Traumatic brain injury (TBI) is a disabling disorder and a major cause of death and disability in the world. Both single and repetitive traumas affect the brain acutely but can also lead to chronic neurodegenerative changes. Clinical studies have shown some dissimilarities in transactive response DNA binding protein 43 (TDP-43) expression patterns following single versus repetitive TBI. We explored the acute cortical post-traumatic changes of TDP-43 using the lateral fluid percussion injury (LFPI) model of single moderate TBI in adult male mice and investigated the association of TDP-43 with post-traumatic neuroinflammation and synaptic plasticity. In the ipsilateral cortices of animals following LFPI, we found changes in the cytoplasmic and nuclear levels of TDP-43 and the decreased expression of postsynaptic protein 95 within the first 3 d post-injury. Subacute pathological changes of TDP-43 in the hippocampi of animals following LFPI and in mice exposed to repetitive mild TBI (rmTBI) were studied. Changes in the hippocampal TDP-43 expression patterns at 14 d following different brain trauma procedures showed pathological alterations only after single moderate, but not following rmTBI. Hippocampal LFPI-induced TDP-43 pathology was not accompanied by the microglial reaction, contrary to the findings after rmTBI, suggesting that different types of brain trauma may cause diverse pathophysiological changes in the brain, specifically related to the TDP-43 protein as well as to the microglial reaction. Taken together, our findings may contribute to a better understanding of the pathophysiological events following brain trauma. Traumatic brain injury (TBI) is one of the most common causes of disability and death in younger individuals ,2,3. It Both single and repetitive exposures to traumatic injury may affect the brain acutely and can lead to chronic neurodegenerative changes, such as chronic traumatic encephalopathy (CTE), a degenerative brain disorder found in individuals with a history of repetitive brain traumas and associated with behavioral problems and the development of dementia . Even thA315T mice, the synaptic function is impaired due to the development of mushroom spines with lower efficiency . A decrease in the nuclear expression of this protein was detected at day 3 following LFPI but only compared to the day 1 level . Further, we wanted to determine whether these TDP-43 expression changes are cell type specific, i.e., if the increased cytoplasmic presence of this protein could be detected in the ipsilateral cortical neurons, microglia, or astrocytes after LFPI. Immunofluorescence analysis of the ipsilateral cortices of injured mice . Next, we investigated the TDP-43 expression patterns following rmTBI. The Western blot analyses showed no difference in cytoplasmic . Levels of the phosphorylated TDP-35 (p = 0.023) in the ipsilateral hippocampus after single moderate LFPI were also higher in comparison to the related control group, as seen in representative immunoblots .Next, we wanted to test whether the LFPI or rmTBI may trigger other post-translational changes of the investigated protein, specifically phosphorylation of TDP-43 and its fragments. As revealed in d TDP-35 I . After detecting the differences in the presence of the pathological TDP-35 fragment as well as in the appearance of P-TDP-43 and P-TDP-35 in the mouse hippocampi after LFPI in comparison to rmTBI animals, at 14 d following the experimental procedures, we wanted to determine whether these changes are related to microglial dysregulation in the same brain region. Namely, it has been suggested that the immune activation, including the activation of the microglia, the brain’s immune system cells, could result from an initial pathological insult. This triggering event could consequently cause an ongoing cytotoxic response, including the accumulation of TDP-43 , and resIn order to test our hypothesis, we evaluated the hippocampal levels of some protein markers of microglia activity by using Western blotting. Densitometric analyses of the protein blots revealed that, both in the LFPI and the rmTBI experiments, there were no changes in the hippocampal levels of the investigated microglial markers, specifically Iba1, iNOS, TLR-2, CD86, and CD206, at 14 d after both brain trauma protocols in relation to their designated control groups . Considering that this molecular approach to phenotypic characterization of microglia activation has garnered much criticism in recent years as being too simplistic and reductive , we alsop = 0.679; rmTBI: p = 0.299) (p = 0.020) (p = 0.744)  (In = 0.299) D,H. Howe= 0.020) G, which = 0.744) C. In the= 0.792) B,F. In cGiven the fact that no change in the hippocampal expression of the pathological TDP-43 species was observed in the rmTBI group of animals, we argue that, at this subacute time point, changes in the microglia activation state are unrelated to TDP-43 protein function and changes. This is also supported by the absence of correlation between the presence of the pathological TDP-35 fragment, the appearance of P-TDP-43 and P-TDP-35, and the microglia state in the hippocampi of mice subjected to LFPI.In this study, we provide evidence from in vivo experiments on some changes in the cortical TDP-43 expression patterns as well as in the levels of the p65 subunit of NF-κB and in the postsynaptic protein PSD-95 in a mouse model of single TBI of moderate severity. We used a model that resulted in brain trauma with characteristics similar to the injuries suffered in traffic accidents or falls. We also detected, in the subacute post-injury period, some differences in the hippocampal expression of the pathogenic TDP-35 fragment, levels of phosphorylated full-length TDP-43 and its 35 kDa cleaved fragment, as well as in the microglial reactivity between the aforementioned model and a TBI model representative of the type of repetitive brain trauma, typical for contact sports or in the military personnel. The brain trauma models we chose for this study represent one of the most used TBI models for their respective types of brain injury and, in both of them, neurobehavioral and cognitive deficits, regularly observed in patients with brain trauma, are detected ,55. HoweIn the animals subjected to a single moderate LFPI, we detected significant upregulation of the cytoplasmic TDP-43 in the ipsilateral cortices of injured animals at 1 and 3 d after TBI, with decreased nuclear TDP-43 content only on the third post-injury day. TDP-43 cellular mislocalization has also been previously described in several studies in which different models of TBI, varying injury severities, and experimental animal species were used ,48,49,50What we also detected, in the ipsilateral cortices of mice following LFPI, is that the TDP-43 cytoplasmic accumulation was present not only in neurons, as was reported by Wright et al. , but alsNF-κB is a major transcription factor and its activation is important for neurogenesis and synaptic plasticity in neurons ,27; howeOur results regarding the TDP-43 and NF-κB cytoplasmic expressions in the animals with a single moderate LFPI also correlate to the findings by Thammissety et al. , in whicSynaptic dysfunction is one of the pathophysiological hallmarks of TBI and numerous chronic neurodegenerative disorders, such as AD, in which synapse loss is not always concomitant with neuronal death, but can also happen in surviving neurons . PSD-95,The victims of TBI often suffer from lasting cognitive deficits, memory difficulties, and behavioral disturbances, and increasing evidence suggests that, even without the presence of evident morphological changes, subtle physiological changes that occur in the hippocampus after TBI are of potentially great importance . Among tIn our experiments, at 14 d following LFPI induction, the presence of the pathological TDP-43 species in the hippocampi of injured animals was not accompanied by changes in the microglial activation. Conversely, in mice subjected to repeated concussions and sacrificed at 14 d after the last mild trauma, we discovered some morphological changes pointing to microglial activation, but with no signs of TDP-43 proteinopathy. Taken together, these results indicate that, in this brain region and at the investigated time point, the TDP-43 protein expression pattern is not related to neuroinflammation. In this study, we did not consider the effects of the biological variables, such as the animal sex, or the investigation bias, e.g., regarding the judgment in cell counting. Moreover, we did not observe behavioral effects and differences in these subacute time points.In conclusion, the present study demonstrated that single moderate TBI causes acute changes in the subcellular TDP-43 expression levels in the ipsilateral cortices of injured animals and a decrease in the cortical PSD-95 levels. PSD-95 expression correlated with the presence of increased cytoplasmic TDP-43 expression in the surviving cortical neurons acutely following LFPI. At 14 d post-LFPI, we also detected some pathological changes of the hippocampal TDP-43, which was not detected in the same brain region in mice following repeated mild brain traumas. Variances in the microglia activity in the hippocampus between the two models of TBI also indicate that there are some differences in the pathophysiological processes employed after different types of brain injury, which might in turn prove to be important for the prognosis and the treatment of TBI.In our future investigations, we plan to examine the molecular background of observed TDP-43 mediated changes, specifically in relation to the expression of TBI specific inflammatory response, changes in different miRNAs expression profiles and the activation of MAPK signaling pathways.n = 50). Male animals were chosen for this study in order to exclude the possible effects of different levels of female sex hormones released during the estrus cycle [Experiments were performed on adult male wild type C57BL/6 mice  apparatus, adjusted to the mouse ,79. In bRepetitive mild brain trauma was induced in mice using the weight drop model as described by Kane et al.  and usedAnimals from the LFPI experiments were sacrificed at 1, 3, or 14 d after the single moderate TBI while the mice from the rmTBI protocol were euthanized at 14 d after the last mild brain trauma. All the sham injured mice, used as the respective control groups for the LFPI and the rmTBI experiments, were sacrificed 1 day after the end of the experimental procedures. We decided to use this single time point after sham TBI procedures by considering the results of our previous research using the LFPI model in rodents as well as the results of our preliminary studies of rmTBI in mice. Namely, in