Rationale: Idiopathic pulmonary fibrosis (IPF) is a complex lung disease characterized by scarring of the lung that is believed to result from an atypical response to injury of the epithelium. Genome-wide association studies have reported signals of association implicating multiple pathways including host defense, telomere maintenance, signaling, and cell–cell adhesion.Objectives: To improve our understanding of factors that increase IPF susceptibility by identifying previously unreported genetic associations.Methods: We conducted genome-wide analyses across three independent studies and meta-analyzed these results to generate the largest genome-wide association study of IPF to date . We performed replication in two independent studies  and functional analyses  to determine putatively causal genes. Polygenic risk scores were used to assess the collective effect of variants not reported as associated with IPF.Measurements and Main Results: We identified and replicated three new genome-wide significant (P < 5 × 10−8) signals of association with IPF susceptibility  and confirmed associations at 11 previously reported loci. Polygenic risk score analyses showed that the combined effect of many thousands of as yet unreported IPF susceptibility variants contribute to IPF susceptibility.Conclusions: The observation that decreased DEPTOR expression associates with increased susceptibility to IPF supports recent studies demonstrating the importance of mTOR signaling in lung fibrosis. New signals of association implicating KIF15 and MAD1L1 suggest a possible role of mitotic spindle-assembly genes in IPF susceptibility. Idiopathic pulmonary fibrosis (IPF) is a devastating disease where the lungs become scarred. It is not known what causes the scarring, but there have been 17 regions of the genome that have been reported as associated with increased susceptibility to IPF from previous genome-wide association studies. These identify host defense (particularly mucus production), cell–cell adhesion, signaling, and telomere maintenance as important processes in the development of lung fibrosis.By combining all previous IPF genome-wide association studies, we have identified three novel regions of the genome identified with IPF risk and confirmed 11 of the 17 previously reported regions. The three novel regions implicate the genes DEPTOR, KIF15, and MAD1L1. These findings support recent research that shows mTOR signaling promotes lung fibrogenesis and also implicate spindle-assembly genes in the development of IPF.Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease characterized by the buildup of scar tissue. It is believed that damage to the alveolar epithelium is followed by an aberrant wound-healing response leading to the deposition of dense fibrotic tissue, reducing the lungs’ flexibility and inhibiting gas transfer . TreatmeMUC5B gene has a much larger effect on disease susceptibility than other reported risk variants with each copy of the risk allele associated with a fivefold increase in odds of disease . All studies were restricted to unrelated individuals of European ancestry, and we applied stringent quality control measures . All studies diagnosed cases using American Thoracic Society and European Respiratory Society guidelines . For individuals in the Genentech study, genotypes were derived from whole-genome sequencing data. Duplicated individuals between studies were removed (see the online supplement).Genotype data for the Colorado, Chicago, UK, and UUS studies were imputed separately using the Haplotype Reference Consortium r1.1 panel  (see theP > 1 × 10−6) and were well-imputed  in at least two studies were included. A genome-wide meta-analysis of the association summary statistics was performed across the Chicago, Colorado, and UK studies using R v3.5.1 (discovery stage). Conditional analyses were performed to identify independent association signals in each locus (see the online supplement).In each of the Chicago, Colorado, and UK studies separately, a genome-wide analysis of IPF susceptibility, using SNPTEST  v2.5.2, P < 5 × 10−8), and nominally significant (P < 0.05) with consistent direction of effect in each study, were further tested in the replication samples. We considered novel signals to be associated with IPF susceptibility if they reached a Bonferroni-corrected threshold  in a meta-analysis of the UUS and Genentech studies . Previously reported signals with P < 5 × 10−8 in the discovery meta-analysis were deemed a confirmed association.Sentinel variants  of the novel signals reaching genome-wide significance in the meta-analysis (see the online supplement).To further refine our association signals to include only variants with the highest probabilities of being causal, Bayesian fine-mapping was undertaken. This approach takes all variants within the associated locus and, using the GWAS association results, calculates the probability of each variant being the true causal variant . The probabilities are then combined across variants to define the smallest set of variants that is 95% likely to contain the causal variant  for each IPF susceptibility signal . Where IPF susceptibility variants were found to be associated with expression levels of a gene, we tested whether the same variant was likely to be causal both for differences in gene expression and IPF susceptibility. We only report associations with gene expression where the probability of the same variant driving both the IPF susceptibility signal and gene expression signal exceeded 80% (see the online supplement).To identify which genes might be implicated by the IPF susceptibility signals, we identified whether any variants in the credible sets were genic coding variants and defined as deleterious (using Variant Effect Predictor [VEP] ). In add= 1,111] –29, the = 4,896] , and 48 = 4,896]  [n betweTo investigate whether the IPF susceptibility variants that were in noncoding regions of the genome might be in regions with regulatory functions , we investigated the likely functional impact of those variants using DeepSEA  . Taking 1, FVC, the ratio FEV1/FVC (used in the diagnosis of COPD), and peak expiratory flow. We applied a Bonferroni corrected P value threshold to define variants also associated with ILAs or lung function.As previous studies have reported shared genetic susceptibility for IPF and other lung traits , 13, 15,r2 ≤ 0.1. As we wanted to explore the contribution from as yet unreported variants, we excluded variants within 1 Mb of each IPF susceptibility locus from the risk score calculation (see the online supplement).The contribution of as yet unreported variants to IPF susceptibility was assessed using polygenic risk scores. For each individual in the UUS study, the weighted score was calculated as the number of risk alleles, multiplied by the effect size of the variant (as a weighting), summed across all variants included in the score. Effect sizes were taken from the discovery GWAS and independent variants selected using a linkage disequilibrium P threshold (PT) such that the variant had to have a P value <PT in the genome-wide meta-analysis to be included in the score. This allows us to explore whether variants that do not reach statistical significance in GWAS of current size contribute to disease susceptibility. We used the recommended significance threshold of P < 0.001 for determining significantly associated risk scores .Following quality control, 541 cases and 542 controls from the Chicago study, 1,515 cases and 4,683 controls from the Colorado study, and 612 cases and 3,366 controls from the UK study were available . For theR2 > 0.5) variants with minor allele count ≥10 in each study and which were available in two or more of the studies .To identify new signals of association, we meta-analyzed the genome-wide association results for IPF susceptibility for the Chicago, Colorado, and UK discovery studies. This gave a maximum sample size of up to 2,668 cases and 8,591 controls for 10,790,934 well-imputed  association with IPF susceptibility in the discovery meta-analysis and were also significant after adjusting for multiple testing (P < 0.01) in the replication stage comprising 1,467 IPF cases and 11,874 controls  and a rare intronic variant in HECTD2 . Conditional analyses did not identify any additional independent association signals at the new or previously reported IPF susceptibility loci .Three novel signals  le blood ), TAF2 . The signal at FAM13A, while genome-wide significant in the discovery meta-analysis, was not significant in the Chicago study. This was the only signal reaching genome-wide significance in the discovery genome-wide meta-analysis that did not reach at least nominal significance in each study in the discovery analysis. Three further previously reported signals at 11p15.5 (near MUC5B) were no longer genome-wide significant after conditioning on the MUC5B promoter variant (Table E1), consistent with previous reports , which was associated with a change in DNase I hypersensitivity in 18 cell types and FOXA1 in the T-47D cell line . The 14 IPF susceptibility signals were found to be enriched in DNase I hypersensitivity site regions in multiple tissues including fetal lung tissue . No enrichment in differential expression in airway epithelial cells between IPF cases and healthy controls was observed for the 14 IPF susceptibility signals when using SNPsea (Table E5).Of the 14 IPF susceptibility signals , the only variant predicted to have a potential functional effect on gene regulation through disruption of chromatin structure or transcription factor binding motifs (using DeepSEA) was rs2013701 (in an intron of P < 0.05) with one or more lung function trait in general population studies (P < 5.2 × 10−4), the previously reported variants at FAM13A, DSP, and IVD were associated with decreased FVC, and variants at FAM13A, DSP, 7q22.1 (ZKSCAN1), and ATP11A were associated with increased FEV1/FVC. Similarly, for the three novel susceptibility variants, all showed at least a nominal association with decreased FVC and increased FEV1/FVC. We observed a nominally significant association of the MUC5B IPF risk allele with decreased FVC and increased FEV1/FVC. The IPF risk alleles at MAPT were significantly associated with both increased FEV1 and FVC. To determine how the variants identified for IPF susceptibility are related to differences in lung function between cases and controls, we investigated whether variants known to be associated with lung function show an association in our IPF GWAS. Of the 279 variants reported .Previous studies have reported an overlap of genetic association loci between lung function and IPF . We unde studies  and E6. reported  as assocKIF15, MAD1L1, and DEPTOR signals were not associated with ILAs .As interstitial lung abnormalities may be a precursor to IPF in a subset of patients, and there have been previous reports of shared genetic etiology between IPF and ILAs , 40, 41,MUC5B). As the PT for inclusion of variants in the score was increased, the risk score became more significant reaching a plateau at around PT = 0.2 with risk score P < 3.08 × 10−23 and explaining around 2% of the phenotypic variation , suggesting that there is a modest but statistically significant contribution of additional as yet undetected variants to IPF susceptibility. Further