Amy genes, population size andgeneration time seem to have played only minor roles in shaping genestructures.Most eukaryotes have at least some genes interrupted by introns. While it is wellaccepted that introns were already present at moderate density in the lasteukaryote common ancestor, the conspicuous diversity of intron density amonggenomes suggests a complex evolutionary history, with marked differences betweenphyla. The question of the rates of intron gains and loss in the course ofevolution and factors influencing them remains controversial. We haveinvestigated a single gene family, alpha-amylase, in 55 species covering avariety of animal phyla. Comparison of intron positions across phyla suggests acomplex history, with a likely ancestral intronless gene undergoing frequentintron loss and gain, leading to extant intron/exon structures that are highlyvariable, even among species from the same phylum. Because introns are known toplay no regulatory role in this gene and there is no alternative splicing, thestructural differences may be interpreted more easily: intron positions, sizes,losses or gains may be more likely related to factors linked to splicingmechanisms and requirements, and to recognition of introns and exons, or to moreextrinsic factors, such as life cycle and population size. We have shown thatintron losses outnumbered gains in recent periods, but that “resets”of intron positions occurred at the origin of several phyla, includingvertebrates. Rates of gain and loss appear to be positively correlated. No phasepreference was found. We also found evidence for parallel gains and for intronsliding. Presence of introns at given positions was correlated to a strongprotosplice consensus sequence AG/G, which was much weaker in the absence ofintron. In contrast, recent intron insertions were not associated with aspecific sequence. In animal  Over thirty years ago, the discovery that eukaryotic genes were split, interrupted bynon-coding DNA e.g. , caused The focus of the debate have shifted today to understanding intron gains and lossesin the course of the history of eukaryotes, and estimating intron density in thegenome of the last eukaryotic common ancestor. Are intron losses more frequent thanintron gains, and if so, how long has this been the case? Or, in contrast, havegains been a high-frequency phenomenon at some time in the past, and thereafter havedecreased to become rare events at more recent periods? Are rates of gains andlosses correlated? Are rates of gains and losses lineage dependent? Genome dataincreasingly show significant levels of corresponding intron positions in conservedorthologous genes between remote eukaryotes, such as plants and animals A particular point that remains to be clarified is the gene-dependence of introndynamics. Many authors have emphasized the importance of introns in the functions ofa number of genes, for example because of the presence of regulatory informationwithin introns e.g. , or becahttp://www.cazy.orgDictyostelium-type, seems to be thecommon and ancestral type in Unikonts, excluding Bilateria. Regarding the focus ofthe present work, it is interesting to note that the animal-type alpha-amylasestudied here may be considered a “recent” gene, because it is assumed tobe of bacterial origin through horizontal transfer Amyintrons have been reported to date in amylases. Thus, amylase genes will beconsidered free of this kind of constraints, so that the observed structuraldifferences may be interpreted more easily: intron positions, sizes, losses or gainsmay be more likely related to factors linked to splicing mechanisms andrequirements, and recognition of introns and exons, or to more extrinsic factors,such as life cycle and population size. Repeated intron losses in amylase genes havebeen reported in Drosophila and other Diptera Investigating intron dynamics in a single gene (or gene family) in various speciesmay be valuable in this respect Pediculus humanus, the flatworms Schmidteamediterranea and Schistosoma mansoni, the leechHelobdella robusta, the aphid Acyrtosiphonpisum. Alpha-amylase genes were either determined experimentally orobtained from databases. In the latter case, the genome data, generally as draftreleases, were searched with TBLASTN or BLASTP Amy sequence could be used. For raw traces archives data,the best BLAST hits were assembled manually, with attention to the fact thatseveral gene copies may occur. For experimental work, DNAs were extracted withclassical methods. Polymerase chain reaction (PCR) amplifications were performedusing combinations of a set of primers spanning a large part of the codingsequence, listed in Asterias rubens , a partially amplifiedAmy gene was used as a probe for screening a mini-libraryThe same method was applied to Ceratitis capitata andApis mellifera . A genome walking strategy was applied to Bibiomarci, Musca domestica, Spodopterafrugiperda (DNA from Sf9 cells), Blaps mucronata,Periplaneta americana, Dysdera crocata,Lithobius forficatus, Corbicula fluminea,Cerastoderma edule, Mytilus edulis,Patella vulgata.The animal species used in this study are listed in Putative introns were identified by translating the DNA sequences in the threeframes and comparing the results with a manual alignment of known amylaseproteins. Discrepancies and premature stops indicated the presence of introns,the boundaries of which were marked by finding donor and acceptor sites, whichled to an in frame coding sequence. There were a few ambiguous cases in variableregions of the protein, where the alignment was uncertain. When possible, ESTdatabases were used to check the predicted intron positions. Searches forremnants of transposable elements or duplicated neighboring exons within intronswere performed using BLASTX . Intron losses orgains were inferred by parsimony, taking into account the phylogeneticrelationships between the species harboring an intron at a given position.Introns were numbered, choosing intron 1 as the most widespread, conservedposition among Bilateria.Amy genes among animals:from no intron in Drosophila melanogaster to 14 introns in onegene of Daphnia pulex. In some documented cases, there is alsoa variation between gene copies of a single species: in D.pulex, we studied three copies, Amy1,Amy2 and Amy3, with a total of 25positions, 21 of which were specific of one of the three copies. In the AnnelideCapitella teleta, three copies harbored a total of 7 intronpositions, four of which were copy-specific. In the amphioxusBranchiostoma floridae, there were a total of 14 positions,among which 5 were found only in the AmyC copy. In contrast, invertebrates, all the intron positions were shared by all gene copies within aspecies. It was for example the case in Xenopus tropicalis,Tetraodon nigroviridis, and human (not shown).We obtained 79 genomic sequences of partial or complete amylase genes from 55Bilaterian taxa. The extent of sequence available for each gene is shown onD. pulex and D. melanogaster beingrepresentative of the two extremes. The Lepidoptera seem to have many introns intheir Amy genes, whereas Diptera have fewer. The case ofDiptera Amy genes has been detailed in Cionaintestinalis has numerous introns but notOïkopleura, a distant Urochordate with a compactgenome Amygenes, but to our knowledge, there is almost no variation in number: 8 inteleost fishes and 9 in other vertebrates.The diversity of intron/exon structures is highest among arthropods, withAmy gene family, whereas other species show strikingdifferences between copies. The copies that share the same structure aregenerally physically close to each others, like in vertebrates. In theamphioxus, AmyC, which has several specific introns, isisolated, whereas the clustered AmyA and AmyBshare most of their intron positions. Divergence in structure is correlated withdivergence in sequence: AmyA and AmyB share78% identity with each other at the protein level, but withAmyC, only 64% (A/C) or 55% (B/C). In theflour beetle Tribolium castaneum, four Amycopies are tandemly arranged, all of which have the same gene structureaccording to the genome annotation. Interestingly, in a previous cloning of twotandem copies [GenBank: U04271], both copies had been found to lackintron 5. In the whole genome sequenced, the four copies have this intron. Thissuggests a polymorphic presence/absence of this intron in T.castaneum, which has been rarely reported Nasonia vitripennis: two tandem copiesAmy2 and Amy3, very close to each other,share the same structure, whereas a third copy Amy1, found on adifferent contig, has a different structure. Thus, the conservation ofintron/exon structure between gene copies, like the coding sequence, may belinked to their physical vicinity, as expected if the duplication events aremore recent than with more distant copies. Additionally, there may be someconcerted evolution (likely gene conversion was observed betweenAmy1 and Amy1' of Daphniapulex). However, although the intron positions are similar intandem copies, it is also frequent that the sizes and sequences of homologousintrons are very different . In thecentipede Lithobius forficatus, there are two close tandemAmy copies. Intron 1 sequences (data not available forother introns in Amy2) are of different sizes (1417 bp vs. 1130bp) and share no similarity at all. In the tandem Amy genes ofNasonia vitripennis, the coding sequences are 90%identical, whereas there is no similarity in introns, except the most distal(3′) intron 63, which is highly conserved.As mentioned above, in some species, the same intron/exon structure is shared bythe whole Capitella, and position −6, which is the onlyintron found in the region encoding the signal peptide. Owing to low sequenceconservation of this region, orthology of this position among species is notsure , 27 are in phaseone (between the first et second base of a codon) and 22 are in phase two(between the second and third base of a codon). This distribution is notsignificantly different from a 1∶1∶1 proportion, and is different from theusual bias observed toward phase 0 introns 2 = 5.09, ns).Intron-rich genes show a spread of the introns along the whole sequence. Incontrast there is no clear trend for intron-poor genes (one or two introns) tohave their introns located at the beginning of the sequence. However in somecases the pattern of distribution does appear unusual: in a number of Molluscanand Annelide sequences, most introns are concentrated in the 5′ part ofthe gene, with the exception of a single 3′ one.All de novo intron insertions are largely unbiased, and thatselection drives the emergence of protosplice-like sequences following introninsertion We also studied intron insertion sites. Exonic nucleotides flanking intronpositions are not random: with observed preference for a consensusAG/(intron)/GT Amy genes is the main goal of this study. Relating thepresence/absence factual data to the loss/gain interpretation is mainly a matterof sampling. From our data set, we have been able to identify numerous intronlosses at various periods in the past, ancient losses basal to a whole phylum,or more recent, phylogenetically more limited losses. For example, Oïkopleura and in Caenorhabditis. Wefocused on Drosophila Amy genes more thoroughly by PCR and weconfirmed that intron 1 was lost independently in several lineages within thelast 30 million years, but only in the subgenus Sophophora, inwhich the number of gene copies is more than one , whereas no intron loss was recorded in thesubgenus Drosophila, in which Amy seems to begenerally