The pooled percentage difference in the proportion of IPF between the UIP and possible UIP patterns was −2%  in the former scenario and 4%  in the latter scenario. The proportion of IPF with the possible UIP pattern was moderately correlated with the prevalence of IPF . There was a negligible pooled percentage difference in the proportion of IPF between the UIP and possible UIP patterns, indicating that IPF diagnosis can be confirmed without biopsy in suspected IPF cases with the possible UIP pattern.This study aimed to determine whether a surgical lung biopsy is essential for IPF diagnosis with the possible UIP CT pattern. We performed literature searches of the MEDLINE and EMBASE databases and included studies that conducted a radiologic-pathologic evaluation of IPF according to the 2011 guideline. Outcomes were pooled using a random-effects model. Twelve studies were included. Pooled proportions of IPF for a UIP pattern were 99% (95%CI, 93% to 100%; I With the recent introduction of anti-fibrotic agents for IPF treatment including nintedanib and pirfenidone, accurate diagnosis of IPF has become more important3. Traditionally, IPF has been confirmed with a histopathologic evaluation of a lung biopsy specimen but the role of high-resolution computed tomography (HRCT) has recently become more central in the diagnostic pathway of IPF5. The official ATS/ERS/JRS/ALAT statement in 2011 specified the HRCT patterns of usual interstitial pneumonia (UIP) into 3 diagnostic categories1: UIP pattern, if honeycombing and reticular abnormality with subpleural, basal predominance exists without features inconsistent with the UIP pattern; possible UIP pattern, if honeycombing is absent but the imaging features otherwise meet the criteria for the UIP pattern; and inconsistent with UIP pattern. IPF can be diagnosed without a pathologic confirmation in patients with the UIP pattern on HRCT scan because it exclusively indicates IPF, while a surgical lung biopsy is recommended for the possible or inconsistent with UIP patterns.Idiopathic pulmonary fibrosis (IPF) is a specific form of chronic, progressive fibrosing interstitial pneumonia of unknown cause characterized by worsening dyspnea and lung function7. Surgical lung biopsy inevitably accompanies morbidity and mortality, especially for nonelective procedures 8. If a reticular abnormality with subpleural basal predominance is highly indicative of IPF regardless of presence of honeycombing, a surgical lung biopsy can be omitted in patients with the possible UIP pattern9 as with the UIP pattern. Indeed, the Fleischner society recently proposed the introduction of probable UIP CT pattern which substitutes for possible UIP CT pattern in the 2011 guideline and the allowance of IPF diagnosis without surgical lung biopsy in case of probable UIP CT pattern based on a selective review of few number of relevant studies. Thus, we performed a meta-analysis of studies to determine whether the surgical lung biopsy is essential for IPF diagnosis in patients with the possible UIP pattern on HRCT scan.Recent studies have shown that basilar predominant reticular opacities without honeycombing on HRCT were strongly associated with pathologic UIP20 with 13 study populations were finally included in our analysis . Pooled proportions of IPF for the possible UIP pattern were 94%  in the scenario inclusive of probable IPF, and 88%  in the scenario exclusive of probable IPF. When compared between studies with a UIP pattern included and those with it excluded  in a scenario inclusive of probable IPF and 4%  in a scenario exclusive of probable IPF without zero-cell corrections  in the scenario inclusive of probable IPF and 4%  in the scenario exclusive of probable IPF . However, in cases with possible and inconsistent UIP patterns, IPF prevalence and the proportion of IPF showed a moderate monotonically increasing relationship   if possible UIP cases probably having IPF actually had IPF in 100% of cases (scenario inclusive of probable IPF). When we assumed that possible UIP cases probably having IPF were not IPF at all, the pooled percentage difference between the patterns was marginal  (scenario exclusive of probable IPF). The pooled proportion of IPF in the possible UIP pattern based on the former and latter assumptions were 94.0%  and 88.0% , respectively, and this depended on IPF prevalence .21. The society introduced a probable UIP pattern as a new category by upgrading the possible UIP pattern and proposed the IPF diagnosis without surgical lung biopsy in patients who had a typical clinical context of IPF with UIP or probable UIP CT pattern. Our systematic review of all relevant publications validated their recommendation.These results imply that IPF can be confidently diagnosed solely based on the HRCT finding in patients with a possible UIP pattern without surgical lung biopsy, if IPF suspects were appropriately identified. This is totally in accord with the latest recommendation of the Fleischner Society on the diagnostic criteria of IPF1. Prior to overt honeycombing, radiologic reticular abnormalities originate from fibrotic thickening of a distorted interstitium and formation of small cysts, which are often beyond the resolution of HRCT and have presumably progressed into honeycombing22. Accordingly, reticular opacities, which typically have a basilar, subpleural predominance on HRCT can correspond to an earlier CT finding of overt radiologic honeycombing23. Indeed, nintedanib had similar therapeutic effects in patients with suspected IPF not only with a UIP pattern but also those with a possible UIP pattern on HRCT scan24.Our finding suggests that radiologic honeycombing is not mandatory for the HRCT diagnosis of IPF. The radiologic honeycombing is one of a spectrum of CT findings, representing the late stage of IPF. It represents advanced fibrotic lung changes which contain clustered cystic airspaces with thick fibrous walls, along with a complete loss of acinar architecture20. The degree of suspicion may be assessed qualitatively by clinicians or quantitatively by the pretest probability model which consists of age, gender, and traction bronchiectasis score on HRCT scan20. Both assessments require sufficient experience with IPF diagnosis, which is why multidisciplinary discussion between experts is emphasized in the current guideline1. In the setting where IPF is highly suspected by experienced clinicians or the pretest probability model, we believe that IPF is sufficiently diagnosed with a possible UIP CT pattern without biopsy21.IPF prevalence was moderately correlated with the pooled proportion of IPF in the cases with a possible UIP pattern. This suggests that assessment of the degree of suspicion of IPF is crucial in patients with a possible UIP pattern before IPF diagnosisThe pooled proportion of IPF was 99.0%  in patients with a UIP CT pattern, supporting the current guideline, which recommends making a diagnosis of IPF without biopsy in those patients. On the other hand, for an inconsistent UIP HRCT pattern, the proportion of IPF varied from 0% to 100% across studies, and it was strongly affected by IPF prevalence . It is appropriate that the current guideline recommends surgical lung biopsy for patients having the inconsistent UIP HRCT pattern.et al.25 Sixth, there was substantial heterogeneity in the IPF proportion with possible and inconsistent UIP diagnoses, presumably because the IPF prevalence in each study was different. In order to resolve this heterogeneity, we pooled the percentage difference of IPF diagnosis between the UIP and possible patterns and the corresponding I-square was 0.0% to 0.1% in Fig. Our study had several limitations. First, as multidisciplinary discussion was not performed in this study, so two different assumptions were introduced for handling probable IPF cases : all the cases were IPF (scenario inclusive of probable IPF) or none were IPF (scenario exclusive of probable IPF). In the clinical setting where multidisciplinary discussion is introduced, we may expect a percentage difference in proportions of IPF diagnosis of −2% (scenario inclusive of probable IPF) to 4% (scenario exclusive of probable IPF) between the radiologic UIP and possible UIP patterns. Second, the study population was defined differently across studies, especially with regard to the inclusion of patients with a radiologic UIP pattern. The prevalence in some of the included studies was underestimated due to the exclusion of patients with typical UIP CT pattern from the study population, and this might have affected the result of correlation analysis between IPF prevalence and the proportion of IPF when biopsied. Likewise, the proportion of IPF in a certain UIP CT pattern might be incorrectly assessed in 4 studies where the confirmatory surgical biopsy was performed in a small portion of IPF patients. The percentage difference of IPF diagnosis between the UIP and possible patterns could be pooled in 6 of 13 included study population in Table In conclusion, our meta-analysis suggests that the pooled percentage difference in the proportions of IPF diagnosis was negligible between radiologic UIP and possible UIP patterns, indicating that IPF diagnosis can be made with a possible UIP pattern without biopsy just as with a UIP pattern in patients highly suspected of having IPF. This enables maintaining an accurate diagnosis while avoiding the morbidity and mortality of surgical lung biopsy in IPF patients having a possible UIP CT pattern who could potentially benefit from anti-fibrotic agents.Two of the authors (K.H.K. and S.H.Y.) independently performed a literature search of the OVID/MEDLINE and EMBASE database to identify relevant publications that conducted a radiologic-pathologic evaluation according to the ATS/ERS/JRS/ALAT guideline in 2011. We used keywords related to ‘idiopathic pulmonary fibrosis’, ‘computed tomography’, and ‘pattern’ from January 2010 until May 2017 (Supplementary Data We applied the following criteria to determine eligibility: (i) study populations consisting of 5 or more patients who were suspected of having interstitial lung disease and underwent a surgical lung biopsy; (ii) a radiologic-pathologic evaluation of IPF based on the combination of HRCT and surgical lung biopsy patterns in accordance with the 2011 statement; (iii) data presented in sufficient detail to assess the proportion of IPF in the possible UIP pattern on HRCT scan.We excluded studies where the proportion of IPF in each HRCT pattern was not extractable due to exclusive inclusion of IPF without inclusion of any other interstitial lung diseases, and studies dealing with patients with specified causes of interstitial lung disease . If study populations were overlapping between studies, the study having enrolled the largest number of patients was included. Review articles, editorials, letters, case reports, and guidelines for management were excluded. When necessary, we contacted the author for additional data related to the full-text article.The primary outcome of our meta-analysis was a percentage difference in the proportion of IPF diagnosis between the UIP and possible UIP patterns on HRCT scan. Secondary outcomes included the proportion of IPF diagnosis and the correlation between prevalence of IPF and proportions of IPF diagnosis according to the 3 HRCT patterns.According to the 2011 ATS/ERS/JRS/ALAT guideline, HRCT could have any of the 3 aforementioned patterns and the result of histopathologic examination of surgical lung biopsy encompassed the following 5 patterns: UIP pattern, probable UIP pattern, possible UIP pattern, unclassifiable fibrosis, and non-UIP pattern. IPF was diagnosed with specific combinations of the HRCT and histopathologic patterns . Subsequent analysis was also performed to explore the results as if none of those cases had IPF (scenario exclusive of probable IPF).et al.10, Casoni et al.12, and Hanley et al.15. Two studies were provided with abstracts, so there was insufficient information to obtain IPF proportions, and the other study did not distinguish pathologic probable UIP from possible UIP.The prevalence of IPF potentially affects the proportion of IPF when biopsied, so we calculated the prevalence of IPF in the included studies. As patients with a radiologic UIP pattern are not subjected to surgical lung biopsy in the current guideline, some studies included those patients, while other studies excluded them from