S375) located in the low-complexity domain, and used it to investigate the presence of S375 phosphorylation in a series (n = 44) of FTLD–TDP and ALS–TDP cases. Immunoblot analysis demonstrated phosphorylation of S375 to be a consistent feature of pathological TDP-43 species, including full-length and C-terminal fragments, in all FTLD–TDP subtypes examined (A–C) and in ALS–TDP. Of particular interest, however, detailed immunohistochemical analysis showed striking differences in the immunoreactivity profile of inclusions with the pTDP-43S375 antiserum among pathological subtypes. TDP-43 pathology of ALS–TDP, FTLD–TDP type B (including cases with the C9orf72 mutation), and FTLD–TDP type C all showed strong pTDP-43S375 immunoreactivity that was similar in amount and morphology to that seen with an antibody against TDP-43 phosphorylated at S409/410 used as the gold standard. In stark contrast, TDP-43 pathology in sporadic and genetic forms of FTLD–TDP type A (including cases with GRN and C9orf72 mutations) was found to be almost completely negative by pTDP-43S375 immunohistochemistry. These data suggest a subtype-specific, conformation-dependent binding of pTDP-43S375 antiserum to TDP-43 aggregates, consistent with the idea of distinct structural TDP-43 conformers  as the molecular basis for the phenotypic diversity in TDP-43 proteinopathies.Aggregation of hyperphosphorylated TDP-43 is the hallmark pathological feature of the most common molecular form of frontotemporal lobar degeneration (FTLD–TDP) and in the vast majority of cases with amyotrophic lateral sclerosis (ALS–TDP). However, most of the specific phosphorylation sites remain to be determined, and their relevance regarding pathogenicity and clinical and pathological phenotypic diversity in FTLD–TDP and ALS–TDP remains to be identified. Here, we generated a novel antibody raised against TDP-43 phosphorylated at serine 375 (pTDP-43The online version of this article (10.1007/s00401-020-02207-w) contains supplementary material, which is available to authorized users. FTLALS–TDP) . FTLD–TD defects . ALS–TDPDespite the common feature of abnormal TDP-43 accumulation, a significant pathological heterogeneity is observed among TDP-43 proteinopathies with respect to the pattern of anatomical distribution and morphology of inclusions, allowing recognition of up to five distinct pathological subtypes of FTLD–TDP –14, 16. The molecular basis explaining the clinical and pathological phenotypic variability of TDP-43 proteinopathies is so far not understood. In analogy to prion diseases and other neurodegenerative diseases such as tau- and alpha-synucleinopathies, a popular hypothesis, supported by an increasing body of evidence, is that the heterogeneity is a reflection of biochemical differences in pathological TDP-43 species that propagate in a prion-like manner with different conformers being associated with distinct seeding/toxicity activities  , 23, 29.TARDBP) either introduce or disrupt potential serine/threonine phosphorylation sites or introduce phosphomimic residues (glutamate/aspartate) [Since its initial discovery as the disease protein in FTLD–TDP and ALS–TDP, aberrant phosphorylation of TDP-43 has been recognized as one major PTM of pathological TDP-43 , 17. Thepartate)  stronglyS409/410 antibodies have facilitated the detection of TDP-43 pathology and are considered to be the most sensitive method for the immunohistological evaluation of TDP-43 pathology. However, to further dissect the potential role of phosphorylation in disease pathogenesis and the biochemical properties and potential differences of aggregated TDP-43 among TDP-43 proteinopathies, more detailed insights into additional TDP-43 phosphorylation sites are essential.TDP-43 has 41 serine, 15 threonine, and 8 tyrosine residues that might act as potential phosphorylation sites. Mass spectrometry analyses of recombinant TDP-43 in vitro phosphorylated with casein kinase 1 and of aggregated TDP-43 isolated from ALS–TDP have revealed several phosphorylated residues . HoweverTARDBP in an ALS patient, the consequences of serine 375 phosphorylation of TDP-43 have recently been analyzed, with in vitro studies demonstrating a potential role for S375 phosphorylation in regulating the nuclear-cytoplasmic distribution of TDP-43 [S375), and employed it for biochemical and detailed neuropathological analysis on a cohort of 44 sporadic and genetic ALS–TDP and FTLD–TDP cases. Phosphorylation at S375 was a consistent finding in all TDP-43 proteinopathies by immunoblot analysis. However, and most excitingly, the results of pTDP-43S375 immunohistochemistry (IHC) distinguished FTLD–TDP with type A pathology from other types of FTLD–TDP and ALS–TDP, most likely due to conformational differences of TDP-43 aggregates; thereby providing further evidence for the existence of distinct subtype-specific TDP-43 strains.Motivated by genetic findings on a p.S375G variant in f TDP-43 . To gainThe rabbit polyclonal antibody against TDP-43 phosphorylated at serine 375 was produced by Biosense/Eurogentec using their custom polyclonal antibody cross-affinity purification service. Briefly, rabbits were immunized with a phosphopeptide corresponding to amino acid 368–379 of human TDP-43: ac-EPNQAFGS(p)GNNS-C-CONH2. Phospho-specific antibodies were purified using affinity chromatography with modified and unmodified peptides. The initial validation for phospho-specificity of cross-affinity purified antibodies was performed by the company by qualitative indirect enzyme-linked immunosorbent assay (ELISA) .Human post-mortem tissues were obtained from the brain banks affiliated with the University of Tübingen, the DZNE Tübingen, and the University of British Columbia. Consent for autopsy was obtained from probands or their legal representative in accordance with local institutional review boards.C9orf72 repeat expansion, and 34 cases with clinical FTD (± ALS) including 15 with FTLD–TDP type A , 11 FTLD–TDP type B (including six with the C9orf72 repeat expansion), and 8 FTLD–TDP type C cases. Control cases included Alzheimer’s disease (n = 3), progressive supranuclear palsy (n = 2), Lewy body disease (n = 2), and neurologically healthy controls (n = 1).A total of 44 cases were selected covering a broad spectrum of clinical and genetic forms of primary TDP-43 proteinopathies Table . These iS375 was first established on two selected cases and regions with robust TDP-43 pathology  by testing different dilutions of the antiserum and antigen retrievals (boiling in EDTA or citrate-based buffers). For comparison, adjacent sections were stained with a phosphorylation-independent rabbit polyclonal TDP-43 antibody (ProteintechGroup).IHC was performed on 2–5 μm-thick sections of formalin fixed, paraffin-embedded (FFPE) tissue using the Ventana BenchMark XT automated staining system with the optiVIEW DAB detection kit (Roche). IHC for pTDP-43S375 IHC protocol (standard IHC) with antiserum dilution 1:400, and boiling in CC1 buffer  for 32 min as pretreatment. Adjacent sections were stained with a rat monoclonal pTDP-43S409/410 antibody (clone 1D3) [From all ALS–TDP and FTLD–TDP cases selected for the study, sections of different CNS regions were then stained with the established pTDP-43one 1D3) . The anaS375 antiserum, adjacent sections of selected cases were treated with 98% formic acid for 5 min before standard IHC.To investigate for conformation-depended binding of pTDP-43S409/410 antibody and the pTDP-43S375 antiserum. To investigate possible cross-reactivity of pTDP-43S375 antiserum with a subset of neurofibrillary tangles, double-label immunofluorescence was performed on AD cases using the pTDP-43S375 antiserum and a mouse monoclonal anti-pS202/205 tau antibody . Secondary antibodies were Alexa Fluor 488 conjugated anti-rabbit and Alexa Fluor 594 conjugated anti-mouse and anti-rat . 4′-6-diamidino-2-phenylindol (DAPI) was used for nuclear counterstaining.Double-label immunofluorescence was performed on select sections and cases using the pTDP-43S409/410 and pTDP-43S375 IHC included neuronal cytoplasmic inclusions (NCI), neuronal intranuclear inclusions (NII), dystrophic neurites (DN), combined threads and dot-like profiles (ThD), white matter threads, and glial cytoplasmic inclusions (GCI). Each type of pathology was graded semi-quantitatively: 0, absent; 1, mild/occasional (easy to find but not present in every medium power field); 2, moderate (at least a few in most fields); 3, severe/abundant . In sections of hypoglossal nucleus and spinal cord, a score of 0.5 was assigned when a single LMN NCI was identified in an entire tissue section. In addition, a cumulative grade was assigned for the total amount of TDP-43-immunoreactive (-ir) pathology in each anatomical region for the respective antibodies. Grading of the pathology demonstrated by pTDP-43S409/410 and pTDP-43S375 IHC was performed blinded to the clinical and genetic information and the assigned FTLD–TDP subtype. Grading of the pathology demonstrated by pTDP-43S375 IHC was performed blinded to the clinical and genetic information and the assigned FTLD–TDP subtype based on pTDP-43S409/410 stainings.The anatomical regions analyzed were precentral gyrus and LMN regions for ALS–TDP cases and hippocampus, temporal cortex, and