Graphene nanostructures have attracted a lot of attention in recent years due to their unconventional properties. We have employed Density Functional Theory to study the mechanical and electronic properties of curved graphene nanoflakes. We explore hexagonal flakes relaxed with different boundary conditions: (i) all atoms on a perfect spherical sector, (ii) only border atoms forced to be on the spherical sector, and (iii) only vertex atoms forced to be on the spherical sector. For each case, we have analysed the behaviour of curvature energy and of quantum regeneration times  as the spherical sector radius changes. Revival time presents in one case a divergence usually associated with a phase transition, probably caused by the pseudomagnetic field created by the curvature. This could be the first case of a phase transition in graphene nanostructures without the presence of external electric or magnetic fields. The experimental isolation of a single graphitic layer (now known as graphene) by means of the so-called “Scotch tape method” by Geim and Novoselov  has undoThe outstanding properties of this 2D material have led to many application proposals. To cite just a few of them: nanocomposites for bone tissue engineering , composiQuantum revivals consist in the temporal periodic reconstruction of a wave packet in systems with a commensurable discrete spectrum . They haOne of the common misconceptions about graphene is that it is flat. In fact, Peierls instability ,29 creatRectangular graphene flakes either free  or subjeWe present in this article the first (to the best of our knowledge) calculation of quantum revivals using Density Functional Theory (DFT) and use it to study the interplay between curvature and regeneration times in a non-planar graphene flake.Since Wallace did the first calculation of the electronic properties of graphene using the tight-binding method , many thIn order to avoid non-trivial magnetic ground states induced by asymmetric edge extensions  we have We have studied (quasi-)spherical nanoflakes of different curvature radii with the three different bending possibilities depicted in We have studied both curvature energy and quantum revivals in this nanostructure. The first one for checking if the macroscopic continuum limit model that predicts that curvature energy is proportional to the Gaussian curvature   holds atE with the inverse squared radius of the sphere R the geometry with only the vertices fixed is energetically more favourable than that with the borders fixed and this in turn is more stable than the one with all the atoms forced to lie on a spherical surface. As expected, for the three geometries the curvature energy increases with curvature but the trend for the fixed surface case is somewhat unexpected. The macroscopic continuum result We present in To check if the macroscopic result holds at least for low curvatures, we present in This deviation from the continuum behaviour is undoubtedly due to the difference between the classical physics laws governing the macroscopic world and the quantum ones ruling at the nanoscale. This difference translates into two distinct aspects. The equilibrium geometry dictated by both sets of laws is different and, even for the same configuration, they lead to different energies. To check which one is more important in this case, we have made a series of non-self-consistent calculations with a hybrid Molecular Mechanics (MM)/DFT model. We have first used LDA calculations to determine a “semiclassical” Hooke potential for the C–C bond. To this end, we have used a graphene flake similar to the one in We have included in R, adopting the formIn order to check if the continuum model is valid for this approach, we have also included the corresponding results in n as R changes calculated using a 3-point finite differences method.We present in It is possible to fit the results in this figure using the simplest Padé approximantThe result of this fitting is presented in n.We can use this Padé approximant to calculate the asymptotic behaviour of This result is very close to 2, which is the value predicted in the continuum model. Therefore, the semiclassical MM/DFT approach tends to the classical continuum macroscopic limit, proving that the main quantum contribution to the deviation from this model is due to the small change in the equilibrium geometry.We can write the initial state of a time-independent Hamiltonian The temporal evolution of this state can be written asSince we have calculated the energy of curved graphene nanoflakes using DFT  we know their energy spectrum and can therefore study the time evolution of a wave packet in these systems. Let us consider a superposition of eigenstates of the Hamiltonian concentrated around a central energy level Taking into account Equations  and 8),,8),(9)ΨEach term in the exponential  defines a characteristic time scale:  and the second one analytical (an.).In principle, calculating quations  and 11)TCl is stpump, creates the initial wave packet, while the second one, called probe, measures its time evolution. By changing the delay between the two pulses, different time scales can be explored. This pump–probe scheme was proposed by Alber, Ritsch, and Zoller [Experiments for measuring quantum regeneration times are based on the use of two consecutive laser pulses . The fird Zoller , and wasd Zoller ,73. In td Zoller ,75. Mored Zoller .We present in The agreement between numerical and analytical estimations is good for all geometries for low curvatures  case, the behaviour is simpler. Numerical and analytical estimations agree perfectly except for the high curvature regime because, as we will see in the next subsection, We show in In all cases, numerical and analytical estimates perfectly agree since superrevival times are much higher than revival ones and do not interfere with them.According to Equation , TRe is Revival times for the fixed surface and fixed borders cases are very similar and decrease monotonically with curvature. n Scilab  and the n Scilab ). The reSimilar behaviours have been found in other 2D Dirac materials in external fields. For instance, planar graphene rings  and grap


Through strong hydrogen-bonding interaction between the phenolic hydroxyl groups on G-TA and primary amine groups on Si–NH2, SiO2 was uniformly loaded to the surface of graphene. Due to the synergistic dispersion effect of graphene and SiO2, which prevents restacking and re-aggregating of both graphene and SiO2, HGS hybrids were distributed evenly in the natural rubber (NR) matrix (HGS@NR). Simultaneously, the surface roughness of graphene after loading SiO2 and the interfacial interaction between the HGS hybrid and NR matrix were substantially improved. Due to the good dispersion and strong interface, the overall properties of HGS@NR nanocomposites are drastically enhanced compared with those of GS@NR nanocomposites prepared by dispersing the blend of unmodified graphene and SiO2 (GS) in NR. The HGS@NR nanocomposites possess the highest tensile strength up to 27.8 MPa at 0.5 wt% and tear strength of 60.2 MPa at 0.5 wt%. Thermal conductivities of the HGS@NR nanocomposites were found to be 1.5-fold better than that of the GS@NR nanocomposites. Also, the HGS@NR nanocomposites exhibit excellent abrasive resistant capacity that is nearly 2-fold better than that of the GS@NR nanocomposites. These results suggest that HGS has great potential in high-performance nanocomposites and a new strategy of constructing the efficient graphene–SiO2 hybrid fillers has been established.A novel graphene–silicon dioxide hybrid (HGS) was prepared by plant polyphenol-tannic acid (TA) functionalized pristine graphene (G-TA) and primary amine-containing silane coupling agent modified SiO 2 (Si–NH2).A novel graphene–silicon dioxide hybrid (HGS) was prepared by plant polyphenol-tannic acid (TA) functionalized pristine graphene (G-TA) and primary amine-containing silane coupling agent modified SiO Generally, a single filler could not meet the variety of performance requirements of polymer products. Meanwhile, the fillers in a composite material system usually are poorly compatible with the polymer matrix. Recently, a hybrid filler consisting of two or more constituents with different performance and functions offers an alternative and novel inspiration for designing high-performance or functional polymer nanocomposites.8–10 Graphene can cause a crucial improvement in the electrical, mechanical, and thermal properties of the resulting graphene/polymer composites at very low doping percentages. However, graphene is an atomically thin nano-plate of sp2 bonded carbon atoms. It is a processing challenge for these hydrophobic fillers to be uniformly dispersed in the polymer matrix. Excellent dispersions and interfacial interactions between graphene and the polymers are vital to the generation and utilization of a strong composite.11 The prevalent strategy to intensify interactions between graphene and the polymer chains is chemical functionalization of the surface of the nanoparticles.12 However, the chemical functionalization strategies are extraordinary comprehensive and poor universal applicability, destruction of graphene intrinsic properties and that the interfaces interaction between graphene and the polymer chains is not obvious.Nowadays graphene is widely used as functional filler in polymer nanocomposites for its extraordinary mechanical, thermal and electrical properties.2, SnO2, Fe3O4)/graphene hybrids have been widely investigated as promising high-capacity anode material for lithium-ion batteries.13,14 A facile ionic self-assembly process has constructed two-dimensional graphene/SnO2/graphene hybrids with SnO2 nanoparticles sandwiched in graphene sheets.15 A nanocomposite comprised of chemically converted graphene and carbon nanotubes can preserve the intrinsic electronic and mechanical properties of both components.16 Baochun Guo et al. have systematically investigated the mechanism for the synergistic reinforcement in an elastomer reinforced by nanocarbon hybrids consisting of 2D reduced graphene oxide and 1D carbon nanotubes.17Recently, various hybrid combinations based on graphene have been supposed to be ideal candidates to construct hybrid fillers towards high-performance polymer nanocomposites. Metal oxides (TiO2) hybrids in elastomer composites is still less research. SiO2 applied in composites have attracted much research attention18–22 in the last few decades owing to the superior properties of SiO2 nanoparticles such as toughness, tensile strength, thermal stability, gas separation performance, high scattered performance, and thermal resistance23 and they have various applications in high-performance coatings,24,25 fibers,26,27 bioactive polymers,28,29 rubber composites etc.30–32 The nanometer-sized SiO2 particles are used not only as a kind of filler, but also as reinforcing33,34 and toughening35 agents that can impart good mechanical properties, thermal stability, and toughness, as well as magnetic and optical properties to the polymers. However, it is usually hard to assure the uniform distribution of SiO2 because of its poor interaction with the organic matrix, which gives rise to poor stability of nanocomposites. Therefore, the strong interface adhesion between the organic matrix and SiO2 nanoparticles is a key to the application of SiO2 nanoparticles as fillers. Ming Tian et al. have prepared graphene oxide (GO)-encapsulated SiO2 hybrids via electrostatic self-assembly. The as-prepared hybrids were introduced into polydimethylsiloxane (PDMS) elastomer to simultaneously increase the dielectric constant (k) and mechanical properties of PDMS.36While these achievements are quite impressive, synergistic effect of graphene and silicon dioxide  by a hydrogen-bonding assembly in favor of overcoming the aggregation of the individual graphene sheet and SiO2 nanoparticle; a schematic