increasing PT beyond 0.2 did not improve the predictive accuracy of the risk score.To quantify the impact of as yet unreported variants on IPF susceptibility, polygenic risk scores were calculated excluding the 14 IPF susceptibility variants . The polygenic risk score was significantly associated with increased IPF susceptibility despite exclusion of the known genetic association signals  kinase activity as part of both the mTORC1 and mTORC2 protein complexes. The IPF risk allele at this locus was associated with decreased gene expression of DEPTOR in lung tissue (Table E2). TGFβ-induced DEPTOR suppression can stimulate collagen synthesis  .The strongest evidence for the new signal on chromosome 8 implicates ynthesis , and theynthesis . MAD1L1,g cancer , 45. Stumutated) . This coted gene , KIF15, RTEL1, a gene involved in telomere elongation regulation, has not previously been identified in an IPF GWAS; however, the collective effect of rare variants in RTEL1 has been reported as associated with IPF susceptibility , and 17q21.31 (MAPT) have also been reported as associated with COPD, although with opposite effects . Spirometric diagnosis of COPD was based on a reduced FEV1/FVC ratio. In an independent dataset of 400,102 individuals, eight of the IPF signals were associated with decreased FVC and with a comparatively weaker effect on FEV1. This is consistent with the lung function abnormalities associated with IPF, as well as the decreased risk of COPD. Of note, only around 3% of previously reported lung function signals  of as yet unidentified variants associated with disease susceptibility.KIF15 and MAD1L1, there was substantial evidence for an association with gene expression in nonlung tissues but not in either of the two (nonfibrotic) lung tissue eQTL datasets. This could reflect cell type-specific effects that are missed when studying whole tissue or effects that are disease-dependent. Finally, our study was not designed to identify rare functional variant associations. As both common and rare variants are known to be important in IPF susceptibility (A strength of our study was the large sample size compared with previous GWAS and the availability of an independent replication dataset. A limitation of our study was that the controls used were generally younger in all studies included, and there were differences in sex and smoking distributions in some of the studies. As age, sex, and smoking status were not available for all individuals in four of our datasets, we were unable to adjust for these variables without substantially reducing our sample size. However, cases and controls in the UUS and UK datasets were matched for age, sex, and smoking. The three novel signals replicated in all of the discovery and replication datasets, providing reassurance that the signals we report are robust despite differences between the datasets. As we had limited information beyond IPF diagnosis status for a large proportion of the individuals included in the studies, we cannot rule out some association with other age-related conditions that are comorbid with IPF. However, other age-related conditions were not excluded from either the cases or controls. For the signals near tibility , this isIn summary, we report new biological insights into IPF susceptibility and demonstrate that further studies to identify the genetic determinants of IPF susceptibility are needed. Our new signals of association with IPF susceptibility provide increased support for the importance of mTOR signaling in pulmonary fibrosis as well as the possible implication of mitotic spindle-assembly genes.


Graphene-metal contact is crucial to fabricate high-performance graphene photodetectors since the external quantum efficiency (EQE) of the photodetector depends on the contact properties, and the influence of the contact properties is particularly dominant in short channel devices for high-speed applications. Moreover, junction properties between the channel graphene and graphene near the contact are also important to analyze the photoresponse because the built-in electric field in the junction determines the EQE of the photodetector. In this study, we investigated a relation between the photoresponse and the built-in electric field induced from the doping level difference in the junction between the channel graphene and graphene near the contact. The photoresponse could be enhanced with a high junction barrier height that is tuned by the doping level difference. In addition, we observed that the improved electrical characteristics of channel graphene do not guarantee the enhancement of the photoresponse characteristics of graphene photodetectors.  Graphene, a two-dimensional honeycomb lattice of carbon atoms, has attracted attention due to its unique electrical and optical characteristics ,2. The uIn this work, we investigated the effect of the doping level difference between the channel graphene and graphene near the metal contact on the photoresponse of a graphene photodetector. We observed that the electrical characteristics and doping levels of channel graphene were changed by the post-annealing process, and we investigated the relation between the photoresponse and the electrical characteristics of channel graphene at various annealing temperatures. Finally, we separately analyzed the photoresponse characteristics of the channel and contact region of the graphene photodetector.2 layer was 100 nm. Photolithography was used for patterning the electrode on the graphene layer. A palladium (Pd)/gold (Au) (20 nm/50 nm) was deposited on the patterned photoresist by an e-beam evaporating system and followed by the lift-off process. Then, we defined the channel region of the graphene photodetectors by using photolithography and oxygen plasma treatment . The photoresist was removed by acetone and isopropanol. A top-view optical image of the fabricated graphene photodetector with a channel length of 15 μm and a channel width of 20 μm is shown in the top of We fabricated graphene photodetectors with various channel lengths using commercially available monolayer graphene . Graphene was transferred on the thermally oxidized silicon substrate. The thickness of the SiO−7 Torr) for two hours. The electrical and photoresponse characteristics of the graphene photodetectors were measured using a semiconductor parameter analyzer  and a 1550-nm-wavelength infrared laser diode . The laser diode was connected to the laser diode controller  and temperature controller  for stable operation.Prior to the electrical measurement, we annealed the fabricated graphene photodetectors in a vacuum probe station (~10D–VG) of the graphene photodetectors measured at a constant drain-source bias (VD = 0.1 V). The gate bias was varied from −20 V to 20 V. The channel lengths of the graphene photodetectors were 2, 5, 10, 15, and 20 μm and the channel width was fixed at 20 μm. The graphene photodetectors annealed at 150°C had a positively shifted Dirac voltage (~ 8 V), whereas the graphene photodetectors reannealed at 300 °C had a Dirac voltage close to 0 V. This indicated that the 150 °C annealing evaporated moisture and the 300 °C annealing effectively removed the polymer residue, which is critical for the practical applications with a low operating voltage of graphene photodetectors. One experimental detail for the graphene FETs is hysteresis. We evaluated the hysteresis of the graphene photodetectors after 300 °C annealing. The hysteresis of the 10-μm- and 20-μm-channel photodetectors were –1.2 V and –1.1 V, respectively. More details for hysteresis characteristics of the graphene photodetectors are shown in mg is the transconductance, chL is the channel length, oxC is the gate oxide capacitance, W is the channel width, and DV is the drain bias [2/V∙s, while the graphene photodetectors reannealed at 300 °C showed an improved value of ~2000 cm2/V∙s. Thus, this annealing process for removing undesirable doping is essential for important material parameters that determine the electrical properties of graphene. We firstly measured the electrical characteristics of the graphene photodetectors with various channel lengths (2–20 μm) after a 150 °C-annealing process. The graphene photodetectors were annealed again at 300 °C, and we remeasured the electrical characteristics after the first measurement. A schematic image of the graphene photodetector with various channel lengths is shown in the bottom of ain bias . The graWe expected that the improved electrical properties of the graphene photodetector would induce enhanced photoresponse characteristics. However, the photoresponse characteristics were degraded after 300 °C annealing rather than 150 °C annealing. G – VDirac of 2 V, whereas that of the 300 °C-annealed photodetector was near 0%. These results show that the 300 °C-annealing process affects the quantum efficiency of the graphene photodetector, thus resulting in degradation of the photoresponsivity. The photoresponse can generally occur in the channel region and near the graphene-metal contact region under light illumination [To find the reason that the photoresponsivity was degraded after the 300 °C annealing even though the electrical properties of graphene had improved, we investigated the optical modulation of the total device resistance of the graphene photodetector at different annealing temperatures. mination . In order to compare the photoresponse characteristics of the channel and contact regions, we extracted the sheet and contact resistance from the total device resistance using the transmission line method (TLM), which has been typically used to evaluate the graphene-metal contact resistance . Figure 2/V∙s (under illumination). G – VDirac = 7 V. Under light illumination, the photogenerated electrons and holes can be separated according to the induced electric field as determined by the doping level difference between the channel and contact graphene . Because the Fermi level of the contact graphene is located in the conduction band, this large doping level difference can induce a large built-in electric field. However, the contact resistance of the graphene photodetector was not significantly modulated by light illumination after 300 °C annealing, as shown in In contrast to the sheet resistance, a noticeable optical modulation of the contact resistance was observed under light illumination. inset of b. After 2/V∙s (under light illumination) after 300 °C annealing.From the electrical measurements b,c, we eWe have investigated the effect of the doping level difference between the channel graphene and graphene near the metal contact on the photoresponse of graphene photodetectors. The photoresponse was found to be related to the junction barrier height because this determines the magnitude of the electric field in the junction. We observed that the post-annealing process for improving the electrical characteristics of channel graphene adversely affects the photoresponse of the graphene photodetector, although the annealing process increased the carrier mobility of graphene and removed the unwanted doping effects. Thus, to fabricate a high-responsivity graphene photodetector, the height of the junction barrier between the channel graphene and contact graphene must be properly designed, considering the graphene doping level in each region.