their study populations. The prevalence of IPF was inevitably underestimated in the latter studies because IPF patients with a radiologic UIP pattern were excluded. We also calculated the number of IPF proportion in possible CT UIP pattern using given data in following 3 studies; Ogawa K 26. All disagreements were harmonized by consensus.Data extraction and quality assessment were performed independently by 2 authors (S.H.Y. and K.H.K). Quality assessment of the included literature was conducted based on the Quality Assessment of Diagnostic Accuracy Studies (QUADAS)-2 tool27. A pooled difference in the proportion of IPF between the UIP pattern and possible UIP pattern was estimated without zero-cell corrections and excluding a study with zero-cell counts in both patterns. A sensitivity analysis was conducted after zero-cell corrections of adding 0.5 to all cells of the study results tables where zero-cells were observed in one or both patterns in a study. Heterogeneity across the included studies was evaluated using I2 statistics. I2 was derived from the Cochran Q statistic with the following equation, I2 = 100% x (Q-df)/Q. An I2 statistic >50% was regarded as indicating substantial statistical heterogeneity28. Spearman correlation analysis was used to assess a monotonic relationship between IPF prevalence in study group and IPF proportion in each HRCT pattern. Publication bias was assessed by visually inspecting the distribution of observed studies on a funnel plot and Egger’s tests for asymmetry was done to quantify the degree of bias. All meta-analyses were done in R version 3.4.0 using the package metafor29.The proportion of IPF was pooled via a DerSimonian-Laird random-effect model according to the HRCT patterns. The proportions were double arcsine transformed to stabilize variances and then back-transformedWritten informed consent was not required for this study.Institutional Review Board approval was not required because this meta-analysis does not involve human subjects.Dataset 1, Dataset 2, Dataset 3, Dataset 4, Dataset 5, Dataset 6


Insoluble protein aggregates are the hallmarks of many neurodegenerative diseases. For example, aggregates of TDP-43 occur in nearly all cases of amyotrophic lateral sclerosis (ALS). However, whether aggregates cause cellular toxicity is still not clear, even in simpler cellular systems. We reasoned that deep mutagenesis might be a powerful approach to disentangle the relationship between aggregation and toxicity. We generated >50,000 mutations in the prion-like domain (PRD) of TDP-43 and quantified their toxicity in yeast cells. Surprisingly, mutations that increase hydrophobicity and aggregation strongly decrease toxicity. In contrast, toxic variants promote the formation of dynamic liquid-like condensates. Mutations have their strongest effects in a hotspot that genetic interactions reveal to be structured in vivo, illustrating how mutagenesis can probe the in vivo structures of unstructured proteins. Our results show that aggregation of TDP-43 is not harmful but protects cells, most likely by titrating the protein away from a toxic liquid-like phase. TDP43 aggregates are a hallmark of amyotrophic lateral sclerosis. By using deep mutagenesis to measure the toxicity of more than 50,000 mutations in the prion domain of TDP43, the authors conclude that mutations that increase toxicity promote formation of liquid-like condensates, while aggregation of TDP43 is protective for the cell. However, both in humans and in animal models, there is often only a weak association between the presence of aggregates and disease progression9. Indeed, multiple therapeutic approaches that reduce the formation of aggregates have failed at different stages of development12. On the other hand, there is increasing evidence that alternative protein assemblies generated during or in parallel to the aggregation process may be toxic17. Despite evidence that cellular damage may be induced either before, after or independent of the formation of insoluble aggregates, the latter are still widely assumed to be pathogenic in many neurodegenerative diseases19.The conversion of specific proteins into insoluble aggregates is a hallmark of many neurodegenerative disorders, including Alzheimer’s, Parkinson’s, Huntington’s, and Amyotrophic Lateral Sclerosis (ALS) with dominantly inherited mutations in aggregate-forming proteins causing rare familial forms of these diseases22 through liquid-demixing. This is a concentration-dependent process through which proteins can separate into two coexisting liquid phases and it has been extensively characterized both in vitro and in the cytoplasm23. In several proteins PRDs are necessary and sufficient for liquid-liquid demixing24. At least in vitro, insoluble aggregates can nucleate from more liquid phases26, leading to the suggestion that liquid de-mixed states can mature into pathological aggregates19.For many proteins, aggregation depends critically on intrinsically disordered regions with a low sequence complexity resembling that of infectious yeast prions. These prion-like domains (PRDs) are also enriched in proteins that can form liquid-like cellular condensates27 and low-complexity sequences28 are also enriched in dosage-sensitive proteins that are toxic when their concentration is increased. At least for one model protein that has been tested, however, it is the formation of a concentration-dependent liquid-like phase—not aggregation—that causes cellular toxicity28. Similarly, the toxicity of two mutant forms of the prion Sup35 could be explained only on the basis of their ability to populate a non-aggregate, liquid-like state29.Disordered regions30. TDP-43 aggregates are also present at autopsy in nearly all cases of frontotemporal dementia (FTD) that lack tau-containing inclusions 31. TDP-43 aggregates also represent a hallmark of inclusion body myopathy, and a secondary pathology in Alzheimer’s, Parkinson’s, and Huntington’s disease33. However, TDP-43 aggregates are also observed—albeit at low frequency—in control samples34 and, in vitro, TDP-43 can form both amyloid aggregates and liquid condensates39. Mutations in TDP-43 cause ~5% of familial ALS (fALS) cases40, with these mutations reported to interfere with nuclear-cytoplasmic transport, RNA processing, splicing, and protein translation46. However, despite extensive investigation, the molecular form of the protein that causes cellular toxicity is still unknown47.Cytoplasmic aggregates of the TAR DNA-binding protein 43 (TDP-43) are a hallmark of ALS, present in 97% of post-mortem samples50. A map of which amino acid (AA) changes increase or decrease the toxicity of a protein to a cell should, if sufficiently comprehensive, clarify both the properties of the protein and its in vivo conformational states associated with toxicity51. The effects of a small number of mutations on TDP-43 toxicity or aggregation have been previously reported55. However, on the basis of a handful of mutations, the relationship between aggregation and toxicity is far from clear.We reasoned that systematic (‘deep’) mutagenesis could be an unbiased approach to identify and investigate the toxic species of proteinsHere we show by quantifying the effects of >50,000 mutations in the PRD of TDP-43 that increasing hydrophobicity and aggregation strongly reduce the toxicity of this protein in yeast. Moreover, mutations that increase the toxicity of TDP-43 actually promote the formation of dynamic liquid-like cytoplasmic condensates. Mutations have their strongest effects in a central ‘hotspot’ region of the PRD TDP-43. The patterns of genetic interactions in double mutants in this region reveal that this ‘unstructured’ region is actually structured in vivo. Our results illustrate how deep mutagenesis can be used to probe the sequence-function relationships and the in vivo structures of ‘disordered’ proteins. We propose that aggregation of TDP-43 is not harmful but actually protects cells, most likely by titrating protein from a toxic liquid-like phase.Saccharomyces cerevisiae cells, induced expression and used deep sequencing before and after induction to quantify the relative effects of each variant on growth in three biological replicates . The dataset therefore allows us to investigate how mutations both increase and decrease toxicity. Very interestingly, ALS TDP-43 mutations increase toxicity, with a strong bias towards moderate effects   Fig. .Plotting the mean toxicity of all mutations at each position in the sequence reveals a 31 AA hotspot (312–342) where the effects of mutations are strongest Fig. . The varTo more systematically identify features associated with changes in toxicity we made use of all 53,468 variants carrying one or two AA substitutions (excluding STOP codon variants). We used principal components analysis (PCA) to reduce the redundancy in a list of over 350 AA physicochemical properties  to characterize the dynamics of TDP-43 variants in the different foci. The large cytoplasmic foci formed by non-toxic variants show little exchange of TDP-43 molecules with the soluble cytoplasmic pool. In contrast, the small foci localized at the nuclear periphery can exchange more protein with the cytoplasm, consistent with a more liquid-like state Fig. . Such diTaken together, these results suggest that mutations that increase the hydrophobicity of TDP-43 result in a re-localization of the protein away from small and dynamic, liquid-like foci at the nuclear periphery to large and more solid aggregates in the cytoplasm. A reduction in hydrophobicity has the opposite effect.36, with hydrophobicity more similar to the globular domains of TDP-43 than to the surrounding hydrophilic disordered regions  spectroscopy of the PRD revealed that residues 321–342 can adopt an α-helical structure in certain conditions47 and four different 6–11 AA peptides from the region could form cross-β amyloid or amyloid-like fibrils whose structures were determined by X-ray crystallography52. However, it is unknown whether any of these structures exist in vivo for full-length TDP-43.The hotspot region of the TDP-43 PRD (AA 312–342) is a conserved regionons Fig. . The hot60. In particular, when a sequence forms an α-helix, the side chains of residues separated by 3–4 AA are close in space and similarly oriented so that mutations in these AA interact similarly with mutations in the rest of the protein. In contrast, in a β-strand, the side chains of residues separated by 2 AA are close and similarly oriented and so make similar genetic interactions with other mutations  interactions between mutations in a protein can report on the secondary structure of that molecule when it is performing the function that is being selected forons Fig. 61.51  in our dataset to identify pairs of mutations that genetically interact. We first identified pairs of mutations that had unexpectedly high or low toxicity . We then quantified the similarity of epistasis enrichment profiles between pairs of positions and compared these patterns to those expected for α-helices and β-strands, scoring significance by randomization51 Fig. .52 typical of protein aggregates  was used in all experiments. Plasmid pRS416 containing TDP-43 or TDP-43-YFP under control of the Gal1 promoter was purchased from Addgene54. Mutagenesis for the characterization of TDP-43 variants was performed through PCR linearization with specifically-designed primers (Supplementary Data E.coli DH5α competent cells (Invitrogen) for plasmid purification and validation through Sanger sequencing. The plasmid used in the co-localization assays contains RNQ1-mCherry under control of the Gal1 promoter was a kind gift from the Rick Gardner lab. Genes coding for the other proteins for which co-localization was tested were cloned in this plasmid by gap-repair.73 could have improved the robustness of sequencing reads, we estimated that the impact of misreads due to the direct sequencing approach here employed would sum up to less than two additional counts per double nucleotide variant attributable to sequencing error (see Variants Toxicity and Error Estimates). Each oligonucleotide was amplified by PCR  for 15 cycles, purified using an E-gel electrophoresis system (Agarose 2%) followed by column purification with a MinElute PCR Purification Kit (Qiagen). In order to introduce the doped sequence in the full-length TDP-43 sequence, the purified oligonucleotide was cloned into 