single-copy. It is possible that increased intron losses could berelated to the number of gene copies. Intron position 11 is also consideredancestral, but would have been lost, according to the data, in Mollusks, inEchinoderms, and in Diptera, Coleoptera and Hymenoptera. Intron 5 is alsowidespread and old, since it is shared throughout Protostomes. It is alsopresent in the amphioxus, but since it is absent from other Deuterostomes, it isnot clear whether it is an ancestral position or represents a case of parallelinsertion, unless one considers intron 4 (vertebrates) as the result of a 5bp-sliding from position 5, a hypothesis for which there is no evidence.Investigating the rates or frequencies of intron losses and gains inCapitella. Thisis more suggestive of parallel gains. Indeed, some of these uncertain casescould be solved with an increased sample. Some cases are however clear: intron35 has been lost in fishes since it is present in the lamprey P.marinus and the tetrapods; intron 34 has been lost in C.elegans, since it is present in C. briggsae,C. brenneri, C. remanei andPristionchus pacificus (not shown). Other examples may befound in The tree in Widely conserved introns andphylum-specific introns above). The assignment of intron losses andgains is thus dependent on sampling. The extreme case is for introns limited toone species or one gene in the data set. For instance, one can infer an introngain for position 25a in the Nematocere A. sarothamni becausethe dipteran sampling is good; intron 53b may have been gained in the waspN. vitripennis, by comparison with other Hymenopterasequences. In Bivalves, the oyster and scallop sequences each show a specificintron , which could be examples of species-specificintron gains. However, this interpretation is weakened because we do not knowall Amy copies of each species. It is thus possible that theseintrons are present in other copies of the other species. The most strikingexample of possible gains is that of Daphnia pulex. Thisspecies has the most split Amy gene (14 introns), and 14species-specific positions, which can be considered gained. Indeed, many likelyparalog-specific gains are inferred. Considering Amy1 andAmy2, which are related to each other, since they areclustered in an animal Amy gene tree (not shown), there areonly two common positions (not shared with Amy3 nor any othersequence), but six additional positions which are unique toAmy1, reflecting either gains in Amy1 orlosses in Amy2. The structure of Amy3 is lessderived, sharing five introns with other Arthropods: 15, 21, 37, 52, 57, 63.This suggests a complex structural history after gene duplications in thislineage. However, though we are confident that these positions are gains, thesampling for Branchiopoda is limited to this species, so that the exactantiquity of these introns is unknown. They could also be shared by other,unsampled Crustacean clades. Overall, we did not find conclusive cases of introngains more recent than 150–200 million years.Cases of gains are also likely, but most often less directly conclusive, andthere is no obvious case of “recent” gain. The seemingly high numberof gains on terminal branches of the tree  does notIn contrast, phylum-specific positions, corresponding to ancient gains stemmingfrom the origin of the phyla, perhaps during the Cambrian or late Precambrianexplosion, seem to be frequent. It is visually clear on D. pulex, positions −2and −1, one base pair apart, harbored by two paralogsAmy2 and Amy1, respectively, create eachwith intron 1, a very small exon of 8 bp and 7 bp, respectively. This wasconfirmed by ESTs (http://wfleabase.org/genomics/est/) and our experimentalresequencing of this gene region. Introns 8 (Amy3) and 9(Amy2) are also one base pair apart. Rather thanindependent gains in paralogous copies, both cases strongly suggest 1 bp intronsliding, the most frequently encountered, according to some authors Intron sliding is suspected to have occurred when two intron positions are veryclose to each other between two gene copies or two species. The phylogeneticsample must be sufficient enough to rule out parallel insertions. In our study,some positions are conspicuous: in Amy arose by horizontal transfer from a bacteriumafter the split of Cnidaria Amy genes cannotbe regarded as “ancient genes” such as those included in clusters oforthologous genes (COG) and used in comparative genomic studies . This suggests thatvertebrates could have conserved ancestral exon-intron structures, while mostother phyla would show derived patterns. However, this reasoning does not holdregarding Amy since we posit that the bilaterianAmy gene originated from bacteria, and then was devoid ofintrons in the early Bilateria. Genome data indicate that 85% of intronpositions in the amphioxus B. floridae are shared withvertebrates Amy genes were shared withProtostomes, and with Protostomes only, whereas few intron positions are sharedamong Deuterostomes. This situation is intriguing. This suggests either repeatedparallel gains, or retention of ancestral positions. Parallel gains arepossible: other Deuterostomes share about one Amy intronposition with Protostomes. But seven parallel gains in this single species raisequestions. On the other hand, retention of ancestral positions implies that thelast ancestor between Protostomes and Deuterostomes had Amygene(s) with a Protostome-like structure, which, in Deuterostomes, was retainedsolely in the amphioxus; but there is no clear pattern of conservation of theamphioxus introns with those of a subset of Protostomes. Instead, thesecoincident positions are scattered among various protostome species. Many lossesare needed for this scenario. Thus, there is still little evidence for thishypothesis. A mixed model is also likely. In any hypothesis, the observedpattern implies numerous intron losses and gains in vertebrates, urochordates,echinoderms. In many genes of the urochordate Oïkopleuradioica, introns have strikingly moved even after the split fromCiona intestinalis, so that most of them arespecies-specific At the genome level, vertebrates share many more intron positions than expectedwith the sea anemone Drosophila, showing that losseswere eight times as frequent as gains Amy genes from various Drosophila species probably lessthan 20 MY ago A. sarothamni may be younger, but it is not possible to dateit. Insect Amy genes have rather undergone mostly intron losses alsofrequently exist in the 5′ part of the gene, and in several species, aproximal, clearly old intron was lost (e.g. intron 1), but not other, moredistal ones. This shows that the mechanism may be more complex, or use more5′ partial retrotranscripts, with internal priming. In our study, genomicdeletions seem not to be responsible for the observed losses, since all intronlosses are accurate, removing or adding no coding sequence, contrary to whatoccurred for example in the jingwei gene of DrosophilateissieriThe most frequently assumed mechanism of intron loss considers a cDNAintermediate produced by endogenous reverse transcriptases Amy genesexperiencing intron losses should be transcribed in the germline, at least at abasal level. Differential levels of germline expression among species couldaccount for the differential variability of intron-exon structure. For example,to explain the intron-rich structures of Amy genes, with littlevariability in the Lepidoptera studied here, since their divergence from eachother over 75 MY ago . Nonetheless, supporting this hypothesis, thecomparison between Amy and its paralog Amyrelwithin flies shows that while the Amy intron (intron 1) waslost several times, there was almost no case of loss in Amyrel(intron 17) in more than 200 species studied, diverged from 0.5 to over 80 MYD. melanogaster,Amy is transcribed in testis whereasAmyrel is not (nor in ovaries).To be transmitted to the progeny, an intron loss or gain must occur in thegermline, as pointed out earlier Amy. This model might apply to other species with shortexons and long introns of our sample, such as Lepidoptera or the shrimpL. vannamei. In some other species, in contrast, intronsare numerous but very short, e.g. in D. pulex, C.elegans and C. briggsae. In these species, thegenome of which has been sequenced, short introns are a general feature. In mammals, nine long introns are present, yet amylaseis produced at a high level. In A. thaliana, Knowles et al.A correlation between the intron number or length and the level of expression ofamylase could be expected as an optimization to lower the cost of transcriptionin highly expressed genes Oïkopleuradioica, whose life cycle is four days long vs. short-cycle animals with fewintrons. For instance, C. elegans probably has dozens ofgenerations a year, and a six-intron Amy gene . The same observation applies to the moth S.frugiperda, which has about 12 generation per year in the wild, andyet, its amylase gene has six long introns, increasing the gene size by150%.Intron richness of organisms may be influenced by generation time and cell cycleduration, as mentioned and discussed earlier Amy gene structures.This suggests that demographic factors had little influence in our case.The influence of population size on shaping gene structures has been proposed asa major evolutionary force Pol genes. As forintron loss, RT would produce partial cDNAs, which would be reinserted in thegenome. Internal duplications at the DNA level could also be a source of intronsif correct splicing sequences were present at the duplicate ends. We foundneither trace of such intronization of duplicated exons in the putatively mostrecently gained introns nor evidence of intron gain through (recent) introntransposition between two genes or within the same gene by reverse splicing. Ourability to detect intron transposition may be scrambled . Recentworks suggest that DNA repair through nonhomologous end joining could generateintrons from any template, explaining that one rarely discover a“parental” sequence. This mechanism creates short direct repeats atthe insertion site Several mechanisms of intron gains have been proposed. But since intron sequencesevolve rapidly, the origin and the insertion mechanism of an intron cannot beidentified, unless the intron gain is recent enough. Indeed, we never found theorigin of gained introns in our data. We found no case of introns created byinsertion of transposable elements, although a few introns contained transposonfragments . Howeverbled see  becauseAmy introns to be surrounded by sequencesmatching the conservative protosplice sequence AG/G; interestingly, thesurrounding bases −2 to +2 fitted the protosplice far less well in“empty” sites Click here for additional data file.Table S1List of the the most useful PCR primers used for partial amplificationof alpha-amylase genes in animals. Position is relative to theD. melanogaster sequence. *: specific forDrosophila Amy gene amplification excluding theAmyrel paralog.(DOC)Click here for additional data file.Table S2Sequences surrounding intron sites. Color code is as in Black: no intron. For unalignable positions, no corresponding intronlesssite could be indicated in other genes (e.g. position 10).(XLS)Click here for additional data file.Table S3Alternative scenarii to the intron gains/losses shown on (DOC)Click here for additional data file.Table S4Amy genes in sequencedgenomes.Accessions of (DOC)Click here for additional data file.Table S5Results of BLASTX searches in long introns of the data set. RT: reversetranscriptase.(DOC)Click here for additional data file.


