Idiopathic pulmonary fibrosis (IPF) is a form of usual interstitial pneumonia (UIP), though its origin is unknown. IPF remains one of the most aggressive and lethal forms of interstitial lung diseases. Extensive basic and clinical research during the past decade has uncovered critical aspects related to the pathophysiological events that lead to the formation of fibrotic scars in the lungs of IPF patients. Our current understanding of IPF pathogenesis includes repetitive/chronic injury to alveolar epithelial type 2 cells (AEC2) that triggers an aberrant wound healing response associated with the activation of fibroblasts and the infiltration of immune cells. This leads to the excessive accumulation of myofibroblasts that deposit extracellular matrix (ECM) proteins, particularly collagen and fibronectin. All aforementioned processes culminate in detrimental scarring of the lung tissue, loss of AEC2 and AEC1, and respiratory failure [bona fide AEC2 (Lysohigh) is the predominant AEC2 subset in the healthy lung, and that this population is largely replaced by an intermediate AEC state (Lysolow) in bleomycin-induced pulmonary fibrosis and in human IPF. Lysolow cells express, in addition to typical AEC2 markers, markers of basal cells, thus highlighting epithelial cell plasticity as a source of cellular heterogeneity in IPF [low cells are also reminiscent of the tdTomato-low (tdTom)low cells, identified using the AEC2 lineage-tracing mouse line  [low cells have been shown to expand during compensatory lung growth following pneumonectomy and were therefore dubbed injury-activated alveolar progenitors (IAAPs) [Fgf10 [In this Special Issue, Wasnick et al. describe two subsets of AEC2 in both healthy and fibrotic lungs [y in IPF ,4,5,6,7.atoflox) ,9,10. Su (IAAPs) ,9. In th (IAAPs) . They al) [Fgf10 . Importa) [Fgf10 . In the ) [Fgf10 . These 3MUC5B promoter rs35705950 polymorphism and the risk of IPF, and the bronchiolization of distal airspaces in IPF lungs. The contribution of airway epithelial cells to IPF pathogenesis is also addressed by Schramm et al., who discuss the role of the ErbB receptor–ligand system in lung fibrosis [Despite the fact that IPF is considered as a “distal” lung disease, with a central role of AEC2 malfunction during its pathogenesis, Chakraborty et al. highlight recent developments that point towards the involvement of airway epithelial cells in IPF pathobiology . The confibrosis . Intrigufibrosis .The cellular heterogeneity of IPF is further discussed by Preisendörfer et al., who stress the potential role of B cells in IPF pathobiology . The patIPF is also associated with a set of metabolic disorders and targeting cellular metabolism, particularly in mesenchymal cells, represents an exciting therapeutic avenue ,19,20,21One of the factors that contribute to the incurable nature of IPF is the fact that patients are already in advanced disease stages at the time of diagnosis. As such, treatment mainly focuses on treating the symptoms and slowing down disease progression rather than treating early events that initiate downstream pathological signaling cascades. Accordingly, efforts that seek to find alternative therapeutic agents against IPF are still ongoing.Current treatment modalities for IPF patients rely on two approved drugs: pirfenidone, which is believed to act as a transforming growth factor beta 1 and hedgehog signaling inhibitor , and ninKorfei et al. contribute to the Special Issue with a comprehensive review on the current knowledge regarding IPF pathophysiology, the associated cellular and molecular mechanisms, clinical management, as well as novel therapeutic targets and therapies in development . ImportaBraubach et al. go beyond IPF and provide a broad characterization of fibroelastotic remodeling (FER), its anatomic distribution, and clinical association . FER is In conclusion, we present a collection of articles in this Special Issue that touch on all aspects of IPF, from the molecular mechanisms driving disease development and progression, to translational models enabling high-throughput drug screening, finishing on clinical studies evaluating adverse effect profiles of drugs approved for IPF treatment. Despite immense improvements in understanding the molecular basis of IPF, further research is urgently needed to pursue innovative translational studies in order to learn more about the enormous repertoire of molecular tricks that lung cells use to adapt to stress conditions and fight against damaging agents.


In addition, the graphene-modified electrode had a considerably larger sensitivity (0.0124 A/M) and lower detection limit (1.81 × 10−6 M), proving the advantages of employing graphene in electrochemical sensing.In this study, a graphene sample (EGr) was synthesized by electrochemical exfoliation of graphite rods in electrolyte solution containing 0.1 M ammonia and 0.1 M ammonium thiocyanate. The morphology of the powder deposited onto a solid substrate was investigated by the scanning electron microscopy (SEM) technique. The SEM micrographs evidenced large and smooth areas corresponding to the basal plane of graphene as well as white lines (edges) where graphene layers fold-up. The high porosity of the material brings a major advantage, such as the increase of the active area of the modified electrode (EGr/GC) in comparison with that of bare glassy carbon (GC). The graphene modified electrode was successfully tested for L-tyrosine detection and the results were compared with those of bare GC. For EGr/GC, the oxidation peak of L-tyrosine had high intensity (1.69 × 10 Tyrosine (Tyr) is a small, electroactive, and aromatic amino acid used to regulate the signal transduction process in proteins . In humaSeveral methods have been developed for the detection of L-tyrosine including surface-enhanced Raman scattering , high-peGraphene, consisting of a monolayer of carbon atoms exhibits excellent mechanical, thermal, electrical, and optical properties ,15. Its In this work, a simple approach was used for manufacturing a modified glassy carbon electrode employing electrochemically exfoliated graphene. The electrochemical properties of the modified electrode were investigated at electro-catalytic oxidation of Tyr. The electrochemically exfoliated graphene exhibits good electro-catalytic activity to the oxidation of Tyr, since it enhances the peak current and lowers the peak potential. In order to demonstrate the possible application of this electrochemical sensor, we used the standard addition method for the determination of Tyr in spiked samples, demonstrating excellent results.In order to investigate the morphological characteristics of graphene sample, a SEM/TEM Hitachi HD2700 instrument  equipped with a cold field emission gun (CSEG), operated at 200 kV and coupled with a double cut windowless 100 EDX detector acted by AZtec Software (Oxford Instruments) was used.α1 radiation (λ = 1.5406 Å). The background corrected pattern was used for the calculation of graphene structural parameters The X-ray powder diffraction (XRD) pattern was obtained with a Bruker D8 Advance Diffractometer using CuK−1 spectral domain; 4 cm−1 resolution).The FTIR spectrum of graphene sample (mixed with KBr) was recorded with a JASCO 6100 FTIR spectrometer . The sample was dispersed by ultrasound in double-distilled water for 3 min, to form a homogeneous suspension. Next, the spectrum was recorded within 200–800 nm range.5 Hz, in solution containing 10−3 M potassium ferrocyanide and 0.2 M KCl.For electrochemical measurements  a three-electrode cell coupled with a Potentiostat/Galvanostat Instrument  was used. The CVs and LSVs measurements were generally run from 0.0 to +1 V vs. Ag/AgCl, at 10 mV/s scanning rate. EIS measurements were recorded between 0.1 and 10−4 M Tyr was prepared in pH 7 PBS. Next, 0.1 mL of the stock was mixed with 4.9 mL pH 7 PBS which gave a final concentration of 1 × 10−5 M Tyr. This was denoted the unknown concentration (Cx). Next, three volumes  of the stock solution (5 × 10−4 M) were added to three beakers, each also containing the unknown concentration Cx .For standard addition method, the following procedure was applied. A stock solution of 5 × 10All chemicals, including: ammonia , ammonium thiocyanate , potassium chloride , potassium ferrocyanide , L-Tyrosine  and Dimethylformamide (DMF)  were used without further purification. The electrolyte solutions were prepared with double-distilled water produced with Fistreem Cyclon equipment.Two electrochemical cells each containing two graphite rods and filled with 100 mL solution of electrolyte (0.1 M ammonia + 0.1 M ammonium thiocyanate) were connected to the exfoliation system (home-made system). A constant voltage of 11 V was applied between the graphite rods (anode and cathode) for about 4 h. In order to avoid the over-heating of the electrolyte, short pulses of current were applied for 0.8 s, followed by short pauses (0.2 s). The black powder deposited after exfoliation at the bottom of each cell was collected, washed with distilled water (10 L) and finally dispersed by ultrasound for 30 min in 125 mL water. Next, the black suspension was filtered on white-ribbon paper to remove the large particles and finally dried by lyophilization. The