our previous experiments, we found no significant differences between the control animals sacrificed at different time points following sham procedures, which is something that can be also supported by a number of previously published studies ,81,82. FBrains from the separate cohorts of mice were used for immunohistological assays. For that purpose, at the same time points , mice we3, 1 mM PMSF, 10 µg/mL aprotinin, 10 µg/mL leupeptin and 10 µg/mL NaPP), homogenized in Dounce homogenizer and incubated for 30 min on ice. After centrifugation at 13,375 rpm for 5 min at 4 °C, the supernatant was collected as the cytosolic fraction and stored at −80 °C. Pellet was resuspended with lysis buffer B (buffer A without Triton-X) and centrifuged at 3000 rpm for 5 min at 4 °C. The supernatant was thrown away and the pellet was resuspended in the nuclear buffer (buffer B with 450 mM KCl and 50% glycerol) and frozen for 30 min at −80 °C. After thawing at RT, the sample was centrifuged at 13,375 rpm for 15 min at 4 °C, and the supernatant was collected as the nuclear fraction and, finally, stored at −80 °C. Cytosolic and nuclear fractions from the isolated cortices and the hippocampi were prepared for the detection of the designated proteins’ expression levels by Western blotting technique, as previously described by Yadavilli et al. . BrieflyEqual amounts of proteins per lane were loaded on a sodium dodecyl sulfate-polyacrylamide gel, separated by electrophoresis, and then transferred to nitrocellulose membranes. According to the antibodies’ manufacturers’ recommendations, membranes were blocked in 5% non-fat dry milk or bovine serum albumin (BSA) as the blocking buffers for one hour at RT. The incubation of the membranes with different primary antibodies, as shown in Frozen brain sections were left to achieve RT and then dried at 50 °C for 10 min. Permeabilization was done with tris-buffered saline (TBS) with 0.025% Triton X-100, for 10 min at RT. Blocking buffer made of 5% normal serum  was prepared in 1% BSA/TBS-Triton X-100 (0.025%), and applied for 2 h at RT. Primary antibodies, according to Cresyl violet staining (Nissl staining) was used for identifying the basic neuronal structure and the brain injury extent. Frozen sections were left to achieve RT and afterwards were dried at 50 °C for 10 min. Slides were immersed directly into 1:1 alcohol/chloroform overnight and were then rehydrated through 100% and 95% alcohol to distilled water. Staining of the slides was performed with warmed acidic 0.1% cresyl violet solution for 10 min. Slides were then rinsed in distilled water and, after differentiation in 95% ethyl alcohol, dehydrated in 100% alcohol and cleared in xylene. Mounting was performed with Entellan .2) were selected. Images were converted to the binary format, processed using the appropriate ImageJ plugins , and then used for the determination of the process length and the number of endpoints/processes per Iba1 positive cell by blind investigator. In the same microphotographs, the number of Iba1 positive cells in the dentate gyrus of the hippocampus was counted. In the animals from the LFPI and the rmTBI experiment, microphotographs of hippocampal sections immunolabeled with a primary antibody raised against ionized calcium-binding adaptor molecule (Iba1) were used for the quantitative analysis of microglia morphology, as previously described by Morrison et al. , in whict-test and comparisons between multiple groups were done using one-way analysis of variance (ANOVA) followed by Duncan’s post-hoc multiple comparison test. Results are expressed as mean ± standard deviation (SD). In all the comparisons, p < 0.05 was considered to indicate statistical significance. To remove the variations between the Western blotting sessions, results of the densitometric analyses were corrected with the Factor Correction program version 16.1 [Data were collected using Microsoft Excel 2016 . For the densitometric analyses of the expression levels of different proteins, the values of the relative optical densities of the bands were corrected for the corresponding β-actin (cytosolic samples) or histone-H3 (nuclear samples) contents and were then expressed as the % of the values from the related control groups. For all the statistical analyses, Statistica software version 13.0  was used. Sample size, i.e., the number of animals per group for the Western blotting and the immunofluorescence analyses, was calculated by using the results of our previous research in which complimentary methods were used ,85. Nameion 16.1 .


We propose a concept of an electrically controllable plasmonic directional coupler of terahertz signal based on a periodical structure with an active (with inversion of the population of free charge carriers) graphene with a dual grating gate and numerically calculate its characteristics. Proposed concept of plasmon excitation by using the grating gate offers highly effective coupling of incident electromagnetic wave to plasmons as compared with the excitation of plasmons by a single diffraction element. The coefficient which characterizes the efficiency of transformation of the electromagnetic wave into the propagating plasmon has been calculated. This transformation coefficient substantially exceeds the unity  due to amplification of plasmons in the studied structure by using pumped active graphene. We have shown that applying different dc voltages to different subgratings of the dual grating gate allows for exciting the surface plasmon in graphene, which can propagate along or opposite the direction of the structure periodicity, or can be a standing plasma wave for the same frequency of the incident terahertz wave. The coefficient of unidirectionality, which is the ratio of the plasmon power flux propagating along (opposite) the direction of the structure periodicity to the sum of the absolute values of plasmon power fluxes propagating in both directions, could reach up to 80 percent. Two different methods of the plasmon propagation direction switching are studied and possible application of the found effects are suggested. As was shown in3, the momentum relaxation time of charge carriers in graphene reaches the value of 2 ps at room temperature. Hence, graphene is a promising platform for excitation and manipulation of plasmons in the THz frequency range4. Possibility of population inversion of free charge carriers in graphene at THz frequencies5 along with the possibility of excitation of highly confined graphene plasmons6 resulted in proposals of THz graphene lasers8 and plasmonic amplifiers10 which may be used for signal processing in THz integrated nanocircuits in the subwavelength regime11.Controlling the propagation the plasma waves (plasmons) as well as switching of plasmon propagation direction is important for designing integrated plasmonic nanocircuits. Noble metals are suitable for exciting and controlling plasmons in the visible and near-infrared frequency range. But at terahertz (THz) frequencies, plasmons on the surfaces of noble metals are weakly confined. Terahertz plasmonic devices can be implemented on other materials, graphene for example. Graphene is a unique material with a zero energy gap and linear spectrum of electrons at THz frequency range12, a single metal gate13 or a thin slit in a metal gate14 which are placed above graphene. One may use the diffraction of external waves on the cantilever of a scanning near-field microscope15. However, the diffraction of THz wave on a single diffraction element couples THz wave to plasmons in graphene with rather low efficiency. Another conventional method for exciting plasmon polaritons in graphene is using the prism coupling17 in the Otto or Kretschmann configurations19. In order to create plasmonic splitters or directional couplers one has to make the plasmon propagate in a desired direction. Diffraction of incident wave on a periodical grating gate with a symmetric unit cell demonstrates high efficiency of transformation of incident wave into standing plasma wave21 but does not allow for launching the unidirectionally travelling plasmon. Creation of travelling plasmon by using attenuated total reflection methods suffers from strong effect of the angle of incidence on the transformation of incident wave into travelling plasmon. To enhance the transformation efficiency of an incident electromagnetic wave into a propagating plasmon, a periodical graphene structure with a dual-grating gate having an asymmetric unit cell was proposed and theoretically investigated in23. Spatially asymmetric plasmon structures can detect THz radiation26. Excitation and amplification of THz plasmons using diffraction of incident wave on a periodical grating gate were experimentally observed in28.There are several methods for excitation of plasmons in graphene. It can be the excitation of surface plasmon polaritons propagating along the graphene using diffraction of incident THz wave on a grating gate29, where the method of switching the graphene conductance between high and low conductance states using appropriate bias voltage pulses was proposed. Trapping the plasmon polaritons on the graphene surface within a broadband frequency range was demonstrated theoretically and numerically in30. A tunable Y-branch graphene plasmonic switch operating at the wavelength of 1.55 mm was proposed and analyzed analytically and numerically in31. Proposed in that paper plasmonic switch supports transverse magnetic and transverse electric graphene plasmons whose propagation characteristics could be controlled by modulating the external electric field and the temperature of graphene. Plasmonic splitter based on two different metal–insulator-metal waveguides with periodic grooves and a subwavelength slit was proposed and theoretically studied in32. Terahertz tunable plasmonic directional coupler based on a graphene and a thin metal film with a nanoscale slit was proposed in14. The possibility of switching the plasmon propagation direction applying different voltages to the graphene layer was numerically shown in that paper.Tunable switches, splitters and couplers are of great importance in optical communication systems and integrated nanocircuits. Graphene-based switch in the near infrared frequency range and its fabrication and operation principles were demonstrated in33 or injection pumping34, or diffusion pumping proposed in36. Passive graphene (without population inversion) is overlayed upon the insulating layer separating the graphene layers. This passive graphene is screened by periodic metal dual grating gate that is placed over the structure. Incident THz electromagnetic wave with the electric field polarized across the grating gate fingers diffracts on the periodic gate