Fancl is a member of the Fanconi Anemia/BRCA DNA repair pathway. Experiments showed that zebrafish fancl was expressed in developing germ cells in bipotential gonads at the critical time of sexual fate determination. Caspase-3 immunoassays revealed increased germ cell apoptosis in fancl mutants that compromised oocyte survival. In the absence of oocytes surviving through meiosis, somatic cells of mutant gonads did not maintain expression of the ovary gene cyp19a1a and did not down-regulate expression of the early testis gene amh; consequently, gonads masculinized and became testes. Remarkably, results showed that the introduction of a tp53 (p53) mutation into fancl mutants rescued the sex-reversal phenotype by reducing germ cell apoptosis and, thus, allowed fancl mutants to become fertile females. Our results show that Fancl function is not essential for spermatogonia and oogonia to become sperm or mature oocytes, but instead suggest that Fancl function is involved in the survival of developing oocytes through meiosis. This work reveals that Tp53-mediated germ cell apoptosis induces sex reversal after the mutation of a DNA–repair pathway gene by compromising the survival of oocytes and suggests the existence of an oocyte-derived signal that biases gonad fate towards the female developmental pathway and thereby controls zebrafish sex determination.The molecular genetic mechanisms of sex determination are not known for most vertebrates, including zebrafish. We identified a mutation in the zebrafish  fancl gene that causes zebrafish to develop exclusively as male due to female-to-male sex reversal. Fancl is a member of the Fanconi Anemia/BRCA pathway involved in the repair of damaged DNA. We find that the sex-reversal phenotype is caused by an abnormal increase of programmed germ cell death during the critical period for zebrafish sex determination in which oocytes progress through meiosis. This abnormal increase in germ cell death compromises oocyte survival, gonadal somatic cells do not maintain the female gene expression profile, gonads become masculinized to testes, and mutants develop into fertile males. Remarkably, we show that the introduction of a mutated allele of the tp53 (p53) tumor suppressor gene into fancl mutants rescues the sex-reversal phenotype by reducing germ cell death. We conclude that Tp53-mediated germ cell death alters gonad fate selection in fancl mutants by compromising oocyte survival, possibly by eliminating a hypothesized oocyte-derived signal, which alters sex determination in zebrafish.Zebrafish has become an important model for understanding vertebrate development and human disease, yet the genetic mechanisms that regulate gonad fate to determine zebrafish sex remain elusive. In this work, we describe a mutation in the  The existence of two differentiated sexes is common among animals and yet the mechanisms that determine sex are amazingly diverse. Among vertebrates, for instance, some species use primarily genetic factors and others rely on environmental factors to cause embryonic gonads to become testes or ovaries. Genetic sex determination (GSD) includes monogenic as well as polygenic systems, and in monogenic systems the sex-determining gene is usually found on sex chromosomes that evolved from a pair of autosomes after acquiring a novel sex-determining allele (reviewed in SRY (Sex determining region Y) is at the top of the sex determination cascade SRY however, does not appear to exist beyond therian mammals Dmrt1 (doublesex and mab-3 related transcription factor 1) is a downstream gene in the male sex-determination pathway, but in medaka (Oryzias latipes), a duplicated copy of dmrt1  is the major sex-determining gene dmrt1 is required for testis development in chickens dmrt1by is absent in most Oryzias species Despite the vast diversity of primary sex-determining mechanisms, genes downstream in the sex determination pathway appear to be broadly conserved among vertebrates. It has been suggested that during evolution, different species recruited different downstream genes to be the major sex-determining gene, sometimes relatively recently, and that changes at the top of the sex-determining pathway appear to be better tolerated than changes at the bottom of the pathway because they are less likely to have deleterious effects Teleost fish show a broad diversity of sex determining mechanisms that range from genetic to environmental, from monogenic to polygenic, and from hermaphroditism to gonochorism (two distinct sexes) fancl zebrafish mutant that develops exclusively as male. Fanconi Anemia complementation group L , along with 12 other Fanconi Anemia proteins, facilitates cellular responses to a variety of stresses, including signals of DNA damage and apoptosis FANCL causes a clinical phenotype that includes hematopoietic and skeletal abnormalities that are similar to, or more severe than, those typically observed in patients suffering from a defect upstream in the Fanconi Anemia pathway . We previously identified the zebrafish ortholog of the human FANCL gene fancl homozygous mutants develop solely as males and that the absence of fancl mutant females is not due to female-specific lethality but to female-to-male sex reversal. Results demonstrated that the sex reversal of fancl mutants is not due to the absence of germ cells, but to an abnormal increase of germ cell apoptosis that compromises survival of developing oocytes and masculinizes the gonads. We found that reducing germ cell apoptosis by introducing a Tp53 (p53 or tumor protein p53) mutation rescues the fancl sex reversal phenotype, and that many double mutants develop ovaries and become females. These results suggest the model that oocytes normally must progress through meiosis to signal the gonadal soma to maintain female development, and point to Tp53-mediated apoptosis of germ cells as a factor that could be targeted by environmental or genetic signals to modify zebrafish sex determination.To broaden our knowledge of the genetic mechanisms involved in zebrafish sex determination, we studied a fancl mutant  was generated by insertional mutagenesis in a Tol2 transposon-mediated enhancer trap screen fancl, thereby disrupting the coding region of the PHD finger domain  PCR-product corresponding to the intact PHD domain found in wild-type siblings , and after genotyping the progeny by PCR, observed that all  HG10A/HG10Afancl homozygous mutants  developed exclusively as males, even though their wild-type and heterozygous siblings developed about as many females as males. Two alternative hypotheses could explain the lack of homozygous fancl mutant females: female-specific lethality or female-to-male sex reversal. To distinguish between these two hypotheses, we crossed female fancl+/− heterozygotes to male fancl−/− homozygotes. We raised 211 progeny to adulthood, determined their phenotypic sex according to sexually dimorphic characters including the color of the anal fin and body shape, and finally scored their fancl genotypes by PCR. Under normal conditions, this cross should give 50% heterozygotes , and 50% homozygous mutants , expecting a 1∶1∶1∶1 ratio of heterozygous females to heterozygous males to homozygous mutant females to homozygous mutant males. The fancl female death hypothesis predicts a 1∶1∶0∶1 ratio, or 66% heterozygotes and 33% homozygous mutants, but the sex reversal hypothesis, predicts a 1∶1∶0∶2 ratio, or equal proportions (50%∶50%) of homozygous mutants  and heterozygotes . Resulting genotypes revealed 46 fancl+/− females: 62 fancl+/− males: 0 fancl−/− females: 103 fancl−/− males, which showed that about half of the progeny were fancl homozygous mutants  and the other half were heterozygous for the fancl mutation  , and while approximately half of fancl heterozygous siblings developed as males , the other half developed as females  . To exclude the possibility that some of the fancl mutants could have ovaries despite their external male phenotypic characters, we dissected the gonads of adult fancl homozygous mutants (n = 45), heterozygous females (n = 11) and heterozygous males (n = 29). In all cases, we found a perfect match between external sexual characters and gonadal sex. These results ruled out the possibility that fancl mutants masqueraded as males externally while having female gonads. We conclude that the HG10A Tol2 insertion into fancl induced a female-to-male sex reversal phenotype in zebrafish.To characterize the 11, 51%) . These r46, 43%) . These sfancl could play a role in zebrafish germ cell development. To address this question, we first tested whether fancl is expressed in germ cells of wild-type zebrafish. We analyzed the expression pattern of fancl by in situ hybridization on sections of gonads at seven developmental time points encompassing representative stages of gonad development (fancl expression in undifferentiated wild-type gonads at 10 dpf (data not shown), but weak expression signal appeared in immature gonads at 17 dpf and 23 dpf , suggesting that fancl is part of the maternal load of messenger RNA transcripts stored in the egg and passed along to embryos. This result agrees with our detection of fancl transcripts by RT-PCR and in situ hybridization experiments even at early developmental stages before the embryonic transcription machinery becomes active fancl expression appeared in spermatocytes  and was up-regulated in early stages of gametogenesis is consistent with the hypothesis that Fancl plays a specific role in germ cell development and suggests that its disruption might lead to the female-to-male sex reversal phenotype displayed by fancl mutants.The finding that dead end (dnd) morpholino (MO) knockdown fancl mutants are fertile, we wondered if the female-to-male sex reversal of fancl mutants could be related to extremely low numbers of germ cells during stages of sex determination in juvenile mutants, or at least in those that otherwise would have developed as females and had been reversed to males. To answer this question, we performed gene expression analyses comparing gonads of fancl homozygous mutants (fancl), wild-type sibling controls (WT) and dnd-MO knockdown animals (dnd) at key stages in sex determination: 19 dpf (vasafancl mutants sectioned (n = 15) (dnd-MO (n = 16) lacked vasa signal  , and 4H'(n = 15) , while aa signal . The prefancl mutants developed as males, we wondered if fancl mutants embark upon the male pathway from the beginning of gonad development, or whether they follow a normal bipotential pathway of development that later derails exclusively to the male pathway. To address these alternatives, we used the expression of cyp19a1a (cytochrome P450 family 19 subfamily A polypeptide 1a) and amh (anti-Mullerian hormone), which are the earliest sex-specific somatic gonadal cell markers known for ovary and testis, respectively, to monitor development before gonads were sexually differentiated at the morphological level Because all fancl mutants, as well as those of wild-type controls and dnd-MO animals, expressed both the female marker cyp19a1a and the male marker amh  lacked expression of cyp19a1a and showed up-regulation of amh  showed clear testes morphology, including the absence of cyp19a1a expression and up-regulation of amh expression  as stage IB oocytes that have not yet decondensed their chromosomes, and “late” perinucleolar oocytes (lpo) as stage IB oocytes that have already formed lampbrush chromosomes and entered the diplotene stage of meiosis I. Gonads of fancl  and WT siblings  at 19–22 dpf both had early  lacked oocytes at late stage IB (fancl mutants failed to progress through meiosis to the diplotene stage. Only one of the twelve fancl mutants showed late stage IB oocytes (lpo in fancl mutants that lacked oocytes (11 of 12) also had numerous pyknotic cells  is an important activator of apoptosis fancl mutants by introducing a tp53 mutation into the fancl mutant line. To generate double mutants, we crossed a zebrafish female carrier of the hypomorphic mutation tp53M214Kfancl mutant, identified double heterozygotes  among F1 progeny by PCR, and in-crossed double heterozygotes to obtain an F2 population containing double homozygous mutants. Among the F2 raised to adulthood, 44/171 (25.7%), or about a quarter, were −/−fancl homozygous mutants. Among these 44 −/−fancl homozygous mutants, 15 were also −/−tp53 homozygous mutants, from which 11 developed as females and four as males (fancl homozygous mutant siblings (n = 