Danio rerio) embryos with T-box transcription factor 5 (tbx5) deficiencies, mimicking human Holt-Oram syndrome.Dysmorphogenesis and multiple organ defects are well known in zebrafish  antisense RNA and mismatched antisense RNA in the 1-cell stage served as controls, while zebrafish embryos co-injected with exogenous growth hormone (GH) concomitant with tbx5-MO comprised the treatment group.Using an oligonucleotide-based microarray analysis to study the expression of special genes in tbx5-MO knockdown embryos were quantified and observed at 24, 30, 48, 72, and 96 h post-fertilization. Though the understanding of mechanisms involving GH in the tbx5 functioning complex is limited, exogenous GH supplied to tbx5 knockdown zebrafish embryos is able to enhance the expression of downstream mediators in the GH and insulin-like growth factor (IGF)-1 pathway, including igf1, ghra, and ghrb, and signal transductors , and eventually to correct dysmorphogenesis in various organs including the heart and pectoral fins. Supplementary GH also reduced apoptosis as determined by a TUNEL assay and decreased the expression of apoptosis-related genes and proteins (bcl2 and bad) according to semiquantitative reverse-transcription polymerase chain reaction and immunohistochemical analysis, respectively, as well as improving cell cycle-related genes (p27 and cdk2) and cardiomyogenetic genes .The attenuating effects of GH in tbx5 knockdown causes a pseudo GH deficiency in zebrafish during early embryonic stages, and supplementation of exogenous GH can partially restore dysmorphogenesis, apoptosis, cell growth inhibition, and abnormal cardiomyogenesis in tbx5 knockdown zebrafish in a paracrine manner.Based on our results,  PCR conditions were comprised of denaturation at 95 °C for 3 min followed by 50 cycles of amplification .Total RNA was prepared from 50 defective or normal embryos (Invitrogen), with amplification of 3 μl of 1st-strand cDNA. Amplification primers for each specific mRNA deduced from published sequences were tbx5-MO (5-GAAAGGTGTCTTCACTGTCCGCCAT-3), was designed against the tbx5 translational start site . Wild-type (WT) embryos, primarily at the 1-cell stage with the chorion intact, were injected with 19.4 ng/4.3 nl of stock MO diluted in Danieau’s solution. Injected embryos were raised at 28.5 °C. Embryos used for analyzing the expression of various markers were fixed with 4 % paraformaldehyde. Otherwise, embryos were scored after 2 days of development for late effects. In our previous study, 4 control groups, including the 3' end of tbx5-MO(2) (5’-GCCTGTACGATGTCTACCGTGAGGC-3’), mismatched tbx5-MO (5’-GTCTCTTGACTCTCCGCGATCTCGG-3’), and embryos with blank microinjection and wild-types without microinjection, were included to identify the specific blockage of tbx5 mRNA translation effect of tbx5-MO [tbx5-MO has been confirmed in previous published articles [The MO antisense oligonucleotide, articles .n = 150/group, with triplicate determinations), accompanied by 19.4 ng/2.3 nl tbx5-MO at the 1-cell stage. Treated zebrafish embryos were placed into plates with wells, and their functional classification was evaluated at 30, 48, 72, and 96 h post-fertilization (hpf).Zebrafish embryos were micro-injected with 1 fM of human GH  . Whole-mount in situ hybridization was carried out essentially as described by Oxtoby and Jowett [amhcvmhc, or cmlc2 probes at 67 °C. An alkaline phosphatase-conjugated anti-digoxigenin antibody  was used to detect zebrafish amhcvmhc, or cmlc2 signals. After staining with NBT/BCIP (Boehringer Mannheim), embryos were re-fixed with 4 % paraformaldehyde and stored in phosphate-buffered saline (PBS).Whole-mount r et al. . The digd Jowett . In brieZebrafish embryos were fixed with 4 % paraformaldehyde in PBS. De-paraffinized sections (3 μm) of zebrafish embryo tissues were placed on slides and processed for immunohistochemistry. After application of a biotin blocking system  for 30 min, sections were incubated with target-purified rabbit primary antibodies, including Bcl2, Bad, Cdk2, and P27  washed in PBS, and then incubated with a rhodamine-conjugated secondary antibody, goat anti-rabbit immunoglobulin G (IgG). After washing in PBS, sections were incubated with mounting medium and kept at 4 °C.4 in Sorenson’s phosphate buffer followed by dehydration through a graded series of ethanol washes, and embedded in Spurr’s EPON. Blocks were heated in an oven for 8 h at 70 °C. Semi-thin (1 μm) sections were