LMN regions for FTLD–TDP cases. The different inclusion types evaluated for pTDP-43The sequential extraction of proteins with buffers of increasing stringency from fresh-frozen post-mortem frontal cortex from FTLD–TDP cases and controls was performed as described previously . BrieflyEscherichia coli BL21 Star DE3 (Invitrogen). The expressed N-terminally His-tagged TDP-43 was purified under denaturing conditions using nickel beads (Qiagen) and dialyzed against NMR buffer . For in vitro phosphorylation, 2 µg of recombinant TDP-43 were incubated with 6 U Casein Kinase 1 δ, active  at 30 °C for 40 min.Human TDP-43 cDNA was subcloned into pRSET expression vector and transformed into S375 antiserum, clone 1D3 anti-pTDP-43S409/410, mouse anti-panTDP-43 , and rabbit anti-panTDP-43 .Proteins were separated by SDS–polyacrylamide gel electrophoresis (SDS-PAGE). Immunoblot analysis was performed using fluorescence detection and the Odyssey® CLx Imaging System (LI-COR Biosciences). Proteins were transferred to nitrocellulose membranes and blocked with Odyssey blocking buffer (LI-COR Biosciences). Antibodies were detected with CF680 or CF770 conjugated anti-rat, anti-rabbit, and anti-mouse IgG (Biotium). Primary antibodies for immunoblot analysis included the rabbit pTDP-43S375 and pTDPS409/410 IHC. Significance level was set at p < 0.05.Statistical analysis was performed with the GraphPadPrism software (version 7.01 for Windows). Wilcoxon test was used to assess for differences of pathology grades between pTDPS375 antiserum for phosphorylated TDP-43 was validated by immunoblot analysis of recombinant TDP-43 before and after in vitro phosphorylation with CK1, a kinase known to phosphorylate S375 residue [S375 antiserum, strong labeling was seen for the in vitro phosphorylated recombinant TDP-43  and the dentate gyrus of an FTLD–TDP type B case . Thus, the novel antiserum is a highly sensitive and specific tool for the further investigation of the S375 phosphorylation status in TDP-43 proteinopathies.Specificity of affinity purified pTDP-43 residue . While uS375 immunoreactivity in the spectrum of TDP-43 pathology in more detail, a cohort of 44 ALS–TDP and FTLD–TDP cases, including FTLD–TDP types A-C and those with GRN and C9orf72 mutations, was examined. Semi-quantitative assessment of distinct types of pTDP-43S375-ir inclusions in selected CNS regions was compared to the amount of TDP-43 pathology present with the pTDPS409/410 antibody as gold standard. As described in detail below, the whole spectrum of TDP-43-ir inclusions present in ALS–TDP, FTLD–TDP type B, and type C  was consistently and strongly labeled by pTDP-43S375 antiserum, whereas the vast majority of inclusions in all anatomical regions examined in FTLD–TDP type A cases  was not detected by pTDP-43S375 antiserum  including those with C9orf72 mutations. Specifically, ALS–TDP showed pTDP-43S375-ir diffuse and compact NCI as well as DN, ThD, and GCI in the spinal cord and affected cortical regions such as the precentral gyrus, hippocampus, and basal ganglia   treatment for 5 min before standard IHC. Notably, only under these harsh denaturing conditions, TDP-43 pathology became pTDP-43S375 in TDP-43 proteinopathies, we performed immunoblot analysis using protein lysates sequentially extracted from frozen brain tissues of cases and controls. The disease-specific TDP-43 signature was observed with pTDP-43S375 antiserum in the urea fraction with specific labeling of abnormal TDP-43 species including hyperphosphorylated full-length TDP-43 (band ~ 45 kDa), C-terminal fragments (bands ~ 20-25 kDa), and a high molecular smear. No band ~ 43 kDa corresponding to physiological TDP-43 was detectable in any fraction in FTLD–TDP or in controls. The biochemical signature observed for pTDP-43S375 resembles that seen with other phosphorylation-specific antibodies, i.e., pTDP-43S409/410 . In contrast, almost all TDP-43-ir inclusions  in sporadic and genetic FTLD–TDP type A cases, including cases with GRN and C9orf72 mutations, were completely negative. Notably, the immunoreactivity profile of TDP-43-ir pathology for pTDP-43S375 was closely associated and determined by the morphological pattern of TDP-43, regardless of the underlying cause.Phosphorylation-specific antibodies with those against pS409/410 as the most widely used are the most sensitive and specific markers for the detection of the complete spectrum of TDP-43 pathology by IHC , 28. WheS375 immunoreactivity by IHC in FTLD–TDP type A is simply a result of absent phosphorylation of TDP-43 at S375 in type A since our biochemical studies demonstrated the consistent presence of pTDP-43S375 in all cases including FTLD–TDP type A. Second, it is known that various factors such as fixation time, fixative, and post-mortem delay might influence the detectability of antigens in tissues. However, this possibility also seems highly unlikely, since these parameters did not differ between FTLD–TDP type A, and in other cases, no correlation was observed between the intensity of immunoreactivity and these factors in ALS–TDP and FTLD–TDP type B/C, and moreover, the same distinction of type A pathology from TDP-43 pathology in other subgroups was seen in tissues sampled at two different brain banks.Several possibilities can be excluded as explanation for the discordant staining properties of TDP-43-ir inclusions between FTLD–TDP type A and others. First, we can exclude that the lack of pTDP-43S375 antibody is buried and not accessible. The interpretation that the novel antibody recognizes TDP-43 in a conformational-dependent and type-specific manner is corroborated by the fact that FTLD–TDP type A inclusions became immunoreactive when FFPE sections were subjected to very harsh denaturing conditions by formic acid treatment for 5 min before IHC. Thus, our data provide strong evidence in support of the popular idea of biochemically distinct TDP-43 species  as the molecular basis of disease heterogeneity among TDP-43 proteinopathies. In this context, it is noteworthy that the postulated distinct biochemical property/conformation of aggregated TDP-43 recognized by our novel antibody is conserved throughout different affected CNS regions, cell types , and inclusion types  in a given subtype, indicating a common mechanism for the formation of all inclusions in an individual, regardless of the cell type and intracellular compartment, consistent with a prion-like propagation of TDP-43 aggregation in a self-templating manner.In fact, our data rather imply that TDP-43 species in type A inclusions have a different structural assembly compared to aggregates in other FTLD–TDP types and ALS–TDP in which the epitope recognized by the pTDP-43S375 to be determined by the morphological pattern of TDP-43, regardless of the underlying gene defect in genetic cases , suggests an involvement of additional factors. Our data are in line with the previous findings by Laferriere et al. in which biochemical/biophysical properties of TDP-43 aggregates were also reported to be better predicted by the pathological subtype than by the genetic cause [Evidence for biochemical heterogeneity of aggregated TDP-43 has been reported previously by description of different patterns of C-terminal fragments, protease-resistant cores, and distinct biophysical properties of aggregated TDP-43 among FTLD–TDP subtypes , 11, 29.VCP mutations and type E) and other genetically based ALS/FTLD–TDP cases  exist and it will be interesting to learn in the future how they will be classified based on pTDP-43S375 immunoreactivity [C9orf72 mutation carriers [The aim of the current study was to investigate pathological subtype-specific differences; therefore, we focused on the most common primary TDP-43 proteinopathies. However, other rarer FTLD–TDP subtypes  and human brain homogenates and lack of physiological nuclear staining in non-inclusion bearing cells by IHC suggest that phosphorylation of S375 is an abnormal event. However, we cannot rule out that phosphorylation at S375 might occur physiologically to a minor extent or under specific stress conditions. Cell culture studies with phosphomimetic TDP-43 mutations suggest that phosphorylation of S375 might modulate nuclear-cytoplasmic shuttling leading to a higher tendency to accumulate in the cytoplasm particularly under cellular stress conditions . A potenS375 antibody will not only be a valuable tool to further address the role of pS375 in disease pathogenesis, but also as an aid in subtyping of FTLD–TDP, particularly for difficult to classify cases. However, we recognize that the latter requires the validation of our discriminatory IHC approach by other research groups in their FTLD–TDP cohorts.In summary, we describe phosphorylation of TDP-43 at S375 as a consistent feature in sporadic and genetic ALS-TPD and FTLD–TDP and demonstrate that standard staining protocols using this novel antibody allows the distinction of TDP-43 pathology of FTLD–TDP type A from other FTLD–TDP types and ALS–TDP, most likely in a conformation-dependent manner, compatible with the concept of alternate TDP-43 conformers as the biochemical basis for phenotypic diversity. The newly generated pTDP-43Supplementary file 1 (PDF 605 kb)Below is the link to the electronic supplementary material.