illustration is shown in 2 by a hydrogen-bonding assembly, but also the strongly interfacial interaction between HGS and polymer due to the increased surface roughness of graphene after loading SiO2. Furthermore, the intrinsic performances of graphene for G-TA are maintained well due to the physical interaction between graphene and TA. The overall performances of HGS@NR nanocomposites are improved drastically compared with that of nanocomposites prepared by dispersing the blend of GS in NR, proving the importance of the strong hydrogen-bonding interaction between phenolic hydroxyl groups on G-TA and primary amine groups on Si–NH2. As a result, the HGS@NR nanocomposites possess the highest tensile strength up to 27.8 MPa at 0.5 wt% and tear strength 60.2 MPa at 0.5 wt%. Thermal conductivities of the HGS@NR nanocomposites were found nearly 1.5-fold better than that of the NR/HGS nanocomposites. The HGS@NR nanocomposites exhibit excellent abrasive resistant capacity with nearly 2-fold better than that of the GS@NR nanocomposites.In this study, we aim to prepare a novel graphene–SiO2.2.12 (average diameter: 300 nm) was supplied by Degussa AG (Germany). γ-Aminopropyltriethoxysilane (KH550) was purchased from Sigma-Aldrich Technology Co., LTD (China). Plant polyphenol-tannic acid (TA) was purchased from Aladdin Technology Co., LTD (China). NR was provided by Sanlux Co., Ltd (China). Zinc oxide, stearic acid, N-isopropyl-N′-phenyl-p-phenylenediamine (4010NA), 2-benzothiazolyl disulfide (DM) and N-cyclohexyl-2-benzothiazole sulfenamide (CZ) were provided by Sanlux Co., Ltd (China).Graphene was supplied by the sixth element (Changzhou) material technology Co. Ltd. (China). Submicron-sized SiO2.22 particles were modified with silane coupling agent γ-aminopropyltriethoxysilane, KH550.38 The mass ratio of coupling agents relative to SiO2 is 0 : 20, 1 : 20, 2 : 20 and 3 : 20. Under vigorous stirring (5000 rpm), a dosage of coupling agents  was respectively dispersed with a mixture solvent (1 mL deionized water and 10 mL ethanol) and added into four different three-necked round-bottom flasks. The reaction was carried out at 50 °C for 30 min for the completion of the hydrolysis reaction. Then a fixed mass SiO2 (20 g) was respectively added into the four three-necked round-bottom flasks mentioned above and vigorously stirred at 5000 rpm at 60 °C for 3 h. The silane coupling agents modified SiO2 nanoparticles were washed with ethanol for three times after the centrifugation separation. The white powder was dried in a vacuum oven at 40 °C for 12 h and designed as 0S, 1S, 2S, and 3S.The SiO2.3−1. The mixture was then sonicated for 30 min in a 30–35 °C water bath using a bath sonicator  to prepare TA functionalized graphene. Then TA functionalized graphene disperse is equally divided into triplicate. Subsequently, all the above prepared 1S, 2S and 3S was respectively added to the three TA functionalized graphene aqueous solution with the same volume, and then the mixtures were sonicated in a 30–35 °C water bath for 1 h and designed as H1GS, H2GS, H3GS respectively. For comparison purpose, a sample of unmodified graphene–SiO2 hybrid (designed as GS) was prepared from 0S and unmodified graphene subjecting to the same procedures that were used to prepare H1GS.3 g of graphene was added into a vial containing 30 mL TA aqueous solution which the concentration of TA is 0.05 g mL2.42 hybrid fillers (H1GS) was all added and compounded into the rubber for 3 min and then the mixture was discharged onto a two roll mill at 80 °C and designed as H1GS@NR, where the sulfur (2.8 phr) was added. H2GS@NR and H3GS@NR were prepared from H2GS, H3GS and NR subjecting to the same procedures that were used to prepare H1GS@NR. For comparison purpose, a sample of NR/unmodified graphene–SiO2 hybrid composite (designed as GS@NR) was prepared from GS and NR subjecting to the same procedures that were used to prepare H1GS@NR.Composite was performed in a 200 mL Banbury mixer at a rotor speed of 60 rpm for the mixing stage at a temperature of 120 °C. The NR was fed into the mixer and premixed for 2 min. This was followed by the sequential addition of zinc oxide (5 phr), stearic acid (3 phr), 4010NA (3 phr), DM (0.1 phr) and CZ (1.4 phr) and mixing the compounding ingredients for another 4 min. Next our above prepared graphene–SiO2.5ζ) was measured on a Malvern Zetasizer Nano-ZS system equipped with a 632.8 nm He-Ne laser. Thermogravimetric analysis was performed with a TGA 209 F1 instrument (NETZSCH) with a heating rate of 10 °C min−1 under nitrogen purging. X-ray diffraction (XRD) pattern was recorded on a Rigaku D-MAX2500-PC diffractometer. Transmission electron microscopy (TEM) images were obtained using a JEM-2100 microscope. Scanning electron microscopy (SEM) was performed with JEOL-JSM-7500F microscopy. Dynamic mechanical analysis was performed on a DMA242 machine (NETZSCH) in tensile mode with a temperature increment of 3 °C min−1. Dynamic rheological tests were used to measure the dynamic viscoelastic properties of hybrid nanocomposites by Rubber Processing Analyzer  with strain sweeps performed from 0.25% to 125% at a frequency of 1 Hz and at a temperature of 333 K. The thermal conductivity was measured by a laser flash system (LFA 447 NanoFlash). Tensile and tear testing were carried out using an AI-7000S Universal Material Tester, with a dumbbell specimen at a tensile speed of 500 mm min−1 according to ISO 528:2009. Compression temperature rise and compression set are determined at 55 °C using rubber compression heat tester. The abrasion test was carried out with the Akron abrader according to GT-7012-A. The abrasion was defined as a volumetric loss of a strip sample.The infrared spectra were obtained with a Bruker Vertex 70 variable temperature Fourier Transform Infrared Spectrometer. Zeta potential  which is attributed to the bending vibration of H2O is represented weaker and weaker for 1S, 2S and 3S. It indicated that the hydrophobicity of SiO2 after modified with KH550 becomes strong. Just the FTIR spectra of 3S show that the double peaks between 3300 cm−1 to 3500 cm−1 which represent the characteristic absorption peaks for the stretching vibration of the primary amine group. It can be expected that the strong signal peaks of SiO2 mask the much weaker signal peaks of primary amine group due to low grafting degree.The FTIR spectra of 0S, 1S, 2S and 3S, are shown in −1), aromatic ring breathing vibration (1640 and 1525 cm−1), are significantly blue shifted to 1383, 1670 and 1570 cm−1, respectively. The broad peak at 3310 cm−1 [via strong physical interaction, which is acknowledged to be the π–π interaction between plenty of aromatic rings of TA and graphene.39–41 The zeta potentials of the G-TA, Si–NH2 and H3GS dispersions respectively reach −0.03 mV, −12.8 mV and −10.7 mV  and (004) of graphite and indicate the multilayer structure in graphene. The intensities of the major peak (002) and (004) monitor the disordered structure and defects inside graphene and hence provides implication for the dispersion status of hybrid filler. And that notes that the broadness of the major peak suggests the presence of small irregularity in the interplanar distances in (002).42 The GS sample demonstrates similar XRD diffraction peaks of graphene, even though the intensities of the major peak (002) and (004) are much weaker, and the broadness of (002) is much wider than that of graphene, which displaying the still existence of multilayer aggregation structure of graphene and poor dispersion even after van der Waals force hybridization process. The XRD diffraction peaks (002) and (004) corresponding to graphene cannot be seen in H1GS, H2GS, and H3GS, indicating that the disaggregation of graphene and the thickness of graphene is less than 3 nm.43 Moreover, it indicates higher hybridization on the surface of hybrid fillers are detected, most likely due to hydrogen bonding interaction between T-GA and Si–NH2. Furthermore, the vanishment of the peaks (002) and (004) of H1GS, H2GS, H3GS is also indicative of a strong interfacial interaction between graphene and SiO2. In our study, this is probably because the Si–NH2 nanoparticles hinder the graphene nanosheets from restacking, disrupt the ordered lamellar structure, and accordingly result in the vanishment of the diffraction peaks of graphene through hydrogen bonding self-assembly hybrid effect.The XRD patterns of graphene, SiO2 nanoparticles are uniformly decorated on the surface of the graphene and well distributed throughout the surface of graphene for them. Moreover, the surface of H1GS, H2GS and H3GS is much rougher than that of GS , and loss factor (tan δ) are given in δ) is usually used to characterize the interaction between filler and polymer as well as filler and filler. The viscoelasticity of hybrid nanocomposites was studied and the response signal of different loss factors was found in GS@NR and HGS@NR nanocomposites.The synergistic intercalation due to the formation of strong network architecture is demonstrated by dynamical mechanical analysis. The results of the measurements of storage modulus    show thaG′′)  at small3.52 is used to bridge the graphene sheet gaps in the in-plane direction that enlarge the phonon transmission channel and benefit the phonon propagation, while the re-aggregation of SiO2 induces larger interfacial thermal resistance that further decreases the thermal conductivity of GS@NR.49 On the other hand, previous studies50–52 indicated that when graphene sheets are over stacked into a paper, its thermal conductivity greatly drops one order of magnitude due to the phonons leakage across the interface and the enhanced umklapp scattering of out-plane acoustic phonons. However, graphene layers in HGS hybrid fillers are intercalated by SiO2 particles. Graphene sheets are partly separated by SiO2 that effectively retards the strong graphene interlayer stacking induced by the strong π–π interactions. Thus phonons transfer thermal energy in the strictly 2D channel with the interference from out-of-plane being eliminated. It also means that effective thermal conduction path of graphene can be formed. The performance of HGS@NR composites would, therefore, be superior to that of GS@NR. Furthermore, thermal conduction path of graphene is more accessible to create promptly with increasing the ratio of KH550 to TA.Concerning with thermal conductivity, compared with GS@NR, this was found to be affected by hybridization forms, where hydrogen bonding self-assembly hybrid interaction resulted in a significant increase in thermal conductivity of composites attributes from the nearly complete connected graphene sheets . On the 2 to graphene. It is clearly seen from the 53 The formation of tight network architecture due to synergistic hydrogen bonding self-assembly can prompt hybrid fillers to disperse well in the polymer matrix and prevent the stress concentration effectively, improving physical properties comprehensively. Furthermore, the formation of a glassy interphase with orders of magnitude slower chain dynamics than that for bulk chains has been explicitly demonstrated in the synergistic hydrogen bonding self-assembly hybrid system.17 Therefore, besides improved dispersion upon hybridization, it is believed the formation of a glassy interphase is another crucial factor in governing the synergistic capability of hybrid composites, as the showed results in DMA (2 hybrid filler by hydrogen bonding self-assembly interaction had higher surface roughness values than that after van der Waals force hybridization.54 Meanwhile, the surface roughness is much higher with increasing the ratio of KH550 to TA as shown in 55 During compounding, due to high contact pressure, the sharp and hard protruding surface asperities of the rough hybrid fillers cause considerable interfacial interaction, leading to unique dispersion in polymer matrix. The strain–stress curve