cut and stained with toluidine blue for adequate preview under a microscope. Ultrathin sections (900 Å) were cut with a diamond knife, stained with uranyl acetate and lead citrate, and examined with an electron microscope.Embryos were fixed at 48 hpf with 2.5 % glutaraldehyde in Sorenson’s phosphate buffer, post-fixed with 1 % OsOBoth whole mount and sectioned TUNEL assays were performed using an ApopTag kit . Zebrafish embryos were briefly fixed with 4 % paraformaldehyde in PBS. Proteinase K-treated whole embryos or de-paraffinized sections (5 μm) of embryos were incubated with the TdT enzyme followed by anti-digoxigenin. Finally, embryos or slides were stained with DAB for 5 min.Embryos were homogenized on ice in lysis buffer (Sigma-Aldrich). Cellular debris was then pelletized by centrifugation at 12,000 rpm for 20 min, and the supernatant was collected and measured. Proteins were mixed with sample buffer before separation in 10 % sodium dodecylsulfate polyacrylamide electrophoresis (SDS-PAGE) gels. The SDS-PAGE was then transferred onto nitrocellulose membranes at 100 V for 1 h. Membranes were blocked with 5 % bovine serum albumin (BSA) buffer at room temperature for 1 h. The Akt and Erk primary antibody  was incubated overnight at 4 °C at a dilution of 1:1000. Nitrocellulose membranes were washed with PBST followed by incubation with a horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000) for 1 h at room temperature before the images were digitized.p < 0.05.Results are given as the mean ± S.D. Where applicable, Duncan’s new multiple range test was used to compare every pair of testing groups. Statistical significance was accepted at tbx5-MO treated (MO) zebrafish embryos, along with curved trunks and abnormal “U-shaped” somites  groups. Formation of the heart  were observed in WT zebrafish embryos Figure A, their s Figure G, and pes Figure L was eits Figure P, trunk s Figure Q, and pes Figure R were vet Figure C, trunk t Figure H, and pet Figure M in the tbx5 using a zebrafish microarray. Genes that were 1.5× downregulated were included. Multiple genes participating in growth were downregulated in zebrafish embryos with the tbx5 deficiency  caused defects of the heart  caused specific defects of the heart   and either the MIS group (n = 50) or the WTGH group (n = 50).Compared to the WT group, microinjection of exogeneous GH into WT embryos (WTGH) did not cause significant phenotypic changes, and no embryonic defects were identified Figure P-R. Therigf1 RNA expression analysis in the heart and pectoral fins , and ghra[tbx5 in embryos diminished GH/IGF-1 pathway mediators, including GH, IGF-1, and GH receptors.Significant dysmorphogenesis Figure  shown inficiency -35. A de and ghra,36. Our via intercellular substance. The expression of igf1ghraghrberk1, and akt2 genes was increasing after co-injection with GH and tbx5 morpholino at 1-cell stage. These results revealed the exogenous GH has activated the downstream signaling pathway. However, it is still unknown how and where the exogenous GH binds with the GH receptors. Whether the injected exogenous human GH is packaged and sent outward to bind with the membranous GH receptors, or it directly binds with the cytoplasmic GH receptors, remains unidentified because the expression of GH receptors could be either in the nucleus or cytoplasm, or both, in different embryonic tissues and cells [Exogenous GH was used in our study to partially restore the anomalies during embryogenesis to ensure the involvement of GH in multiple organ defects by tbx5 knockdown. In the study, we injected human recombinant GH, which human GH instead of zebrafish GH is reported to have full activity in fish as well as in human , into thnd cells .tbx5 deficiency could activate expression of GH receptor genes, to induce an increase in igf1 levels, and to elevate downstream Akt and Erk systems, coinciding with restoration of morphological anomalies and transcriptional cascades. It could be hypothesized that GH is a factor that may act in a paracrine manner within the tbx5 functional pathway to modulate embryonic development.Exogenous GH in zebrafish embryos with Tbx5 is essential for regulating the progression of the cell cycle by controlling the length of the embryonic cardiac cell cycle [septum primum[tbx5 downregulated genes, which contributes to developmental delays in organogenesis including the cell cycle (p27 and cdk2) and apoptosis (bcl2 and bad). Early administration