29) that were either homozygous wild type for +/+tp53 (n = 8) or heterozygous for the +/−tp53 mutation (n = 21) developed exclusively as males  is linked to the tp53 genotype. Histological analyses of −/−;tp53−/−fancl females corroborated the conclusion that external female sex characteristics were accompanied by ovaries filled with normal oocytes at all stages of development similar to +/+fancl; +/+tp53 wild-type female siblings  or wild-type for the +/+tp53 mutation (n = 5) at 25 dpf, a critical stage for sex determination  had an average number of apoptotic germ cells approximately three fold lower  fancl in zebrafish causes homozygous mutants to develop exclusively as males due to female-to-male sex reversal rather than female-specific lethality. This is the first demonstration, to our knowledge, that a mutation in a Fanconi gene can cause female-to-male sex reversal.Fancl protein helps mediate cellular responses to a variety of stresses, especially DNA damage and apoptosis fancl in germ cells during zebrafish gonad differentiation, which is consistent with a role of Fancl in germ cell development. Other species, such as mouse, also express fancl in their germ cells dead end, nanos, ziwi, or zilifancl mutation, which rules out the possibility that male development in fancl mutants that otherwise would have become females is due to lack of germ cells. Work presented here shows specifically that the mere presence of germ cells is insufficient to feminize gonads, but rather, it suggests that oocytes passing through meiosis are essential to support differentiation of ovaries. Our results are in agreement with previous suggestions that zili mutants all become phenotypic males probably due to the lack of oocytes at week 4 during the window of sex determination rather than due to the total loss of germ cells at week 8 fancl mutants, in which germ cells are always present, provide a useful tool to better understand the role of germ cell-soma signaling that tips gonad fate towards the male pathway.Our work revealed expression of fancl mutants, WT controls and dnd-MO animals, which lack germ cells, reveals that the onset of expression of the female marker cyp19a1a and the early male marker amh in individual undifferentiated gonads at 19 dpf is similar in all genotypes. This result supports the conclusion that the onset of early somatic makers is independent of germ cell signaling fancl mutants initially develop as normal bipotential “juvenile ovaries” containing oocytes at early stage IB with no obvious histological differences from gonads of WT controls.Comparison of sex-specific gonadal markers among fancl mutant gonads become morphologically different from wild-type gonads. Wild-type animals have perinucleolar oocytes that progress through meiosis from early stage IB to late stage IB with obvious lampbrush chromosomes, indicating that recombination is complete and oocytes are at the diplotene stage of meiosis I, in which homologous chromosomes begin to separate but remain attached at chiasmata fancl mutants lack late stage IB oocytes, indicating that oocytes fail to progress beyond pachytene stage, when recombination occurs, and do not enter diplotene. Our results show that the levels of fancl transcripts are regulated during the process of gametogenesis because fancl expression up-regulates in oocytes transitioning from early to late stage IB , however, stage IB . Consistfancl mutants correlates with the observation that most mutant gonads do not express the female somatic marker cyp19a1a, but instead up-regulate the male somatic marker amh. Interestingly, we found a few fancl mutants with some late stage IB oocytes accompanied by expression of cyp19a1a, but also showing high expression levels of amh; we interpret these animals as females whose progress towards ovary development was being derailed due to the mutation of fancl. These results would be expected if oocytes are essential to maintain cyp19a1a expression.The failure of oocytes to progress through meiosis in fancl mutants, the absence of oocytes progressing through meiosis alters oocyte signaling to the soma that maintains the female program. Without this signal, somatic cells do not maintain the expression of cyp19a1a, do not suppress amh expression, and as a result, gonads do not become ovaries but instead become masculinized and form testes. It is likely that this signal arising from meiotic oocytes is essential for somatic pre-granulosa cyp19a1a-expressing cells to proliferate and to differentiate as mature granulosa cells. In mammals, it has been suggested that meiotic oocytes reinforce ovarian fate by antagonizing the testis pathway fancl mutant gonads, our results are consistent with the hypothesis that fancl mutants, which lack oocytes at the diplotene stage of meiosis, can not prevent the trans-differentiation of pre-granulosa cyp19a1a-expressing cells into Sertoli-like amh-expressing cells. This hypothesized mechanism could explain the disappearance of cyp19a1a-expressing cells and the maintenance and proliferation of amh-expressing cells in fancl mutant gonads that results in gonad masculinization. Future transcription profiling analyses comparing wild-type animals and fancl mutants lacking oocytes will help to identify genes involved in oocyte-soma signaling essential for ovary development.We hypothesize that in juvenile fancl mutants during the time-window of sex determination (25 dpf) is accompanied by an abnormal increase of Caspase-3-mediated apoptosis of germ cells compared to wild-type siblings. This result suggests the hypothesis that the disappearance of meiotic oocytes in fancl mutants is due to an increase in germ cell apoptosis, which provides a cellular mechanism for the female-to-male sex reversal phenotype of fancl mutants. To test this hypothesis, we suppressed cell death in fancl mutants by making them homozygous for a tp53 mutation. We show that the reduction of apoptosis in −/−;tp53fancl−/− double mutants is sufficient to promote the survival of developing oocytes and to rescue the female-to-male sex reversal phenotype of fancl mutants. Our result showing that only −/−;tp53fancl−/− double mutants developed any females, while their −/−;tp53fancl+/− and −/−;tp53fancl+/+ sibling controls developed exclusively as males, indicates that the amount of germ cell apoptosis alters sex determination in fancl mutants.We observed that the loss of oocytes in fancl mutation. Doubly homozygous −/−;tp53−/−fancl rescued females were fertile and developed normal ovaries full of oocytes maturing through all stages of oogenesis. Active Caspase-3 results show that the amount of germ cell apoptosis is lower in double homozygous −/−;tp53−/−fancl individuals than in their fancl−/−;tp53+/+ mutant sibling controls, which further supports the hypothesis that the abnormal increase of apoptosis in fancl mutants that compromises the survival of meiotic oocytes is the mechanism responsible for the female-to-male sex reversal.The double mutant experiments further show that Tp53 activity mediates increased apoptosis associated with the tp53M214K mutant line. This allele, however, is hypomorphic, and may possess levels of apoptosis compatible with the male pathway. This conclusion is supported by our finding that a few −/−;tp53−/−fancl double mutants developed as males. An alternative explanation is that mechanisms of apoptosis independent of Tp53 might occur in male gonads that promote oocytes to disappear in developing testes.We did not notice a sex ratio biased towards females in the fancl zebrafish mutants is consistent with the increase of apoptosis in a variety of cell types reported in Fanconi Anemia knockout mice. For instance, Fanca−/−, Fancc−/−, and Fancg−/− knockout mice show increased apoptosis of hematopoietic or neuronal cells, which might lead to a progressive loss of stem and progenitor cells −/−Fanca knockout mice also show increased male germ cell apoptosis Fancl−/− knockout mice, in contrast to fancl mutant zebrafish, do not show sex reversal but initially develop as sterile males and sterile females. Fancl−/− knockout male mice – but significantly, not Fancl−/− knockout female mice – can recover fertility and become fertile adult males. These results suggest that Fancl is necessary for germ cell proliferation in mouse embryos and for the maturation of oocytes, but not for the proliferation or maturation of spermatogonia in adulthood fancl mutant males are fertile and that −/−;tp53−/−fancl rescued females are also fertile indicates that Fancl function is not essential for the maturation of zebrafish spermatogonia and oogonia to become sperm or mature oocytes, but rather that Fancl function affects specifically germ cell survival.Our finding of increased germ cell apoptosis in fancl mutants suggests that Fancl function is involved in the survival of developing germ cells through meiosis, and that when Fancl is mutated, developing oocytes cannot survive due to an inappropriate increase of Tp53-dependent germ cell apoptosis. This idea is consistent with the fact that genetic deletion of Tp53 can rescue the TNF-alpha dependent apoptosis caused by accumulation of the pro-apoptotic protein kinase PKR resulting from a mutation of the human FANCC gene fancl mutants might identify compounds of therapeutic importance for Fanconi Anemia patients.The loss of oocytes progressing through meiosis in fancl mutants suggests a model in which oocyte survival regulated by Tp53-mediated apoptosis is a central element that can tip gonad fate towards the male or the female pathway (gradient red box in cyp19a1a) and male (amh) specific markers .fancl mutation  was generated by insertional mutagenesis by Tol2 transposon-mediated enhancer trap tp53 mutant line zdf1tp53 causing the amino acid substitution M214K was obtained from ZIRC (http://zebrafish.org/zirc/home/guide.php) tp53 animals was performed as described http://zfin.org/zf_info/nomen.html).The zebrafish fancl cDNA was previously described fancl wild-type or mutant alleles were: WT_F:CTGGTCTTTATTGACTGTAATGGC; WT_R:TAGATAAGCTCCAGATTTGGCTTG; Mutant_F:GTCAGCCCATCCAGATCAGCAG; Mutant_R:CATGACGTCACTTCCAAAGGACC. PCR conditions were: 5′94°C; 32 cycles of: 30″94°C, 30″55°C, 1′72°C; followed by 10′72°C. Sizes of PCR-amplified bands: Wild type: 479 bp; Mutant: 370 bp.The full-length zebrafish GACGGCTTCATCACAGTGCTG; R1:CATGACGTCACTTCCAAAGGACC; F2:GAACCCTGACTGCACTGTCCTAC; R2:GCTTTGGCGACTGGTTGGCAGAC. PCR conditions were: F1-R1: 3′94°C; 40 cycles of: 30″94°C, 30″58°C, 1′30″72°C; followed by 10′72°C; F2-R2: 3′94°C; 37 cycles of: 20″94°C, 30″60°C, 45″72°C; followed by 10′72°C. Sizes of PCR-amplified bands: F1-R1: 1239 bp F2-R2: 232 bp.Total RNA isolation from dissected adult testes and cDNA synthesis were performed as described dead end as described dnd MO-injected embryos were fixed at 24 hours post-fertilization to confirm the presence or absence of germ cells by whole-mount in situ hybridization using vasa probe as described To obtain animals lacking germ cells, wild-type zebrafish embryos from the AB strain were injected at the 1–2 cell stage with antisense morpholino oligonucleotide  directed against amh and cyp19a1a were made as described vasa was made from its 3′end as described fancl cDNA fragment of 786 nt containing the PHD domain (nucleotides 646-1431 of AY968598) was cloned in TOPO vector (Invitrogen) and used to synthesize DIG-labeled riboprobe (Boehringer Mannheim). For gonad histology, euthanized animals were fixed in Bouin's fixative for about 24–48 hours and washed repeatedly in 70% ethanol. Animals were dehydrated and embedded in paraffin, sectioned at 7 microns, and stained with hematoxylin and eosin.Animals were reared and collected under standard conditions fancl and wild-type animals was scored in 840 sections: 70 sections containing gonads per each animal (n = 12).Animals were fixed at 25 dpf in 4% PFA ON at 4°C, dehydrated, embedded in paraffin, and sectioned at 7 microns. Apoptotic cells were detected by immuno-fluorescence using anti-active Caspase-3 as primary antibody  and Alexa-Fluor594 goat anti-rabbit as secondary antibody  following an immuno-histochemical protocol . Gonads were screened for positive signal by DIC-fluorescence microscopy. The number of positive cells in gonads of