and excites transverse magnetic (TM) plasmons (the magnetic field of the plasmon is polarized in the y-direction) in both graphene layers. We have studied the structure with equal widths of the grating-gate fingers in both subgratings comprising the dual grating gate. Thus, the structure under consideration has a geometrically symmetric unit cell. It is known that for effective excitation of the propagating plasmon one needs an asymmetry in the unit cell of the structure21. Applying constant dc voltage U or V between the gate and upper graphene layer, one can change the Fermi energy of the free charge carriers in the gated regions of the upper graphene by the value of eU or eV, respectively, where e is the electron charge. Change of the equilibrium electron concentration in graphene applying the constant voltage to the gate subgratings of the dual grating gate structures is experimentally and theoretically described in28. As was shown in37, the voltage applied to the gate almost does not affect the concentration of the free charge carriers in the lower graphene layer. Carrier concentration in the lower graphene could be changed if only the upper graphene concentration is depleted that was not realized for applied voltage values used in our calculations. Applying different dc voltages to different subgratings of the dual grating gate one can create spatial asymmetry in the unit cell of the studied structure (forming different Fermi energy levels under different gate subgratings) which allows for switching of plasmon propagation direction. Active graphene allows for amplification of the propagation plasmon.In this paper, we theoretically investigate electrically controllable THz plasmonic directional coupler based on a periodic graphene structure with a dual grating gate, termed dual grating gate graphene heterostructure (DGGGH), which is schematically shown in Fig. T = 300 K (assumed the same in both graphene layers), and free carrier momentum relaxation time is 38. The quasi-Fermi energy value in the lower (active) graphene  was taken to be 7. The frequency was fixed at 7.The coefficients of absorption of the electromagnetic wave and transformation of this incident wave into the propagating plasmon in the studied DGGGH are presented in Fig. entclass1pt{minimaU  are caused by the presence of the lower active graphene.The coefficient of absorption of the incident wave in the DGGGH is defined as mentclass2pt{minimx-direction, respectively , and x-direction (see red regions on the map), while negative values of this coefficient correspond to excitation of the plasmon travelling against the x-direction (blue regions on the map). Away the mode intercrossing regions, the incident electromagnetic wave excites rather weak plasmons in graphene. The transformation coefficient The coefficient of transformation of the electromagnetic wave into a propagating plasmon  by changing the gate voltages values. The plasmon propagation direction could be switched when the Fermi energy value under one of the sub-gratings remains fixed while the other Fermi energy value is changed  across the mode intercrossing  in dependence on the Fermi energy value x-direction. Positive x-direction to the sum of the absolute values of these power fluxes:P exceeds 6 in value). For the method of type I of plasmon direction switching, the unidirectional coefficients values after swapping the voltage values corresponding to mode intercrossing remain as high as 80 percent while the transformation coefficient P exceeds 6. This could be seen from Fig. We start from the method of type We have shown the possibility to realize electrically tunable terahertz active plasmonic directional coupler based on the periodical DGGGH. Proposed directional coupler allows for switching the plasma wave propagation direction between two opposite directions along the graphene layers. Important features of this coupler is high efficiency of transformation of incident THz wave into propagating plasmon and high directionality of the plasmon due to using the dual-grating-gate and the active graphene to excite undumped and/or amplified propagating plasmons.x-component of the electric field amplitude over the unit cell of the DGGGH in the passive graphene layer for the fixed Fermi energy values a little shifted from the values corresponding to the plasmon mode intercrossing  for frequencies exceeding 5 THz. The higher-frequency plasmon mode in this case has a lower radiative damping, therefore it becomes an amplified mode due to the gain provided by the active graphene, while the other mode remains absorbing39.An important feature to be discussed is the structure of the plasmon modes near the intercrossing regimes where the switching of the plasmon propagation direction occur. In Fig. Finally, the concept of the electrically controllable active plasmonic directional coupler of THz signal based on a periodical dual grating gate graphene structure is proposed. The advantage of effective energy transformation by using dual grating gate periodical graphene structure allows for excitation of unidirectionally propagating plasmon with great transformation efficiency and high directionality. One can control the plasmon propagation direction by applying different dc voltages to the subgratings of the grating gate. High transformation efficiency occurs due to amplification of plasmons in the structure by using pumped active graphene. Such electrically controllable directional coupler is of high importance for application in THz plasmonic integrated nanocircuits.Numerical calculations were performed using the electromagnetic approach based on the integral equation method. Basic steps of this approach are the following. (i) Maxwell equations, Ohm’s law and boundary conditions are written in the Fourier representation. (ii) The integral equations in respect of oscillating currents in the conducting areas of DGGGH are formed. (iii) The integral equations are numerically solved using the Galerkin procedure. The described method allows for calculation the field of plasma wave at any point of the structure. For more detail, see the Supplemental material.Supplementary Information.


Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease with a significant unmet medical need. Development of transformational therapies for IPF is challenging in part to due to lack of robust predictive biomarkers of prognosis and treatment response. Importantly, circulating biomarkers of IPF are limited and none are in clinical use.We previously reported dysregulated pathways and new disease biomarkers in advanced IPF through RNA sequencing of lung tissues from a cohort of transplant-stage IPF patients (n = 36) in comparison to normal healthy donors (n = 19) and patients with acute lung injury (n = 11). Here we performed proteomic profiling of matching plasma samples from these cohorts through the Somascan-1300 SomaLogics platform.Comparative analyses of lung transcriptomic and plasma proteomic signatures identified a set of 34 differentially expressed analytes (fold change (FC) ≥  ± 1.5, false discovery ratio (FDR) ≤ 0.1) in IPF samples compared to healthy controls. IPF samples showed strong enrichment of chemotaxis, tumor infiltration and mast cell migration pathways and downregulated extracellular matrix (ECM) degradation. Mucosal (CCL25 and CCL28) and Th2 (CCL17 and CCL22) chemokines were markedly upregulated in IPF and highly correlated within the subjects. The mast cell maturation chemokine, CXCL12, was also upregulated in IPF plasma  and significantly correlated  to lung function (%predicted FVC), with a concomitant increase in the mast cell Tryptase, TPSB2. Markers of collagen III and VI degradation (C3M and C6M) were significantly downregulated (C3M p < 0.001 and C6M p < 0.0001 IPF vs control) and correlated, Pearson r = 0.77) in advanced IPF consistent with altered ECM homeostasis.Our study identifies a panel of tissue and circulating biomarkers with clinical utility in IPF that can be validated in future studies across larger cohorts.The online version contains supplementary material available at 10.1186/s12931-021-01860-3. Idiopathic pulmonary fibrosis (IPF) is a progressive, chronic and fatal lung disease with a huge unmet medical need –3. DespiPast studies have profiled gene expression in lung tissues, peripheral blood and isolated cells through microarray and bulk/single cell RNA-sequencing analyses, identifying aberrant cell populations as well as molecular signatures of progressive IPF –12. ReceWe hypothesized that a combined analysis of lung and plasma gene/protein signatures will identify robust biomarkers with potential clinical utility. Here, we have performed unbiased proteomic analyses of matching plasma from the advanced IPF cohort through the Somascan-1300 aptamer platform and compared the plasma proteome signature to the previously reported lung transcriptome signature. Our data reveal a striking enrichment of pathways involved in chemotaxis/Th2 chemokine and T-cell signaling, Wnt signaling, mast cell migration and activation, and extracellular matrix degradation in both tissue and plasma of advanced IPF. Notably, the Th2 chemokines CCL17 and CCL22 as well as mucosal chemokines CCL25 and CCL28 were robustly upregulated in IPF and correlated within subjects. The mast cell maturation chemokine CXCL12 was also increased in IPF together with a concomitant increase in the mast cell protease, TPSB2. Neoepitopes of collagen type III and VI degradation (C3M and C6M) were strongly downregulated and highly correlated in advanced IPF subjects. Our data provide a comprehensive signature of IPF tissue and plasma that could be potentially validated and utilized for clinical assessment of advanced IPF.All human subject sample acquisitions and experiments were conducted with the appropriate approval from the Institutional Review Board . The clinical profile and demographics of IPF, ALI and control subjects used in this study have been previously described . The IPFDetails on the RNA-sequencing method and analyses have been described previously in detail .Plasma samples were collected in Citrate EDTA tubes in operating room prior to explant, centrifuged at 4 ℃ at 3521 RPM for 10 min, and stored at − 80℃. Plasma samples were analyzed on the SOMAscan V2 multiplex proteomic assay —an aptamer-based quantitative proteomic biomarker discovery platform which measures 1033 analytes , 19. TheEach sample in the study was normalized by aligning the mean to a common reference. Inter-plate and inter-run calibration were achieved by applying a multiplicative scaling coefficient to each SOMAmer. These scaling factors were calculated using the eight reference calibrators on each plate. Sample data were first normalized