Exaggerated fibroblast proliferation is a well‐known feature in idiopathic pulmonary fibrosis (IPF) which may be – in part – due to insufficient autophagy, a lysosome dependent cellular surveillance pathway. Bcl2‐associated athanogene 3 (BAG3) is a pivotal co‐chaperone of the autophagy pathway. Here, we studied whether therapeutic modulation of BAG3‐mediated autophagy can rescue insufficient autophagy and impact IPF fibroblast proliferation.BAG3.Primary interstitial fibroblasts or precision cut lung slices (PCLS) of IPF lungs were treated with (1) the antifibrotic drug pirfenidone (Pirf), (2) the demethylating agent 5‐azacytidine (Aza), (3) the BAG3 modulator cantharidin (Ctd). Autophagy flux was measured following pretreatment with the autophagy inhibitors or by GFP‐RFP‐LC3B transfection followed by drug treatments. Proliferation was measured by 5‐bromo‐2′‐deoxyuridine assay. BAG3, filamin C (FLNC), proliferating‐cell‐nuclear‐antigen (PCNA), collagen1A1 (COL1A1) and autophagy proteins were assessed by immunoblotting or immunofluorescence. Loss of function experiments were performed by siRNA mediated knockdown of BAG3 per se in IPF fibroblasts. Similarly, PCLS of IPF patients showed a significant decrease in collagen deposition in response to these drugs, either alone or in a more potent form in combination with Pirf.In comparison with healthy donors, increased BAG3 protein was observed in IPF lung homogenates and IPF fibroblasts. In addition, the substrate of BAG3‐mediated autophagy, FLNC, was increased in IPF fibroblasts, implying insufficient activation of BAG3‐dependent autophagy. Therapeutic modulation of this pathway using Aza and Ctd alone or in combination with the IPF therapy drug Pirf rescued the insufficient BAG3‐mediated autophagy and decreased fibroblast proliferation. Such effects were observed upon therapeutic modulation of BAG3 but not upon knock down of Our study reveals that repurposing drugs that modulate autophagy regulating proteins render therapeutic benefits in IPF. Fine tuning of this pathway may hence signify a promising therapeutic strategy to ameliorate antifibrotic properties and augment the efficacy of current IPF therapy. BAG3 per se in IPF fibroblasts does not ameliorate BAG3‐mediated autophagy. Therapeutic intervention (pirfenidone+5‐azacytidine or pirfenidone+cantharidin) sensitizes IPF fibroblasts to BAG3‐mediated autophagy. Drug‐induced sensitization of BAG3‐mediated autophagy in precision cut lung slices of IPF patients alleviates fibroblast proliferation and collagen deposition.IPF‐fibroblasts are refractory to BAG3‐mediated autophagy. Knock down of  BAG family contains about six proteins (BAG1‐6), which contain at least one copy of their characteristic ‘BAG’ domain at the C‐terminus, allowing them to interact with the ATPase domain of the eukaryotic chaperone 70‐kilodalton heat shock protein (Hsp70).Given the role of the BAG3‐mediated autophagy under pathological conditions,Here, we studied the role and function of BAG3‐dependent autophagy in IPF fibroblasts and found that therapeutic induction of this pathway by different drugs reduced collagen deposition and fibroblast proliferation.22.1Explanted lungs were obtained from sporadic IPF patients or nondiseased control subjects/healthy donors/organ donors (HD) from the Department of Thoracic Surgery, Vienna. Patient information and patient samples used for different experiments are tabulated in Table 2.2n = 8) and control lungs (n = 8) following an outgrowth technique as described before,Primary human lung fibroblasts were isolated from explanted IPF  was filled in each segment of explanted human IPF lung and was allowed to cool on ice for 30 min for the agarose to solidify. Vibrating blade microtome (ThermoFisher Scientific) was used to section blocks of tissue filled with agarose. About 500‐µm thick sections were made and were cultured in RPMI medium without phenol red supplemented with 2% FCS, 1% penicillin/streptomycin and 1% L‐glutamine. PCLS were left for 24–48 h in cell culture incubator. PCLS were cultured in 6‐well plates, by pooling about three to five slices in one well, depending on the size of the PCLS used, and three such technical replicates were performed per each IPF lung used . Cells were grown to 60% confluency and transfected with ptf‐LC3 plasmid using X‐tremeGENE HP DNA‐transfections reagent (Merck) as per manufacturer's instructions. Next day, drug treatments were performed as indicated for 24 h. Sterile‐filtered DMSO served as vehicle control. ‘n’ of 5–8 IPF patient fibroblasts were used and three technical replicates were performed for each treatment. Nontargeting control