Knowledge of the entire protein content, the proteome, of normal human cerebrospinal fluid (CSF) would enable insights into neurologic and psychiatric disorders. Until now technologic hurdles and access to true normal samples hindered attaining this goal.We applied immunoaffinity separation and high sensitivity and resolution liquid chromatography-mass spectrometry to examine CSF from healthy normal individuals. 2630 proteins in CSF from normal subjects were identified, of which 56% were CSF-specific, not found in the much larger set of 3654 proteins we have identified in plasma. We also examined CSF from groups of subjects previously examined by others as surrogates for normals where neurologic symptoms warranted a lumbar puncture but where clinical laboratory were reported as normal. We found statistically significant differences between their CSF proteins and our non-neurological normals. We also examined CSF from 10 volunteer subjects who had lumbar punctures at least 4 weeks apart and found that there was little variability in CSF proteins in an individual as compared to subject to subject.Our results represent the most comprehensive characterization of true normal CSF to date. This normal CSF proteome establishes a comparative standard and basis for investigations into a variety of diseases with neurological and psychiatric features. Knowledge of the entire protein content, the proteome, of normal human cerebrospinal fluid (CSF) would provide a critical standard to allow meaningful comparisons with and between neurologic and psychiatric disorders. CSF contains both cellular and soluble components providing insights into processes occurring in the central nervous system (CNS). As much as 30 to 40% of CSF is formed by the extracellular fluid of the brain and spinal cord. CSF contains both normal and disease specific components, and provides an accessible liquid window into the brain. In fact, recent data suggest CSF may provide more relevant evidence for initial or propagating pathology than the brain parenchyma itself in certain neuropsychiatric diseasesUntil recently, technological limitations have prevented full characterization of the CSF proteome. Comprehensive analysis of CSF has been challenged by low protein levels (0.3 to 0.7 mg/ml) compared to plasma, protein concentration variability up to twelve orders of magnitude, potential masking of brain-specific proteins by highly abundant proteinsTowards our principal purpose of establishing a comprehensive list or proteome of normal CSF, we have prepared CSF samples from healthy normal people for analysis by using immunoaffinity depletion of abundant proteins  to enhance coverage and detection of low abundance proteins3 and protein levels ranged from 14 to 40 mg/dl with a median of 25 mg/dl). We also examined pairs of individual serial CSF samples, obtained at least 4 weeks apart, from 10 additional normal healthy volunteers to assess the potential variability of particular CSF protein levels in an individual from one time point to another.We examined pooled CSF from 11 normal healthy volunteers  who reported their health as excellent or good and were taking no medications. Standard clinical laboratory testing on their CSF was normal . Four sets of different types of normal CSF samples were analyzed. The first set, designated as true  normals was comprised of pooled CSF from 11 healthy normal individual volunteers from the United States  was used for the comprehensive analysis using immunoaffinity depletion and 2D-LC-MS/MS. A second set, also true normals included pairs of serial CSF aliquots taken at least 4 weeks apart from 10 healthy volunteers from the United States . A third set, designated as non-neurologic surrogate-normals, was a pool of 200 subjects from Sweden ). Ages ranged from 16 to 65 years with a median of 44 years; 50∶50 female:male. They were used in the direct LC-MS analysis using the AMT tag approach. These samples were collected on ice and cells removed by centrifugationAll specimens had normal clinical laboratory values with respect to microbiology, chemistry (including protein levels), and cell counts  using a 12.7×79.0 mm Seppro® IgY14 LC10 affinity LC column  on an Agilent 1100 series HPLC system , followed the protocols described previouslyA total of 18 mL of the pooled CSF sample  was subjected to the separations of 14 high-abundance proteins  at a trypsin/protein ratio of 1∶50. Sample cleanup was achieved using a 1-mL SPE C18 column  as described previouslyThe CSF proteins were incubated in 8 M urea and 10 mM dithiothreitol at 37°C for 60 min, followed by alkylation with 40 mM iodoacetamide in the dark for 30 min at room temperature. The samples were diluted 10-fold with 50 mM ammonium bicarbonate (pH 8) and 1 mM CaClFor tryptic digests of the IgY14 bound and flow-through fractions , 300 µg of tryptic peptides from CSF samples were resuspended in 300 µL 10 mM ammonium formate, 25% acetonitrile and fractionated by strong cation exchange chromatography as described previouslyThe SCX fractions were analyzed using a custom-built automated four-column high pressure nanocapillary LC system coupled on-line to either a linear ion trap mass spectrometer  or a linear quadrupole ion trap-orbitrap mass spectrometer , both modified in-house with an electrodynamic ion funnelwww.ebi.ac.uk/ipi). The search parameters used were: 3-Da tolerance for precursor ion masses and 1-Da tolerance for fragment ion masses with no enzyme restraint and a maximum of 2 missed tryptic cleavages. Static carboxyamidomethylation of cysteine and dynamic oxidation of methionine were used during the database search. LTQ-Orbitrap MS/MS data were first processed by an in-house software DeconMSnThe LTQ LC-MS/MS raw data were converted into .dta file by Extract_MSn (version 3.0) in Bioworks Cluster 3.2 (Thermo) and the SEQUEST algorithm (version 27 revision 12) was used to independently search all the MS/MS spectra against the human International Protein Index (IPI) database with a total of 69,731 total protein entries  from the CSF study by Zougman et al., reveals a 92% overlap see .In order to assess the CSF protein variability from serial sample collections, we next examined individual (non-pooled) samples from another group of 10 healthy volunteers  who had two CSF samples obtained at least 4 weeks apart using the AMT tag approach. Inter-subject differences were far greater than intra-subject differences . We perfAs an example of how CSF proteomic databases may be used to better understand disease states, we compared the proteomes of two similarly processed see  pooled sThis study provides the most comprehensive CSF protein coverage and list reported to date for healthy normal individuals including serial lumbar punctures. The protein set has immediate utility for investigators interested in using CSF to study neurological or psychiatric diseases. It will serve as a normative base to which disease states may be compared. Our study also suggests CSF protein variability over a short time is relatively limited in an individual. If this observation is supported by larger scale studies, it would further facilitate the utility of disease-state sample comparative analyses.The other major previous investigations of CSF from healthy individuals were published by Zhang et al Because of the challenge in obtaining CSF from healthy people, most previous studies may have used CSF from “surrogate-normals,” that is CSF collected from people with neurological complaints such as headache but with normal clinical CSF laboratory values.We compared proteomes of two different surrogate-normal groups. We found significant differences between the two groups. This study supports the potential usefulness of the normal human CSF proteome data library as an invaluable tool in investigating pathophysiological abnormalities in neurological and psychiatric disorders.Proteomic databases can be used in several ways. One of our own perspectives on using these proteomic databases for studying diseases involves a stepwise strategy. The first step would be a comparison of pooled samples representative of the disease to normal subjects or a comparator disease. The second step involves the selection of specific candidate proteins. The selection of candidate proteins is not likely to be predicted in advance and may require bioinformatic strategies and knowledge related to the disease under study. A third step would involve analysis of the individual samples contributing to the pool to ascertain how many of the samples actually contained one or more of the candidate proteins. This step provides a check in the event that a single individual in the pool disproportionately contributes a protein compared to other subjects. We would subject the results to statistical analyses. In the case of a search for biomarker proteins we strive to select those that meet clinically useful criteria, such as presence, absence or relative abundance in a large percentage of disease subjects and not so in most subjects without the disease under consideration. The fourth step would involve verification of the previous results using independent individual samples with the same disease. A final validation step may involve analyzing a larger number of subjects with the disease and controls using assays targeted to the candidate proteins. In contrast to the discovery phases, it would be advantageous, if feasible, to use assay platforms already having wide clinical use. Immunobased assays such as ELISA and Western blots may serve this purpose being relatively inexpensive. Steps 3 and 4 will likely employ a type of mass spectrometry which targets selected candidate proteins, such as Multiple Reaction Monitoring (MRM) using triple quadrupole instrumentation.The availability of the data presented here, detailing the normal human CSF proteome, should prove to be a critical base on which to compare proteins, both qualitatively and quantitatively, in studies of patients with a variety of neurological or psychiatric diseases.Figure S1Immunoaffinity depletion of plasma and CSF samples using the IgY14 LC10 column.(0.30 MB TIF)Click here for additional data file.Figure S2Comparison of the distributions of the gene ontology terms for all proteins identified from the healthy normal CSF sample and those for the 3654 plasma proteins reported by us previously (Text Reference 21). Biological process.(0.74 MB TIF)Click here for additional data file.Figure S3Comparison of the distributions of the gene ontology terms for all proteins identified from the healthy normal CSF sample and those for the 3654 plasma proteins reported by us previously (text reference 21). Cellular component.(0.85 MB TIF)Click here for additional data file.Figure S4Comparison of the distributions of the gene ontology terms for all proteins identified from the healthy normal CSF sample and those for the 3654 plasma proteins reported by us previously (text reference 21). Molecular function.(0.71 MB TIF)Click here for additional data file.Figure S5Unsupervised hierarchical clustering analysis of 88 proteins found to be present at significantly different levels  comparing serial CSF samples from 10 individuals . Log2 transformed protein abundances were used. M: male; F: female; numbers right after the hyphen indicate the two serial samples from the same individual.(0.31 MB TIF)Click here for additional data file.Figure S6Unsupervised hierarchical clustering analysis of 9 proteins found to be present at significantly different levels  comparing serial CSF samples from 10 individuals . Log2 transformed protein abundances were used. M: male; F: female; numbers right after the hyphen indicate the two serial samples from the same individual.(0.28 MB TIF)Click here for additional data file.Figure S7Unsupervised hierarchical clustering analysis of 2 proteins found to be present at significantly different levels  comparing serial CSF samples from 10 individuals . Log2 transformed protein abundances were used. M: male; F: female; numbers right after the hyphen indicate the two serial samples from the same individual.(0.26 MB TIF)Click here for additional data file.Table S1Peptides detected in CSF from healthy normal individuals using immunoaffinity depletion and 2D-LC-MS/MS.(2.36 MB PDF)Click here for additional data file.Table S2Analysis of overlap between proteins identified in normal CSF, plasma and previous CSF  proteomic study.(0.24 MB PDF)Click here for additional data file.Table S3Proteins identified and quantified from direct LC-MS analysis of CSF from non-neurologic and neurologic (headache) surrogate-normals.(0.02 MB PDF)Click here for additional data file.