The characterization showed that the surface morphology of five graphene-like magnetic sawdust biochars prepared with different preparation conditions were quite different. The specific surface area and pore structure increased with the increment of K2FeO4 addition. The results have shown that graphene-like magnetic sawdust biochar (1:1/900 °C) had the best removal on E2. The experimental results indicated that pseudo-first-order kinetic model and the Langmuir model could describe the adsorption process well, in which the equilibrium adsorption capacity  of 1:1/900 °C were 59.18 mg·g−1 obtained from pseudo-first-order kinetic model and the maximum adsorption capacity (qmax) of 1:1/900 °C were 133.45 mg·g−1 obtained from Langmuir model at 298K. At the same time, lower temperatures, the presence of humic acid (HA), and the presence of NaCl could be regulated to change the adsorption reaction in order to remove E2. Adsorption capacity was decreased with the increase of solution pH because pH value not only changed the surface charge of graphene-like magnetic sawdust biochar, but also affected the E2 in the water. The possible adsorption mechanism for E2 adsorption on graphene-like magnetic sawdust biochar was multifaceted, involving chemical adsorption and physical absorption, such as H-bonding, π-π interactions, micropore filling effects, and electrostatic interaction. To sum up, graphene-like magnetic sawdust biochar was found to be a promising absorbent for E2 removal from water.The occurrence of environmental endocrine disrupting chemicals (EDCs) in aquatic environments has caused extensive concern. Graphene-like magnetic sawdust biochar was synthesized using potassium ferrate (K AgriMohammad ,17. Furt2FeO4 as an activator, which possessed a positive electrochemical performance as electrode materials for supercapacitors. Zhou et al. stated that K2FeO4 was a promising reagent because it could not only improve the pore structure of the material, but also generate new active sites, such as FeO42− and Fe2O3 [2FeO4 could load the iron oxide compound on the surface of the material, which provided convenience for the magnetic ionization technology, and at the same time overcame the disadvantages of the difficulty of collecting biochar after adsorption, making the recovery of biochar a reality.There are several methods for the modification of the biochar surface, including physical or chemical activation, steam activation, and coating for the contaminants’ removal from wastewater . Among tnd Fe2O3 . In addi2FeO4 could fulfil the synchronous carbonization and graphitization for converting biomass to porous graphitic carbon biochar, which have been used as advanced electrode materials [2FeO4 to produce graphitic biomass carbon (GBC) by the simultaneous carbonization and graphitization of sawdust biochar. K2FeO4 was utilized as graphitization catalyst (Fe), activating agent (KOH) and magnetizing agent (Fe) [Sawdust is an abundant biomass waste resource everywhere and sawdust biochar has great application potential in the removal of water environmental pollutants ,25. Prepaterials ,26, cataaterials , and adsaterials . Based oent (Fe) . The syn2FeO4 dosage; (2) to analyze the surface characteristics of the adsorbent with multiple characterization methods, such as scanning electron microscope (SEM), transmission electron microscope (TEM), X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS); (3) to explore the adsorption performance of graphene-like sawdust biochar on E2; (4) to discuss the possible adsorption mechanism of graphene-like sawdust biochar removal E2 from water.The main research objectives of this study are: (1) to produce graphene-like sawdust biochar with different pyrolysis temperatures and different K18H24O2) was purchased from Sigma–Aldrich. K2FeO4, HA, and NaCl were obtained from Shanghai chemical corporation. Other chemicals including sodium hydroxide (NaOH), hydrochloric acid (HCl) and pH buffer solution , used to adjust the solution pH, were purchased from Shanghai Chemical Corp. Deionized water was obtained from Millipore Milli-Q water purification system. All reagents used were of analytical grade or higher and prepared with ultra-pure water.E2  and annealed temperature  were prepared to explore the excellent preparation conditions. 2.0 g BC was dispersed in aqueous 0.1 M K2FeO4 solution whose volume was 20 mL, 50 mL, and 100 mL, respectively with continuous stirring for 12 h, and then dried at 65 °C to obtain. The mass ratios of BC and K2FeO4 were 1:0.2, 1:0.5 and 1:1 respectively. Subsequently, the solid mixture  were heat-treated in a tube furnace at 900 °C for 2 h under a nitrogen atmosphere with a heating rate of 7 °C min−1. The mass ratio of 1:1 was heat-treated at a constant annealed temperature  for 2 h under a nitrogen atmosphere with a heating rate of 7 °C min−1. The obtained samples were washed with deionized water, followed by drying at 65 °C. These resultant samples were notated as 1:0.2/900 °C, 1:0.5/900 °C, 1:1/900 °C, 1:1/800 °C, and 1:1/1000 °C (mass ratio (BC: K2FeO4)/ annealed temperature). The mass ratios (BC: K2FeO4) and annealed temperature are also in Several samples with different mass ratios . The graphitic structure was analyzed by high-resolution transmission electron microscopy . The surface chemical composition was determined by X-ray photoelectron spectroscopy . Raman spectrometer was performed on a Raman Microscope  with YAG solid-state laser under wavelength of 532 nm. An X-Ray Diffractometer  was used to study the crystallinity. The magnetic properties of samples were examined using a vibratory probe sample magnetometer . The specific surface areas were determined using the Brunauer–Emmett–Teller .2FeO4) and annealed temperature on adsorbents properties were studied. Subsequently, the sample 1:1/900 °C was used for batch adsorption experiments based on the superbly adsorptive properties. In the beginning, three mass ratios  and different annealed temperature  have been used for adsorption experiments. The experiments were performed in a 100 mL conical flask with 50 mL aqueous solution containing the fixed concentration of E2 and adsorbents . More details on the selection of E2 and adsorbents concentrations were presented in The effects of mass ratios  were used to investigate the effect of salinity on adsorption behavior [−1) [Batch adsorption experiments included kinetics adsorption experiments, isothermal adsorption experiments, pH effect, ionic strength effect, and OM effect on E2 adsorption which were conducted at 25 °C with a shaking speed of 160 rpm for 24 h in the vibrational chamber excluding kinetic adsorption experiments . The soll flasks . The pH ior [−1) . The desx/Em = 280 nm/310 nm [E2 concentration was determined using an F-4500 fluorescence spectrophotometer . The emission source is a 450 w xenon lamp which collected the emission matrix spectra of 5 nm in the excitation range of 200–400 nm and 300–500 nm, respectively. Fluorescence intensity of E2 was measured at Em/310 nm . The dat2FeO4, the surface of