siRNA and BAG3 siRNA were obtained from Santa Cruz Biotechnology. siRNA transfections were performed using Lipofectamine 2000 following manufacturer's protocols and as described beforePirf, and Ctd were dissolved in sterile‐filtered DMSO. Aza was dissolved in cold culture medium and was prepared freshly for each treatment. Hence, sterile‐filtered DMSO was used as vehicle‐treated control group (Veh). IPF fibroblasts or PCLS were treated for 24 h with pirfenidone  as described before,2.5https://www.unige.ch/medecine/bioimaging/files/1914/1208/6000/Quantification.pdf).Denatured lung homogenates and cell lysates were used for western blotting following standard protocols as described before2.6T‐method. Threshold cycles (CT) of target genes were normalized to a housekeeping gene (ACTB). The resulting ΔTC were compared to control samples (Veh), and relative mRNA expression was calculated by R  =  2−ΔΔCT.Total RNA was isolated using standard protocols as described before using RNeasy kit from Qiagen according to manufacturer's instructions and as described before.2.7p‐value summary: *p≤0.05, **p≤0.01, ***p≤.001 or §p≤.5 or §§p≤.01, §§§p≤.001, §§§p≤.001.Data are expressed as means, ± SD. At least two independent experiments were performed from eight IPF and eight HD fibroblasts. About three to five PCLS from each IPF patient were pooled and PCLS from three IPF patients were used. The data did not follow normal distribution; hence statistical significance was assessed using nonparametric tests: Kruskal‐Wallis test followed by the Dunn's multiple comparison test for comparison between multiple groups and Mann–Whitney U test for comparing two groups. 33.1While BAG3 is involved in the autophagy pathway, BAG1 has been reported to play important role in the ubiquitin‐proteasomal pathway.3.2In an attempt to investigate whether drugs interfering with autophagy may also affect the cell fate of IPF fibroblasts, we used two drugs: (1) Aza, a cytostatic drug with autophagy‐inducing properties, besides its well‐documented demethylation effects.3.3We next analysed whether Aza, Ctd or Pirf would also influence the protein levels of BAG3 and its substrate FLNC. Pirf‐treated IPF fibroblasts did not reveal any changes in BAG3, but its substrate FLNC was significantly decreased. Treatment with either Aza alone or Ctd alone or in cells co‐treated with Prif–Aza or Pirf–Ctd, a significant decrease in BAG3 and its substrate FLNC was observed. In addition, the fibrosis marker α‐SMA as well as the proliferation marker, PCNA were also decreased Figure . Immunof3.4BAG3 followed by drug treatments in these cells. As shown in Figure siBAG3. In IPF fibroblasts with BAG3 knockdown, Pirf alone, Aza alone or Ctd alone or Pirf in combination with Aza or Ctd did not reduce the protein levels of FLNC as significantly as in the cells without BAG3 knockdown in all treatment groups. Interestingly, the protein levels of LC3BII as well as the myofibroblast marker α‐SMA also did not decrease upon BAG3 knockdown in any of the treatment groups  in PCLS treated with Aza, Ctd or co‐treated with Pirf–Aza, Pirf–Ctd. These experiments also showed a decrease in protein levels of BAG3 and its substrate FLNC. Although Pirf treatment alone showed a significant decrease in BAG3, PCNA and COL1A1 protein levels, its effects were milder, especially as compared to Pirf+Aza or Pirf+Ctd groups. Figure . Further4In this study, we identified insufficient BAG3‐dependent autophagy in fibroblasts of IPF patients. Administration of Aza and Ctd in both fibroblasts and in PCLS of IPF patients rescued BAG3‐dependent autophagy and decreased fibroblast proliferation alongside with a significant decrease in the fibrosis marker COL1A1 in PCLS from IPF patients Figure . To our Reports in the past have revealed defective autophagy in both alveolar epithelial cells type II (AECII) and in fibroblasts of IPF patients.The molecular mechanisms responsible for the defective macroautophagy in IPF fibroblasts are not clear. Possible errors may occur in BAG3‐dependent selective degradation of cargo such as FLNC a process that occurs in an ubiquitin‐dependent manner, by closely cooperating with the HSP70‐associated E3 ubiquitin‐protein ligase CHIP.Defective autophagy has been implicated in several neurodegenerative diseases including Alzheimer's disease, Parkinson's disease and Huntington's disease.A note of caution here is the apoptosis inducing nature of Aza and Ctd. It has been reported that Aza induces a dose‐dependent increase in apoptosis. In that, at concentrations between 2 and 6 µM, only cells at G1 phase preferentially underwent apoptosis.BAG3 silencing