Listeria monocytogenes (LM) or vesicular stomatitis virus (VSV). PD-L1 blockade impaired antigen-specific CD8 effector T cell expansion in response to LM, but not to VSV infection, particularly limiting short-lived effector cell differentiation. Simultaneous CD4-T cell depletion and anti-PD-L1 blockade revealed that PD-L1 provided costimulation even in the absence of CD4-T cells. Most importantly, specific blockade of PD-L1 binding to CD80 or to PD-1 did not recapitulate PDL-1 blockade. The results suggested that PD-L1 plays an important costimulatory role for antigen-specific CD8 T cells during LM infection perhaps through a distinct receptor or interaction epitope.Programmed death ligand-1 (PD-L1) is an important negative regulator of T cell immune responses via interactions with PD-1 and CD80. However, PD-L1 can also act as a positive costimulator, but the relevant counterreceptor is not known. We analyzed the role of PD-L1 in CD8-T cell responses to infection with  Optimal T cell activation requires three signals: 1) interaction between TCR and the cognate peptide-MHC complex, 2) positive costimulation of antigen-specific T cells to promote expansion and survival Listeria monocytogenes (LM), but not to vesicular stomatitis virus (VSV) infection. PD-L1 signaling augmented the proliferation of responding CD8 T cells and modulated differentiation of the short-lived effector cell subset via a CD4 T cell independent mechanism. Moreover, PD-L1 signals appeared to be delivered through a PD-1 and CD80 independent pathway, thereby suggesting the possible existence of an additional PD-L1 ligand.In contrast to the inhibitory roles played by the PD-1 pathway, PD-L1 can also serve as a positive costimulator. PD-L1 interactions promote bacterial clearance 3 cfu LM-OVA or 1×105 pfu of VSV-ova i.v.C57BL/6 mice were purchased from the National Cancer Institute. All animal protocols were approved by the University of Connecticut Health Center Animal Care Committee. Mice were infected with 1×10Mice were treated with 200 µg mAb specific for PD-L1 10F.9G2 , PD-L2 , PD-1 R or 43H12Mice were treated with 1 mg BrdU i.p. 16 hr before sacrificing. Staining of BrdU incorporation followed the BrdU Flow kit protocol (Becton-Dickinson).6 cells/ml) were stained with peptide:MHC tetramers, and other antibodies as indicated. The LLO-I-Ab tetramer Single-cell suspensions were prepared by collagenase digestion as previously described t-test. For bacterial counts statistical significance was determined with a Mann-Whitney test, and for data containing more than 2 groups, a one-way ANOVA test was applied by GraphPad Prism.Statistical significance was determined with unpaired LM infection induced higher levels of PD-L1 on bulk CD8 T cells as compared to levels induced by VSV infection , anti-PD-L2 (TY25), or anti-PD-1 (RMP1-14) blocking mAb throughout the infection. The pMHCI tetramer-OVAD-1 mAbs . Interesblockade , indicatblockade . Howeverblockade . Thus, PEffector T cell heterogeneity is a hallmark of CD8 T cell responses to infections b-specific CD8 T cells that did incorporate BrdU during PD-L1 blockade the level of incorporation was reduced (To further understand the mechanism of PD-L1 costimulation we examined early proliferation of antigen-specific CD8 T cells. To this end, we administered BrdU to infected mice 16hrs before sacrifice with or without PD-L1 blockade. Incorporation of BrdU into CD8 T cells was analyzed on day 5 post-infection. While m reduced . Using a reduced . Thus, Phigh CD8 T cells was not different between the groups (PD-L1 preferentially costimulated the CD8 T cell response with little effect on the CD4 T cell response . Since te groups , suggestThe two known counter-receptors of PD-L1 are PD-1 and CD80, both of which are well documented to transduce negative regulatory signals during T cell activation While much research has focused on the inhibitory effects of the PD-L1/PD-1 axis, positive costimulatory effects of these and other predominantly negative regulators have also been described. The underlying reasons that determine negative versus positive regulatory events are not clear. Our studies show obvious contextual cues that control the requirement for PD-L1 mediated costimulation since the CD8 T cell response to VSV infection was PD-L1 independent while the response to LM infection integrated positive signals from PD-L1 costimulation . PreviouOur data also indicated that both positive and negative regulation were occurring simultaneously through PD-1 and PD-L1. Thus, while PD-L1 inhibition reduced the overall CD8 T cell response and decreased protection, PD-1 blockade enhanced bacterial clearance without consistently affecting the overall magnitude of the CD8 T cell response. The latter result suggested that PD-1 may be inhibiting the functional abilities of CD8 T cells or was affecting innate immune system components. Of additional significance was the demonstration that PD-L1 costimulation operated cooperatively, but independently of CD4 T cell help. Thus, the summation of the positive and negative signaling events mediated through PD-1/PD-L1 family members served to fine-tune the overall immune response to provide protection while maintaining the integrity of the host.


The first developed a rapid cortical neurodegenerative phenotype in the early postnatal period, characterized by fragmentation of TDP-43 and loss of endogenous murine Tdp-43, but entirely lacking aggregates of ubiquitin or TDP-43. A second, low expressing line was aged to 25 months without a severe neurodegenerative phenotype, despite a 30% loss of mouse Tdp-43 and accumulation of lower molecular weight TDP-43 species. Furthermore, TDP-43 fragments generated during neurodegeneration were not C-terminal, but rather were derived from a central portion of human TDP-43. Thus we find that aggregation is not required for cell loss, loss of murine Tdp-43 is not necessarily sufficient in order to develop a severe neurodegenerative phenotype and lower molecular weight TDP-43 positive species in mouse models should not be inherently assumed to be representative of human disease. Our findings are significant for the interpretation of other transgenic studies of TDP-43 proteinopathy.The majority of cases of frontotemporal lobar degeneration and amyotrophic lateral sclerosis are pathologically defined by the cleavage, cytoplasmic redistribution and aggregation of TAR DNA binding protein of 43 kDa (TDP-43). To examine the contribution of these potentially toxic mechanisms  TARDBP, in ALS, which are principally though not exclusively found in the glycine rich C-terminus TAR DNA binding protein of 43 kilodaltons (TDP-43) is the major pathological substrate in frontotemporal lobar degeneration with TDP-43 pathology (FTLD-TDP) and most cases of amyotrophic lateral sclerosis (ALS) in vitro confers toxicity and knockout results in lethality in vivo, both in utero in constitutive −/−Tardbp mice and in conditional knockout animals in which deletion is postponed until adulthood It is unclear if one or several of these pathological TDP-43 alterations is a dominant driving mechanism in disease, and there is evidence to support the toxicity of each in FTLD-TDP. Pathological C-terminal fragments of the protein are more aggregate-prone, undergoing phosphorylation, ubiquitination and ultimately demonstrating cytotoxicity in cells in vivo, a host of constitutive and inducible transgenic rodents expressing human or mouse (wild type and mutant) TDP-43 under the control of various promoters have been created, which recapitulate some of the hallmark pathology of FTLD-TDP/ALS including inclusions of ubiquitinated, phosphorylated TDP-43 and TDP-43 immunoreactive lower weight molecular species To determine if one mechanism is more dominant than others All procedures were conducted according to the National Institutes of Health guide for animal care and approved by the Institutional and Animal Care and Use Committee at University of Florida or Mayo Clinic.BsrBI digestion, gel purified followed by β–agarase digestion (NEB), filtration and concentration. The modified TDP-43 transgene was injected into the pronuclei of donor FVB/NCr embryos (Charles River). 14 founders were positive for the TDP-43 responder transgene. These were then bred with 129S6 mice (Taconic) with the tetracycline transactivator (tTA) transgene downstream of calcium calmodulin kinase II alpha (CaMKIIα) promoter elements iTDP-43 mice were generated similarly to a previously described protocol Antibodies used in this work are described in table s1. Epitopes recognized by TDP-43 antibodies are detailed in After euthanasia via cervical dislocation, brains were harvested and divided along the midline. The right hemisphere was flash-frozen on dry ice, while the left hemisphere was drop fixed in 10% neutral buffered formalin for histological analyses. Brains were embedded in paraffin and cut into 5 µm sagittal sections. For hTDP-43, caspase 3 and ubiquitin antibodies, tissues were immunostained using the DAKO Autostainer  with DAKO Envision HRP System. For p403/404 detection, sections were deparaffinzed and hydrated through a graded alcohol series prior to antigen retrieval in citrate buffer  for 30 minutes in a steamer with water at a rolling boil. Blocking was performed using DAKO Protein Block for 1 h followed by incubation overnight with primary antibody at 4°C. Peroxidase conjugated secondary antibody was visualized with diaminobenzidine . Hematoxylin and eosin (H&E) staining was performed by standard procedures.Sections were prepared as above, except visualization of primary antibody was performed using Alexa Fluor 488 secondary antibody (Invitrogen). Slides were dipped in Sudan Black to reduce background autofluorescence and vectashield with DAPI (Vector Laboratories) was used to stain nuclei. Images were captured using an Olympus BX60 microscope (Olympus).g for 20 minutes at 4°C. For experiments using HEK293T cells, cells were washed once with and then harvested in ice cold PBS. Cells were then pelleted by brief centrifugation at 500×g and lysed in lysis buffer as described above. 30–50 µg protein was loaded onto 10% or 15% Tris–glycine polyacrylamide gel (Novex). Following electrophoresis and transfer, nitrocellulose membranes were blocked with 5% milk in TBS-T , incubated with appropriate primary and HRP-conjugated secondary antibodies and visualized using ECL reagent (Perkin-Elmer). Images were captured on the ProteinSimple FluorChem E . In all cases where blotting was to be quantified, lysate prepared fresh from homogenate was used, and following probing with antibody of interest the blot was stripped (70 mM SDS in Tris HCl (pH 6.8) with 0.7% BME for 30 minutes at 55°C) and re-probed with loading antibody. Densitometry was performed using AlphaView (ProteinSimple), and following normalization to loading controls, fold changes calculated and unpaired t-test used to assess significance.To isolate SDS-soluble fractions, brain or dissected cortex was homogenized in lysis buffer ) at 6 ml/g tissue, and aliquots of homogenate stored at −80°C. Lysate was prepared via brief sonication, addition of SDS to 1% and centrifugation at 40K×g. The supernatant containing the high salt fraction was snap frozen on dry ice and the pellet washed and re-homogenized in 5 ml/g myelin floatation buffer . This homogenization, centrifugation and fraction collection sequence was repeated with sarkosyl buffer , and finally in 1 ml/g urea buffer . 