also indicates the discrepancy in toughness among the four nanocomposites. Here toughness is defined by the area of strain–stress curve which represents the absorbing energy without fracturing. H1GS@NR, H2GS@NR and H3GS@NR lead to improved toughness compared to GS@NR. Moreover, with increasing the ratio of KH550 to TA, it exhibits a significant increase in toughness. This is attributed to the improved dispersion and strong interface interaction between HGS and NR upon hydrogen bonding self-assembly hybridization. This leads to higher energy absorption and higher toughness.The results obtained for the mechanical properties of the variously compounded samples are presented in s in DMA  and RPA s in DMA . It can 4.2 hybrid filler through hydrogen bonding self-assembly interaction has been demonstrated. The SiO2 nanoparticles are uniformly decorated on the surface of the graphene and are well distributed throughout the surface of graphene. The formation of tight network architecture due to synergistic hydrogen bonding self-assembly intercalation effect between silane coupling agents modified SiO2 and TA modified graphene can prompt hybrid fillers to disperse well in the polymer matrix and prevent the stress concentration effectively, improving comprehensive performances. An effective thermal conduction path of graphene for HGS@NR can be formed to dramatically improve thermal property of composites. The superior synergistic hydrogen-bonding interaction can make the filler–rubber interfacial interaction strongly, which, in turn, contributes to the significant enhancement in the mechanical performances of composites by the incorporation of graphene–SiO2 hybrid filler into NR matrix. Also, it was noted that HGS has a higher reinforcing effect than those of GS. Particularly, the tensile strength and tear strength for H3GS@NR are dramatically increased by about 21.4% and 35.0% as compared with those of GS@NR, respectively.In conclusion, a novel method for fabricating graphene–SiOThere are no conflicts to declare.RA-008-C8RA01659C-s001


Idiopathic pulmonary fibrosis (IPF) is a form of chronic and irreversible fibrosing interstitial pneumonia of unknown etiology. Although antifibrotic treatments have shown a reduction of lung function decline and a slow disease progression, IPF is characterize by a very high mortality. Emerging evidence suggests that IPF increases the risk of lung carcinogenesis. Both diseases show similarities in terms of risk factors, such as history of smoking, concomitant emphysema, and viral infections, besides sharing similar pathogenic pathways. Lung cancer (LC) diagnosis is often difficult in IPF patients because of the diffuse lung injuries and abnormalities due to the underlying fibrosis. This is reflected in the lack of optimal therapeutic strategies for patients with both diseases. For this purpose, we performed a proteomic study on bronchoalveolar lavage fluid (BALF) samples from IPF, LC associated with IPF (LC-IPF) patients, and healthy controls (CTRL). Molecular pathways involved in inflammation, immune response, lipid metabolism, and cell adhesion were found for the dysregulated proteins in LC-IPF, such as TTHY, APOA1, S10A9, RET4, GDIR1, and PROF1. The correlation test revealed a relationship between inflammation- and lipid metabolism-related proteins. PROF1 and S10A9, related to inflammation, were up-regulated in LC-IPF BAL and serum, while APOA1 and APOE linked to lipid metabolism, were highly abundant in IPF BAL and low abundant in IPF serum. Given the properties of cytokine/adipokine of the nicotinamide phosphoribosyltransferase, we also evaluated its serum abundance, highlighting its down-regulation in LC-IPF. Our retrospective analyses of BAL samples extrapolated some potential biomarkers of LC-IPF useful to improve the management of these contemporary pathologies. Their differential abundance in serum samples permits the measurement of these potential biomarkers with a less invasive procedure. Idiopathic pulmonary fibrosis (IPF) is a specific form of chronic, progressive, and fibrosing interstitial pneumonia of unknown cause, which mostly occurs in elderly males who have a smoking habit ,2,3. It Antifibrotic therapies decelerate the disease progression and, consequently, increase the survival of IPF patients. However, increased survival due to delayed disease progression leads to a higher prevalence of comorbidities and complications . Indeed,Both IPF and LC often affect the periphery of lower lung lobes and share common risk factors such as smoking, environmental or occupational exposure, viral infections, and chronic tissue injury. Moreover, IPF and LC studies also report common pathogenic mechanisms such as epigenetic and genetic alterations, abnormal expression of microRNAs, and cell and molecular aberrations ,11,15. IThe main demographic, clinical, respiratory functional and BAL data of IPF, LC-IPF, and healthy controls are reported in Histological and/or cytological evaluation of sampling obtained through CT-guided needle aspiration, transbronchial biopsy, and drainage of malignant pleural effusion showed a pattern of lung adenocarcinoma in all LC-IPF cases.Proteomic analysis was performed matching all 2DE-gels obtained from BALF samples of the three analysed conditions . Spot data by proteomics permitted us to perform an unsupervised multivariate analysis by heatmap and principal component analysis (PCA), as shown in p-value ≤ 0.05 and percentage of relative volume (%V) mean ratio ≥1.8. Sixty-one spots satisfying these parameters are shown on the reference gel images in In order to extrapolate differential abundant spots with high significance among the conditions, we performed Kruskal–Wallis analysis taking into consideration only spots with a Based on the 61 differentially abundant spots (DASs), we elaborated a further supervised multivariate analysis by heatmap and PCA, as reported in Enrichment analysis was performed by MetaCore software uploading the identified proteins in BALF samples by proteomic analysis, divided into different lists based on their abundance trend.In order to understand the interaction between the highly abundant proteins in IPF or in LC-IPF, we performed two independent protein network analyses. Furthermore, in order to distinguish experimentally validated biological functions and canonical maps associated with the highly abundant proteins in IPF or LC-IPF, we performed the Process Network and Pathway Maps comparisons by MetaCore software . The mosGiven the role of centrals hub of PROF1, highly abundant in LC-IPF, in the protein net in Moreover, S10A9 was found to be a central hub in the protein nets A,B, and HDL dyslipidemia and lipoprotein metabolism are common dysregulated pathways suggested by the up-regulated proteins in IPF and in LC-IPF. APOA1 plays a central role in these pathways, and interestingly we identified an increase in abundance in IPF and LC-IPF with respect to CTRLs of different proteoforms of APOA1. To this purpose, we tested APOA1 abundance by WB analysis in a new cohort of BALF samples and respective serum samples, reporting an interesting result. In IPF BALF, we visualize the up-regulation of a band at 25 kDa, corresponding to the differential protein species that we found by proteomics, while in serum samples the same band shows an opposite trend A. MoreovDue to the interesting finding regarding APOA1 and our previous evidence regarding the contribution of dysregulated lipid metabolism in fibrotic processes and in IPF development ,26, we ap < 0.05). Correlation analysis reveals a co-regulation of proteins involved in organization of the actin cytoskeleton, cell motility, and adhesion. In particular, PROF1 was positively correlated with S10A6 and GDIR1, corroborating our bioinformatic results. Moreover, proteins involved in inflammation, such as S10A9, S10A6, B2MG and CYTB, were positively correlated with each other and to proteins engaged in lipid metabolism and transport, for example to FAB4; on the other hand, CFAB and FETUA were co-regulated with APOA1. TTHY and RET4 were interrelated and negatively correlated to immune and inflammatory response proteins, such as FRIL, IGHA1, and IGKC. Furthermore, we performed a Pearson’s correlation analysis, aiming to investigate whether the identified proteins present a linear relationship about their regulation. We constructed a correlation matrix containing pairwise relationships between all identified proteins  and the Enrichment analysis by process networks combined with correlation analysis reveals that the up-regulated proteins in both IPF and LC-IPF are particularly involved in inflammatory pathways and also in lipid metabolism. Given these results, we propose to evaluate the nicotinamide phosphoribosyltransferase (NAMPT) in BALF and serum samples. In fact, the extracellular NAMPT is reported to exhibit cytokine-/adipokine like properties, suggesting that it stands at the crossroad between lipid metabolism and inflammation . Figure p < 0.05) (p < 0.05)  is correlated with the %V of significant proteins detected by proteomics. PROF1 and Rho GDP-dissociation inhibitor 1 (GDIR1) were negatively correlated with KCO%  ( < 0.05) A,B. On t < 0.05) C.IPF is a pathology associated with poor prognosis and patients that are also diagnosed with LC have a further reduced mean survival time 1.6–1.7 years)  years 7,13. For . For 13.Proteomic analysis, performed on BALF samples of patients with diagnosed IPF and LC-IPF, together with healthy controls, represents a good strategy to identify a pool of proteins that, changing in abundance, could characterize the presence of LC in IPF lung. By the unsupervised multivariate analysis, BALF proteome comparison in these three conditions  immediately shows that the protein pattern from all IPF patients, including LC, is distinctive from healthy controls. On the other hand, both the supervised PCA and heatmap show a distinction between IPF and LC-IPF protein profiles suggesting that the presence of a tumor induces a change in protein abundance, distinctive from IPF pathology alone. Enrichment analysis reveals several terms related to HDL-mediated reverse cholesterol and sphingolipid transport and lipoprotein metabolism, response to hypoxia and oxidative stress, and inflammation, associated with both IPF and LC-IPF. These results confirmed the pivotal role of the lipid metabolism and inflammation in the pathogenesis of IPF, as already found by Landi et al. in 2014 and 2020 , while LIn order to explore connections between the identified proteins in a complementary way to functional interrelation, we generated a correlation matrix by Pearson’s coefficient. By proteomic analysis, TTHY and RET4 were strongly up-regulated in LC-IPF condition. They were not only interrelated, but also presented the same behaviour with respect to FRIL, HPT, IGHA1, and IGKC, suggesting their strong co-regulation. In addition to the interrelation found between TTHY and RET4, we interestingly highlight a canonical path starting from TTHY to E2F by protein network analysis. All these results could suggest the pivotal role of TTHY in LC-IPF. Indeed, TTHY is well-known to form a complex with RET4 that, in turn, binds retinol required for RET4-mediated transport from the liver to target tissues ,30. RET4Furthermore, our correlation and bioinformatic analyses highlight that several proteins involved in inflammation present a direct correlation with proteins associated with lipid metabolism and transport, strengthening the coexisting functional link between metabolic dysregulation and inflammation, which occur in IPF and LC-IPF. Additionally, we conducted a correlation test between dysregulated proteins and KCO% as clinical functional parameter, because it better reflects the pulmonary physiology . ILEU isOn the other hand, two proteins were negatively correlated with KCO%, such as PROF1 and GDIR1, which were co-regulated and associated