of exogenous GH improves the outcome of tbx5-deficiency-mediated heart defect embryos probably by inducing cardiac cells to re-enter the cell cycle. It also reduces aberrant apoptosis because GH works in a similar way to stimulate the cardiomyocyte to re-enter the cell cycle and thereby increases the number of cardiac myocytes in ischemic and infarcted myocardia [ll cycle  and reguum primum, all of um primum. In our yocardia ,26.cmlc2, amhc, and vmhc). Exogenous GH restored the expression levels of amhc, vmhc, and cmlc2 in our tbx5 morphant embryos with cardiac defects. Inactivation of tbx5 diminished amhc, vmhc, and cmlc2 expressions, and although it also reduced heart size, exogenous GH reversed that result and enhanced cardiac formation in zebrafish embryos. Our results indicate not only that GH is necessary for the growth of these cardiac structures, but also that supplementary exogenous GH restores tbx5 knockdown-mediated defects, including dysmorphogenesis and cardiomyogenesis, via cell proliferative and apoptotic pathways. It could be concluded that knockdown of tbx5 in early zebrafish embryogenesis causes functional GH deficiency and leads to dysmorphogenesis. The comorbidity of morphologic defects and functional GH deficiency could be observed in early embryogenesis of tbx5 morphants and implied that GH may involve a role in embryogenesis including cardiomyogenesis through transcriptional regulation of tbx5.The GH signaling pathway governs cell growth, proliferation, and apoptosis by controlling key regulatory genes that execute these processes. Herein, we also provide the first evidence that tbx5 acts together with GH to regulate cardiac myogenetic pathway-responsive genes  without subsequent effects, but the phenomenon didn’t happen in the expressions of ghra, ghrb, erk1, and akt2. It may aggressively assume that GH effects may involve IGF-1 dependent and independent pathways in embryonic development to switch on the cascade reactions. Thus the role of IGF-1 attracts attention for further exploration. Moreover, the expressions of ghra and ghrb in WTGH group significantly increased than MOGH group, but the expressions of erk1, akt2, amhc, vmhc, cmlc2 in WTGH group had no remarkable change than MOGH group. It implies that excess GH works inefficiently in individuals without tbx5 deficiency, or GH deficiency. GH receptors could be reactivated and responded to exogenous GH, but downstream signals and cardiomyogenesis-related genes didn’t markedly act in response to overload of GH. The results are compatible with the biological functions of GH in mature adult individuals. Surly, supplementary designs of control groups, for example, such as use of BSA with equivalent amount as a blank control, could be launched to access the authentic GH effects by our experimental model.We established four control groups in order to verify the specific GH effects without interference of morpholino and technical influences of micro-injection. However, interesting phenomena were disclosed by careful interpretation among those different control groups. First, tbx5 morphants, it cannot completely rescue those changes. Proper timing of GH treatment and optimal dosing might be found to enhance its therapeutic capability. Conversely, this approach may be limited by GH being partially significant to the complex functioning of the tbx5 network or because it compensates for only a small part of the chronological effects of tbx5 deficiency. Additional research is required to determine whether it is practicable to introduce GH to mend developmental defects in early embryogenesis.Although GH is able to partially restore dysmorphogenesis and cascade gene expressions in tbx5 deficiency. We suggest that the functional knockdown of zebrafish tbx5 results in a failure to develop a complete or functional heart, trunk, and pectoral fins and might be due to a functional GH deficiency induced by the tbx5 deficiency because it is a key factor causing abnormal organogenesis. Exogenous GH experiments in zebrafish embryos with tbx5 deficiency led to the conclusion that intrinsic growth-control mechanisms, including apoptosis, cell cycle, and cardiomyogenesis that control organic growth, depend on local GH and growth factors between cells and their neighbors. They also indicate that these interactions include controlling cardiac loop formation and the development of trunk and pectoral fins. The improvement of abnormal embryonic organogenesis in zebrafish embryos with tbx5 deficiency by the administration of exogenous GH suggests its potential application in human congenital anomalies.In summary, our work provides novel insights into the possible role of GH in contributing to developmental defects in zebrafish embryos with The authors declare that they have no competing interests.JHL and JKL conceived of the study, participated in its design, coordination. TCT participated in its design and drafted the manuscript. SLC and SYY carried out the molecular genetic studies. RBT and HYL participated in its design and coordination. All authors read and approved the final manuscript.


