immunomagnetic technique and immunofluorescence microscopy. Phosphatidylserine-positive microparticles were evaluated by flow cytometry. Microparticle-mediated procoagulant activity was measured by the endogen thrombin generation assay. Results: pre- and posttreatment levels of markers were higher in patients than in controls (p<0.0001). Elevated levels of microparticles were associated with longer survival. Elevated pretreatment levels of circulating endothelial cells were associated with shorter survival.Circulating levels of microparticles and circulating endothelial cells correlate with prognosis, and could be useful as prognostic markers in patients with advanced non-small cell lung cancer. Lung cancer is one of the malignant tumors with the highest incidence worldwide. Non-small-cell lung cancer (NSCLC) represents more than 80% of lung cancer cases, and most of them have advanced or metastatic disease at diagnosis Platinum-based chemotherapy is the standard treatment for advanced lung cancer. Several platinum doublets are currently used with similar efficacy, though different toxicity profiles Circulating endothelial cells (CECs) are markers of vascular damage Microparticles (MPs) are small vesicles (100 nm–1 µm) which directly bud from the plasma membrane The aim of this study was to assess CECs, MPs and MP-mediated procoagulant activity in patients with advanced NSCLC, before and after standard chemotherapy, to analyze their prognostic value and to establish their possible association with tumor response.This prospective study included consecutive patients with NSCLC treated at the Medical Oncology Department of La Fe University Hospital with first-line chemotherapy of platinum-based doublets. Bevacizumab was added to treatment when indicated. To be included in the study, patients were required to meet the following criteria: age >18 years; stage IIIB or stage IV histologically confirmed NSCLC; life expectancy ≥12 weeks; ECOG Performance Status (PS) ≤2 and adequate renal, hepatic, and bone marrow function.Staging was determined by chest and abdominal computed tomography (CT) and body proton emission tomography (PET) scanning. Three weeks after the third cycle of treatment, CT-based clinical assessment was carried out using the Response Evaluation Criteria for Solid Tumors (RECIST) Blood samples were obtained by cubital non-traumatic vein puncture, and collected into Vacutainer ™ plastic tubes (BD Diagnostic) with two different anticoagulants: K3 EDTA (4.3 mg) for CECs, and sodium citrate (129 mM) at a ratio of 1∶9  for MPs and ETG. Samples were kept at 4°C and processed between 2 and 4 hours after collection. Citrated blood samples were centrifuged at 1,500 xg for 30 minutes at 4°C to obtain platelet-free plasma, and then stored at −80°C to allow later batch analysis.2 (pH 7.4). Samples were then incubated for 60 minutes at 4°C in darkness with 1 µL of FITC-Annexin V . The acquisition of events was quantified in an EPICS XL- cytometer (Beckman Coulter) at high flow rate for 2 minutes during which 325 µL of this suspension were analyzed. Standard fluorescent beads of different diameters were used for size calibration  and to set the gate of MP detection at a diameter from 0.5 to 1 µm, following a consensus guideline on MP measurement Plasma samples containing MP suspensions were analyzed by flow cytometry. Annexin V-FITC conjugates were used to detect accessible phosphatidylserine on MP membranes. Ten µL of frozen plasma samples were diluted in 500 µL of HEPES buffer containing 2.5 mM CaClETG assays were carried out to assess the in vitro thrombin generating capacity of plasma by using the calibrated automated thrombogram . Eighty µL of frozen plasma samples were dispensed into 96-well round-bottom microtiter plates . Each sample requires two wells. Twenty µL of standard thrombin calibrator  were added to one well, and 20 µL of fluorogenic thrombin substrate with calcium (TS50.00 Fluka Kit Thrombinoscope BV) were added to both wells to start ETG with continuous comparison to the thrombin calibrator reagent. Thrombin activity was measured as fluorescence in a Fluoroskan Ascent reader fluorimeter (Thermo Labsystems) equipped with a 390/460 filter set. No exogenous tissue factor or phospholipids were added. Thrombin generation curves were calculated using the Thrombinoscope software and results were expressed as thrombin peak (nM) (height of the curve).The isolation and quantification of CECs were performed in whole blood by an immunomagnetic technique, following a consensus protocol Results are given as mean values ± standard deviations (SD) for continuous variables, and as percentages for categorical variables. Kolmogorov-Smirnov test was used to evaluate whether each parameter followed a normal distribution. Analysis of variance (ANOVA) with the Bonferroni post