to remove hybridization variation within a run followed by median normalization across all samples to remove other assay biases within the run and finally calibrated to remove assay differences between runs. Log transformed RFU values were used to analyze differential expression of biomarkers across cohorts by using the Limma linear modeling framework for differential expression , 21. PatNeoepitopes of collagen III and VI degradation (C3M and C6M) respectively were analyzed by previously described ELISA methods , 23.Statistical analyses of differential gene expression data using the R package has been described previously . SomaLogPlasma samples from the IPF, ALI and healthy control cohorts were analyzed using the Somascan 1300-plex aptamer platform. Normalized log-transformed data were used to generate protein expression intensities. Expression data were visualized using t-scholastic neighborhood enrichment (t-SNE), that showed a robust separation of the IPF samples from the control and ALI samples , we identified 53 genes commonly regulated in lung and plasma, with 34 of them moving in the same direction Fig. a and plaSince pathway analyses of IPF plasma revealed a strong dysregulation of proteins involved in ECM homeostasis and remodeling, we measured C3M and C6M, neoepitopes of Collagen III and Collagen VI degradation respectively. Interestingly, we found a marked decrease in the levels of both C3M and C6M in IPF compared to healthy controls Fig. a, b. AddWe describe here for the first time a simultaneous comprehensive analyses of plasma proteome and lung transcriptome of a unique cohort of advanced IPF patients in comparison to that of normal healthy donors. Our studies suggest a strong dysregulation of T-cell activation, chemokine signaling, mast cell maturation, Wnt signaling and ECM homeostasis pathways in lung tissue as well as peripheral blood of these patients and identify new biomarkers that could have clinical utility. Although numerous profiling studies in the past have identified dysregulated genes, proteins and pathways in early and progressive IPF, there are currently no diagnostic or prognostic biomarkers in clinical practice , 24–26. Unbiased hierarchical clustering of protein expression across the cohorts shows that the IPF samples were clearly separated from control and ALI samples. Consistent with this separation, we did not find major differences between the ALI and control cohorts, although our previously published RNA-seq data identified significant differences between the cohorts at the gene expression level. In the ALI samples, the degree of diffuse alveolar damage varied with the majority having extensive areas of pathology, while some had more focal areas. This degree of sample heterogeneity is expected and may have potentially contributed to the similarities seen between the ALI and donor sample groups in our study.Plasma data from the IPF cohort not only confirmed and extended our previous lung transcriptome findings in the same cohort , but alsAnother key finding from our study was the upregulation of the mast cell chemokines, CCL5, CCL21 and CXCL12, and the mast cell protease tryptase-B2. Prior studies have shown increased infiltrating mast cell numbers and tryptase activity in human IPF , 36, andAberrant reactivation of developmental pathways including that of Wnt signaling is known to play a role in the pathogenesis of IPF . In our Consistent with an advanced fibrotic state, we found ECM remodeling and proteolysis pathways strongly dysregulated in the IPF cohort. The matricellular protein, SPARC, plays a key role in promoting collagen assembly into the ECM, and implicated as a biomarker in previous studies , 45. SPAAlthough RNA-seq and SomaLogics represent distinctly different platforms that limit robust comparison, our analyses clearly confirmed many analytes dysregulated at gene level to be differentially regulated in plasma as well. It is possible that many other markers of interest may be missed due to the targeted 1300-plex analyses. Future studies with the currently available expanded Somascan platform  could help further identify and validate additional biomarkers of disease. Interestingly, new emerging data indicates that the use of a multi-omic approach such as ours could be valuable in identifying molecular disease signatures and biomarkers of IPF . AnotherIn summary, we have presented a unique comparative transcriptome-proteome signature of advanced IPF and identified key tissue and circulating biomarkers that could be predictive of progressive/worsening IPF. Further validation of these findings in larger cohorts will help develop a comprehensive panel of biomarkers with clinical utility to address the current unmet need in the diagnosis and management of IPF.Additional file 1. Differential protein expression in plasma of IPF patients compared to healthy controls.


Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive disorder with an estimated median survival time of 3–5 years after diagnosis. This condition occurs primarily in elderly subjects, and epidemiological studies suggest that the main risk factors, ageing and exposure to cigarette smoke, are associated with both pulmonary and extrapulmonary comorbidities . Ageing and senescence, through interactions with environmental factors, may contribute to the pathogenesis of IPF by various mechanisms, causing lung epithelium damage and increasing the resistance of myofibroblasts to apoptosis, eventually resulting in extracellular matrix accumulation and pulmonary fibrosis. As a paradigm, syndromes featuring short telomeres represent archetypal premature ageing syndromes and are often associated with pulmonary fibrosis. The pathophysiological features induced by ageing and senescence in patients with IPF may translate to pulmonary and extrapulmonary features, including emphysema, pulmonary hypertension, lung cancer, coronary artery disease, gastro-oesophageal reflux, diabetes mellitus and many other chronic diseases, which may lead to substantial negative consequences in terms of various outcome parameters in IPF. Therefore, the careful diagnosis and treatment of comorbidities may represent an outstanding chance to improve quality of life and survival, and it is necessary to contemplate all possible management options for IPF, including early identification and treatment of comorbidities. Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive disorder with an estimated median survival time of 3–5 years after diagnosis. The diagnosis of IPF requires the presence of a typical pattern of usual interstitial pneumonia (UIP) by either high-resolution computed tomography (HRCT) or histology in an appropriate clinical setting and the absence of an identifiable aetiology .Cohort studies suggest that different IPF phenotypes exist , with soTwo antifibrotic drugs, pirfenidone and nintedanib, have been shown to slow the decline in lung function in patients with mild to moderate disease , 9, but IPF is primarily a disease of elderly people, and epidemiological features suggest that multiple risk factors including ageing and genetic alterations that enhance susceptibility to the disease, as well as environmental factors, especially cigarette smoke exposure, could underlie the pathogenesis of the disease and its comorbidities. Furthermore, although IPF is considered a disease limited to the lung, its risk factors are shared with a number of comorbidities  thaAgeing is defined as a progressive decline in physiological function leading to an increase in the age-specific mortality rate ; it is aThe two main risk factors for IPF, i.e., ageing and cigarette smoke, are associated with common comorbidities, defined as the occurrence of two or more disorders in a single individual  Table 11.Table 1Early evidence suggests that the prevalence and clinical impact of these comorbidities are likely to increase as survival in IPF improves with current and future interventions. However, evidence from pharmaceutical trials is not always fully applicable to the general IPF population because the most influential selection bias in pharmaceutical cohorts is the exclusion of patients with significant comorbidities .In this review, we will highlight the importance of ageing; its mechanisms; and its interaction with other risk factors, such as cigarette smoke, in determining IPF and its comorbidities, highlighting how conditions directly or indirectly related to pulmonary fibrosis may have a similar origin and how their management requires specific diagnostic approaches and combined interventions.Ageing is a complex process that includes genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, deregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and altered intercellular communication . In partCurrently, the most characteristic pathogenetic IPF features are considered the aberrant activation of alveolar epithelial cells, probably determined by environmental factors such as cigarette smoke or air pollution , and theA variety of cell types in the lungs of IPF patients undergo ageing and its consequences , 25. AgeCurrent evidence suggests that the main hallmarks of ageing occur prematurely in IPF and primarily affect epithelial cells . In factMore recently, in addition to alveolar epithelial cells, fibroblasts/myofibroblasts within fibroblastic foci showed markers of cell senescence . SenesceIn conclusion, ageing and senescence, through interaction with environmental factors, may contribute to the pathogenesis of IPF through dual mechanisms, causing the abnormal secretory pattern of the lung epithelium and increasing the resistance to apoptosis in myofibroblasts, resulting in extracellular matrix accumulation and pulmonary fibrosis Fig. .Fig. 1MoTelomere length physiologically shortens with age and predicts the onset of replicative senescence. Telomerase is a significant enzyme that maintains chromosome ends . FamiliaAccording to the current concept, mutations in telomerase and telomere components predispose adults to a broad spectrum of diseases characterized by pulmonary fibrosis, liver fibrosis and haematological features , with agMUC5B) gene was associated with an approximately sevenfold increased risk for IPF development in a genome-wide linkage study [Among other genes that may have a relationship with cell senescence, the rs35705950 variant in the promoter region of the mucin 5B (ge study . This MUge study .TOLLIP) gene were found to be associated with IPF, one of which (rs5743894) was associated with a decreased risk of IPF but increased mortality in those with the disease [Using genome-wide association technology, three common variants  in the Toll-interacting protein  by Fingerlin et al. enrolled a total of 2492 patients with