Beta diversity, which involves the assessment of differences between communities, is an important problem in ecological studies. Many statistical methods have been developed to quantify beta diversity, and among them, UniFrac and weighted-UniFrac (W-UniFrac) are widely used. The W-UniFrac is a weighted sum of branch lengths in a phylogenetic tree of the sequences from the communities. However, W-UniFrac does not consider the variation of the weights under random sampling resulting in less power detecting the differences between communities.We develop a new statistic termed variance adjusted weighted UniFrac (VAW-UniFrac) to compare two communities based on the phylogenetic relationships of the individuals. The VAW-UniFrac is used to test if the two communities are different. To test the power of VAW-UniFrac, we first ran a series of simulations which revealed that it always outperforms W-UniFrac, as well as UniFrac when the individuals are not uniformly distributed. Next, all three methods were applied to analyze three large 16S rRNA sequence collections, including human skin bacteria, mouse gut microbial communities, microbial communities from hypersaline soil and sediments, and a tropical forest census data. Both simulations and applications to real data show that VAW-UniFrac can satisfactorily measure differences between communities, considering not only the species composition but also abundance information.VAW-UniFrac can recover biological insights that cannot be revealed by other beta diversity measures, and it provides a novel alternative for comparing communities. The assessment of differences between communities is an important problem in ecological studies. By comparing the compositions of natural communities from different environments, locations or time periods, we can learn how specific factors affect community assembly and how species or individuals associate with each other -3. The dMany statistics have been proposed to compare communities based on genomic sequence data of a specific gene sampled from the communities. These include LIBSHUFF , ∫-LIBSHLozupone et al. proposed a novel statistical method termed UniFrac  and a weDespite the many studies on statistical methods to compare communities, there had been some confusions about the hypotheses being tested for the different statistics. Schloss  addresseNote that in W-UniFrac the length of a branch is weighted by the difference of relative abundances of the two communities for that branch of the tree. Under the null hypothesis that the two communities are the same, the weights for the different branches in W-UniFrac have difference variances and we provide a formula for calculating the variance in this paper. Based on the variance formula, we propose a new weighting scheme for the branch length in W-UniFrac by taking the variation of the weight into consideration. The new resulting statistic is termed Variance Adjusted Weighted UniFrac (VAW-UniFrac). The statistical significance of VAW-UniFrac is evaluated by randomizing the labels of the sequences along the leaves of the tree. Similar to UniFrac and W-UniFrac, the VAW-UniFrac can be used to evaluate if two communities are different. More precisely, it tests the hypothesis that the sequences from the communities are randomly distributed along the leaves of the tree. To study the power of this new statistic, we first carried out simulation studies similar to that in  to detecGiven a phylogenetic tree composed of all individuals from two communities, UniFrac distance  is definn is the number of branches in the tree, i bis the length of branch i, i Aand i Bare the numbers of individuals that descend from branch i in communities A and B, respectively, and T Aand T Bare the total numbers of individuals in communities A and B, respectively. In the