Systematic first-principles calculations were performed to investigate the adsorption and diffusion of Li on different graphene layers with B/N-doping and/or C-vacancy, so as to understand why doping heteroatoms in graphene anode could significantly improve the performance of lithium-ion batteries. We found that the formation of single or double carbon vacancies in graphene are critical for the adsorption of Li atoms. While the N-doping facilitates the formation of vacancies, it introduces over binding issue and hinders the Li diffusion. The presence of B takes the excessive electrons from Li and N and reduces the energy barrier of Li diffusion on substrates. We perceive that these clear insights are crucial for the further development of graphene based anode materials for lithium-ion batteries. His/her work confirmed that the N-decorated graphene with single and double vacancies could greatly improve the reversible lithium capacityet al. found that the substitutional B atoms cause slight electron deficiency, which makes the adsorption of Li on the C3B monolayer easyMany approaches have been pursued to functionalize graphene as an anode material and doping graphene with nitrogen (N) and/or boron (B) atoms is one of the most effective ways. For example, Reddy C-decorated graphene geometries, as well as the Li adsorption and diffusion on them. We sorted out the roles of doping and vacancy decoration on the improvement of Li-adsorption on graphene, and identified several structures that have good adsorption energy, low diffusion barrier and easy preparation in experiments. Our theoretical results provide instructive guidelines for the further development of high performance C-based anode materials for LIBs.In the present work, we performed systematic density functional theory (DFT) calculations for the structures and energetics of different B/N/Vx N-atoms and y VC, G_NxVy (x = 0–4 and y = 0–2), corresponding to a N-concentration of ~1.4–5.6%, comparable with the experimental range for graphene samples synthesized via the thermal reaction between graphene oxide and NH3 at high temperaturey = 0, i.e., N-substituted graphene without VC, N atoms prefer to stay far away from each other to minimize the perturbation to the graphene π-electron bonds, as depicted in y = 1 and 2, i.e., N-doped graphene with VC, the N dopants prefer to connect with two C atoms and form pyridine-like structure, which introduces a strongly localized donor states near the Fermi level34We first studied the adsorption configurations and energies of one Li atom on the decorated graphene cells with Ead, are shown in Ead of Li on the hollow site (H) of a 6 × 6 graphene supercell, 0.27 eV, is lower than that on the top site (T), 0.57 eV. The distance between Li and substrate, dLG, of the H-site, 1.72 Å, is also smaller than that of the T-site, 1.92 Å. These results indicate a single Li adatom prefers the hollow site on pristine graphene but Li atoms are more likely to form Li clusters since the value of Ead is positive, in consistent with previous results241V0 and G_N2V0 systems, we found that Li prefers to stay on the H-site of graphene rather than any places near the N atoms. The Ead of Li on G_N1V0 (G_N2V0) systems, 0.39 (0.34) eV, are also positive, and the corresponding values of dLG are 1.75(1.76) Å, indicating that pure N-dopings in graphene don’t improve the lithium adsorption. Strikingly with the presence of VC on pristine graphene, the Ead decreases to negative values of −1.36 eV for single VC and −0.73 eV for double VCs, as shown in 0V1 have no obvious changes compared with perfect graphene due to the weak intraplanar relaxation0V2 with decreased C-C bond lengths near vacancies from 2.46 Å to 1.81 Å, as depicted in the insets in Ead values decrease further for Li on the N and VC-codoped graphene sheets. For example, Ead becomes −3.12 eV for Li/G_N3V1, which implies a strong lithium adsorption on this substrate, as also demonstrated in previous experimental and theoretical studies31C number doesn’t significantly change the Ead value, and the lowest Ead is −3.59 eV, which occurs in Li/G_N4V2. The detailed analyses of adsorption structures show that the vertical dLG is 1.43 Å for Li/G_N3V1, and decreases to 0.10 Å for Li/G_N4V2, implying that the Li atom is in the hole position surrounded by four pyridine N atoms and almost in the same plane with the N-doped graphene. Overall, the red curve in Ead of Li on different graphene cells suggests that the carbon vacancies play the key role in the adsorption of Li on graphene.For each system, we explored different Li adsorption sites cf.  in SM anC on graphene, Efor(VC), shown in Efor(VC) drops to 4.69 eV on N-doped graphene, and the trend of decrease continues with the further increase of N atoms, as shown in the red line in Efor(VC) of G_N3V1 has a small negative value, indicating the easy formation of the N-VC structure as a group. Similar trend appears for the formation of double-vacancies in graphene, with a very low Efor(VC) of −2.89 eV for G_N4V2. The formation energy of single N-dopant on graphene, Efor(N), is 0.91 eV, comparable with the previous DFT studies of 0.79 eVEfor(N) of single N-dopant on the G_NxV0 substrates (shown in the insets) that were used in Efor calculations of single C-vacancy, and the error bar illustrates the Efor(N) window for different doping configurations of N on G_NxV0 , changes from −5.30 eV under B-rich conditions to +8.09 eV under oxidation conditions, as shown in Efor(B) under typical experimental conditions, ranging from −0.23 eV with B4C precursor to +1.47 eV with NH3BH3 precursor, are a little higher than Efor(N), the calculated Efor(VC) of single C-vacancy on G_B3V1 is comparable with that of G_N3V1, and interestingly, its value for the B/N co-doped graphene, G_BxNx3-V1 (x = 0–3), is even lower than the corresponding value for the pure N-doped graphene by ~1.20 eV, as presented by the green stars in As we mentioned above, the graphite-like B/C/N layered materials prepared using CVD or other methods showed good performances as the anode matrix of LIBs3940Ead for Li on several adsorption sites of G_BxNx3-V1 substrates, as depicted in 1N2V1, we found that Li in the ground state is located at the vacancy site, close to the B dopant with a dLG of 1.65 Å. A similar adsorption structure was obtained for Li/G_B2N1V1, but the Li atom is much closer to the N dopant instead of B. Strikingly, the most stable adsorption site of Li on G_B3V1 substrate is on the hollow site of the hexagonal ring adjacent to the B atom. Overall, the B and N co-doped graphene substrates give negative adsorption energies, and the values of Ead of one Li atom on G_BxNx3−V1 are in the order: G_N3V1 (−3.12 eV) < G_B2N1V1 (−1.77 eV) < G_B1N2V1 (−1.20 eV) < G_B3V1 (−0.99 eV). Obviously, all of them are more active toward the adsorption of Li, compared with pristine graphene. Using G_B2N1V0 as an example, we also studied the x-dependence of Ead  − Ead(H). For each adsorption site, we fixed the lateral coordinates of Li and optimized all the other atomic positions. The energy barrier for the movement of one Li atom on G_B3V1 is only 0.41 eV, and the largest difference in dLG is about 0.27 Å. The low energy barrier and the small change in dLG indicate a relatively flat potential energy surface of G_B3V1 for the Li movement. Similarly, we studied the diffusion of Li on G_B2N1V1 and obtained ΔEad of 0.80 eV and 0.48 eV along two different pathways, and the corresponding difference in dLG of 0.83 Å and 0.56 Å, respectively . The adsorption of Li atom keeps the DOS profile almost unchanged, but the Ef moves to the conduction states due to the addition of 1e from Li. As was well known, the Ef of B(N)-doped graphene is shifted to the valance(conduction) band, as shown in the middle panels in 1(G_N1) is a hole (electron)-rich material. The adsorption of Li on G_B1 substrate compensates the hole attraction and the Dirac state of Li/G_B1 moves back to Ef, making the system stable with a negative Ead of −1.4 eV1, we see an obvious peak existing on the Ef, coming from the N-pz orbital. For these three systems, there is little hybridization between Li and substrate/dopant. For example, the B-px,y states in G_B1 locate at ~−2.9 eV below the Ef, but the Li-s and -pz states appear at ~2.1 eV above the Ef. The attractive/repulsive electrostatic interactions between Li and substrates lead to a negative/positive Ead values. Strikingly, for G_V1 substrate, as shown in the lower panel in Ef caused by the hybridization of Li-px,y and C-px,y in-plane orbitals, and the adjacent peak at −0.7 eV below the Ef is composed of the Li-s and C-px,y orbitals. Thus the interaction between Li and G_V1 substrate is very strong, leading to a low Ead value.We performed detailed analyses on the electronic properties of various graphene substrates with/without Li adsorption, so as to understand the role of doping and/or vacancy on the Li adsorption. The total and partial density of states (DOS) of pristine graphene (G), G_B3V1 substrate in 1, with peaks of B-pz orbital near the Ef. The adsorption of Li makes the B-pz states shift to lower energies, but the hybridization of Li-s,p and B-p states is still relatively weak near the Ef. Similar with G_V1, the DOS of G_N3V1 also has a big peak right at the Ef, coming from the dangling bonds at the N atom sites. The orbital hybridizations of N-p and Li-s,p orbitals can be observed around −1.8 eV and −3.5 eV, which are responsible for the lower Ead of G_N3V1 than G_B3V1. The interaction between Li and substrate can be described more clearly by the electron redistribution, rn = nLi/sub(r) − nsub(r) − nLi(r), obtained from electron densities of Li on substrates, pure substrates and Li atom. As displayed in the insets in 3V1 and G_N3V1 manifest through the influence of N extending to the second nearest neighbors of C atoms due to the symmetry breaking of the π-electron system, whereas B appears to only affect the