fibrotic IIPs (most of them IPF) and compared these with over 6000 control subjects. This study showed seven new loci related to genes controlling host defence, cell–cell adhesion and DNA repair, which contributed to the risk of developing fibrosis  and are A recent systematic review confirmed a high prevalence of a variety of comorbidities in IPF patients compared to the general population . AdditioRecent studies have shown that coronary artery disease (CAD) is significantly more prevalent in lung transplant candidates with IPF than in those with emphysema  . In a siHigh-resolution CT of the chest is recommended for all patients with (suspected) IPF as a mandatory tool for an IPF diagnosis; it can therefore also become an appropriate tool of screening for CAD in high-risk populations. The detection of moderate-to-severe coronary artery calcifications on high-resolution CT had a high sensitivity and specificity for the diagnosis of CAD . This raThe high prevalence of this comorbidity in IPF patients is partially explained by a shared genetic predisposition to the development of both IPF and CAD by detection of specific interleukin (IL)-17 immune responses to the alpha 1 chain of collagen type V and by their correlation with human leukocyte antigen (HLA)-D15 alleles . HoweverLow-level evidence suggests that other cardiac comorbidities, including arrhythmias, such as atrial fibrillation, and cardiac or congestive heart failure, may also be frequent in patients with IPF . Data frIPF is associated with a prothrombotic state that may be important in pathogenesis . ConsequMutations in the hTERT and hTR genes are also associated with haematological abnormalities, such as aplastic anaemia, macrocytosis, thrombocytopenia and myelodysplasia/leukaemia , 66. TheA few studies have explored the relationship between IPF and bone status, showing that reduced bone mineral density has been observed in the thoracic vertebrae of patients who were not receiving corticosteroid treatment . In a rePlatelet-derived growth factor (PDGF) subunit B has been linked to both osteoporosis  and fibrVitamin D has also been related to bone metabolism. In a recent study, IPF patients exhibited low serum vitamin D concentrations, and such deficiency correlated with all-cause mortality, suggesting its possible role as a prognostic factor and therapeutic target .Various studies have shown an increased prevalence of diabetes mellitus in IPF patients –75 compaInterestingly, abnormal telomere shortening has been found to be associated with type 2 diabetes mellitus, insulin resistance and impaired glucose tolerance .Advanced glycosylation end-products are considered critical for arterial and vascular damage and inflammation in diabetes . RAGE poFurthermore, the nuclear RAGE isoform (nRAGE) seems to be essential to preserve DNA double-strand break repair, and the modulation of this nuclear mediator coupled with an ATM kinase shows the potential to reverse fibrosis in mice . The relIn a retrospective case–control analysis, Oldham et al. showed that hypothyroidism was common and markedly more prevalent (13% of men and 28% of women affected) among IPF patients than among the general population (1–2% of men and 5–9% of women) . The potThe relationship between the thyroid and the lung is complex, and its deep mechanisms are still unknown; mutations in thyroid transcription factor NK2 homeobox 1 (NKXS2-1) are linked to brain-thyroid syndrome, affecting neonates and children with histological features of surfactant disruption . FurtherCombined pulmonary fibrosis and emphysema (CPFE) defines the coexistence of upper lobe emphysema and basilar fibrosis. This term was established by Cottin et al. in 2005 , althougInterestingly, telomere length and mutations in the TERT/TERC system have been linked to both emphysema susceptibility  and to CBecause no clinical trial specifically targeting CPFE patients has been performed, treatment recommendations in these patients are based on expert opinion. Smoking cessation, vaccinations, supplemental oxygen, and pulmonary rehabilitation should be prescribed when appropriate. Treatment of CPFE patients with inhaled long-acting anti-cholinergic drugs, inhaled long-acting beta agonists and/or inhaled corticosteroids is of unclear benefit . PatientSince IPF is a chronic and progressive condition with a significant impact on morbidity and mortality, it is frequently associated with anxiety and depression. Specifically, worsening dyspnoea, declining pulmonary function, and hypoxemia are characteristic features of IPF progression, and they have been shown to correlate with depression and anxiety , which, Pulmonary rehabilitation has been shown to result in sustained improvements in depressive symptoms and should be recommended to all patients with IPF who have depression and functional impairment .In advanced fibrotic lung disease, a palliative care intervention showed a positive effect on both anxiety and depression, confirming the importance of best supportive care in these patients .Pulmonary hypertension is defined as a resting mean pulmonary artery pressure (mPAP) greater than or equal to 20 mmHg as assessed by right heart catheterization . A normaHowever, a study performed on more than 6500 patients indicated that even mild pulmonary hypertension in patients with IPF increases the risk of death . Some stThe frequency of PH further increases with some comorbidities, such as obstructive sleep apnoea, thromboembolism or cardiac diastolic dysfunction .Generally, the degree of PH in IPF patients is mild to moderate. However, some patients (approximately 10%) show disproportionate pulmonary arterial pressure compared to the severity of lung disease as measured by lung function tests  or HRCT Symptoms of PH in patients with IPF are non-specific, and the presence of PH can be easily missed or the diagnosis delayed. In fact, the occurrence of PH in IPF may be characterized by increased dyspnoea, reduction of gas exchange at rest, markedly low DLCO, rapid desaturation upon exercise, high brain natriuretic peptide (BNP) levels, right heart dilation on chest radiography, impaired quality of life, lower exercise tolerance, and greater supplemental oxygen requirements . Rapid pAlthough right heart catheterization (RHC) is considered the gold standard for PH diagnosis, transthoracic echocardiogram is usually utilized as the initial screening tool for pulmonary hypertension, although its accuracy in chronic lung disease, including IPF, is still a matter of debate .Supplemental oxygen is indicated for the prevention and therapy of PH due to hypoxia, although no data are available supporting the beneficial effect of oxygen on survival in this group of patients.There are no approved targeted therapies for PH in IPF , and recSildenafil, a phosphodiesterase-5 inhibitor, was studied in patients with advanced IPF. In a large double-blind, placebo-controlled trial called “STEP-IPF”, IPF patients were randomized to receive oral sildenafil or placebo. The primary outcome measure was the presence of at least a 20% improvement on the 6-min walking test (6MWT) at 12 weeks: it did not meet statistical significance, as only 10% in the sildenafil arm versus 7% in the placebo arm showed improvement (P = 0.39). However, small but clinically significant differences in the secondary outcomes of arterial oxygenation, DLCO, degree of dyspnoea and quality of life in patients receiving sildenafil were observed . A post A recent phase 2b/3 trial with pulsed inhaled nitric oxide in patients with fibrotic ILDs complicated by PH showed preliminary promising results on a beneficial impact of this treatment on physical activity with a good overall safety profile and tolerability .Gastro-oesophageal reflux (GER) is the reflux of gastric contents into the oesophagus, with some degree of GER occurring physiologically. GER can be acidic or non-acidic with oesophageal pH values below or above 4.0, respectively. Gastro-oesophageal reflux disease (GERD) is a condition that develops when the reflux of stomach contents causes symptoms or comorbidities .It is important to note that GERD is not synonymous with GER or simple heartburn and that GERD syndrome, by definition, involves adverse downstream effects from the reflux .GERD is commonly observed in patients with IPF , 127 andRecently, Ghebre and Raghu  hypothesThe typical GERD symptoms, including heartburn, are present in only 25–65% of IPF patients with a confirmed GERD diagnosis; therefore, the absence of symptoms does not preclude a diagnosis of GERD in IPF patients .Although 24-h pH monitoring and oesophageal manometry can be useful in identifying GERD, the best diagnostic flowchart to diagnose GERD in IPF patients is undefined and should therefore be personalized. Mainly for this reason, GERD therapy is often started on the basis of GER symptoms .GERD also seems to be a risk factor for acute exacerbation of IPF (AE-IPF): micro-aspiration is in fact believed to be one of the mechanisms triggering AE-IPF, as demonstrated by Lee et al., who showed a high level of pepsin in bronchoalveolar lavage fluid of AE-IPF patients. The detection of pepsin in bronchoalveolar lavage fluid of these patients was proposed as a biomarker of micro-aspiration and correlated with AE-IPF even if its increased level was not predictive of mortality .The benefit of antacid medication on IPF progression is still debated. Case series and uncontrolled trials have shown beneficial effects of the treatment of GERD in IPF, including lung function stabilization in IPF patients affected by GER and treated with antacid therapy . FurtherIn contrast, a recent pooled, post hoc analysis including patients with IPF from the placebo groups of three trials showing the efficacy and safety of pirfenidone  could not replicate the effects of PPIs and reported a potential association with an increased risk of infection in those with advanced disease . In a siWhile anti-acid treatments target the acidic component alone, surgical interventions include repair of hiatal hernia and Nissen fundoplication to treat GERD by suppressing the anatomical predisposition to reflux .This strategy acts by suppressing the reflux of both acidic and nonacidic gastric material from the stomach, and thus, the reduction risks of aspiration are clearly reduced . Raghu eVarious studies have shown that obstructive sleep apnoea (OSA), defined by an apnoea-hypopnoea index > 15 events/hour, is frequent in IPF patients –144, witSeveral sleep alterations have been detected in IPF patients, including changes in sleep architecture, such as abnormal sleep stage distribution, multiple awakenings, decreased percentage of total sleep time, low sleep efficiency, and increased wake time after sleep onset, in