denominator, n' is the number of different individuals in the two communities, j dis the distance from the root to individual j, while j αand j βare the numbers of times the sequences were observed in communities A and B, respectively . The same annotation will be used in the rest of the paper.In the numerator, Note thatWU is always larger than, or equal to, the numerator. Equality holds when either i A= 0 or i B= 0, which means that the two communities are totally separated. The numerators of both UniFrac and W-UniFrac can be written asTherefore, the denominator of In W-UniFrac,and in UniFrac,i in communities A, and Where i-th branch length assuming that the sequence labels are randomly distributed along the leaves of the tree. By ignoring the variance of i ωin W-UniFrac, the true relationships between communities may not be well characterized. Hence, we propose to adjust the weight i ωas follows. Given individuals from two communities, A and B, we first generate a phylogenetic tree composed of all the T A+ T Bindividuals in communities as leaves. Each leaf is labeled "A" or "B" to represent the community from which it comes. We test the hypothesis that the labels of the individuals are randomly distributed on the phylogenetic tree.From the definition of W-UniFrac given above, we note that it does not consider the variance of the weight i-th branch of the phylogenetic tree. Let i m= i A+ i Bbe the total number of individuals belonging to the i-th branch. Let m = T A+ T B. We randomly choose T Aindividuals from the total of m individuals, label them as being from community A and label the other leaves as being from community B. Then, iA, the number of individuals in community A that belong to the i-th branch, is hypergeometric with parameters  under the null hypothesis. That isConsider the Therefore,LetT Aindividuals are randomly sampled from the m individuals, we haveUnder the null hypothesis that the andi-th branch of the tree,From the above derivation, we propose the following variance adjusted weight (VAW) for the length of the We standardize the resulting statistic so that its value is between 0 and 1. The final VAW-UniFrac is defined asT , we also consider a variation by taking the weight as In addition to the statistic WU), VAW-UniFrac (T ) or SqT , are more powerful in detecting the relationship between two communities if they are related.Similar to UniFrac and W-UniFrac, the VAW-UniFrac aims to test if the two communities are different and, more specifically, if the sequences are randomly distributed along the leaves of the tree. The statistical significance of VAW-UniFrac is evaluated by randomizing the labels of the sequences. We are interested in which methods, including UniFrac, W-UniFrac  or the distribution patterns of samples in circles (ellipses) represented changing the differences between the communities. The maximum distance between any two points in one community was designed to be 0.3 units according to the distance between sequences from different phyla . Three cFor one comparison, we first sampled 200 points from each community, resulting in a total of 400 points. Second, Euclidean distances among all 400 points were calculated. Third, a phylogeny tree was generated based on this distance matrix using the neighbor joining method in the neighbor program in PHYLIP . The fourth dataset involves tropical forest census data in three plots across a precipitation gradient in central Panama [We applied UniFrac, W-UniFrac and VAW-UniFrac to reanalyze three datasets consisting of 16S rRNA sequences and one tropical forest census data. First, Costello et al.  investigr et al.  studied r et al. . All thel Panama -28. The l Panama . DiffereFor each dataset, we first calculated the distances between each pair of communities using the three statistics: UniFrac, W-UniFrac and VAW-UniFrac. Then the unweighted pair group method with arithmetic averages (UPGMA) clustering  was usedIn order to study our new methods and compare their performance to UniFrac and W-UniFrac, we carried out simulation studies according to the simulation methods developed in . We thenWe carried out three classes of simulations for two communities: 1) both were uniform samples from two circles with different centers and radii; 2) both were uniform samples from two ellipses with different orientations; and 3) one community was a uniform sample, while the other was an uneven sample from the same circle.In this simulation, communities