nearest neighbor C atoms.Interestingly, we found that the DOS profile of G_BC sites (<3% in this study) rather than the dopants serve as attractive centers for Li adsorption on graphene. While the formation energy of one VC on graphene is as high as 8.01 eV, the N-doping drastically decreases this value to as low as −0.14 eV for G_N3V1. The co-doping of B keeps negative adsorption energies for Li, and significantly reduces the energy barrier of Li diffusion, e.g., 0.41 eV for Li/G_B3V1 and 0.48 eV for Li/G_B2N1V1. The electronic structure analyses show that the interaction between Li and B states is rather weak, whereas Li has a strong orbital hybridization with N-p states, causing a high diffusion barrier. Our theoretical studies provide clear insights for the understanding of the individual roles of doping and vacancy decorations for the performance of enhancement of N(B)-doped graphene as electrode materials in LIBs, and provide guidelines for the design of new battery materials.In summary, we performed systematic first-principles calculations to study the adsorption and diffusion of Li on decorated graphene with B and/or N dopants and C vacancies. Our results indicate that Vk-points sampling in the Brillouin zone (BZ). The crystal constant and positions of the ions were fully relaxed until the final force on each atom is smaller than 0.01 eV/Å. The optimized in-plane lattice constant of graphene primitive cell is 2.47 Å, in good agreement with the experimental values of 2.46 Å derived from the X-ray crystallography of AB graphite and with previous theoretical predictionsOur density functional theory calculations were performed using the Vienna Ab initio Simulation Package (VASP)4245Ead) of a Li adatom as followsTo assess the stability of Li adsorbed on graphene, we calculate the adsorption energy  of defects, including N-doping and C-vacancy, on graphene are calculated as follows:The formation energies .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Eutreptiella pomquetensis, and the spread and peculiarities of introns.Over the last few years multiple studies have been published showing a great diversity in size of chloroplast genomes (cpGenomes), and in the arrangement of gene clusters, in the Euglenales. However, while these genomes provided important insights into the evolution of cpGenomes across the Euglenales and within their genera, only two genomes were analyzed in regard to genomic variability between and within Euglenales and Eutreptiales. To better understand the dynamics of chloroplast genome evolution in early evolving Eutreptiales, this study focused on the cpGenome of Etl. pomquetensis cpGenome was sequenced, annotated and afterwards examined in structure, size, gene order and intron content. These features were compared with other euglenoid cpGenomes as well as those of prasinophyte green algae, including Pyramimonas parkeae.The Etl. pomquetensis, a basal taxon in the phototrophic euglenoids, was considerably larger than the two other Eutreptiales cpGenomes sequenced so far. Although the detected quadripartite structure resembled most green algae and plant chloroplast genomes, the gene content of the single copy regions in Etl. pomquetensis was completely different from those observed in green algae and plants. The gene composition of Etl. pomquetensis was extensively changed and turned out to be almost identical to other Eutreptiales and Euglenales, and not to P. parkeae. Furthermore, the cpGenome of Etl. pomquetensis was unexpectedly permeated by a high number of introns, which led to a substantially larger genome. The 51 identified introns of Etl. pomquetensis showed two major unique features: (i) more than half of the introns displayed a high level of pairwise identities; (ii) no group III introns could be identified in the protein coding genes. These findings support the hypothesis that group III introns are degenerated group II introns and evolved later.With about 130,561 bp the chloroplast genome of  Euglena, Monomorphina, Euglenaformis, Colacium, Strombomonas and recently Phacus  in addition to small cpGenomes with low intron numbers like M. aenigmatica  have been largely used to retrace evolutionary steps of phototrophic euglenoids. Members of the genera y Phacus  cpGenomeectively . Fittingectively . This hydetected . Howeverigmatica . AlthougEutreptiella pomquetensis  Marin & Melkonian in In the present study Eutreptiella da Cunha, with ten described species, and Eutreptia Perty, with eight known species. They are regarded as basal phototrophic euglenoids in aspects of morphology  Marin & Melkonian in Three-hundred mL of cell culture were harvested by centrifugation and submitted to cell cleaning and chloroplast isolation protocol as described in n homology search  and a Long Amp Taq DNA Polymerase .Using a BLASTy search  the fourThe final annotation of the chloroplast sequence was performed with Geneious 9 Pro ” and Emboss Sixpack Sequence translation (EMBL- EBI 2015) “Codon Table” set at “Bacterial” and added to the annotation. The introns within protein coding genes were analyzed for the presence of potential twintrons as described in abcdef (3–8 nucleotides) f′e′d′A∗c′b′a′ (four nucleotides)). Afterwards, all 51 introns were searched for the conserved 5′ insertion sequence GUGYG. RNA secondary structure for group II introns was created by RNA folding via Mfold web server using default settings  for being a previously identified chloroplast protein-coding gene were included in the annotation. ORFs were named according to the number of amino acids in the coding region. To evaluate the proportion of short repeated sequences the variable number of tandem repeats was scanned with the online version of REPuter  from the beginning of the 16S to the rpl32 gene. Primers were created manually by Primer3Plus  and C. vesiculosum  were found for each protein coding gene, except rpoA, where an alternative start codon (ATA) was accepted. Four protein coding genes were annotated with alternative start codons in Etl. gymnastica and Et. viridis (P. parkeae (Etl. gymnastica (59), where psaI was missing. Et. viridis lacked psaM, ycf12 (psb30) and ycf65 and hence counted only 57 protein coding genes. No mat5 or mat2 genes have been identified in Etl. pomquetensis, but mat1 (ycf13) was detected, as was expected from results in other Eutreptiales  to 51 introns in Etl. pomquetensis , but additionally to an increased intergenic space in Etl. pomquetensis. The intergenic space of Etl. pomquetensis comprised more than 23 kb, which was more than twice in that of Etl. gymnastica. While most of the blocks in Etl. gymnastica were quite compact with little intergenic or intron space in blocks C, E and G, all of the identified clusters showed heavily fragmented blocks in Etl. pomquetensis, except A and B . As the phototrophic euglenoids have a reduced amount of protein coding genes in contrast to the green algae, this high number of clusters was expected.Three additional Mauve analyses using O. tauri . A compaEtl. pomquetensis. A BLASTp analysis was performed against the NCBI nonredundant protein sequences (nr) database to determine whether any of the ORFs had functional similarity to previously sequenced genes (psbD gene of Etl. pomquetensis contained two ORFs (orf585 and orf532). The intron encoded orf585 of Etl. pomquetensis psbD I2 shared strong similarity with the orf583 of atpB I1 in the chloroplast genome of Pycnococcus provasolii  were found in ed genes . The psbovasolii , with anindracea . The opete 2.4.0  and a maThere is no evidence of a VNTR (variable number of tandem repeat) sequence, though this could be a result of our inability to circularize the genome.psaA contained the highest count with six introns (Et. viridis (27), nearly eight times higher than found in Etl. gymnastica (7), and consequently constitutes the highest intron number known in the Eutreptiales  with the presumed “branch-point” *A for splicing at position eight in domain VI, where the first transesterification takes place  in Etl. pomquetensis with high pairwise identity were closely related and arose from a single ancestor proliferating via retrotransposition and moved horizontally into DNA target sequences, which resembled the homing site. According to Etl. pomquetensis could be explained.We assume that all of these 28 introns of Etl. pomquetensis used host encoded proteins to promote splicing, reverse splicing and mobility, which is typical for most mitochondrial and plant chloroplast group II introns (Chlamydomonas reinhardtii even utilized nuclear-encoded maturases for splicing of the trans-spliced group II introns (Still the question remained of how these introns could be spliced without an ORF including maturase activity in domain IV. One possibility was that they rely on trans-acting RNAs or proteins with two feasible splicing mechanisms: (1) The introns of  introns . Chlamyd introns .orf1311 in Oedocladium (Chlorophyceae), with an intron encoded maturase, could function as promoter for splicing the ORF-less group II introns. G. planctonica without ORFs, which may reflect an evolutionary pressure for a smaller and more compact intron structure enabling increased efficiency of splicing and mobility, when maturase activity is provided from elsewhere. Furthermore, it might be assumed that an early event in the Etl. pomquetensis cpGenome was the deletion of an intron encoded ORF, which appeared to have occurred prior to the spreading of introns across the genome and that other group II introns with encoded IEPs or freestanding ORFs acted in trans to promote splicing and mobility of ORF-free introns . The gene psaA contained the most of these introns, and five of six introns contained high similarity.