GBC became more rough and uneven  [2 and K2O could further react with carbon over 700 °C to K. During physical and chemical activations, the carbon lattices expanded irreversibly and resulted in many micropores. Since these chemical reactions occurred at over 700 °C, the specific surface area was mainly related to the addition ratio of the K2FeO4 [2FeO4 made the microporous structure more powerful.Surface morphology and microporous structure of carbonaceous materials were characterized by SEM. The surface of the BC was relatively smooth a. With td uneven b–d. Moreon sites . During  700 °C) . Additioe K2FeO4 . More mi2·g−1. This indicated high BET surface area and total pore volume of 1:1/900 °C in the 2·g−1 and a tiny total pore volume of 0.020 cc/g. Nevertheless, the BET surface area was corresponding increase with K2FeO4 activation of the materials (1:0.2/900 °C and 1:0.5/900 °C). Definitively, the sample 1:1/900 °C demonstrated a powerfully abundant porous structure. The large accessible surface area and its suitable pore size distribution are beneficial for adsorption [The N2 adsorption-desorption isotherms and mesoporous size distribution of carbonaceous materials were presented in sorption .2FeO4 and annealed with high temperature. The graphitization was testified by Raman spectra  is generally used to reflect graphitization of samples. The ID/IG value of BC, 1:0.2/900 °C, 1:0.5/900 °C, 1:1/900 °C, 1:1/800 °C and 1:1/1000 °C were 0.81, 1.01, 1.08, 1.02, 0.98, and 1.01, respectively. The increasing of the ID/IG value indicated that the graphitization of the samples has been shown via the K2FeO4 excitation and high-temperature calcination. The 2D band of the samples 1:1/900 °C and 1:1/1000 °C also verified the formation of few-layered graphene rather than single-layer graphene [2FeO4 and annealed with high temperature. Moreover, the higher graphitization temperature was also beneficial to graphitization [The crystallization and graphitization of the samples were observed by XRD and Raman. The XRD patterns were shown in  spectra b. All saon atoms . The 2D graphene ,34,35. Ttization .The TEM images of BC and 1:1/900 °C have been compared in order to further understand the properties of the materials before and after modification. In 2FeO4. The XPS peak deconvolution for C1s, O1s, and Fe2p spectra of samples was shown in 3+ species on the surface of 1:1/900 °C [0 and Fe2+, respectively [0 was only 9.2%, while the proportion of Fe3+ and Fe2+ was 79.9% and 10.9%, respectively. The 1:1/900 °C become magnetic biochar with the presence of Fe species and produced more oxygen-containing functional groups accompanied by the oxidation of Fe3+ species. From the above characterization results, it can be confirmed that the 1:1/900 °C sample was successfully synthesized by one-step process [−1 which suggested that the adsorbent was enough to achieve solid-liquid separation by a permanent magnet. These results reflected that biochar after modification had excellent magnetic properties and could be easily separated and recycled.To get an insight into the surface chemical composition, the full survey scan XPS a spectra1/900 °C . While tectively . The pro process . SupplemAs presented in R2)  , E2 adsoace area . Moreove process ,41. In t , E2 adsIn −1, while the maximum adsorption capacity of BC was 107.78 mg·g−1 at 298 K. The results indicated that low temperature was more conducive to the removal of E2 by graphene-like sawdust biochar. The relative parameters for two models were summarized in R2), which suggested that the adsorption of E2 by graphene-like sawdust biochar occurred on a homogeneous surface were a monolayer adsorption process [n of the Freundlich adsorption isotherm model  [Kf value related to the adsorption capacity of the Freundlich adsorption isotherm model  [However, the adsorption capacity of 1:1/900 °C was obviously better than that of BC. In the  process ,39,42. Rfficult) , we coulsorbent) , it can The pH value of the solution was crucial to the adsorption process, which not only changed the surface charge of biochar, but also affected the E2 in the water. In Furthermore, to investigate the effect of ionic strength on adsorption of E2 on graphene-like sawdust biochar, different concentrations of NaCl (0.0 M–0.6 M) were added to the E2 solution, owing to the popular existence of this ionic compound in wastewater. In Natural organic matters (NOMs) have been regarded as an important factor affecting the adsorption process since they widely existed in the natural environment . They miAdsorbent recovery is an important consideration as it relates to the operating cost and the feasibility of its practical application. In Different characterizations and experiments were carried out to explore the possible E2 adsorption mechanisms onto 1:1/900 °C. In this work, the graphene-like magnetic structure of 1:1/900 °C have been observed by characterization. On the one hand, the sample 1:1/900 °C had a high surface area, abundant micropores, and high crystallinity properties. These surface structures were beneficial to E2 adsorption, while similar conclusions were also reported in ,43,48. M(1)2FeO4 was 1:1 and the second pyrolysis temperature was 900 °C, the graphene-like magnetic carbon sawdust biochar had the best adsorption capacity on E2.During the carbonization and graphitization process, when the mass ratio of BC to K(2)2FeO4 dosages and pyrolysis temperatures had an important impact on the morphology and structure of the graphene-like magnetic carbon sawdust. The surface area of graphene-like magnetic sawdust biochar was affected by the K2FeO4 dosage. The more addition, the larger the specific surface area. However, under the condition that BC and K2FeO4 was 1:1, the surface area of graphene-like magnetic sawdust biochar showed a positively obvious result.Different K(3)−1 obtained from Langmuir model. Adsorption kinetics and adsorption isotherm indicated that the E2 adsorption by graphene-like sawdust biochar occurred on a homogeneous surface were a monolayer adsorption process and controlled by multiple processes.The maximum adsorption capacity at 298 K was 133.45 mg·g(4)The pH, NaCl, and HA were crucial to the adsorption process because the adsorption capacity was affected by physical-chemical properties caused by the change of surface charge and the salting-out effect.(5)The possible adsorption mechanism for E2 adsorption on graphene-like magnetic sawdust biochar was multifaceted, involving chemical adsorption and physical absorption, such as H-bonding, π-π interactions, micropore filling effects, and electrostatic interaction.Based on the above conclusion, graphene-like magnetic sawdust biochar was found to be a promising absorbent for E2 removal from water as well as wastewater treatment, and the preparation method of graphene-like magnetic sawdust biochar was a green and feasible process. However, as a green technology, the economic viability might be a consideration and there should be some industrial viability of that process/product to be considered strictly.