with cell adhesion , as suggConsistent with our proteomic analysis, an increased level of GDIR1 has been found in multiple types of human cancers, including lung cancer tissues ,56,57. HAnother interesting differential protein is calgranulin B (S10A9), a central hub of our interactomes and already found up-regulated in IPF in our previous works ,59. InteLooking at the network resulting from this comparison, HNF-4α interestingly emerges as a pivotal transcriptional factor. In particular, it is a nuclear receptor whose ligands are fatty acids and generally HNF-4α target genes encoded proteins are involved in metabolic processes . In our While the pathways related to cellular metabolism and adhesion and retinoic acid regulation are statistically relevant in LC-IPF, suggesting dysregulation of cellular differentiation, the lipid metabolism and transport acquire a similar significance in both LC-IPF and IPF. Therefore, it is interesting to verify whether lipid dysregulation could be a biomarker for IPF with respect to LC-IPF by testing APOA1 and APOE. Both proteins were found to be highly abundant in BALF IPF and lower in LC-IPF. Interestingly, the behaviour of APOA1 was opposite in serum samples of the same IPF BALF donors. While APOA1 could be a characteristic biomarker in both BALF and serum samples, APOE does not change its abundance at the serum level. Of note, we report an unexpected result for APOA1 by 1DWB showing differential protein species at a higher MW (50 kDa) in IPF BALF and serum, subsequently also evidenced by 2DEWB. On one hand, the proteins suggesting a dysregulated lipid metabolism profile are characteristic in distinguishing IPF to LC-IPF and, on the other hand, up-regulated proteins in LC-IPF are particularly linked to inflammatory pathways. Given this, we propose the evaluation of NAMPT level on BALF and serum samples in IPF, LC-IPF, and CTRLs, considering that extracellular NAMPT is reported to exhibit cytokine-/adipokine like-properties, suggesting that it stands at the crossroad between metabolism and inflammation . Our resThe main limitation of the present study is the limited number of samples; therefore an expansion of these studies to a larger cohort could confirm our findings and further demonstrate the utility of TTHY, RET4, PROF1, S10A9 and GDIR1, and NAMPT as biomarkers of LC onset in IPF patients.The study group included 23 patients diagnosed with IPF . They were picked from patients who were naïve at the moment of the diagnosis. None of them ever received previous treatment with anti-fibrotic drugs. Seven patients diagnosed with both IPF and lung cancer , without any prior exposure to chemotherapy or radiotherapy, were selected for the study. IPF diagnosis was performed according to the ATS/ERS guidelines at Siena Regional Referral Centre for Interstitial Lung Diseases. BAL test was performed for diagnostic purposes, according to multidisciplinary evaluation. The histocytopatological assessment of LC was perfomed through cytological analysis of malignant pleural effusion, transbronchial biopsy, and CT-guided needle aspiration . Demographic data and information such as smoking habits, onset symptoms and comorbidities were recorded in a database, as well as BAL differential cell count, blood gas analysis data, and lung function tests. In addition, functional, radiological, histological, and immunological data were also recorded. According to ATS/ERS guidelines, pulmonary functionality tests were performed in order to obtain FEV1, FVC, TLC, and DLCO percentages -1-propanesulfonate hydrate (CHAPS), 40 mM Tris base, and 1% w/v dithioerythritol (DTE). Protein concentration was determined by Bradford assay [BALF samples were dialyzed for 12 h at 4 °C against four changes of distilled water, in order to clean samples from salts. As a second step, they were first lyophilized and then dissolved in lysis buffer, composed of 8 M urea, 4% rd assay , 60 µg ow/v SDS, 2% w/v DTE, 30% v/v glycerol and 0.5 M Tris–HCl pH 6.8 and a second incubation for 5 min—in a solution composed of 6 M urea, 2% w/v SDS, 2.5% w/v iodoacetamide, 30% v/v glycerol, 0.5 M Tris–HCl pH 6.8 and traces of bromophenol blue. Second dimension by SDS/PAGE was performed at 9 °C at 40 mA/gel constant current, using 9–16% SDS polyacrylamide linear gradient gels (18 cm × 20 cm × 1.5 mm in size). Then, ammoniacal silver staining was used for analytical gels, whereas MS-compatible silver staining was used for preparative gels. The resulting gels were scanned with Image Scanner III laser densitometer run by the LabScan 6.0 software . Image Master 2D Platinum 6.0 software  was used to carry out 2D image analysis. Two-dimensional electrophoresis (2DE) was performed by the Immobiline polyacrylamide system. The first dimension was carried out on pH 3–10 nonlinear, 18 cm long, immobilized pH gradient strips  using an Ettan™ IPGphor™ system . Analytical runs consisted of an overnight rehydration of strips with 350 µL of lysis buffer and traces of bromophenol blue at room temperature and a loading of the samples at the cathodic end of the strips by cup-loading. Electrical conditions were as follows: 200 V for 8 h, from 200 to 3500 V in 2 h, 3500 V for 2 h, from 3500 to 5000 V in 2 h, 5000 V for 3 h, from 5000 to 8000 V in 1 h, 8000 V for 3 h, from 8000 to 10,000 V in 1 h, 10,000 V, for a total of 90,000 VhT  at 16 °C. Preparative runs were carried out by rehydration loading with 350 µL of the sample at 16 °C at 30 V overnight, then 100 µL of the sample were loaded at the cathodic end of the strips, with the following electrical conditions: 200 V for 8 h, from 200 to 3500 V in 2 h, 3500 V for 2 h, from 3500 to 5000 V in 2 h, 5000 V for 3 h, from 5000 to 8000 V in 1 h, 8000 V for 3 h, from 8000 to 10,000 V in 1 h, 10,000 V, for a total of 90,000 VhT  at 16 °C. Traces of bromophenol blue and carrier ampholyte at 0.2% for the analytical runs and at 2% for the preparative ones were added to the samples. After isoelectric focusing, an equilibration of the strips was performed as follows: first, incubation for 12 min in a solution composed of 6 M urea, 2% p-value ≤ 0.05. Unsupervised and supervised Principal Component Analysis (PCA) and Heatmap analysis were performed by XLSTAT 2021.1.1. Venn diagram was performed by Venny 2.0 free online software . A correlation test by Pearson’s correlation test was performed to investigate the linear correlations between %V of the proteins of interest, and to determine the linear relationship between the percentage of carbon monoxide transfer coefficient  and the %V of the proteins of interest, by using software XLStat.Proteomic data were statistically analyzed by XLSTAT 2021.1.1  applying non-parametrical tests . Moreover, the percentage of relative volume (%V) comparison of the 2DE protein spots among the three conditions was performed taking into consideration at least a 1.8-fold change in the ratio of the %V means and a v/v ACN and 5% v/v trifluoroacetic acid (TFA), then dried again. UltrafleXtreme™ MALDI-ToF/ToF instrument  was used for MS analysis, according to Landi C et al. [http://www.matrixscience.com, accessed on 22 September 2021), setting the search parameters as follows: Swiss-Prot/TrEMBL and NCBInr were chosen as databases, Homo sapiens as taxonomy, a peptide mass tolerance of 100 ppm, only one missed cleavage site, carbamidomethylation  of cysteine as fixed modification, and oxidation of methionine as a variable modification. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [Differential electrophoretic spots were manually excised from MS-preparative gels. Spots were subjected to destaining by using a solution of 30 mM potassium ferricyanure and 100 mM sodium tiosulphate anhydrous and then in 200 mM ammonium bicarbonate. The destained spots were then dehydrated in 100% acetonitrile (ACN). Overnight rehydration in trypsin solution at 37 °C of spots was performed to digest proteins. The obtained peptide solution was spotted on the MALDI target (0.75 µL), dried, and covered with 1 µL of matrix solution, composed of 5 mg/mL α-cyano-4-hydroxycinnamic acid (CHCA) dissolved in 50% he PRIDE  partner http://portal.genego.com, Clarivate Analytics, Philadelphia, PA, USA, accessed on 5 January 2022). MetaCore works by a revised database of human protein–protein and protein–DNA interactions, transcription factors, signaling and metabolic pathways, disease and toxicity, and the effects of bioactive molecules. Specifically, we first performed enrichment analysis obtaining a hierarchical list of pathway maps and network processes, organized by their statistical significance (p ≤ 0.001). Furthermore, we performed a network analysis processing our proteins list by shortest path algorithm, which builds a hypothetical network of the potential interactions between the experimental proteins and the MetaCore database proteins.Enrichment analysis was performed by submitting the list of the identified proteins by their accession numbers to the MetaCore version 22.1 build (w/v) SDS, 20% (v/v) glycerol, 4% (v/v) β-mercaptoethanol) and heated at 95 °C for five minutes. Specifically, twenty-five μg of proteins for each sample were loaded and separated on 10% polyacrylamide gel. In order to increase resolution and patients’ number, for calgranulin B validation a bigger 1D-WB (18 cm × 20 cm × 1.5 mm in size) was performed, 40 μg of proteins for each sample were loaded and separated on 10% polyacrylamide gel. As described by Towbin [w/v Ponceau S in 3% w/v trichloroacetic acid, in order to confirm correct protein transfer. Hybridization with primary antibodies was carried out overnight at room temperature. Goat-anti-mouse or anti-rabbit HRP-conjugate secondary antibody was incubated for 2 h at room temperature. Immunostained bands and spots were visualized by chemoluminescence. Band and spot density were quantified by Image Master 2D Platinum and statistical analysis was performed by XL STAT tool.Mono-dimensional Western blot analysis was performed with a cohort of BALF and serum samples of CTRL, IPF, and LC-IPF patients. Tested proteins were apolipoprotein A1 , apolipoprotein E (sc53570- Santa Cruz Biotechnology), profilin 1 , calgranulin B (S10A9) (SAB1409623-Sigma-Aldrich), and nicotinamide phosphoribosyltransferase (NAMPT). Additionally, a two-dimensional Western blot for apolipoprotein A1 was performed. Aliquots of BALF samples were prepared as previously described. Serum samples were prepared as reported by Landi C et al. . Considey Towbin , gels wey Towbin . HybridiThe proteomic analysis of BALF from IPF, LC-IPF, and CTRL patients provided insights into the molecular pathways and processes differently regulated in the two pathologic conditions. Most of the enriched pathways common in the IPF and LC-IPF conditions were related to HDL-mediated reverse cholesterol and sphingolipid transport and lipoprotein metabolism, response to hypoxia and oxidative stress, and inflammation. Moreover, the correlation test stressed the relationship of inflammatory response and lipid metabolism and transport in pathologic conditions, further confirmed by the APOA1, APOE, and NAMPT dysregulation. In addition, the down-regulation of the latter in LC-IPF could be considered as potential prognostic biomarker. This study also emphasizes the different proteomic patterns that occur in LC-IPF compared to IPF and CTRL. In particular, the marked up-regulation of RET4 associated with TTHY may represent a potential biomarker of LC development in IPF disease, principally related to metabolic dysfunction and to the activation of pathways dependent on retinoic acid receptors. The correlation analysis between proteomic data and functional parameters provided a functional and clinical context for data interpretation. The KCO% was negatively correlated with PROF1 and GDRI, and we confirmed the up-regulation of PROF1 in LC-IPF BALF, which could represent an interesting biomarker of LC associated with IPF.