breathing pattern, such as increased respiratory frequency during sleep and rapid and shallow breathing, especially during rapid eye movement (REM) sleep, and in nocturnal oxygenation parameters, such as oxygen desaturation (during both REM and non-REM sleep) and desaturation due to respiratory events (apnoeas and hypopneas) .The variety of obstructive sleep-disordered breathing in IPF patients comprises a spectrum of respiratory events during sleep ranging from simple snoring to complete cessation of airflow (apnoea) . ConsideThe proposed connection between IPF and OSA is probably based on tracheal traction theory. In fact, decreased lung volumes, observed in restrictive pulmonary diseases, can reduce upper airway stability and increase resistance because of decreased traction on the upper airway, therefore facilitating upper airway collapse .Patients with IPF with OSA should be treated with continuous positive airway pressure (CPAP), as are all other patients with OSA, improving quality of life and decreasing mortality in this population .However, a high rate of continuous positive airway pressure (CPAP) nonacceptance or poor compliance, mainly because of claustrophobia, irritating cough during sleep, insomnia, and depression, has been reported in IPF, suggesting the need for follow-up by a well-organized sleep centre .Supplemental oxygen is generally prescribed to correct nocturnal hypoxaemia. However, no studies have demonstrated an effect on long-term survival or on the development of pulmonary hypertension .The risk of developing lung cancer (LC) is markedly higher in patients with IPF than in a control population . This inIn IPF, lung cancer often presents with nonspecific symptoms, including haemoptysis, weight loss, and other constitutional symptoms; when detected in IPF patients, they should therefore prompt further evaluations . AdditioAlthough the studies that reported mortality and survival among IPF patients with lung cancer were limited by small sample sizes, survival was decreased in those with concurrent LC compared to patients with either idiopathic interstitial pneumonia  or IPF aIPF patients with lung cancer are generally not good candidates for standard therapy, particularly because of the decreased tolerance of these patients to cancer therapies. Management of lung cancer in IPF should therefore be approached on a single-patient basis, balancing the likelihood of treatment against the potential for therapy comorbidities and considering the frequent poor prognosis of IPF . PatientAE-IPF was also observed in patients treated with chemotherapy for lung cancer or following radiotherapy , 161. A Deconditioning is a physical and/or psychological decline in function commonly experienced in all chronic diseases. Prolonged inactivity or reduced activity can affect nearly all systems of the body and can be regarded as an important comorbidity of chronic respiratory diseases such as COPD . These cSarcopenia, defined as a progressive loss of muscle mass and strength, is associated with many adverse outcomes such as disability, poor QoL and death . It can The complex clinical syndrome of sarcopenia and deconditioning in IPF may clearly benefit from multimodal interventions, including a tailored therapeutic approach based on distinct wasting phenotypes. Early referral and participation in pulmonary rehabilitation programs may represent an important intervention to improve symptoms, physical activity and QoL in IPF . PrelimiVarious studies found that nutritional abnormalities, such as low body mass index (BMI), body weight loss, and vitamin D deficiency, seem to have negative prognostic significance in patients with IPF . SeveralDespite the importance of nutritional abnormalities, their prevalence and impact in IPF is largely understudied.According to the mean BMI values reported in multiple studies, in most studies, a high proportion of overweight/obese patients may be expected , 180. InThe prevalence appears to be lower in underweight patients than in overweight patients; however, alterations in nutritional parameters suggestive of malnutrition were observed in nearly one-third of cases, particularly in advanced stages, in a recent study on French IPF patients .The two currently available antifibrotic drugs nintedanib and pirfenidone can cause adverse events that may interfere with food intake and absorption, including diarrhoea, nausea and appetite loss .Efforts should be made to reduce these adverse events to minimize the effect of chronic antifibrotic therapies on nutritional status.Currently, the role of genetic testing in IPF is uncertain and limited to familial forms of pulmonary fibrosis, with a prevalence between 2 and 20% of all cases of IPF , 183. InFurthermore, there is no consensus on the optimal workup with which to diagnose hTERT/hTR disease-associated variants. Relative simple assays such as flow fluorescence in situ hybridization (flow-FISH) or monochrome multiplex quantitative polymerase chain reaction (MM-qPCR)  can incrIPF is a progressive fibrotic disease limited to the lungs; approved antifibrotic therapies may modify its relentless course by acting on a multitude of cellular pathways. However, IPF can also be viewed as a disease due to the interaction between ageing, genetic predisposition and environmental exposures such as cigarette smoke and is therefore often accompanied by comorbidities that affect the clinical phenotype and survival. Many comorbidities have substantial negative impacts on various outcome parameters in IPF. Therefore, the careful diagnosis and treatment of comorbidities may represent an excellent opportunity to optimize the survival of IPF patients. The broad range of comorbidities in IPF will require a holistic approach, contemplating all possible management options, including early identification and treatment.It is possible that, in the future, elucidation of the fine genetic mechanisms underlying IPF and its comorbidities will enable better management of the disease, especially for familial forms.


In this paper, graphene-coated spherical nanoparticles are arranged around an infinite length dielectric cylinder to enhance its extinction cross-section. Initially, a single longitudinal one-dimensional periodic array is considered in different loci concerning the transverse electric (TE) incident plane wave. It is observed that regardless of the position of the particles, the extinction cross-section of the dielectric cylinder is considerably enhanced with respect to the bare one. Later, by increasing the number of longitudinal plasmonic arrays around the cylinder, each residing in a different azimuthal direction, the extinction cross-section is further manipulated to observe double pronounced Fano resonances. The origin of the Fano resonances is described by considering their planar counterparts constructed by the periodic assembly of plasmonic oligomers. Finally, the hexamer configuration is considered as the prototype, and the effect of various optical, geometrical, and material parameters on the optical response is investigated in detail. Interestingly, due to the spherical symmetry of the cells, the extinction cross-section is also enhanced for the transverse magnetic (TM) incident wave, which is unattainable using a continuous plasmonic cover made of metal or graphene. The potential application of our proposed structure is in the design of reconfigurable conformal optical absorbers and sensors. Also, core–shell cylindrical particles made of noble metals can be used to enhance luminescence, nonlinear optical activities, and for deep sub-wavelength-scale optical wave-guiding2. Moreover, a triple-layered tube consisting double metallic and single dielectric layers is proposed as a super-scatterer, designed using the dispersion engineering method3. By further increase of the stacked metal-dielectric layers, multiple super-scattering and invisible cloaking states can be observed for the possible application in electromagnetic tagging4. For the metal-based cylindrical scatterers, the plasmonic effects are mostly observed for TE polarized wave fields with respect to the cylinder axis5.Scattering analysis of cylindrical objects covered by patterned metallic elements or plasmonic metals has been the subject of many pieces of research because of the wide range of applications that can be provided by them. For instance, an infinite length dielectric cylinder coated with a sub-wavelength conformal array of slotted Jerusalem crosses can be used as a mantle cloak for both TE and TM polarizations of the incident waves6. Moreover, reconfigurable plasmonic waveguides and refractive index sensors are realized using localized surface plasmons of graphene shells around cylindrical objects8. As another application, double graphene shells around the dielectric cylinders are used to design single and double band super-scatters10. By attaining multiple peaks and dips in the scattering response using multiple plasmonic layers, an optical device for simultaneous super-scattering and super-cloaking is proposed11. Moreover, flatten conformal hyperbolic metamaterials are presented using graphene strips around a cylinder under magnetic bias12. The main limitation in all above-mentioned plasmonic devices is that they function only under TE polarized waves13. The goal of this paper is to introduce a dual-polarized graphene-based plasmonic cover for cylindrical wires.By the emergence of two-dimensional (2D) graphene material, the aforementioned applications are implemented with the graphene plasmons due to their exotic properties. For instance, graphene patches are wrapped around a cylindrical object to attain an invisible cloak14. By tailoring the corresponding intrinsic surface resistance, optical-resistive sheets and Salisbury absorbers have been implemented15. Moreover, by properly adjusting the homogenized surface reactance of the arrays made of silver particles wrapped around a dielectric cylinder, the dipole scattering mode has been canceled16. It should be noted that Clausius–Mosotti's effective medium approximation and bi-dimensional model have been used to theoretically demonstrate the functionality of the above meta-surfaces17. As further instances, different distributions and orientations of silver ellipsoidal particles have been considered around a dielectric sphere to implement a plasmonic cloak in the visible spectrum18. Another scheme for cloaking of the cylindrical objects has been proposed by core–shell nanoparticles around the cylinder and it is proved that perturbation of the periodic alignment does not significantly affect the performance of the structure19.Optical covers can be constructed by a periodic arrangement of plasmonic nanoparticles rather than