obtained sample was following denoted EGr.In 2 mL solution of N,N-dimethylformamide (DMF) were dispersed by sonication 2 mg of graphene powder . Next, a volume of 10 µL from the dispersion was deposited by drop-casting onto the clean surface of a glassy carbon (GC) electrode and dried at room temperature for 24 h. After that, the modified electrode (EGr/GC) was employed for the electrochemical detection of Tyr.The morphology of graphene powder deposited onto a solid substrate was investigated by scanning electron microscopy technique. Two representative micrographs are presented in The structure of graphene sample was next investigated by X-ray powder diffraction technique. In −1 is assigned to the O-H stretching vibrations from the adsorbed water molecules, while the two adsorption peaks at 1383 and 1057 cm−1 are assigned to the C-O stretching vibrations. Their weak intensity is in excellent agreement with the XRD pattern of the sample where a small GO peak is evidenced. The peaks at 1577 cm−1 and 1638 cm−1 represents the vibration modes of sp2-hybridized C=C. Complementary to this, the UV-Vis spectrum of the sample  values were determined after fitting the Nyquist plots with the appropriate equivalent electrical circuit. The first circuit characterizes the bare GC electrode and contains the following elements: the solution resistance (Rs) which depends on the concentration of supporting electrolyte, the Warburg impedance (ZW) that appears due to the diffusion of ions within the double-layer at low frequency, the charge-transfer resistance (Rct) that reflects the easiness of electron transfer across electrode-solution interface and a constant phase element (CPE) that replaces the double-layer capacitance (Cdl). For graphene-modified electrode, due to the high porosity of graphene layer the circuit additionally contains a transmission line (T) [ct values were determined to be: 75.5 kΩ for bare GC and 1.16 kΩ for EGr/GC, indicating that the graphene layer highly favors the transfer of electrons when deposited on top of a conductive substrate.In order to fully characterize the bare and graphene-modified electrodes, the EIS spectra were recorded and the experimental data were represented as Nyquist plots . The chaline (T) . After f−4 M) was investigated in buffer solutions of various pH (3.6–8), using the LSV technique . In highly acidic solution (pH 3.6) a clear oxidation peak appears at around +0.85 V which shifts towards lower potentials, in neutral and basic solutions. The linear regression equation that describes the variation of peak potential, Ep, versus pH is given by: y = 1.019 − 0.053 × pH. In this case, the slope value is 0.053 V/pH being close to that obtained from a Nernstian plot (0.059 V/pH) and indicating an equal number of protons and electrons involved in the electrochemical reaction. The maximum peak current, Ip, was observed in pH 7 PBS, so all of the experiments were next performed at this pH  and appears at higher potential (+0.84 V) in comparison with that of graphene-modified electrode (+0.64 V). The increased sensitivity of EGr/GC towards Tyr oxidation may be due to the enhanced electron transfer rate at the edges of graphene sheets. In addition, the π–π stacking interaction between the aromatic ring of Tyr and graphene may help with the orientation of OH group towards graphene, favoring its oxidation.In order to evidence the electro-catalytic properties of the synthesized material towards L-tyrosine oxidation, the comparison between the graphene-modified electrode and the bare GC electrode is shown . The LSV−6 M Tyr, while with bare GC the lowest signal was obtained at a higher concentration, of 3 × 10−5 M Tyr. This indicates an increased sensitivity of the graphene-modified electrode, in comparison with bare GC.Next, Tyr was quantitatively analyzed by LSV and the corresponding recordings at various concentrations are plotted in p) versus Tyr concentration, the corresponding calibration plots were obtained and represented in p = 1.88 × 10−7 + 0.0124 × C, R2 = 0.988, while for bare GC is Ip = 8.8 × 10−8 + 0.00265 × C, R2 = 0.968. As can be seen in this figure, the EGr/GC electrode has a considerably higher sensitivity (0.0124 A/M) and lower detection limit (1.81 × 10−6 M), in comparison with those of bare GC: 0.00264 A/M sensitivity and 9 × 10−6 M detection limit. The detection limit was obtained by dividing the limit of determination (LOD) by 3.3.By representing the peak current (I−4 M each). In the case of a single analyte (ascorbic acid) the oxidation signal appears as a very broad peak, at low potential . The dopamine peak is well defined and appears at higher potential  while uric acid appears at +0.35 V (pink curve). When Tyr is mixed with all of these analytes (10−4 M each), an increase of the capacitive current is observed but no changes are noticed for the peak potential (see the olive curve in comparison with the brown curve). For the Tyr peak intensity, a slight increase is observed, from 1.042 × 10−6 A  to 1.129 × 10−6 A (mixture of TYR + AA + DOP + UA), indicating a weak influence of the interfering species.The selectivity of EGr/GC electrode towards Tyr oxidation was evaluated in the presence of other biomolecules, such as ascorbic acid (AA), dopamine (DOP) and uric acid (UA). In −5 M, giving an excellent recovery (98.5 %). In order to confirm the results five standard addition measurements were performed and the recovery range was between 98.5% and 103.1%, proving the efficiency of the EGr/GC electrode.The EGr/GC electrode was also tested for Tyr detection using the standard addition method . The peaSeveral advantages of the employed electrode can be mentioned such as the simple approach used for manufacturing the graphene-modified electrode, the very wide linear range of L-Tyrosine with good sensitivity and detection limit, and the excellent recovery results obtained with the standard addition method in spiked samples .In addition, it is important to mention that the graphene sample employed as electro-catalyst has an excellent time stability. If stored in a closed recipient it can be used in electrochemistry for over a year. As concerned the modified electrode, it shows an excellent working sensitivity. More than 20 measurements were recorded with the same electrode and the signal was >95% of its original value. Between the measurements, the electrode was kept in distilled water for 15 min to desorb the surface contaminants. The longtime stability was also investigated over a 10 days period, indicating that the analytical performance of the modified electrode has not significantly changed , 2 configuration of carbon atoms is predominant within the synthesized material. The graphene sample was used for glassy carbon surface modification (EGr/GC) and next tested towards Tyr detection. By representing the peak current (Ip) versus Tyr concentration, the corresponding calibration plots were obtained. The linear regression equation for EGr/GC electrode was: Ip = 1.88 × 10−7 + 0.0124 × C, R2 = 0.988, while for bare GC was Ip = 8.8 × 10−8 + 0.00265 × C, R2 = 0.968. As expected, the EGr/GC electrode had higher sensitivity (0.0124 A/M) and lower detection limit (1.81 × 10−6 M), in comparison with those of bare GC: 0.00264 A/M sensitivity and 9 × 10−6 M detection limit.A graphene sample was prepared by electrochemical exfoliation of graphite rods in solution containing 0.1 M ammonia + 0.1 M ammonium thiocyanate. The porous morphology of the sample was revealed by SEM technique while the structural parameters were determined from the XRD pattern. Hence, the average number (n) of layers within the graphene crystallites, the inter-layer spacing (d), the mean crystallite size (D) of graphene and the amount of GO, FLG, and MLG (expressed as %) were determined. In addition, FTIR and UV-Vis complementarily characterized the sample indicating that the sp


Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease with limited therapeutic options, and there is a huge unmet need for new therapies. A growing body of evidence suggests that the histone deacetylase (HDAC) family of transcriptional corepressors has emerged as crucial mediators of IPF pathogenesis. HDACs deacetylate histones and result in chromatin condensation and epigenetic repression of gene transcription. HDACs also catalyse the deacetylation of many non-histone proteins, including transcription factors, thus also leading to changes in the transcriptome and cellular signalling. Increased HDAC expression is associated with cell proliferation, cell growth and anti-apoptosis and is, thus, a salient feature of many cancers. In IPF, induction and abnormal upregulation of Class I and Class II HDAC enzymes in myofibroblast foci, as well as aberrant bronchiolar epithelium, is an eminent observation, whereas type-II alveolar epithelial cells (AECII) of IPF lungs indicate a significant depletion of many HDACs. We thus suggest that the significant imbalance of HDAC activity in IPF lungs, with a “cancer-like” increase in fibroblastic and bronchial cells versus a lack in AECII, promotes and perpetuates fibrosis. This review focuses on the mechanisms by which Class I and Class II HDACs mediate fibrogenesis and on the mechanisms by which various HDAC inhibitors reverse the deregulated epigenetic responses in IPF, supporting HDAC inhibition as promising IPF therapy. B. B165]. +-expressing basal cells in response to fibrotic signalling in the lung mesenchyme in hAECII-derived organoids ex vivo and in hAECII xenotransplantation experiments with bleomycin-treated mice in vivo , w, w165],  in vivo . AlthougI in IPF ,118,302.In contrast to all other Class II HDACs, isoform-selective inhibitors have been developed for HDAC6 and not only evaluated in cancer but also lung fibrosis  174,190,235.HDAC6, pharmacological inhibition of HDAC6 deacetylase acitivity by the small molecule inhibitor tubacin resulted in the abrogation of TGF-β-induced Notch1 signalling and EMT in A549 cells [HDAC6, tubastatin was shown to lead to α-tubulin hyperacetylation (and thus successful HDAC6 inhibition) and to repress TGF-β-induced expression of type-I collagen (COL1A1) in lung fibroblasts by inducing AKT dephosphorylation at Ser43 through increasing the association of AKT with the specific phosphatase PHLPP (PH domain and leucine-rich repeat protein phosphatase), with consequent AKT inactivation [Similar to silencing of 49 cells ,191. Howtivation . In additivation .Hdac6(−/−) knockout mice were not protected against bleomycin-induced lung fibrosis despite pronounced hyperacetylation of α-tubulin. Similarly, TGF-β-induced collagen expression was not decreased in murine lung fibroblasts isolated from Hdac6(−/−) knockout mice compared to TGF-β-stimulated normal fibroblasts from wild-type mice. As mentioned above, RNAi-mediated knockout of HDAC6 in human lung fibroblasts did also not repress TGF-β-induced collagen expression. Together, these results suggested that tubastatin might have ameliorated bleomycin-induced lung fibrosis by targeting the PI3K-AKT pathway, likely through an HDAC6-independent mechanism, and that tubastatin might have significant off-target effects aside from HDAC6 [Hdac6(−/−) knockout mice, the additional loss of HDAC6 function to transport cytotoxic polyubiquitinated (misfolded) proteins into autophagosomes for their degradation, may aggravate bleomycin-induced lung fibrosis by causing defective autophagy [However, homozygous om HDAC6 . On the om HDAC6 . Thus, tutophagy .ACTA2, COL1A1, COL3A1 and FN. The authors of this study concluded that HDAC6 confers the pronounced hyperproliferative and profibrotic effects to bronchiolar basal cells in IPF, thereby underscoring that inhibition of HDAC6′s deacetylating function plays an important role in the treatment of IPF [Very recently, novel specific inhibitors for human HDAC6 enzyme have been developed as promising pharmaceutical tools for the treatment of IPF, with the aim of also investigating the role of HDAC6 in the abnormal bronchiolization process in IPF, as this enzyme isoform was found to be robustly overexpressed in bronchiolar basal cells of IPF lungs . It coult of IPF . HoweverTaken together, HDAC6 deacetylase activity appears to mediate profibrotic responses and signalling through non-histone protein deacetylation, leading to altered cellular signalling, presumably involving HSP90 chaperone (a substrate of HDAC6)-mediated signalling and sustained AKT activation, which can be blocked by HDAC6 selective inhibitors. The selectivity of tubastatin to inhibit HDAC6 deacetylase activity should be re-checked.The summarised data provides evidence that abnormally increased HDAC activity in lung fibroblasts and bronchiolar basal cells versus a lack of HDAC activity in AECII is critical in the pathogenesis of lung fibrosis, which can be overcome by treatment with HDAC inhibitors. In particular, compelling evidence reveals a favourable therapeutic efficacy of pan-HDAC inhibitors in preclinical models of lung fibrosis . Pan-HDAPGC1A and the suppression of various cytokine and ECM genes in TGF-β-stimulated IPF fibroblasts, which was, in part, attributed to the abrogation of HDAC7-mediated TGF-β signalling [By employing an image-based screening assay with the use of α-SMA immunofluorescence intensity as the primary readout parameter of in vitro fibroblast activation (induced by TGF-β), Jones and coworkers (2019) tested and rank-ordered 99 modulators of epigenetic-regulating enzymes and identified the pan-HDAC inhibitor pracrinostat as the most effective small molecule in downregulating TGF-β-induced α-SMA expression . Interesgnalling . Howevergnalling ,167. Thegnalling . SAHA wagnalling . These fFAS expression [ACTA2 and COL1A1 expression [In IPF fibroblasts in vitro, SAHA suppressed genes associated with ECM and anti-apoptosis and upregulated proapoptotic genes through the modulation of chromatin acetylation and specific histone modifications associated with such genes and induced significant apoptosis in these cells ,167. In pression  and abropression .PLAT) in the vasculature of mice and men while downregulating PAI-1 [PLAT promoter [As outlined in ng PAI-1 ,306. Thepromoter . VPA alspromoter . In humapromoter . Considepromoter .Recently, a novel clinical-stage HDAC inhibitor, CG-745, specific for Class I HDACs and Class IIB HDAC6, revealed favourable therapeutic efficacy in bleomycin-treated mice as it significantly reduced inflammatory cell populations in BALF and lowered the collagen contents back to the levels of the control saline group. Interestingly, CG-745 also efficiently attenuated severe lung fibrosis in mice induced by polyhexamethylene guanidine (PHMG) .+ IPF-AECII but abnormally upregulated in KRT5+ bronchiolar basal cells [+high progenitors in fibrotic lungs [+ basal cells. Interestingly, the administration of pan-HDAC inhibitors did not exert “deleterious” effects on fibrotic AECII in various rodent models of lung fibrosis but changed their abnormal phenotype. In the mouse model of bleomycin-induced lung fibrosis, Ota and coworkers (2015) showed that administration of the pan-HDAC inhibitor TSA from day 7 to 21 after bleomycin instillation restored Sftpc expression in FACS-isolated AECII in vivo [+ AECII re-differentiation, proSP-C+ AECII survival and proper re-epithelialization of the damaged alveolar epithelium as a therapeutic strategy. In renal fibrosis, it could be demonstrated in the murine model of unilateral ureteral obstruction that TSA led simultaneously to the inactivation of renal interstitial fibroblasts and the inhibition of renal tubular epithelial cell death [per se are still underexplored.However, in vitro and experimental studies for lung fibrosis/IPF described in this review were mainly restricted to the assessment of inflammation status and fibroblast/myofibroblast apoptosis, but not on the effects of HDAC inhibitors on AECII injury/AECII death and the aberrant bronchiolar re-epithelialization process. Although nearly all Class I and Class II HDAC enzymes appeared to be actually absent in differentiated proSP-Cal cells , it can ic lungs ,309,310, in vivo . Thus, a in vivo ,168, thell death . HoweverInterestingly, a recent in vitro study showed that the weak pan-HDAC inhibitor 4-phenyl-butyrate (4-PBA) alleviated the aggregation and improved the secretion of IPF-associated mutant SP-A2 proteins in A549 cells through the upregulation of glucose-regulated protein (GRP)78 . 4-PBA iHowever, the clinical use of pan-HDAC inhibitors in cancer patients has been, in part, associated with several challenges and side effects in some patients ,313,314 CIP1p21 expression, and the knockdown of each of these HDACs resulted in the derepression of CIP1p21 expression. However, in each case, the magnitude of CIP1p21 induction was markedly less than that induced by pan-HDAC inhibitors [The discussions about the side effects and improvement of therapeutic strategies have led to the development of HDAC isoform-selective inhibitors to overcome undesired effects . Until nhibitors . In agrehibitors ,318. On hibitors ,235,258.hibitors  remains Taken together, it can be suggested that specific inhibition of single HDACs may yield some therapeutic benefit, whereas the use of pan-HDAC inhibitors is likely to yield a stronger therapeutic response. In conclusion, the published findings summarised in this review indicate that HDACs offer novel molecular targets for IPF therapy and that FDA-approved Class I and pan-HDAC inhibitors for cancer treatment may also be promising therapeutic agents for the treatment of IPF. The putative antifibrotic effects of HDAC-inhibitor treatment on IPF are illustrated in + basal cells, directly correlate with mortality in IPF [Idiopathic pulmonary fibrosis is associated with a progressive loss of lung function and a poor prognosis. Loss of AECII, myofibroblast expansion and the ectopic appearance of basal cells in the alveoli are the hallmarks of IPF. The number of myofibroblast foci, as well as the extent of alveolar KRT5y in IPF ,319. Appy in IPF ,169,271,We believe that pan-HDAC inhibitors can not only reverse the aberrant epigenetic response in (myo)fibroblasts and bronchiolar basal cells in IPF but also the activated senescent phenotype in the AECII of IPF lungs despite pro-apoptotic events and a lack of many HDACs in IPF–AECII. The mechanisms of HDAC inhibitors towards the injured alveolar epithelium  in IPF have not yet been addressed and should be elucidated in future studies.