100 ng of linearized pRS416 Gal TDP-43 by a Gibson approach (Supplementary Data E. coli (NEB), by electroporation in a Bio-Rad GenePulser machine . Cells were recovered in SOC medium (NEB) for 30 min and plated on LB with ampicillin. A total of ~ 2.7 × 106 transformants were estimated. The plasmid library was purified with a GeneJET Plasmid Midiprep Kit (Thermo Scientific).Two 186 nt oligonucleotides were purchased from TriLink. Each consisted of a ‘doped’ region of 126 nt, corresponding to TDP-43 AA 290–331 or AA 332–373, flanked by 30 nt of the WT TDP-43 sequence on each side. Each position in the mutated area, was doped with an error rate of 1.59%. The target frequency for each library was 27.0% for single mutants and 27.3% for double mutants. With this approach, the WT sequence was represented with a frequency of 13.3%. Although a barcoding strategyYeast cells were transformed with the TDP-43 doped plasmid in 4 independent biological replicates for each library. One single colony was grown overnight in 30 ml YPDA medium at 30 °C for each replica. Cells were diluted to 0.3 optical density at a wavelength of 600 nm (OD600) in 175 ml of YPDA and incubated for 4 h at 30 °C. Cells were then harvested, washed, re-suspended in 8.575 mL SORB  and incubated for 30 min at room temperature. For the transformation, 10 mg per mL of salmon sperm DNA and 3.5 μg TDP-43 plasmid library were used. Cells were mixed to 100 mM LiOAc, 10 mM Tris-HCl pH 8.0, 1 mM EDTA/NaOH pH 8.0 and 40% PEG 3350. Heat-shock was performed for 20 min at 42 °C. YPD with 0.5 M sorbitol was used to recover the cells, incubating them for 1 h at 30 °C. After recovery, cells were resuspended in SC-URA 2% raffinose medium, while an aliquote was plated to calculate transformation efficiency.After ~50 h of growth, cells were diluted in SC-URA 2% raffinose medium and grown for 4.5 generations. At this stage, 400 mL of each replica were harvested, washed, split into two tubes and frozen at −20 °C for later extraction of input DNA. To induce plasmid expression, for each replicate two cultures were diluted in SC-URA 2% galactose medium. After 5–6 generations, 2 × 400 mL for each replicate were harvested to obtain output pellets for DNA extraction.Input and Output pellets were resuspended in 1.5 mL extraction buffer . Two cycles of freezing in an ethanol-ice bath and heating at 62 °C were performed. Deproteinization was performed using 25:24:1 phenol-chloroform-isoamyl alcohol and glass beads. After centrifugation, the aqueous phase, containing the DNA, was recovered and treated again with phenol-chloroform-isoamyl alcohol. The samples were incubated for 30 min at −20 °C with 1:10 V 3 M NaOAc and 2.2 V 100% ethanol for DNA precipitation. At this stage and after centrifugation for 30 min, the pellets were dried overnight at room temperature. RNA was eliminated by incubation with RNAse 10 mg per mL for 30 min at 37 °C. DNA purification was achieved with a QIAEX II Gel Extraction Kit (Qiagen) and DNA was eluted in 375 μL of elution buffer. DNA concentration was measured by q-PCR, with primers annealing to the Ori site of the pRS416 plasmid (Supplementary Data The TDP-43 library was then prepared for deep sequencing by PCR amplification in two steps using Q5 High-Fidelity DNA Polymerase (NEB). In step 1, 300 million plasmids were amplified for 15 cycles using frame-shifted adaptor primers with partial homology to standard Illumina sequencing primers . Plates were kept constantly shaking at 30 °C. Growth curves were fitted in order to extrapolate growth rates that correspond to the maximum slope of the linear range of the LN(OD600) curve over time.Imaging was performed by using a Confocal TCS SP8 and a Confocal TCS SP5 (Leica) equipped with PMT detectors both for fluorescence and transmitted light images. AOBS beam-splitter systems are in place on both instruments. 63X oil immersion objectives and the LAS AF software were used for all imaging. YFP fluorescence was excited with a 488 nm laser, while mCherry fluorescence with a 561 nm laser. Ranges for emission detection were 495–554 and 637–670 nm respectively. Image depth is 8-bit in all cases and pixel size equals 120.4 nm. The LUT is linear and covers the full range of the data.https://github.com/lehner-lab/tardbpdms_cellprofiler_scripts).Yeast cells expressing TDP-43 selected variants were grown in SC-URA 2% raffinose non-inducing medium and then transferred to SC-URA 2% galactose medium to induce protein expression for 8 h. They were then imaged under a Confocal TCS SP8 microscope (Leica). Counting of foci was conducted both manually and by automated pipelines using the CellProfiler software where quantification of fluorescent intensity was tracked for each focus. The coordinates of the center of each focus and nucleus were derived from CellProfiler and used to calculate distances using a custom R script  where bleaching was achieved with 488 Laser Power at 70% for three frames (1.3 s per frame) while fluorescence recovery was recorded for 50 frames. The curves were then fitted to a single exponential, following normalization, with the EasyFrap package7 cells were collected and re-suspended in 200 μL EtOH and 2.5 μL PMSF. Samples were vortexed with glass beads for 15 min at 4 °C and frozen overnight at −80 °C. The samples were dried in a speed vacuum for 20 min and resuspended in 200 μL solubilizing buffer . After boiling for 5 min, the lysate fraction was run on a NuPAGE 4–12% Bis-Tris gels (Novex) and transferred to PVDF membranes in an iBlot (Invitrogen). Membranes were blocked with 5% milk powder in TBS-T and incubated overnight at 4 °C with primary antibodies: anti-GFP mouse antibody (Santa Cruz sc-9996) and anti-PGKD1 mouse antibody (Novex 459250) diluted 1:1000 and 1:5000 in 2.5% powder milk respectively. Secondary antibody anti-proteinG was incubated for 1 h at room temperature. Proteins were detected with an enhanced chemi-luminescence system (Millipore Luminata) and visualized using an Amersham Imager 600 .Single yeast colonies were grown overnight in non-inducing medium and then diluted to 0.2 OD600 in galactose medium to induce protein expression for ~8 h. At this stage, 6 × 10https://github.com/lehner-lab/DiMSum, manuscript in prep.). DiMSum is an R package that wraps common biological sequence processing tools including FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) , cutadapt (for demultiplexing and constant region trimming), USEARCH75  and the FASTX-Toolkit (http://hannonlab.cshl.ed/fastx_toolkit/). First, 5′ constant regions were trimmed, but read pairs were discarded if 5′ constant regions contained more than 20% mismatches to the reference sequence. Read pairs were aligned (reads that did not match the expected 126 bp length were discarded) and Phred base quality scores of aligned positions were calculated using USEARCH. Reads that contained base calls with Phred scores below 30 (290–331 DMS library) or below 25 (332–373 DMS library) were discarded. Approximately five and seven million reads passed these filtering criteria in each sample corresponding to the 290–331 and 332–373 libraries respectively. Finally, unique variants were counted and merged into a single table of variant counts  per DMS library. One out of four input replicates  from each DMS library were discarded due to considerably lower correlations with the other replicates  libraries before (‘input’) and after selection (‘output’) were processed using a custom pipeline  are often not observed in the output samples  variants in each DMS library and scaled such that the error-weighted means of single STOP codon variants coincided for both DMS libraries  for the 290–331 library and 0.0004 (sd = 4 × 10−4) with little variability depending on the wild-type base. By multiplying these frequencies by the length of the doped region we calculated the probability of a misread in each variant (0.0126 for the 290–331 library and 0.0504 for the 332–373 library). Single nucleotide substitutions account for ~2 × 106 reads in a typical input sample of the 290–331 library, of which we estimate 98.74% to be “true” single nucleotide variants on the basis of a 0.0126 misread probability. Therefore, we estimate an additional 2 × 104 misreads originate from single nucleotide variants (2 × 104 = 0.0126/0.9874 × 2 × 106). In the 126 bp mutagenized region, a total of 7875 × 3 × 3 = ~7 × 104 possible double nucleotide variants exist, since each base in each pair can be mutated to one of the three other nucleotides. We therefore estimated that, even in a scenario in which single nucleotide variants are solely distributed among all possible double nucleotide variants, the additional count due to sequencing errors in the 290–331 library would be ~0.5 as it follows from the estimated additional 2.6 × 104 misreads over a total of 7 × 104 possible doubles. Similarly, additional counts due to sequencing errors would not reach 2 even in the 332–373 library, where the misread frequency was higher (0.0004).The impact of misreads (i.e. sequencing errors) was evaluated by measuring the per base error frequency in the WT sequence 10 bp upstream and 10 bp downstream of the mutagenized (doped) region. The frequency of an incorrect base call in these regions is 0.0001 (sd = 6 × 10We used simple linear regression to predict variant toxicity from (i) a collection of AA property features, (ii) a panel of scores from aggregation/structure algorithms and (iii) location with respect to the toxicity hotspot.http://www.genome.jp/aaindex/). From a total of 539 indices, we retained 379 high confidence indices with no missing values , individual residue-level scores were summed to obtain a score per AA sequence . The entire PRD AA sequence was supplied to AGADIR and all unique six-mers to ZipperDB. For the remainder, the full-length AA sequence was used.Similarly, aggregation, disorder, structure and other feature values for single mutant variants represent the difference between scores obtained using WT and single mutant AA sequences. AGADIR, Variants inside the hotspot were defined as those with mutant residue positions in the range of 312–342. Change in absolute charge (regardless of sign) is shown in Fig. 60. In brief, double mutants were classified as epistatic if they had more extreme toxicity values (below 5th percentile or above 95th percentile) than other double mutants with similar single mutant toxicities, which was estimated from non-parametric surface fits of double mutant toxicity as a function of a two-dimensional single mutant toxicity space  were excluded from epistasis quantification. We calculated position-pair enrichments for epistatic double mutants resulting in a pair-wise enrichment matrix. Diagonal entries on this matrix were imputed as column-wise mean enrichments. An epistasis correlation score matrix was then derived from this enrichment matrix by calculating the partial correlation of epistasis interaction profiles (columns of the enrichment matrix) between all pairs of positions. The rationale for the correlation score is that structurally close positions within a protein should have similar epistatic interactions with all other positions in the protein. Calculating partial correlations additionally removes transitive interactions and was found to be superior over epistasis enrichments in estimating secondary structures51. Significance of secondary structure propensities was assessed by comparison to propensities derived from 104 randomized epistasis correlation score matrices.Secondary structure propensities were calculated by testing for agreement of epistasis correlation score patterns with the stereotypical periodicities of an α-helix and β-strand, using two-dimensional kernels at each position along the diagonal of the epistasis correlation score matrixhttps://www.rcsb.org/structure/5WHN]) and mutant sequences (PDB 5WHP [https://www.rcsb.org/structure/5WHP] and 5WKB [https://www.rcsb.org/structure/5WKB]). Contact matrix values were normalised to have zero sum. Association score matrix values were normalised to have mean of zero and unit variance. Significance of LARKS structure propensities was assessed by comparison to propensities derived from 104 randomized epistasis correlation score matrices, where randomization was restricted to within-LARKS interactions, i.e., distances compatible with a six-mer.Similarly, LARKS structure propensities were calculated using PDB-structure derived contact matrices based on a minimal side-chain heavy atom distance of 4.5 Å (Supplementary Fig. Further information on research design is available in the Supplementary InformationPeer Review FileReporting SummaryDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Source Data