We balance the interplay of the absorption, electrical and thermal conductivity of graphene via the device geometry. This approach yields remarkable device performance featuring room temperature high sensitivity  photodetectors is to funnel light into a small nanoscale active area and efficiently convert it into an electrical signal. Here, the authors couple a plasmonic antenna to hyperbolic phonon-polaritons in hexagonal-BN to highly concentrate mid-IR light into a graphene pn-junction. In fact, novel nano-optoelectronic platforms can be attained by merging HPPs functionalities with other 2D materials-based devices, such as graphene photodetectors governed by the photothermoelectric (PTE) effect. This mechanism generates a photoresponse in graphene pn-junctions19 driven by a temperature gradient and Fermi level asymmetry across the channel. Nevertheless, one of the limitations of these detectors is the low light absorption of graphene, especially for mid-IR frequencies where the photon energy becomes comparable to the typical doping level of graphene reaching the Pauli blocking regime21. This is further exacerbated by the small photoactive area of graphene pn-junctions22, limited by the cooling length of the hot carriers (0.5–1 μm)23. These limitations can be overcome by exciting HPPs and focusing them towards the photoactive area and consequently absorbing them in graphene. However, efficient exploitation of HPPs for mid-IR photodetection still remains unexplored25. In this work, we embed hBN and graphene within metallic antennas in order to couple their plasmonic interactions with HPPs and achieve highly concentrated mid-IR light on a graphene pn-junction for sensitive and fast mid-IR photodetection.Hyperbolic phonon-polaritons (HPPs) are hybridized modes of ionic oscillations and light present in polar dielectric materials, such as hexagonal-BN (hBN)λ ≈  5–7 μm  range 26 and heat diffusion away from the junction area. As a result, a symmetric electronic temperature profile Te(x) is produced in the graphene junction24, giving rise to a thermoelectric voltage VPTE ∝ S(x) ∇Te(x), where x runs along the graphene channel and S(x) represents the Seebeck coefficient which is tunable by the gates. Since  ∇Te(x) is antisymmetric, an antisymmetric S(x) is also needed to maximize the net PTE response, which is achieved by applying opposite voltages to the two gates  heat capacitance mismatch between graphene electrons and lattice, the HPP absorption does not amount to any meaningful temperature rise and thus does not contribute to the device PTE response.The absorption process in the graphene is mediated mostly by interband transitions, which mainly occur in the regions within the gap of the gates where the graphene doping is sufficiently small to avoid Pauli blocking  as shown in Fig. μW/μm2. Next, we investigate the photoresponse as a function of the two gate voltages (VL and VR), shown in Fig. 28. The highest values of photocurrent occur at pn or np configuration, specifically at VL = 1.6 V (170 meV) and VR = −0.82 V (−130 meV), which are relatively low doping levels. We note that when applying a voltage bias in the graphene channel, the photocurrent remains constant while the source-drain current increases linearly with bias  and a second peak (3.5 mA/W) around 8 μm. We also plot the simulated responsivity that is extracted from the multiphysics simulations, which considers the exact geometry of the photodetector and the whole device photoresponse  across the channel in the x direction  = Absdevice/Absair, which is the ratio between the graphene absorption incorporating all the elements of the device  to that of suspended graphene as a function of λ and the position vector r. G and responsivity are proportionally related via the electronic temperature gradient as shown in Supplementary Fig. We first identify the behavior of the resonant mechanisms, in terms of field intensity enhancement and spatial localization by studying the absorption enhancement in graphene . The value of G peaks around 6.8 μm due to the hybridization of the hBN HPPs with the antenna LSPR and to the constructive interference of the propagating HPPs occurring at x ~ ±100  nm. In fact, the different spatial patterns of G arise from the wavelength dependence of the HPP propagation angle in hBN following the equation 29). For longer wavelengths, we find a negligible G between 7 and 7.3 μm that corresponds to the hBN transverse optical (TO) phonon. We observe that the highest G values are only found for the spatially confined region (from x ~ −100 to 100 nm) where the antenna and gates overlap, which is designed to coincide with the graphene pn-junction  hybridization with the gate LSPR as presented in Supplementary Figs. For TE-polarization Fig. b, we finG, we simulate the system without the contribution of the HPPs using wavelength-independent refractive index values for the hBN  due to the complex shape of the gates and their interactions with the source–drain contacts  such that its LSPR peak fully overlaps with the hBN HPPs peak, thus obtaining 65 mA/W.To evaluate the coupling between the bow-tie antenna LSPR and the hBN HPPs, we study the responsivity as a function of the antenna length for TM-polarization as shown in Fig. λ = 6.5 μm with TE-polarization on the responsivity and NEP  by varying the width and length of the gate tip and graphene, while keeping the source-drain distance and width fixed as indicated in Fig. μm, which is larger than the experimental one (855 nm), pointing to future design and performance improvements  detector. We plot in Fig. 33), assuming the graphene thermal noise as the dominating noise source35. We emphasize that the zero-bias operation leads to low noise levels and a very low power consumption, which is given by the voltage applied to the gates. Furthermore, our design allows sensitive detection in different polarizations  of infrared imaging systems36.The sensitivity of the detector is best expressed in terms of external responsivity, which the maximum measured value is 27 mA/W . Additional tuning and wavelength sensitivity can be realized by controlling the hyperbolic material’s thickness42 or shape45.The device concept introduced in this work can be extended to detectors for other wavelengths or more specific functionalities such as hyperspectral imaging and spectroscopy. Our approach can also be combined with HPPs in other regions of the mid-IR and long-wave infrared range such as MoOμm, a total width of 2 μm and a narrow width region (tip) of 500 nm on a Si/SiO2 substrate using electron beam lithography (EBL) followed by evaporation of titanium (2 nm)/gold (30 nm). The gap between the gates is 155 nm. Afterwards, we transfer a hBN/graphene/hBN stack onto the metallic gates. We cleave and exfoliate the top and bottom hBN and the graphene onto freshly cleaned Si/SiO2 substrates, stack them following the Van der Waals assembly technique47 and release onto the gates. We then use EBL with a PMMA 950 K resist film to pattern source and drain electrodes and expose the device to a plasma of CHF3/O2 gases to partially etch the Van der Waals stack. Subsequently, we deposit side contacts of chromium (5 nm)/gold (80 nm) and lift off in acetone as described in ref. 46. We then etch the hBN-encapsulated graphene into an H-shape using a CHF3/O2 plasma and deposit 17 nm of Al2O3 using atomic layer deposition (ALD). Finally we pattern the bow-tie antenna of 2.7 μm total length (L) and with a small gap of 200 nm between its branches with EBL and deposit titanium (2 nm)/gold (80 nm). We point out that the bow-tie antenna and gate dimensions were selected based on preliminary optical simulations based on a simplified device, ignoring bow-tie antenna interactions with gates and metal electrodes, resulting in the non-optimal performance as evident from Fig. VL and VR both at the same potential), we attain 12,000 cm2 V−1 s−1 as a lower bound of the estimated mobility . We focus the mid-IR light with a reflective objective with a numerical aperture (NA) of 0.5. We measure the mid-IR power using a thermopile detector from Thorlabs placed at the sample position.We use a pulsed QCL mid-IR laser (LaserScope from Block Engineering) that is linearly polarized and has a wavelength tuning range from λ = 6.6 μm with a pulse width of 496 ns. We use a MCT as a reference detector from VIGO System model PCI-2TE-13. We measure the photoresponse using a current amplifier from FEMTO model DHPCA-100 with switchable gain and acquire the signal with an oscilloscope from Teledyne Lecroy model HDO6000.For the time-resolved measurements, we set the QCL wavelength to IPTE/Pin) × (Afocus/Adiff)49, where Pin is the power measured by the commercial power meter, Afocus is the experimental beam area at the measured wavelength and Adiff is the diffraction-limited spot size. We measure the photocurrent IPTE from the output signal of the lock-in amplifier VLIA considering 49), where ξ is the gain factor in V/A (given by the lock-in amplifier). We use the ratio Adiff/Afocus for estimating the power reaching our photodetector since Adiff is the most reasonable value one can attain when considering the detector together with an optimized focusing system  and it is widely used in the literature for comparing the performances among photodetectors49. Note that this simple geometrical scaling is not used here to predict the exact performance at a hypothetical diffraction-limited spot but rather as clear performance benchmark so to consistently compare different device architectures measured at differed focusing conditions. We usually have a ratio of Afocus/Adiff ≈ 7. This ratio is given by w0,x and w0,y we use our experimental observation that the photocurrent is linear in laser power and measure the photocurrent while scanning the device in the x-  and y-direction. Consequently, the photocurrent is described by Gaussian distributions w0,x and w0,y are the respectively obtained spot sizes  and RD the device resistance.The external responsivity is given by: Responsivity = (Supplementary Information