In situ production of a secreted therapeutic protein is one of the major gene therapy applications. Nevertheless, the plasmatic secretion peak of transgenic protein may be deleterious in many gene therapy applications including Epo gene therapy. Epo gene transfer appears to be a promising alternative to recombinant Epo therapy for severe anaemia treatment despite polycythemia was reached in many previous studies. Therefore, an accurate level of transgene expression is required for Epo application safety. The aim of this study was to adapt posology and administration schedule of a chosen therapeutic gene to avoid this potentially toxic plasmatic peak and maintain treatment efficiency. The therapeutic potential of repeated muscular electrotransfer of light Epo-plasmid doses was evaluated for anaemia treatment in β-thalassemic mice.Muscular electrotransfer of 1 μg, 1.5 μg, 2 μg 4 μg or 6 μg of Epo-plasmid was performed in β-thalassemic mice. Electrotransfer was repeated first after 3.5 or 5 weeks first as a initiating dose and then according to hematocrit evolution.Muscular electrotransfer of the 1.5 μg Epo-plasmid dose repeated first after 5 weeks and then every 3 months was sufficient to restore a subnormal hematrocrit in β-thalassemic mice for more than 9 months.This strategy led to efficient, long-lasting and non-toxic treatment of β-thalassemic mouse anaemia avoiding the deleterious initial hematocrit peak and maintaining a normal hematocrit with small fluctuation amplitude. This repeat delivery protocol of light doses of therapeutic gene could be applied to a wide variety of candidate genes as it leads to therapeutic effect reiterations and increases safety by allowing careful therapeutic adjustments. One drawback to be avoided in such therapeutic strategy is the potentially deleterious secretion peak of therapeutic protein following DNA administration. The aim of this study was to adapt dosage and administration schedule of a chosen therapeutic gene to avoid this potentially toxic plasmatic peak.Therapeutic protein secretion by an ex-vivo strategies using engrafted transduced myoblasts or other cell types [Recombinant erythropoietin (rhEpo) injections are commonly used to treat anaemia linked to cancer treatment or chronic renal failure. However, rhEpo injections remain an expensive treatment which requires frequent delivery injection repeats and which can lead to anti-Epo antibodies production by the patient . Therefoll types -4, viralll types  adeno-asll types ,7, helpell types , or non-ll types , poloxamll types  or nakedll types ,11-13. Ill types ,9-13 whill types . TherefoTemporal control systems of transgene expression have already been used in gene therapy preclinical experiments, including for Epo gene use ,10,14,15In order to avoid the toxic Epo plasmatic peak and to reduce plasmatic fluctuation amplitude, we decided to test different doses and administration schedules of an Epo encoding plasmid in anaemia treatment of β-thalassemic mice. Considering electrotransfer advantages in terms of safety, efficiency and cost, we chose this well-handled gene transfer method. Our previous experiment with β-thalassemic mice using intramuscular electrotransfer of an Epo encoding plasmid  led to aConsidering those results, we have presently tested the therapeutic potential of repeated electrotransfer of suboptimal low Epo-plasmid doses in the β-thalassemic mouse model to restore and maintain a normal hematocrit without reaching toxicity.The pCMV-Epo plasmid used for experiments was a pCOR plasmid  containiAnimal experiments were conducted following NIH recommendations. The β-thalassemic Hbb-thal1 mice  from the® kit (Medac™) based on cross-reaction with human Epo.Blood samples were collected by retro-orbital puncture of anaesthetised mice at desired time after plasmid electrotransfer. Hematocrits were measured using a standard micro-hematocrit method . Mouse EAnalysis of variance (ANOVA) and Fisher PLSD were used.Our previous study of β-thalassemic mice demonstrated that electrotransfer of 1–10 μg Epo-plasmid doses were sufficient to induce a significant hematocrit increase. However, after a hematocrit burst depending on the dose of injected DNA during the first month after treatment, the hematocrit of treated mice started to decrease, and finally stabilised two months after electrotransfer. Surprisingly, this plateau was the same whatever the DNA dose used for gene transfer, and hematocrit still remained different from controls for at least 4 months . MoreoveTo avoid a possible hematocrit busrt following the electrotransfer treatment, we decided to raise the hematocrit step by step by repetitive treatments with small doses of plasmid DNA. In our mind, the first treatment should be performed with a small dose of the plasmid that would be insufficient to reach a normal hematocrit value, but which should just raise it a little. The purpose of this first dose was to initiate the treatment. The following treatments would then performed to assess the possibility to raise the hematocrit a little bit more, closer to a normal value, and to maintain it to an almost constant value. To assess the DNA dose appropriate to this aim, we first evaluated Epo plasmid doses of 2, 4 and 6 μg per mouse which were electrotransfered at days 0 and 25 fig . MaximumRegarding those results, we decided to decrease plasmid doses down to 1 μg and 1.5 μg and to increase the time interval between electrotransfer treatments fig . ElectroA hematocrit decrease of approximately 3% was observed in the control group between the beginning and the end of the experiment fig  p < 0.0 and 2-B.This over one year study indicates that an appropriate administration schedule to treat β-thalassemic anaemia in mice could consist in a 1.5 μg Epo-plasmid dose electrotransfer firstly repeated after 5 weeks as an initiating dose to restore a normal hematocrit, and then repeated every 3 or 4 months to maintain this hematocrit level. The present experiment shows that repeated electrotransfer of low Epo-plasmid doses allows fine tuning of hematocrit response on a more than one year period. Looking at individual data, it appears that the hematocrit can be maintained at an almost constant level for each of the treated animal. This strategy allows to avoid the deleterious initial hematocrit peak and to maintain a normal hematocrit with small fluctuation amplitude. Furthermore, we may hypothesise that this administration schedule which leads to low Epo endogenous production, may limit humoral response which has been clearly correlated to transgene expression level . TherefoRegarding possible clinical applications of the electrotransfer technology, one may argue that repetitive use of electric pulses might be painful. As far as we know, no significant discomfort related to the electrotransfer technology in humans has been reported so far. Several clinical trials of electrochemotherapy were reported with a good tolerance to the electric pulses delivery. Electrochemotherapy has recently been evaluated in an European project (ESOPE) and validated for clinical use.As far as muscle electrotransfer is concerned, at least two clinical trials have been approved and are being conducted in the area of cancer vaccination by two different companies, Ichor and Inovio (vaccination using tumor antigen). The results of these first in man studies should give us more details about the discomfort linked to this procedure.The present work indicates that plasmids can be delivered repetitively with little or none impairment of transgene delivery and expression, in opposite to viral vector mediated gene delivery. This repeated delivery protocol allows careful adjustments to reach the clinical endpoint and feedback for subsequent dose delivery. This safe treatment protocol could be applied to another anaemic context and extend to a wide variety of gene therapy applications using many candidate therapeutic genes such as growth factor genes.The author(s) declare that they have no competing interests.YB, DS, PB and EP carried out the design of the study. EEF and EP performed experimental protocols, assays and data collection. All the authors participated in data analysis. EEF drafted the manuscript with advices provided by PB. All the authors read and approved the manuscript.™ kit was used to measure mouse Epo based on cross-reaction (detection limit of 25 mU/ml for human Epo). Data are presented as mean Epo levels with standard error of the mean (SEM).Changes in erythropoietin (Epo) levels after repeated muscular electrotransfer of 1 μg and 1.5 μg of Epo-plasmid. the data provided shows the mean EPO level reached in mice following the electrotransfer treatments, for all three groups of mice . Mouse Epo changes in β-thalassemic mice electrotransfered with NaCl 150 mM solution for control group (solid diamond) or with 1 μg (solid sphere) and 1.5 μg (solid square) Epo-plasmid doses for the other groups. Electrotransfer was performed at day 0, 34, 112 and 215 for the three groups. One additional electrotransfer was performed at day 77 for the 1 μg group. Arrows indicate electrotransfer applications. The EPO ELISA MedacClick here for file


Peroxisome proliferator-activated receptor (PPAR)-γ is a nuclear hormone receptor that regulates gene expression, cell proliferation and differentiation. We previously described airway epithelial cell PPARγ deficient mice that develop airspace enlargement with decreased tissue resistance and increased lung volumes. We sought to understand the impact of airspace enlargement in conditionally targeted mice upon the physio-mechanical properties of the lung.We measured elastic recoil and its determinants, including tissue structure and surface forces. We measured alveolar number using radial alveolar counts, and airspace sizes and their distribution using computer-assisted morphometry.Air vs. saline-filled pressure volume profiles demonstrated loss of lung elastic recoil in targeted mice that was contributed by both tissue components and surface tension, but was proportional to lung volume. There were no significant differences in surfactant quantity/function nor in elastin and collagen content between targeted animals and littermate controls. Importantly, radial alveolar counts were significantly reduced in the targeted animals and at 8 weeks of age there were 18% fewer alveoli with 32% more alveolar ducts. Additionally, the alveolar ducts were 19% larger in the targeted animals.Our data suggest that the functional abnormalities, including loss of recoil are secondary to altered force transmission due to differences in the structure of alveolar ducts, rather than changes in surfactant function or elastin or collagen content. These data further define the nature of abnormal lung maturation in the absence of airway epithelial cell PPARγ and identify a putative genetic determinant of dysanapsis, which may serve as a precursor to chronic lung disease. Genetic heritability of airway structure and function in humans has been described ,2. FurthPeroxisome