hoc analysis was used to evaluate marker level differences between sample types . Bivariate correlation was performed using the Spearmańs correlation test. OS and PFS were analyzed by means of the Kaplan-Meyer method, and survival curves of subgroups were compared using the log-rank test. All statistical analyses were performed with the SPSS computer software, version 15.0 for Windows . Probability values <0.05 were regarded as statistically significant.Between January 2009 and September 2010, sixty patients and sixty healthy subjects were included. Levels of CECs, MPs and ETG in patients and controls are shown in The correlations between levels of circulating markers showed that MP levels positively correlated with ETG levels, both before  and after treatment .th percentile in controls) were associated with better PFS  and OS . No such association was found for CECs or ETG (th percentile in patients) were associated with a worse OS   .The results of the current study show that the CEC, MP, and ETG levels are elevated in patients with NSCLC in comparison with healthy controls. Chemotherapy did not change the levels of these markers. We have also found an association between high MP levels, both before and after treatment, and survival. Moreover, we have observed an association between pretreatment levels of CECs and survival.Our results show an association between high pre- and posttreatment levels of MPs and longer survival. To our best knowledge, this is the first study showing such an association in patients with NSCLC. There is growing interest in the characterization of MPs and in their potential role as biomarkers in different diseases A strong correlation was observed between MP levels and ETG. This finding is in accordance with previous data, indicating that the presence of MPs in plasma significantly affects ETG in both normal We observed an association between high pretreatment levels of CECs and poor survival. There is no conclusive data on the prognostic value of CECs in cancer. Most studies agree that high levels of CECs are associated with a poor prognosis Our findings provide new data on the prognostic value of MPs and CECs in patients with NSCLC. The role of MPs and CECs as potential biomarkers in patients with NSCLC, treated with conventional chemotherapy or targeted therapies, warrant further study, since a better understanding of their role can open new perspectives in the fields of diagnosis and treatment.


Serological markers of cardiac injury are elevated following intense exertion. Whether myocardial tissue is actually injured remains unresolved, although we have found evidence for systolic and diastolic dysfunction following exertion. Furthermore, we have also observed that a significant proportion of well-trained elite endurance athletes present with evidence of myocardial fibrosis at rest.Myocardial edema reflects an acute reversible injury that may be associated with reduced ventricular compliance. Accordingly, the purpose of the current investigation was to determine whether myocardial edema develops acutely in active men challenged with an incremental test to exhaustion and a high-intensity interval exercise training session, as visualized by T2-weighted imaging.16 physically active men (age 33±11 years) were recruited for a 2-day study. LV function and T2-weighted imaging were performed on a 1.5T MRI system, prior to and within an hour of exertion. During study day 1, participants completed an incremental test to exhaustion on a braked cycle ergometer, where load was increased by 15-25 Watts every 2 minutes until physical exhaustion. The point of exhaustion was considered to be the maximal power output. During study day 2, a high-intensity interval training session was performed where athletes completed 15 repetitions of cycling at maximal power output for 1 minute followed by 2 minutes of light recovery.The extent of myocardial edema was assessed quantitatively using semi-automated detection, where myocardial regions that had signal intensity (SI) above a threshold of twice the mean SI of skeletal muscle were considered to be edematous. Global edema was measured by normalizing myocardial SI to skeletal muscle SI, generating a T2-ratio.The ratio of global myocardial edema increased following both incremental exertion to exhaustion  and intense interval training .The spatial extent of myocardial edema also increased following both incremental exhaustion challenge  and high-intensity interval training  (Figure We provide first evidence for the development of myocardial edema following an acute incremental challenge to exhaustion and high-intensity interval exertion in healthy men. The extent of edema may be related to diastolic dysfunction. These findings have implications for the safety of sport for athletic and non-athletic populations.