Background: Left ventricular (LV) and right ventricular (RV) dysfunction is recognized in idiopathic pulmonary fibrosis (IPF). Little is known about cardiac involvement in non-idiopathic pulmonary fibrosis (no-IPF). This issue can be explored by advanced echocardiography. Methods: Thirty-three clinically stable and therapy-naive fibrotic IPF and 28 no-IPF patients, and 30 healthy controls were enrolled. Exclusion criteria were autoimmune systemic diseases, coronary disease, heart failure, primary cardiomyopathies, chronic obstructive lung diseases, pulmonary embolism, primary pulmonary hypertension. Lung damage was evaluated by diffusion capacity for carbon monoxide (DLCOsb). All participants underwent an echo-Doppler exam including 2D global longitudinal strain (GLS) of both ventricles and 3D echocardiographic RV ejection fraction (RVEF). Results: We observed LV diastolic dysfunction in IPF and no-IPF, and LV GLS but not LV EF reduction only in IPF. RV diastolic and RV GLS abnormalities were observed in IPF versus both controls and no-IPF. RV EF did not differ significantly between IPF and no-IPF. DLCOsb and RV GLS were associated in the pooled pulmonary fibrosis population and in the IPF subgroup , independently of confounders including pulmonary arterial systolic pressure. Conclusion: Our data highlight the unique diagnostic capabilities of GLS in distinguishing early cardiac damage of IPF from no-IPF patients. Interstitial lung diseases (ILDs) include more than 200 disorders, characterized by a variable degree of inflammation and fibrosis leading to an often irreversible loss of lung function, wide spectrum in the clinical course, treatment, and prognosis. ILDs can be broadly divided in those without any identifiable cause, that is, idiopathic interstitial pneumonias, and those with identifiable factors such as environmental/occupational exposure, infections, autoimmune systemic diseases, drugs, and radiations . Among iBecause of its worse prognosis and challenging treatment, pneumological and cardiac aspects of IPF have been deeply investigated. Respiratory function declines along with disease progression, and changes in lung diffusion capacity of carbon monoxide (DLCO) and forced vital capacity (FVC) are both independent predictors of worse prognosis . PulmonaSpeckle Tracking Echocardiography (STE) has shown to be suitable for diagnosing early cardiac dysfunction in IPF patients ,13. Convn = 14), chronic hypersensitivity pneumonia (n = 9), and smoking-related desquamative interstitial pneumonia (n = 5). Diagnosis of IPF was made according to the 2011 international diagnostic criteria [The study population included 61 patients with clinically stable and therapy-naive fibrotic ILDs, referred at the time of first diagnosis to the Respiratory Diseases Division of the Federico II University, Monaldi Hospital , between October 2016 and October 2018. Thirty-three patients were affected by IPF (IPF group) and 28 had fibrotic ILDs other than IPF (no-IPF group), including idiopathic nonspecific interstitial pneumonia . All patients gave their written informed consent at enrollment. Patient data were collected in an anonymous way.sb) were performed using a computer-assisted spirometer  according to international standards [Spirometry, lung volume measurement, and determination of the haemoglobin-adjusted single-breath diffusing lung capacity of carbon monoxide , using a 2.5 MHz transducer with harmonic capability, according to the standards of our laboratory ,19,20 anLV quantitative analysis was performed according to guidelines . RelativIn a non foreshortened apical four-chamber view oriented to obtain the maximal RV internal chamber size, RV diameters  were measured at end-diastole. RV systolic function was assessed measuring tricuspid annular systolic excursion  by M-mode echo. Pulsed Doppler RV inflow was recorded to measure tricuspid early diastolic (E) and atrial (A) peak velocities (m/s) and E/A ratio . RV GLS Notably, LV and RV GLS values were considered positive (sign +) to strengthen the clinical meaning: the higher the values, the better the strain deformation.3D echocardiographic examination of the right ventricle was performed according to previously described procedures ,30. A fusb values and RV after adjusting for confounders such as age, LVEF, and PASP. The null hypothesis was rejected at p ≤ 0.05.Statistical analysis was performed by SPSS package, release 12 . Data are presented as mean value ± SD. Intergroup comparisons were performed using ANOVA and Bonferroni post-hoc test. Pearson’s correlation was used to evaluate univariate correlates of a given variable. Multivariable linear regression analyses were performed to examine the independent correlates between DLCOSince the data are normally distributed, they are presented as mean with standard deviation. Demographics, clinical features, and main baseline heart and lung function parameters of the study population are summarized in p = 0.004), but not in the no-IPF group, compared to controls. LV EF did not differ significantly between IPF, no-IPF, and controls, whereas LV GLS was lower in IPF than in no-IPF (p = 0.003) and controls (p < 0.0001).LV standard echocardiographic and STE assessment are reported in n = 21) in the IPF group and 75% (n = 21) in the no-IPF group but significative PAH  was observed only in four patients (12.5%) with IPF, and in two patients (7%) with no-IPF. RV standard, STE, and 3D analysis are summarized in p = 0.028) and midcavity (p = 0.051) diameters and lower TAPSE (p = 0.007). Both IPF and no-IPF patients had lower tricuspid inflow E/A ratio  and higher PASP  versus controls. Both IPF and no-IPF patients had also lower RV GLS in comparison with controls  but RV GLS was lower in IPF than in no-IPF (p < 0.05). Three-dimensional-derived RV EF was lower in IPF and no-IPF compared to controls (both p < 0.002) but did not differ significantly between IPF and no-IPF (p = 0.987). The prevalence of PAH was 65% , whereas the relation between LV GLS and DLCOsb was not significant  . The relations of RV GLS with pO2 , pCO2 , FVC , and TLC  did not achieve statistical significance. Of note, RV EF was not significantly related with any of the spirometric and pulmonary functional parameters. 6-MWT did not correlate with any of the echocardiographic parameters.In the pooled ILDs population, DLCO = 0.32) . RV GLS sb was independently associated with RV GLS . These results were substantially confirmed in the IPF subgroup  whilst no independent association between RV GLS and DLCOsb was found in the no-IPF population  LV diastolic dysfunction, which is detectable even in no-IPF patients, and a subclinical LV systolic dysfunction, testified by the reduction of LV GLS but not of LV EF, which cannot be observed in no-IPF patients; (II) a clear alteration of RV geometry and of both systolic (RV EF and GLS) and diastolic function, and a substantial PASP increase in comparison with controls, whereas no-IPF patients present only an alteration of RV diastolic dysfunction and a lower degree of PASP increase. Moreover, (III) an independent association between DLCOLV diastolic dysfunction, characterized by a reduction of transmitral E/A ratio and e’ mitral annular velocity and increase of E/e’ ratio, was already observed in IPF patients . Our datRV abnormalities have been demonstrated to be evident and predict prognosis in IPF. In a retrospective study on 135 IPF patients referred for lung transplantation, an increase of RV/LV ratio, right atrial and RV dilation, and moderate to severe RV dysfunction (TAPSE <1.6 cm) were all associated with an increased risk of death, independently of lung function parameters . RV abnosb reduction and RV GLS impairment, found in the pooled ILDs and in IPF but not in no-IPF, was independent of confounders such as body mass index, heart rate, and PASP. DLCOsb is more strictly associated with pulmonary fibrotic changes and represents, therefore, the best index of the IPF disease status and extension [Univariate and multivariate associations provided additional insights. The lack of association between FVC and RV GLS was partially unexpected as FVC is more feasible and reproducible than DLCO and largely adopted in the clinical practice and in clinical trials as a predictor of mortality in ILDS . Conversxtension  and reprxtension ,42,43. IFirst, despite being realized in a highly specialized reference setting, the present study is representative of the effort of a single center with a relatively small sample size of ILDs. As we mainly focused on IPF and smoke-related no-IPF pneumonias, our results certainly do not allow driving definite considerations in other forms of no-IPF including those related to autoimmune, connective tissue diseases. Indeed, patients with connective tissue disease-derived fibrotic ILDs likely merit separate analysis because of their high frequency of concomitant pulmonary vascular involvement ,45. In asb. Unlike FVC, DLCO changes are not specific as they depend on both ventilation and perfusion and are an expression of the integrity of the alveolar-capillary membrane. In addition, DLCOsb has a marked within-session and intersession variability [sb is widely recognized as a surrogate measure of pulmonary fibrosis extent and its changes have been demonstrated to be predictive of IPF disease progression over a 12-month period [sb are not feasible in ILD but this is true in the advanced disease stages and in particular in those requiring long-term oxygen therapy at high oxygen flows, whereas our population included clinically stable ILD patients at the time of first diagnosis.Another limitation could correspond to the use of DLCOiability . Neverthh period . SomeoneThe novel findings of the present study point out the diagnostic capabilities of strain imaging in distinguishing early cardiac damage in IPF from no-IPF patients, a task which cannot be attributed to both standard and 3D echocardiography. RV GLS is a key parameter for detecting early cardiac damage in ILDs, with a likely prognostic power, in this clinical scenario. Future studies could be addressed to investigate if RV GLS alterations are also associated with fibrotic heart rearrangement .