were represented by circles, and individuals were uniformly sampled from each circle. It is reasonable to assume that the magnitude of a circle's radius can reflect the diversity of the community that it represents. In order to investigate the type 1 error rates and statistical power of the four statistics, UniFrac, W-UniFrac, VAW-UniFrac, and SqT, different levels of overlaps between communities were simulated by changing the distance between two centers, known as offset, and by changing the radius of one of the two circles  of bacterial 16S rRNA sequence data from Costello et al.  to underWe first assigned each sequence in samples to its closest relative in a phylogeny of the Greengenes core set  using BLFigure We wondered why the 8 native samples from individual F3 were clustered together and separated from other native samples of the same habitats according to VAW-UniFrac, while the clustering of native samples from F3 was not detected at all by UniFrac and W-UniFrac. To investigate this phenomenon in more depth, we then applied the three statistics to all 72 native microbiotas in that study. The UPGMA results are shown in Figure We then applied the three statistics to analyze the 16S rRNA sequences from mouse gut communities . LozuponAs observed in , both PCIn order to see how the statistics perform when they are applied to sequence collections derived by different technologies, but from the same sample, we analyzed the 16S rRNA sequence data from soil and sediments from . The samAfter applying the three statistics to each pair of the 16 samples, we used PCoA and UPGMA to analyze the results and investigated the performance of the three statistics  neighbor-joining, 2) maximum parsimony, 3) maximum likelihood, and 4) BLAST assignments of the sample sequences to the closet relatives on a known phylogenetic tree. The effects of the different tree construction methods on the results of UniFrac, W-UniFrac, and VAW-UniFrac are presented.Click here for file


Local curvature, or bending, of a graphene sheet is known to increase the chemical reactivity presenting an opportunity for templated chemical functionalisation. Using first-principles calculations based on density functional theory (DFT), we investigate the reaction barrier reduction for the adsorption of atomic hydrogen at linear bends in graphene. We find a significant barrier lowering (≈15%) for realistic radii of curvature (≈20 Å) and that adsorption along the linear bend leads to a stable linear kink. We compute the electronic transport properties of individual and multiple kink lines, and demonstrate how these act as efficient barriers for electron transport. In particular, two parallel kink lines form a graphene pseudo-nanoribbon structure with a semimetallic/semiconducting electronic structure closely related to the corresponding isolated ribbons; the ribbon band gap translates into a transport gap for electronic transport across the kink lines. We finally consider pseudo-ribbon-based heterostructures and propose that such structures present a novel approach for band gap engineering in nanostructured graphene. Nanostructures based on graphene have an enormous potential for applications. Especially in future electronic devices compatible with and extending silicon technology, due to the outstanding electronic transport properties of graphene . However3 hybridisation [A common approach towards engineering the electronic structure is to form quasi-1D graphene in the form of nanoribbons (GNR) . The eledisation . Periodidisation . Finallydisation , or a nadisation –10 can hdisation  down to disation .Graphene consists entirely of surface atoms and is thus exceedingly sensitive to the surroundings. In particular, the van der Waals (vdW) interaction with the substrate is of importance. The substrate interactions, which make graphene cling to small features, may be exploited by manufacturing nanostructures in the substrate. Periodic steps in a Cu substrate has been used to induce “wrinkles” or ripples in graphene with period and height on the order of 10 nm . Recentl2 nanoparticles induced local regions of strain and increased reactivity in a selective manner. Atomic hydrogen or other chemical species do not easily react with flat graphene when dosed from a single side [The ability to accurately control sharp local curvatures of graphene presents opportunities for strain-assisted modification of the local electronic structure and chemical