proliferator-activated receptor (PPAR)-γ is a nuclear hormone receptor that regulates gene expression, cell proliferation and differentiation. PPARγ is one of three PPARs, which cumulatively function as regulators of cellular lipid trafficking and metabolism . They fuWe previously described mice with conditional PPARγ deficiency within the conducting airway epithelium . These cWe sought to better understand the impact of the airspace enlargement in conditionally targeted mice upon the physio-mechanical properties of the lung, such as elastic recoil, and how surface tension and tissue elasticity contribute to total lung recoil. Recoil is the tendency of a tissue to return to its resting shape when an external force is removed . It is aHere, we report that alterations in lung structure and physiology in airway epithelial cell PPARγ targeted mice are accompanied by a loss of absolute tissue elastic recoil. These recoil effects are not attributable to changes in surfactant quantity or function, or extracellular matrix content or distribution. However, we find a significant abnormality in the distribution of airspace size, with mature conditionally targeted animals displaying a reduction in alveolar number and increase in alveolar duct number and size.floxed/floxed, Cre+) and littermate controls  by appropriate matings. Animals were genotyped for the presence of the Cre and PPARγ floxed alleles by tail biopsy, which provides a presumptive genotype predicting conditional targeting and confirmed by postmortem analysis of lung DNA. Targeted animals were specifically defined as those displaying the recombined allele, identified at 400 bp. All studies were performed on 8-12 week old mice unless otherwise noted comparing conditionally targeted animals with littermate controls.The generation and use of genetically modified animals was performed according to approved Harvard Medical School Institutional Animal Care and Use Committee protocols. Airway epithelium-specific PPARγ deficient mice were generated as described previously . Brieflyγ) and tissue elasticity (Ptis) to lung recoil (Pel) were determined by measuring recoil with deflation pressure-volume curves in degassed lungs filled first with air and then with saline as previously described [tis). Recoil pressure in the air-filled state (Pel) is equal to the combined contributions from tissue (Ptis) and surface tension (Pγ). Pγ, which includes surface tension contributions from both the alveoli and alveolar ducts, is calculated from the expression Pel = Ptis + Pγ at iso-volume [The contributions of surface tension  by radioimmunoassay and the collagen marker hydroxyproline (HP) by amino acid analysis, as previously described . Total p2. Surface film interfacial properties were recorded using a pulsating bubble surfactometer  at 20 oscillations/min, 37°C, and 100% relative humidity as previously described [A subset of animals underwent bronchoalveolar lavage (BAL) with 1 ml of saline × 3. The lung lavage fluid was pooled for each animal, and cells removed by low speed (250 × g for 6 minutes) centrifugation. The surfactant pellet was isolated by high speed centrifugation , and total phospholipid content determined as previously described . Values escribed . Films w2) and alveolar ducts , which were determined based upon empirical values of airspace sizes in control animals  using computerized morphometry methods as previously described ,29. BrieHematoxylin and eosin stained slides of inflated, fixed lung tissue were analyzed to calculate radial alveolar counts (RAC) using the method originally described by Emery and Mithal . BrieflyTwo-tailed unpaired Student's t-test was performed for individual comparisons between conditionally targeted and littermate control animals.γ)and tissue elasticity (Ptis) to recoil (Pel). Saline-filled data measures the tissue contribution (Ptis) to lung elasticity. The difference between these is the surface film contribution (Pγ).The contribution of surface tension and tissue forces to lung recoil can be assessed by measuring deflation pressure-volume curves of the degassed lung filled first with air, followed by saline ,28. Air-max - (Vmax - Vmin) e-kP, where Vmax is defined as the theoretical lung volume predicted for infinite distending pressure, Vmin the lung volume at 0 distending pressure, k the parameter that determines the curvature of the exponential function describing the relationship between pressure and volume, and P the transpulmonary pressure. Results for each group were summarized in terms of mean values for Vmax, Vmin, and k and representative group mean P-V relationships constructed. Figure el) in control (blue) and conditionally targeted animals (red). At iso-volume, lung recoil pressure is significantly reduced in KO mice relative to controls, as demonstrated by a leftward shift in the QSDPV curve. Results further indicate that the iso-volume reductions in overall lung recoil in the KO group are the result of decreases in both tissue (Ptis) and surface tension (Pγ) forces relative to control animals, as both curves show a relative leftward shift. Normalized to lung volume, the relative contributions of surface  and littermate control  mice were evaluated using this approach. Quasi-static deflation pressure-volume (QSDPV) data were fit to the exponential expression of Salazaar-Knowles, V(P) = Ve Figure  and tissThe observed airspace enlargement and physiological changes in the lungs of conditionally targeted mice could be explained by defects in airway-airspace tethering, primarily contributed by the extracellular matrix (ECM). Elastin histochemistry did not show any apparent changes in quantity, distribution or orientation of elastin fibers in the lungs of conditionally targeted or littermate controls at 8 weeks of age Figure . SimilarBiochemical analysis for desmosine (Des) showed no difference in elastin content when corrected for total protein content (P) in conditionally targeted animals compared with littermate controls . Similarly, biochemical analysis for hydroxyproline (HP) showed similar collagen content between conditionally targeted and control animals .As the content and distribution of ECM appeared not to be the cause of altered lung mechanics in conditionally targeted mice, we investigated surfactant quantity and function. Surfactant was isolated from the lungs of mature conditionally targeted (n = 9) and littermate control (n = 7) mice by bronchoalveolar lavage. Analysis of lavage surfactant demonstrated a similar content of total lung phospholipids .max), minimum (γmin), and equilibrium (γequil) surface tensions measured under dynamic and static conditions. There were no differences in γmax (37.67 ± 2.7 vs. 37.14 ± 1.8 dyn/cm), γmin (9.44 ± 4.7 vs. 10.29 ± 3.5 dyn/cm) or γequil (25.56 ± 0.7 vs. 25.57 ± 0.8 dyn/cm) in conditionally targeted animals compared with littermate controls.Surfactant function was characterized in terms of interfacial properties measured by pulsating bubble surfactometry. Characterization was expressed in terms of maximum  and airspace area  . We obse2), and a consistent increase in the frequency of airspaces >1000 μm2   in the targeted animals  Figure  in the c) Figure . Additios Figure .2, p = 0.01)  Figure .We sought to understand the role of PPARγ in the regulation of lung development and homeostasis in gene targeted mice. Due to the early fetal lethality of complete PPARγ deficiency , we usedfloxed/WT, Cre+) and the CCtCre transgenic mice were analyzed and found to have comparable airspace size with the conditionally targeted littermate controls [There have been reports suggesting that Cre recombinase is toxic to mammalian cells . Considecontrols .Recoil is the tendency of a tissue to return to its resting shape when an external force is removed  and is dWe observed that conditionally targeted mice have enlarged lungs that display reduced recoil properties, but were proportional when normalized to total lung volume. Such changes could be explained by abnormalities in lung ECM-mediated tethering. We previously reported changes in the basal expression levels of specific collagen  and elastin genes in adult conditionally targeted lung tissue, after the time when most of the matrix has been produced and has accumulated. However, analysis of lung elastin and collagen protein content indicated there were no significant differences in the distribution of ECM in the lungs of conditionally targeted mice. These biochemical methods measure the accumulation of protein throughout the heterogeneous tissue over time, and are a more robust indication of the physiological contributions of the ECM. The data reported in the current study thus clarifies that differences in expression of these genes at this time-point are not sufficient to alter whole lung tissue content of these markers. Thus, changes in lung recoil could not be accounted for by a decrease in total tissue content of the two major constituents of the lung's ECM. The ECM of the lung is highly complex, consisting of dozens of collagen molecules and numerous molecules necessary for elastin fiber formation, with unique distributions. We do not report, nor have we tested, for changes in the local distributions  of specific molecules. Whether our previously defined changes in expression reflect ongoing repair, alterations in homeostatic mechanisms or affect local distributions of ECM components is currently unknown.in vivo.Since PPARγ contributes to cellular lipid metabolism and trafficking, it is also reasonable to hypothesize that a deficiency in PPARγ may affect surfactant lipid production and/or secretion. Analysis of surfactant isolated from the lungs of conditionally targeted mice did not reveal any differences in absolute phospholipid quantity or surfactant surface tension. These data suggest that abnormalities in surface forces from surfactant are not a direct cause for the loss of elastic recoil in the conditionally targeted animals and do not support a role for airway epithelial cell PPARγ in regulating surfactant expression It is appreciated that abnormalities in alveolar duct structure, distribution or function can alter surface forces and ultimately affect elastic recoil . ComputeDifferences in lung structure and/or function may be a contributing factor for the development of lung disease . Airway-We report airway epithelial cell PPARγ deficiency results in airspace enlargement accompanied by a reduction in total lung elastic recoil. Our data indicate that these physiological abnormalities are secondary to altered force transmission due to differences in the structure of the lung parenchyma, rather than changes in elastin or collagen content or surfactant function. These data further clarify the special role of lung epithelial cell PPARγ in contributing to the establishment of normal airspace structure. Finally, our study describes how genetics can contribute to the determination of lung structure and function, which may predispose to chronic lung disease.The authors declare that they have no competing interests.DMS organized the studies, performed or assisted with all experiments and contributed to writing the manuscript. LWT performed physiology and lipid biochemistry experiments. EPI conceived the studies and participated in physiology and lipid biochemistry experiments and contributed to data interpretation. BCS performed extracellular matrix biochemistry experiments. TJM conceived the studies, contributed to data analysis and interpretation, and wrote the manuscript. All authors read and approved the manuscript.Supplemental Methods. This file provides detailed methodology on the computer-assisted determination of alveolus and alveolar duct number and size.Click here for fileSupplemental Figure. This figure shows the frequency distribution for airspaces of individual sizes for both control and conditionally targeted mice.Click here for file