In microwave frequency band the conductivity of graphene can be varied to design a number of tunable components. A tunable phase shifter based on commercial graphene nanoplatelets is introduced. The proposed configuration consists of a microstrip line with two stubs connected with a taper. On each side of the stubs there is a gap, short circuited through a via, where the commercial graphene nanoplatelets are drop casted. By applying a DC bias voltage that alters the graphene resistance the phase of the transmitted signal through the microstrip line can be varied. In order to maximize the phase shift of the transmitted signal and minimize the insertion loss, the length of the taper and the stubs are optimized by the help of circuit model and full-wave simulations. A prototype working at 4GHz is fabricated and measured. A phase variation of 33 degrees is acquired with an amplitude variation of less than 0.4 dB. Among the various carbon based materials, graphene is the most notable ,2,3. DueGraphene has remarkable electrical, mechanical and thermal properties. Due to the remarkable properties of graphene, it has found inwards into several applications including electrochemical sensors ,9, bioseCommunication systems involve a number of components working at different frequencies. For efficient working, there needs to be an interconnection between components that form an entire communication system. This interconnection can be facilitated if the components are able to tune their working frequency. Therefore, the tuning of microwave components that form a communication system is vital to efficient functionality. Graphene being tunable with the application of a DC bias, is a good contender for being deployed in microwave communication systems. The acquisition of monolayer graphene is technologically demanding and not very cost effective. Multilayer graphene on the other hand bears tunable conductive behavior similar to monolayer graphene albeit with reduced cost and very low technological complexity. Until recently, multilayer graphene has been grown in laboratory. The availability of commercial graphene nanoplatelets on a large scale takes the ease of fabrication and commercialization of tunable components based on graphene one step further. Recently, a tunable attenuator  and anteIn this paper, a tunable phase-shifter based on commercial graphene nanoplatelets is designed. The proposed configuration consists of a microstrip line with two stubs connected with a taper. On each side of the stubs there is a gap, short circuited through a via, where the commercial graphene nanoplatelets are drop casted. By applying a DC bias voltage that alters the graphene resistance the phase of the transmitted signal through the microstrip line can be varied. The lengths of the tapered line and open line sections are optimized by the help of a circuit model. The phase shifter is further optimized with a full-wave simulator. A prototype of the tunable phase shifter is fabricated and measured. A variable phase shift of 33 degree is obtained with a degradation of the insertion loss of less than 0.4 dB. The type of graphene used in this work is graphene nanoplatelets based on multiple graphene layers. The graphene nanoplatelets are produced by Nanoinnova. Raman and FESEM (Field Emission Scanning Probe Microscope) are used for morphological characterization of the graphene nanoplatelets. TM 40 microscope was used. The FESEM images are shown in For FESEM analysis of the commercial graphene nanoplatelets, a ZEISS SUPRA −1 contains the D (defects) and G (graphitization grade). The second spectral range from 2200–3500 cm−1 is the second order Raman spectral range containing overtones. The ratios of the peaks are ID/IG = 0.15, ID/I2D = 0.27 and IG/I2D = 1.81. According to guidelines described in -Rin [Rg = 70 Ω] and ΔXin = Xin [Rg = 700 Ω]-Xin [Rg = 70 Ω] are found as shown in s the value of ΔXin is increased while increasing the value of Lt, the value of ΔRin is reduced up to length Ls = 0.3 λ0. Increasing the length, Ls further reduces the value of both ΔRin and ΔXin. For the case of Ls = 0.15λ0, in which there is no impact of the variation of Rg on Zin. The best case from this analysis is thus: Ls = 0.3 λ0 and Lt = 0.08 λ0.For each set of values of Lr = 3.5 and loss tangent is tanδ = 0.0015. The thickness of copper is 35 μm. The width of the main line is w = 3.2 mm, corresponding to a characteristic impedance of 50 Ω. The stub is shown in detail in t and an open ended line section of length, Ls. In order to realize the grounds, two grounded metallic vias are symmetrically placed on each side of the line section in the middle of a metallic pad. The metallic pad has length, Lp = 1 mm and width, wp = 2 mm. In between the metallic pad and the line section, graphene is deposited. The length of graphene deposition is equal to the length of the metallic pad, Lp = 1 mm. The graphene deposition has width, Wg = 0.2 mm and length, Lg = 1 mm. The aspect ratio is kept low to ensure lower resistance since commercial graphene nanoplatelets possess higher sheet resistance value. The operating principle of the phase shifter is to have a variable reactance on a transmission line caused by the optimized stubs of the s  for graphene resistance values ranging between 350 Ω/sq. and 3500 Ω/sq. (the graphene sheet resistance is measured in Ohm/square). The amplitude and phase variation of the phase shifter versus graphene sheet resistance are shown in s = 0.25 λ0 to Ls = 0.3 λ0. Increasing the value of Ls further to 0.35 λ0 results in increased variation of |S21|. The phase variation is shown in 21 increases with increasing Ls. The maximum phase variation is attained in the case of Ls = 0.35 λ0. The optimum length is Ls = 0.3 λ0 because it provides minimum amplitude variation with reasonable phase variation.The phase shifter is simulated with a full-wave simulator Ansys HFSS. In order to further optimize the structure, the phase shifter has been simulated at a frequency of 4 GHz with three different lengths of the open-ended line section, LThe structure of the phase shifter with optimized dimensions resulting from 21|. For an AR of 0.2, the maximum and minimum |S21| is −4.5 dB and −5.8 dB respectively. For the maximum AR value of 0.8, the maximum and minimum |S21| is −3.3 dB and −5.0 dB respectively. The value of the phase of the transmission coefficient increases with a reduction in the aspect ratio. The maximum value of ∠S21 for an AR of 0.2 is 73° while the minimum value is 22°. This shows that a reduction of the AR reduces the variation of the amplitude of the transmission coefficient and increases the variation of the phase of the transmission coefficient, a highly desirable trait of tunable phase shifters. The aspect ratio of the gap with graphene can be defined as: AR = Wg/Lg see b inset. 21 / insertion loss variation (dB)) is observed at a frequency of 4.3 GHz. The measured phase shift at the frequency of 4.3 GHz is almost 33 degrees with a measured amplitude variation less than 0.4 dB. Hence the maximum figure of merit is 82.5 degree/dB.In order to compare measured and simulated values, simulations are performed with graphene sheet resistance values corresponding to the measured graphene resistance values. The simulated amplitude (dashed lines) of the SAt this frequency the insertion loss and phase variation are simulated for different Rg values as shown in The phase shifter presented is a dynamically tunable phase shifter that varies its phase upon an application of a voltage bias. The phase shifter deploys commercial graphene nanoplatelets and is a step towards mass-production of tunable microwave components based on graphene. The phase shifter provides almost 33 degrees of phase shift with negligible variation of the insertion loss. A comparison of the phase shifter with other similar phase shifters based on novel materials is shown in As shown in The effects of a negative bias applied to the graphene nanoplatelets are predicted to change the carrier charge, the Fermi level and the electron mobility  resultinA voltage controlled dynamically tunable phase shifter based on commercial graphene nanoplatelets is presented. The phase shifter is composed of a microstrip transmission line connected to a tapered line and an open stub. Graphene connected to grounded metallic vias are symmetrically placed at the interconnection between the tapered line and the stub. The electrical conductivity of graphene is tuned by a voltage bias, which results in the variation of the insertion loss and phase of the signal transmitting through the microstrip line. In order to maximize the phase shift and minimize the insertion loss, optimization of the lengths of the open stub, tapered lines and the dimensions of the graphene’s depositions are performed by the help of circuit models and full wave simulations. A prototype is fabricated and measured. The measured phase shift of the phase shifter is almost 33 degrees with a variation of the insertion loss of less than 0.5 dB at the frequency of 4.3 GHz.