We reported an innovative transparent, elastic and flexible conductive composite materials P(SSNa-BA-St)/PEDOT/graphene which were prepared by using P(SSNa-BA-St) latex as template for PEDOT polymerization and graphene doping. This P(SSNa-BA-St)/PEDOT/graphene film exhibited highly transparent, good water resistance, low moisture adsorption, highly elastic and highly conductive properties, which can serve as a practical approach to fabricate the flexible, conductive and transparent films for wearable and implantable electronic devices, and photovoltaic cells. We reported an transparent, water resistant, and flexible conductive materials P(SSNa-BA-St)/PEDOT/graphene and their conductivity are due to the large surface area to polymerize the extended PEDOT chain on the nanoparticles. Basically, EDOT can be chemically polymerized in a PSS solution to form PSS/PEDOT dispersion.5 However, PSS as an insulator could strongly decrease the conductivity of the PSS/PEDOT.6 Therefore, increasingly intensive work is going on to enhance the conductivity of PSS/PEDOT by organic compounds, salts, acid, zwitterions and anionic surfactant, etc.7–9 Secondary doping by polar solvents is also used to increase the conductivity, which is attributed to morphological changes. Doping the polar solvents could trigger phase separation between the conducting PEDOT chains and insulating PSS chains, leading to an interconnected network of elongated PEDOT grains and removal of insulating PSS chains for electrical conductivity enhancement.10–12The polyelectrolyte complex poly (PEDOT)/poly(styrene sulfonate) (PSS) is one of the most well-known organic conductors with highly conductive, largely transmissive to light, processible in water and flexible.13 Directing mixing the water soluble graphene oxide (GO) with PEDOT/PSS was limited by the poor conductivity of GO,14 therefore, the water insoluble reduced graphene oxide (RGO) was carefully in situ polymerized with PEDOT/PSS to make the highly conductive PEDOT/PSS/graphene composite.15Graphene has also been used as the candidates for making conductive composite with PEDOT/PSS.16 Blending with polymers is one of the most efficient route to increase the flexibility of PSS/PEDOT. Poly(ethylene glycol) (PEG), poly(ethylene oxide) (PEO), poly (PVA), and polyurethane (PUR) have all been used.16–19 Chiu et al. designed highly flexible PEDOT:PSS–P(styrene-co-butyl acrylate) (P(St-BA)) dispersions by using PEDOT:PSS as stabilizer for P(St-BA) latex, but the P(St-BA) latex were micro-sized range with polydispersity due to the weak stabilizing capability of PEDOT:PSS.20,21 Chiu et al. also designed PEDOT:PSS–(P(St-BA)) dispersions by applying dodecylbenzene sulfonic acid (DBSA) as surfactant.22 However, one disadvantage of these polymer blended film is the decrease in conductivity with increasing amounts of insulating polymer. And the blending approach is even more difficult with hydrophobic elastomers.On the other hand, much work has been devoted to increasing the flexibility and stretchability of PEDOT/PSS which is a characteristic required for a range of applications such as wearable, implantable and large-area electronic devices.Herein, P(SSNa-BA-St) latex were used as template for both graphene and PEDOT to prepare the transparent, highly elastic and conductive P(SSNa-BA-St)/PEDOT dispersions. SSNa served as both stabilizer and monomer for P(SSNa-BA-St) copolymerization latex, therefore, no extra surfactant is needed and monodispersed nanoparticles are formed. The graphene locating on the surface of P(SSNa-BA-St)/PEDOT particles through the hydrogen bonding and stacking interaction between not only PSS and graphene but also PSt and graphene. P(SSNa-BA-St) latex with approximately 128 nm sizes have large surface area to polymerize the extended PEDOT chain on the surface of the nanoparticles, which can provide much higher conductivity than PSS/PEDOT that having grains with hydrophobic coiled PEDOT chains in the core and hydrophilic insulating PSS as the shell. Moreover, the P(SSNa-BA-St)/PEDOT/graphene presented here exhibited transparent, good water resistance, low moisture adsorption and highly elastic properties, which can serve as a practical approach to fabricate the flexible, conductive and transparent films for wearable and implantable electronic devices, and photovoltaic cells.2.2.1n-butyl acrylate , potassium persulfate , dimethyl sulfoxide  and methanol (≥99%) were purchased from Aladdin.Sodium styrenesulfonate  and iron(iii) ferric sulfate (Fe2(SO4)3, AR 99%) were purchased from Macklin. Reduced graphene oxide was purchased from Suzhou Tanfeng Graphene Tech Co., Ltd.3,4-Ethylenedioxythiophene , styrene (St), 2.22 with stirring in a round bottom flask, followed by addition of the 0.1 g KPS through a constant flow pump. After stirring at 78 °C for 4 h, the emulsion polymerization was finished. The NPs were washed twice by centrifugation at 10 000 rpm for 30 min and ready to use.A total of 3 g SSNa, 4.9 g BA and 2.1 g St were mixed with 78 g water at 78 °C for 15 min under N2.32, followed by stirring overnight at 30 °C. Then the P(SSNa-Ba-St)/PEDOT elastic conductive dispersions was prepared.15 g of the prepared P(SSNa-Ba-St) emulsion and 0, 1, 2, 4, 5 g EDOT  were separately mixed with 15 g water followed by 15 min sonication. 0.005 g ferric sulfate in 10 g water and 2.85 g KPS in 60 g water were separately added into two constant pressure dropping funnels. Extra water was added into the flask to keep the total mass of the content as 135 g. Then the two mixture in funnels were dropped into the flask for 2 h at the same time under N2.4−4, 1.5 × 10−3, 3.0 × 10−3, 3.7 × 10−3 wt%) was added into the flask with 30 min sonication and 1 h stirring at 30 °C under N2. 0.005 g sulphate heptahydrate in 10 g water and 2.85 g KPS in 60 g water were separately added into two constant pressure dropping funnels. Extra water was added in to the flask to keep the total mass of the content as 135 g. Then the two mixture were dropped into the flask for 2 h at the same time under N2 and stirring followed by stirring overnight at 30 °C. A flow of N2 was used to prevent the overoxidation of PEDOT which might lead to conductivity deceasing. Then the P(SSNa-Ba-St)/PEDOT elastic conductive dispersions was prepared.15 g of the prepared P(SSNa-BA-St) emulsion and 0, 1, 2, 4, 5 g EDOT  were mixed with 15 g water followed by 30 min sonication. Then 0, 1, 2, 4, 5 g graphene /PEDOT or P(SSNa-Ba-St)/PEDOT/graphene dispersion in advances.The mixed solution was dropped cast on a glass plate and then dried at 50 °C for 3 h to form the P(SSNa-Ba-St)/PEDOT or P(SSNa-Ba-St)/PEDOT/graphene elastic conductive composite film.2.6−1 resolution in the spectral range 4000–400 cm−1. The glass transition temperature (Tg) of each sample was determined using a differential scanning calorimeter (DSC) . 12 mg of dispersions dried powders were sealed into a 40 μL aluminium pan. An empty pan was used as a reference sample. The temperature scan for each sample was measured from −50 °C to 50 °C at a heating rate of 5 °C per minute. The data acquired by the DSC were analysed by STAR Thermal Analysis software (NETZSCH 200F3) to determine the onset and endset glass transition temperatures.To determine the hydrodynamic diameter and zeta potential of the P(SSNa-Ba-St) latexes, dynamic light scattering (DLS) and zeta potential measurements were carried out using a Zetasizer Nano ZS system . DTS Application 5.10 software was employed to analyse the data obtained. The chemical groups of the nanoparticles were analysed by Fourier transform infrared (FTIR) (Thermo NICOLET iS10) in transmission mode. For that, dried material powders were mixed with KBr (40 mg) and then formed into a disc in a manual press. Transmission spectra were recorded using at least 32 scans with 4 cm, where R represents the surface resistance (Ω sq−1), D is the film thickness (cm). Equal width and length were used throughout this study. The optical transmittance spectra of the conductive composite films in the visible light wavelength range (300–700 nm) were detected using a UV-visible spectrometer. The contact angle was measured by Powereach JC 2000D.The surface resistance was measured using an ohmmeter. The conductivity of the composite films was evaluated by the following equation: Bending tests of the PSS/PEDOT and P(SSNa-Ba-St)/PEDOT/graphene film were performed to detect the flexibility. The conductive dispersions were coated on flexible poly substrates to form the specimens. The bending test was executed by bending the specimens back and forth (180°) 100 times.3.ca. 128 nm by intensity (PDI: 0.08) and the average zeta potentials was ca. −50 mV. With the help of efficient stabilizer SSNa, the size of the latex was in nanometre range with low PDI which provide large surface area to polymerize EDOT on the nanoparticles. And the strong negative surface charges make them serve as good charge compensator of positively charged PEDOT and also provide the high colloidal stability of the nanoparticles. Then the P(SSNa-BA-St) latex were applied as a template for both EDOT polymerization and graphene–water dispersion. As shown in 13,23The P(SSNa-BA-St)/PEDOT/graphene were synthesized as shown in −1, 1080 cm−1, 1335 cm−1, and 1519 cm−1 are corresponding to the C–S bond in thiophene rings; –SO2, and –SO3− in PSS; C–C stretching in thiophene rings and C PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>C asymmetrical stretching in thiophene rings, respectively, indicating the successful incorporating the PSS in the system.24 The peaks at 1725 cm−1 of PEDOT/P(SSNa-BA-St) and PEDOT/P(SSNa-BA-St)/graphene was associated with CO stretch of BA.25 The extra peaks at 3152 cm−1 and 3432 cm−1 for PEDOT/P(SSNa-BA-St)/graphene are attributed to the remaining –OH group on the surface of the graphene, which are typical peaks for reduced graphene oxide, and it confirms the formation of graphene dispersed PEDOT/P(SSNa-BA-St) dispersions.23,26In order to investigate the components of the conductive film, the typical FTIR spectrum of the extracted composite polymer was shown in −1, owing to the first-order scattering of the E2g phonons and the D band exhibited at 1343 cm−1 which is attributed to the breathing mode of κ-point phonons of A1g symmetry.27 Based on the empirical equation, ωG = 1581.6 + 11/(1 + n1.6),28 where ωG is the band position in wavenumbers and n is the number of layers present in the sample, the number of layers of the graphene can be estimated as