Secretion of gonadotropin-releasing hormone (GnRH) produced in neurons in the basal forebrain is the primary regulator of reproductive maturation and function in mammals. Peptidergic signals relating to circadian timing and energy balance are an important influence on the reproductive axis. The aim of this study was to investigate the innervation of GnRH neurons by peptidergic neurons.Immunohistochemistry and confocal microscopy were used to detect appositions of peptidergic fibers  associated with energy balance and metabolic status in transgenic mice expressing a green fluorescent protein reporter construct in GnRH neurons. The frequency of these appositions was compared to those of vasoactive intestinal peptide (VIP), a hypothalamic neuropeptide likely to convey circadian timing information to the GnRH secretory system. The majority of GnRH neurons (73–87%) were closely apposed by fibers expressing NPY, β-endorphin, or MCH, and a significant proportion of GnRH neurons (28%) also had close contacts with VIP-ir fibers.It is concluded that GnRH neurons in the mouse receive a high frequency of direct modulatory inputs from multiple hypothalamic peptide systems known to be important in conveying circadian information and signalling energy balance. Gonadotropin-releasing hormone-1 decapeptide (henceforth referred to as GnRH) is the final common signalling molecule in the brain that regulates reproductive function in all vertebrates In mammals, reproduction is one of the most energy costly processes, hence it is imperative that the mechanisms controlling energy balance integrate with those that control reproduction ad libitum. Studies were approved by the University of Nottingham Local Ethical Review Committee and conducted in accordance with the United Kingdom Animals (Scientific Procedures) Act, 1986.Adult male and randomly-cycling female GnRH-GFP mice (B6/GnRHhGFP2) GnRH-GFP mice were terminally anesthetized with Euthatal . The chest cavity was opened, and 500 units of heparin  were injected intracardially. Mice were then perfused via gravity flow through the ascending aorta with PBS  for 3 minutes followed by 4% paraformaldehyde  for 5 minutes. Brains were dissected out and placed in 4% PFA for 3–4 hours then cryopreserved by overnight incubation in 20% sucrose in PBS. Brains were sectioned in the coronal plane on a freezing microtome at a thickness of 60 µm.Immunohistochemical procedures were carried out on free-floating sections at room temperature. For NPY and MCH antisera, sections were placed into small jars of citrate buffer  and placed in a 650 W microwave on high power for ∼12 seconds to promote antigen retrieval prior to staining. Sections were allowed to cool for 10 minutes and washed twice in PBS. Sections were incubated for 1 hour in 2% normal goat serum , then in primary antisera (in buffer 1) overnight at 4°C. Additional sections were incubated in normal rabbit serum or no serum as control procedures. Primary antisera were i) rabbit polyclonal anti-GnRH antiserum which recognizes amino acids 6–10 of GnRH in pro-GnRH and GnRH , used at a final dilution of 1∶15,000, ii) rabbit polyclonal anti-VIP raised against N-terminal of rat VIP peptide  sections were incubated in biotinylated goat anti-rabbit IgG  at 1∶400 (in buffer 2) for 1 hour at room temperature. Following a further 2 washes in buffer 2, and 1 wash in PBS, sections were incubated with avidin-TRITC conjugate  at 1∶200 (in buffer 2). Sections were then washed again; twice in buffer 2 and once in PBS prior to mounting on gelatin-subbed slides and air-drying. Slides were briefly washed in distilled HSections were initially viewed under a Leica DMRB fluorescence microscope to identify the locations of GnRH-GFP neurons and immunostained peptidergic perikarya and fibers. Sections were examined using filters appropriate for visualisation of GFP and TRITC, and images were captured and superimposed using Openlab software . Each GnRH-GFP cell soma was scored by a single investigator for the presence and number of ‘close appositions’, defined as immunostained peptide fibers or putative terminals which appeared to either directly contact the GnRH-GFP cell or which overlapped the cell and were in the same plane of focus.To confirm that the overlap or contact of peptide-immunostained structures with GnRH-GFP cell soma and dendrites identified using fluorescence microscopy truly represented close contacts, sections with putative appositions of each peptide were examined further using a confocal laser scanning microscope (Leica SP2), with a Leica IRE2 inverted fluorescence microscope to help relocate GnRH-GFP neurons. GFP was excited using the 488 nm argon laser line (emission 500–550 nm) and TRITC-labelled structures were sequentially excited  using the 561 nm helium-neon laser line (emission 570–620 nm). Each cell was examined through its entirety in 0.5 µm steps (∼20 slices) and all appositions were assessed. Images were displayed as maximum intensity projections of the entire z-series or as single 0.5 µm slices using the associated Leica LCS software.In order to confirm that the distribution of GFP-expressing neurons accurately represents the distribution of GnRH positive neurons, a series of sections from 2 transgenic adult male mice were immunostained with an anti-GnRH antibody (LR1) using a TRITC fluorescent tag. There was a very high incidence of colocalization of GnRH immunoreactivity and GFP . Of the p<0.01 vs rostral areas, χ2 test).VIP-ir varicose fibers and terminals were scattered throughout the GnRH continuum. Of 510 GFP-positive neurons examined in sections from 3 female mice, 28% were contacted by VIPergic axons , Table 1p = 0.38, χ2 test). The mean number of appositions per GnRH-GFP neuron was 2.45, and there was no statistical difference in the number of NPY appositions per neuron between regions . There was some overlap between NPY-ir fibers and GnRH neuroterminals in the caudal median eminence . β-endorphin-ir fibers were also apposed to GnRH neuroterminals in the external layer of the median eminence . In addition, there was some overlap between MCH-ir fibers and GnRH neuroterminals in the external layer of the ME  of immunohistochemically labelled GnRH neurons also expressed GFP. In the same strain of transgenic mice and using the same antiserum to identify GnRH neurons, Spergel et al. originally observed GFP fluorescence in only 65% of GnRH immunoreactive neurons A significant body of evidence implicates VIP in the regulation of the preovulatory LH surge in rodents Three peptidergic families known to be of considerable importance in the central control of energy balance, NPY The distribution of β-endorphin-ir fibers in the basal forebrain and the localization of β-endorphin-ir perikarya within the arcuate nucleus of the mouse that we report here are consistent with previous findings in the rat The distribution of MCH-ir fibers in the basal forebrain and the localization of MCH-ir perikarya in the lateral hypothalamic area and zona incerta in the mouse are also consistent with previous reports in the rat ex vivo experiments, which showed the addition of MCH to explants of the ventral hypothalamus from proestrous rats increased GnRH release This neuroanatomical circuitry revealed in the mouse in the current study is likely to be of functional significance. It is clear that there is a circadian rhythm of VIP abundance in the mouse SCN The current studies reveal that GnRH neurons in the mouse receive a high frequency of close appositions from peptidergic systems known to be important in conveying circadian information (VIP) and signalling energy balance . The current study adds to the evidence that GnRH perikarya and terminals have a plethora of different peptidergic inputs, as other studies have generated either neuroanatomical or functional evidence that a wide range of other peptides also regulate GnRH secretion . This huge diversity of peptidergic inputs raises the question of their individual significance. The complete infertility in genetically modified mice lacking kisspeptin


Management of endometrial precancerous lesions has been of much debate due to inconsistencies in their classification, natural history and histologic diagnosis. Endometrial hyperplasia constitutes a wide range of histomorphologic features associated with high intra and interobserver diagnostic variability.Although traditional microscopic diagnosis is by far the most applicable method and the gold standard for histomorphologic diagnosis, digitized image analysis has been used as a powerful adjunct to maximize the histologic data retrieval and to add some detailed objective criteria for correct diagnosis in difficult cases.A series of 100 endometrial curettage specimens with diagnosis of endometrial hyperplasia or well differentiated adenocarcinoma were blindly reviewed by 5 pathologists; their intra and interobserver reproducibility determined and further compared to the objective morphometric data i.e. D-score and volume percent of stroma (VPS).The results were assessed using the weighted kappa statistics. Mean intraobserver kappa value was 0.8690 (99.44% agreement). Mean interobserver kappa values by diagnostic category were: simple hyperplasia without atypia: 0.7441; complex hyperplasia without atypia: 0.3379; atypical hyperplasia: 0.3473, and well-differentiated endometrioid carcinoma: 0.6428; with a kappa value of 0.5372 for all cases combined.Interobserver agreement was in substantial rate for simple hyperplasia (SH) and well differentiated adenocarcinoma (WDA) but was in fair limit for complex hyperplasia (CH) and atypical hyperplasia (AH). Intraobserver agreement was almost perfect. The specimens were divided in two groups according to the computerized morphometric analysis: Endometrial Hyperplasia (EH) ( D Score ≥ 1 or VPS ≥ 55%) and Endometrial Intraepithelial Neoplasia (EIN) (D-Score < 1 or VPS < 55%). Morphometric findings were closely compatible with routine WHO classification made by one expert pathologist; however; diagnosis of (CH) and (AH) made by other pathologists were not concordant with morphometric data.It may be necessary to make some revisions in WHO classification for endometrial hyperplasia and precancerous lesions. Endometrial hyperplasia which is believed to increase the risk of endometrial carcinoma, is a common disease and comprises a wide spectrum of histological changes from simple aggregation of the normal-looking proliferate glands at one extreme to the changes that are difficult to distinguish from carcinoma at the other end of the spectrum. The current classification, introduced by Kurman et al 1985, has been accepted by WHO and ISGP. This classification considering two criteria  there are four diagnostic categories of endometrial hyperplasia: simple hyperplasia (SH), complex hyperplasia (CH), simple atypical hyperplasia (SAH) and complex atypical hyperplasia (CAH). -4The wide range of histomorphologic presentation of endometrial hyperplasia is accompanied by high intra and interobserver variability in diagnostic classification. Previous studies have shown that only 10–20% of endometrial hyperplasias progress to carcinoma when left untreated. [The lack of criteria that could accurately predict the disease outcome may have been an important cause of over and under treatment and need for establishment of a new classification composed of three groups: endometrial hyperplasia (EH), endometrial intraepithelial neoplasm (EIN) and endometrial carcinoma.  