50 ug of high salt extract and equivalent volumes of all other fractions underwent Western blotting as described above.To analyze solubility of TDP-43 fragments, cortex from P5 animals was homogenized in 5 ml/g of high salt buffer , incubated at 4°C for 15 minutes and clarified by centrifugation at 110K×(−1/slope), where slope was determined by plotting the Cq (quantification cycle) values against the log10 input of a cDNA dilution series. A 1/50 dilution of each experimental cDNA sample was run. From this, the relative quantities (RQ) were calculated for each primer pair in each sample and normalization of Tardbp performed using the geometric mean of the two reference genes Gapdh and β-actin, giving the Normalized Relative Quantity (NRQ) Tardbp1 F (5′AAAGGTGTTTGTTGGACGTTGTACAG 3′), Tardbp1 R (5′ AAAGCTCTGAATGGTTTGGGAATG 3′), Tardbp2 F (5′ GATTGGTTTGTTCAGTGTGGAGTATATTCA 3′), Tardbp2 R (5′ ACAGCAGTTCACTTTCACCCACTCA 3′), Tardbp3 F (5′ GGTGGTTAGTAGGTTGGTTATTAGGTTAGGTA 3′), Tardbp3 R (5′ AAATACTGCTGAATATACTCCACACTGAACA 3′), Gapdh F (5′ CATGGCCTTCCGTGTTCCTA 3′), Gapdh R (5′ CCTGCTTCACCACCTTCTTGAT 3′), β-actin F (5′ GATGACCCAGATCATGTTTGAGACCTT 3′) and β-actin R (5′ CCATCACAATGCCTGTGGTACGA 3′). Single PCR products were verified by melt curve analysis. Statistical significance was assessed using unpaired t-test.Total RNA was isolated from dissected hippocampus or cortex using TRIzol reagent (Life Technologies) and Pure Link RNA Mini Kit (Life Technologies). During this step, DNA was removed using on-column DNase digestion (Life Technologies) and the resulting RNA purity and integrity was assessed on spectrophotometer  and agarose gel electrophoresis. 2 µg of RNA was used to synthesize cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). All samples were run in triplicate on the ABI 7900 HT Real-Time PCR Detection System using SYBR green PCR master mix (Applied Biosystems). Primer efficiencies (E) were calculated using E = 10208–414 and TDP-431–280. The primers used were TDP-43208–414 F (5′-TACGGATCCCACCATGGAACAAAAACTCATCTCAGAAGAGGATCTGCGGGAGTTCTTCTCTCAGTACGG-3′); TDP-43208–414 R (5′- GAATCGCGGCCGCCTACATTCCCCAGCCAGAAGACT-3′); TDP-431–280 F (5′-TACGGATCCCACCATGGAACAAAAACTCATCTCAGAAGAGGATCTGATGTCTGAATATATTCGGGTAACCGAA-3′); TDP-431–280 R (5′-GAATCGCGGCCGCTCATGGATTACCACCAAATCTTCCAC-3′). Products were cloned into pcDNA3.1 (Life Technologies) using BamH1 and Not1 sites and plasmids were verified by sequence analysis.Full-length human TDP-43 complementary cDNA in plasmid pEGFP-C1 Human Embryonic Kidney 293T cells were maintained in Dulbecco’s Modified Eagle’s Medium (Lonza) supplemented with 10% Fetal Calf Serum (Sigma) and penicillin-streptomycin (Life Technologies). Transfection in 6 well plates was performed for 48 hours using 2.0 ug of plasmid and Lipofectamine 2000 (Life Technologies) following the manufacturer’s guidelines.8A animals developed normal brain structure, there was obvious reduction in the cortical volume of iTDP-4314A brains compared to non-transgenic (NT) littermates  to produce bigenic iTDP-43 mice expressing human mutant TDP-43. We initially screened iTDP-43 mice at two months of age for expression of human TDP-43 and selected the two highest expressing lines (14A and 8A) for further analysis. Transgene expression in bigenic animals from both lines was limited exclusively to the brain, predominantly in the cortex, hippocampus and striatum , consisttermates .14A, we examined postnatal ages from P0 to P60 to determine the time point of initial phenotypic onset. During this period, brain weights of NT mice experienced a rapid phase of growth between P0 and P12, followed by more modest increases into adulthood  by P5 and iTDP-4314A brain weight never reached that of non-transgenic litter mates , total TDP-43 (tTDP-43 Ab1) and TDP-43 phosphorylated at residues 403/404 (p403/404) and 409/410 (p409/410) to investigate the contribution of aggregation to the cell death observed in P5 iTDP-43g cortex . Despiteg cortex . We did 14A mice. To validate the antibodies used, we overexpressed either a C-terminal pathological fragment identified in human cases of FTLD-TDP (TDP-43208–414) 1–280), both with an N-terminal myc tag, in HEK293T cells. Subsequent western analysis of lysate verified that the C-terminal antibody detected TDP-43208–414, but not TDP-431–280. Conversely, tTDP-43 Ab2, which is raised to residues 181–198, detected TDP-431–280 but not TDP-43208–414. Human specific antibody and myc antibody detected both. TDP-43 3–12 detected TDP-431–280 but not the C-terminal fragment  and 25 kDa (TDP-25) using total TDP-43 Ab1 and total TDP-43 Ab2 . As transgenic lines driven by the CaMKIIα promoter or bearing the M337V mutation have already been reported that do exhibit these phenotypes 14A mouse line occurred in the complete absence of aggregation. This observation is consistent with previous research demonstrating that aggregation does not appear to be a major driver of cell death in TDP-43 transgenic animals 14A, and this has been implicated elsewhere as a potential driver of neurodegeneration where it correlates with phenotypic progression 8A – in which we observed downregulation of mTdp-43 of 30% until 25 months of age, the oldest age examined – indicates that if loss of murine Tdp-43 is capable of driving aggressive neurodegenerative phenotypes, a critical threshold must be reached. Interestingly, we failed to detect downregulation of murine Tardbp transcript in either young or aged iTDP-438A cortex despite the reduction in mTdp-43 protein. This contrasts with findings from our lab and elsewhere demonstrating in vivo, hTDP-43 autoregulated reduction in Tardbp as the underlying mechanism of reduced Tdp-43 8A mice is sufficient to cause a measurable reduction in Tdp-43.The human neuropathology of FTLD-TDP - consisting of loss of nuclear TDP-43, TDP-43 aggregation, phosphorylation and fragmentation to C-terminal lower molecular weight species - has driven hypotheses as to TDP-43 toxicity both in human disease and in mice expressing transgenic TDP-43. Herein, we found that early neurodegeneration in the iTDP-4314A may be fragmentation of TDP-43 to the low molecular weight species TDP-35 and TDP-25. At the earliest phenotypic time point studied here, we observed accumulation of these species at least partially derived from the human TDP-43 transgene and found evidence of detergent insoluble, urea soluble TDP-35. Although these species may be inherently neurotoxic and contribute to cell death in iTDP-4314A, we also determined that they are derived from a central portion of TDP-43 and are thus may not be biochemically identical to the C-terminally derived fragments of human disease. Consequently, we cannot be certain that any neurotoxicity these species invoke is relevant to human pathology. More study is required to determine if these fragments are identical to those almost universally present in other TDP-43 transgenic animals. Elsewhere, these fragments have been described as C-terminal in nature, based on the use of antibodies raised to the C-terminal 154 residues of TDP-43 A potential additional source of neurotoxicity in iTDP-4314A is aggressive and occurs prior to full development of the brain, an effect that we have documented in a previous line constitutively expressing wild type TDP-43 and that may be the case in several other high expressing transgenic lines with early phenotypes in vivo models that faithfully recapitulate age dependent accumulation of disease relevant TDP-43 pathology.Consequently, our data here add to prior studies suggesting that neurodegenerative phenotypes in existing TDP-43 transgenic animals might be influenced by a number of factors depending on the transgenic line, and that it may prove difficult to separate toxicity of potentially disease relevant phenomena from one another or from global RNA dysregulation that occurs upon mutant TDP-43 overexpression Figure S1TDP-43 antibodies used in this study. N-terminal, C-terminal, mTDP-43 and tTDP-43 Ab2 are raised to the indicated epitopes. hTDP-43 and tTDP-43 Ab1 antibodies have been mapped to the indicated epitopes in previous studies (TIF)Click here for additional data file.Figure S214A phenotypes. (A) Brain weights of NT, monogenic hTDP-4314A and monogenic tTA mice at P5 and 2 months of age were identical. (B) Western analysis of P5 brain lysate demonstrated no increase in activated caspase 3 in tTA only mice compared to NT mice. However, elevated activated caspase 3 was observed in iTDP-4314A.tTA expression is not responsible for iTDP-43(TIF)Click here for additional data file.Figure S31–280 or myc-hTDP-43208–414. Blots were probed with the indicated antibodies.Validation of TDP-43 antibody specificity. Western analysis of lysate from HEK-293T cells transfected with empty vector, myc-hTDP-43(TIF)Click here for additional data file.Figure S48A mice covering the postnatal period and aged cohorts. No significant difference in weight was observed in the time period to 2 months; tTA and iTDP-43 mice in 10 month and 25 month cohorts showed a small, significant decrease relative to NT mice (N(P0) = 4 per genotype; N(P12) = 3 per genotype; N(P21) = 4 per genotype; N(2 M) = 5 per genotype; N(10 M) = 5 NT, 8 iTDP-43, 7 tTA; N(25 M) = 7 NT, 7 iTDP-43, 5 tTA. Error bars are SD, *p<0.05, one way ANOVA, Bonferroni post hoc). (B) Hematoxylin and eosin staining of 25 M cohorts from line iTDP-438A show no gross morphological differences between tTA and iTDP-438A cortex. There was a trend toward smaller dentate gyri in tTA mice compared to NT mice. (C) Long exposure of western blot of 11 M NT and tTA cortical lysates using antibody to total TDP-43 (tTDP-43 Ab2), mouse specific TDP-43 antibody was used to probe same lysates, β-actin for loading. (D) Densitometric quantitation of blots in (C) confirmed no change in TDP-43, TDP-35 or TDP-25 in tTA mice versus NT mice; values are represented as fold change relative to NT mice following sample normalization to β-actin loading controls.Expression of tTA trangene does not affect TDP-43 metabolism. (A) Brain weights of iTDP-43(TIF)Click here for additional data file.Table S1Primary antibodies used in this study.(DOCX)Click here for additional data file.