Timely and accurate detection of microclimates is extremely valuable for monitoring and stimulating exchanges of mass and energy in forest ecosystems under climate change. Recently, the rapid growth of wireless sensor networks (WSNs) has provided a new approach for detecting microclimates in a complex environment at multiple temporal and spatial scales. However, applications of wireless sensors in forest microclimate monitoring have rarely been studied, and the corresponding observation accuracy, error sources and correction methods are not well understood. In this study, through field experiments in two typical subtropical forest ecosystems in Zhejiang Province, China, the accuracy of the temperature and humidity observed by the wireless sensors was evaluated against standard meteorological data. Furthermore, the observation error sources were analyzed and corresponding correction models were established. The results showed that the wireless sensor-based temperature and humidity values performed well within the total observation accuracy. However, the observation errors varied with season, daily periodicity and weather conditions. For temperature, the wireless sensor observations were overestimated during the daytime while they were underestimated during the nighttime. For humidity, the data observed by the wireless sensors generally appeared as overestimates. Adopting humidity as the corrected factor, correction models were established and effectively improved the accuracy of the microclimatic data observed by the wireless sensors. Notably, our error analysis demonstrated that the observation errors may be associated with the shell material of the wireless sensor, suggesting that shading measures for the wireless sensors should be considered for outdoor work. The exact and timely acquisition of forest meteorological information, especially on forest microclimates, is very valuable for monitoring and investigating carbon and water cycles, energy balance and interactions between the canopy and ground within forest ecosystems under climate change5.Climate warming, variabilities in precipitation intensity and frequency, and resulting climatic events  have already significantly influenced the physiological and ecological processes of forest ecosystemsin situ observations using a thermometer screen or auto weather station. This approach usually ensures the observational accuracy of microclimatic variables. However, it is too expensive and requires too much labor to meet the need for continuous time monitoring over a relatively wide region due to limitations in power supply, etc. More importantly, it is infeasible to place the sensors in a complex terrain or at multiple layers and to adjust the layout regularly based on demand and supply6. Although the spatial pattern of the microclimates  of the canopy or land surface could be derived from remote sensing data, large uncertainties may be associated with the spatial and spectral resolution of the satellite7. Similarly, successive observations cannot be achieved due to the limitations in satellite transit time.Currently, the main means of monitoring forest microclimates is through 17. Wireless sensors are characterized by their low costs, high monitoring accuracy, flexible layouts and high-frequency continuous data19. These advantages make detecting microclimatic variables in a complex ecosystem  possible21. Hence, wireless sensors could provide valuable data for investigating and simulating carbon and water cycles and energy balance within an ecosystem24. Although applications of wireless sensors for forest microclimate monitoring and the corresponding evaluations have been explored25, the observational accuracy of the sensors, sources of observational bias and correction approaches are less understood28.With the developments of wireless sensor network (WSN) technology, also known as the Internet of Things (IOT), wireless sensors have been increasingly used in monitoring the ecological environmentTo fill these gaps, this study investigated the suitability of wireless sensors for forest microclimate monitoring. Two typical subtropical forest sites, a mixed broadleaf forest site and bamboo forest site, were chosen as the study sites. Compared to observations from standard meteorological instruments in the study sites, the accuracies of the temperature and humidity data observed by the wireless sensors and the error sources were detected. Then, correction models of the climatic variables were established to improve the monitoring effectiveness of the wireless sensors in forest microclimates. Achieving these goals is beneficial to guiding sensor placement in forest ecosystem and provide a scientific basis for improving the designation and data correction of wireless sensors.2 of the regression between the gradient and sensor observations were 1.04 and 0.94, respectively, and the Root Mean Square Error (RMSE) and Evaluation Accuracy (EA) were 1.94 and 87%  were removed. The valid data were compared to the corresponding standard meteorological observations at both the mixed broadleaf forest site and bamboo forest site, as shown in Fig. 87% Fig. . For humely Fig. .Figure 1Diurnal variabilities of errors of temperature and humidity observed by the wireless sensor are shown in Fig. The seasonal variations in the errors of temperature and humidity observed by the wireless sensor were detected, shown in Fig. In addition to the diurnal variations in meteorology, the variations in air humidity with weather conditions, such as cloudy and rainy, may lead to some random errors observed by the wireless sensor. Here, we further analyzed the diurnal differences between the wireless sensors and gradient system observations in clear and cloudy/rainy conditions. The cloudy/rainy condition was defined as a 5-hour duration in which the humidity was over 80% RH.The results Fig.  showed t29. The plastic was more able to dissipate heat, resulting in a lower temperature and more dew in the shell. Hence, the wireless sensor may underestimate air temperature and overestimate air humidity.In summary, the temperature and humidity errors in the wireless sensors were affected by variabilities in temperature and humidity with day and night, seasons and weather. The error characteristics implied that the observation errors may be associated with the sensor shell. The shell used in this study is plastic, and the sensor is wrapped in the shell. During a clear day, the shell was rapidly warmed by solar radiation, leading to a higher temperature in the shell than that of the ambient air and consequently an overestimate of the temperature. Nevertheless, during a clear night, a weak atmospheric counter radiation caused a low temperature and a corresponding low saturated vapor pressureAccording to the evaluations mentioned above, the errors in temperature and humidity observed by the wireless sensors may be related to the air humidity within the diurnal period, which partly resulted from the weather and the sensor in the shell. Correlations (r) between errors and the corresponding humidity observed by the gradient system were detected during the daytime and nighttime. The results showed that for temperature the daytime errors significantly and negatively related to humidity , while the nighttime errors showed a significant positive correlation with humidity . In contrast, for humidity, the daytime errors showed a significant positive relationship with humidity , while the nighttime errors exhibited a significant negative correlation with humidity . Hence, to correct the observation error in the wireless sensor, humidity was selected as the correction factor, and the error correction model of the wireless sensor was established by hour based on the linear regression function.i and bi indicate the slope and interception of linear regression at the hour, respectively.The relationships between the observation errors in the wireless sensors temperature and humidity are shown as follow:The parameters of the correction models are shown in Fig. raw (RHraw)) and ΔT (ΔRH), which was calculated considering formula The corrected T (RH) was the difference between the raw temperature (humidity) observed by the sensors . The other study site is the flux-observation site in the moso bamboo (Phyllostachys pubescens) technology park in Anji, which represents the Mao bamboo forest ecosystem31. Like the other study site, a 40-meter-high tower stands in the park , where the annual temperature, precipitation and relative humidity are approximately 16.6 °C, 1270 mm and over 70%, respectively.In this study, two typical subtropical forest flux-observation sites in Zhejiang Province, China were selected as the study sites. One study site is the Tianmushan forest ecosystem research site in the Chinese Forest Ecosystem Research Network (CFERN) in Hangzhou, which represents an evergreen and deciduous mixed broadleaf forest ecosystemThe meteorological gradient observation system (hereafter referred as the gradient system) on the two flux towers were similar, including wind speedometers, thermometers and moisture meters placed at 2 m, 7 m, 11 m, 17 m, 23 m, 30 m and 38 m along each tower. These gradient system observations were treated as the standard meteorological observations with high observation accuracy. Additionally, two infrared temperature instruments (SI-1111) were placed at 2 m and 23 m along each tower to capture the land surface and canopy temperatures, respectively. The meteorological information was automatically collected every 30 minutes.32. The measurement range for temperature is from −40 °C to 123.8 °C, and the accuracy of the measurement is ±0.3 °C at a 0.01 °C resolution. The measurement range for humidity is from 0 to 100% (RH), and the observational accuracy is ±3.0% (RH) at a 0.01% (RH) measurement resolution.The wireless sensor used in this study is the Telos-Rev.B sensor node, which loads the temperature/humidity sensor module SHT1133. Three types of nodes were implemented in this study, including wireless sensor nodes equipped with the temperature/humidity sensor (abbreviated to sensor nodes), sink notes and task manager node. The information was collected by each sensor node and then transferred to sink notes through adjacent routers. Finally, the information was transferred to the task manager nodes by the bridging ability of the sink nodes, so that it could be handled in the user terminal.In this study, the wireless sensor network was based on the GreenOrbs deploymentsThe wireless sensors were placed along the flux tower at the Tianmushan and Anji sites, and the accuracies were evaluated by the corresponding gradient system observations. The details of the experiments are described as follows. Two or more wireless sensors were placed at each layer of the flux towers and placed as close as possible to the instruments of the gradient system to reduce observation uncertainties. The layouts of the wireless sensors on the flux towers are shown in Fig. Each field experiment generally lasted 10~20 days due to the limitation of the battery life of the wireless sensor. In total, seven and ten field experiments were carried out in the Tianmushan site from September 2013 to May 2015 and Anji site from December 2011 to October 2012, respectively. Excluding the missing data due to power or sensor failures and severe weather, there were 97 site-days of valid data samples  and evaluation accuracy (EA) were also used, which are shown as the following:In this study, the overall accuracies of the temperature and humidity observed by the wireless sensors were first evaluated. Considering the potential impacts of climatic conditions and plant seasonal activities on equipment precision, observational accuracy of the wireless sensors were further detected during different seasons, at different daily periodicities and in different weather conditions. Meanwhile, the error characteristics and causes were analyzed. Then, an optimized correction factor was selected to establish correction models for adjusting the original sensor-based observations. Finally, the data before and after the correction were compared.