resulted in a functional correction of disease associated cystic fibrosis transmembrane conductance regulator (CFTR) variants including F508del‐CFTR.It was suggested that BAG3‐mediated aggregation of critical cellular components that are required for an efficient autophagy‐lysosomal system may result in a defect in this pathway, providing a rational for the observation that decreased BAG3 leads to increased autophagy.5To conclude, our study reveals that insufficient BAG3‐mediated autophagy is seen in IPF fibroblasts and may result their exaggerated proliferation. The BAG3 modulating drugs used here, namely Aza and Ctd, restored the autophagic flux to reduce fibroblast proliferation and collagen deposition and re‐established cellular homeostasis. These agents may thus be potentially usable in combination with clinically used Pirf and may represent a novel treatment approach in IPF by exerting further inhibitory effects on IPF fibroblast proliferation, altogether eliciting desirable therapeutic implications of targeted autophagy‐modulating drugs for IPF and maximize the efficacy of current IPF therapy.The authors declare no conflicts of interest.Supporting informationClick here for additional data file.FigureS1Click here for additional data file.FigureS2Click here for additional data file.FigureS3Click here for additional data file.


Stroke is a major cause of death and disability across the world, and its detrimental impact should not be underestimated. Therapies are available and effective for ischemic stroke ; however, there are limitations to therapeutic interventions. Recanalization therapy has developed dramatically, while the use of adjunct neuroprotective agents as complementary therapies remains deficient. Pathological TAR DNA-binding protein (TDP-43) has been identified as a major component of insoluble aggregates in numerous neurodegenerative pathologies, including ALS, FTLD and Alzheimer’s disease. Here, we show that increased pathological TDP-43 fractions accompanied by impaired mitochondrial function and increased gliosis were observed in an ischemic stroke rat model, suggesting a pathological role of TDP-43 in ischemic stroke. In ischemic rats administered rapamycin, the insoluble TDP-43 fraction was significantly decreased in the ischemic cortex region, accompanied by a recovery of mitochondrial function, the attenuation of cellular apoptosis, a reduction in infarct areas and improvements in motor defects. Accordingly, our results suggest that rapamycin provides neuroprotective benefits not only by ameliorating pathological TDP-43 levels, but also by reversing mitochondrial function and attenuating cell apoptosis in ischemic stroke.  Stroke is the second leading cause of death in the World Health Organization’s 2-2-global health estimate, leading to severe long-term disability and death worldwide, and incurring huge social costs. Stroke is therefore a serious health problem that requires further attention to attenuate death and disability. Among all strokes, approximately 87% are ischemic strokes, which are usually caused by blood clots that occlude the blood supply from the cerebral artery in the brain and initiate ischemic cascades . ReperfuTDP-43 pathology is identified as the major component of the UBIs aggregated in numerous neurodegenerative diseases, especially in frontotemporal lobar degeneration (FTLD-U) and amyotrophic lateral sclerosis (ALS) ,10. TAR It has been shown that the mammalian target of the rapamycin (mTOR) cascade is associated with I/R injury . The mTOThe pathological hallmark of insoluble TDP-43 proteinopathy has been shown to be the major component of UBIs aggregated in numerous neurodegenerative diseases, especially in 45% frontotemporal lobar degeneration (FTLD-U) patients and 95% amyotrophic lateral sclerosis (ALS) patients . HoweverDuring ischemia reperfusion, ROS release substantially contributes to cell damage and death via caspase3-mediated apoptotic signals. The mitochondrial antioxidant enzyme superoxide dismutase 2 (SOD2) is the first-line defense against mitochondrial ROS release and, subsequently, reduces superoxide radicals and infarction volumes after cerebral ischemia . Thus, wRapamycin, a selective inhibitor of the mammalian target of rapamycin (mTOR), has been reported to be beneficial in TDP-43-related neurodegenerative diseases, such as FTLD-U. Since we observed the induction of the TDP-43 protein after ischemic stress , we furtThe therapeutic efficacy of rapamycin in eliminating TDP-43-mediated pathology after ischemic stroke was further examined. After 60 min of ischemia, rats were injected intraperitoneally with rapamycin (250 μg/kg) for 24 h and then killed for further analysis. The control animals were injected with PBS (vehicle) in parallel. We then further analyzed the infarction size in the ischemic brain regions of the rats, and found that the infarction size in the right ischemic cortex was significantly decreased after rapamycin treatment compared to the vehicle control group . In addiTo determine whether the improvement in ischemic stroke with rapamycin treatment was due to the reduction in neuronal apoptosis, we examined the expression levels of active caspase-3 by using Western blot analysis. The protein lysates were assayed by immunoblotting and collected from the SH-SY5Y cells treated with different doses of rapamycin  for 24 h after 4 h of OGD hypoxia or normoxia incubation. Caspase-3 is activated in apoptotic cells, and we found that the levels of caspase-3 proteins were decreased in the SH-SY5Y cells treated with rapamycin under OGD hypoxia, while no significant change was observed in the cells under normoxia administered with different doses of rapamycin . In addiIt is known that a high percentage of ischemic stroke patients die from extensive brain damage caused by reperfusion ,5. The oRevascularization and the limitation of secondary neuronal injury are the primary goals of advanced stroke management. IV thrombolysis and endovascular therapy are the two main forms of revascularization for selected patients. A limited number of acute ischemic stroke (AIS) patients are suitable for these treatments, largely owing to the narrow therapeutic window. Only 3.2–5.2% of all AIS patients are treated with IV-tPA within 3 h of suffering an acute ischemic stroke in the United States . The extIt has been shown that the mammalian target of the rapamycin (mTOR) cascade is associated with I/R injury . The mTOThe nuclear protein TDP-43, which forms detergent-insoluble cytosolic aggregates in the central nervous system, including the hippocampus, cortex and spinal cord motor neurons, was recently identified as a major component of the UBIs aggregated in numerous neurodegenerative diseases ,10. TDP-Our results showed a time-dependent increase in phosphorylated and insoluble TDP-43 fractions in the ischemic stroke brain region, but not in the cortex contralateral to the ischemic side. The longer ischemic time caused greater pathological TDP-43 accumulation, including insoluble and phosphorylated TDP-43, suggesting that pathological TDP-43 plays a role in ischemic stroke. In the right cortex of an MCA  occlusion rat model, the insoluble full-length TDP-43 in the urea fraction was increased immediately in the right ischemic cortex region after 15 min of ischemia, showing that the acute rise in TDP-43 is synchronized with acute ischemic stroke. We discovered that rapamycin could rescue cell viability after OGD hypoxia and decrease the infarction volume after ischemia/reperfusion injury, prompting us to evaluate whether TDP-43-related pathology is involved in rapamycin’s therapeutic mechanism in ischemic stroke. It has been clearly demonstrated that pathological TDP-43 is significantly degraded through the autophagy pathway, and that the activation of autophagy accelerates the clearance rate for the insoluble TDP-43 fraction and attenuates the neuropathy caused by TDP-43 ,54,55. WIt is well known that aging is the most critical, comorbid, and unmodifiable risk factor for stroke and it affects subsequent treatment responses. In addition, lifestyle including high sugar diets, alcohol and tobacco addiction or high fat diets as well as ageing, brain injury, oxidative stress and neuroinflammation all negatively influence the onset, severity and duration of neurodegenerative diseases. As suggested, choosing a variety of healthy dietary components, such as polyunsaturated fatty acids and the antioxidants curcumin, resveratrol, blueberry polyphenols, sulphoraphane and salvionic acid, as well as caloric restriction and physical activity, will help us counteract ageing and associated neurodegenerative diseases via increasing autophagy and neurogenesis in the adult brain. In our study, we did not consider these influencing factors such as age, eating habits and lifestyle on stroke mechanism, and these influencing factors will be very good directions for our further research in the future.After 4 h of OGD (oxygen–glucose deprivation)-hypoxia incubation, SH-SY5Y cells were treated with different doses of rapamycin  for 24 h, after which the cell viability, ROS production and protein expression levels were determined. The normoxia groups were also treated with different doses of rapamycin  for 24 h.6 cells per