two. And also, the 2D band in Fig. S128,29 However, the D band exhibited very high intensity (ID/IG = 1.1) indicating the disorder or the defect present in the graphene.29,30 For the P(SSNa-BA-St)/PEDOT in C anti-symmetric stretching (1570 cm−1), CC asymmetrical stretching (1502 cm−1), CC symmetrical stretching (1437 cm−1) and single C–C stretching (1366 cm−1).31 Comparing with the PEDOT/P(SSNa-BA-St), the graphene doped PEDOT/P(SSNa-BA-St) exhibited slightly shifted  according to the strong π–π interaction of aromatic structures of PEDOT/P(SSNa-BA-St) and electron-rich graphene, illustrating the successful doping of graphene into PEDOT/P(SSNa-BA-St).23,32The protection effect of PSS and electron-stacking interaction between graphene and P(SSNa-BA-St)/PEDOT are confirmed by Raman spectroscopy. The Raman spectrum of graphene applied in this work was consistent of the typical reduced graphene oxide spectrum as shown in Fig. S1.The conductive dispersions were drop casting on glass coverslips and PSS/PEDOT , PSSNa- and PSS films al−1 to 0.025 S cm−1 owing to the concentrations of conducive component increasing, followed by the decreasing to 0.006 S cm−1 owing to the precipitation formation of excessive PEDOT, indicating the best doping concentration of EDOT to PSS here was 0.06. More important, instead of PSS, the conductivity trend of applying P(SSNa-BA-St) as the template for PEDOT polymerization follows the same with PEDOT/PSS but exhibit much higher conductivities than them. The sample of EDOT to PSS(SSNa-BA-St) ratio as 0.06 provides the highest conductivity as 0.25 S cm−1 which is 10 times higher than PEDOT/PSS with the same content ratio. The improvement of conductivity is owing to the small nanoparticles sizes (approximately 128 nm) of P(SSNa-BA-St) latex providing larger surface area to polymerize the extended PEDOT chain on the surface of the nanoparticles, leading to higher conductivity than PSS/PEDOT that having grains with hydrophobic coiled PEDOT chains in the core and hydrophilic insulator PSS as the shell.In order to investigate the optoelectronic properties, P(SSNa-BA-St)/PEDOT were drop casting on the glass substrate to form the well mixed uniform conductive films. The conductivity of PSS/PEDOT and P(SSNa-BA-St)/PEDOT films were shown in −1 to 2.500 S cm−1 which is higher than the reported PEDOT/graphene composite layers (0.13–0.20 S cm−1).33,34 The 10 times conductivity increasing compared with P(SSNa-BA-St)/PEDOT and 100 times increasing comparing with PSS/PEDOT are due to the help of unique electrical properties of graphene and also indicates that the graphene were successfully doped on the surface of P(SSNa-BA-St)/PEDOT particles through hydrogen bonding and strong π–π interaction of graphene with PSS and PSt. Then the conductivity dropped down to 0.625 S cm−1 by reaching the graphene to EDOT ratio at 0.005, owing to the excess of graphene precipitated out of the solution. In addition, the conductivity of P(SSNa-BA-St)/PEDOT/graphene films can also be enhanced by applying the solvent effect. As shown in By controlling the EDOT to PSS ratio as 0.06, the conductivity of the graphene doped P(SSNa-BA-St)/PEDOT were shown in 35 However, the P(SSNa-BA-St)/PEDOT films exhibited elongated grains and form longer connected networks of PEDOT-rich area and the phase segregate of P(SSNa-BA-St) and PEDOT was shown in 36 This phenomenon demonstrated that the conductivity improvement from PSS/PEDOT to P(SSNa-BA-St)/PEDOT is also attributed to an increase of phase separation. The graphene sheet of P(SSNa-BA-St)/PEDOT/graphene film was observed by AFM images which illustrated the graphene sheet was stabilized in the system and located in the P(SSNa-BA-St)/PEDOT matrix, as shown in Atomic force microscopy (AFM) was applied to reflect the morphological changes in the films. 37 indicating their excellent waterproof ability due to the copolymerization of water insoluble monomers BA and St, as shown in In addition, the hydrophobicity of as prepared films was also investigated. The high hydrophilic PEDOT:PSS film exhibit the water contact angle at 30°, however, the contact angle of P(SSNa-BA-St)/PEDOT/graphene film can reach to 78° which is much higher than the reported 4.8–7.0° for PSS/PEDOT/graphene,Tg but the DSC results showed that the Tg for P(SSNa-BA-St)/PEDOT was around −8.1 °C and P(SSNa-BA-St)/PEDOT/graphene was around −10.6 °C which can be attributed to the introduction of the lower Tg material BA and St. The decreases of Tg could provide the excellent flexibility and elasticity to the films. The bending tests were also performed in σ0/σ) was used, where σ0 and σ were the surface conductivity before and after bending, respectively. For the pure PSS/PEDOT specimen, the surface conductivity decreases to 15% of the original after 100 times bending cycles while P(SSNa-BA-St)/PEDOT/graphene remain 83% of the original. This indicated that the introduction of P(SSNa-BA-St) latex could strongly enhance the flexibility of conductive films compared with the conventional PSS/PEDOT film.4.Tg of (SSNa-BA-St)/PEDOT/graphene film provide the excellent flexibility and elasticity to the films. Therefore, the P(SSNa-Ba-St)/PEDOT/graphene presented here exhibited transparent, good water resistance, low moisture adsorption, highly elastic and highly conductive properties, which can serve as a practical approach to fabricate the flexible, conductive and transparent films for wearable and implantable electronic devices, and photovoltaic cells.We reported an innovative transparent, elastic and flexible conductive composite materials P(SSNa-Ba-St)/PEDOT/graphene which were prepared by using P(SSNa-Ba-St) latex as template for both graphene and PEDOT. No extra surfactant is needed and monodispersed nanoparticles are formed. The conductivity of P(SSNa-BA-St)/PEDOT/graphene exhibit 220 times higher than the untreated PSS/PEDOT, which are due to the phase separation between the conducting PEDOT chains and insulating P(SSNa-Ba-St) chains. And P(SSNa-BA-St) latex have large surface area to polymerize the extended PEDOT chain on the surface of the nanoparticles, which provide higher conductivity than PSS/PEDOT that having grains with hydrophobic coiled PEDOT chains in the core and hydrophilic insulating PSS as the shell. Furthermore, the water contact angle of P(SSNa-BA-St)/PEDOT/graphene film can reach to 78° and remain the original shape on the substrate under water flow, and also, the moisture absorption of P(SSNa-BA-St)/PEDOT/graphene films is 13.3 times lower than of PSS/PEDOT film after leaving in air for 12 h, which indicates an excellent water resistance ability due to the copolymerization of water proof BA and St monomers. The relatively low There are no conflicts to declare.RA-009-C9RA09099A-s001


Background: Long-Interspersed Nuclear Element (L1) retrotransposons are silenced in healthy tissues but unrepressed in cancer. Even if L1 reactivation has been associated with reduced overall survival in breast cancer (BC) patients, a comprehensive correlation with clinicopathological features is still missing. Methods: Using quantitative, reverse-transcription PCR, we assessed L1 mRNA expression in 12 BC cells, 210 BC patients and in 47 normal mammary tissues. L1 expression was then correlated with molecular and clinicopathological data. Results: We identified a tumor-exclusive expression of L1s, absent in normal mammary cells and tissues. A positive correlation between L1 expression and tumor dedifferentiation, lymph-node involvement and increased immune infiltration was detected. Molecular subtyping highlighted an enrichment of L1s in basal-like cells and cancers. By exploring disease-free survival, we identified L1 overexpression as an independent biomarker for patients with a high risk of recurrence in hormone-receptor-negative BCs. Conclusions: Overall, L1 reactivation identified BCs with aggressive features and patients with a worse clinical fate. The “Junk DNA” definition was coined to describe the large fraction of the non-coding, redundant and repeated human genome ,2. The LAPC (in a colon cancer patient) [MYC (in a breast tumor patient) [In healthy adult human cells, this process is negatively monitored, and retro-transpositions are rarely identified. However, in stressing cell conditions such as cancer, the deregulation of global DNA methylation favors L1 reactivation ,8. Alongpatient)  and MYC patient) , L1 sequpatient) . L1 reacpatient)  or the Lpatient) . Confirmpatient) , L1 de npatient) . Accordipatient) ,16 and tpatient) , with popatient) . DespiteTo assess the poorly known correlation between L1 reactivation and the clinical and molecular characteristics of breast carcinomas, we evaluated the expression of these retrotransposons in pre-clinical and clinical settings, investigating BC and non-transformed mammary epithelial cells along with a cohort of 210 BCs and 47 normal mammary glands. To this purpose, we assessed the expression of L1 transcript (mRNA) by using two independent qRT-PCR assays encompassing the ORF1 and ORF2 sequences. The mRNA expression results were then compared with several molecular and clinical data, including promoter methylation, molecular subtyping and disease-free survival (DFS).We profiled 14 cell lines, 2 non-transformed and 12 breast cancer cells. HMEC non-transformed cells were grown in DMEM/F15 with 10% FCS plus 10 ng/mL insulin, 0.5 μg/mL hydrocortisone and 5 ng/mL EGF. MCF-10A cells were grown in DMEM/F12 with 5% HS, 10 μg/mL insulin, 10 ng/mL EGF and 500 ng/mL hydrocortisone. MDA-MB-453 cells were grown in Leibovitz’s L-15 Medium  with 10% FBS  in free-gas-exchange atmospheric air; SKBR3 and MDA-MB 231 in high-glucose DMEM  with 10% FBS; MCF-7 in high-glucose DMEM with 10% FBS supplemented with 10 µg/mL insulin; T47D, ZR751, MDA-MB 468, BT549, HS-578T, HCC70 and HCC1187 in RPMI (Sigma-Aldrich) with 10% FBS; BT474 in RPMI with 20% FBS and 10 µg/mL insulin. MDA-MB-468, T-47D, HS-578T, MDA-MB-231 and BT549 cell lines were obtained from the NCI-60 panel, while MDA-MB-453, SKBR3 and ZR751 cells were derived from the Interlab Cell Line Collection (ICLC) and MCF-7, HCC70, HCC1187, HMEC and MCF-10A from the American Type Culture Collection (ATCC). Lastly, BT474 cells were obtained from the “Deutsche Sammlung von Mikroorganismen und Zellkulturen” . BC cell lines are reported in ® 16 HS System, Promega, Madison, WI, USA) evaluated in September 2021.The genetic identity was confirmed by short tandem repeat profiling , as reported . Patient™ DNase  treatment was