(2) All these introns could be spliced by a single IEP (Intron-Encoded Protein) that could either be free-standing or located in a functional intron. This would provide an accessible splicing apparatus and allow all but one intron to lose its own IEP . Brouard introns . To gainrocesses , thus heEtl. gymnastica, Et. viridis and P. parkeae and all other euglenoid cpGenomes did not reveal any sequential or positional homology. Insertion sites found in Etl. pomquetensis were unique to that taxon.A search for related introns in E. gracilis var. bacillaris with 56.7%, but only for three small 97 bp long group III introns . Also, outside of euglenoid chloroplast introns, very few species showed high pairwise similarity. For instance, Oedocladium carolinianum with high levels of nucleotide identities, which displayed over 80% pairwise identity. As well ycf3 and psbH in Gloeotilopsis sarcinoides were 85.6% identical. To our knowledge, Etl. pomquetensis is the first organism with more than 50 introns within protein coding genes and half of those sharing a pairwise identity of 90%.The highest pairwise identity of introns was found in Etl. pomquetensis is the absence of group III introns. Group III introns are believed to be the descendants of group II introns which only retained domains DI and DVI (abcdef (3–8)f′e′d′A∗c′b′a′ . Furthermore, the intron size was even larger than group III twintrons (group III introns within group III introns), which were found in the chloroplast genome of E. gracilis . It is clearly a group II intron/ twintron in all Eutreptiales, but a group III twintron in E. gracilis with an open reading frame  within the internal group III intron  that is at least three times larger than the group III twintron (I4) including mat1 of E. gracilis  act in trans to promote splicing and mobility of ORF-less introns.Summarizing, we presume that group II introns appeared first in an intron-less ancestral genome and gave rise to group III introns and from there on degeneration went on independently in different lineages. Further on, either the petB (I1) and that this intron/twintron may be a synapomorphy for at least the Euglenaceae. petB I1 of P. orbicularis and discussed this intron/ twintron as a putative synapomorphy for the order Euglenales. However, in the cpGenome of Etl. pomquetensis two introns were detected in petB. The first was found at the identical insertion site, but nearly two times larger than that of E. gracilis strain Z and five times larger than that of P. orbicularis. All petB I1 introns started with a typical group II 5′ -GUGYG  with only one 3′ motif and only one 5′ -GUGYG prior to the identified 3′ motif. Two of these potential group II twintrons (rpoB I1 and psbD I4) were those which share strong nucleotide identity with half of the introns detected in Etl. pomquetensis. We assume that all 28 introns (petG I1 and psaC I2) with equal intron organization  are potential twintrons with an external and internal group II intron  near the base of the stem of domain V were detectable, but the secondary structure showed a slightly altered terminal loop and no branch-point A∗ was detectable in domain VI  shared 88% pairwise identity with the other 27 potential twintrons . It is ln search for the external intron of psaC I2 (psbC I2 (containing a still unspecified maturase) of Etl. gymnastica. Secondary structure analysis of domains V and VI of psbC I2 from Etl. gymnastica, realigned by psaC I2 in Etl. pomquetensis revealed highly conserved structures of domains V , Pycnococcus provasolii (B) and Ostreococcus tauri (C). In the Mauve alignment the repeat regions of rRNA were not included, because Mauve will not align repeat regions, which have multiple matches on both genomes.Each box represents a cluster of homologous genes between Click here for additional data file.10.7717/peerj.3725/supp-3Figure S3Etl. pomquetensis based on the model proposed by Michel, Umesono & Ozeki, (1989) and on comparative analysis of other euglenoid group II introns  with branch-point A* at position 8 of domain VI and conserved three base pairs (5’- …AGC …-3’) near the base of the stem of domain V (red box). Domain V and VI of r po B I1 of Etl. gymnastica (B) with slightly altered base pairs (5’- …AGUC …-3’) of domain V (red box) but without branch-point A* at position 8 of domain VI.Domain V and VI of Click here for additional data file.10.7717/peerj.3725/supp-5Figure S5rpo B I1 of Etl. pomquetensis (A) with conserved three base pairs (5’- …AGC …-3’) near the base of the stem of domain V (red box). Internal intron of psb D I4 of Etl. pomquetensis (B) with conserved three base pairs (5’- …AGC …-3’) near the base of the stem of domain V (red box).Internal introns of Click here for additional data file.10.7717/peerj.3725/supp-6Figure S6psa C I2 external intron of Etl. pomquetensis (A) with branch-point A* at position 7 of domain VI and conserved three base pairs (5’- …AGC …-3’) near the base of the stem of domain V (red box). Secondary structure model of putative domain V and VI of psb C I2 of Etl. gymnastica (B) with slightly altered branch-point AU* at positions 7 and 8 of domain VI and conserved three base pairs (5’- …AGC …-3’) near the base of the stem of domain V (red box).Secondary structure model of putative domain V and VI of Click here for additional data file.10.7717/peerj.3725/supp-7Table S1* Chloroplast circle not closed. a Twintrons were counted as single insertion sites. b Including rRNA repeats and intermediate tRNAs. c Includes the identified 5S, for S. acuminata the two identified 5S. d Includes the two introns in rps18. e Includes the one intercistronic intron rps4-rps11. f First exon could not be identified, so gene length is a minimum. g Realigned, but with alternative start codon. h Start codon not determined, due to undetermined exon1 i: New intron start after re-analysesClick here for additional data file.10.7717/peerj.3725/supp-8Table S2Click here for additional data file.10.7717/peerj.3725/supp-9Supplemental Information 1Click here for additional data file.10.7717/peerj.3725/supp-10Supplemental Information 2Click here for additional data file.


Our results suggest pT3N0M0 ESCC clinical outcomes correlate with CD8+ and FOXP3+ TIL densities and PD-L1 levels. Moreover, an intrinsic mechanism for induction of PD-L1 overexpression may be occurring during early tumor oncogenesis. This information may be useful for stratifying patients and guide the application of checkpoint blockade therapy in ESCC.Data describing relationships between the tumor immune microenvironment and patient outcome are limited for esophageal squamous cell cancer (ESCC). The present study investigated the prognostic values of programmed death-ligand 1 (PD-L1) expression and CD8+ or forkhead box protein 3+ (FOXP3+) tumor-infiltrating lymphocytes (TILs) in 133 pathological T3N0M0 stage ESCC patients who underwent radical resection without neoadjuvant or adjuvant therapy. CD8+ and FOXP3+ TIL densities as well as PD-L1 levels in tumor cells and lymphocytes, were assessed through immunohistochemical staining. Patient survival was not associated with CD8+ or FOXP3+ TILs alone, but PD-L1 expression and the CD8+/FOXP3+ ratio were independent predictors of both disease-free and overall survival. PD-L1 expression correlated with age ( Esophageal squamous cell carcinoma (ESCC) remains the predominant type of esophageal cancer (EC) in the “EC Belt”, which extends from China via central Asia to northern Iran . DespitePatients with pathological T3N0M0 stage (pT3N0M0) disease represent a considerable fraction of middle-stage patients. Although they have the same TNM stage, they are divided into stages IB, IIA, and IIB based on tumor location and histologic grade in the 7th edition of the AJCC manual . Howeveret al. proposed stratifying tumors according to PD-L1 status, and TIL presence or absence in the microenvironment might predict patient response to anti-PD-1 therapy [As an immune checkpoint, the programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1) axis facilitates T-cell exhaustion and enables tumors to avoid immunosurveillance . PD-L1-P therapy . However133 patients with pathological T3N0M0 stage ESCC who underwent radical Ivor-Lewis esophagetomies were included in this study  were negatively correlated (PD-L1 showed a predominantly membranous or cytoplasmic (or both) focal or scattered staining pattern , longer tumor length , poorer histological differentiation (p = 0.002) and tumor recurrence (p = 0.037)  Table . There w7) Table .p = 0.013, 0.001 respectively; Figure p = 0.002, 0.001 respectively; Figure p = 0.043 and 0.022, respectively) and OS  compared to patients with negative PD-L1 expression  and OS   but was not an independent prognostic factor in multivariate analyses  for PD-L1 IHC detection (SP142) and validated our results via qRT-PCR in 10 tissue samples . We asseMolecular mechanisms underlying immune avoidance appear to be distinct among tumors. To date, two phenotypes have been identified: T cell-inflamed  and non-T cell-inflamed  . The twoOur study had several limitations. First, while pT3N0M0 stage ESCC patient optimal management strategies are still controversial, surgery alone is not standard treatment for such patients. We analyzed tumor original immune states, which are not affected by chemotherapy and radiotherapy. However, preoperative chemoradiotherapy may alter the tumor immune microenvironment, inducing T cell influx and PD-L1 upregulation, and this requires further study , 36, 37.Within the context of these limitations, our study identified an inverse relationship between CD8+ TILs and PD-L1 expression, suggesting that an intrinsic induction-type mechanism is active in ESCC. Our findings also suggest that differences in TIL density and PD-L1 expression may partly explain the diversity of clinical outcomes observed in patients with similar ESCC stages. These findings may be suggestive of the optimal treatment choice and checkpoint blockade therapy in patients with pT3N0M0 ESCC.The study cohort included 133 patients with pathological T3N0M0 stage thoracic ESCC who had radical esophagectomies in Shandong Cancer Hospital and Institute since June 2005. No patient exhibited evidence of distant metastases in preoperative examination, none had received any prior anticancer treatments or postoperative adjuvant therapy, and all underwent complete macroscopic tumor removal. All tumor tissues were confirmed as ESCC by hematoxylin and eosin (H&E) staining after surgical resection. Only patients who survived more than three mo post-surgery were included in the study. Tumor stage was determined according to the American Joint Committee on Cancer  TNM  staging system . Each tuIHC analyses were performed using the streptavidin-biotin-peroxidase method with postoperative specimens from all patients. 