EIN is dThis alternative strategy that is intended to recognize the precancerous lesions earlier provides through multivariate analysis, a subset of objectively measured morphometric parameters which may predict the subsequent development or concurrent carcinoma. Several attempts have been made to improve the microscopic tissue diagnosis by the aid of the modern digitized image technology. For example, Kayser et al worked on a method of automatically scanning and analyzing routinely stained glass slides known as virtual microscopy that provides fast and reproducible data about the object-associated  and non-object-associated (background) tissue components or so-called texture analysis. Studying on 896 lung cancer slides and using virtual microscopy they produced non-overlapping compartments on each slide that were subsequently subjected to texture analysis. With certain calculations performed at different objective magnifications, they conclude that this system is a fast and reliable procedure for automated pre-screening of lung tumor pathology with diagnostic accuracy of 96–100% that can be made on only 10% of the original image field without increasing error rate. [An additional advantage of digitized image technology is its application in the web-based internet communications also known as telepathology. Improved image analysis incorporates computer-measure architectural as well as cytological features into a cancer predictive formula (D-Score) which is useful for patient management. The D-Score has been developed in the early 1980s and its essential features are of architectural (volume percentage stroma and outer surface density of glands) and cytological (standard deviation of the shortest nuclear axis) nature. Retrospective studies in the USA, the Netherlands and Norway confirmed the prognostic value of the D-Score greatly exceeding the WHO 94 criteria. D-Score have higher sensitivity (100%), specifity (82%), positive predictive value (PPV 38%) and negative predictive value (NPV 100%) compared to WHO 94 with sensitivity (91%), specifity 58%), PPV (16%) and NPV (99%). [8%, PPV  stained histological sections were made.Five pathologists with varying experiences in the field of gynecologic pathology who worked at hospitals in Tehran University of medical sciences contributed to this study.The cases were selected to represent four diagnostic categories including simple hyperplasia(SH) which shows glands are irregular in size and shape with occasional dilated, cystic glands lined by pseudostratified uniform and oval nuclei showing orientation toward the basement membrane and separated by abundant stroma fig  and 2, cTwenty-five cases from each category were included and one representative H&E slide was selected of each case. To assess interobserver variability slides were randomly labeled from 1 to 100, evaluated by 5 pathologists and presumptive diagnoses were recorded in a checklist. For intraobserver evaluation one expert gynecopathologist examined all of the slides twice within a period of two months.The checklists included four diagnostic categories . After data collection the checklists were coded and statistically analyzed using the STATA-8 statistical software and weighted kappa test. Data analysis evaluated interobserver and intraobserver agreement using the (Kappa) statistic, a measure of agreement between observers that attempts to correct for chance agreement. Within the positive values of kappa, given interpretations used in this study were scaled as: 0.00–0.20 = slight, 0.21–0.40 = fair 0.41–0.60 = moderate, 0.61–0.80 = substantial and 0.80–1.00 = almost perfect The correlation coefficient between morphologic data and results of morphometricanalysis is about 80%,  so 55 ou® (V.4.R117) software incorporated into the digitized light-microscope.Computerized morphometric analysis of delineated regions on H&E stained sections was performed using the Leica IM 500For each sample the D-Score was calculated, using three features include 1) volume percentage of stroma (VPS), which assesses the percentage of endometrial tissue composed of stroma  2) standard deviation of shortest nuclear axis (SDSNA), which reflects nuclear pleomorphism and 3) gland outer surface density (out SD), which is a measurement of basement membrane length about the endometrial glands (measurement of gland complexity) and the following formula:-5,14 MeaIn brief, VPS was measured on histological images (40 × objective magnification (field diameter 340 μm) with a 88 point grid or graticule (weibel grid with 2-point length 28.3 μm), and the tissue underlying each point was scored visually from the ocular lens of microscope as stroma, epithelium or gland lumen. Results from a total of 400–600 points were tallied and the VPS was calculated as the number of stromal points divided by the total points counted. (Range of 14–75%)Intersections of gland outer surfaces with calibrated horizontal lines of the weibel grid were tallied and the outer surface density was calculated by underlying formula:® program followed by nuclear mean and SD determination. Measurement was terminated when the coefficient of variation went below 5% (range of 0.68–1.52)Nuclear morphometry was preformed on at least 150 randomly selected nuclei and the shortest nuclear axis was calculated by sending results to Microsoft ExcelFor interobserver diagnostic agreement, using the diagnosis given by each pathologist on each diagnostic round, kappa results show significant differences in diagnostic groups, with highest agreement in SH and WDA groups and lowest agreement in CH and AH groups. Table Table After pathologist agreement assessment, mean difference of computerized morphometric data (VPS and D-Score) with pathologist diagnosis subgroup, statistically analyzed with post HOC test and shuffle exam for three pathologists.According to diagnosis in 01t1 (Observer 1 Time 1), O2t1 (Observer 2 Time 1) & O3t1 (Observer 3 Time 1)Compared assessment of different diagnostic groups with D-Score results show high concordance and ability in O1t1 (Observer 1 Time 1) for classification and differentiation of endometrial hyperplasia subgroups but overlapping results in differentiation of CH with AH and AH with WDA groups in O2t1 (Observer 2 Time 1) and CH with AH in O3t1 (Observer 3 Time 1).Case by case comparison of computerized VPS (cut-off range 55%) and D-Score (cut-off range 1) with pathologist diagnosis in four diagnostic variables analyzed with kruss-kall Wallis test are shown in tables Endometrial carcinoma is the most common female genital tract malignancy in developed countries.  EndometrEndometrioid subtype may arise in background of endometrial hyperplasia at a younger age while the high grade in an older age group. The WHO 94 endometrial hyperplasia classification system will continue to play an active role in the daily practice of many pathologists but is plagued by poor diagnostic reproducibility and the lack of a solid statistical foundation on therapeutic context.It is important to characterize high or low risk groups before initiation of therapy, because about 1–28% of hyperplasias progress to carcinoma, depending on the degree of severity. Considering the combined interobserver agreement level of "moderate" attained in this study and the previously reported results as "fair" by Skov (1997); "substantial" by Kendall (1998) and "moderate" by Bergeron(1999) it seems that WHO 94 classification system needs essential improvements by an entirely new approach rather than minor revisions. -18EIN clThis new molecular genetic-based and morphometric-based classification differs from the WHO 94, which is based entirely on histological findings. Diagnosis of EIN is possible with assessment of D-Score and VPS morphometrical parameters i.e. lesions with D-Score<1 or VPS<55% are classified as EIN. It should be emphasized that morphometric studies of endometrial hyperplasia have identified a unique multivariate prognostic combination of quantitative architectural and nuclear features that corresponds well with both cancer risk and biologic lesion properties ,9 but ouOne with high concordance and 95% rate of confidence interval in all subgroups but those two others with overlapping results, especially in diagnoses of CH with AH that show lowest rate of reproducibility in all studies.Compared VPS and D-Score results rendered highly concordant replicate results.Case by case comparison of VPS (cut-off range 55%) and computerized D-Score (cut-off range 1) with pathologist diagnosis is shown in tables In conclusion, diagnosis of endometrial hyperplasia and carcinoma with WHO-defined nomenclature may be problematic, mainly due to stylistic differences between individuals and inherent poor reproducibility of the broad range of diagnoses from benign to malignant.Limitation of borderline or precancerous lesions into one category (EIN) recognized by objective morphometry will probably simplify the diagnosis and improve the patient's management.Measurement of VPS – by far the most predictive component of the D-score – can be accomplished simply by applying an inexpensive ocular grid into an ordinary microscope eyepiece and counting the specified points on glandular and stromal components.The authors declare that they have no competing interests.NIM is study designer, contributed in histological diagnosis and writing of manuscript, performed morphometrical analysis. MY Writing of the manuscript and performing morphometrical analysis. SAA Histological diagnosis, writing, revising and editing of manuscript. GI and HH Histological diagnosis and writing of the manuscript. APM Data analysis, interpretation of statistical data and writing of the manuscript. MK histological diagnosis. All authors read and approved the final manuscript.