additionally performed, according to the manufacturer’s protocol, to avoid the problem of genomic contamination in qRT-PCR.DNA was extracted from cells using the Blood & Cell Cultured Mini Kit , while the RNeasy Mini Kit  was used for RNA extraction. TURBO® 2.0 fluorometer .After review with hematoxylin and eosin (H&E), FFPE tumoral sections were dissected to obtain nucleic acids from tissue areas containing at least 100 cells with >50% tumoral cells. Maxwell RSC FFPE RNA Kit (Promega) was used to obtain RNA from the 210 FFPE BC samples and the 47 FFPE BC normal mammary glands. Genomic DNA was purified from a subset of 41/210 FFPE tissues using the GeneRead DNA FFPE Kit . Nucleic acids were quantified with the Qubit™ IV VILO™ reverse transcriptase .One μg of purified RNA was reverse-transcribed in a mix containing both oligo(dT) and random hexamers using SuperScriptThe expression of L1-ORF1 and ORF2 transcripts were evaluated in triplicate by qRT-PCR using two independent couples of primers and specific thermal profiles, reported in ® assay  based on the classification algorithm PAM50 [Molecular subtyping was performed using the Prosignahm PAM50 ,22,23,24® system  based on the MALDI-TOF (matrix-assisted laser desorption ionization time-of-flight) method, as previously reported [The 41 BCs with available DNA were mutationally characterized with a 147-hotspot breast cancer gene panel using the MassARRAYreported .L1 methylation was studied for both cancer cells and the 41 BCs with available DNA by performing quantitative bisulfite pyrosequencing in triplicate using the PyroMarkQ96 pyrosequencing , as previously reported . BrieflyData analysis was carried out with the SPSS version 20.0 software  by three independent authors .p-values are reported within the text and/or in the tables. p < 0.05 was considered statistically significant. ROC curves were produced with the pROC package (R v4.0.2).Student’s t-distribution was used to determine the distribution of L1-ORF1 and L1-ORF2 expression and the Fisher’s Exact Test to evaluate contingency tables for nominal variables. Survival analysis of disease-free survival (DFS) was evaluated with the Kaplan–Meier method, and groups were compared with the log-rank test. DFS was calculated as the interval between the beginning of the therapy to the time of progression. The Cox regression model  was used to perform the multivariate analysis for tumor relapse. All the available (RQ): 0.05 and ORF2(RQ):0.03; ORF1(RQ): 0.08 and ORF2(RQ): 0.09, respectively), whereas all BC cells showed higher but heterogeneous levels of both ORF1 and ORF2 (ORF1(RQ)mean: 0.59, range 0.03–1.83; ORF2(RQ)mean: 0.81, range 0.09–2.54, p < 0.01, ρ Spearman = 0.91, p = 0.019, p = 0.09, We first assessed L1 expression by evaluating the relative quantification RQ (ΔCt) of ORF1 and ORF2 in twelve BC and two non-transformed mammary gland cell lines. L1 transcript levels were barely detectable in the non-transformed mammary gland cells, namely, HMEC and MCF-10A  , which is the cut-off to discriminate methylation/hypomethylation ,31, wher= −0.65) D.(RQ): 0.05, range: 0.005–0.09; ORF2(RQ): 0.06, range: 0.006–0.07, To confirm the BC-exclusive expression of ORF1 and ORF2, we compared the RQs (ΔCt) of both transcripts in 210 BCs and 47 normal mammary glands. Unsupervised clustering revealed a clear separation between BCs and non-tumoral tissues, the latter being characterized by barely detectable levels of expression for both ORFs (ORF1(RQ): 0.55, range: 0.11–3.47; ORF2(RQ): 1.32, range: 0.09–11.75), supporting the tumor-specific expression of both transcripts  mean: 17.79, range: 0.37–108.13; ORF2(RQ) mean: 27.69, range: 1.24–235.24).None of the tumor samples showed an ORF amount lower than the most expressive normal tissue mean: 34.54, range: 3.43–107.49; ORF2(RQ)mean: 58.76, range: 7.10–235.24) ((RQ) for each subgroup is reported in Each of the PAM50 molecular subtypes had a comparable number of patients . Similar to the scenario detected in the BC cell lines, we observed the enrichment of L1 transcripts in the basal-like subtype (ORF1–235.24) C. Lumina–235.24) C. The HEp < 0.01) ((RQ)p < 0.01 and ORF2(RQ)p < 0.01) ((RQ)p = 0.02, ORF2(RQ)p = 0.01), with a higher L1 expression in tumors with higher TIL scores ((RQ) for each group is reported in L1-ORF expression was increased in poorly differentiated (G3) BCs compared to G1/2 tumors (ORF1 and ORF2 both  < 0.01) D. BCs ch < 0.01) . FollowiL scores E. The men = 8, Luminal B: n = 11, basal-like: n = 12, and HER2-enriched: n = 10). In addition to a mean methylation percentage of 57% (range 27–82%), we observed a negative correlation between demethylation and ORF2 expression , together with a weaker trend for ORF1  (TP53 (29%), eight PIK3CA (20%), two AKT1 (5%), and one CDH1 pathogenic variants (2%)   . Hotspotnts (2%) . Sevente < 0.01) .(RQ) and ORF2(RQ) showed a trending polarization of the patients with recurrence according to high L1 expression . A heatmap for both ORF1pression . Patient(RQ) median level as the cut-off. The BCs highly expressing ORF1 were characterized by a median of 32 months of DFS compared to 49 months for the low expressors (p < 0.01), with a two-times higher risk  of relapse. Similarly, high ORF2 values identified patients with an increased risk of relapse  to assess whether L1 expression was associated with differential DFS. As shown in Data concerning the stratification with the mean levels of ORF1 and ORF2 expression are reported in (RQ) values over the median level, whereas “L1 low-expressors” included patients with at least one ORF(RQ) below the median level. The derived Kaplan–Meier curve identified a strong level of significance (p < 0.01), with an HR for L1 high expressors of 1.9  for both cut-offs , node involvement (0 vs. 1–3 nodes vs. >3 nodes), Ki67 expression (high vs. low), PAM50 subtypes, TILs  and tumor size. Of note, only L1 expression (both stratified for average and median) and lymph-node involvement retained an independent level of significance .p-values, HR and CI95% in Since we described increased L1 mRNA expression as a negative prognostic factor, we focused on the DFS of L1 high expressors (cut-off: median L1(RQ)). In this subgroup, patients with a basal-like (median: 46 months) or with a HER2-enriched subtype (median: 52 months) had a worse prognosis, whereas Luminal patients with a tumor showing high L1 expressions were characterized by a longer DFS . No other correlations were identified between L1 expression and recurrence in other therapeutic approaches , compared with those without recurrence (21/50) (LINE-1 (L1) sequences are usually silenced in human tissues through several mechanisms, including hypermethylation of their promoter . DeregulHere, we report for the first time a detailed characterization of RNA-based L1-ORF expression in a large cohort of BC patients in the attempt to contextualize L1 transcription among BC patients and to correlate L1 reactivation with clinical outcome. We demonstrated a high level of tumor-specific L1 transcripts in 12 BC cell lines and in a retrospective BC cohort, enhanced in HR-negative BCs. Moreover, the association between ORF expression and a high risk of tumor recurrence, confirmed by multivariate analysis, proposes L1 as a biomarker for BC prognostic stratification.The tumor-specific activation of L1 mRNA can be considered crucial to define L1 as a biomarker . The absL1-ORF expression using IHC on FFPE tissue is complex. A pioneering work about L1 expression in BC was based on IHC staining, allowing us to also evaluate the localization of the expression as a marker ; howeverThe application of an RNA-based test was not free from issues, mainly associated with the overlapping of L1 DNA–RNA sequences  and withMYC gene showed the correlation between the hypomethylation of these sequences and a retrotransposon-linked carcinogenic effect [L1 reactivation in BC has historical proofs: in 1988 the insertion of L1 into the c effect . Over thc effect ,15,42,44TP53 mutated tumors, recently confirmed in breast, ovarian and colon cancers [Our results in the BC cell lines are in line with previous reports ,45, thus cancers . The pre cancers .Among all BC pathological factors, we highlighted a possible cross-link between L1 expression and immune-cell infiltration, since L1-high BCs showed high levels of TILs in the stroma. Histologic evaluation of TILs has reached level IB evidence as a prognostic marker in TNBC and level 2A with respect to prediction of response to chemotherapy. Some authors have shown a favorable outcome in TNBCs with TILs > 30% treated by surgery alone , thus suWe also explored the possible clinical impact of L1 expression levels. High L1 levels significantly correlated with a higher risk of BC recurrence in terms of DFS. In the multivariate analysis, lymph-node involvement and L1 expression were the only two independent/significant variables. Node involvement is recognized as one of the main markers for the risk of relapse in BC ,55,56. BBy stratifying the risk according to the BC subtype, the relationship between HR-negative BCs and L1 expression showed stronger results, whereas HR-positive/L1-high lesions did not show a reduced DFS, suggesting poor biological and prognostic significance of L1 retrotransposons in Luminal tumors. HER2-enriched BCs showed great heterogeneity for L1 mRNA level, both in cell models and in the retrospective cohort. HER2-addicted tumors were also characterized by a significant, albeit less robust, L1 stratification of the risk of relapse.Interestingly, L1 has also been considered from a therapeutic perspective. The use of reverse-transcriptase inhibitors (efavirenz) has been shown as particularly effective in TNBC preclinical models and is associated with the metabolism of fatty acids . ConversOur work presents two main issues, both associated with the retrospective design. First, given the heterogeneity of the population, it is impossible to recover data about the overall survival. Second, the small amount of archival material precluded high-throughput DNA analysis for all tumors in the study cohort.L1 expression could be interpreted as a strong negative prognostic marker, especially for HR-negative BCs. When considering the impact on treatment, our data may lead to a hypothesis generating further studies. Indeed, whether L1 expression may be exploited to modulate the treatment of TNBCs or to predict response to chemotherapy or to the combination of chemotherapy and ICIs warrants further investigation.