CX3CR1, FKBP1A, NOG, RBM12B, SENS3, TSHZ2; T cell/JAK-STAT pathway genes such as AKT3, CBLB, DLG1, IFNG, IL2RA, IL7R, ITK, JUN, MALT1, NFATC2, PLCG1, SPRED1). In summary, this validated peripheral blood molecular gene signature appears to be a valuable biomarker in identifying cases with sarcoidoisis and predicting risk for complicated sarcoidosis.Sarcoidosis, a systemic granulomatous syndrome invariably affecting the lung, typically spontaneously remits but in ∼20% of cases progresses with severe lung dysfunction or cardiac and neurologic involvement (complicated sarcoidosis). Unfortunately, current biomarkers fail to distinguish patients with remitting (uncomplicated) sarcoidosis from other fibrotic lung disorders, and fail to identify individuals at risk for complicated sarcoidosis. We utilized genome-wide peripheral blood gene expression analysis to identify a 20-gene sarcoidosis biomarker signature distinguishing sarcoidosis (n = 39) from healthy controls  and which served as a molecular signature for complicated sarcoidosis (n = 17). As aberrancies in T cell receptor (TCR) signaling, JAK-STAT (JS) signaling, and cytokine-cytokine receptor (CCR) signaling are implicated in sarcoidosis pathogenesis, a 31-gene signature comprised of T cell signaling pathway genes associated with sarcoidosis (TCR/JS/CCR) was compared to the unbiased 20-gene biomarker signature but proved inferior in prediction accuracy in distinguishing complicated from uncomplicated sarcoidosis. Additional validation strategies included significant association of single nucleotide polymorphisms (SNPs) in signature genes with sarcoidosis susceptibility and severity (unbiased signature genes -  Individuals with sarcoidosis, a systemic inflammatory and non-caseating granulomatous disease of unknown origin affecting multiple organs and invariably the lung BTNL2 (butyrophilin-like 2) gene to be associated with sarcoidosis development Previous sarcoidosis candidate gene studies focused on granuloma formation and immune response pathways implicated several genes linked to sarcoidosis susceptibility Our study was designed to identify novel genomic biomarkers by comparing genome-wide gene expression data in African American (AA) and European descent ancestry (EA) sarcoidosis cases. We identified a universal gene signature that differentiates sarcoidosis patients from healthy controls and distinguishes complicated sarcoidosis  from uncomplicated sarcoidosis. This gene signature was superior in prediction accuracy in each of the AA and EA populations when compared to a second signature comprised of genes within the T cell receptor–innate immunity pathway that includes genes previously associated with sarcoidosis. These signatures distinguished sarcoidosis patients from idiopathic pulmonary fibrosis (IPF) cases with signature validation provided by significant association of genetic variants within signature genes with sarcoidosis susceptibility. These results highlight the utility of peripheral blood molecular gene signatures as valuable biomarkers for predicting individuals at risk for complicated sarcoidosis and for potentially facilitating individualized therapies in this enigmatic disorder.PBMC samples were collected from subjects with sarcoidosis (n = 39) and healthy controls (n = 35) . The cliAll cases with diagnoses of cardiac, neurologic, or severe pulmonary sarcoidosis (FVC<50%) comprised the cohort labeled as ‘complicated sarcoidosis’. At the specified significance level , 316 genes were differentially expressed between all sarcoidosis cases and healthy controls in the combined samples (pooled AAs and EAs). For individual populations, 118 genes were differentially-expressed between all AA cases and controls, whereas 861 genes were differentially expressed between all EA cases and controls. In contrast, 1124 genes were differentially expressed between complicated sarcoidosis cases and healthy controls in the combined samples. For individual population, 730 and 980 genes were differentially expressed between AA and EA cases with complicated sarcoidosis and healthy controls, respectively with the TCR signaling pathway significantly enriched among complicated sarcoidosis-associated genes in both populations (adjusted P<0.05) .HBEGF (heparin-binding EGF-like growth factor) and SAP30 , were strongly up-regulated in complicated sarcoidosis whereas the remaining 18 signature genes were down-regulated in complicated sarcoidosis . Finally, as sarcoidosis and IPF represent the most common interstitial lung diseases (ILDs) of unknown etiology, the capacity for the unbiased 20-gene and TCR/JS/CCR sarcoidosis gene signatures to distinguish sarcoidosis cases from IPF cases (GEO - GSE38958) was assessed. Each signature performed with comparable prediction accuracy in IPF and sarcoid with the 20-gene signature (77.2%) slightly superior to the TCR/JS/CCR signaling pathway signature (76.5%) in distinguishing sarcoidosis from IPF cases (−5 by t-test).As the T cell receptor pathway (TCR), the JAK STAT signaling pathway (JS) and the cytokine-cytokine receptor signaling pathway (CCR) have all been implicated in sarcoidosis We evaluated the performance of our gene signatures in two different independent sarcoidosis blood gene expression datasets. One dataset (GEO - GSE19314) from University of California, San Francisco (UCSF) NOG [noggin], RMB12B [RNA binding motif protein 12B], SESN3 [sestrin 3], TSHZ2 [teashirt zinc finger homeobox 2]). The most highly significant signature gene SNP in AAs was rs629508 (P = 1.7×10−3) in SESN3, whereas in EA cases, the most significant SNP was rs2618134 (P = 4.7×10−5) in RBM12B. Interestingly, several SNPs were also significantly associated with complicated sarcoidosis, including rs629508 (P = 5.4×10−5) and rs1294689 (P = 3.6×10−5) in the AA samples and rs10485815 (P = 2.8×10−5) in the EA samples (−5) in AKT3, whereas in EA cases, the most significant SNP was rs7614488 (P = 7.8×10−7) in CBLB. Several TCR/JS/CCR signature gene SNPs, rs2953040 and rs6791765 in CBLB  and rs2131817 in AKT3 were significantly associated with sarcoidosis in both EA and AA sarcoidosis cases (P<0.01) (A genome-wide association study (GWAS) (Affymetrix 6.0 SNP array) involving 407 sarcoidosis cases including 212 AAs (including 68 complicated cases) and 195 EAs (including 46 complicated cases) was performed and allele frequencies of ∼1,300 common SNPs residing in unbiased sarcoidosis signature genes analyzed in sarcoidosis cases and healthy controls (see Supplementary  samples . In comp samples . The mos(P<0.01) .http://pubmatrix.grc.nia.nih.gov) tool was next used to evaluate the relevance of sarcoidosis signature genes in the published biomedical literatures (PubMed). Each signature gene was searched against a series of terms related to lung fibrosis or sarcoidosis including: “sarcoidosis”, “tuberculosis”, “granulomatous disease”, “hypersensitivity pneumonitis”, and “pulmonary fibrosis”. The majority of 20-gene signature genes were highly novel to these terms . Of the 31 TCR/JS/CCR-gene signature genes, 8/31 genes were cited in the sarcoidosis literature with CD28, IFNG, IL7R, AKT3, IL2RA, IL2RB, and STAT4 demonstrating a robust relationship with these terms  with each signature superior in accuracy to signatures with fewer genes  . The exp−15, t-test). We speculate that the unbiased nature of the 20-gene signature allows better capture of the characteristics of complicated sarcoidosis compared to the more restrictive TCR/JS/CCR signaling pathway signature genes. The potential role of TCR/JS/CCR signaling pathways genes in the development of sarcoidosis was confirmed by the capacity of this signature to successfully differentiate the majority of sarcoidosis and healthy controls. However, we speculate that either sarcoidosis disease progression or the development of complicated sarcoidosis likely requires the participation of genes and pathways extending beyond the TCR/JS/CCR pathway. These findings underscore the complex pathobiology of this disorder and implicate the necessity of global and unbiased approaches.Gene products encoded by TCR/JS/CCR signaling pathway genes have been implicated in sarcoidosis pathogenesis We further evaluated the classification accuracy of the 20-gene sarcoid signature separately in EA and AA samples and found the 20-gene signature to demonstrate >85% accuracy for classifying either EA or AA sarcoidosis cases (complicated and uncomplicated) from healthy controls. In contrast, the 20-gene sarcoidosis signature differentiated complicated sarcoidosis and uncomplicated sarcoidosis cases with an accuracy >80% in AA cases, but only ∼60% in EA cases, potentially the relative smaller complicated EA sample size or a bias for AA expression dysregulation driven by greater genetic variation, an issue which requires further examination. Both the 20-gene signature and TCR/JS/CCR-gene signature successfully discriminated sarcoidosis cases from IPF patients with similar prediction accuracies reflecting the differences in immunopathogenesis, clinical course, prognosis, and response to steroid treatment cis-eQTLs. From ∼1,300 SNPs in our 20 signature genes, we identified 30 SNPs (corresponding to 6 signature genes) which were significantly associated with sarcoidosis in either EA or AA samples, suggesting a potential role of these cis-acting SNPs in regulating the expression of sarcoidosis signature genes. Similarly, from ∼3,800 SNPs in TCR/JS/CCR signature genes, relationships between SNPs and sarcoidosis were observed. While these findings serve to validate the potential importance and relevance of signature genes, a direct association between these SNPs and expression is necessary to validate these relationships. Our results suggest that genetic variants via cis-acting eQTLs may contribute to the variation in expression of sarcoidosis signature genes. We further recognize that additional factors, such as trans-acting eQTLs, environmental factors, or epigenetic pathways, may contribute substantially to signature gene expression variation. Further investigations involving genome-wide genotypic data  and expression data on the same samples could potentially provide greater insights into the contribution of genetics to the identified gene signature.As evidenced by the paucity of PubMed citations (PubMatrix results), the 20-gene signature is comprised of highly novel candidate genes in sarcoidosis susceptibility and severity of disease. As a complementary method to validate our findings HBEGF, a member of the EGF family of growth factors, is a potent mitogen and chemoattractant for many cell types including fibroblasts, smooth muscle cells and epithelial cells HBGEF plays a role in wound healing and response to injury HBEGF may represent a target involved in the pathobiology of chronic lung sarcoidosis and a novel therapeutic target, an observation supported by the PubMatrix search results.Quantitative abnormalities in T cells have been described in the peripheral blood of patients with sarcoidosis LOC100132356 was most cited in PubMed literatures, though it only codes a hypothetical protein. This gene was linked to the terms such as sarcoidosis, tuberculosis, granulomatous disease, hypersensitivity pneumonitis, and pulmonary fibrosis. However, the detailed function of this gene is still unclear.Among our 20-gene signature, Recently, lung gene expression profiles were compared between patients with self-limiting sarcoidosis and those with progressive restrictive fibrotic disease Furthermore, our sarcoidosis gene signatures performed well in two independent validation cohorts (UCSF and Oregon) In summary, despite significant limitations including a relatively small size of the EA complicated cases in the analysis set, an unbiased 20-gene molecular gene signature was identified as a potential novel molecular biomarker in the diagnosis of sarcoidosis as well for the presence of complicated sarcoidosis with substantial accuracy in both EA and AA sarcoidosis cases. With validation in a replicate sarcoidosis cohort and testing against other granulomatous disorders like Wegener’s disease, hypersensitivity pneumonitis, and tuberculosis, this sarcoidosis gene signature may represent a novel universal gene signature for complicated sarcoidosis and serve as a springboard to individualized therapies in this enigmatic disorder.The study was approved by the Institutional Review Board (IRB) of the University of Illinois at Chicago (UIC) with written informed consent obtained from all subjects. The UIC’s IRB committee members (Chairs) include: Indru Punwani, D.D.S., Susan Labott, Ph.D., Paul Heckerling, M.D., and Kathryn Rugen, Ph.D. The DNA samples provided by the Johns Hopkins University investigators, and their use in this study, were approved by the IRB of the Johns Hopkins University. PBMC samples were collected from subjects with sarcoidosis (n = 39) and healthy controls (n = 35) . The diaTotal RNA was isolated from PBMCs using standard molecular biology protocols (n = 74) without DNA contamination or RNA degradation. Sample processing  was performed by the University of Chicago Functional Genomics Facility per manufacturer’s instructions.http://www.affymetrix.com/) , EA, and AA samples, respectively. False discovery rate (FDR) was controlled using the q-value method Human Exon 1.0 ST arrays were summarized using the Affymetrix Power Tools v.1.12.0  using a linear kernel, was applied in combination with recursive feature elimination (RFE) for generating a predictive model . The red line shows the average accuracy for each RFE step.(PDF)Click here for additional data file.Figure S2Distribution of classification accuracies of the 20-gene signature. X-axis: the classification accuracy from a five-fold cross-validation . The dashed lines indicate the average classification accuracy. (A) All sarcoidosis patients versus healthy controls in the AA samples; (B) Patients with complicated sarcoidosis versus patients with uncomplicated sarcoidosis in the AA samples; (C) All sarcoidosis patients versus healthy controls in the EA samples; and (D) Patients with complicated sarcoidosis versus patients with uncomplicated sarcoidosis in the EA samples.(PDF)Click here for additional data file.Figure S3Comparison between the 20-gene signature and the TCR/JS/CCR signaling pathway gene signature in individual populations. The distribution of accuracy is based on 1,000 times of five-fold cross-validation. The dashed lines indicate the average classification accuracy for the 20-gene signature or the TCR/JS/CCR signaling pathway gene signature. HC: healthy controls; US: patients with uncomplicated sarcoidosis; and CS: patients with complicated sarcoidosis.(PDF)Click here for additional data file.Figure S4Capability of the the 20-gene signature and the TCR/JS/CCR signaling pathway gene signature in separating sarcoidosis patients from IPF patients. The distribution of accuracy is based on 1,000 times of five-fold cross-validation. The dashed lines indicate the average classification accuracy for the 20-gene signature or the TCR/JS/CCR signaling pathway gene signature.(PDF)Click here for additional data file.Table S1SNPs significantly associated with sarcoidosis within the 31 TCR/JS/CRR signature genes (P<0.01).(PDF)Click here for additional data file.Table S2PubMatrix search results for the TCR/JS/CCR signature genes against sarcoidosis-related search terms.(PDF)Click here for additional data file.Table S3Patient therapy description.(PDF)Click here for additional data file.Text S1Supplementary methods.(PDF)Click here for additional data file.