reactivity in the graphene sheet, and may open a route to band gap engineered devices ,18–21. Vgle side . Howevergle side ,23. So fgle side –26. HoweIn this paper we consider the reactivity of linear bends in a graphene sheet, and the electronic transport properties of kinks resulting from the hydrogenation of bends. Our starting point is the generic graphene structure shown in L, while the rest of the atoms are allowed to relax. A separation of L = 90 Å is chosen in order to obtain realistic curvatures [The bend we consider in rvatures . We firsk-points. We use a mesh cut-off of 500 Ry throughout. When calculating reactivity in the form of reduced reaction barriers the unit-cell is chosen so that the distance between single hydrogen atoms is larger than 8.5 Å. This ensures low hydrogen–hydrogen interaction, which is known to impact reaction barriers [k-points). We use a TZP basis-set for hydrogen and a SZ basis for carbon, except in the reaction barrier calculations where we compare calculations using a DZP and SZP basis for the four carbon atoms nearest to the hydrogen. In the barrier determinations we furthermore use spin-polarised calculations because of unpaired electrons. For the relaxed geometries a force tolerance of 0.01 eV/Å is used, and the final energies are corrected for basis-set-superposition errors (BSSE) [The atomic and electronic structure calculations are based on density functional theory (DFT) using the SIESTA  code, anbarriers . In the s (BSSE) .S1 and S2 in S1 and S2. In the conductance calculations we employ a dense transverse k-point grid of 400 points.Based on the computed atomic and electronic structures we subsequently use the TranSIESTA  method tAdsorption of hydrogen on graphene involves a reaction barrier that needs to be overcome before the single hydrogen atom sticks to the graphene sheet. Several investigations based on DFT calculations show that atomic hydrogen adsorbs on-top on flat graphene with a barrier about 0.2 eV and binding energy in the range of 0.7–1.0 eV ,32–34. THere, we first focus on the trends in the change in adsorption barrier as a function of the local curvature of the graphene sheet. To this end we have considered atomic hydrogen absorption at the on-top carbon positions at points with different curvature on the bent structure, see 60 molecules, CNTs, and graphite to show that reactivity is increased with curvature. In our case we find that the local electronic density of states changes little for the atoms on the pristine bent graphene sheet (as in For the positions (I-VIII) we obtain the reaction barrier for adsorption of hydrogen as a function of the local radius of curvature (RoC) shown in et as in . Thus, w3-binding of a single H makes the graphene curve even more in its vicinity, which in turn, preferentially lowers the barrier for absorption of a H along the linear bend. This suggests a mechanism in which the hydrogen adsorption is propagating and leads to the decoration of the entire linear bend turning it into a kink line. It may be viewed as analogous to crack-formation mechanisms, where the breaking of a bond increases the stress on neighbouring bonds; only in this case, the graphene is hydrogenated rather than broken or destroyed.We may understand the reaction barrier and its change with curvature by considering the changes in carbon bond lengths. The barrier is due to the fact that the reacting carbon atom has to be pulled out of the graphene plane, stretching the strong carbon–carbon bonds, when reacting with the incoming hydrogen atom. When the graphene sheet is curved the carbon atom is already slightly out of the plane, and thus the energy required to pull the atom further out of plane is decreased compared to flat graphene. The ortho- and para-locations in the graphene hexagon have been shown to be the preferred locations for hydrogen adsorption in studies of flat graphene ,29,40. WS1 and S2 in 3 configuration where 2φ = 109.5°. The adsorption of H causes local changes in the geometry, i.e., only the carbon atoms very close to the kink are moved, while the remaining structure remains unperturbed. For this reason, the adsorption of hydrogen atoms can be considered as a process that locally pins the bend.Next, we examine the energetic and transport properties of kink-lines in the armchair direction. We first consider a single kink with angle φ, e.g., between sections E = 0), e.g., T0E. We find similarly that the calculated