Position-sensitive detectors (PSDs) are of great significance to optical communication, automatic alignment, and dislocation detection domains, by precisely obtaining the position information of infrared light spots which are invisible to human eyes. Herein, a kind of PSD based on graphene/germanium (Ge) heterojunction architecture is proposed and demonstrated, which exhibits amplified signals by unitizing the charge injection effect. Driven by the graphene/Ge heterojunction, a large number of photogenerated carriers diffuse from the incident position of the light spot and subsequently inject into graphene, which ultimately generates a photoresponse with high efficiency. The experimental results show that the device can exhibit a fast response speed of 3 μs, a high responsivity of ~40 A/W, and a detection distance of 3000 μm at the 1550 nm band, which hints that the graphene/Ge heterojunction can be used as an efficient platform for near-infrared light spot position sensing. PSDs are optoelectronic position sensors that can accurately detect the light spot position, distance, incident angle, and other related physical parameters ,2,3,4. TThe silicon-based PIN  and SchoTwo-dimensional materials are those in which electrons can move freely in only two dimensions, including graphene, black phosphorus, and transition metal dichalcogenides . Two-dimIn this paper, a photoconductive type of PSD based on graphene/Ge heterojunction by using the charge injection effect is presented. Under near-infrared illumination, the photogenerated carriers in Ge diffuse laterally to the graphene/Ge heterojunction region and then inject into graphene. During this process, the graphene/Ge heterojunction plays a critical role in efficiently collecting the photogenerated carriers. As a result, the graphene/Ge photoconductive PSD shows a responsivity of ~40 A/W and a response speed of 3 μs/1.9 μs. More importantly, the device shows a dependent photocurrent variation with the position change of the 1550 nm light spot, and its working distance can reach 3000 μm.−28 A2/Hz. When calibrating the power of the laser, a commercial power meter from Thorlabs was utilized. Throughout the measurement, the bias voltage of the device is 1 V.Raman measurements were performed under ambient conditions using a Renishaw inVia system with a 532 nm excitation wavelength. During the electric characteristic measurement, the graphene/Ge photoconductive PSD was attached to a printed circuit board and inserted into a socket located at a scanning Galvo System. The printed circuit board was connected to a semiconductor device analyzer Keithley 4200A-SCS for electrical signal read-out. The 1550 nm and 980 nm lasers were focused and vertically irradiated the device through the refraction optical path. The spot diameter of the 1550 nm laser is about 2 μm, and the spot diameter of the 980 nm laser is around 1 μm. The laser and microscope share the same optical path. By exploiting a charge-couple device (CCD) camera, the 980 nm laser light spot (with an overlapping incident position with 1550 nm laser) and the device can be seen in the software, in which the light spot of 980 nm laser is marked with visible color. With the assistance of the software, the alignment of the light spot and device can be realized. To measure the position sensitivity, the scanning system carried the graphene/Ge photoconductive PSD and moved accurately with a given value. To measure the response speed, a signal generator was used to connect the laser and generated a modulated light signal at a frequency of 1 kHz. Then, a SourceMeter (FS-Pro) with a high sampling rate was used to measure the transient response of the device. For the noise measurement, a noise analyzer was connected to the device and recorded the noise current of the device. The measurable value of the noise analyzer is as low as 2 × 10−1 (G band) and 2700 cm−1 (2D band), respectively. The intensity of the 2D band is twice as high as that of the G band, indicating that the monolayer graphene used in this work is of good quality. The insert of To prove the improvement effect of introducing graphene, the detection distances of the devices with and without graphene are compared. As shown in netI) is defined bypI is the photocurrent and darkI is the dark current. The noise current of the device is shown in −16 A2/Hz, which provides a strong guarantee for weak light detection and a large working area. oP is the incident light power. Under different light power densities, the average responsivity is about 40 A/W, and the minimum noise equivalent power is 7.15 × 10−12 W·Hz−1/2. The noise equivalent power is defined bynI is the noise current. The high responsivity and low noise equivalent power can be attributed to two factors, namely, high photoconductive gain brought by the high carrier mobility of graphene [In addition to the position sensitivity characteristics, the photoresponse performance of the graphene/Ge photoconductive PSD was also measured. As shown in graphene ,32,33 angraphene ,35.Note that the high carrier mobility of graphene and the built-in field of the graphene/Ge heterojunction are vital for the response speed. The former can ensure the rapid transport of carriers through graphene channels, while the latter can accelerate the charge injection. The response speed of the device is shown in In summary, PSDs that can detect the position change of the 1550 nm laser spot have been realized by integrating graphene with Ge. The introduction of graphene can produce a strong built-in field at the graphene/Ge heterojunction region, which is helpful for effectively collecting photogenerated carriers and driving these photogenerated carriers to inject into graphene. Thereby, the graphene/Ge photoconductive PSD achieves a fast response and linear near-infrared detection. Moreover, the working distance of the graphene/Ge photoconductive PSD is up to 3000 μm, which is three times higher than that of the bare Ge device. These results provide a promising opportunity for infrared PSDs based on low-dimensional materials and traditional bulk semiconductors.


RSC Advances article. These have been given below as ref. The authors are publishing this correction to draw the reader’s attention to three related papers that should have been cited as ref. 43–45 in this 43”On page 4464, a citation to ref. 43 should be added to the end of the sentence beginning “Ultrapure water…”. Therefore, the sentence should be changed to “Ultrapure water obtained from a Millipore water purification system  was used in all runs.F1/F0 are selected to evaluate the effects of the reaction conditions on the sensing performance of the assay.43,44”On page 4465, citations to ref. 43 and 44 should be added at the end of the sentence beginning “The fluorescence intensity…”. Therefore, the sentence should be changed to “The fluorescence intensity and the value of 45”On page 4467, a citation to ref. 45 should be added to the end of the sentence beginning “Aptamer can recognize…”. Therefore, the sentence should be changed to “Aptamer can recognize MB specifically, and thus provide a promising method for its assay, this promising strategy might prove feasible as an MB assay for real samples.The authors also regret that there are portions of unattributed text overlap in the Results and discussion and Conclusion sections with other papers, including ref. 43–45.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers.


The paper presents the results of research on the use of flake graphene as an additive to plastic grease in order to improve its tribological properties. The influence of concentration (0.25–5.00 wt.%) and the form of graphene  on selected properties of the base grease were investigated. It has been found that the addition of graphene flakes improves the anti-wear properties of the lubricant. The greatest improvement in the properties of the lubricant was achieved by using graphene at a concentration of 4.00 wt.%; the reduction in the average diameter of the wear scar was almost 70% for GO and RGO, compared to the base lubricant without the addition of graphene. The factor driving the growth of the market for these products in the world is the dynamic technological development. Lubricant manufacturers have to keep pace with more and more advanced vehicles as well as innovative machines and devices, where precision and smooth operation are crucial for their efficient and profitable operation. During mechanical processes, as a result of strong friction between the surfaces, wear of the mechanical elements of the friction junction occurs and heat is generated, which ultimately reduces the efficiency of work and shortens the life of the device. It is estimated that about 23% 119 EJ) of global energy consumption is due to anti-friction and regeneration of friction elements [19 EJ of missions . Therefomissions ,6.Flake graphene, which is a known 2D carbon nanomaterial, is obtained from graphite. At the same time, it should be emphasized that graphite is characterized with greasing properties and it is widely implemented, i.e., as an anti-wear agent. Graphene is another allotropic type of carbon, next to graphite. GO is the oxidized form of graphene, decorated with oxygen functional groups . GO particles are highly hydrophilic and form stable dispersions in water and some organic solvents. RGO is obtained by reduction of oxygen functional groups of GO. Contrary to GO, rGO is hydrophobic and show high mechanical strength, large specific surface area and high electric and thermal conductivity .The use of flake graphene is currently the subject of much scientific research in various areas, such as composites , functioAs part of the research described in the literature, the tribological properties of various lubricants  with the addition of various forms of graphene flakes  were analyzed. The tribological properties of polyalphaolefin-2 (PAO2) base oil with the addition of multilayer graphene containing 0.05 wt.%, 0.10 wt.% and 0.50 wt.% were investigated using a 4-ball tester. The tests were carried out at loads from 120 N to 400 N and rotational speeds in the range of 100–400 rpm . It was Graphene as an anti-wear additive to plastic greases was also tested. The properties of mineral plastic grease thickened with lithium saponified, enriched with the addition of graphene nanoplatelets with a concentration of 0.50 wt.%, 1.00 wt.%, 5.00 wt.% and 10.00 wt.