Background and Objectives: Progressive pulmonary fibrosis (PPF) is a recently described term reserved for patients with fibrotic ILD other than idiopathic pulmonary fibrosis (IPF) with fast clinical deterioration. Here, survival and prognostic biomarkers at the time of diagnosis for PPF are investigated in a fibrotic ILD other than IPF cohort (non-IPF). Materials and Methods: Patients diagnosed during the period of 2012–2018 at the ILD Center of Excellence  with a fibrotic ILD were included in this study. The presence of PPF was investigated using the criteria from the updated IPF/PPF guideline during the first year after diagnosis. Logistic regression analysis was used to determine risk factors for PPF. A Kaplan–Meier survival analysis with log-rank test was conducted to analyze survival in patients with and without PPF. Results: This study included 304 non-IPF patients and, for comparison, 379 IPF patients. In non-IPF patients, 146 (46%) fulfilled ≥2 criteria for PPF. These patients had a median transplant-free survival rate of 2.9 ± 0.4 years, which was worse than non-IPF patients without PPF . The risk for PPF was increased in patients with FVC < 50% (odds ratio (OR) of 2.50, 95% CI = 1.01–6.17, p = 0.047) or DLCOc ≤ 35% . In the first 3 years after diagnosis, survival in PPF and IPF is the same, while in the following years IPF has a significantly worse survival. Conclusions: The non-IPF cohort with PPF had a significantly worse transplant-free survival compared with the non-IPF cohort without PPF. Independent risk factors for PPF in non-IPF were FVC < 50% and DLCOc ≤ 35%. Interstitial lung disease (ILD) is a heterogeneous group of >200 pulmonary diseases, which are characterized by an inflammatory and/or fibrotic process located at the pulmonary interstitium . PatientMUC5B gene , which is associated with improved survival in IPF [The hallmark of progressive, fibrotic ILD is idiopathic pulmonary fibrosis (IPF), a disease with an unknown etiology and predominantly diagnosed in older males with a positive smoking history ,4. One ol in IPF . Telomerl in IPF . Previoul in IPF ,12.Fibrotic ILDs have a more erratic clinical behavior than IPF. For instance, in fibrotic hypersensitivity pneumonitis (fHP), patients may survive up to 8–9 years after diagnosis. However, some patients deteriorate rapidly and have a disease course as seen in IPF ,14. HyldThe recently updated ATS/ERS/JRS/ALAT clinical practice guideline presents a definition of progressive pulmonary fibrosis (PPF) in fibrotic ILD other than IPF. In short, patients having at least two of the following three criteria occurring without an alternative explanation are labelled as PPF: worsening of respiratory symptoms, lung functional deterioration and/or radiological increase in ILD/fibrosis . This deMUC5B (rs35705950) genotype and telomere length were investigated as potential risk factors for PPF and the results were compared with an IPF cohort.In this study, we evaluated the proportion and survival of patients with a fibrotic ILD other than IPF who will develop PPF within the first year after diagnosis. Covariates, such as demographics, baseline characteristics, lung function, radiological and histopathological patterns, Screening was performed on patients diagnosed with a fibrotic ILD between January 2012 and January 2018 during a multi-disciplinary discussion at the ILD Center of Excellence . The included diagnoses in which the analyses were IPF, fHP, unclassifiable ILD, CTD-ILD and smoking-related ILD (SR-ILD). Patients with IPF were diagnosed according to the 2011 ATS/ERS/JRS/ALAT IPF criteria . All patients had signs of pulmonary fibrosis on baseline high-resolution computed tomography  as documented by an experienced ILD thoracic radiologist. Furthermore, all patients had provided written informed consent .Patients were evaluated for the presence of PPF using the definition from the recently published ATS/ERS/JRS/ALAT clinical practice guideline on IPF and PPF . Patient(1). Worsened respiratory symptoms (cough and/or dyspnea);(2). Either ≥ 5% absolute forced vital capacity (FVC) decline and/or ≥10% absolute diffusing capacity for carbon monoxide (DLCO) decline;(3). Increase in fibrosis on HRCT.We are well aware that the PPF definition is only intended for non-IPF patients with a fibrotic ILD. However, we did include an analysis of PPF in IPF for comparison purposes with the non-IPF cohort. Furthermore, diffusing capacity for carbon monoxide corrected for hemoglobin (DLCOc) was used and patients who could not perform the DLCOc maneuver due to coughing or dyspnea were noted as fulfilling the ≥10% absolute DLCO decline criterion. Furthermore, data on increase or stability in pulmonary fibrosis on HRCT were retrieved from rapports from ILD thoracic radiologists. HRCT scans performed within one year after diagnosis were compared to the baseline HRCT. In patients with clinical, lung functional or radiological progression, it was verified whether there was not an alternative explanation for the deterioration. If so, this event was not considered for the PPF analysis.In non-PPF, follow-up time was ≥1 year for respiratory symptoms and lung function. For HRCT, we decided that a follow-up time of ≥1 year was not mandatory, because it is illogical to perform additional HRCT scans in clinically stable patients.MUC5B (rs35705950) T-allele carriership was determined using a pre-designed taqman single nucleotide polymorphism (SNP) genotyping assay and the QuantStudio® 5 Real-Time PCR system  on DNA isolated from whole blood. Telomere length  was measured using a monochrome multiplex, quantitative polymerase chain reaction as described elsewhere [lsewhere . This waBaseline categorical and continuous variables were analyzed with a Chi-squared test or Fisher’s exact test where appropriate and independent Student’s t-test, respectively. Binary logistic regression analysis was used to identify covariates associated with the odds ratio (OR) for PPF. This was conducted in a non-IPF and IPF cohort. Age and percentage predicted FVC and DLCOc were stratified according to the strata used in the ILD-GAP study by Ryerson et al. . The ratKaplan–Meier survival analysis was performed in order to obtain transplant-free (TPF) survival. Date of diagnosis was used as time point zero and lung transplantation and death were treated as events. Differences in survival between groups were determined with the log-rank test.All analyses were conducted with SPSS version 24.0  and figures were created with Graphpad Prism version 9 .n = 1152), 683 patients were included in this study (see After screening (tudy see . The nonn = 379), there were 167 patients (44%) with PPF. Baseline characteristics and clinical parameters of the non-IPF without PPF, non-IPF with PPF and IPF cohorts are provided in MUC5B minor allele frequency was also significantly lower in the non-IPF with PPF cohort compared with the IPF cohort.In the IPF group . Furthermore, an increased odds for PPF was found for patients with a baseline DLCOc ≤ 35%  or patients who could not perform the DLCOc maneuver  compared with patients with a DLCOc >55%. There was a trend toward significance in patients diagnosed with CTD-ILD  and patients with a UIP or probable UIP on HRCT or LB  [The term progressive fibrosing ILD (PF-ILD), which was coined in the INBUILD trial , is diff = 0.95) . In accoFurthermore, our study replicates two findings reported in two previous PF-ILD studies. First, a lower baseline lung function (FVC < 70% and DLCO < 75%) was also associated with a higher risk for PF-ILD reported in a large prospective, Canadian registry study . Second,MUC5B (rs35705950) minor allele or decreased telomere length were not predictive for PPF. Prior studies on disease progression and survival have found similar results. In a cohort of patients with RA-ILD (n = 261), Juge et al. could not detect a significant association between MUC5B (rs35705950) genotype and annual FVC decline/survival [n = 217), a significant association between survival and MUC5B (rs35705950) genotype was not found [MUC5B (rs35705950) minor allele was associated with worse TPF survival, but not to changes in FVC percentage predicted per year [MUC5B minor allele was associated with better survival [Carriership of -ILD n = 1, Juge eot found . In a stper year , while isurvival ,29.PPF has created the possibility for the introduction of antifibrotics in non-IPF patients. In patients with PPF, it seems a plausible treatment approach as IPF and non-IPF with PPF patients have a similar disease behavior, as shown here. Additionally, there is accumulating evidence that antifibrotics appear to be beneficial in ILD primarily driven by inflammation, fibrosis or a mixture of them ,30,31. FA couple of limitations need to be addressed. The retrospective nature of this study has resulted in missing data and patient exclusions. Selection bias may have resulted in a higher percentage of PPF in the non-IPF cohort, which was discussed above. Another limitation from this study is that sarcoidosis patients were not included in this study. A significant proportion of these patients are suspected to develop PPF .n = 304), it was demonstrated that 46% of the patients have PPF. The transplant-free survival of these patients is significantly impaired compared with non-IPF patients without PPF . FVC < 50% and DLCOc ≤ 35% or patients who cannot perform the DLCOc maneuver are risk factors for PPF. Future studies are needed to validate our results and evaluate whether the identified predictors for PPF can also be applied to predict PPF in later stages of follow-up (>1 year after diagnosis).In conclusion, this study is the first to report on PPF in a real-world setting. In non-IPF (


P6̄2m space group, named azugraphene, was predicted and its 38 atoms in the unit cell can be fragmented into three 5–7 rings of azulene, one hexagon, and two remainder atoms. Azugraphene is a low-energy graphene-like hexagonal carbon allotrope with six Dirac cones in the first Brillouin zone. Due to its stability and the existence of its elementary fragments, azugraphene is potentially synthesizable. In addition, the stable AB stacking bilayer azugraphene is also a Dirac cone material with a band gap of 2.5 meV. Therefore, both the monolayer and bilayer azugraphenes have great potential in physics, chemistry, and nanoelectronics.Classic two-dimensional graphene possesses outstanding properties due to Dirac cone structures so that many Dirac cone materials had been predicted. Using the first principle symmetric search algorithm, a new graphene-like carbon allotrope with  Azugraphene is a new graphene-like hexagonal carbon allotrope with Dirac cones and is potentially synthesizable from its fragments: azulene. Up to now, numerous 2D carbon allotropes were predicted with distinct electronic properties including metallic, semimetallic, semiconducting, and topologically insulating in nature. In addition, there are many predicted 2D carbon allotropes with exotic geometric structures, such as the penta-graphene composed of only carbon pentagons5 and the PCF-graphene with a finite thickness.6Graphene, a two-dimensional (2D) array of planar hexagonal units ,13 was predicted, which is only 141 meV per atom less stable than graphene.Among all these 2D allotropes, the Dirac cone materials attract more attentions because of its extraordinary electrical properties. From 2012, a series of 2D carbon allotropes with Dirac cones were predicted using the first principle methods, such as α-graphdiyne,via a cross coupling reaction using its elementary fragment, hexaethynylbenzene,14 though it is very unstable in thermodynamics. On the other hand, to find the more stable 2D carbon allotropes, one of the feasible bottom-up approaches is to mix