Furthermore, the CVD graphene substrates beneath the microstructure LEDs facilitated transfer of the LED arrays onto any foreign substrate. In this study, Cu foil was used for flexible LEDs. The flexible devices exhibited stable electroluminescence, even under severe bending conditions. Cyclic bending tests demonstrated the excellent mechanical stability and reliability of the devices.We report flexible and monolithically integrated multicolor light-emitting diode (LED) arrays using morphology-controlled growth of GaN microstructures on chemical-vapor-deposited (CVD) graphene films. As the morphology-controlled growth template of GaN microstructures, we used position-controlled ZnO nanostructure arrays with different spacings grown on graphene substrates. In particular, we investigated the effect of the growth parameters, including micropattern spacings and growth time and temperature, on the morphology of the GaN microstructures when they were coated on ZnO nanostructures on graphene substrates. By optimizing the growth parameters, both GaN microrods and micropyramids formed simultaneously on the graphene substrates. Subsequent depositions of InGaN/GaN quantum well and  Organic materials have long been studied for such applications, but higher device performance is expected when using inorganic materials due to their excellent electrical and optical characteristics as well as long-term stability and reliability4. Notably, GaN based micro-LEDs are promising candidates due to their excellent brightness and long life. However, integration of micro-LEDs for full-color displays remains to be resolved. Previous integration methods using multiple transfer steps (pick-up and drop-off) onto a flexible substrate and self-assembly limit inorganic LEDs to high-resolution displays and are neither reliable nor reproducible for mass production. The integration issue would be resolved using monolithically integrated micro-LEDs to demonstrate multicolor emissions via tuning their bandgap by varying the In composition of the InGaN/GaN quantum well (QW)7. Meanwhile, recent studies on GaN based micro- and nanostructures on chemical-vapor-deposited (CVD) graphene films have demonstrated their suitability for flexible light-emitting-device applications9. Accordingly, monolithic integration of various GaN microstructures on CVD graphene films is a critical step for the fabrication of flexible and high-resolution micro-LED displays11. Herein, we report the simultaneous growth of GaN microrods and micropyramids on CVD graphene films and the fabrication of flexible monolithically integrated multicolor micro-LEDs using the microstructures.Highly efficient and flexible multicolor micrometer-scale light-emitting diodes (micro-LEDs) are required for mobile and wearable optoelectronic devices and displays with high resolution and low power consumption2/Si substrates. For selective growth of inorganic materials on a CVD graphene film, a 300-nm-thick SiO2 layer was deposited on the CVD graphene film and patterned by conventional lithography. Then, ZnO nanotubes were selectively grown on the CVD graphene film by catalyst-free MOVPE using high-purity diethylzinc and oxygen (O2) as the reactants for Zn and O, respectively. Herein, we prepared ZnO nanostructures with different spacings of 2, 4, 6, and 8 µm and a fixed height and diameter of 3.8 and 0.6 µm, respectively magnesium as the doping source10. The typical resistivity and carrier concentration of the n-GaN were measured to be 6 × 10–3 Ω cm and 4 × 1018 electrons cm−3, respectively; those of the p-GaN were 5–10 Ω cm and 0.8–1.3 × 1017 holes cm−3 after p-type activation at 700 °C.The basic strategy for the growth of GaN layers on ZnO-nanostructured graphene films was as follows. The graphene thin films were synthesized on Cu foil and transferred onto SiOely Fig. . The grop-GaN overlayer by PI etching using O2 plasma treatment, Au/Ni layers (20/20 nm) were deposited as p-contact electrodes of the micro-LEDs. Ohmic contact was achieved by rapid thermal annealing in ambient air at 300 °C for 5 min. Subsequently, the entire device structures fabricated on the CVD graphene films were released from the SiO2/Si substrate by selective etching of the SiO2 interlayer using buffered oxide etchant. The LEDs on the CVD graphene films were transferred onto plastic and Cu foil.To fabricate the LED devices, metal electrodes were made on the GaN microstructures by evaporating metals on a patterned area. Before depositing the metal thin films, a PI layer was spin-coated on the microstructures to support the metal electrodes. Then, after exposure of the The surface morphology of the ZnO nanostructures, GaN microstructures, and the monolithically integrated micro-LEDs was investigated using field-emission scanning electron microscopy .I–V characteristics and EL spectra of the devices were measured while applying a DC voltage to the device using a Keithley 2400 source meter. The EL spectra were measured using a monochromator  and a detection system equipped with a charge-coupled device .The 2/Si substrates. Using graphene films as the substrate for the GaN microstructure growth was essential to prepare high-quality GaN microstructures and to facilitate transfer of the microstructures to arbitrary substrates for flexible device applications15. For position-controlled growth of ZnO nanotubes on the graphene films, a 300-nm-thick SiO2 layer was deposited on the CVD graphene films and patterned to 0.6-μm-diameter hole arrays with different spacings of 2, 4, 6 and 8 μm by conventional lithography. Then, ZnO nanotubes were selectively grown on the graphene films and GaN layers were coated on the ZnO nanotubes by metal–organic vapor-phase epitaxy (MOVPE)12. For simultaneous and morphology-controlled growth of GaN microstructures, we investigated the effect of the GaN growth time and the spacing between ZnO nanotubes in their arrays on the morphology of GaN microstructures when GaN was coated on the ZnO nanostructures prepared on graphene. In this study, we prepared ZnO nanostructures with different spacings of 2, 4, 6, and 8 μm and a fixed height and diameter of 3.8 and 0.6 μm, respectively  is smaller than that of the semipolar –2), growth along the semipolar facet would be energetically more favorable, thus resulting in the formation of micropyramids.Simultaneous growth of different GaN microstructures was achieved by heteroepitaxial growth of GaN layers on position-controlled ZnO nanotube arrays with different spacings prepared on graphene films. For the graphene substrate preparation, graphene thin films were synthesized on Cu foil using conventional CVD, lifted-off by wet-chemical etching, and transferred onto SiO17, so the growth rate could be significantly affected by the ZnO nanostructure spacing when it is narrower than 10 μm. When the spacing was wider than 10 µm, smaller micropyramids with poor growth selectivity were obtained (not shown). Accordingly, for larger spacing that was less than the surface diffusion length of 10 μm, more adatoms reached each microstructure, which enhanced the microstructure growth rate. The effect of surface diffusion of Ga adatoms on the surface morphology of the GaN microstructures was also confirmed by our supplementary study on the change in the surface morphology of the GaN microstructure as a function of growth temperature  layer filling, and exposure of the PI layer on the p-contact area, integrated micro-LED arrays were fabricated by depositing Ni/Au metal contact layers with an area of 50 × 50 µm2 on the top region of the as-grown LEDs. For flexible device applications, the LEDs were removed from the SiO2/Si substrate by chemical etching of the SiO2 layer using buffered oxide etchant and transferred to Cu-coated polyethylene terephthalate films. The Cu layer was also used to make an electrical connection for flexible LEDs. Figure The morphology-controlled growth of GaN microstructures enabled the fabrication of monolithically integrated GaN microstructure LEDs that displayed multicolor light emission. The basic approach for the integrated micro-LED fabrication is shown in Fig. The optical characteristics of the microrod- and micropyramid-based LEDs were further investigated by electroluminescence (EL) spectroscopy. The dominant light emission of these LEDs appeared at different wavelengths. For the microrod-based LEDs, the dominant EL emission at an applied current of 0.11 mA was observed at 506.3 nm with a shoulder peak centered at 536.9 nm. As the current level increased to 0.25 and 0.50 mA, the main EL peak shifted to 505.3 and 504.8 nm, respectively. The dominant EL peaks in Fig. The EL spectra at various current levels also indicated that the shift in the dominant EL peak with increasing current level for the microrod LEDs was much smaller than that for micropyramid LEDs. This behavior presumably resulted from the reduced quantum-confined Stark effect due to the dominant formation of QWs on nonpolar planes for microrod LEDs but on semipolar planes for micropyramid LEDs.5. Their observation of photoluminescence (PL) and cathodo-luminescence (CL) shows the similar tendency to our data demonstrating wavelength shift from blue to green and red as the structure changes from microrod to micropyramid and microdisk. In addition, Hong et al. reported the appearance of additional blue emission peak from microrod-based LED at high applied bias voltages with qualitative explanation using the field distribution model7. Based on their investigation, EL spectra in Fig. The optical characteristics of the GaN microstructure LEDs can be analyzed by comparing them with previously reported result. Robin et al. reported simultaneous growth of GaN microrod, micropyramid, and microdisk by controlling the diameter of the growth mask opening and fabricated different LEDs by subsequent deposition of InGaN/GaN QWs on those structuresThe monolithically integrated multicolor micro-LED arrays fabricated on graphene substrates demonstrated excellent mechanical stability and reliability under severe bending conditions. Figure I–V) characteristic curves of the microrod- and micropyramid-based LEDs. The I–V characteristic curves show typical rectifying behavior LEDs with a turn-on voltage of ≈ 3 V and a leakage current of 3 × 10−6 A at –3 V, originated by flows of electrons and holes from n to p and p to n layers when a forward bias voltage is applied across the junction resulting in reduction in the potential barrier.Figure Severe bending did not significantly affect the curves, which exhibited similar rectifying behavior without appreciable differences in the device parameters, such as the turn-on voltage or leakage current.Figure n-GaN microstructures on graphene films. The morphology-controlled growth of the GaN microstructures was realized by optimizing the growth parameters, including the spacing of the ZnO nanotube arrays used as the growth template, and the GaN growth time and temperature. GaN microrod and micropyramid arrays were grown on ZnO nanotubes spaced at 2 and 6 µm at 1000 °C for 10 min. The microrod and micropyramid-based LEDs emitted blue and green light, respectively. Additionally, blue shifting of the EL peaks was observed at higher applied currents from 0.1 to 0.5 mA. The EL peak shift was much smaller for microrod LEDs than for micropyramid LEDs, presumably as a result of the reduced quantum-confined Stark effect. Furthermore, the LEDs fabricated on CVD graphene films were readily transferred onto flexible substrates and exhibited reliable operation during bending. Our study investigates simultaneous growth of non-polar and semi-polar GaN microstructures on CVD graphene films and their fabrications to flexible LEDs to demonstrate multi-color light sources with a minimized blue shifting issue in a flexible form. We believe that the monolithic integrated flexible multicolor LEDs will pave the way for the development of next-generation optoelectronic devices using flexible inorganic LEDs, such as large-scale, high-resolution, and full-color flexible displays.We fabricated monolithically integrated multicolor LEDs on CVD graphene films for flexible devices, using simultaneous growth of morphology-controlled Supplementary Information.