% were investigated. Various loads were applied: 5 N, 10 N and 15 N . It was Flake graphene offers great research opportunities because its surface can be chemically modified and its properties can be controlled in this way. For example, the lubricating properties of monolayer graphene oxide when used as an additive to water-based lubricants were investigated . TriboloBased on the literature review, it can be concluded that the addition of graphene to lubricants improves their tribological properties. However, graphene still remains the object of tribological research and the obtained results are not always unequivocal. This may result from the use of various forms of graphene , different sizes of flakes, different number of carbon layers, different content of oxygen functional groups on the surface and different media in which graphene and its derivatives are dispersed, such as also different test conditions. Therefore, the available information is still incomplete and requires further research. The complexity of the application of graphene in terms of improving the tribological properties of lubricants is also indicated by Larrson et al. . MoreoveBearing in mind the above-mentioned limitations of the available literature data, this work proposes an innovative approach to the implementation of graphene materials as a nanoadditive with anti-wear properties to greases. For the purposes of the work, graphene materials were designed and manufactured, representing two types of flake graphene: graphene oxide and reduced graphene oxide. The paper presents the comparative results obtained for both materials. In order to obtain the best knowledge about the influence of graphene materials on the anti-wear properties of the grease, the tests were carried out for a wide range of concentrations of graphene materials in the grease, amounting to 0.25–5.00 wt.%. The experiment designed in this way allowed to obtain a detailed quantitative description of the issue under study. In addition, the article attempts to answer which of the two tested types of graphene materials will better meet the requirements for carbon nanoadditives for lubricants, taking into account their practical application.® —The research was conducted using graphene oxide powder and reduced graphene oxide powder by G-Flake® CrossBeam® Workstation, Carl Zeiss, Oberkochen, Germany, at a voltage of 1 kV, energy selective backscattered EsB detector), Raman spectroscopy , elemental analysis . Regarding the SEM experiment, it was implemented in order to visualize GO and RGO materials. The powders were placed on a silicon wafer and then glued with a carbon tape to an SEM table. No coating with conductive agent was conducted prior to visualization. As for the Raman examination, collected raw data were analyzed with Wire 3.54 software provided by the spectrometer producer. The analysis was carried out in order to discriminate the position of D and G peaks using a Gaussian/Lorentzian. Prior to determination of the peaks, position baseline subtraction was implemented. Detailed characterization of the purified GO water suspension, from which GO powder and RGO powder were then obtained, is presented in our earlier publication [Graphene oxide was produced using a modified Hummers method. This method is based on the oxidation of flake graphite  in sulfuric acid VI  with the addition of potassium permanganate  and potassium nitrate . After the oxidation process, the graphite oxide in the form of an aqueous suspension is exfoliated and then purified by filtration. After the purification process, an aqueous suspension of graphene oxide is obtained. In order to produce reduced graphene oxide, graphene oxide undergoes a reduction reaction, which takes place at high a temperature in the presence of a reducing agent—hydrazine . After the reduction process, the reduced graphene oxide is purified by filtration. To obtain GO or RGO in powder form, the aqueous material dispersion was dried. The obtained graphene materials (GO and RGO) were then characterized using the following methods: scanning electron microscopy (SEM)  and lithium stearate  thickener. It was prepared in laboratory conditions by adding the thickener lithium stearate to the paraffinic base oil, mixing and then heating with constant stirring until the thickener completely dissolved (to approx. 220 °C). The dissolved mixture was allowed to cool completely at room temperature.The lubricant compositions were prepared in laboratory conditions by adding the appropriate amount of GO or RGO in the form of a powder to the lubricant base. The samples were then mixed using a planetary mixer  to obtain a homogeneous dispersion . Mixtures of grease and flake graphene were subjected to tribological tests, in which the concentration of graphene  was from 0.25 wt.% up to 5.00 wt.%. It was observed that as a result of the shear stresses arising when mixing pure base grease and base grease with the addition of graphene, the viscosity of the grease decreased due to the shear-thinning phenomenon .The authors of the study  describe3). These balls are pressed down with a ring and fixed with a tightened clamping nut. The fourth ball (movable) is mounted in the upper holder. During the test, it rotates at a speed of 1450 ± 50 rpm, which corresponds to the speed of the rubbing surfaces of 0.55 m·s−1. The balls in the lower holder are pressed with the force P against the ball mounted in the upper holder by means of a lever. The friction linkage diagram is shown in The tests of the lubricating properties of the obtained plastic greases with the addition of flake graphene were determined by measuring the average diameter of the wear scar. The tests were performed with the use of a 4-ball tester . In this tester, the elements of the tribological couple are in point contact and a sliding movement is performed. The tribological couple in the tester consists of four balls with a diameter of 12.7 mm, made of bearing steel with a hardness of 62.7 HRC. Three balls (stationary) are placed in the lower holder, into which the tested lubricant is also introduced  results from the presence of vacancies or dislocations in the graphene layer and at the edge of this layer. This band is also related to the presence of defects in the material. The G band (located near 1590 cm−1) corresponds to the sp2 hybridization of the carbon network and is attributed to the first-order scattering from the doubly degenerate E2g phonon modes of graphite in the Brillouin zone center as well as bond stretching of sp2 carbon pairs in both rings and chains [−1 for GO and ~1347 cm−1 for RGO, respectively, and a G peak at ~1586 cm−1. The ID/IG ratio for GO is 1.05 and for RGO 1.20. The value of the ID/IG parameter increases with the increase in the degree of disorder in the structure of the graphene material.d chains . The ID/d chains . The anaThe results of elemental analysis of graphene materials are presented in The grease is a colloidal system, where the dispersing phase is oil and the dispersed phase is thickeners. The role of the thickener is to ensure the proper spatial structure of the plastic grease and the specific, required rheological and tribological properties. The spatial structure formed in the lubricant by the thickener fibers, as a result of the action of shear forces and the temperature increase during the operation of the tribological node, disintegrates ,32. An ePrepared lubricants with different contents of graphene material  were subjected to anti-wear tests. In the case of lubricant samples containing the additive in the form of RGO, the trend observed was similar to the compositions with the addition of GO. In this case, an increase in the wear of the test elements by 24% (4.03 ± 0.27 mm) was also observed compared to the base lubricant (3.26 ± 0.28 mm) for the lowest additive content of 0.25 wt.%. Increasing the concentration of graphene to 0.50 wt.% results in a wear scar with an average diameter close to the scar obtained when lubricating the node with base grease without additive. Only higher concentrations of the nanoadditive result in a significant reduction in consumption. The lowest wear was observed at 4.00 wt.%. RGO concentration (scar reduction by approx. 70%). Increase in RGO concentration in the lubricant from 1.00 to 4.00 wt.% reduces the average diameter of the wear scar from 2.51 ± 0.23 mm (23%) to 1.01 ± 0.05 mm (69%), respectively, compared to the grease without additive. Further increase of the additive content to 5.00 wt.% may increase the wear of the balls (1.97 ± 0.09 mm). It can be concluded that the RGO concentration of 4.00 wt.% in the grease is the limit concentration, exceeding which the antiwear properties of the grease may deteriorate. Even though 5.00 wt.% RGO concentration increases the consumption in relation to the concentration of 4.00 wt.%, in relation to the grease without the addition of graphene, the average diameter of the wear scar was reduced by 40%. Deterioration of lubricating properties for the highest concentration of the additive 5.00 wt.% has also been observed as a result of the use of GO. In the case of high concentrations of graphene, agglomerates form in the lubricant, which negatively affect the friction surface protection. The resulting tendency to change the wear properties of a lubricant with an increase in graphene concentration is consistent with the results of the research carried out by Fu et al. . AccordiDuring the tests of lubricants, the color of the lubricant with the addition of GO changed from brown to clearly darker, which may suggest that the graphene oxide is reduced during friction ,38. The Tests were also carried out on lubricants with the addition of RGO for a higher load of 100 kgf for 1 min and compared with the results obtained in the tests with a load of 60 kgf and a duration of 60 min, and the results of the measurements are presented in Grease samples were produced with the addition of GO and RGO with a concentration in the range of 0.25–5.00 wt.%. It was found that the addition of flake graphene improves the anti-wear properties of the lubricant, while the concentration and type of graphene have an impact on the tested lubricant properties and its stability. For lower contents of graphene additive (0.25 wt.% and 0.50 wt.% for GO and 0.25 wt.% for RGO), an increase in wear is observed as compared to the base grease. For low RGO concentrations (0.50 wt.%), the grease properties are similar to those of the base grease without additive. The introduction of flake graphene to the base of the plastic grease may disturb the internal network of the grease due to the high activity of the surface of the graphene flakes. This phenomenon may be the cause of increased node wear at low additive concentrations (0.25 wt.% and 0.50 wt.%), as these contents are probably too low to compensate for the breakdown of the lubricant’s inner network. This effect is particularly evident in GO containing numerous oxygen functional groups on the surface of the flakes.The use of a higher concentration of graphene 1.00–5.00 wt.% results in a reduction in the average diameter of the wear scar, which is greatest for an additive content of 4.00 wt.% . A further increase in the concentration of the additive to 5.00 wt.% results in a renewed increase in wear, which may result from the agglomeration of the graphene material and a significant increase in viscosity, so that the lubricant is not able to produce a lubricating film that will protect the friction surface well.Out of the two tested forms of flake graphene, RGO is a recommended additive for lubricants due to its stability under operating conditions, while GO was reduced during friction. The RGO powder tested in operation can therefore be successfully used as a material improving the tribological properties of lubricants.The use of flake graphene in lubricants is not a thoroughly researched and explained issue. In order to further develop the application of graphene flakes as a lubricant additive, it is necessary to continue research, including in the direction of chemical modification of the flake surface, and to explain the mechanisms of graphene operation during friction, and also to maintain the properties of lubricants unchanged in subsequent work cycles.