Nine men  randomly performed two CrossFit® sessions (shorter: ~4 min and longer: 17 min) with a 7-day interval between them. The response of LAC and HR were measured pre, during, immediately after, and 10, 20, and 30 min after the sessions. RPE was measured pre and immediately after sessions. Lactate levels were higher during the recovery of the shorter session as compared with the longer session . There were no significant differences between protocols on HR during . The LAC was significantly higher throughout the recovery period for both training sessions as compared to pre-exercise. The RPE was increased immediately after both sessions as compared to pre-exercise, while there was no significant difference between them . These results demonstrated that both shorter and longer sessions induced elevated cardiovascular responses which met the recommendations for gains in cardiovascular fitness. In addition, both training sessions had a high metabolic and perceptual response, which may not be suitable if performed on consecutive days.The aim of this study was to analyze blood lactate concentration (LAC), heart rate (HR), and rating perceived exertion (RPE) during and after shorter and longer duration CrossFit The regular practice of exercise is associated with the reduction of morbidity and mortality . NeverthAlternative training modalities have gained popularity in recent years. According to the American College of Sports Medicine (ACSM), high intensity interval training (HIIT), training with body weight, resistance training, and group training are the main fitness trends of 2019 , showing® that consists of performing functional and sports exercises. This type of training utilizes Olympic weightlifting exercises, such as snatch and clean, basic exercises including squats, deadlifts, and bench press, rowing, running and cycle ergometer cyclic exercises, and gymnastic movements, such as parallels, rings, and bars [® modality. High intensity training also has been associated with a more pronounced participation of the general population as compared with moderate aerobic training and resistance training [An alternative to traditional HIIT is the CrossFitand bars . These etraining .® training [® training  with high intensity treadmill training (20 min at 90% of HRmax). The mean HR for 20 min was above 93% of HRmax, and significantly higher in the high intensity treadmill training. This HR response meets the intensity recommendations from ACSM to improve cardiorespiratory fitness [® includes a dynamic of movements with elevated repetitions and high intensity, training protocols have distinct duration, from shorter protocols of only some minutes (2 to 8 min) to longer sessions (20 to 30 min), which may directly affect cardiovascular, metabolic, and rating of perceived exertion responses. We are not aware of any study evaluating these responses during shorter versus longer CrossFit sessions.Although some research has evaluated physiological responses to CrossFittraining ,11,12,13training  has comp fitness ,17. Alth® sessions. The hypothesis is that the cardiovascular response is higher than the recommended values by the ACSM with a greater metabolic and RPE response for both protocols.Thus, the aim of the present study was to compare blood lactate (LAC), HR and rating of perceived exertion (RPE) responses during and after shorter versus longer duration CrossFit® practice. The study was approved by the Research Ethics Committee from the local University .Nine apparently health men participated in the present study . The inclusion criteria were as follows: No muscle, joint, or bone injuries, presence of any disease that could compromise health during the study; negative response to the Physical Activity Readiness Questionnaire; signed the written informed consent document; have a minimal experience of six months with CrossFit® session . During the tests, HR was measured, while before and after the protocols, RPE and LAC were evaluated. Subjects were instructed to avoid meals two hours before sessions, to avoid alcoholic drinks, and stimulating drinks  in the 2 days preceding the experimental session. Medication or supplementation that could change LAC and HR were also suspended for 2 days preceding the experimental session. When medication could not be suspended, the subjects were excluded. The subjects were counterbalanced in regards to the order of the training sessions; the sessions were randomly chosen and completed at the same time of day in the presence of the same researchers under controlled room temperature. The subjects were adapted to both CrossFit® sessions and they recognized Fran and FGB sessions as described elsewhere [Subjects visited the laboratory on two occasions with 7 to 9 days apart. They completed a shorter (Fran), and a longer CrossFitlsewhere , as they® session was Fran, which consists of 21-15-9 repetitions of thrusters (43 kg) and pull-ups as fast as possible , which consists of three sets of 5 min per set, during which, as many repetitions as possible in five exercises are attempted , sumo deadlift high-pull (34 kg), box jump (50 cm), push-press (34 kg), and rowing , respectively; The longer duration CrossFitBefore training protocols, subjects remained seated for 10 min to collect blood samples for LAC measures by a portable device  . LactateThe monitoring of HR was done by Polar H10 HR-monitor , with recording intervals of 1 s. All subjects were monitored during both sessions: Shorter duration varying from 2 min 20 s to 9 min 01 s; and longer duration with a fixed time of 17 min . Moreover, after the protocols, subjects remained seated for 30 min for the analysis of HR as follows: Immediately after, 10, 20-, and 30-minutes following exercise. Maximal heart rate was estimated using the age-predicted equation (220-age).The RPE was measured before and immediately after exercise by the RPE CR10 Borg scale adapted by Foster et al. , as instBriefly, the following information was provided: “The perceived exertion is defined the effort intensity, stress, discomfort, and fatigue felt during exercise. Utilize the numbers of this scale to report how your body feels during exercise. The number zero in the scale describes “minimal effort” and represents your lowest imaginable effort. The number 10 described “maximum effort” and represents the highest imaginable effort. If you feel an exertion between extremely easy and maximum effort indicate a number between 0 and 10. At the end the tests we will require you to point a number to inform us about how your body is feeling in general. There are no right or wrong numbers. The verbal descriptors may help you to choose a number” .During the anchoring procedure, a RPE scale was presented to the subject by another evaluator that was present in the evaluation room. Subjects also received a copy of the scale with the respective instructions for anchorage. This was provided for subjects to read during the general warm-up for each session .®. Compound sphericity was verified by the Mauchley test. When the assumption of sphericity was not met, the significance of F-ratios was adjusted according to the Greenhouse–Geisser procedure. Tukey’s post-hoc test with Bonferroni adjustment was applied in the event of significance. Paired sample t test was used to compare HR during exercise between CrossFit® sessions. The achieved power of the sample size was calculated based on the interaction LAC between short and long sessions of CrossFit®. The effect size f was 0.302 and the achieved power was 0.801. The level of significance was p ≤ 0.05 and SPSS version 20.0  software was used.The data are expressed as mean value ± standard deviation (SD). Shapiro–Wilk test was applied to check for normal distribution of study variables. In case of non-normal distribution, logarithmic transformation was performed. A two-way repeated measures ANOVA was used to compare the LAC, HR, and RPE between short and long sessions of CrossFitThe average time for Fran was 4.11 ± 2.36 min, the fastest time was 2.20 min and the slowest time was 9.01 min. The FGB was a fixed time of 17 min and the average number of repetitions were 294.1 ± 32.3 repetitions.® sessions and recovery time on LAC  than the recovery time of FGB. The 30 min area under the curve of LAC after Fran was also significantly higher than the area under the curve of LAC after FGB .There was a statistically significant two-way interaction between CrossFit® sessions. When the average and peak HR during sessions were compared, there was no statistically significant difference between CrossFit® sessions . There was also no statistically significant difference between the average HR during Fran and average HR during the first set of the FGB , second set , and third set . During Fran CrossFit® session, 90% of HRmax was achieved 30 s after the start of the session, on the other hand, 90 s was required to achieve 90% of HRmax during FGB CrossFit® session.