Idiopathic pulmonary fibrosis (IPF) is a chronic disease mainly associated with aging and, to date, its causes are still largely unknown. It has been shown that dietary habits can accelerate or delay the occurrence of aging-related diseases; however, their potential role in IPF development has been underestimated so far. The present review summarizes the evidence regarding the relationship between diet and IPF in humans, and in animal models of pulmonary fibrosis, in which we discuss the bioactivity of specific dietary food ingredients, including fatty acids, peptides, amino acids, carbohydrates, vitamins, minerals and phytochemicals. Interestingly, many animal studies reveal preventive and therapeutic effects of particular compounds. Furthermore, it has been recently suggested that the lung and gut microbiota could be involved in IPF, a relationship which may be linked to changes in immunological and inflammatory factors. Thus, all the evidence so far puts forward the idea that the gut-lung axis could be modulated by dietary factors, which in turn have an influence on IPF development. Overall, the data reviewed here support the notion of identifying food ingredients with potential benefits in IPF, with the ultimate aim of designing nutritional approaches as an adjuvant therapeutic strategy. Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, irreversible and heterogeneous disease characterised by the excessive deposition of extracellular matrix associated with progressive decline in lung function and respiratory failure . IPF occThe UIP pattern characteristic of IPF includes temporal and spatially heterogeneous fibrosis, advanced scarring and microscopic honeycombing, clusters of fibroblasts and myofibroblasts (fibroblastic foci), the accumulation of hyperplastic type II alveolar epithelial cells, the reduction of type I alveolar epithelial cells and exaggerated deposition of disorganised collagen and extracellular matrix (ECM), resulting in the distortion of normal lung architecture . In thisAs a response to sustained injury, epithelial cells acquire features of mesenchymal cells, a process termed epithelial-mesenchymal transition (EMT), which is initiated by the transforming growth factor β1 (TGF-β1) ,15. ThesActivated epithelial and endothelial cells promote an aberrant epithelial-mesenchymal crosstalk ,15. CircBoth oxidative stress and immunological factors may play a pathogenetic role in IPF ,18. An iAging is the most evident host factor that influences IPF occurrence. The main molecular and cellular hallmarks of aging are present in IPF, which include telomere attrition, genomic instability, mitochondrial dysfunction, epigenetic alterations, cellular senescence, loss of proteostasis, dysregulated nutrient sensing, stem cell exhaustion and altered intercellular communication . FurtherHowever, the development of pulmonary fibrosis in individuals with no identifiable mutations, or that have not been exposed to the abovementioned environmental inputs, means that other factors must be considered. Since it is well known that diet influences the risk of age-related diseases, and that the microbiome is highly implicated in a wide range of conditions, we decided to delve into the data published so far, in order to elucidate their potential involvement in IPF.Nutrition is intimately linked to health and disease. Numerous epidemiological studies support the premise that unhealthy dietary habits increase the risk of age-related chronic diseases and accelerate mortality . On the The best-known nutritional strategy to increase longevity in several animal species is the reduction of food intake without reaching malnutrition. Dietary restriction exerts a protective effect against obesity, type 2 diabetes, inflammation, hypertension and cardiovascular disease, and reduces the metabolic risk factors associated with cancer . A lowerHowever, the effects of dietary manipulations on respiratory function and age-related respiratory chronic diseases have been hardly investigated. Hegab et al.  recentlyUp until now, most studies on IPF have mainly focused on factors that can damage the airway. So far, the role of nutritional status in the development of IPF has been poorly evaluated in humans and, to the best of our knowledge, few epidemiological studies have analysed diet in IPF. Two case-control studies in a Japanese population found a lower fruit and higher meat consumption, together with a higher intake of saturated fat, in IPF patients ,35. DietInterestingly, among IPF patients, cases of cardiovascular diseases, obesity, diabetes mellitus and dyslipidaemia are frequently reported . Such asThe exposure to diets rich in lipids triggers the occurrence of pulmonary fibrosis per se ,43 and wHowever, the effect of dietary lipids could depend on the type of fatty acid or the lipid source. Accordingly, one of the first experiments addressing these issues revealed that the intake of coconut oil or beef tallow as lipid sources triggered lower increases in lung hydroxyproline content and lipid peroxidation after the administration of bleomycin (a DNA-interactive antitumor agent commonly used to induce pulmonary fibrosis), indicating that alterations in dietary lipids can, indeed, reduce the severity of pulmonary fibrosis . Here, wExposure to a high-fat diet has been shown to trigger the occurrence of incipient lung fibrosis , whereby1, which inhibits EMT in human alveolar type II cells [Following the observation that IPF patients show lower relative contents of oleic acid surfactant phospholipids , it has II cells , and thrII cells . In all Ophiocordyceps lanpingensis [Ganoderma lucidum [Dendrobium officionale [The addition of fructose to a high-fat diet enhanced the development of pulmonary fibrosis without the participation of another intentional agent in mice , suggestingensis  and Gano lucidum . Other p lucidum , and chi lucidum ,61. Antiicionale  or from icionale .IPF patients show an alteration in amino acid metabolism, characterised by higher amounts of proline, 4-hydroxyproline, alanine, valine, leucine, isoleucine and allysine, detected in both lung tissue and breath . This haIn vivo studies have addressed this issue and, in fact, oral treatment with L-arginine improved arginine metabolism in bleomycin-treated mice and reduced lung damage . SimilarTaurine is a cysteine-derived amino acid that naturally occurs in the body. Interestingly, its anti-fibrotic potential has been documented in animal models of pulmonary fibrosis, which is explained, in part, by its antioxidant and immunomodulatory abilities ,69. In hEucheuma, named EZY-1, exhibited anti-fibrotic properties, with a superior potency and safety than pirfenidone [Recently, it was demonstrated that a small peptide isolated from the edible seaweed fenidone , by altefenidone . The admfenidone .The levels of vitamers with antioxidant activity are increased in the BALF of IPF patients, in an attempt to restore oxidative balance . Such vi(1)Vitamin ARetinoic acid, the bioactive metabolite of vitamin A, is an important signalling molecule during normal early lung development, and has anti-fibrotic and anti-inflammatory properties . All-tra(2)Vitamin BNiacin is a vitamin B3 form involved in DNA repair, as a precursor of the coenzymes nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate. As such, its ability to enhance DNA repair caused by bleomycin has been demonstrated, being one of the first described agents able to attenuate bleomycin-induced lung fibrosis . Subsequ(3)Vitamin CVitamin C is involved in tissue repair and collagen secretion, and its deficiency leads to impaired collagen synthesis. In pulmonary fibrosis induced by chromium, paraquat or LPS, the damage was reduced by the subsequent or simultaneous administration of vitamin C ,81,82. T(4)Vitamin DSerum vitamin D has been proposed as a biomarker of IPF prognosis . IPF pat(5)Vitamin EThe role of vitamin E in the development of pulmonary fibrosis has been shown in both vitamin E deficient animals, which show a more severe interstitial pneumonitis and emphysematous lesions , and vit(1)IronThe lungs of IPF patients show numerous aspects of dysfunctional iron metabolism, including increased iron levels, the presence of iron-laden macrophages, and iron-induced oxidative stress . Fibrosi(2)CopperExcess of copper plays an important role in the aetiology and pathogenesis of many diseases, including IPF , and its(3)Sodium ChlorideIt has been suggested that dietary salt may influence IPF, based on the fact that its intake is associated with cardiac and renal fibrosis and, in cultured cells, promotes the trans-differentiation of monocytes into fibrocytes ,108. DesAn increasing number of studies have analysed the anti-fibrotic effect of various phytochemicals in animal models of lung fibrosis. Quercetin, curcumin, resveratrol, epigallocatechin-3-gallate, S-allyl compounds and several lignans are the most studied phytochemicals in the