In the event of a terrorist-mediated attack in the United States using radiological or improvised nuclear weapons, it is expected that hundreds of thousands of people could be exposed to life-threatening levels of ionizing radiation. We have recently shown that genome-wide expression analysis of the peripheral blood (PB) can generate gene expression profiles that can predict radiation exposure and distinguish the dose level of exposure following total body irradiation (TBI). However, in the event a radiation-mass casualty scenario, many victims will have heterogeneous exposure due to partial shielding and it is unknown whether PB gene expression profiles would be useful in predicting the status of partially irradiated individuals. Here, we identified gene expression profiles in the PB that were characteristic of anterior hemibody-, posterior hemibody- and single limb-irradiation at 0.5 Gy, 2 Gy and 10 Gy in C57Bl6 mice. These PB signatures predicted the radiation status of partially irradiated mice with a high level of accuracy (range 79–100%) compared to non-irradiated mice. Interestingly, PB signatures of partial body irradiation were poorly predictive of radiation status by site of injury (range 16–43%), suggesting that the PB molecular response to partial body irradiation was anatomic site specific. Importantly, PB gene signatures generated from TBI-treated mice failed completely to predict the radiation status of partially irradiated animals or non-irradiated controls. These data demonstrate that partial body irradiation, even to a single limb, generates a characteristic PB signature of radiation injury and thus may necessitate the use of multiple signatures, both partial body and total body, to accurately assess the status of an individual exposed to radiation. In the event of a terrorist-driven detonation of an improvised nuclear device (IND) in a populated U.S. city, it is expected that hundreds of thousands of people could be exposed to ionizing radiation, with even larger numbers fearful that they have been exposed We have applied genome-wide analytical methods and high-throughput computational tools to determine whether “signatures” of radiation injury can be identified in the peripheral blood (PB) of mice and humans following exposure to several dose levels of gamma irradiation While these studies have clearly identified PB metagenes that can predict radiation status and dose of exposure after total body irradiation (TBI), an important refinement to these signatures would be incorporation of analysis of partially-exposed individuals; this is particularly important in the development of an biodosimetry assay for acute radiation injury since it is expected that a large percentage of radiation victims in a mass casualty scenario will have heterogeneous exposures due to partial shielding Twelve week old female C57Bl6 mice  were housed at the Duke Cancer Center Isolation Facility and all protocols in this study were approved by the Duke University Animal Care and Use Committee (Protocol Number A037-10-02). Six to seven mice/group were treated with partial body irradiation to either the anterior hemibody (AH), posterior hemibody (PH), or hind limb (HL) regions with an X-ray source at doses of 0.5, 2, or 10 Gy using a filter of 0.1 mm Cu and 2.5 mm Al. After dosimetry studies were performed to assess absorbed dose in the body and in the hind limb of the animal, anterior and posterior regions of the mice were irradiated at an average of 1.49 Gy/min and the hind limbs at an average of 1.25 Gy/min. Six hours post-irradiation, approximately 500 ul peripheral blood was collected by cardiac bleed from both irradiated and control mice. PB mononuclear cells (PB MNCs) were isolated by Lymphoprep density gradient centrifugation  and total RNA was extracted with a Qiagen RNEasy Mini Kit  as previously described https://www.operon.com/arrays/oligosets_mouse.php) contains 35,852 oligonucleotide probes representing 25,000 genes and approximately 38,000 transcripts. In comparing to previously published total body irradiation dataset Mouse and human oligonucleotide arrays were printed at the Duke Microarray Facility using Operon's Mouse Genome Oligo sets (version 4.0). Operon's Mouse Genome Oligo set (version 4.0) (http://www.stratagene.com) were amplified and used in probe preparation as previously described http://microarray.genome.duke.edu/services/spotted-arrays/protocols).Briefly, MNCs were pelleted, and total RNA was isolated using the RNeasy mini spin column as previously described http://www-stat.stanford.edu/tibs/PAM/) was used to impute missing values. k-nearest neighbor was used where missing values were imputed using a k-nearest neighbor average in gene space.Genespring GX 7.3 (Agilent Technologies) was used to perform Lowess normalization of the data and then the data was filtered in which spots whose signal intensities below 70 in either the Cy3 or Cy5 channel were removed. To then account for missing values, PAM software  a supervised analysis using binary regression methodologies as described previously Prediction analysis of the expression data based on the supervised binary regression analysis was performed using MATLAB software as previously described Analysis based on unsupervised factor models was carried out with publicly available software as previously described Mice were irradiated at the doses described previously . At 6 hours post irradiation, PB MNCs were isolated and stained for flow cytometry using rat anti mouse APC-Ter-119, APC-B220, PE-Mac-1, PE-Gr-1, and FITC-Thy1.2 antibodies . Cell subsets were analyzed as a percentage of the live cell population. For the survival analysis, mice were irradiated with the X-ray source at 10 Gy to either AH (n = 10) or PH (n = 10) as described above. Mice were followed for 60 days post-irradiation to assess differences in survival.We first sought to determine if irradiation of one-half of the body could produce a PB gene expression response that was characteristic of that level of radiation exposure and whether irradiation of the AH (head to T12), PH (below T12) or HL (single hind limb) produced unique PB gene expression profiles. Of note, irradiation to the AH encompasses the spleen, whereas irradiation to the PH encompasses the pelvis and both femurs. Twelve week old C57Bl6 female mice (n = 6–7 per group) were irradiated with single fractions of 0.5 Gy, 2 Gy or 10 Gy and PB was collected at 6 hours post-irradiation for analysis. We chose these dose levels since they reflect medically distinct exposure levels which require different levels of intervention  We also irradiated a group of mice (n = 7 per dose level) to the PH and identified PB gene expression profiles for each dose level that reflected PH irradiation . A leaveSince we were able to identify PB signatures of radiation that reflected AH and PH irradiation, we sought to determine if irradiation to a single limb produced a PB gene expression profile that could comparably predict radiation status versus non-irradiated controls. Interestingly, we identified patterns of PB gene expression that appeared to distinguish HL-irradiated mice from non-irradiated control mice using a binary regression analysis . When wearyl hydrocarbon receptor (AHR), neuropsin (Prss19), R-spondin, lectin-galactose binding-soluble 3 , NTF2-related export protein, and nuclear factor-IL3 regulated; no genes were in common between the 3 dose levels in the PH condition and Pscd3 and Slc41a3 were in common at all dose levels in the HL group  at the 10 Gy dose level . In contrast, there was nearly uniform distribution of p-values generated from the partial body radiation, suggesting a much smaller response. This analysis does not preclude a groups of genes, each having a small response, from being used to build predictors, but does suggest that there are no strong single gene predictors for this phenotype. At the same time, there are none from partial body group which pass this test, and in addition, the smallest p-value in this group is more than an order of magnitude too large to qualify as significant. Taken together, these results indicate that PB signatures of radiation generated from TBI-recipients are unlikely to accurately predict radiation status in partially irradiated individuals.As an alternative approach to compare the PB gene expression profile from TBI versus partial body irradiation, all genes were tested for association with total body and partial body radiation exposure (ANOVA) and p-values signifying the strength of association were generated . In ordeCdkn1a and Dcxr) were found to be in common between the 10 Gy TBI signature and the 10 Gy signature from PH-treated mice  compared to partially irradiated mice. These differences between PB cell content in TBI- and partial body irradiated-mice may be explained, in part, by the generalized mobilization of hematopoietic cell subsets which occurs following partial body irradiation Since partial body irradiation produced significantly different PB gene expression profiles compared to TBI, we analyzed PB from partially irradiated versus TBI-mice to determine if changes in PB cell content contributed to these differences. TBI caused a 33% decrease in PB MNCs within 6 hours of exposure , but parGene expression profiles of solid tumors have been successfully applied to predict patient prognosis and the responsiveness of various cancers to different chemotherapies We have sought to develop a PB assay for ionizing radiation exposure using gene expression profiles. Recently, we demonstrated that PB signatures of TBI exposure were capable of predicting both radiation status and dose level of exposure in mice with 96% accuracy Despite the fact that we were able to identify PB signatures of partial body exposure that predicted radiation status within the AH, PH and HL exposure groups, we also found that the PB signatures of partial body irradiation failed to predict radiation status based upon site of exposure. For example, the PB signature of 2 Gy AH irradiation failed to predict the radiation status of mice treated with 2 Gy to PH. Similarly, the PB signature of 10 Gy PH failed to predict the radiation status of mice irradiated with 10 Gy to HL. Taken together, these results suggested that the PB molecular response to ionizing radiation is distinct depending upon which portion of the body is exposed. Examination of the genes which comprise the PB signatures of partial body irradiation provide a possible explanation for the lack of predictions across different partial irradiation conditions. We found no overlapping genes between the 3 partial body irradiation conditions at 0.5 or 2 Gy dose levels and only 1 non-annotated gene, RIKEN cDNA 6330579B17, which was in common between the AH, PH and HL groups at 10 Gy dose level. Taken in a broader context, these results demonstrate that partial body irradiation induces unique PB molecular responses as a function of the extent of the exposure and that caution should be applied when applying PB gene signatures to diagnose partially irradiated individuals. Furthermore, the lack of ability to discriminate dose levels across different partial body conditions suggests that distinct reference gene expression profiles may be necessary to accurately predict the radiation status of partially irradiated individuals.Cdkn1a, in common with a PB signature of human TBI exposure Recently, we and others have sought to develop and validate PB signatures of radiation exposure generated from TBI treatment of mice and/or humans for the purpose of biodosimetry in a mass casualty radiation event In a broader context, the PB radiation signatures that we have developed have the potential to serve as biomarkers of individual susceptibility to radiation-induced toxicity in patients undergoing large volume therapeutic irradiation or TBI. Moreover, sufficient molecular overlap has now been established between normal hematopoietic stem cells and cancer stem cells Table S1Gene lists for partial body irradiation signatures.(0.42 MB DOC)Click here for additional data file.Table S2Overlapping genes between partial irradiation conditions.(0.16 MB DOC)Click here for additional data file.Table S3Overlapping genes between TBI and Partial Body Signatures.(0.05 MB DOC)Click here for additional data file.


Adamantane-resistant influenza A is an emerging problem, but infections caused by resistant and susceptible viruses have not been compared. We identified adamantane resistance in 47% of 152 influenza A virus (H3N2) isolates collected during 2005. Resistant and susceptible viruses caused similar symptoms and illness duration. The prevalence of resistance was highest in children. The study was approved by the Marshfield Clinic institutional review board, and all participants provided written informed consent.Study participants were derived from a study of influenza vaccine effectiveness conducted within a 14–Zip code region surrounding Marshfield, Wisconsin, during January–March 2005. Eligible participants included children 6–23 months of age, adults Influenza virus was isolated by inoculation and incubation in monolayered rhesus monkey kidney cells. Cultures were examined for cytopathic effect, and cultures showing no cytopathic effect were tested twice for hemagglutination titers. Influenza A and B were confirmed by immunofluorescence . All influenza A isolates were tested for adamantane resistance at the Centers for Disease Control and Prevention (CDC). Procedures for RNA extraction and pyrosequencing have been previously described .>65 years of age (39%) .Influenza virus was isolated in 167 (20%) of 818 cultures from ill cohort members; 153 (92%) were influenza A (H3N2) and 14 (8%) were influenza B . Influen>65 years of age who had a high-risk condition. In a secondary analysis, no association was found between adamantane resistance and number of symptoms, duration of illness, or hospitalization.Number of symptoms and duration of illness were similar for patients with adamantane-resistant and -susceptible influenza isolates . The proThis study provides epidemiologic evidence that the point mutation (S31N) conferring adamantane resistance has not altered influenza A (H3N2) virulence as measured by clinical symptoms and duration of illness. Hospitalization rates were also similar, although the power to detect a difference was low. These results are consistent with prior research in ferrets, which demonstrated that virulence was similar for adamantane-resistant and -susceptible influenza A (H3N2) isolates (–Factors contributing to rapid emergence of adamantane-resistant influenza viruses are not fully known. Resistance has been observed in a variety of outbreak and institutional settings for many years –,7. The pThe 2004–05 influenza season provided an opportunity to learn more about the epidemiology of adamantane resistance during a period when drug resistance was increasing in Wisconsin but was not yet universal. Children had the highest risk of acquiring an adamantane-resistant influenza infection and are an important reservoir for transmission of influenza to adult household members and the community (,CDC continues to recommend that adamantane drugs not be used for influenza treatment in the United States. Given the recent emergence of oseltamivir-resistant influenza A and B infections (