No studies have addressed the impact of lung cancer (LC) on prognosis of patients with idiopathic pulmonary fibrosis (IPF) in Upper Egypt. We aimed to evaluate the prevalence and risk factors for LC among IPF patients and its impact on their outcomes and survival in Upper Egypt.A total of 246 patients with IPF who had complete clinical and follow up data were reviewed. They were categorized into 2 groups: 34 patients with biopsy-proven LC and IPF (LC-IPF) and 212 patients with IPF only (IPF). Survival and clinical characteristics of the two groups were compared.p = 0.001). Survival in patients with LC-IPF was significantly worse than in patients with IPF without LC; median survival, 35 months vs 55 months; p = 0.000. LC accompanying IPF was one of the most significant independent predictors of survival in IPF patients . Mortality in LC-IPF patients was mainly due to LC progression in 36% and LC therapy-related complications in 22%.Prevalence of LC was 13.8%. Pack/years was the most significant predictor for LC development in IPF (Odds ratio; 3.225, CI 1.257–1.669, Prevalence of LC in IPF patients was 13.8%. Lung cancer has significant impacts on patients with IPF in Upper Egypt, in terms of clinical outcomes and survival. Smoking is the most significant independent predictor of LC development in IPF patients. A poorer survival was observed for patients with IPF developing LC, mainly due to LC progression, and to complications of its therapies. Further prospective, multicenter and larger studies are warranted. Idiopathic pulmonary fibrosis (IPF) is the most common of the idiopathic interstitial pneumonias and carries the worst prognosis . DespitePrevious studies have addressed the clinical risk factors associated with LC development in IPF patients , 5–7. A Previous studies had compared LC profiles in patients with and without IPF –10. HoweAssiut University Hospital (AUH) and South Egypt Cancer Institute (SECI) are two large tertiary hospitals serving a large number of populations at Upper Egypt.A systematic search of the patient database at the Chest Department of AUH revealed 267 patients who fulfilled the international guidelines on the diagnosis and management of IPF , during These 246 cases were categorized in two groups: (1) the group of 34 patients who had (at the time of diagnosis of IPF) or developed LC (biopsy-proven) during the follow up period, named the LC-IPF group. Once diagnosed with LC, the patients were referred to the Medical Oncology Department, SECI, for further oncologic management, and (2) the group of 212 patients with IPF only (IPF group). The 2 groups were compared for clinical features, outcomes, and survival analysis. The later was carried out in all cases from the time of IPF diagnosis.The medical records of all IPF patients were reviewed to obtain data regarding age, smoking history, the method used to diagnose IPF, time of IPF diagnosis, PFT, HRCT findings, comorbidities, follow up duration, and outcome. Pack/years value was calculated by multiplying the number of packs of cigarettes smoked per day by the number of years the person has smoked . The finFor the LC-IPF group, the time of diagnosis and location of LC including the IPF-associated area were analyzed. All cases of LC were biopsy-proven. Lung cancers were classified according to the World Health Organization classification. Staging of LC has been established by the TNM system current at the time of diagnosis. Side effects of treatments were assessed using the National Cancer Institute Common Terminology Criteria for Adverse Events  . EvaluatThe study was approved by the Ethical Committee of AUH and SECI.χ2 test for categorical variables. Logistic regression analysis was performed to assess the risk of LC in IPF patients. Survival was estimated using Kaplan-Meier curves. The survival rates of patients in the IPF only group and the IPF with lung cancer (LC-IPF) group were compared using a log-rank test. Cox’s proportional hazards regression analysis was used to identify significant variables that affect the survival of IPF patients and to estimate hazard ratios (HR) and 95% confidence intervals (CI) for predictors of survival. All tests were two-tailed, and p < 0.05 were considered statistically significant. SPSS software Version 22.0  was used for all statistical analyses.Patient demographics were compared using the unpaired t-test for continuous variables and the Pearson’s 1), diffusing capacity of the lung for carbon monoxide (DLCO), and AEIPF.The prevalence of LC in IPF patients was 13.8%. Table Among the 34 patients with LC-IPF, 12 (35.3%) were diagnosed as having primary pulmonary LC at the same time of IPF diagnosis. The other 22 (64.7%) patients developed LC 19.4 ± 16.8 months  after diagnosis of IPF during the follow up period. The cumulative incidence of LC at 1 and 3 years was 37.2 and 62.5%, respectively.Most of the cancerous lesions were located in peripheral areas of the lung (73.5%). Two-thirds (67.6%) of the lesions were in the lower lobes. Also, two-thirds of the cancerous lesions were located in a fibrotic background (IPF-associated areas) in the same patients. Squamous cell carcinoma (44%) and adenocarcinoma (41%) were the most common histological findings in LC-IPF patients.Eight 23.3%) patients had early stage I lung cancer. Five (15%), 9(26.4%), and 12(35.3%) patients had stage II, III, and IV, respectively. Table 3.3% patiLCO were analyzed. Multivariate regression after adjustment for significant factors revealed that pack/years and male gender were significant independent predictors of LC in IPF patients  patients experienced therapy-related complications. Surgery for early LCs consisted of four lobectomies. Three patients (75%) had complications, mainly pneumonia. Estimated 30-day mortality was 25%. Complications were reported in 3/5 (60%), 1/2(50%), and 10/13(77%) of patients who underwent radiotherapy, radio-chemotherapy, and chemotherapy, respectively. Those who received radio- or radio-chemotherapy were among the 11 patients with non-fibrotic lung pathology. Among 13 patients treated with chemotherapy only, 5 (38.5%) patients died with a median survival of 7 months . Table p = 0.000, log-rank test). One- and 3-year survivals among the two groups were 90% and 52% in the LC-IPF group and 98 and 83% in the IPF group, respectively. Survival curves of patients with IPF with and without LC are displayed in Fig. Among the 212 patients with IPF without LC, 93(43.8%) died, median survival was 55.0 months, while in the group of patients with LC-IPF, 28(82.3%) patients died, median survival was 35.0 months. Survival time was significantly different between the 2 groups (p = 0.000). The other significant predictor was smoking . Table Lung cancer accompanying IPF was one of the most significant independent predictors of survival in IPF patients  patients died of lethal complications related to LC therapies. Four (14%) died of AEIPF. Ten (36%) patients died of LC progression, and eight (28%) patients died of respiratory failure related to IPF.p = 0.087. After adjusting for age, gender, smoking status, % predicted DLCO, and % predicted FVC, there was no difference .Median follow up duration was 32 months  in the LC-IPF group and 42.6 months  in the IPF group. During the follow up period, 24 patients among 34 with LC (70.6%) and 122 among 212 without LC (57.5%) experienced IPF progression. Median time-to-disease progression was 24 months in the LC-IPF group and 27 months in the IPF group. There was not a significant difference in progression-free survival between the two groups; p = 0.032, respectively.  experienced AEIPF during the study period, with a significant difference between the 2 groups; 18/34 (53%) were in the LC-IPF group, and 69/212 (32.5%) were in the IPF group, To the best of our knowledge, this is the first study that addresses the impact of LC on IPF patients in the Middle East and Egypt. Results showed that LC has significant impacts on clinical outcomes and survival of patients with IPF in Upper Egypt. Demographic features of patients with IPF and/or LC could be different in our locality. Mohamed and Ibrahim , in theiOur data revealed that the prevalence of LC in IPF patients is 13.8% in all (8.9% in LC onset during the IPF follow up). The cumulative incidence of LC among IPF patients at 1 and 3 years was 37.2 and 62.5%, respectively. Our results are consistent with the worldwide reported prevalence range of 4.8 to 48%  and TomaIt has been observed that LC was more common among IPF patients who are older, males, and smokers. These findings are consistent with those of previous reports enrolling different populations –7, 9, 10Similarly, there was no consensus regarding the most prevalent histologic type of LC-IPF –10. SquaConsidering the clinical, radiologic, and pathologic findings of our cohort together may support the current hypothesis that considers IPF as a disease of premature aging with several links to LC , 8, 24. Previous studies have highlighted the clinical risk factors associated with LC development in IPF patients –7, 25. Ip = 0.000). LC accompanying IPF was one of the most significant independent predictors of survival in IPF patients .Survival analysis of our cohort showed interesting results. Survival time was significantly different between LC-IPF and IPF only , while Tomassetti et al [p = 0.001).Our results are in strong agreement with previous reports –7. Lee ati et al , observeThe majority of deaths among LC-IPF patients seen in the current study was not due to worsening of pulmonary fibrosis, but to both LC progression and complications of LC treatment; in 36 and 22% of patients, respectively. Moreover, it was observed that 50% of AEs in patients with LC were triggered by therapies for LC. These risks make the decisions about LC management in IPF, despite undertaken by a multidisciplinary team, still challenging to the clinician.Furthermore, two-thirds of patients with LC-IPF 23/34, 67.6%) had advanced cancer and received either chemotherapy (with a high rate of complications) or best supportive care. Despite improvements in our understanding of pathogenetic mechanisms for the development of LC among IPF patients , 67.6% h.Several studies had addressed the impact of surgical resection of LC in patients with IPF. Early postoperative mortality ranged from 0 to 18.2% and postoperative morbidity ranged from 7.1 to 40.7% , 28. NotThis high rate of surgery-related complications, again highlights the importance of annual HRCT for patients with IPF, for early detection of LC which might give the patient better surgical therapeutic options, hence better outcomes.There are only sparse data concerning radiotherapy in LC with IPF . DespiteAcute exacerbations have significant impact on IPF patients, particularly those with LC-IPF, with considerable morbidity and mortality. In the literature, the incidence of treatment-related AEs in patients with LC-IPF ranged between 12.5 and 30% and mortality of treatment-related AE ranged between 9 and 16% . HoweverDespite the current study is the first one in Egypt and the Middle East that describes the impact of LC on IPF in a unique group of populations, yet it is to be considered that the inherent disadvantage of being a retrospective study, together with its single-center design, and the relatively limited number of patients are important limitations. The impact of LC on IPF and the optimal management of LC in patients with IPF needs to be addressed in larger prospective multicenter studies. Finally, our data from Upper Egypt are generally similar to those previously reported from Western and Asian populations.Lung cancer has significant impact on patients with IPF in Upper Egypt, in terms of clinical outcomes and survival. Smoking and male gender were significant independent predictors of lung cancer development in IPF patients. A poorer survival rate was observed in patients with IPF developing LC, mainly due to LC progression, and to complications of its treatments. Chemotherapy and surgery had a high incidence of fatal complications. Careful monitoring of the patients for these complications is needed. Further prospective, multicenter and larger studies are warranted.