® sessions and recovery time on HR  The shorter duration protocol displayed a higher LAC response during the recovery period as compared with the longer duration; (ii) both protocols reached a mean superior to 90% HRmax during training, without differences between them, and (iii) the RPE was close to the maximum for both protocols, without differences between them.The present study compared LAC, HR, and RPE during and after shorter versus longer CrossFit® sessions (shorter vs. longer) on cardiovascular, metabolic, and RPE responses in trained men. Previous studies have reported these results, but only separately, and in response to different training models [® workouts. They showed that both workouts analyzed are high intensity workouts achieving near maximal physiological  and perceptual levels . Recently, Kliszczewicz, et al. [® sessions with different duration (< 5 min and 15 min). The results demonstrated no difference in cardiovascular response between sessions (92.7 ± 4% vs. 91.3 ± 3% HRmax). Similarly to Kliszczewicz, et al., [®, with high levels of blood lactate, should consequently have attentional performances somewhat limited. Thus, reaction time, execution time, number of errors, and number of omissions that exhibited a significant worsening concomitantly with the increase in blood lactate, could be changed during both short and long sessions.To the best of our knowledge, this is the first study to compare different durations of CrossFitg models ,12,13,14g models ,12 analy, et al.  analyzed et al.,  the resu et al.,  also sho® training sessions meet the intensity recommended by the ACSM to improve cardiovascular fitness [® sessions.An important finding of the present study is that CrossFit fitness , regardl fitness ,17, despDespite the interesting findings of this study, some limitations need to be mentioned. First, the reduced number of subjects. Second, it should be noted that these results should be considered only for trained male subjects. Therefore, our findings may not be directly transferable to the untrained or to females.Results revealed that both shorter and longer duration sessions elicited high cardiovascular responses, which meets the recommendations to improve cardiovascular fitness. The elevated metabolic and RPE responses observed in both protocols indicate that they may not be suitable on consecutive days without sufficient recovery.


Idiopathic pulmonary fibrosis (IPF) is a lethal, agnogenic interstitial lung disease with limited therapeutic options. To investigate vital genes involved in the development of IPF, we integrated and compared four expression profiles , including 87 IPF samples and 40 normal samples. By reanalyzing these datasets, we managed to identify 62 upregulated genes and 20 downregulated genes in IPF samples compared with normal samples. Differentially expressed genes (DEGs) were analyzed by gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis to illustrate relevant pathways of IPF, biological processes, molecular function, and cell components. The DEGs were then subjected to protein–protein interaction (PPI) for network analysis, serving to find 11 key candidate genes . The result of RT-qPCR and immunohistochemical staining verified our finding as well. In summary, we identified 11 key candidate genes related to the process of IPF, which may contribute to novel treatments of IPF. Idiopathic pulmonary fibrosis (IPF) is a chronic, devastating, progressive, and fibrotic interstitial lung disease of unknown etiology with few treatment options. IPF occurs primarily in middle-aged and elderly adults, and its median survival time varies from three to five years after diagnosis . The maihttp://www.ncbi.nlm.nih.gov/geo/ (accessed on 2 December 2020)). The GEO database, a repository that contains adequate independent studies, was a place where we could find datasets containing the results of gene expression profiles of clinical samples. During the research, we downloaded four original datasets, GSE110147, GSE53845, GSE24206, and GSE10667, which involved 87 IPF lung samples and 40 normal lung tissue samples. Despite that these datasets are independent, we used bioinformatics methods to integrate the data, combined with gene expression analysis techniques. Thus, we identified the common DEGs of the four different datasets, which are representative to the development of IPF to some extent. Furthermore, gene ontology (GO) and pathway enrichment analysis were performed, contributing to insights into the functional aspects of relevant genes. The common DEGs could be divided into upregulated genes and downregulated genes. Meanwhile, the common DEGs were collected to construct a protein–protein interaction network (PPI), serving to provide a network among these genes according to their interaction. The analysis showed that there are 11 core genes participating in the pathological changes of IPF, in which ANXA3 and STX11 are downregulated genes while THBS2, MMP1, MMP9, MMP7, MMP10, SPP1, COL1A1, ITGB8, and IGF1 are upregulated genes. To confirm the results, we conducted RT-qPCR and immunohistochemistry experiments on normal and IPF clinical samples, accordingly.To break the limits of inadequate patient samples and obtain an overall pattern in the development of IPF, we sought four different datasets from the Gene Expression Omnibus . The interaction relationships among the common DEGs were visualized by using Cytoscape software. A total of 82 common DEGs (62 upregulated genes and 20 downregulated genes) were filtered into the PPI network complex. Each node represents a gene, and the edges stand for the interactions between nodes (the disconnected nodes are not shown); the final networks are shown in https://imagej.nih.gov/ij/ (accessed on 2 December 2020)) showed that IGF1 and ITGB8 were also upregulated in the lung tissues of IPF patients. Additionally, ANXA3 is downregulated in the lung tissues of IPF patients.Finally, we identified 11 candidate genes according to the significant changes in expression levels among the common DEGs and the degree of connectivity within the PPI network. To validate the result, 11 key DEGs  were chosen to test in clinical samples. The result of RT-qPCR A shows tThe mechanisms underlying the pathogenesis of IPF are quite complex and remain incompletely understood. Increased proliferation, migration, and activation of fibroblasts, as well as inflammation and oxidative stress are all involved in the etiology of IPF. Excessive production of extracellular matrix components also contributes to pulmonary fibrosis. The currently available treatments for IPF are limited; however, the incidence of this disease is rising globally, with associated high morbidity, mortality, and growing economic healthcare burden. Therefore, there is a drive to identify novel biomarkers to find potential therapeutic targets for IPF. We integrated four expression profiles, including 87 IPF samples and 40 normal samples and successfully found 11 key candidate genes  related to the pathogenesis of IPF.Matrix metalloproteinases (MMPs) are proteases that degrade all extracellular matrix and many non-matrix protein components. It was initially thought that MMP could limit pulmonary fibrosis by degrading ECM proteins. Nevertheless, recent studies reported that MMPs are involved in the regulation of protein activity other than ECM proteins, including latent growth factors, inflammatory mediators, cleavage of cell surface molecules, antifibrotic growth factors, and receptors . AdditioBased on the clinical report that the levels of MMP1, MMP7, MMP8, and MMP9 in IPF blood and/or lung samples are elevated, previous studies proposed the role of MMPs in IPF mechanisms. Those include: (1) promote the transition from epithelium to mesenchyme (MMP3 and MMP7); (2) increase the activity or lung levels of the profibrotic mediators or decrease lung levels of antifibrotic mediators ; (3) promote aberrant migration of epithelial cells and other processes of abnormal repair (MMP3 and MMP9); (4) induce lung macrophage phenotypes to switch from M1 to M2 types (MMP10 and MMP28); and (5) promote migration of fibrocyte (MMP8) [In our study, the upregulated expression of MMP1, MMP7, MMP9 was consistent with previous research. Moreover, as a complement to other studies, our finding suggests that MMP10 may also serve as an IPF biomarker.MMP7 is expressed by mononuclear phagocytes, pulmonary epithelial cells, and fibrocytes , and it MMP9 is predominantly expressed by all fibroblasts, epithelial cells, leukocytes, and endothelial cells. Without being produced by resident cells in healthy lungs, MMP9 can be produced by bronchial epithelial cells, Clara cells, alveolar type II cells, fibroblasts, smooth muscle cells, and endothelial cells under various forms of stimulation . FurtherInsulin-like growth factors (IGF1 and IGF2) are insulin-related polypeptides that are significant for regulating cell proliferation and inhibiting programmed cell death ,19. Thesp < 0.001), suggesting that IGF1 is likely to play an extensive role in inducing cell proliferation instead of rigidly promoting fibroblasts proliferation or differentiation. IGF1 induced lung fibroblasts migration in mice. In pulmonary fibrosis, it can be observed that fibroblasts migrate to the intra-alveolar space with extracellular matrix protein deposition. Through the transwell filter system, it was found that IGF1 promoted the