using the metallic sheets with patterned elements which suffer from the practical realizably at high frequencies20. The surface plasmons of a periodic assembly of graphene-coated spherical particles are engineered to design different types of absorbers23. Following the design procedures of the particle coated optical curved structures, in the present research, a graphene-based optical cover is proposed using spherical nanoparticles to enhance the extinction cross-section of an infinite length dielectric cylinder. The potential of the structure for the double Fano resonance generation is also discussed. In particular, in the plasmonic oligomers, the nanoparticles with optimized sizes, shapes, and inter-particle distances are clustered in a way that the corresponding scattering cross-section contains two peaks (bright modes) having a dip (dark mode) between them, as a result of plasmonic hybridization24. Fano resonances can be possibly used for bio-sensing, surface-enhanced Raman scattering (SERS), molecular fluorescence, and solar energy conversion.Recently, the capability of the localized surface plasmons of graphene-wrapped spherical nanoparticles in the design of tunable optical meta-surfaces has been investigated25. In another approach, a wide dipole resonance is overlapped with narrower higher-order modes spatially and spectrally to give raise to plasmonic induced transparency (PIT)26. Moreover, the interface of electric and magnetic dipole modes can easily excite Fano resonances in all-dielectric oligomers27. Also, magnetic plasmon modes constructed by circulating currents in the rings of plasmonic nanoparticles lead to the Fano resonances with low radiation loss29. Another approach is anapole modes which are non-radiating configurations obtained by spectrally overlapped composition of electric and toroidal dipoles30. Symmetry breaking is another efficient approach for the Fano resonance generation that can be achieved in various ways including but not limited to the size, position, and incident angle engineering34. Our proposed device benefits from the incident angle symmetry breaking for the excitation of Fano resonances.In general, the generation of Fano modes can be related to the coupled excitation of various types of resonances. For instance, the destructive interaction between the narrowband and broadband dipolar plasmonic resonances in a gold-graphene sensor results in the plasmonic electromagnetic induced transparency (EIT) with a Fano line-shapeThe paper is organized as follows. In “35. This model is consistent with the well-known Kubo model of local surface conductivity σ in which graphene is characterized by the relaxation time τ and chemical potential μc36. Based on this model, graphene is considered as an infinitely thin spherical shell using the convert to sheet option in the software. Note that because of the large number of carbon atoms in the simulated shells, the curvature of the spherical nanoparticles does not affect the surface conductivity model available for the planar sheets37. To simulate an infinite length cylinder, image theory is used to set the top, and bottom faces of the solution domain with respect to the cylinder axis to perfect electric conductor (PEC) under TM waves and to perfect magnetic conductor (PMC) for TE waves. The details of image theory can be found in classic electromagnetic books38.In this section, various combinations of graphene-based plasmonic nanoparticles are considered around a cylindrical dielectric core. Later, the influence of the array position and configuration on the extinction cross-section of the structure is investigated in detail. All the simulations are carried out with the frequency domain solver of the CST 2017 commercial software using the built-in surface conductivity model of graphene materialrc = 500 nm and core permittivity εc = 2 under plane wave illumination, as shown in Fig. z-axis and the linearly polarized wave is transverse electric (TE) with respect to it. It is also assumed that a single one-dimensional periodic array with the periodicity along the cylinder axis covers the wire. The array is constructed by graphene-coated hollow spherical nanoparticles with the longitudinal periodicity of h = 250 nm. The possible locations of the array with respect to the plane wave are denoted by L (Left), R (Right), U (Up), or D (Down). The initial parameters are as follows: the radii of the spheres are rs = 100 nm, relaxation times of graphene shells are τ = 1.5 ps, and chemical potentials of graphene shells are μc = 0.5 eV.The structure under consideration is a dielectric cylinder with the radius 39 which are calculated using broadband far-field monitors in the simulations. It is observed that the presence of a one-dimensional periodic array of plasmonic nanoparticles enhances the extinction cross-section of the dielectric cylinder, considerably. This effect is due to the excitation of localized surface plasmons of the graphene shells and it depends on the position of the array with respect to the incident wave, which alters the incident angle of the illuminating wave. Therefore, as Fig. Initially, a one-dimensional periodic array is considered in R, L, U, or D locations concerning the incident wave, as illustrated in Fig. 40. These structures, mainly have 2D periodicity which is attained by pattering the oligomer on the planar surface44. The generation of the Fano resonances in our proposed structure benefits from the same strategy using a cylindrical substrate. Symmetry breaking provided by the different incident angles that each particle experience is the main mechanism for the Fano resonance generation. Note that for hexamer, heptamer, and octamer configurations, there are double Fano dips in the extinction cross-section. The meta-molecules can be engineered for the modification of the plasmon line-shape in double spectral positions, simultaneously, which have great potential in dual plasmonic sensor applications45. In this regard, a metallic double Fano-resonant nano-cluster is proposed as a four-wave mixing device46 and our proposed structure presents a novel double Fano resonant structure using graphene plasmons.The impact of increasing the number of one-dimensional periodic arrays around the cylinder on its extinction cross-section is investigated in Fig. 10. The main drawbacks of this method are its extreme sensitivity to the fabrication tolerance and material losses due to its highly resonant behavior. Therefore, very limited realizations of them are available in the low-frequency window11. Our proposed device performs more robustly considering these two aspects, as will be further clarified by the parametric analysis of the performance. Moreover, the invisible cloak design using plasmonic particle covers with different morphologies is a well-established topic19. Interestingly, the extinction cross-section enhancement can be considered as the opposite side of cloaking, where sensing is of interest rather than hiding. This purpose is attained using the same geometry as the previously published ones for the cloaking. The aforementioned cloaks can be fabricated by the self-assembly technique and have a robust performance against fabrication imperfections19. Since the graphene-coated hollow particles can be realized chemically47, the proposed cover can also be fabricated with the self-assembly technique.Note that a well-known approach for the extinction (scattering) cross-section enhancement of the cylindrical objects is exploiting the super-scattering phenomenon23. These resonances are the main mechanism of the performance.To further investigate the performance of the cylindrical wire with a cover constructed by graphene-coated particles, the hexamer configuration is considered as a prototype, and the impact of various material, optical, and geometrical parameters on the extinction cross-section of the wire is investigated. Before proceeding to the next sections, the excited localized surface plasmons on the three peaks of the extinction cross-section of the hexamer Fig. b are ill48. Based on Fig. 50. The proposed sub-wavelength plasmonic cover can be simply reconfigured by changing the bias voltage/doping density of the graphene covers. This fact is illustrated in Fig. c = 0.4–0.7 eV. As the figure shows, by increasing the chemical potential, the resonance frequency experiences a blue shift. This feature is unattainable with metal-based plasmonic oligomers and has only been implemented using a planar assembly of graphene-based disk-shaped particles51.This subsection studies the influence of the core material and graphene optical parameters on the extinction cross-section of the hexamer. As Fig. 52. In order to obviate the need for further modification of the geometry, the ion-gel method is proposed as another approach, which has been widely used for planar structures55. As Fig. ngel = 1.43 covers the particles54. The resulted extinction cross-section and its comparison with the initial design are provided in Fig. 7.Modulating the chemical potential of graphene shells is an important issue in practice. A simple approach for this purpose is to electrically connect the particles using cross-hair geometry or reside them on top of another simply biased structure37. Note that the longitudinal periodicity is fixed in all the previous simulations. Moreover, cylinders with larger core radii exhibit a remarkable Fano dip at high frequencies, as illustrated in Fig. The impact of the geometrical parameters on the modulation of the Fano dips is illustrated in Fig. 57 and this outstanding feature is attained thanks to using a spherically symmetric cell.An outstanding feature of our proposed cylindrical structure is its dual-polarized optical performance. To discuss this feature, in Fig. n = 1.333–1.363 to investigate the sensing performance of the generated Fano resonances. The extinction cross-section of the structure is illustrated in Fig. 58 are included in Fig. −159, our proposed structure performs better in all three plasmonic resonances due to the excitation of higher order modes  and scattering cross-section (RCS) of the hexamer are compared with the bare dielectric wire in Fig. One-dimensional and two-dimensional arrays of polarizable nanoparticles constructed by graphene-coated spherical particles have the capability of enhancing the extinction cross-section of a dielectric cylinder. By adjusting the number of particles, the amount of the extinction cross-section can be tuned. The structure has many degrees of freedom for optical response manipulation and it performs for both TE and TM polarizations. Our proposed structure can be possibly used is in the design of sub-wavelength reconfigurable absorbers for sensing applications.