4-μm thin sections were deparaffinized with xylene, rehydrated with graded alcohol solutions, and then exposed to the antigen retrieval system under high pressure for two min. Sections were stained with anti-CD8 , anti-FOXP3  and anti-PD-L1  in a humidified chamber at 37°C for 60 min. Slides were incubated with a secondary anti-rabbit and anti-mouse antibodies (Beijing Zhongshan Golden Bridge Biotechnology Company) at 37°C for 15 min. Diamino-benzidine (DAB) was then added to visualize CD8, FOXP3 and PD-L1 staining and slides were counterstained with hematoxylin.Quantitative evaluations of CD8+ or FOXP3+ TILs were performed by examining at least five non-overlapping high-power fields (40× objective and 10× eyepiece) with the most abundant TILs in each stained section. PD-L1 expression was considered positive if distinct membranous or cytoplasmic staining was observed in tumor or stromal cells. PD-L1 staining intensity was recorded as follows: negative for no staining, positive for membranous or cytoplasmic staining in tumor cells or lymphocytes. Sections were evaluated independently by two experienced pathologists blinded to clinicopathologic information. Variations of > 5% were reassessed and a consensus was reached. As other cell populations can stain with CD8 and FOXP3 antibodies, CD8+ or FOXP3+ cells with apparent different morphological appearances were excluded from cell counts. FOXP3+ and CD8+ TILs were counted both in the cancer cell nest and tumor stroma. The CD8+/FOXP3+ TILs ratio was defined as the number of CD8+ TILs divided by the number of FOXP3+ TILs (Tregs). The mean numbers of CD8+ TILs and FOXP3+ TILs per field and the ratio of CD8+/FOXP3+ TILs were calculated. ESCCs were classified into high and low groups for CD8+ TILs, FOXP3+ TILs and the CD8+/FOXP3+ TILs ratio using a cut-off value defined as the median number of infiltrating cells per field, as described previously Figure .−ΔΔCT) method, and normalized to with the endogenous control, GADPH. The following primers were used in this study: HIF-1α, 5′-GAACGTCGAAAAGAAAAGTCTCG-3′(forward) and 5′ -CCTTATCAAGATGCGAACTCACA-3′ (reverse); PD-L1, 5′ - TTGCTGAACGCCCCATACAA-3′ (forward) and 5′ - TCCAGATGACTTCGGCCTTG-3′ (reverse); and GADPH, 5′ - GCACCGTCAAGGCTGAGAAC-3′ (forward) and 5′ - TGGTGAAGACGCCAGTGGA-3′ (reverse).To validate the PD-L1 IHC detection method, PD-L1 mRNA was quantified via qRT-PCR in 10 tissue samples. Total RNA was extracted using the RNA extraction kit spin column method  following the manufacturer's introductions. Total RNA (5 ng) was reverse transcribed with cDNA Reverse Transcription kits  according to the manufacturer's instructions. qPCR was conducted in a SYBR Green PCR Master Mix (ROX). Relative gene expression was calculated using the comparative cycle threshold (CT) . Cumulative survival time was calculated using the Kaplan-Meier method and compared using the log-rank test. Univariate and multivariate analyses were performed according to the Cox proportional hazards model. Associations among variables were evaluated using Fisher's exact test or the χ


Expression of programmed cell death receptor ligand 1 (PD-L1) has been shown to be up-regulated in some gastric cancer patients and to correlate with the density of tumour infiltrating lymphocytes (TILs). However, conflicting results have been reported regarding TILs and the expression of PD-L1 as a prognostic marker for gastric cancer. We investigated the correlation of PD-L1 and TILs expression with clinicpathological characteristics in 105 well characterized gastric cancer patients. PD-L1 expression and CD3+ and CD8+ TILs were evaluated by fluorescent multiplex immunohistochemistry (mIHC) analysis. PD-L1 positive staining on tumour cells was observed in 35% cases and 48% cases showed PD-L1 expression on immune cells. Up-regulated PD-L1 expression on tumour cells and immune cells was associated with high density of pre-existing tumour infiltrating CD3+ and CD8+. In additional, more than 70% tumor infiltrating CD3+ cells were CD3+CD8+ cells. More than 60% PD-L1+ immune cells were PD-L1+CD3+CD8+ cells. PD-L1 expression in tumour cells was associated with poor prognosis and high density CD3+ and CD8+ TILs indicated improved overall survival in gastric cancer patients. Increased PD-L1 expression with low density CD3+ and CD8+ TILs had the shortest overall survival. In accordingly, PD-L1 absence with high density CD3+ and CD8+ TILs indicated the best prognosis. Combination of PD-L1 with pre-existing TILs may be more precise than PD-L1 alone for predicting survival in gastric cancer. Gastric cancer (GC) is the fourth most common cancer and second leading cause of cancer associated death for men and women worldwide , 2. AlthPD-1 is present on the surface of active T and B cells, and PD-L1 is expressed on many types of immune cells. Several tumour types express PD-L1, including gastric cancer , 9. The There are several reports suggesting that PD-L1 expression is associated with poor prognosis of gastric patients –18. GastWe investigated the expression of PD-L1, CD8 and CD3 in gastric tumour specimens and evaluated the relationship of these factors to clinicopathological characteristics and patient survival. These results may serve as a surrogate marker for PD-L1-positive GCs and may help in validating biomarkers to select patients for immune checkpoint treatment strategies.We examined PD-L1 expression levels in tumour and immune cells in 105 gastric cancer specimens, but not in non-neoplastic gastric epithelium. We observed that PD-L1 was mainly expressed on the membrane and cytoplasm of tumour and immune cells Figure . In the We also evaluated PD-L1 expression on immune cells. PD-L1 staining in ≥1% of immune cells was considered PD-L1 positive. Fifty of 105 cases (48%) were PD-L1 expression Table  and 15 cClinicopathological features and molecular characteristics according to PD-L1 expression on tumour cells and immune cells are summarized in Table n = 105) included 84 males (80%) and 21 females (20%). According to age, patients were classified into <65y (49%) and ≥65y (51%) subsets. Expression of PD-L1 in tumour cells was associated with location (P = 0.012), tumour differentiation (P = 0.016), Ki67 status (P = 0.022) and HER-2 status (P = 0.022). In 30 cases (29%), tumours were located in the gastric cardia and body (17% had PD-L1 positive tumour cells). In 75 cases (71%), tumours were located in the gastric antrum (43% had PD-L1 positive tumour cells). Forty-two cases were moderate to well differentiated (21% had PD-L1 positive tumour cells), and 63 cases were poor differentiated (44% had PD-L1 positive tumour cells). Expression of PD-L1 in tumour cells was significantly associated with the high Ki67 and HER-2 positive cases. In Ki67 high status cases approximately 45% had PD-L1 positive tumour cells. However, in Ki67 low subsets 23% had PD-L1expression in tumour cells. In HER-2 positive cases approximately 58% had PD-L1 positive tumour cells. However, in HER-2 negative subsets less than 30% had PD-L1 positive tumour. Expression of PD-L1 in immune cells was also associated with antral location (P = 0.007) and high Ki67 subtype (P = 0.004). Interestingly, in Ki67 high cases approximately 60% had PD-L1 positive immune cells. However, in Ki67 low cases 32% had PD-L1 positive immune cells. Unlike in the tumour cells, expression of PD-L1 in immune cells was not significantly associated with the poor differentiation or HER-2 status.The study cohort (Staging was classified according to the tumour-node-metastasis (TNM) classification of the American Joint Committee on Cancer . PD-L1 expression was not significantly associated with age, gender, disease stage or tumour growth pattern on either tumour cells or on immune cells.Increased PD-L1 expression on tumor cells and immune cells both positively correlated with CD3+ and CD8+ cell infiltration in gastric cancer. PD-L1 positive in tumour cell subset had a high density of tumor infiltrating CD3+ cells and CD8+ cells Figure . And morTumor infiltrating CD3+ and CD8+ cells were counted, scored as 1 (<1%), 2 (1–9%), 3 (10–20%), or 4 (>20%) and classified into low and high subsets. The correlation between density of different TIL types and PD-L1 expression was summarized in Table These results demonstrated that PD-L1 expression in tumor cells and immune cells was positively associated with the density of CD3+ and CD8+ TILs.P = 0.025). There was no significant difference (P = 0.138) in cumulative survival time between PD-L1 positive and PD-L1 negative immune cell subsets in the whole population  for 10 min at room temperature to block endogenous enzyme. Then, 5% BSA  was used to block unspecific binding for 20 min at room temperature. For IHC, sections were incubated with 1:250 diluted primary antibody PD-L1  incubated at 4°C over-night. Anti- Rabbit IgG SABC kit  and DAB detection kit  were used. For Fluorescent Multiplex immunohistochemistry (mIHC), firstly, slides were incubated in 1:250 diluted primary antibody CD8  for 60 minutes at room temperature. After wash, slides were incubated in HRP labeled anti-mouse secondary antibody  for 30 minutes at room temperature. Wash slides by TNT buffer . Sections were keep in dark place and incubated with 1:50 diluted TSA Plus Cyanine 5  for 5 minutes at room temperature. Slides were