The ecological insurance hypothesis predicts a positive effect of species richness on ecosystem functioning in a variable environment. This effect stems from temporal and spatial complementarity among species within metacommunities coupled with optimal levels of dispersal. Despite its importance in the context of global change by human activities, empirical evidence for ecological insurance remains scarce and controversial. Here we use natural aquatic bacterial communities to explore some of the predictions of the spatial and temporal aspects of the ecological insurance hypothesis. Addressing ecological insurance with bacterioplankton is of strong relevance given their central role in fundamental ecosystem processes. Our experimental set up consisted of water and bacterioplankton communities from two contrasting coastal lagoons. In order to mimic environmental fluctuations, the bacterioplankton community from one lagoon was successively transferred between tanks containing water from each of the two lagoons. We manipulated initial bacterial diversity for experimental communities and immigration during the experiment. We found that the abundance and production of bacterioplankton communities was higher and more stable  for treatments with high initial bacterial diversity. Immigration was only marginally beneficial to bacterial communities, probably because microbial communities operate at different time scales compared to the frequency of perturbation selected in this study, and of their intrinsic high physiologic plasticity. Such local “physiological insurance” may have a strong significance for the maintenance of bacterial abundance and production in the face of environmental perturbations. Marine bacterial communities are recognized as major players in the ecology of coastal ecosystems, i.e. nutrient cycling, production and decomposition of organic matter, and the regulation of major biogeochemical cycles Marine bacterioplankton consist of drifting microorganisms that often inhabit large volumes of water. This particularity necessitates the inclusion of their spatial distribution as a central element in understanding the relationship between their diversity and ecosystem functioning. Indeed, while it is intuitive that in connected aquatic systems “everything is everywhere” In a temporally fluctuating environment, diversity may increase the stability of a community through different mechanisms a concentrations, and in their bacterial community structure. Bacterioplankton communities were transferred between lagoons to mimic changes in environmental conditions experienced by natural aquatic bacterial assemblages We investigated how coastal bacterial assemblages with different initial levels of diversity and immigration responded to temporal environmental variation. We used bacterial communities from two distinct Mediterranean lagoons, the Thau and Bagnas lagoons. These two lagoons differ in their salinity, chlorophyll Testing the insurance hypothesis with natural bacterioplankton communities requires the whole community to be subjected to a temporally fluctuating environment. This was achieved by successive transfer of bacterioplankton lagoon communities, with differing levels of diversity and immigration over two contrasting environments.a concentrations (Chl a), over a short geographic distance (<10 km): the Bagnas (43°24′53′′N - 3°41′16′′E) and the Thau (43°19′47′′N - 3°31′13′′E) lagoons. Salinity measured with a Cond probe 197i were 8 and 34 respectively, and Chl a concentrations  and rinsed in deionized water, and were immediately transferred to a field laboratory located at the edge of Thau lagoon. We filled two 900-L polypropylene tanks with either 1 mm filtered water from the Thau or the Bagnas lagoons and allowed a constant water flow (1 day retention time). Our experimental approach consisted of transferring the Thau bacterial community between tanks containing water from the Thau and the Bagnas lagoons to experimentally mimic temporal variation in environmental conditions , most of which are connected to the Mediterranean Sea nditions . In pracnditions . These mPrior to filling with bacterioplankton communities, diffusion chambers were acid-washed (24 h in 10% HCl) and rinsed thrice in deionized water and dried. Chambers were filled with bacterioplankton communities from the Thau Lagoon with three different levels of diversity (see below). −3) and “low” (dilution 10−5) diversity. Measuring band number in a denaturing gradient gel electrophoresis  confirmed that a fraction of the community had been eliminated. As DGGE is effective for detecting dominant bacterial operational taxonomic units (OTU), a decrease in DGGE band number between two dilutions is consistent with a reduction in community diversity. Dilutions of bulk bacterial communities were done with sterile lagoon water (0.22 µm filtrated plus two autoclave cycles at 121°C for 20 min). The dilution treatment could itself influence initial abundances within incubated communities. We thus controlled for bacterial abundances by allowing a 48 h acclimation period in Thau lagoon water before the first environmental transfer.The dilution-to-extinction approach was used to obtain three different starting levels of bacterioplankton diversity. Dilution removes the rare species and thereby different dilutions create communities that differ in their diversity. This approach allowed us to explore the link between community diversity and functioning Identical series of initial diversity levels were prepared for each of the three immigration treatments: no immigration, medium  and high  immigration. Immigration events were performed twice for each chamber, at each transfer  using a FACSCalibur flow cytometer (BectonDickinson) after nucleic acid staining with green fluorescent SYBR®GreenI (Invitrogen). Light scattering was used in conjunction with fluorescence to discriminate bacterial cells -samples .The initial bacterial community diversity was assessed by denaturing gradient gel electrophoresis (DGGE) analysis of the 16S rRNA genes after touchdown PCR amplification Time series for bacterial abundance and production were first analyzed by a three factor analysis of variance (ANOVA) with time, diversity and immigration included as explanatory variables. No interactions among factors were included. Then, we ran a second fully factorial ANOVA on the residuals (thus taking account of the effect of time) with diversity and immigration as explanatory factors. This reveals the effect of diversity and immigration on the abundance and production of our bacterial communities independently of the effect of time. We performed Tukey-Kramer post-hoc tests  to test for differences between diversity and immigration levels on temporal averaged abundance and production.We also calculated the temporal coefficient of variation (CV) for both bacterial abundance and production, for each replicate population, from 48 h (i.e. after acclimation) to the end of the experiment (240 h). The coefficient of variation is a common and convenient metric for temporal stability The diffusion rate of the solutes through the 0.22 µm chamber membranes must be greater than the incubation time between two transfers (48 h) so that communities experience changes in their environment. When chambers containing Thau water were immersed in Bagnas water, the changes in salinity inside the diffusion chambers were gradual and reached equilibrium after 15 h of incubation . ClogginLagoon bacterial cells did not contaminate the experimental bacterial communities within each chamber during a 10 days submersion period . This coA pre-requisite of the experiment is that bacterioplankton from both lagoons differed in their bacterial community composition. One week before the experiment, triplicate water samples were collected from the Bagnas and Thau lagoons to explore differences in the bacterioplankton composition based on DGGE banding pattern analysis. Both lagoons had a similar number of OTUs, but they differed in their OTUs composition .The DGGE analysis of the diluted Thau bacterial community confirmed that our dilution treatments resulted in a bacterial diversity gradient .Overall, bacterial abundances increased during the first 100 h and then decreased through time . We founBacterial production also varied trough time . Temporaperformance-enhancing effect characterized by an increase in temporal mean bacterioplankton abundance and production, and second, a buffering effect characterized by a reduction in the temporal variance of production The insurance hypothesis predicts a buffering effect of both diversity and immigration on ecosystem functioning based on species asynchrony and compensatory dynamics. Using an experimental approach, we observed two effects of diversity when the bacterial communities are confronted with temporal environmental fluctuations. First, a in situ hybridization with microautoradiography, or nano-scale secondary-ion mass spectrometry, which have been shown as promising tools for correlating microbial identity with specific metabolic functions, for individual cells, within heterogeneous bacterial communities Click here for additional data file.Figure S2We assayed how much our diffusion chambers were resistant to potential contamination from the surrounding waters in a pilot experiment performed before the main experiment. This control of sterility was determined by incubating for 10 days four chambers (2 completely waterproof and 2 with 0.2 µm membranes) containing sterile Thau lagoon water into a flow-through natural Thau lagoon water tank. The sterile Thau lagoon water was obtained after filtration through a 0.22 µm polycarbonate membrane plus two cycles of autoclave at 121°C during 20 min. Using this procedure, bacterial abundance was abated by 98.4% (from 2.3 106 cells ml−1 to 3.6 104 cells ml−1). Most of the persisting cells were considered as dead cell. Two milliliters of water were withdrawn from the chambers at t = 0, t = 1 h, t = 24 h and t = 240 h. Bacterial abundance was determined by flow cytometry as described in the experimental procedures section. Bacterial abundance did not significantly change over time and between the waterproof and 0.22 µm membranes diffusion chambers Click here for additional data file.Figure S3One week before the experiment, 200-ml of water from both Thau and Bagnas lagoons were collected with a 500-ml acid washed glass bottle to determine the DGGE banding pattern of the bacterioplankton communities. The DGGE method is describe in the experimental procedure section. The number of OTUs was similar with 17 and 20 OTUs for Thau and Bagnas bacterioplankton, respectively. However, both communities shared only 5 OTUs (y 5 OTUs . This re(DOC)Click here for additional data file.