context of lung fibrosis. Studies in which phytochemicals were orally administrated or supplemented in the diet are included in The flavonoid quercetin is found in vegetables and fruits, particularly in onions, apples and broccoli. Quercetin presents anti-aging properties, due to its antioxidant activity and ability to induce apoptosis in senescent cells, thereby mitigating the severity of several age-related chronic diseases . In expeCurcuma longa), and is known for its antioxidant and anti-inflammatory effects mediated by its ability to inhibit NF-kB. Curcumin arises as a potential anti-aging molecule [The flavonoid curcumin is present in turmeric , its ability to activate serine/threonine kinase AMP-activated protein kinase complex (AMPK) signalling, and its capacity to extend lifespan in several species, including mammals, by activating SIRT1 ,29. In aSchisandra chinensis, known for its diverse beneficial health effects, was also shown to attenuate pulmonary fibrosis in experimental animals [Plant lignans are fibre-associated polyphenolic compounds that form lignin, a constituent of the plant cell wall. They are particularly abundant in flaxseed, rye bran and oat bran, and are known for their antioxidant properties. The supplementation of the diet with flaxseeds triggers anti-fibrotic effects, both before and after lung damage ,129, dou animals ,132.The polyphenol epigallocatechin-3-gallate (EGCG) is a major component of green tea, which aroused interest due to its anti-aging and immunomodulatory properties . The admGarlic possesses antioxidant activity, and has been traditionally used for the treatment of ailments associated with aging. Garlic bioactive compounds include S-allyl-cysteine and S-allylmercaptocysteine, organosulphur compounds that target the Nrf2 and NF-kB pathways, which are involved in the in vivo anti-inflammatory and anti-fibrotic activities ,139,140.The role of the microbiota, which refers to the microbial  community found in the human body, in health and disease is a topic of great interest, and it is now fully established that it is crucial in human wellbeing, and how an imbalance, or dysbiosis, can lead or contribute to a wide range of diseases . In thisHistorically, the lung has been considered as a sterile organ. However, with the development of non-culture dependent techniques, it has been shown that there is a diverse bacterial microenvironment ,158. EveIn line with this new area of research, it has been observed that patients presenting some pulmonary conditions, including inflammation or impaired lung function, also exhibit gastrointestinal (GIT) diseases. Furthermore, even though the GIT and the respiratory tract present different environments and functions, they have the same embryonic origin and structural similarities, suggesting a potential interaction . Thus, aIt has been suggested that three main features regarding lung microbiota are of importance when considering IPF prognosis, pathogenesis and progression: bacterial burden, microbial diversity and overall composition .On one hand, those individuals presenting decreased lung microbial diversity have a worse prognosis, and that an increased bacterial burden at diagnosis is associated with a higher progression of IPF and mortality risk ,12,13. MHowever, while these data are promising, they do not prove a causal role for lung microbiota in IPF. In order to elucidate this, a range of studies have attempted to determine bacterial composition and how its manipulation can impact IPF pathogenesis. The major bacterial phyla found in lungs is the same as in gut, thus Firmicutes and Bacteroidetes are the predominant bacteria followed by Proteobacteria and Actinobacteria . It has Streptococcus and Staphylococcus in the lungs of patients with a poorer disease outcome, together with Prevotella spp., Veillonella spp. and Escherichia spp. [Streptococcus, Haemophilus, Neisseria and Veillonella spp. have all been observed to be more abundant in patients with IPF (vs. controls) [Gemella spp., which could in turn be altering community composition and promoting injury, due to mucin overexpression and defective mucocillary clearance [At the species level, microbial communities differ significantly between the gut and lung. One of the first studies to show an association between specific bacteria or operational taxonomic units (OTUs) and disease progression in IPF reported the presence of hia spp.  This wasontrols) ,13. A molearance .Manipulation of lung microbiota has been achieved in other pulmonary diseases by means of antibiotic treatment; this, however, still needs to be determined whether it occurs in IPF . For thiThroughout the entire life span, there is a close connection between the bacterial composition of the lung and gut microbiota, leading to the hypothesis of the existence of a host-wide network. This is evidenced by studies showing that changes in a newborn’s diet alter lung microbiota composition, and that fecal transplantation in rats also modifies bacteria in lungs ,169. An In disease, an inflammatory inter-organ cross-talk seems to occur between the lung and the intestine, with a clear association between airway-related diseases and GIT conditions . Even th13 microbial cells, which live in a mutualistic relationship with the host and carry out a wide range of functions key to maintain host metabolism and homeostasis of the immune system [To date, the microbiota present in the gut, particularly in the intestine, is the most studied by far. This is not surprising, considering that it is the largest and most diverse community of the human microbiome, with over 4 × 10e system . In compe system .The impact of gut microbiota on the immune system has been extensively studied, demonstrating so far that bacteria and its by-products interact with the host using pro-inflammatory and regulatory signals, as well as influencing immune cell responses . CurrentPrevotella and Staphylococcus abundance in the lungs of individuals with IPF is negatively correlated with the expression of immune response genes, and that changes in the microbial community structure are associated to changes in the phenotypes of circulating leukocytes [The effect of gut microbiota on immunity, and its further impact on lung disease, has been researched in conditions such as COPD, asthma, cystic fibrosis and influenza virus, among others . Moreoveukocytes . Furtherukocytes . This paukocytes .However, as with most data regarding host microbiota, discerning the causal relationship between pulmonary and gut microbiota and IPF progression and pathogenesis is still complicated and needs further study, in order to fully understand the mechanistic pathways. It is still unknown whether the impact observed after microbiome manipulation in IPF are due to indirect, off-target effects, mainly on gut microbiota; for this, more studies are required, that can selectively alter lung microbiota without changing gut bacterial composition , which iThe potential roles of bioactive food ingredients and microbiota on IPF may be considered as inter-dependent factors, although their purported role as independent influencing players may also be taken into account. The bioactivity of a given food ingredient cannot be solely exerted by the chemical form it is presented in food, but by a more active by-product derived from gut microbiota. Therefore, its bioactivity in host cells may be dependent on the presence or abundance of particular bacteria capable of metabolizing such compounds. The opposite relationship between food ingredients and gut microbiota could also explain the bioactivity of a compound, in which a nutrient modifies gut bacteria composition, and such changes could determine host health. In this sense, there is accumulating evidence that supports the role of interactions between bioactive food ingredients and gut microbiota, which have been primarily investigated in the context of metabolic diseases ,177,178.Pseudomonas aeruginosa infection were fed a diet enriched in acidic oligosaccharides derived from pectin [In the context of chronic lung diseases, the interaction between bioactive food ingredients and gut microbiota, although to a lesser extent, has also been analyzed. Thus, in individuals with cystic fibrosis, the intake of antioxidant vitamins and various flavonoids correlated with the amount of particular gut bacteria, and such variations could potentially influence immune function and inflammation, which are important in cystic fibrosis disease and co-morbidity management ,180. Them pectin . This dim pectin . Thus, im pectin ,183. Intm pectin . The modDietary manipulations can also impact lung microbiota composition. For example, dietary fiber is able to modify the lung microbiome , potentiOverall, the evidence presented in this review supports a role for diet and particular bioactive food ingredients in IPF, as has been previously discussed for other chronic lung diseases , and sug