DDX11, E2F1,NPTX1, and PLXNA4) which might be repressed by the upregulated hsa-miR-486-3p. According to the proposed functions of DDX11, E2F1, and PLXNA4 reported in previous studies, these gene expression changes together might contribute to decreased proliferation of fibroblasts and decreased angiogenesis in the microenvironment of IPF. Our findings need further studies to confirm.Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and fatal interstitial lung disease. Therapeutic options for IPF remain limited. Nintedanib, a tyrosine kinase inhibitor approved for IPF treatment, is known to inhibit fibroblasts proliferation, migration and transformation to myofibroblasts. However, how nintedanib changes gene regulations in IPF has never been systematically investigated. We conducted a next-generation sequencing and bioinformatics study to evaluate the changes of mRNA and miRNA profiles in IPF fibroblasts treated with 2 µM and 4 µM nintedanib, compared to those without treatment. We identified 157 upregulated and 151 downregulated genes and used STRING and DAVID databases for analysis of protein–protein interactions, biological pathways, and molecular functions. We found strong protein–protein interactions within these dysregulated genes, mostly involved in the pathways of cell cycle and mitotic cell cycle. We also discovered 13 potential miRNA–mRNA interactions associated with nintedanib treatment. After validation using miRDB, TargetScan, and RT-qPCR, we identified 4 downregulated genes ( Idiopathic pulmonary fibrosis (IPF) is a chronic and fatal interstitial lung disease, characterized by rapid progression of lung fibrosis with usual interstitial pneumonia (UIP) as the typical histologic pattern ,2. The pTo date, therapeutic options for IPF remain limited. Nintedanib and pirfenidone are the only two drugs demonstrated to be effective for IPF . NintedaCTGF and SRF may play key roles in lung fibrogenesis [IPF is considered as a complex genetic disorder . Severalogenesis . How antogenesis . They foThe rapid evolution of next-generation sequencing (NGS) technology provides a powerful tool for systematic analysis of transcriptomic profiles ,21. AfteThe flowchart of study design is illustrated in 2-containing incubator in FGM™-2 Fibroblast Growth Medium-2 Bulletkit™  containing 0.5 mL human fibroblast growth factor-basic (hFGF-B), 0.5 mL insulin, 10 mL fetal bovine serum, and 0.5 mL GA-1000. The medium was changed every 2–3 days and the cells were passaged at 80–90% confluence for the following experiments.Human IPF lung fibroblasts , purchased from Lonza Inc. , were incubated at 37 °C in a 5% CONintedanib , purchased from Selleckchem , was dissolved in dimethyl sulfoxide (DMSO)  to obtain various concentrations. The reagents were stored at −20 °C until use in the experiments. In nintedanib-treated cultures, cells were treated with 1 µM, 2 µM, and 4 µM nintedanib for 24 h, 48 h, and 72 h. In control cultures, cells were treated with the carrier solvent (0.1% DMSO). All experiments were performed in triplicate. The selection of nintedanib concentrations of 1–4 µM in this study was mainly based on previous study evaluating the effects of nintedanib on IPF fibroblasts .5 cells/plate). When the confluence reached 80%, carrier solvent (0.1% DMSO), 1 µM, 2 µM, or 4 µM nintedanib were added into plates. Cell morphological changes were observed and photographed with an inverted-phase contrast microscope at 24 h, 48 h, and 72 h after nintedanib treatment.Human IPF lung fibroblasts were incubated in 6 cm plates (1 × 103 cells/well) were incubated to confluence of 80% and treated with carrier solvent (0.1% DMSO), 1 µM, 2 µM, or 4 µM nintedanib for 24–72 h. Then 10 µL Premixed WST-1 reagent was added into each well, followed by incubation at 37 °C for 30 to 150 min. Quantification of cell proliferation and viability was measured by detecting the absorbance at 450 nm using a multi-well plate reader . Cell proliferation was expressed as percentage, compared with controls .Cell proliferation and viability were assessed using the Premix WST-1 Cell Proliferation Assay System , according to the manufacturer’s instructions. In brief, the Premix WST-1 assay measures cell proliferation and cell viability with a colorimetric assay, based on cleavage of tetrazolium salts by mitochondrial dehydrogenase in viable cells. Human IPF lung fibroblasts seeded in the 96-well plates (5 × 105 cells/well) were incubated to confluence of 80% and treated with carrier solvent (0.1% DMSO), 1 µM, 2 µM, or 4 µM nintedanib for 24–72 h. Harvested cells were double stained with Annexin V and propidium iodide (PI), then analyzed by the BD Accuri™ C6 Plus personal flow cytometer . Cell apoptosis was expressed as the percentage of cells with Annexin-V and PI double staining in total cells.Cell apoptosis was assessed using the Annexin V-FITC Early Apoptosis Detection Kit , according to the manufacturer’s instructions. In brief, human IPF lung fibroblasts seeded in the 6 cm plates  according to the instruction manual. Purified RNA was quantified at OD260 nm by using a ND-1000 spectrophotometer  and qualitated by using a Bioanalyzer 2100  with RNA 6000 LabChip® kit . The small RNA library construction and deep sequencing were carried out at the biotechnology company Welgene . Samples were prepared using Illumina sample preparation kit according to the TruSeq Small RNA Sample Preparation Guide. The 3′ and 5′ adaptors were ligated to total RNA and reverse transcription followed by PCR amplification. The enriched cDNA constructs were size-fractionated and purified on a 6% polyacrylamide gel electrophoresis and the bands containing the 18–40 nucleotide RNA fragments (140–155 nucleotide in length with both adapters). Libraries were sequenced on an Illumina instrument (75 single-end cycles). Sequencing data was processed with the Illumina software. Small RNA sequencing data was also obtained and analyzed. Initially, the sequences generated went through a filtering process to obtain qualified reads. Trimmomatic (version 0.36) [The expression profiles of miRNAs and mRNAs were examined by using an NGS platform. Total RNA was extracted by TRIzolon 0.36)  was implon 0.36)  to clip p values were calculated by cuffdiff (Cufflinks version 2.2.1) with non-grouped samples using the “blind mode”, in which all samples were treated as replicates of a single global "condition" and used to build one model for statistical test [p values were calculated. Genes with FDR p values <0.05  > 1.3) in 4 µM nintedanib-treated IPF fibroblasts and >2-fold changes (FPKM in 4 µM nintedanib-treated IPF fibroblasts / FPKM in controls) in a dose-dependent manner (fold change in 4 µM nintedanib-treated IPF fibroblasts > fold change in 2 µM nintedanib-treated IPF fibroblasts) were considered as significantly dysregulated genes. For analysis of functions and pathways associated with nintedanib treatment, we uploaded all significantly dysregulated genes into the STRING and DAVID database.For transcriptome sequencing, the Agilent’s SureSelect Strand Specific RNA Library Preparation Kit was used for preparing libraries. Then the libraries were sequenced on a Solexa platform (150 paired-end cycles), using the TruSeq SBS Kit . Similar with small RNA sequencing data, reads with low quality score were trimmed and removed using the Trimmomatic (version 0.36). Qualified reads were analyzed using HISAT2 (version 2.1.0) , and gencal test ,27. To chttp://mirmap.ezlab.org/) [miRNA have the ability to repress the expression of protein-coding genes. miRmap is an open-source software library which can provide the prediction of comprehensive miRNA targets (ab.org/) . By calchttp://www.targetscan.org). This is an online database. The results of predictions are ranked by the predicted efficacy of targeting or by their probability of conserved targeting [TargetScan predicts the target of miRNA by searching for the presence of conserved 8mer, 7mer, and 6mer sites matching the seed region of each miRNA (argeting . TargetShttp://mirdb.org) [miRDB provides web-based microRNA-target prediction and functional annotations (rdb.org) ,31. In mhttps://string-db.org/) has collected and integrated this information, by consolidating known and predicted protein–protein association data for many different organisms. STRING offers the information of the associations include direct  interactions and indirect  interactions. Interactions in STRING are derived from five main sources: Conserved Co-Expressions, High-throughput Lab Experiment, Genomic Context Predictions, Automated Text-mining, and Previous Knowledge in Database [The functional interactions between expressed proteins in cells are very important and complicated. The Search Tool for the Retrieval of Interacting Genes (STRING) database (Database .https://david.ncifcrf.gov/) [p value. The reference score represents how specifically the user genes are involved in the category . In this study, we selected EASE score = 0.1 as default and define pathways with a Bonferroni p value < 0.05 as significant.The Database for Annotation, Visualization and Integrated Discovery (DAVID) is a powerful tool for gene functional classification (rf.gov/) . It inte® Reagent (Thermo Fisher Scientific). The mRNAs and miRNAs were reverse transcribed using the PrimeScript RT reagent Kit   and the Mir-X miRNA First-Strand Synthesis Kit , respectively. The mRNA and miRNA expressions were assessed using real-time analysis with Fast SYBR Green Master Mix  catalog number: 4385612)) on the QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific). The primers used are listed in −ΔΔCt) was used to determine relative mRNA and miRNA expression levels.The expression levels of mRNAs and miRNAs in IPF fibroblasts treated with 0.1% DMSO (control) and 4 µM nintedanib for 24 h were assessed with RT-qPCR. In brief, total RNA was extracted using TRIzolThis study aimed to evaluate the gene expression changes associated with nintedanib treatment in IPF fibroblasts. To determine the optimal concentrations and treatment duration of nintedanib, we first performed cell studies to test the effects of 1 µM, 2 µM, or 4 µM nintedanib with treatment duration of 24 h, 48 h, 72 h, on cell morphology, proliferation, and apoptosis in IPF fibroblasts. p < 0.05) shown in red (upregulation) and green (downregulation). In summary, 84 upregulated genes and 32 downregulated genes were identified in IPF fibroblasts treated with 2 µM nintedanib; while 161 upregulated genes and 206 downregulated genes was identified in IPF fibroblasts treated with 4 µM nintedanib. As expected, more dysregulated genes were identified in IPF fibroblasts treated with 4 µM nintedanib than in fibroblasts treated with 2 µM nintedanib. The density plots of deep sequencing results among 3 groups were showed in The raw sequencing data in this study were uploaded to the GEO repository with the GEO accession number of GSE124786, GSE124787, and GSE124788. The volcano plots of gene expression data in IPF fibroblasts associated with nintedanib treatment were shown in p value of <1.0 × 10−16 , cell cycle process , mitotic cell cycle process , mitotic cell cycle .The 157 significantly upregulated and 151 significantly downregulated genes were used for protein–protein interactions, biological pathway, and molecular function analysis. Firstly, we uploaded these 308 dysregulated genes into the STRING database for protein–protein interaction (PPI) network analysis. We set the minimum required interaction score as 0.900 (highest confidence) and