stripped in 10mM sodium citrate buffer, pH 6.0 at a sub-boiling temperature for 10 min by microwave. Secondary, 1:250 diluted primary antibody CD3  were add on the sections and incubated in dark for 60 minutes at room temperature. HRP labeled anti-rabbit secondary antibody  and 1:50 diluted TSA Plus Cyanine 3  were used. After stripping, slides were incubated with 1:250 diluted PD-L1  for 60 minutes at room temperature. HRP labeled anti-rabbit secondary antibody  and 1:50 diluted TSA Plus Fluorescein  were used. Finally, mount sections with coverslips using DAPI . Leica Laser confocal microscopy was used for Multi-Color Fluorescence Imaging.The diagnosis was verified for all samples using H&E staining. For IHC, sections were deparaffinised, rehydrated and incubated in Antigen retrieval Citra solution, pH 6.0  at 95°C-99°C for 20 min, then cooled to room temperature. Slides were incubated in 3% HAll IHC and mIHC analyses were performed by two independent observers (Shen and Huang) who were unaware of the patients’ record of clinicopathological features. PD-L1 expression in tumour cells was graded into three groups: 0 (no staining), 1 (weak staining), 2 (moderate to intense staining). We counted PD-L1 staining cells per 1000 tumor cells. The percentage of PD-L1 expression tumuor cells was determined based on 10 random areas (HPF 400 × magnification) in each section and the values were average for statistical analysis. Cases in group 1 or 2 with ≥5% PD-L1 in tumour cells were considered positive as previously. Immune cells were scored separately. PD-L1expression immune cells  were counted per 1000 immune cells, and the percentage was determined based on 10 random areas (HPF 400 × magnification) in each section and the values were average for statistical analysis. Cases with ≥1% PD-L1 in immune cells were considered positive. The number and percentage of CD3 and CD8 expression TILs were determined as PD-L1. Expression of CD3 and CD8 in TILs was scored as 1 (<1%), 2 (1–9%), 3 (10–20%), or 4 (>20%). Subsequently, the grades were classified into low (scores 1–2) and high (scores 3–4) subsets. The IHC results for Ki67 and HER-2 expression were obtained from the pathology department of the First People’s Hospital of Zhenjiang.t-test was used to analyze the significance of differences between the mean values of two variables. All comparisons were calculated using a two-tailed test. Significant differences were considered at P < 0.05.All statistical analyses were performed using SPSS 22.0 . Pearson’s chi-square test and Fisher’s exact test were performed to evaluate the correlation between PD-L1 expression, density of TILs and other clinicopathological features. Kaplan-Meier methods were used to calculate cumulative survival and 95% confidence intervals (CI) were computed. Log-Rank test was used to determine significant difference between survival curves. Student


Nowadays, many types of materials are elaborated for microwave absorption applications. Carbon-based nanoparticles belong to these types of materials. Among these, graphene presents some distinctive features for electromagnetic radiation absorption and thus microwave isolation applications. In this paper, the dielectric characteristics and microwave absorption properties of epoxy resin loaded with graphene particles are presented from 2 GHz to 18 GHz. The influence of various parameters such as particle size  and weight ratio (from 5% to 25%) are presented, studied, and discussed. The sample loaded with the smallest graphene size (3 µm) and the highest weight ratio (25%) exhibits high loss tangent (tanδ = 0.36) and a middle dielectric constant ε′ = 12–14 in the 8–10 GHz frequency range. As expected, this sample also provides the highest absorption level: from 5 dB/cm at 4 GHz to 16 dB/cm at 18 GHz. It presents interesting thermal, mechanical, and electrical properties  along wider form  and σ = yer form ). Anotheyer form ,6,7,8 anyer form . GraphenThe present paper deals with graphene particles embedded into an epoxy resin for microwave absorption applications, and more specifically for the microwave isolation of closer antennas attached on the same composite panel and working between 2 GHz and 18 GHz. It is organized as follows. In Samples were fabricated from pre-dispersals of graphene particles in epoxy resin and a related hardener (masterbatch). The graphene particles consist of the agglomeration of single-layer graphene flakes, each about 0.34-nm-thick corresponding to the interlayer spacing of graphite from sp2 carbon chemistry . Graphen11 reflection coefficient measured via a commercial dielectric kit (High Temperature probe (19 mm in diameter) associated with the N1500A Material Measurement Suite software, both supplied by the Keysight Technologies company, Les Ulis, France). The set-up consisted in a probe in close contact with the surface sample. After an air/short/water probe calibration, results between 2 GHz and 18 GHz were presented to be consistent with the forthcoming reflection and absorption characterizations. To restrict any implementation error and microwave cable effect, a homemade bench-test was fabricated  were retrieved from the Sbricated a. At lea11 and the transmission S21 coefficients, the material absorption was computed from the linear power conservation Equation (1) expressed as follows (Equation (2) [The second and third studied parameters here are the microwave reflection and absorption of the materials. To this end, 3 pairs of high matching horn antennas (4.65–8 GHz // 8–12 GHz // 12.5–18 GHz) were successively used to measure the S-parameters in a near-field setting. The largest apertures of the horn antennas (109.25 mm × 79 mm) had imposed the sample dimensions  were first studied. Other samples fabricated with different weight ratios and the three particle sizes  were studied . MaximumSamples loaded with the largest particle sizes at the maximum weight ratio exhibit closer dielectric constant and loss tangent values . The use11|2 and the electromagnetic transmitted power |S21|2 by and through the composite material under test, respectively. With Equation (2), it is easy to compute the electromagnetic absorption α of the composite material.The assessment of the electromagnetic absorption was made using an homemade near field free-space bench as described in 11|2 ≈ 0 dB), |S11|2 values of the composite materials remain lower than −3 dB in the 8–18 GHz frequency range, evidencing the penetration of the electromagnetic wave into the materials. Reflected power and complex permittivity are closely interrelated with respect to Equation (3) applied with a normal incident electromagnetic wave in air striking a dielectric slab, as follows [air = 1, tanδair = 0) is the dielectric characteristics of air, and  is the dielectric characteristics of the composite material with thickness d.Electromagnetic absorption of the graphene composite materials depends on different parameters. The influence of the graphene particle size in epoxy resin was first investigated.  follows :(3)|S1111|2 between −0.8 dB and −6.4 dB, as observed in 11|2 ≤ −10 dB is explained by the total reflection of the electromagnetic waves by the composite material, the guide wavelength λg into the composite material ; the sample loaded with a 3-µm graphene particle size presents a resonance peak at 11.7 GHz (n = 1); those with 6-8-µm graphene particle size at 7.6 GHz (n = 1) and 15.0 GHz (n = 2); those with 15 µm at 5.7 GHz (n = 1) and 11.4 GHz (n = 2)—in agreement with the increase of the ε′ value when the graphene particle size increases (The variation of ε′ between 5 and 24 a and tantion (4) :(4)n×λgncreases a.n = 1); those with 20% weight ratio at 10.8 GHz (n = 1). The magnitude peak is damped by the increase of the graphene weight ratio, due to the rising of the related tanδ value  to convert α in dB/cm. In order to check the relevance of the ε′ and tanδ values provided by the dielectric kit method  from :(5)α=A×t method a,b, α vat method , despite11|2 ≤ −10 dB). Samples with a 6–8-µm graphene particle size and 10% weight ratio shows two resonance peaks at 7.6 GHz (n = 1) and 15.0 GHz (n = 2). With the increase in the weight ratio to 15%, these peaks shift at 5.6 GHz (n = 1) and 10.5 GHz (n = 2). This behavior is consistent with that of samples loaded with a 15-µm graphene particle size at 5% and 10% weight ratios. The second point is a behavior similar to these samples. The reflected power of a sample loaded with a 6–8-µm graphene particle size with a 10% weight ratio and that of a sample loaded with a 15-µm graphene particle size with a 5% weight ratio are alike. In the same way, a sample loaded with a 6-8-µm graphene particle size with a 15% weight ratio presents a similar |S11|2 to that loaded with a 15-µm graphene particle size with a 10% weight ratio. We attribute these results to the close dielectric constants and loss tangents of the two sets of samples  and the graphene weight ratio (ranging from 5% to 25%). For these, the dielectric constant and loss tangent , and the microwave absorption α of each sample, were measured using a dielectric kit (from 2 GHz to 18 GHz) and a homemade near field free-space bench (from 4 GHz to 18 GHz), respectively. The epoxy resin loaded with the smallest graphene size (3 µm) and the highest weight ratio (25%) exhibits the highest loss tangent (tanδ = 0.36) and a middle dielectric constant value (ε′ ≃ 12–14) in the 8–10 GHz frequency range. Filling the epoxy resin with the smallest graphene particles promotes high loss tangent at microwaves and thus the microwave absorption of the electromagnetic waves by the composite material. Moreover, a lower dielectric constant will be favored to restrict the impedance mismatch at the interface between air and surface samples and thus to restrict the electromagnetic wave reflectivity at this interface. The composite material then provides a high absorption level: from 5 dB/cm at 4 GHz to 16 dB/cm at 18 GHz.