some 5–7 rings into the hexagonal array. From 2015, a series of 5–6–7 2D carbon allotropes were predicted, such as phagraphene,11 twin graphene,15 ψ-graphene,16 C-57 carbon,17 which are generally more stable than the graphyne allotropes. However, most of them are no Dirac cones in their band structure, except that the Cmmm SW-graphene is a semimetal with distorted Dirac cones. Therefore, it is desirable to explore the stable 2D carbon allotropes with Dirac cones, just like the hexagonal graphene.Beside graphene, another 2D carbon allotrope, graphdiyne (graphyne with diacetylene groups) was successfully synthesized on the surface of copper In this work, using the first principle symmetric search algorithm, we predicted a new 2D hexagonal carbon allotrope, which consists of 38 carbon atoms in its unit cell. This allotrope contains mainly three 5–7 rings of azulene to be named as azugraphene. As far as we know, azugraphene is a new stable graphene-like hexagonal allotrope with Dirac cones so that it has great potential in physics, chemistry, and nanoelectronics.18,19 At first, five 2D unit cells were defined by γ = 120° and a = b = 4, 6, 8, 10, and 12 Å, respectively. Secondly, given a 2D groups P6, P6mm, P3, P3m1, or P31m, these predefined cells were filled by randomly putting the finite-sized atoms and their symmetric equivalent atoms step-by-step. In each step, all distances between two carbon atoms must be greater than 1 Å in its 3 × 3 supercell. At last, these filled cells were optimized using the first principle method.The first principle symmetric search program were performed with 3–40 carbon atoms per unit cell.20,21 using the projector augmented wave (PAW) method22 in conjunction with the PBE functional.23 The plane wave cut-off energy was 500 eV throughout our calculation. Each calculation was considered to be converged when the energy precision during the geometry optimization process were smaller than 10−6 eV per atom and the forces components on each atom were less than 0.01 eV Å−1. For geometric optimization, both the lattice constants and positions of atoms were fully relaxed. The structures is represented by a unit cell with a 15 Å vacuum region in the normal direction to avoid the interactions between neighboring layers. The Brillouin zone is sampled using k-points with 0.02 Å−1 spacing in the Monkhorst–Pack scheme24 for all calculations. The van der Waals interactions between the bilayer sheets are corrected by the many-body dispersion energy method (MBD@rsSCS) of Tkatchenko et al.25,26The density functional theory calculations were performed by the VASP5.4.4 package27 implemented in the VASP5.4.4 package, and phonon frequencies were calculated from the force constants using the Phonopy 1.13.2 code.28,29Phonon calculations were performed using the supercell approach. Real-space force constants of supercells were calculated in the density-functional perturbation theory (DFPT)P6̄2m space group with the lattice constant of 10.845 Å, which belongs to hexagonal symmetry similar to graphene. Because of the rotation–inversion axis 6̄, azugraphene has two alternative unit cells: A with the lattice vectors of  and  (black rhombus), and A′ with the lattice vectors of  and  (red rhombus). In each unit cell, their 38 atoms can be fragmented to three 5–7 rings of azulene , one hexagon (6 atoms), and two remainder atoms. In the unit cells of A and A′, the orientations of all 5–7 rings are just reverse. In the following, we will mainly investigate the unit cell A in detail.The first principle symmetric search found 17 new 2D carbon allotropes, including some hexagonal structures and the other structures , in whice to +0.10e, grey spheres), and some atoms have considerable charges: C3 atoms , C8 and C9 atoms . Therefore, the C3 atoms should be the promising nucleophilic sites, and the C8 and C9 ones should be the promising electrophilic sites in chemical reactions. Contrast with the unreactive graphene, azugraphene has many ordered chemical active sites to be an interesting chemical template.As shown in According to the typical C–C lengths of the single (1.54 Å), double (1.34 Å), and aromatic (1.42 Å) bonds, the bonding pattern of azugraphene is also plotted in −1, distinctly larger than about 1600 cm−1 for graphene.Because there is no imaginary mode in the phonon spectra of azugraphene in the entire Brillouin zone , azugrapCmmm SW-graphene13 is not similar to the hexagonal graphene in symmetry. Therefore, it is safe to predict that azugraphene is a new stable graphene-like hexagonal carbon allotrope, as shown in Fig. S1.†Among all hitherto reported 2D carbon allotropes, graphene has the lowest energy to be set as zero Table S1. AzugrapIn experiment, graphdiyne had been synthesized from its elementary fragment: hexaethynylbenzene. The main fragment of azugraphene is the 5–7 ring of azulene, an isomer of naphthalene, so that azugraphene should be synthesized from azulene. Because of its high stability and its existing elementary fragment, azugraphene is potentially synthesizable.k-points: Γ → M → K → Γ (k-point (K) within the first Brillouin zone. In particular, these bands are linearly dispersed with the k-vector in the proximity of the Fermi level. The valence and conduction bands meet the degenerate point with a slope (∂E/∂k) of ±25.2 eV Å along a line that passes through the high symmetric point K along both M and Γ directions. The 3D band structure of azugraphene was plotted on an energy scale of −0.18 to 0.18 eV (E(q)/ħ|q| relation, the Fermi velocity of azugraphene is 6.09 × 105 m s−1, which is comparable to those of other 2D carbon allotropes, such as graphene (8.22 × 105 m s−1),2 α-graphyne (6.77 × 105 m s−1),8 β-graphyne (3.87–6.77 × 105 m s−1),8 phagraphene (3.43–6.48 × 105 m s−1),11 and α-2 graphyne (6.36–7.13 × 105 m s−1).12The electronic structure of azugraphene is presented in 0, 0.00) . It can  0.18 eV . It suggx + py of azugraphene disappears at about −3.0 eV, and the PDOS on pz gradually decreases to zero around the Fermi level. This feature corroborates that the azugraphene is semimetallic in nature. It is evident from the band decomposed charge densities  were plotted in ensities  of VB an directions one C–C bond length, respectively. Their interlayer binding energy were calculated by subtracting the energies of two monolayers from their corresponding energies of bilayers. The binding energies per atom of AA, AB, AA′, and AB′ are 20 meV, 24 meV, 21 meV, and 24 meV at the interlayer distances of 3.641 Å, 3.434 Å, 3.586 Å, and 3.447 Å, respectively. It is just as 20 meV per atom and 23 meV per atom of AA and AB stacking bilayer graphene. Consequently, there are two stable stacking bilayer azugraphene with the same stability, AB and AB′.Because azugraphene has two alternative unit cells of A and A′, its bilayer sheets have four stacking modes: AA, AB, AA′, and AB′ , in whicThe electronic properties of four bilayer azugraphenes were investigated and were compared with that of graphene. The low-energy bands of AA stacking bilayer azugraphene are linear and almost isotropic near the K-point . The intP6̄2m space group, azugraphene, was predicted. The 38 atoms in its primitive cell can be fragmented to three 5–7 rings (30 atoms), one hexagon (6 atoms), and two remainder atoms. Compared with the other 2D carbon allotropes, the azugraphene is a new stable graphene-like hexagonal one, and has six Dirac cones at the high symmetric K and K′ points in the Brillouin zone. In addition, the stable AB bilayer azugraphenes is also a Dirac cone materials with a band gap of 2.5 meV. Therefore, both the monolayer and bilayer azugraphenes have great potential in physics, chemistry, and nanoelectronics.Using the first principle symmetric search algorithm, a new graphene-like carbon allotrope with The authors declare that there is no conflict of interests.RA-009-C9RA07953J-s001


Animals predominantly use salient visual cues (landmarks) for efficient navigation. When the relative position of the visual cues is altered, the hippocampal population exhibits heterogeneous responses and constructs context-specific spatial maps. Another critical factor that can strongly modulate spatial representation is the presence of reward. Reward features can drive behavior and are known to bias spatial attention. However, it is unclear whether reward features are used for spatial reference in the presence of distal cues and how the hippocampus population dynamics changes when the association between reward features and distal cues is altered. We systematically investigated these questions by recording place cells from the CA1 in different sets of experiments while the rats ran in an environment with the conflicting association between reward features and distal cues. We report that, when rewards features were only used as local cues, the hippocampal place fields exhibited coherent and dynamical orientation across sessions, suggesting the use of a single coherent spatial map. We found that place cells maintained their spatial offset in the cue conflict conditions, thus showing a robust spatial coupling featuring an attractor-like property in the CA1. These results indicate that reward features may control the place field orientation but may not cause sufficient input difference to create context-specific spatial maps in the CA1. Animals use multimodal information to encode space, and the hippocampal system receives information from various sensory modalities . The hipWhen animals encounter different environments, the hippocampus responds to them with a sparse, orthogonal representation, where different combinations of place cells are recruited to represent each environment . In addiHowever, motivational stimuli like rewards drive the behavior and reorganize the spatial representation  and may Nevertheless, it is unclear how hippocampal representation is driven by these two different cue systems when the association between the distal and reward features is altered. In the present study, we systematically investigated the dynamics of hippocampal representation in a dynamically changing reward feature-distal cue association by recording from the CA1 region of the dorsal hippocampus. We report that CA1 ensembles exhibited a highly coherent yet unstable spatial representation in the presence of reward features, thus exhibiting attractor network dynamics. In the absence of localized reward, the hippocampal neurons show orthogonal activity in a dynamically changing environment defined by distal cues and local textures, destabilizing the attractor network.n = 12) were housed separately on a reversed light-dark cycle (12:12 h). Food and water were provided ad libitum. All the experiments were conducted on the dark phase of the cycle. All procedures related to surgery, animal care, and euthanasia were approved by the Institutional Animal Ethics Committee (IAEC) of National Brain Research Center at Manesar, Haryana, constituted by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India, and were conducted in accordance with NIH guidelines.Male Long-Evans rats  on a circular track to receive specific reward flavors at specific spokes on the track. During training and experimental sessions, the rats were maintained to no less than 85% of their body weight.All the surgical procedures were performed under aseptic conditions. At the time of surgery, the rats were initially anesthetized with ketamine (60 mg/kg b.w.) and xylazine (8 mg/kg b.w.) and subsequently switched to isoflurane gaseous anesthesia. A custom-built microdrive containing 9–20 independently moving tetrodes (1–2 references in each microdrive) was surgically implanted over the right hemisphere to access dorsal CA1 of the hippocampus . To relieve pain, Meloxicam was administered intramuscularly (1 mg/kg b.w.) on the day of the surgery. During the post-surgical recovery period, Meloxicam was also provided through the oral route (1 mg/kg b.w.). During the post-surgical care of 7 days, food and water were provided via the commutator cables to the data acquisition system using specific tethers. The multi-unit activity was obtained by filtering the brain signals between 600 and 6 kHz. Spike waveforms were sampled for 1 ms at 32 kHz. The spikes waveforms were differentially recorded against a reference electrode in the corpus-callosum (a cell-free layer). Local field potentials (LFP) were differentially recorded against a ground screw on the skull above the frontal cortex. LFP signals were recorded as continuous signals with a bandwidth between 0.1 Hz and 1 kHz and sampled at 4 kHz. In this study, only the spike data are used. LFP signals were only used to visually observe ripples and complex spiking patterns, while the tetrode tips reached the hippocampus, and no analysis was performed on it. The rat position was tracked during experimental sessions using LEDs connected to the headstages, acquired at 25 Hz using a ceiling-mounted camera .VG bonded platinum-iridium wire (17-μm) was used to make tetrodes . The tips of individual wires were cleaned to remove debris by passing current (0.2 μA). Then, the tetrode tips were electroplated with the platinum black solution to reduce the impedance to 100–150 kΩ with 0.2 μA current. We used 96 channel data acquisition system to acquire electrophysiological recordings . The custom-made microdrive was attached to the EIB-27 board. The tetrodes and the references were connected to this EIB board. Headstage preamplifier (HS-27) was connected to the EIB-27 board. HS-27 was used to amplify the brain signals. HS-27 was connected After post-surgical recovery (∼7 days), the tetrodes were slowly advanced to reach the CA1 layer of the hippocampus over 12–21 days. The rats were trained for an average of 10 days (∼30 min/day) in a behavioral room to run clockwise (CW) on a centrally placed plain circular track/textured track  elevated 90 cm from the floor. Whenever the rat tried to turn back and run in a counter-clockwise (CCW) direction, the path was blocked using a cardboard sheet. A circular LED light mounted over the ceiling delivered the required illumination for the experiment room. A white noise generator placed centrally below the track apparatus was used to mask external noise and thus prevent any bias in behavior or/and neural activity due to any external noise. The experimental session as described below began once the tetrodes reached the CA1 region, and the animal was trained to complete 15–20 laps per session. Some tetrodes were adjusted each day to maximize the number of clusters recorded.Four rats were trained to run clockwise to fetch chocolate sprinkles placed at arbitrary positions on a textured circular track . The surface textures on the track served as local cues. There were four types of visually distinct textured surfaces , each enveloping the track’s quadrant. Six salient visual cues served as distal cues in which four cardboard cues were hung on a curtain, and two boxes of different sizes were positioned on the floor at the boundary of the curtain. Distal cues were entirely visual modality, whereas local cues were from visual and somatosensory modalities. This experimental paradigm precludes any representational bias due to reward anticipation as the reward was provided at random locations.The Tex experimental paradigm followed the protocol shown in multiple studies . Each daThe experimental paradigm is similar to the Tex experiments except that the reward is not placed randomly but at a particular location (at the junction of brown sandpaper and a black-textured cue). Each day of the experiments comprised of three standard sessions separated by two mismatch sessions. The cue configuration of the STD session is the same as that of the training sessions. In the mismatch sessions, cue conflict was created in which the distal cues were rotated CW, and the textured circular track was rotated CCW by either 90° (MIS1) or 45° (MIS2). The reward was placed at the exact location relative to surface textures in all the sessions. These experiments were conducted in the same four rats after completing double rotation experiments with the textured track as the only cues (Tex).Each day of the experiment comprised of five sessions where three standard (STD) sessions were separated by a mismatch session (MIS1) and a flip session (FLIP). The configuration between the local cues (distinct reward magnitudes) and the distal cues in the STD sessions was preserved the same way as in the training sessions. The black circular track  had three visible spokes separated by 120° projecting outward from the track. The three spokes were separated by 120°. In STD sessions, the spoke that provided two pellets (high reward) was aligned (0°) with the stripe cue card, the spoke that provided no pellets (no reward) was aligned (0°) with the white cue card, and the spoke that provided one pellet (low reward) was aligned (0°) with the triangle cue card. In the MIS1 session, the track was rotated 120° CCW, and the distal cue was rotated 120° CW. In the FLIP session, the distal cues were not rotated. However, the spoke that provided high reward was provided with no reward, and the spoke with no reward was provided with high reward. Four rats were used in this experiment.The black circular track is the same as that of RwdMag experiments. Three distinct flavors of reward pellets, namely, banana flavored, sucrose, and chocolate-flavored pellets, were provided at each spoke . Each day of the experiment comprised of five sessions where three standard (STD) sessions were separated by two mismatch (MIS) sessions. The configuration between the local cues (distinct reward flavors) and the distal cues in the STD sessions was preserved the same way as the training sessions. In STD sessions, the banana flavor spoke was aligned (0°) with the stripe cue card, the sucrose flavor spoke was aligned (0°) with the white cue card, and the chocolate flavor spoke was aligned (0°) with the triangle cue card. In the first mismatch (MIS1) session, the track was rotated 60° CCW, and the distal cues were rotated 60° CW. This change in association led to a local-distal cue conflict due to a change in the configuration between the local cue  and the distal cues. In MIS1, the banana flavor spoke was aligned (0°) with a triangle cue card, the sucrose flavor spoke was aligned (0°) with a stripe cue card, and the chocolate flavor spoke was aligned (0°) with a white cue card.All STD sessions have the same configurations. In the second mismatch session (MIS2), the track was rotated 120° CCW, and the distal cues were rotated 120° CW. This rearrangement led to a distinct configuration. Here, the banana flavor spoke was aligned (0°) with the white cue card, and the sucrose flavor spoke was aligned (0°) with the triangle cue card. The chocolate flavor spoke was aligned (0°) with the stripe cue card. The animals ran 15 laps in each session. There is no waiting time for the animal to receive the reward. Five animals were used in this experiment. Reward flavors act as local cues because the animals experience having rewards with distinct flavors at different spokes. Each flavor was exclusively available at a specific spoke only, i.e., one spoke received only one particular flavor of reward. The distal cues were visual, whereas local cues, here reward flavors, had distinct gustatory and olfactory features. Pilot experiments have shown that the rats differentiated reward flavors when they were restricted to 90–95% of their initial body weight. However, while the animal was near 85% b.w., no preference was observed, and we believe it might not have significantly impacted the results. Reward delivery systems were not intentionally used as they might act as a visual cue and may interfere with experiment results. Thus, rewards were provided manually. Distinct reward flavors were kept at the base of the track, hidden from the animals. To minimize the diffusion of odors, it was closed in three small boxes. Additionally, only 15–20 pellets were kept and were replenished after each session. These methods likely minimize odor diffusion during reward delivery. Moreover, fingers were cleaned regularly with water-based wet wipes to remove any mixing of odors while keeping rewards. After completing each session, the animals were provided with water so that the animals did not confuse between reward flavors due to saturation of taste buds and could identify reward flavors on the next session.In these experiments, the animals were trained in the STD configuration for just 2 days to familiarize themselves with the textured track as the animals already associated reward flavors with distal cues. This experiment was conducted to study whether the introduction of the textured track would stabilize standard sessions. The experimental paradigm is similar to RwdFlav experiments, except that the track is textured. The circular track had three different surface textures, each covering 1/3 of the track: a striped surface (black and white duct tapes), a black surface, and a rubber surface (blue colored). The center of the striped surface of the track was aligned with the banana flavor spoke, the center of the black surface was aligned with the sucrose flavor spoke, and the center of the rubber surface was aligned with the chocolate flavor spoke. The alignment of the distal cue and reward spoke was the same as in the RwdFlav experiment. Only the alignment between the textures and the spoke (reward flavor) was maintained when the track was rotated. The STD configuration is changed to create a local-distal cue conflict by rotating the textured track and reward flavors (CCW) and the distal cues (CW) by either 60° or 120° in MIS1 and MIS2 conditions, respectively. A total of 108 neurons from four animals were used in this experiment.via the head stage. The rat was released at a random location on the track every session. After completing 15–20 laps, the rat was disconnected and transferred to the box for disorientation. During this period, the distal cues and the track were rotated to change the configuration. We repeated the same procedure for all the experiments. After completing all sessions on an experimental day, the circular track was wiped with 70% ethanol to remove any olfactory traces that could potentially bias the behavior and neural representation on the next day of recording. Rat droppings during experimental sessions, if any, were removed with tissue paper. In most of the animals, more than one experiment was conducted. Details of the experiments conducted on each rat are shown below.At the start of the experiment, the rat was carried by the experimenter into the behavioral room inside a covered cardboard box to disrupt the animal’s sense of direction. The rat was then transferred on a pedestal placed at the center of the track. The tethers were connected to the EIB boards The data analysis and statistical tests were performed in Matlab (MathWorks R2019a), Circular Statistics , and Graph Pad Prism.All data analysis, including spike sorting, was performed offline. Single units from tetrode recordings were isolated using custom-written spike-sorting software  spatial information score > 0.5 with a clear place field in at least one of the sessions on an experimental day.Have a statistically significant  is the mean firing rate in each pixel (position), λ is the mean firing rate of the cell, p(x) is the probability of the rat to be in the pixel x [occupancy time in pixel (x)/total occupancy time]. The spatial information score was computed on a smooth