A total of 19 SNPs on chromosomes (SSC) 3, 5, 8, 10, 14, and 18 were significantly associated with OC lesion scores . The SNPs MARC0098684, MARC00840086, MARC0093124, and ASGA0062794 at SSC14 36.1–38.2 Mb encompass a region of six linkage disequilibrium (LD) blocks. The most significant SNP ASGA0062794 is located in a LD block spanning 465 kb and covering the gene encoding T-box transcription factor 5 (TBX5). A SNP (c.54T > C) identified in TBX5 was significantly associated with OC lesion scores in a single-marker analysis. TBX5 c.54T > C showed highest LD with ASGA00627974 (r2 = 0.96) and superior association with OC lesion scores over other SNPs when included in the genome scan, whereas its treatment as an additional fixed effect in the GWAS statistical model led to a drop of significance of nearby markers. Moreover, real-time PCR showed different transcript abundance of TBX5 in healthy and defect cartilage. The results imply that the association signal obtained on SCC14 is largely attributable to TBX5 c.54T > C likely to be in LD with a regulatory polymorphism of TBX5. The transcription factor TBX5 interacts with GJA5 and MEF2C both being involved in vascularization. This study provides evidence for epistatic interaction of TBX5 and MEF2C, thus supporting deficiency of blood supply to growth cartilage as being fundamental for the initiation of OC.Osteochondrosis (OC) is an orthopedic syndrome of the joints that occurs in children and adolescents and domestic animals, particularly pigs, horses, and dogs. OC is the most frequent cause of leg weakness in rapidly growing pigs causing animal welfare issues and economic losses. In this study, a genome-wide association study (GWAS) was performed using the Porcine 60k SNPChip in animals of the breed Large White ( Osteochondrosis (OC) is a joint disorder characterized by the disturbance of endochondral ossification in the articular cartilage and the epiphyseal growth plates that is common in human beings and several animal species including pigs  gene which represents a functional and according to the GWAS, also a positional candidate gene for OC.In general linkage studies based on genotyping a limited number of highly polymorphic markers, mostly microsatellites, in special family designs have revealed numerous QTL regions for various traits  from SUISAG during 2005–2008. The animals used were selected from the two ends of the distribution of OC lesion scores  recorded from 2,622 animals. In order to avoid bias due to any obvious parameter that might affect OC scores, selection was done accounting for sex, year, body weight, and ancestry; in fact, selected animals represent offspring of 141 boars and 264 dams. The animals were kept in the performance test station of SUISAG and fed ad libitum during the whole testing period from 30 to at average 103 kg of body weight. The animals were examined for osteochondral lesions at seven positions of the carcass after dissection  and the epiphyses was examined at the cutting surface. Scores from 1 to 4 were defined . Osteochondral lesions at the DEU and CMF are scored from 1 to 6, because these lesions were more variable  or OCcat low (n = 162) .Genomic DNA and phenotypic data were obtained from animals of the breed Large White  to 4 (=severe)   and approved standard techniques as recommended by the manufacturer. This array contains 62,163 SNPs distributed throughout the porcine genome. Raw data were normalized and assigned a cluster position for genotyping with the GenomeStudio software . SNPs were filtered with call rates >99%. Markers were excluded if they had low minor allele frequency <5%. Deviation from Hardy-Weinberg equilibrium and the degree of LD were analyzed using HaploView 4.2  and T-box transcription factor 5 (TBX5) both located in close proximity to each other and a number of significant markers, were screened for SNPs in the coding region by comparative sequencing of PCR fragments of eight individuals. The PCR products were purified and comparatively sequenced in both directions using an ABI3130 DNA Analyzer. We detected a single SNP at g.38,175,026T > C or position 54 relative to the start codon (c.54T > C) of TBX5. Then, TBX5 genotyping was performed by PCR-RFLP technique using the restriction enzyme PfeI. The following primers were used: forward 5′-AAACTGCTGGTGGAAGCCTA-3′ and reverse 5′-GCAGAGAAAGGTGGGATTGA-3′. The PCR was performed in 20 μl volume containing 50 μg of genomic DNA, 1 × PCR buffer (with 1.5 mM MgCl2), 0.25 mM of dNTP, 0.2 μM of each primer, and 0.5 U of Taq DNA polymerase . The PCR procedures were performed under the following conditions: initial denaturing at 95°C for 5 min followed by 40 cycles of 30 s at 95°C, 30 s at 62°C, and 1 min at 72°C, and final elongation of 10 min at 72°C. After PCR amplification, PCR products were checked for specific amplification on 1.5% agarose gels  and digested by PfeI enzyme. The digested products were separated using 3% agarose gels.Two genes, namely T-box transcription factor 3 . Although the subset of animals of a total of 2,622 animals was built in order to combat any bias due to population stratification, the success of these measures of the selection procedure in this regard was tested. Accordingly, to determine population stratification, pairwise clustering of identity by state (IBS) distance of the samples was computed using the IBS and multidimensional scaling (MDS) approach within JMP Genomics 5.0. MDS analysis show that all samples cluster together within indication of any subclusters, suggesting that population stratification is not prominent Figure . We obtap-value < 10−5 was used, equaling a false discovery rate (FDR) based q-value ≤ 0.1  was applied in order to define LD blocks across a 3.4 Mb region on chromosome 14. The D′ and 2r coefficient  were used to calculate pairwise LD estimates. Finally, LD measure D′  between pairs of SNPs was estimated to create blocks of consecutive SNPs based on those in strong LD  and control (OCcat low) for testing trait association by a χ2 test with one degree of freedom. Whereas, for the GWA analysis to assess the association between SNPs and the ordinal traits of OC lesion scores, a logistic regression model  was used with the fixed effect of line, sex, and slaughter weight as a covariate. Genotype effects were estimated as odds ratios (ORs) and 95% confidence intervals (95% CIs). ORs were estimated for homozygotes of the minor allele compared with homozygotes for the major allele and heterozygotes (dominant model). OR value less than 1 imply the desired effect of lower OC lesion scores. A significance threshold of  Storey, . The samGene annotations for 1 Mb genomic intervals around the significantly associated SNPs were identified by the Ensemble genome browser (Build9)GAPDH) gene. All RNAs were stored at -80°C for further analysis.Total RNA was isolated from the articular cartilage of CMF of four divergent sib pairs using TRI reagent  according to manufacturer’s protocol. After DNase treatment a column-based purification using the RNeasy Mini Kit  was done. The RNA samples were visualized on 1% agarose gels containing ethidium bromide to check RNA integrity. RNA was quantified by spectrometry with a NanoDrop ND-1000 spectrophotometer . In addition, absence of DNA contamination was checked by using the RNA as a template in standard PCR amplifying fragments of the glyceraldehyde-3-phosphate-dehydrogenase . The 2−ΔCt and 2−ΔΔCt methods were used to calculate relative expression of the target gene TBX5. For the 2−ΔCt method, mean Ct values of TBX5 in each sample was normalized to housekeeping gene values  and affected  tissues, where the ΔCt values in the affected tissues was normalized to the ΔCt values in the normal tissues to obtain the ΔΔCt values. Statistical analysis was performed by mixed model analysis using Proc Mixed of SAS version 9.2  with the fixed effects of sex and OC lesion score; family was used as a random effect and slaughter weight as covariate. Differences were considered significant at p-value ≤ 0.05.RNA was reverse-transcribed into cDNA using random primers and an oligo dT primer in the presence of Superscript III reverse transcriptase . Quantitative real-time RT-PCR (qPCR) was performed on LightCycler480 system using the LightCycler 480 SYBR Green I Master kit . Two common housekeeping genes The phenotype distribution of OC lesion scores at different joints among the animals used is shown in Table p-value ≤ 10−5, q-value ≤ 0.1) with either one or more of the OC lesion score traits. Significant SNPs were distributed along chromosomes (SSC) 3, 5, 8, 10, 14, and 18. The Manhattan plots of −log10 for OC lesion scores are shown in Figure ASGA0060524) was found significantly associated with OC lesion scores at DEU that mapped to position 3.4 Mb. As for CMH, two SNPs (M1GA0019792 and M1GA0019802) were identified and located on 144.9 and 145.2 Mb, respectively. Most interesting, significant associated with OC lesion scores of CMF were found for five SNPs  at 25.4 and between 36.1 and 38.2 Mb. In addition, there were 11 significantly associated SNPs which located on SSC3, 5, 8, 10, and 18  to ASGA0062794. Therefore, the TBX3 and TBX5 gene were selected to screen of sequence variation and to assess LD with the significant SNP-markers and association with OC in the same population used for the GWAS and LD study.We investigated the region between 36.0 and 38.5 Mb on SSC14 where multiple significant SNPs were located, that were associated with OC lesion scores at CMF. LD was calculated between the SNPs in this region across 2.5 Mb. Figure TBX3 revealed no polymorphisms. In the TBX5 gene, a single synonymous SNP was detected at position 54 relative to the start codon (c.54T > C) of TBX5. The SNP TBX5 c.54T > C showed a high LD of 2r = 0.96 and D′ = 1.0 with ASGA0062794. The single-marker association analysis between TBX5 c.54T > C with OC lesion scores is shown in Table p < 0.0001, OR = 0.40, 95% CI = 0.26–0.61), DEU , and OCcat . Moreover, when integrating the SNP TBX5 c.54T > C in the GWAS together with the 48,716 SNPs of the SNPchip, it showed highest statistical significance of association with CMF and DEU among the markers of the haplotype block and even reached the significance threshold for the trait OCcat  of all markers in the region below the significance threshold indicating that the SNP TBX5 c.54T > C explains much of the variation in OC lesion scores depending on this genomic region . This may be simply due to the reduction of residual variance. However, it might also be a hint for epistatic interaction between TBX5 and a gene located in the region on SSC2. Indeed, Ingenuity Pathway Analysis revealed a network of indirect and direct effects between TBX5 and a number of genes all being located between 87.3 and 115 Mb on SSC2 , ELL2 , GLRX [glutaredoxin (thioltransferase)], ERAP1 (endoplasmic reticulum aminopeptidase 1), ERAP2 (endoplasmic reticulum aminopeptidase 2), PAM , TNFAIP8 , FAM170A , FTMT , LOX (lysyl oxidase), and SNCAIP . The logistic regression model complemented with an appropriate interaction term of TBX5 c.54T > C and SNPs on SSC2 revealed pairs of SNPs with significant interaction effects .Comparative sequencing of the coding region of TBX5 did not reveal any SNP leading to amino acid exchange we performed qPCR of CMF cartilage samples of divergent sib pairs with either low or high OC lesion scores in order to test the hypothesis that TBX5 may exhibit a regulatory polymorphism in LD with the SNP TBX5 c.54T > C. Significant differences were found (p < 0.05) with 1.63-fold higher transcript abundance in the normal cartilages compared with OC cartilages . Among these, 12 SNPs were located in previously reported QTL regions for leg weakness and OC related traits in pigs on SSC3, 5, 14, and 18.TBX3 (T-box 3) and TBX5 (T-box 5) as candidate genes for the liability to develop OC lesions. TBX3 and TBX5 belong to the T-box gene family that were demonstrated to function as transcriptional activators and repressors  and the MGMT (O-6-methyguanine-DNA methyltransferase) gene, respectively. PTPRE plays a major role in regulating bone structure and metabolism  gene and 0.13 kb downstream of the PLEKHH2  gene, respectively. There are no obvious functional links between MOB1A, or PLEKHH2 and OC lesions. However, the region (63.3–90.0 Mb) is consistent with the QTL for leg weakness and OC related traits of previous studies  which may play a role in the endothelial inflammatory disease, as atherosclerosis. The mapping site of KRT8 on the porcine SSC5 matches in a QTL region for OC lesions of fissure between cartilage and bone and OC  and the MKLN1 (muskelin 1) gene, respectively. These sites match a QTL for front leg conformation detected by ] which was identified as a target gene of TBX5 and harbors binding site for transcriptional factor of TBX5 in its promoter  was not significant associated with OC lesion (p = 0.18).TBX5 with OC lesion scores. This study provides further positional and genetic-statistical evidence for the candidacy of TBX5. However, beside TBX5 also two other genes (TBX3 and ZSCAN21) reside in the same haplotype block and may potentially be responsible for the association with OC lesions. TBX3 is expressed in the anterior and posterior margins of both fore limb and hind limb buds. The posterior expression of TBX3 is crucial for development of the more distal limb elements  the high LD of TBX5 c.54T > C with the significant SNP-markers at 36.0–38.5 Mb of SSC14, (2) its association with OC lesion scores confirmed by single-marker analysis, (3) the superior association of TBX5 c.54T > C when included in the genome scan, and (4) the LOD-drop when fitting the GWAS model using the TBX5 c.54T > C genotype as an additional fixed. The results imply that the association signal obtained on SCC14 is largely attributable to TBX5 c.54T > C, or other as-yet-unspecified polymorphisms in LD that most likely affect the expression of TBX5. The functional role of TBX in synergy with GJA5 and MEF2C promote deficiency of blood supply to growth cartilage as being fundamental for the initiation of OC.In summary, we identified The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.http://www.frontiersin.org/Livestock_Genomics/10.3389/fgene.2013.00078/abstractThe Supplementary Material for this article can be found online at Click here for additional data file.