Fibroblasts were isolated from diseased (n = 8) and non-diseased (n = 4) lungs. Global gene expression analysis was carried out at the initial point of isolation and after 3 weeks of culture. We identify several genes that are altered by removal of the fibroblast from the IPF environment. Comparison of this subset of genes to four previously published whole lung analyses refined our list to a small subset of key fibroblast specific genes important in IPF. Application of STRING database analysis and confirmation via in-vitro and histological assay highlights the CXCL14/CXCR4 chemokine axis with a possible role in the progression and/or activation of fibroblasts within the IPF lung. Our findings, present a possible therapeutic target for IPF and a model for the study and discovery of novel protein and processes in this terrible disease.Idiopathic Pulmonary Fibrosis (IPF) is a progressive disorder that is marked by an over accumulation of activated fibroblast populations. Despite the improved understanding of many mechanisms within this disease, global gene expression analysis has few focused studies on the fibroblast, the central effector cell of progressive fibrosis. We present a unique analysis of IPF pulmonary fibroblasts as they transition through cell culture and identify  The overabundance of these cells and their excessive extracellular matrix (ECM) deposition aids in the progressive fibrosis observed. This results in tissue distortion, impaired gas exchange, and ultimately organ failure6. The exact source of the IPF fibroblasts population remains unclear, however there are four potential reservoirs, including resident lung fibroblasts, circulating fibrocytes, epithelial cells that have undergone epithelial-mesenchymal-transition (EMT), and pleural mesenchymal cells9. As the fibroblast is so integral to the pathogenesis of IPF, much effort has been directed toward characterizing the specific processes within this cell. However, a comprehensive global understanding of the IPF fibroblast remains elusive.Idiopathic pulmonary fibrosis (IPF) is a lethal form of interstitial lung disease of unknown etiology. IPF has been defined as a disease of unregulated wound repair. This is likely driven by an abnormal epithelium and propagated by a dysregulated overabundant, heterogeneous, fibroblast population in various states of activationin vitro fibroblasts as a model system has revealed many important IPF mechanisms and molecules including transforming growth factor beta (TGF-beta1), tumor necrosis factor, and mediators of the Wnt pathway15. Despite this improved understanding of possible pathogenic mechanisms of IPF, only two therapeutic options have emerged each of which serve to slow disease progression. Global gene expression analysis is a powerful method for investigating the transcriptome and cellular diseased states, however analysis of isolated IPF and normal fibroblasts has shown no discernible genomic differences between populations18. Some data exists on the global genomic phenotype of IPF whole lung tissue in comparison to hypersensitivity pneumonitis, non-specific interstitial pneumonitis (NSIP) and emphysema. In addition, differential gene expression in familial and sporadic interstitial pneumonia has also been described20. However, the use of whole lung tissue in these experiments does not allow for characterization of the specific cellular contributions to disease.Investigation of this disease using in vitro models of disease; specifically the observation that once primary cells such as fibroblasts, chondrocytes, melanocytes, and hepatocytes are placed in tissue culture they undergo phenotypic changes affecting biological processes, cell motility, cytokine production, and cytoskeletal arrangements28. These changes often result in the primary cells bearing little resemblance to their cells of origin.The pace of progress is hampered in part by the imperfections of 17 at the time of isolation. Here we observed and analyzed these cells after three passages (P3) in culture. We expanded our analysis to include our initial list of 1700 differentially expressed genes and four previously published lists that identified differences in genomic expression between IPF and normal whole lung isolates32. These data revealed that through culture the genomic profiles of these cells change in vitro and we hypothesized that characterization of the adaptive processes evident in the transition to in vitro culture may enable the identification of novel IPF related mechanisms or proteins that may be suitable for therapeutic intervention.We have previously reported the development of a method for the isolation of IPF and normal fibroblasts  directly from lung tissue without the confounding effects of tissue culture. This method revealed over 1700 significantly differential expressed genes between normal-F and IPF-F17. However, in this investigation we found that once these cells transition to the in vitro state, this differential profile is lost. Specifically, gene expression analysis, using Statistical Analysis of Microarray ) software, of cultured IPF-F (n = 8) and normal-F (n = 4) demonstrated no statistically significant differential gene expression between these two populations.We have previously demonstrated that IPF-F and normal-F have significantly different global gene expression patterns when freshly isolatedin vitro, revealed 2889 and 1118 genes to be significantly altered, respectively  and normal-F (n = 4) from initial isolation (non- cultured) to 3 passages in vitro (P3) fibroblasts for both normal  and principle component analysis demonstrated clear clustering and separation between the non-cultured fibroblasts (P0) and the mal Fig.  and IPF mal Fig. . This hiAnalysis of the fold change in genes associated with myofibroblast differentiation and subpopulations reveals that the IPF-F and normal-F adapt to tissue culture by the promotion of markers typically associated with fibroblast activation such as ACTA2, CTGF, FGFR1 34. This analysis revealed enrichment for several well characterized processes . A result of particular interest is the CXCR4/CXCL12/CXCR14 axis as our string analysis indicates a possible role of these genes as part of a linker axis for the WNT pathway, VEGF angiogenetic pathway and the HIF1 regulated oxidative stress response. To demonstrate the value of refining the whole lung data sets with our derived IPF list, we performed overlap analysis solely with the data from the public lists. This analysis resulted in 704 genes found in at least 3 of the 4 public data sets  for 24 hours to simulate the oxidative stress present in the IPF lung. We then examined the expression of six genes with close connections to the CXCR4/CXCL12/CXCL14 axis found in the string pathway  regardless of the in-vivo origin, IPF or normal. This analysis also highlights the many genes whose transcriptional control is directly influenced by the IPF environment and are separate from the intrinsic propensity of the fibroblast itself.Analysis of whole lung tissue in IPF has resulted in several important findings identifying many of the key underlying processes in IPF; however, identifying disease specific processes that are primarily related to and/or regulated by the fibroblast is difficult in the whole tissue background. Our hypothesis is predicated on the idea that some fibroblast genomic adaptation to the disease environment is lost through the process of isolation and tissue culture. To address this issue, we documented the differential expression profile of IPF and normal fibroblasts from isolation and during culture. This unique approach demonstrated that there are a number of cellular pathways that are altered by the process of To demonstrate the utility of this approach we first compared four broad IPF whole lung tissue profiles to find overlapping processes within these lists. As expected, there is substantial concordance within these publically available data lists resulting in more than 700 genes overlapping Table . RefinemBy comparing our fibroblast specific list to the same four broad IPF whole lung tissue profiles, we identified a narrow subset of 59 IPF specific genes that we believe are indicative of the environmental impact on the fibroblast phenotype in disease. Many of these genes correspond to signaling peptides indicative of a dynamic crosstalk between the IPF environment and the IPF fibroblast. Using a smaller focused set of genes, we generated a string pathway which resulted in significant enrichment for biological pathways that control cell migration and developmental processes Fig. . As comp39  and migratory associated processes42  that respond to the IPF environment. Through our STRING analysis  and the receptor CXCR4L12 Figs  and 8 inion Figs  and 7 ou49. Prostate tumors develop rapidly and display increased angiogenesis in the presence of CXCL14 overexpressing cancer associated fibroblasts50. We speculate that in the IPF lung the CXCL14/CXCR4 interaction may be a driving force for the recruitment or migration of fibroblasts to the sites of fibrosis and that once the foci form, there is decreased need for this axis and therefore a decrease in expression. This hypothesis is further supported by recent studies into both CXCL12 and CXCL14. Like CXCL12, CXCL14 is over expressed in whole lung tissue of IPF patients51 and also found at higher levels in the serum of IPF patients52 strengthening its potential role as a recruitment mediator. In-vivo studies targeting CXCL12 in lung fibrosis models have overall been inconclusive, however they have demonstrated some success in the slowing of disease progression54. We theorize that inclusion of CXCL14 may reveal a complementary role for this ligand in the axis that when addressed may effectively target this pathway. Further studies into the significance of the co-localization of CXCL14/CXCR4/ACTA2 within the IPF lung needs to be performed, however, we speculate that it may be possible to slow or inhibit fibroblast migration and activation by pharmacological intervention directed at the CXCR4/CXCL14 complex.The role of CXCL14 in fibroblast biology is poorly understood. However CXCL14 has been identified in cancer associated stromal fibroblasts as a promoter of migration and ERK-dependent proliferation55. There is general agreement that therapeutic intervention in fibrotic disease is complicated by the heterogeneity of both the IPF lung and the disease as a whole. Our study highlights an added layer of complexity which further can cloud or even misdirect our understanding of fibroblast biology. Our unique approach highlights the transcriptomic changes observed through the first weeks of tissue culture that has enabled us to identify a subset of genes not previously reported as significant in IPF.IPF is a progressive disease that presents in acute, progressive, and stable variations based on many yet unknown environmental and genetic factorsin-vitro fibroblast culture model both before and after challenge. Finally, we identified co-expression of our most interesting gene, CXCL14, with ACTA2 in vivo in areas of