migration of lung fibroblasts in mice compared with serum-containing medium. Furthermore, blocking antibody treatment of IGF1R (A12) can abrogate the increased migration [Extracellular matrix synthesis and collagen deposition are the hallmarks of pulmonary fibrosis. Elevated collagen deposition has been verified to be an indicator of unsatisfied prognosis for patients who have fibrotic lung disorders. It has been confirmed that during the fibro-proliferative stage, the increased expression of collagen III in the lung is related to the development of fibrosis, while the production of collagen I is related to the remodeling of fibrous tissue. Studies have reported a significant correlation analysis of the relationship between IGF1 reactivity and collagen I and collagen III in lung biopsy specimens . Cellulaigration . The expigration .Integrins are a large family of heterodimeric cell–surface adhesion transmembrane receptors, which mediate cell–extracellular matrix, cell–cell, and cell–pathogen interactions. In mammals, specific and noncovalent bindings of 18 α-subunits to 8 β-subunits were contributed to form 24 diverse integrins. When integrins bind to extracellular ligands, they will activate a series of signaling pathways related to regulating cell cycles, moving receptors to cell membranes and organizing intracellular cytoskeleton. Almost all integrins are linked to the actin cytoskeleton via kindlin-binding motifs and talin in their β-subunit cytoplasmic domains, promoting cell migration, except integrin αvβ8  through its β8 subunit binding), and integrin α6β4 (linked to the intermediate filament through its β4-subunit). Among the 24 integrins, 8  bind ligands via the arginine−glycine−aspartic acid (RGD) sequence .TGF-β is the central cytokine that mediates the homeostasis of mesenchymal, epithelial, and immune cells. Excessive TGF-β signaling by fibroblasts has been shown in numerous fibrotic conditions in the lung, such as pulmonary and airway fibrosis, by regulating the profibrogenic critical components of fibrosis . IntegriIntegrin αvβ8 is expressed by epithelial cells and fibroblasts in the lung—the immunohistochemistry staining results in our study also confirmed this—while αvβ6 is expressed exclusively by epithelial cells. Patients with fibrosis have overexpressed αvβ6 receptors in the lung tissue, and inhibit receptors with a monoclonal antibody can prevent pulmonary fibrosis in mammals. Both αvβ6 and αvβ8 are important in stimulating latent complexes of the growth factor TGF-β. Several integrins can bind to the latent TGF-β complex via RGD binding motifs in the extracellular domains. This interaction enables integrins to function as a direct link between cells and the latent TGF-β complex in the ECM and allows integrins to activate the latent TGF-β complex. Most TGF-β activating integrins  activated TGF-β via mechanical transduction of intracellular forces to the latent TGF-β complex . HoweverOsteopontin  is a phosphorylated acidic glycoprotein secreted by various cells, including activated T cells, osteoclasts, and activated macrophages . OPN conAccording to reports, the development of fibrosis is associated with increased expression of OPN in the lungs. OPN increased more than 20-fold in IPF lungs, and it was one of the most upregulated genes among the genes that distinguished IPF lungs . MeanwhiOPN has a profibrotic effect on molecules involved in the remodeling of the extracellular matrix. It promoted the increase of type I collagen expression in fibroblasts and MMP7 expression in alveolar epithelial cells. The increased expression of type I collagen genes indicates that OPN can promote the fibrotic environment through its effects on epithelial cells and induce a nondegradative microenvironment similar to the one observed in experimental pulmonary fibrosis and IPF . OPN andAs shown in previous studies, in vivo administration of anti-αv integrin antibodies (RMV-7) that interfere with the binding of OPN and fibroblasts significantly attenuated bleomycin-induced pulmonary fibrosis. Moreover, RMV-7 almost completely eliminates the influence of OPN on in vitro adhesion, proliferation, and migration of fibroblasts. Those findings strengthened the relationship between the OPN–αv integrin interaction and the pathogenesis of pulmonary fibrosis .The proliferation and migration of fibroblasts and the accumulation of extracellular matrix are important in the pathogenesis of pulmonary fibrosis, and the interaction of OPN with collagen, fibronectin, and proteoglycan has been confirmed . StudiesThe high-quality study of Xu et al. has updated and enriched our knowledge about the genes involved in the molecular mechanism of IPF . They scCompared with other similar studies, the genes screened by integrating different databases in our study are more representative. More importantly, we validated the candidate genes using RT-qPCR and immunohistochemistry staining to make the results more credible. Although some of the candidate genes have been explained in previous studies, their clinical applications are still lacking. Our functional enrichment analysis and PPI network indicate that combining multiple markers rather than a single protein intervention may be more helpful for clinical diagnosis and treatment. SPP1, THBS2, ITGB8, and some other genes screened out in this study have rarely been studied in the pathogenesis of pulmonary fibrosis, which also suggests possible approaches for subsequent pathogenesis studies.To sum up, this study identified several genes associated with IPF through bioinformatics methods, which may provide valuable new ideas for diagnosis and treatment. However, the accurate and direct connections with IPF are still lacking and further studies are needed.p-value < 0.05 and logFC (>1 or <1). Hierarchical clustering analysis and the Genesis program were employed to generate volcano plots to visualize the results mentioned above. Furthermore, different common expression genes were figured out using the Morpheus Website (available online: https://software.broadinstitute.org (accessed on 2 December 2020)); a Venn diagram was generated to represent the result. The Limma R package was used for DEG screening, while the intersect function in R was employed for identifying the common DEGs.The original gene-expression-profiling datasets were obtained from NCBI GEO databases. On the GEO home page, “IPF” was used as the search term. We selected datasets according to the following criteria: (1) the samples for the study contained lung tissues from healthy donors and patients with IPF; and (2) the datasets provided raw data. Finally, we selected four datasets, GSE110147, GSE53845, GSE24206, and GSE10667. In addition, although GSE10667 and GSE110147 contained samples from patients with usual and nonspecific interstitial pneumonia, respectively, we excluded that information from the analysis, and 17 IPF samples from GSE24206 were derived from only 11 IPF patients . Four gene expression profiles were analyzed with the online tool GEO2R, the accession numbers of which were GSE110147, GSE53845, GSE24206, and GSE10667, respectively. Among these datasets, GSE110147 was generated from Canada, while the others were generated from the United States. All the profiles could be divided into two groups according to sample types—normal and IPF. The GSE110147 data included 11 normal pulmonary tissues and 22 IPF tissues; the GSE53845 data included 8 normal lung tissues and 40 IPF tissues, and the GSE24206 data included 6 normal lung tissues and 17 IPF tissues. The GSE10667 data included 15 normal pulmonary tissues and 8 IPF tissues. Accordingly, to screen out differentially expressed genes (DEGs) between two sample groups in each gene expression profile, cut-off criteria were set at https://david.ncifcrf.gov/ (accessed on 2 December 2020)). Meanwhile, the functional enrichment analysis tool ) was employed to achieve pathway enrichment analysis. A p-value < 0.05 was considered as statistically significant, with the GO results ranked by p-value.To realize the gene ontology enrichment analysis, we input the common DEGs into The Database for Annotation, Visualization and Integrated Discovery ) to construct the PPI network, and then we visualized the results using Cytoscape software (https://cytoscape.org (accessed on 2 December 2020), v.3.7.0). the k-means clustering, only selecting experimentally validated interactions with a combined score > 0.4, which is regarded as crucial.The common DEGs were submitted into the FunRich tool by group, which included 62 upregulated genes and 20 downregulated genes, accordingly. Therefore, we managed to construct the protein–protein interaction (PPI) network and figure out central genes participating in the process of IPF. We input the common DEGs in the STRING database . The qRT-PCR analysis was carried out using SYBR Green I mix . The mRNA expression was measured as previously described . Primer The immunohistochemistry staining was performed as described previously . Details