kink-transmission curves are also linear, and therefore we express the results for the transmissions in terms of the roughly energy-independent ratio T/T0 = const. The kink breaks the electron–hole symmetry, and we fit E > 0 and E < 0 separately, as shown in T/T0 is close to 0.17 in both regions . The close correspondence between the electronic band structure for the GNR and the transmission gap for the double-kink system allows us to consider the structure between two kinks as a pseudo-ribbon.Band-structure calculations show that periodic nanoscale rippling of the graphene is not sufficient to create a band gap  due to t integer . In FiguEgap = 0.4 eV and Egap = 0.5 eV, are in reasonable agreement with the power-law scaling of Egap with width found for aGNRs [N = 18 case a larger van Hove resonance is seen at the valence band edge, while for N = 19 a larger resonance is seen at the conduction band edge. There are small transmission values within the electronic band gap due to leakage through the barriers, which we expect to introduce shifts in the energies between the real and pseudo aGNR.For the semiconducting pseudo-ribbons transport gaps surrounded by van Hove-type 1D behaviour are seen in the transmission functions , in the band structure along the pseudo-ribbon direction. The weight on S3 is represented by a circle of radius Rnk,In order to analyse the transmission we single out the band structure projected on to the top section, kx (kz = 0) with n (i) being the band  index. The obtained projected band structures are shown in the left parts of each subfigure in S3 and other sections.Here Ψ is the wave function at S2/S3/S4 being M/M/M and SC/M/SC, respectively. For the all-metal pseudo-ribbons, M/M/M, an almost energy-independent transmission function is seen with a transmission close to that of the metallic double-kink in the previous subsection. The SC/M/SC structure shows a transport gap similar to that of the single SC pseudo-ribbon with van Hove resonances, while the S3-projected band structure reveals isolated metallic states within the gap. We note that the transmission at the resonances for the SC/M/SC structure is larger than the corresponding M/M/M transmission. For the case of semiconducting top pseudo-ribbons in S2, S4, i.e., there is a good correspondence between the side section band gaps  and the transmission gaps.In The presented investigations show that linear kink-line structures may form in graphene by reacting with atomic hydrogen along a linear bend in the sheet. The adsorption barrier is lowered in the close vicinity of the bend, which can be exploited to form the kink. In particular, we have shown that a radius of curvature of ≈20 Å reduces the hydrogen adsorption barrier by roughly 16% compared to H adsorption on pristine graphene. The calculations suggest that once a single hydrogen atom has been adsorbed, the induced local kink and resulting increase in local curvature makes it easier for the following H to adsorb, thus creating a propagating kink formation. A full line of hydrogen atoms pins the structure and divides the electronic systems into different regions. We have shown that the electronic transmission through a single kink is reduced by 83% compared to pristine graphene, meaning that the kink-line acts as an efficient barrier for electron motion. We have demonstrated how two close-by parallel kinks form a pseudo graphene nanoribbon with similar behaviour of the electronic structure to that for isolated nanoribbons. The transmission function displays transport gap features corresponding to the isolated nanoribbon band gaps.The present work thus suggests that it may be feasible to template functional electronic nanostructures by using the conformation of graphene, e.g., to the substrate, and that this in turn induce changes in local reactivity. Our work clearly calls for extensions in a number of directions. First of all more calculations are needed in order to investigate the kink-line propagation reaction proposed by our results. To this end it is important to include a realistic description of the actual substrate. It is also interesting to consider other adsorbate species, possibly introducing doping of the pseudo-ribbons and electronic gating. Finally, decoration and pinning of the edges of other geometries such as “bubbles” or “blisters” is of interest, e.g., in order to produce GAL-like structures  without


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.