fibrosis. Identification of CXCL14 as a gene of interest in IPF has been recently been reported56, however, to our knowledge, this is the first time that CXCL14/CXCR4/ACTA2 has been demonstrated together in the IPF lung and attributed to disease. This observation has not been previously described, which we hypothesize is due to the environmental limitation inherent to in-vitro tissue culture. We therefore suggest that our data set and model system can be applied to other IPF associated pathways to help identify potential therapeutic targets.A potential limitation to this study may emanate from what we have termed as the initial fibroblast population isolated from the IPF lung. Criticism of this technique, which is widely employed in other fields of study, may call into question the purity of the population at initial isolation. We addressed this concern in three ways: first, we used previously published data sets and string pathway analysis to refine the large number of genes in the IPF list. Second, we confirmed expression in an in-vitro tissue culture model. In doing so we identify a potential role for CXCL14 in IPF within the CXCL12/CXCL14/CXCR4 axis that is in accordance with our data and four published data sets. As a soluble ligand that is upregulated in IPF, CXCL14 serves as a candidate for further study that may also lead to a therapeutic intervention. Finally, we propose that further application of this data set may result in the identification of novel processes of interest in the IPF lung.In conclusion, we have taken a wide range of novel and published data and focused it through a model system that reduces the confounding effects of the traditional IPF lung tissue was obtained through Inova Fairfax Hospital (VA). All normal control lungs were obtained through the Washington Regional Transplant Community (WRTC). Appropriate written informed consent was obtained for each patient and donor by Inova Fairfax hospital and the WRTC. This study was approved by the Inova Fairfax Hospital Internal Review Board (IRB #06.083) and the George Mason University Human Subject Review Board (Exemption #5022). All experiments were performed in accordance with relevant guidelines and regulations. Additionally, no organs/tissues were procured from prisoners.57. Briefly, samples were dissected into 1–2 mm2 pieces and subjected to enzymatic digestion in 0.4% collagenase P  complete media  containing 10% fetal bovine serum (FBS), penicillin (100 I.U/ml), streptomycin (100 MCH/ml), amphotericin B (0.25 M.C.G./ml P/S/A) and 0.1% DNase1, at 37 °C and 5% CO2 for 2 hours. The resulting material was passed through sterile cell filters  to remove undigested tissue and remaining cells were pelleted by centrifugation at 1000 g for 5 min. The pelleted cells were then suspended in complete media and seeded onto tissue culture treated plastic at 37 °C and 5% CO2 for 45 minutes. The attached fibroblast population was vigorously washed with PBS to remove any unattached cells. P0 cell populations were extracted for RNA immediately. P3 populations were continued in culture and maintained in complete media at 37 °C and 5% CO2. All demographic information for the patients is listed below  and 4 normal  controls as previously describedRNA was extracted from IPF-F and Normal-F fibroblasts at P0 and P3 using a Qiagen RNeasy kit  and DNase treated using DNase free . The quality and quantity of the RNA was determined using RNA 6000 nanochips and an Agilent Bioanalyzer. For analysis, 1 µg of RNA was amplified and amino-allylated using the MessageAmp II aRNA kit (Ambion). All microarrays were carried out by the Duke Institute for Genome Sciences and Policy, Microarray Facility using HO36K human chip representing 33,791 transcripts from the Ensembl Human Build (BI-35C) including 22,169 unique genes (Operon Human Oligo set V4). Using two color amino-allylated amplified RNAs: cy5 corresponding to patient and normal samples, and cy3 for Stratagene human reference RNA. Microarray results were confirmed by QPCR analysis using a random selection of 11 genes  data was analyzed using statistical analysis of microarray (SAM)34. Any resulting transcript without a corresponding Unigene ID was not considered in this analysis. Additionally, some unigene IDs did not have a corresponding ID in the Database for Annotation, Visualization, and Integrated Discovery as noted in the discrepancy between Unigene and David IDs below. Table The resulting genepixs files were analyzed using the TM4 software suite  and normalized by locally weighted linear regression (lowess)34. Functional categories were clustered using the Functional Annotation Clustering tool, and clusters with a score of greater than 3.0 were selected for discussion. Protein-protein interactions were obtained using STRING  database. Interactions were parsed in three steps. Initial input of 59 proteins corresponding to differentially expressed genes were entered into STRING. All genes with predicted associations were curated manually and any closely linked non-associated proteins were also selected. The second step was simply the redefined string network that only included the manually curated proteins. Finally, predicted functional partner network linker nodes (string score > 0.995) were added to complete the network.Ontological analysis and classification of the genes identified in each analysis was carried out using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) as previously describedFibroblast populations isolated as described above were seeded in 6-well falcon tissue culture plates for 24-hours in complete media. After this attachment cells were placed in serum free DMEM for 24-hours and then challenged with serum free DMEM containing 250 µM hydrogen peroxide. Cells were then harvested for RNA analysis as described above. Assays were carried out on cells in passages 4–7. Two of the four IPF cell lines selected for this assay were derived from the pool of cells isolated for the microarray analysis and another two cell lines were derived from IPF donors not included in the microarray data set. All non-disease cell lines challenged by hydrogen peroxide were derived from normal donors in the microarray dataset.59  and Quantifast SYBR Green PCR Kit (Qiagen). QPCR was carried out in triplicate and normalized to 18 S expression levels using the delta delta CT methodFormalin fixed paraffin-embedded IPF and normal tissue sections (3–5 µm) were deparaffinized by serial washings in xylene, 100, and 90% ethanol prior to rehydration in nuclease free water. Antigen retrieval was performed by boiling in sodium citrate buffer (pH 6). Slides were blocked for 1 hour in 1% BSA prior to overnight incubation with primary antibodies: anti-actin smooth muscle (Thermo #PA5–16697), EpCAM , CXCR4 (Santa Cruz #sc-53534), CXCL12 (Santa Cruz#P-159X), CXCL14 (Thermo# PA5-528820). Primary antibody incubation was followed by 1 hour secondary incubation in Alexa Fluro 594 or 488 conjugate . Counter staining with DAPI was performed prior to imaging using Life Technologies EVOS FL. All antibodies were tested for cross reactivity prior to use and secondary antibodies were added in the absence of the primary antibody to confirm lack of non-specific binding. No cross reactivity was identified and minimal background florescence was detected at very high light levels which were not used during imaging. All images were taken from three different IPF and three different normal lung sections derived from the lower peripheral lobes. Select images are representative of whole section observations.https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE87175.The microarray data sets used in this analysis are available in the Gene Expression Omnibus repository, Supplmentary Information


Specific corticostriatal structures and circuits are important for flexibly shifting between goal-oriented versus habitual behaviors. For example, the orbitofrontal cortex and dorsomedial striatum are critical for goal-directed action, while the dorsolateral striatum supports habits. To determine the role of neurotrophin signaling, we overexpressed a truncated, inactive form of tropomyosin receptor kinase B [also called tyrosine receptor kinase B (TrkB)], the high-affinity receptor for Brain-derived Neurotrophic Factor, in the orbitofrontal cortex, dorsomedial striatum and dorsolateral striatum. Overexpression of truncated TrkB interfered with phosphorylation of full-length TrkB and ERK42/44, as expected. In the orbitofrontal cortex and dorsomedial striatum, truncated trkB overexpression also occluded the ability of mice to select actions based on the likelihood that they would be reinforced. Meanwhile, in the dorsolateral striatum, truncated trkB blocked the development of habits. Thus, corticostriatal TrkB-mediated plasticity appears necessary for balancing actions and habits. Nevertheless, the mechanisms by which the brain balances actions and habits are still being identified.Flexible action requires shifting between familiar and novel behavioral strategies. Extensive response training and exposure to stressors and certain drugs of abuse can lead to a bias towards habit-based behaviors that are by contrast inflexible. Maladaptive habits may contribute to illnesses characterized by impulse control deficits, such as addiction and obsessive-compulsive disorder5. The posterior dorsomedial striatum (DMS) and orbitofrontal prefrontal cortex (oPFC) are necessary for goal-directed actions, while the dorsolateral striatum (DLS) controls habits8.During the initial acquisition of an instrumental behavior, organisms are typically sensitive to the predictive relationship between actions and their outcomes, and goal-directed action selection strategies dominate. After continued training, reward-related stimuli can gain control over behavior, and behavioral response strategies become automated, or “habitual,” and insensitive to action-outcome associationsBdnf knockdown causes failures in action-outcome decision making and a deferral to habit-based behaviors9. Where, precisely, stimulation of the high-affinity BDNF receptor tyrosine receptor kinase B (TrkB) is important remains unclear, however, given that BDNF is subject to anterograde transport. For example, oPFC-selective Bdnf knockdown reduces BDNF protein in the dorsal striatum7, suggesting that TrkB activation in the oPFC, DMS, or both could support goal-directed action. Resolving these possibilities is important because upon BDNF binding, the intracellular domain of TrkB auto-phosphorylates, creating docking sites for effector proteins that initiate intracellular signaling cascades, e.g., the ERK42/44 and Akt pathways. TrkB impacts a diverse array of neuronal functions including cell survival and differentiation, axonal and dendritic growth and arborization and synapse formation and plasticity.The primary neurotrophin Brain-derived Neurotrophic Factor (BDNF) appears to be a key cortical substrate coordinating goal-directed action selection, given that oPFC-selective Here, mice were trained to generate two food-reinforced behaviors in operant conditioning chambers, then