removed protein nodes without interactions. This analysis obtained a highly interactive PPI network of 307 nodes (nodes represent proteins) and 1227 edges , with PPI enrichment  × 10−16 . AccordiSecondly, we used DAVID to analyze possible mechanisms of the top dysregulated genes in IPF fibroblasts related to nintedanib treatment. In biological process A, cell dDDX11, E2F1, NPTX1, and PLXNA4, and interaction of hsa-miR-92a-1-5p with SLC25A23 were validated in both TargetScan and miRDB database. The details of possible binding sites in these 5 validated miRNA–mRNA interactions were provided in the Using the miRmap database with a criteria of miRmap score ≥99.0, we predicted 568 target mRNAs from 46 significantly downregulated miRNAs and 640 target mRNAs from 36 significantly upregulated miRNAs. Venn diagram analysis of the 568 predicted targets (of 46 downregulated miRNAs) and 157 upregulated mRNAs identified 7 upregulated genes with potential miRNA–mRNA interactions in nintedanib-treated IPF fibroblasts; while analysis of the 640 predicted targets (of 36 upregulated miRNAs) and 151 downregulated mRNAs identified 5 downregulated genes with potential miRNA–mRNA interactions . Table 1DDX11, E2F1, NPTX1, and PLXNA4 were significantly lower in nintedanib-treated IPF fibroblasts than in controls, and the expression levels of SLC25A23 were significantly higher in nintedanib-treated IPF fibroblasts than in controls  and 4 µM nintedanib for 24 h. For mRNAs, the expression levels of controls A. For micontrols B. The dyIPF is the most common among idiopathic interstitial pneumonias. The incidence of IPF ranges between 0.22 and 17.4 per 100,000 population and the patients with IPF are usually in the sixth or seventh decade of life . BecauseSLC25A23 (might be de-repressed by downregulated hsa-miR-92a-1-5p) and downregulated DDX11, E2F1, NPTX1, and PLXNA4 (might be repressed by the upregulated hsa-miR-486-3p) were validated as the dysregulated genes with potential miRNA–mRNA interactions associated with nintedanib treatment. The dysregulations of these mRNAs and miRNAs were further validated with RT-qPCR, except for hsa-miR-92a-1-5p.The STRING analysis showed strong protein–protein interactions within the top dysregulated genes. Interestingly, most genes in the PPI network were related to biological pathways of cell cycle, cell cycle process, mitotic cell cycle process, and mitotic cell cycle. The DAVID analysis revealed that the role of nintedanib in treating IPF fibroblast might most possibly relate to the biological process of cell division, mitotic nuclear division, sister chromatid cohesion, and DNA replication. We also identified 7 upregulated genes and 5 downregulated genes with potential miRNA–mRNA interactions. After validation in TargetScan and miRDB, upregulated DDX11 was downregulated after nintedanib treatment. DDX11 is the predicted target of hsa-miR-486-3p, which is upregulated in nintedanib-treated IPF fibroblasts. DDX11, also known as CHL-1, is able to promote cell proliferation, metastasis, and migration in human glioma cells [DDX11 in IPF fibroblasts, our data suggest that nintedanib may suppress DDX11 in IPF fibroblasts and therefore suppress the proliferation of IPF fibroblasts. According to our NGS data, the expression change of DDX1 in nintedanib-treated IPF fibroblasts was in a dose-dependent manner. Therefore, we hypothesized that nintedanib might decreased proliferation of IPF fibroblasts through regulation of hsa-miR-486-3p-DDX11.Our data showed that ma cells . AlthougPLXNA4. PLXNA4 is also the predicted target of the upregulated hsa-miR-486-3p in nintedanib-treated IPF fibroblasts. PLXNA4 may promote progression and angiogenesis of tumor, possibly by enhancement of VEGF and bFGF signaling pathways [PLXNA4 may be associated with decreased angiogenesis. Therefore, our data suggest the changed expressions of hsa-miR-486-3p-PLXNA4 associated with nintedanib treatment may contribute to decreased angiogenesis in IPF.We found that nintedanib treatment was associated with downregulation of pathways . DownregE2F1 was downregulated after nintedanib treatment. Similar to DDX11 and PLXNA4, E2F1 is the predicted target of hsa-miR-486-3p. E2F1 protein is a member of the E2F family of transcription factors. The E2F1 transcription factor play critical roles in the regulation of cell cycle and apoptosis [E2F1 [E2F1. Our interaction analysis suggests that upregulation of hsa-miR-486-3p might have an interaction with downregulation of E2F1 in nintedanib-treated IPF fibroblasts. However, this hsa-miR-486-3p-E2F1 interaction was predicted from the bioinformatic database. How hsa-miR-486-3p, E2F1 transcription factors, and growth factors interact with each other in nintedanib treatment need further studies to confirm and elucidate.Our data also showed that poptosis . It is cpoptosis . Severalis [E2F1 ,39,40,41NPTX1 was also the predicted target of hsa-miR-486-3p. The protein encoded by NPTX1 is a member of the neuronal pentraxin family. The expression of this gene is mainly localized to the nervous system [SLC25A23, which might have an interaction with the downregulated miRNA hsa-miR-92a-1-5p based on our bioinformatic prediction. SLC25A23 is a member of the mitochondrial transporter family SLC25 embedded in the inner mitochondrial membrane [SLC25A23 has been shown to augment mitochondrial matrix Ca2+ influx, and may induce oxidative stress-mediated cell death [SLC25A23 and enhance cell death of fibroblasts. However, the downregulation of hsa-miR-92a-1-5p in nintedanib-treated IPF fibroblasts was not confirmed by the RT-qPCR method.Another downregulated gene s system . The onlmembrane . SLC25A2ll death . AlthougDDX11, E2F1, and PLXNA4. These gene expression changes together might contribute to decreased proliferation of fibroblasts and decreased angiogenesis in the microenvironment of IPF (To the best of our knowledge, this is the first study using NGS and bioinformatics to scan mRNA and miRNA changes following nintedanib treatment. We identified 308 significantly dysregulated genes associated with nintedanib treatment in IPF fibroblasts. We found strong protein–protein interactions within these dysregulated genes, mostly involved in the pathways of cell cycle and mitotic cell cycle. We also identified 1 upregulated and 4 downregulated genes with potential miRNA–mRNA interactions. According to our data from nintedanib-treated IPF fibroblasts and the literatures, we proposed that upregulation of hsa-miR-486-3p following nintedanib treatment might be associated with decreased expression of t of IPF . Our finDDX11, E2F1, and PLXNA4 expressions. These together might contribute to decreased proliferation of fibroblasts and decreased angiogenesis in the microenvironment of IPF. However, this is a screening study using primary IPF lung fibroblasts and bioinformatics approaches. Our findings need further studies to confirm.We identified 308 significantly dysregulated genes associated with nintedanib treatment in IPF fibroblasts. We found strong protein–protein interactions within these dysregulated genes, with most genes involved in the pathways of cell cycle and mitotic cell cycle. In IPF fibroblasts, we found nintedanib treatment was associated with upregulation of hsa-miR-486-3p, which might repress


Exhaled biomarkers may be related to disease processes in idiopathic pulmonary fibrosis (IPF) however their clinical role remains unclear. We performed a systematic review to investigate whether breath biomarkers discriminate between patients with IPF and healthy controls. We also assessed correlation with lung function, ability to distinguish diagnostic subgroups and change in response to treatment.MEDLINE, EMBASE and Web of Science databases were searched. Study selection was limited to adults with a diagnosis of IPF as per international guidelines.alvNO) levels were significantly higher in IPF (8.5 ± 5.5 ppb) than controls (4.4 ± 2.2 ppb). Markers of oxidative stress in exhaled breath condensate, such as hydrogen peroxide and 8-isoprostane, were also discriminatory. Two breathomic studies have isolated discriminative compounds using mass spectrometry. There was a lack of studies assessing relevant treatment and none assessed differences in diagnostic subgroups.Of 1014 studies screened, fourteen fulfilled selection criteria and included 257 IPF patients. Twenty individual biomarkers discriminated between IPF and controls and four showed correlation with lung function. Meta-analysis of three studies indicated mean (± SD) alveolar nitric oxide  contains supplementary material, which is available to authorized users. LCO) of 15% has been shown to be an accurate predictor of mortality in IPF , markers of nitrosative stress (nitrite and nitrate), metallic elements, and cytokines. Two studies found significantly higher levels of 8-isoprostone in IPF patients (25-74 pg/mL) compared to controls (5-33 pg/mL) [21H44N2O but were unable to identify a metabolite from known databases.Eight studies measured biomarkers present in EBC in patients with IPF and healthy controls , 58–64. 3 pg/mL) . One stu3 pg/mL) . There wTwo studies assessed VOCs in IPF patients and healthy controls , 66. YamalvNO levels in 27 IPF patients whose lung function had been declining in the previous 6–12 months to those with stable lung function. CalvNO levels were noted to be significantly higher in the 16 patients with declining lung function. Psathakis et al [LCO % predicted with both H202 and 8-isoprostane although no association was noted with other measures of pulmonary function. Yamada et al [LCO. Ethylbenzene had a negative correlation with %DLCO/VA (diffusion capacity per litre lung volume). No correlation was noted with levels of cysteinyl leukotrienes (CysLTs), Docosatetraenoyl (22:4) LPA or metallic elements in EBC and lung function parameters in IPF [Six studies assessed correlation between breath biomarkers and lung function in IPF patients , 66–68. is et al  found a s in IPF , 63, 67.alvNO levels in IPF patients receiving treatment with immunosuppression  and untreated patients. Corradi et al [Two studies compared breath biomarkers in IPF patients receiving treatment. Kotecha et al , found ndi et al  found thalvNO levels in IPF patients and controls and were included in meta-analysis [alvNO using both the Tsoukias and George method [alvNO using the Tsoukias and George method was 4.4–11.8 ppb for IPF patients and 2.9–5.1 ppb for healthy controls. Using the Condorelli method the range was 3.9–10. 