A rough estimation of the density of states is given from temperature measurements.Using high-temperature annealing conditions with a graphite cap covering the C-face of, both, on axis and 8° off-axis 4H-SiC samples, large and homogeneous single epitaxial graphene layers have been grown. Raman spectroscopy shows evidence of the almost free-standing character of these monolayer graphene sheets, which was confirmed by magneto-transport measurements. On the best samples, we find a moderate  It is now widely accepted that graphene-based devices are promising candidates to complement silicon in the future generations of high-frequency microelectronic devices. To this end, the most favourable technique to produce graphene for industrial scale applications seems to be epitaxial graphene (EG) growth. This can be done by chemical vapour deposition on a metal ,2 or by n-type doped, with a low-carrier mobility. On the contrary, on the C-face of the same SiC substrates, there is no need of a C-rich buffer layer at the interface before growing the first graphene layer [2/V s in the work of Ref. [On the Si-face of 6H or 4H SiC substrates, graphitisation at high temperature in an Ar atmosphere close to atmospheric pressure shows promising results for on-axis substrates. In this way, single-layer epitaxial graphene (SLEG) has already been grown at the full wafer scale ,8 but anne layer -12. In t of Ref. .2/Si [For a long time, whatever the growth technique, the uniformity and quality of the EG was not good enough to find evidence of the so-called "half integer" quantum Hall effect (QHE). However, recently, large SLEG areas have been produced on the C-face of on-axis SiC substrates and, on such monolayer graphene, the carriers were holes with mobility close to the one found in mechanically exfoliated graphene films on SiO2/Si . Consequ2/Si . This sh2/Si .In this work, we compare the results of graphene growth on semi-insulating, on axis and and 8° off-axis, 4H-SiC substrates. The quality, uniformity and size of the growth products will be compared using optical microscopy (OM), scanning electron microscopy (SEM), atomic force microscopy (AFM) and micro-Raman spectroscopy (μR). Then, Hall effect measurements will be done at different temperature in order to extract the density of states in the epitaxial monolayers.To produce SLEG, in both cases of on axis and 8° off-axis SiC substrates, we used the recipes of Ref.. On the In Figure 2/Si [-1) which is strong evidence that there is no strain at the layer to substrate interface . Third, this 2D-band can be fitted with a single Lorentzian shape with a FWHM of 30 cm-1 [I2D/IG between the integrated intensities of the 2D-band and the G-band is high, which suggests weak residual doping in the order of 3 to 6 × 1012 cm-2 [Tens of similar monolayer islands grown on, both, on axis and off-axis substrates were probed by Raman spectroscopy. We used the 514 nm laser line of an Ar-ion laser for excitation and got very similar features. At the micrometer size, all spectra reveal that the islands are of the same nature and very homogeneous. First, the D-band, which usually indicates the presence of disorder or edges defects, is very weak and the Raman signature is extremely close to the one found for exfoliated graphene on SiO2/Si . Second, 30 cm-1 . Fourth,012 cm-2 . Altoget012 cm-2 .Gold alignment marks were used to select some SLEG position by OM. Then, they were contacted by e-beam lithography and subsequent deposition of a contact layer made of Cr/Au in Hall bar configuration. A typical example is shown in Figure 12 to 1 × 1013 cm-2 at low temperature, with a weak temperature dependence.Then transport measurements were done at low temperature on the different samples, using a maximum magnetic field of 13.5 T. The contact geometry allowed simultaneous measurement of, both, the longitudinal and transverse voltages with the current flowing between two injection contacts at the flake extremities. In both series of samples, from the sign of the Hall voltage, we found that the carriers were holes (in agreement with other results published on the C-face ). The ho12 cm-2, no QHE could be detected and only Shubnikov-de Haas (SdH) oscillations were found. This is shown in Figure For carrier concentrations larger than 3 × 10RK/4(N + 1/2) in which RK = h/e2 is the Von Klitzing constant [N = 0, 1, 2... As already known, this peculiar sequence of resistance values is the well-known quantum transport signature of the monolayer graphene Landau levels [ns = 1.2 × 1012 cm-2 and mobility μ ~5000 cm2/V s at T = 1.6 K. At B = 12 T, the longitudinal resistance cancels while the transverse resistance tends to 12.9 kΩ which is the expected value for the N = 0 plateau (RK/2).For the low doped layers, the transverse resistance exhibits now quantized Hall plateaus, clearly governed by the sequence constant  and N = u levels . In Figuns = 8 × 1011 cm-2 and a mobility μ ~11,000 cm2/V s. The mobility is high enough and the concentration low enough to make the N = 0 and N = 1 plateaus well resolved and stable up to 13.5 T. The experimental results of Figure xy ~(V(B)+V(-B))/2I, where the voltage V is measured between a lateral probe and the current drain. At high magnetic fields, we identify V(+B)/IG as ρxx, where G~4 is the geometric factor and I is the current.In Figure ρxx(B) is shown in Figure ρxx ~exp(-Ea/kBT) of the N = 0 plateau. This activation energy Ea is the energy separation between the Fermi energy EF and the delocalised states of the N = 1 Landau level. In Figure xx taken at different magnetic fields in the vicinity of the RK/2 plateau. The activation energy Ea varies from 0.7 to 3.3 meV between B = 10 and 13 T, which remains much smaller than the distance between the first and the second Landau level (~120 meV at B = 10 T). This indicates that the Fermi energy is firmly pinned by localised states. Ea has been calculated by taking into account only temperatures above 6 K. At lower temperatures, there is an additional contribution to the conductivity, which is visible in Figure The temperature dependence of E). The filling factor is calculated from B = 10 to 13 T, each filling factor change Δν at a given magnetic field corresponding to a density variation Δns = nsΔν/ν. The Fermi energy shifts by ΔEa to compensate for the density variation and the mean value for the density of states at energy ~Ea is given by ρ(E) = Δns/ΔEa.In principle, from the activation energy, we can reconstruct the density of state ρ increases and becomes one order of magnitude larger than the density of states ρ0(E) without magnetic field at a comparable energy EF ~100 meV: ρ0(E) ~15 × 109 cm-2 meV-1. The shape of ρ(E) gives a rough upper bound of the half-width at half-maximum (HWHM) of the N = 1 Landau Level. We find HWHM ≤ 3 meV. This value is in good agreement with results obtained recently on EG by STM [0 over the whole investigated energy range. This observation, combined with the fact that hopping was neglected, indicates that more detailed investigations are still needed.Following this procedure, already used in the early times after the discovery of the integer QHE , we findG by STM . HoweverB = 13.5 T. This magnetic field is far from the filling factor υ = 2 and; therefore, the breakdown occurs at relatively low current: I = 0.5 μA, which corresponds to a current density j = 0.025 A/m. By comparison, for III-V heterostructures, critical current values of 1 A/m are reported.Finally, since EG has recently been proposed for metrological application, we plot, in Figure To summarize, we have shown the possibility to grow large islands of monolayer graphene on the C-face of on-axis and 8° off-axis commercial 4H-SiC wafers. The graphene layers are continuous, almost free-standing and show quantum transport properties comparable with high-quality, low-doped, exfoliated graphene. We show evidence of half-integer QHE specific of graphene monolayer and give a first estimate of the density of states in the magnetic field.AFM: atomic force microscopy; EG: epitaxial graphene; HWHM: half-width at half-maximum; QHE: quantum Hall effect; SEM: scanning electron microscopy; SdH: Shubnikov-de Haas; SLEG: single-layer epitaxial graphene.The authors declare that they have no competing interests.NC and AC carried out the Graphene growth, the Hall Bars fabrication, the AFM, SEM and Raman characterisation. AT carried out the Raman investigation and interpretation. BJ, BJ and CC carried out the magnetotransport measurements. Finally PG and JC participated in the design and the coordination of this work. All authors read and approved the final manuscript.