speed-filtered rate map.where I is the spatial information score, λ. The population is further grouped based on the orientation of neuronal ensembles. To quantify the change in the firing field of place cells in the hippocampus, we conducted rotational correlation analysis on the ensemble recorded on each day of the recording. Two-dimensional occupancy and spike data were linearized to obtain a one-dimensional firing rate array of 72 bins each (5° each per bin). One-dimensional firing rate arrays were constructed by dividing the number of spikes fired when the rat was in a particular bin by the amount of time spent (in seconds) in that specific spatial bin on the circular track. To exclude behavioral and neural epochs when the rat was immobile , positional sampling in which the velocity of the rat was <1 cm/s was removed. We have quantified the rotation of the preferred firing field of individual cells between STD sessions or between STD and MIS sessions using Pearson’s product-moment correlation between linearized firing rate arrays of a cell between two corresponding sessions. We did this procedure 71 times by shifting the session being correlated by one bin increment (5°) to obtain shifted correlation . The angTo identify any patterns in the mean angle of orientation across ensembles in different comparisons, we used the k-means elbow method to determine the optimum number of clusters based on the mean vector length and the mean angle of rotation of place fields from all the ensembles. The optimum number of clusters was found to be three across all the comparisons. We then used the k-means algorithm to group the ensembles based on their mean direction and mean vector length into three different clusters.To measure the population activity of the hippocampus, 2D spatial correlation matrices were created from firing rate vectors (1D) at each position on the track . PopulatOne of the characteristic features of attractor dynamics in the spatial neural network is the maintenance of the spatial offset between place fields of co-recorded cells. We studied the attractor dynamics of the hippocampus in different experiments by comparing the spatial offset between co-recorded place cells across standard and mismatch sessions. We modified the analysis performed by Marker lesions were made on a selected few tetrodes by passing current (10 μA for 10 s). The rats were perfused transcardially on the following day using 4% formaldehyde solution. After perfusion, the brain was extracted and preserved in 30% sucrose-formalin solution till it sank. Later, brains were sectioned , mounted, and left to dry. Sections were then stained through the Nissl stain procedure using 0.1% Cresyl Violet. Images of the sections were acquired using a Leica DFC265 digital camera connected to a Leica M165-C stereomicroscope. The tetrodes on the sections were identified using tetrode arrangements in the microdrive. Finally, depth reconstruction was conducted on each tetrode track to identify the brain region from which electrophysiological recordings were conducted each day based on the tetrode tip distance. We also considered the shrinkage factor (15%) of the brain tissue due to histological processing.We studied the dynamics of the spatial representation in the hippocampal CA1 population when the animals experienced a change in the association between reward features  and distal cues in the dynamically changing environment in different sets of experiments. (STD1 vs. STD2) = 0.92, MVL(STD2 vs. STD3) = 0.95, Rayleigh’s test p < 0.01 in both comparisons]. During both mismatch sessions, place fields were oriented to either local or distal cues . These results replicate findings from other studies that CA1 exhibits heterogeneous representation  = 0.89, MVL(STD2 vs. STD3) = 0.89, Rayleigh’s test p < 0.01 in both comparisons]. During both mismatch sessions, place fields were oriented to either local or distal cues .Next, we checked whether localizing reward on a particular location alters the hierarchical representation of the space by the hippocampus. In the Tex experiment, the reward was provided at random locations to motivate the animals to run CW on the track. But, in this experimental paradigm, the reward (the chocolate sprinkle) was provided only at one specific location on the circular track. The experimental paradigm is shown in p < 0.0001) and MIS2 (p < 0.0001) sessions in Tex + Rwd experiments. These results demonstrate that providing reward at a specific location biased the spatial representation of the hippocampus toward local cues.We then checked whether localizing reward alters the distribution of place field orientation . We founFurthermore, these results also raise another interesting question–Can place cells orient their firing fields to reward features and locations? Two additional experiments were conducted to understand the specific role of different reward features like reward magnitude and reward flavor. In one set of experiments, rewards with different magnitudes were considered local cues. Different reward flavors act as local cues in the other set of experiments. In both these experiments, plain tracks were used, and the rewards were provided on visible reward locations (spokes) and were kept in conflict with distal cues in the cue-conflict sessions.In this set of experiments, different reward magnitudes were kept in conflict with distal cues. The rats were trained to run CW on a plain black track with three spokes (black) separated 120° apart from each other. In this experiment, chocolate pellets  with different magnitudes were provided at specific spokes, and each spoke was associated with distinct distal cues. Here, rewards with different magnitudes act as local cues. Each day of the experimental session comprised three standard sessions (STD), separated by a mismatch session (MIS1) and a flip (FLIP) session. In the MIS1 session, the track was rotated CCW, and distal cues were rotated CW by 120°, leading to a change in the association between reward magnitude and distal cues. In the FLIP session, the distal cues were not rotated. However, the spoke, which provided a high reward, was provided with no reward, and the spoke, which provided no reward, was provided with a high reward. A total of 188 CA1 place cells were analyzed from four rats. The experimental paradigm is shown in (STD1 vs. STD2) = 0.93, p < 0.001; MVL(STD2 vs. STD3) = 0.86, p < 0.001]. MVL values of MIS1  and FLIP  sessions also indicate that place fields from all the co-recorded neurons exhibited a coherent rotation  separated 120° apart (the same track as RwdMag experiments). Three different flavors of pellets, namely, sucrose, chocolate, and banana-flavored pellets , served as rewards and were manually delivered at each specific spoke. In this experiment, reward flavors were considered local cues as the animals directly experienced these cues. Each day of the experimental session comprised three standard sessions (STD) separated by two mismatch sessions (MIS). The experimental paradigm is shown in p < 0.01), indicating a coherent structure in the orientation.Individual ensembles across comparisons that exhibited a coherent structure with a high mean vector length also showed distinct orientation across sessions. We investigated if any underlying pattern exists in these ensembles. We used the k-means algorithm to optimize the number of clustered ensembles obtained based on each ensemble’s mean vector length and mean direction across sessions. p < 0.002). This result shows that the orientation of ensembles in a cue mismatch environment is essentially dynamic and slightly biased toward the reward cues. On other days (across rats), the ensembles showed a dynamic shift in orientation (in at least one session) from one cue to another or shifted based on the position of spokes. We observed a dynamic shift in the orientation during some standard sessions, leading to a reorientation of place fields. However, the place fields coherently rotated away from the initial location even during this rapid reorientation. The cluster groups obtained organized the ensembles into three groups in all STD vs. STD and STD vs. MIS comparisons, and rotational analysis was performed in the entire neural population in each group. All the active neurons were pooled together from each ensemble in a particular cluster group from their respective STD vs. STD and STD vs. MIS comparisons. Circular statistics were performed on all ensemble data from each group, and the distribution also showed a coherent orientation , indicating similar responses in both STD vs. STD and STD vs. MIS comparisons. These results show that the hippocampus maintains a location-specific coherency despite the dynamic change in the orientations. We have also checked whether reward alters the distribution of place fields across the tracks. We found that the place field distribution was not uniform as significantly more place fields were present at the reward locations  were introduced in the same experimental paradigm . We hypop < 0.02, Kruskal–Wallis test, after Bonferroni correction), indicating more coherency in the presence of reward, possibly suggesting an attractor-like representation in the hippocampus. A spatial or head direction system exhibits an attractor-like network when the neurons maintain their spatial offset across sessions. We then calculated the mean direction of co-recorded neurons across subsequent sessions to study the spatial offset between different experiments. p < 0.05, Kruskal–Wallis test, after Bonferroni correction) when reward features alone were presented as local cues. This result indicates that co-recorded neurons maintained spatial offset under these conditions (see p < 0.05), indicating a coherent structure  and replicated the finding that co-recorded place cells exhibited heterogeneous responses. Then, we ran the experiments with localized reward (Tex + Rwd). We found that, compared to Tex experiments, a significantly higher number of place fields were controlled by the local cues, suggesting the prominent role of reward in modulating the spatial orientation of the hippocampal place cells. Additional experiments (RwdMag and RwdFlav) were conducted to study how specific reward features modulate the hippocampal population. In these experiments, reward magnitudes and reward flavors act as local cues. In both conditions, the hippocampus maintained a coherent and dynamic response. We observed coherent rotation of place fields, following the rotation of either reward features or distal cues in many sessions. On many days, the place fields were only controlled by reward features. On some occasions, the place fields were oriented away based on the reward location. Still, they exhibited coherency, indicated by the maintenance of spatial offset by co-recorded neurons. The spatial offset is not maintained when surface textures act as local cues (Tex). However, introducing textured tracks increases the stable firing of place cells in standard sessions (RwdFlav + Tex), implying the role of surface textures in recognition memory. Our novel findings indicate distinct functioning of CA1 as an attractor-like network under conditions when only localized reward features were presented as local cues in visible reward locations.via EC  and CA3 pathways, providing information about the organism’s current state and mnemonic representation  of National Brain Research Center at Manesar, Haryana, constituted by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India.IN conceived and designed the study and performed the data analysis. IN and GB performed the experiments and histology. IN and DR interpreted the data and wrote the manuscript. All authors revised and edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.