tested for sensitivity to action-outcome associations using a contingency degradation procedure. In this task, food pellets associated with one familiar behavior are delivered non-contingently (“for free”), regardless of the animal’s actions, while the other response remains reinforced Fig. . Mice thTrkB.t1, which lacks an intracellular domain and therefore cannot initiate intracellular signaling pathways (from10). We infused lenti-TrkB.t1 with an HA tag, lenti-Green Fluorescent Protein , or a half-and-half mixture of the two in order to generate multiple lenti-TrkB.t1 “doses”      Fig. , as expe13) Fig. , as alsoure Fig. . Specifi64) Fig. .TrkB.t1 mice were simply unable to energize a response that was reinforced . We feel that this is unlikely, however, given that across all groups, response rates during the reinforced phase of testing were higher than those generated on the last day of training   t5 = 4.385, p = 0.007), even though responding was not reinforced. By contrast, the other groups did not (GFP control 1-sample t-test against no change (0) t4 = −2.414, p = 0.073; Half TrkB.t1 1-sample t-test against no change (0) t5 = −1.233, p = 0.272).Given low response rates during instrumental response training Fig. , it is cns) Fig. . Groups ns) Fig. . Again, TrkB.t1 overexpression in the oPFC block, or instead delay, action-outcome learning and memory? To answer this question, we exposed mice to non-contingent pellet delivery for 2 additional sessions. Ultimately , full-titer TrkB.t1 mice inhibited nonreinforced responding in a goal-directed manner, indicating that TrkB.t1 overexpression delayed, but did not fully occlude, action-outcome-based decision making   Fig. .TrkB.t1 in the oPFC. We euthanized them 3 weeks following viral vector infusion and extracted the oPFC using a tissue punch. In this case, tissue samples would be expected to contain both infected and uninfected cells. Nevertheless, Trkb.T1 protein levels were detectably elevated in mice bearing the lenti-TrkB.t1 virus, as would be expected     , while total ERK42/44 was unchanged     Fig. . Phospho01) Fig. . Meanwhi58) Fig. . Consist26) Fig. . Similar61) Fig.  and no c34) Fig. .Figure 217. To address the possibility that TrkB.t1 overexpression led to an accumulation of cortical BDNF , or alternatively, diminished local BDNF levels, we also quantified BDNF. Neither mature nor pro-BDNF were significantly affected, despite large group sizes     Fig. . To addr27) Fig. . Our fin57) Fig.  further TrkB.t1 overexpression here7. Interfering with oPFC-striatal interactions also impedes an organism’s ability to modify instrumental behaviors when reward value changes8. The striatum contains very little Bdnf mRNA18, but abundant BDNF protein anterogradely transported from cortical sources19. We thus next examined whether TrkB in the dorsal striatum is similarly important for flexible action selection. In this case, we overexpressed TrkB.t1 selectively in the DMS or DLS    Fig. . TrkB.t117) Fig. . Thus, Tinduced habit behavior using a random interval schedule of reinforcement   Fig. .TrkB.t1 overexpression in these striatal sub-regions causes response patterns that bear remarkable resemblance to those following inactivation of each respective structure21. Although TrkB is expressed in both the DMS and DLS22, these patterns were nevertheless somewhat unexpected, given that systemic administration of a putative TrkB agonist blocks habits induced by extensive response training9 and excess glucocorticoids16, rather than facilitating this DLS-dependent behavior. TrkB stabilizes dendritic spine densities and morphologies throughout multiple brain regions23 and is essential for corticostriatal long-term potentiation24. The switch from goal-directed action to habits is thought to reflect a transition in the coordinated control of response strategies by multiple cortico-striatal regions to a predominantly DLS-controlled output . Thus, broad-spread TrkB stimulation  may energize goal-directed action by stimulating multiple cortico-striatal structures 12 competing with the DLS for control over behavior. Further understanding the molecular mechanisms mediating the balance between actions and habits could shed light onto treating disorders characterized by impairments in flexible action and decision making, such as obsessive-compulsive disorder and addiction2.To summarize, TrkB appears to be essential to the functions of both the DMS  and DLS (supporting habits). Indeed, ad libitum access to water and food, except during instrumental conditioning when body weights were maintained at ~90% of baseline. Procedures were approved by the Emory University Institutional Animal Care and Use Committee and were performed in concordance with The Guide for the Care and Use of Laboratory Animals.Experiments used adult male wild-type C57BL/6 mice  . Mice were housed 2–5 per cage and maintained on a 12-hour light cycle (on at 0800) and were experimentally naïve. Mice had TrkB.t1 and an HA tag or GFP under a CMV promotor were generated by the Emory University Viral Vector Core and have been described in detail previously10. Viral vectors were infused at a rate of 0.1 μL/minute, with a total volume of 0.5 μL and the microsyringe left in place for 5 minutes following infusion. In experiments targeting the oPFC, viral vectors were infused at +2.6 mm anteroposterior (AP), −2.85 mm dorsoventral (DV) and +/−1.2 mm mediolateral (ML). Viral vectors targeting the DMS were delivered to +0.74 mm AP, −3.0 mm DV and +/−2.2 mm ML. DLS coordinates were +0.5 mm AP, −3.5 mm DV and +/−2.7 mm ML.Mice were anaesthetized with ketamine/dexdomitor and then mounted onto a digital stereotaxic apparatus . Lentiviral vectors expressing Mice were trained to nose poke for food pellet reinforcers  in Med-Associates  operant conditioning chambers. Mice were trained to nose poke on 2 available apertures using a fixed ratio 1 (FR1) schedule of reinforcement for 5 sessions. Next, mice were trained for 2 additional days using a random interval 30 second (RI30) schedule of reinforcement. Sessions lasted for 70 minutes or until the maximum 60 pellets (30 per nose poke) had been delivered.26. Briefly, during the ‘non-degraded’ session, one nose poke aperture was occluded and responding on the other nose poke aperture was reinforced using a variable ratio 2 (VR2) schedule of reinforcement. The next day, during the ‘degraded’ session, pellets were delivered non-contingently at a rate yoked to the reinforcement rate from the previous session. Responses were recorded, but had no programmed consequences. The location of the ‘degraded’ aperture was counterbalanced across subjects. Mice that decrease their response rates during the ‘degraded’ session are considered goal-directed. Equivalent response rates during the ‘non-degraded’ and ‘degraded’ sessions are thought to reflect habitual responding4.Next, mice were tested for sensitivity to action-outcome contingencies using a modified version of classical action-outcome contingency degradation, the details of which are further discussed in refsIn experiments with oPFC infusions, mice were tested in the modified contingency degradation procedure 3 consecutive times. In experiments with striatal infusions, following the first contingency degradation test, mice were trained for an additional 4 days with 2 available nose poke recesses using an RI60-second schedule of reinforcement. Then, mice were again tested for sensitivity to action-outcome contingency degradation, as above.TrkB.t1 virus, or GFP was imaged. To visualize HA, sections were blocked, then incubated with the primary antibody  overnight at 4 °C. The next day, sections were incubated with secondary antibody  and then mounted with Permount  for fluorescence imaging. Mice with mislocalized infusions were excluded from analysis, resulting in the omission of 1 mouse from each of the TrkB.t1 groups and 2 mice from the GFP control groups in the oPFC infusion experiment and 2 mice from each group in the dorsal striatal infusion experiment.Mice were anesthetized by isoflurane and euthanized by rapid decapitation. Brains were stored for 48 hours in 4% paraformaldehyde and then transferred to a 30% w/v sucrose solution. Brains were then sectioned at 50 μM. To verify infusion sites, sections were immunostained for the HA tag on the Sections were immunostained for the HA tag (as above). Sections were imaged on a Nikon 4550 s SMZ18 stereo microscope . All images were collected in the same session with settings held constant. A sampling area was drawn around the infusion site and the mean integrated intensity was quantified in NIS Elements (Nikon Instruments).TrkB.t1 or GFP as above. Approximately 3 weeks following infusion, matching the onset of behavioral studies, mice were rapidly decapitated and brains were stored at −80 °C, then later sectioned into 1-mm thick sections. The oPFC was dissected using a 1 mm tissue core. Tissue was homogenized in lysis buffer  and protein concentrations were determined by a Pierce BCA Protein Assay kit (Thermo Fisher Scientific). 15 μg of each sample was separated by SDS-page on a 7.5% gradient Tris-glycine gel . Next, samples were transferred to a PVDF membrane (Bio-Rad) and blocked with 5% nonfat dry milk for 1 hour. The membrane was incubated overnight at 4 °C in primary antibodies. Primary antibodies were TrkB , phospho-Trk (Y706/Y707) , Akt , phospho-Akt (T308) , ERK42/44 , phospho-ERK42/44 (T202/Y204) , BDNF , GFAP , PSD-95  and HSP70 . Following 1 hour of incubation in secondary antibodies [goat anti-mouse and anti-rabbit peroxidase labeled IgG ], immunoreactivity was assessed using a chemiluminescence substrate (Thermo Fisher Scientific) and a ChemiDoc MP Imaging System (Bio-Rad). Immunoblot comparisons were generated at least twice.Behaviorally-naïve mice received oPFC-targeted infusions of full-titer lenti-e.g., refs9). Behavioral response rates were compared by 2-factor mixed-design ANOVA and Bonferroni post-hoc comparisons in case of significant interactions. In an additional analysis, response rates during the instrumental contingency degradation testing phases were normalized to response rates associated with the same nose poke port generated on the final day of training. Fold-change values were compared by 2-factor ANOVA, as well as 1-sample t-tests against no change (0).All mice were randomly assigned to condition, and sample sizes were in line with prior reports using the same approaches  in the same lane and then to the control sample mean on the same gel to accommodate fluorescence variance Throughout, normality was confirmed using the Shapiro-Wilk test. Values >2 standard deviations above or below the mean were considered outliers and excluded, resulting in the omission of 1 mouse each from the fold-change calculations in Fig. Behavioral experiments were not performed blind to the condition, but response rates were collected via automated photobeam-based systems, minimizing bias. Similarly, equivalent amounts of protein were loaded in western blotting experiments, also minimizing bias.Data can be made available upon reasonable request.