7 ppb for IPF patients and 1.7–4.5 ppb for healthy controls. We performed meta-analysis to compare mean difference in CalvNO (ppb) levels between IPF patients and healthy controls, using both Tsoukias and George and Condorelli methods. Figure alvNO was significantly higher in patients with IPF using either method, however the total number of patients included was small (n = 101) and significant heterogeneity between the studies was noted .Three studies compared Canalysis –57. Eache method  and corre method . The ranThis systematic review has highlighted the heterogeneity of results from breath research in IPF. Over 60 known biomarkers and multiple unknown molecules have been investigated within a total patient cohort of less than 300 patients using a wide variety of analytical techniques. In this context, it is difficult to draw any firm conclusions regarding potentially clinically relevant markers. Twenty individual biomarkers were reported to discriminate between IPF and healthy controls however most of the studies did not include a validation cohort and there was an absence of longitudinal data. Only two of these biomarkers, exhaled NO and 8-isoprostane, have been studied more than once.50 is the most common method used for measuring and reporting NO in breath. NO measured at this flow rate contains a higher proportion of airway and lower proportion of alveolar NO [50 did not show consistency in IPF, [50 levels in IPF patients ranged from 20.8–31.5 ppb [50 in healthy adults ranges from 30.3–50.6 ppb depending on age and gender [Exhaled NO is a breath biomarker which is easily measurable and already used in asthma care –35. NO ieolar NO . FeNO50  in IPF, , 57 or o in IPF, , 74. FeN31.5 ppb , 57, 67.d gender .50 is unlikely to be of use in IPF, CalvNO may have potential as a clinical biomarker. Meta-analysis in this review, while limited by small sample size and significant heterogeneity, suggests that CalvNO is higher in IPF patients compared to controls. Average CalvNO levels for patients with IPF were 8.5 (±5.5) ppb compared to 4.4 (±2.2) ppb for healthy controls. In a recent study of 433 healthy individuals, the upper limit of CalvNO reported was 3.88-3.93, depending on age [alvNO, but not FeNO, has also been reported to be elevated in ILD related to systemic sclerosis [alvNO may be in disease monitoring. Kotecha et al [alvNO levels were reflective of deteriorating lung function. This is consistent with other studies suggesting that CalvNO levels are inversely related to lung function parameters in fibrotic ILD, a phenomenon not seen with FeNO [alvNO levels between patients treated with immunosuppressive therapy and untreated patients [Although, FeNOg on age . Elevateg on age , 77. Calclerosis –80. Incrclerosis , 81–85. ha et al  suggesteith FeNO , 81, 83.patients . Lower lpatients . In bothpatients . No studRespiratory Research has identified all the relevant markers of oxidative stress investigated in IPF [202, a reactive oxygen species, and 8-isoprostane, a prostaglandin-like compound formed by lipid peroxidation. Both were found to be elevated in EBC in patients with IPF [Several studies investigated biomarkers in EBC implicated in the pathogenesis of IPF –64, 67. d in IPF . These iwith IPF , 62. 8-iwith IPF , 89 MDA,with IPF . Howeverwith IPF .Another study reported differences in concentration of metallic elements in EBC of patients with IPF compared to controls . Metal wCytokines, such as tumour necrosis factor-α (TNF-α) and TGF-β, have been strongly implicated in cell signalling in IPF , 95. How2O2 did appear to have a negative correlation with DLCO % predicted in IPF but no relationship with other lung function parameters [2O2 and lung function in 21 patients with mixed ILD including eight patients with IPF [2O2 have both been reported to be elevated in airways disease [2O2 levels have been reported to be lower in patients with mixed ILD compared to obstructive lung disease [The evidence for EBC biomarkers in IPF is limited. These were primarily exploratory studies involving small cohorts and apart from 8-isoprostane, have not been reproduced. Currently a biomarker with diagnostic potential has not been identified. There is no evidence that EBC biomarkers distinguish between IPF and other forms of fibrotic ILD. Likewise, evidence is limited regarding their value as a prognostic marker. Levels of 8-isoprostane and Hrameters . Anotherwith IPF . EBC is  disease –103, whi disease . H2O2 le disease . Identif disease . UnfortuVOCs offer a promising alternative source of breath biomarkers in IPF. The advantage of breath volatiles is that they could potentially travel from the blood or interstitium unaltered before detection in exhaled breath and therefore contain metabolites with high specificity for the fibrotic process. VOC work has been limited in IPF to date. Yamada et al  undertooThe process of biomarker development is often described as the “biomarker pipeline” and thus far none of the potential candidates have progressed past the biomarker discovery phase . It can The hope is that biomarker discovery studies inform our understanding of the pathophysiology of IPF and improve accuracy of diagnosis. In addition, novel biomarkers may be able distinguish some of the phenotypes that appear to exist within IPF . ProgresThere are several limitations to this systematic review. We defined IPF based on international consensus guidelines , 50. ThialvNO levels are significantly higher in patients with IPF compared to healthy controls. CalvNO, 8-isoprostone, hydrogen peroxide and p-cymene demonstrated correlation with lung function parameters in IPF patients. There was a lack of studies investigating relevant treatment options in IPF. None of the studies compared breath biomarkers in patients with IPF in relation to the presence of honeycombing. Recent studies have indicated that a “breathomic” approach may identify biomarkers with specificity for IPF [In this PRISMA-compliant systematic review we identified a heterogeneous group of studies investigating a wide variety of breath biomarkers in IPF using a range of analytical techniques. Evidence for the use of specific biomarkers was inconclusive. Twenty individual biomarkers were reported to discriminate between IPF and healthy controls. Meta-analysis of three studies indicated that C for IPF , 66. ThiAdditional file 1:Search results per database. (DOCX 12 kb)Additional file 2:Studies excluded following full text review (DOCX 18 kb)Additional file 3:Summary of QUADAS-2 assessment for each study. (DOCX 15 kb)


In particularly, the different fabrication protocols, and the electrochemical performance of the composites are summarized in detail. The structural and morphology characters of graphene sheets that may affect the property of the composites are discussed briefly. The possible ongoing researches in area are speculated upon.This mini-review highlights selectively the recent research progress in the composites of LiFePO A 3D cross-sectional view of LFP formation is shown in Fig. Significantly, the LFP/graphene composites assuming more complicated 3D hierarchical structure have been prepared through a facile template-free sol–gel procedure, as shown in Fig. −1 (in comparison with 10−9 S cm−1 of pure LFP), a high lithium-ion capacity of 168 mAh g−1  at the 0.5 C rate, and a high rate capacity of 115 mAh g−1 at the 10 C rate. Moreover, the as-prepared LFP/graphene composite shows high cycling stability over the composites prepared through other methods.As mentioned above, no matter using the physical mixing of LFP with graphene sheets or the in situ growing of the LFP on graphene sheets, the preparation of individual graphene sheets is prerequisite. Additionally, the individual graphene sheets usually aggregate easily, which affects the distribution of the graphene sheets within the composites that influence finally the electrochemical performance of the composites , 63. To 4 as catalyst, Li et al. [2 (95:5) at 750 °C, see Fig. −1, and shows high rate performance and prolonged cycling life as shown in Fig. Similarly, using glucose as carbon source and FeSOi et al.  prepared+ diffusion through a defect-free perfect graphitic plane is rather limited [+ to be very easily inserted and extracted via the holes, which was experimentally verified by the HRTEM analysis of the graphene used. To get insight the lithium diffusion pathway through the basal plane of graphene layers and the influence of defect population, Yao et al. [It is well known that the graphene is a typical anisotropic material. The electron and lithium-ion transportations along the longitudinal and horizontal (in plane) directions should be different , 66. Thu limited . However limited  proposedo et al.  prepared+ diffusion through the planar structure of graphene. This indicates additionally that the steric hindrance from the coating layer on the LFP for ion diffusion should be considered in further design of the surface modification of LFP.Actually, the lateral size of graphene sheets is also a key factor influencing the electrochemical performance of LFP/graphene composite as cathode in LIBs. For instance, Yang and colleagues  showed tBesides the orientation and the lateral size, the edge structure and thickness of the graphene sheets can also affect the property of LFP. Uthaisar and Barone  demonstr−1. Through testing the coin cells with the cathodes made by 0 and 1.8 wt% graphene flakes on silica particles, they disclosed that the excess capacity is attributed to the reversible reduction–oxidation reaction between the lithium ions of the electrolyte and the exfoliated graphene flakes, where the graphene flakes exhibit a capacity higher than 2,000 mAh g−1. The highly conductive graphene flakes wrapping around carbon-coated LFP also assist the electron migration during the charge/discharge processes, diminishing the irreversible capacity at the first cycle and leading to ~100 % Columbic efficiency without fading.Although the graphene sheets can improve the electrical and electrochemical performance of LFP as cathode in LIBs, however, it is hard to control specifically on the orientation, the size, and the edge structure in practice. Therefore, many researches were explored to using the synergistic effects of graphene and other carbon materials to increase the performances of LFP cathode materials , 72–74. In summary, we have reviewed the recent progresses in the studies on the composites of LFP/graphene. As one of the most promising cathode materials for LIBs, LFP has been exploited overwhelmingly; however, the poor electrical conductivity limited deadly the application of bare LFP in the LIB cathode. Graphene, a novel 2D material, has unique morphology and incomparable electrical conductivity and other attractive properties. It has been illustrated that the graphene sheets could be coated on the LFP surface through several controllable manners, including the simple physical mixing, in situ growth of the graphene onto LFP, and, alternatively, the in situ growth of the LFP onto graphene sheets. After the formation of the composites of LFP/graphene, the electrical and electrochemical performances of the LFP can be improved significantly. The structural characters of graphene sheets that may affect the properties of the composites have also been elucidated. The composites were found applicable as cathode for LIBs.However, for the practical application of LFP/graphene composite, several fundamental issues remain to be solved and some possible solutions are as follow: first, the graphene sheets used so far for preparation of LFP/graphene composites were generated usually through the chemical reduction of the GO in which there are various oxygen containing groups, thus, the effects of the surface functionality of the graphene sheets on the interaction between the graphene sheets and LFP and the influences on the properties needs to be studied. Second, a reasonable theoretical model should be developed to describe the electron and Li-ion transportation through the LFP/graphene, the structure and morphology changes of LFP/graphene during the charge/discharge should be studied systemically, that will help to understand the mechanism of the storage mechanism of the Li-ions or atoms, which may also help for further rationally designing and preparing the composites with desired properties for practical application. Third, the scalable preparation procedures of the composites with homogeneous composition and controlled morphology need to be developed, and liquid process is recommended.