Graphene oxide (GO) is an oxygenated functionalized form of graphene that has received considerable attention because of its unique physical and chemical properties that are suitable for a large number of industrial applications. Herein, GO is rapidly obtained directly from the oxidation of graphene using an environmentally friendly modified Hummers method. As the starting material consists of graphene flakes, intercalant agents are not needed and the oxidation reaction is enhanced, leading to orders of magnitude reduction in the reaction time compared to the conventional methods of graphite oxidation. With a superior surface area, the graphene flakes are quickly and more homogeneously oxidized since the flakes are exposed at the same extension to the chemical agents, excluding the necessity of sonication to separate the stacked layers of graphite. This strategy shows an alternative approach to quickly producing GO with different degrees of oxidation that can be potentially used in distinct areas ranging from biomedical to energy storage applications. The presence of functional groups attached to graphene extends the functionalities of this two-dimensional 2D) material. Graphene oxide (GO) is an important form of functionalized graphene that disperses in water and other polar solvents. Because of the presence of oxygenated groups, such as carboxyl, hydroxyl, and epoxy, GO can be further functionalized with various organic molecules, making it a versatile chemical functionalization platform with an extensive potential for a wide range of applications in some of the greatest challenges of our time, such as water treatment, energy storage, environment, and medicine ,2.D materiaBeyond the chemical synthesis, the choice of the carbon source also plays an important role in the final quality of GO. Specifically, different types of graphite and synthesis methods can lead to significant modifications in the physical characteristics of GO, such as the lateral size of the flake, the number of layers, and disorder. As a result, the final properties of GO are sensitive to the materials and methods of production, making every structure suitable for specific applications. For example, highly oxidized GO flakes with sub-micrometer size are of great interest for biomedical applications , whereas4) and sodium nitrate (NaNO3) in a concentrated sulfuric acid (H2SO4) solution [2/N2O4) due to the presence of NaNO3. Additionally, the Hummers method uses not only large amounts of concentrated H2SO4 and KMnO4 to ensure sufficient oxidation of graphite flakes but also huge amounts of water to remove the excessive ions after oxidation. As a result, the process is costly in terms of time, energy, and waste treatment, with several safety and health concerns. Besides, the oxidation is not homogeneous, with the oxidation degree being extremely difficult to control, and some residual graphite is still left unless long sonication periods are used, to produce just a few layers of GO [The Hummers method is the most-used approach for preparing GO and consists of the oxidation of graphite using potassium permanganate (KMnOsolution . Despiters of GO .2FeO4) [3 to avoid the formation of toxic gasses and to facilitate the disposal of wastes due to the absence of Na+ and NO3− ions. However, the elimination of NaNO3 usually demands compensatory strategies to guarantee the efficiency of the Hummers method. Reports including increased amounts of KMnO4 and H2SO4, the introduction of new components, such as phosphoric acid (H3PO4), to the reaction [Recently, numerous strategies have been developed to minimize the issues mentioned above. For example, to reduce the oxidation time, stronger oxidizing agents, such as potassium ferrite (K2FeO4)  or elect2FeO4) . Other sreaction , or reacreaction  have beeN-methyl-2-pyrrolidone, is used to produce monolayer graphene and platelets originally from graphite on large scales [3 is not required, this chemical route is considered eco-friendlier and much less harmful to health and the environment compared to the traditional modified Hummers method. 3 and, consequently, the absence of toxic gasses (NO2/N2O4) and the reduced oxidation time.The liquid phase exfoliation (LPE) in organic solvents, such as e scales . The dire scales  allows ue scales ,13. Here2SO4 , with subsequent cooling up to 2 °C. Next, 2.25 g of KMnO4  was slowly added to the suspension. The system was kept under stirring at room temperature for different periods of time, in which the graphene flakes were oxidized, followed by cooling to 2 °C and further dilution in water. Lastly, the resulting GO suspensions were cleaned with 3 cycles of washing using HCl  10% and dialysis until pH 5.Graphene flakes were supplied by 2D Materials Pte. Ltd. (2DM) (Singapore). The chemical route to obtain graphene oxide directly from graphene consisted of an environmentally friendly simplified Hummers method in which 0.5 g of graphene flakes were added to 17 mL of concentrated HGO with different degrees of oxidations were obtained from Sigma Aldrich  and Abalonyx . The materials were used without further purification and are labelled as commercial GO I and II .2) for Raman spectroscopy and atomic force microscopy (AFM). For XPS analysis, the measurements were performed in a Kratos AXIX Ultra  equipment with a mono-chromatic source Al Kahv = 1486.81 eV. The calibration, Shirley-type background, peak fitting, and quantification were carried out using Casa-XPS software (version 2.1.19) . For the SEM analysis, a FESEM VERIOS 460  with an accelerating voltage of 2.0 kV and a current of 100 pA was used. For Fourier transform infrared (FTIR) spectroscopy, an ALPHA Platinum-ATR , instrument was used. For this, the aqueous suspensions of GO were freeze-dried, and prepared by the KBr disc method, and the spectra were obtained in a in attenuated total reflection (ATR) mode. Atomic force microscopy (AFM) measurements were carried out in a Bruker Dimension Icon Microscope  operated in ScanAsyst tapping mode and scan lines of 512 under ambient conditions. Confocal Raman spectroscopy was performed in a WITec Alpha 300R  with an excitation wavelength of 532 nm and a 100× objective.The samples were drop casted on silicon (Si) substrates for XPS and SEM analyses and on silicon substrate with 300 nm silicon dioxide , as implemented in Quantum ESPRESSO v. 6.5 , with thThe evolution of the increase in oxygenated groups in the graphene structure as a function of the oxidation time is demonstrated in The thickness and lateral size of GO obtained from atomic force microscopy (AFM) images did not change significantly after 5 min of reaction, indicating that most of the modifications occur very quickly, beyond our limit of observation due to the restrictions of the experimental setup conditions. Looking closer at the spectra and comparing the percentages shown in −1 are attributed to the C–OH  stretching, C–O (epoxy) stretching, and O–H (hydroxyl) deformation vibrations, respectively. These are compared with first- principles calculations, which find C–OH bending modes at 1089–1095 cm−1, the epoxy symmetric stretch at 1254 cm−1, and C–OH hydrogen wag mode at 1509 cm−1, which may be the origin of the resonance observed at 1418 cm−1. The carboxylic acid group appears at 1733 cm−1 with the carbonyl (C=O) stretching calculated to lie at 1691 cm−1. The peak at 1620 cm−1 is usually attributed to the non-oxidized domains in the graphene structure [−1. These modes are very close in frequency with a graphene vibrational mode and therefore have significant localization in the carbon atoms as well , which can result in aggregation. After the oxidation, the sharp 2D band at ~2683 cm−1 disappears, and some broad features between 2600 and 3000 cm−1, typical for GO [As expected, after the oxidation, the degree of disorder of the graphene backbone was higher, corroborating the higher relative intensity of the D band and a broader G band in the Raman spectra b. After GO modes . As the ntations . The gral for GO  to the eBy comparing our GO obtained directly from graphene with two commercially available GO obtained from graphite , we see However, as expected, GO from graphite presents larger sheets  compared to GO from graphene since the graphene sheets used in this study had a small lateral size of 1 µm . Importa3, that are commonly used to expand the graphite structure are not required, and toxic gasses, such as NO2/N2O4, are not generated, diminishing several safety and health concerns. Consequently, the reaction time is reduced by the orders of magnitude when compared with conventional methods. As graphene has a comparatively larger surface area per volume, the oxygenated groups are rapidly and homogeneously distributed over the graphene lattice, eliminating steps of sonication to further separate the layers.We proposed an approach to synthesize graphene oxide with a controllable degree of oxidation. The route consists of an environmentally friendly modified Hummers method, in which the carbon source is graphene, not graphite flakes as in the standard processes. For this reason, intercalant agents, such as NaNOOur study opens up new avenues to explore environmentally friendly production routes as well as novel mild oxidation approaches to expand industrial applications of GO, such as chemical sensors , solar c