The use of SERS for real-world bioanalytical applications represents a concrete opportunity, which, however, is being largely delayed by the inadequacy of existing substrates used to collect SERS spectra. In particular, the main bottleneck is their poor usability, as in the case of unsupported noble metal colloidal nanoparticles or because of the need for complex or highly specialized fabrication procedures, especially in view of a large-scale commercial diffusion. In this work, we introduce a graphene paper-supported plasmonic substrate for biodetection as obtained by a simple and rapid aerosol deposition patterning of silver nanowires. This substrate is compatible with the analysis of small (2 μL) analyte drops, providing stable SERS signals at sub-millimolar concentration and a detection limit down to the nanogram level in the case of hemoglobin. The presence of a graphene underlayer assures an even surface distribution of SERS hotspots with improved stability of the SERS signal, the collection of well-resolved and intense SERS spectra, and an ultra-flat and photostable SERS background in comparison with other popular disposable supports. Two-dimensional arrays of plasmonic nanoparticles are gaining consensus within the scientific community in surface-enhanced Raman scattering (SERS) detection ,2 of molIn this work, we introduce a highly responsive SERS substrate specifically designed for biodetection of small sample drops and aimed at overcoming some limitations of the previous system as a result of the use of a simplified fabrication procedure and the introduction of a graphene underlayer to improve the SERS response. On the one hand, the fabrication setup was implemented with a common nebulizer for aerosol therapy, assuring a facile deposition of AgNWs without the need for expensive, time-consuming, or highly specialized procedures. Several studies proposed the direct deposition of sprayed colloids on sample surfaces to probe the surface composition of atmospheric particles , inks, aw 40000), isopropanol (99.5%), myoglobin from horse skeletal muscle (Mb), and hemoglobin (Hb) were purchased from Sigma-Aldrich . Ethylene glycol  was purchased from Carlo Erba . Silver nitrate (AgNO3) and silver chloride (AgCl) were obtained from Cabro S.p.A. .Polyvinylpirrolidone  and UV-Vis spectroscopy . The concentration of the as-obtained AgNWs suspension was finally determined as 1 mg/L by gravimetric determination.AgNWs were synthesized by the polyol method. Briefly, 80 mL of EG was heated and thermally stabilized at 170 °C in a flask. Once the temperature had been stabilized, 112.5 mg of AgCl was added to the flask. Meanwhile, 495 mg of AgNO® 454 polyester film, thickness 125 µm, DuPont, Wilmington, DE, USA ) patterned with 1.5-mm in size round holes, obtained by mechanical punching. SERS substrates were fabricated by aerosol deposition of AgNWs on 50-μm thick graphene-based paper  as achieved by mechanical compression of small stacks of graphene, which in turn was obtained by exfoliation of expanded graphite. Different AgNWs dispersions, as obtained by sequential dilutions in isopropyl alcohol of the original solution, were aerosolized on varying the exposition time within the 1–15 min range by using a common compressor nebulizer emitting micron-sized AgNWs drops for aerosol therapy . A fixed spacing between the nebulizer output and the graphene paper was maintained by using a third-hand support clip. Before deposition, the graphene was wetted in ethanol and then adhered to a PET mask  . The morphology of AgNWs on graphene paper at the nanoscale was analyzed by tapping mode AFM by using a JPK NanoWizard III Sense  scanning probe microscope at a 250–300 kHz drive frequency and a 0.5 Hz scan rate and equipped with single-beam uncoated silicon cantilevers (μMash HQ:NSC15 Cr-Au BS).The as-fabricated substrates were analyzed using a micro-Raman spectrometer  working at 532 nm with 1200 grooves/mm grating, an integration time of 5 s, and laser power at the sample of 150 μW, unless otherwise specified. A 10× objective with 0.25 NA (7 μm waist) was used.The electric field distribution in the near proximity of the AgNWs had been evaluated using a commercial FEM package, the wave optical module of COMSOL multiphysics , and the MNPBEM MATLAB  toolbox for the simulation of metallic nanoparticles, using a boundary element method approach .0| values were averaged over 5 different polarization angles of the incident light. A further configuration including a single AgNW laying on graphene and surmounted by another crossed wire was presented in the We chose to depict the AgNW as a cylinder with hemispherical ends, a radius of 25 nm, a total length 5 μm, and a refractive index for silver taken from Rakić et al. . An eval2 piece of thin graphene paper (G-paper)  . The sys−6 M hemoglobin (Hb) once excited at 532 nm , while lower time values resulted in an uneven distribution a–c. Oncet 532 nm e. We wert 532 nm  down to t 532 nm e. The abWe point out that the as-fabricated substrates allow to support the analysis of minimal quantities (2 µL) of biomolecule solution, which were initially dropped onto an array spot. They were then confined by a high contact angle formed with the surrounding hydrophobic graphene barrier  and fina−1) and G (1584 cm−1) band signals (2), and flexibility , which makes it even more attractive. Our initial aim in choosing graphene as a low-cost support for nanoparticles was dictated by three main considerations: (1) superior hydrophobicity behavior with respect to other popular supports for disposable substrates as cellulose paper, thus enabling analyte confinement and concentration enrichment, as discussed above; (2) low Raman signals in the fingerprint region as compared to other candidate hydrophobic substrates as polytetrafluoroethylene (PTFE) and polydimethylsiloxane (PDMS) and limited to D :The amplification provided by the as-fabricated AgNWs@G-paper was evaluated by calculating the SERS enhancement factor (EF). EF is defined as the ratio between EF value of 4 × 106 was calculated, proving an enhancement ability of the same order of magnitude or higher than that from recently proposed low-cost disposable SERS substrates including those based on AgNWs assemblies [0|)graphene/AgNW/(|E|/|E0|)AgNW/AgNW = 1.2), could be explained by taking into account the higher refractive index of graphene in comparison to other popular supports, which boosts the near field within the gap between metal and graphene, as shown in Ex = 532 nm appears ∼2-fold larger (|E|/|E0| = 44) than that at a PTFE/AgNW (|E|/|E0| = 24) or cellulose/AgNWs (|E|/|E0| = 19) interfaces. As SERS enhancement is proportional to the fourth power of the E-field, an average EF = 3.7 × 106 is obtained in the former case. A large matching between simulated and calculated EF let us hypothesize that the electromagnetic mechanism (EM) largely prevailed in AgNWs@G-paper substrates over a chemical mechanism (CM), as previously reported, as a possible additional effect in the SERS response of graphene-based substrates [An semblies ,44,45. Wsemblies ,46,47. Tbstrates .−1 [Another significant aspect in the choice of a SERS substrate resides in its background contribution to the overall SERS signal and generated at the interface between plasmonic layer and underlying support, which becomes critical especially in the detection of species with reduced SERS response, such as biomolecules. In −1  by the n−1 , generat−5 M to 1 × 10−9 M (1.3 μg to 0.3 ng) (2 > 0.99) between the band area of the 1378 cm−1 mode and the amount of protein was observed , suggesting a high sensitivity of AgNWs@G-paper that overcomes previous detection systems based on unsupported colloidal particles for protein detection including hemoglobin [We finally evaluated the detection sensitivity of the AgNWs@G-paper substrate by decreasing Hb concentration from 1 × 10 0.3 ng) a. A sigmobserved b, which moglobin ,52.SERS substrates proposed so far are often unsuitable for the practical detection of biomolecules due to the lack of simultaneous presence of preferred characteristics, such as low manufacturing costs, disposable characteristics, and simplicity for routine use in turn limiting their use at or near point-of-need settings. In this work, we introduced a highly responsive SERS substrate relying on a simple aerosol deposition of AgNWs on graphene paper. The substrate was specifically designed for biodetection of small sample drops at submicromolar concentration. The proposed fabrication procedure relied on low-cost and facile steps that overcame a number of weak points frequently encountered in substrate preparation and patterning. These included the use of contaminating stabilizers to impart sufficient viscosity to nanoparticle inks as usually required in screen or ink-jet printing, as well as the need for dedicated instrumentation for top-down nanoparticle fabrication. Additionally, the presence of graphene improved the SERS response, conferring superior signal stability, low SERS background, and photostability. Overall, the proposed substrate exhibited high SERS efficiency, reliability, and sensitivity, as well as easy handling and usage aspects, meeting many of the requirements for effective and successful SERS detection of biomolecules. Future experiments on AgNWs@G-paper substrates will be aimed at exploring the SERS sensing of larger biological entities as cells for rapid and tag-free screening of tumor diseases  or to monitor light treatments  for therapeutic applications.