well) and allowed to adhere overnight. After cell incubation under normoxia or OGD conditions in the presence or absence of emodin for 4 h, cells were incubated with DCFDA for 30 min at 37 °C and washed with PBS. DCF fluorescence was determined using a multi-well fluorescence spectrophotometer  with 490 nm excitation and 525 nm emission filters.Cytosolic ROS production was measured using 2’,7’-dichlorofluorescien diacetate (DCFDA) as previously described , which iWe used 15 adult male Sprague Dawley rats (250~300 g) and randomly assigned three rats to different time points of ischemic injury . In addition, we used a further 24 adult male SD rats for analyzing body asymmetry, infarct brain volume and TUNEL staining, 12 of which were designated as a vehicle, while a further 12 rats were administered with rapamycin. Animals were housed under a 12-h light/dark cycle and provided with food and water ad libitum. All animal procedures were conducted in accordance with the National Institutes of Health under the approval of the Animal Care and Use Committee of the Taipei Medical University, Taipei, Taiwan (protocol: LAC-2016-0495).Adult male Sprague Dawley rats were anesthetized with chloral hydrate . The right MCA was ligated with a 10-O suture, using methods previously described ,58. The After 60 min of ischemia and reperfusion injury (60 min I/R injury), rats received intraperitoneal injections of rapamycin (250 μg/kg) for 24 h and were then killed for further analysis ,62,63. TThe size of infarction was evaluated by triphenyltetrazolium chloride (TTC) staining, as described by . After rI/R-injury rats with or without rapamycin treatment were analyzed using an elevated body swing test ,65,66,673VO4). Next, the lysates were centrifuged at 4 °C for 15 min at 13,800× g. The supernatants were the RIPA-soluble fraction. The RIPA-insoluble pellets were washed twice in the RIPA buffer, re-sonicated and re-centrifuged. The washed pellets were finally dissolved in the urea buffer as the urea fraction. The total protein concentration was determined according to the Micro BCA procedure , using bovine serum albumin as standard. Equal amounts of proteins from each sample were electrophoresed on 4–12% polyacrylamide gels  under reducing conditions and then transferred by electrotransfer onto polyvinylidene-difluoride membranes. The membranes were incubated in blocking buffer (20 mM Tris (pH 7.5), 137 mM NaCI, and 0.1% Tween-20 (TBST) with 5% (w/v) nonfat dry milk) overnight at 4 °C. The membranes were then incubated with primary antibody in TBS-T containing 1% nonfat dry milk for 1 h at room temperature or overnight at 4 °C. After a series of washes with TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody  for 1 h at room temperature. The immunoblots were visualized using the ECL chemiluminescence-detection system. The animals experiencing sham surgery were regarded as experimental controls for comparisons with other experimental groups. The following primary antibodies were used: rat polyclonal anti-GFAP , anti-TDP-43 antibodies , anti-phospho-Ser409/410-TDP-43 antibodies , anti-SOD2 , anti-mTFA , anti-BDNF , anti-active Cap3  and β-actin .For preparation of the RIPA-soluble and urea fraction , the braAnimals were killed and their brains removed. One side of the brain was placed in 4% paraformaldehyde after transferring from 20 and 30% sucrose in PBS overnight. Brains were then sectioned. The brain was sectioned at 10~15 μm thickness and rinsed with PBS (3 × 10 min). TUNEL staining was performed by using a Roche assay kit , according to the manufacturer’s protocolt-test. All the experiments were conducted at least in triplicate, and the results are expressed as the mean ± standard error of the mean (SEM). Statistical analyses were conducted using one-way analysis of variance (ANOVA) or Student’s


In Norway spruce, as in many other conifers, the germination capacity of somatic embryos is strongly influenced by the desiccation phase inserted after maturation. The intensity of drying during desiccation eminently affected the formation of emblings . Compared to non-desiccated embryos, the germination capacity of embryos desiccated at 100% relative humidity was about three times higher, but the reduction of relative humidity to 95 and 90% had a negative effect on the subsequent embryo development. The water loss observed in these embryos did not lead to an increase in lipid peroxidation, as shown by malondialdehyde levels. Another metabolic pathway in plants that mediates a response to abiotic stresses is directed toward the biosynthesis of polyamines (PAs). The activities of PA biosynthetic enzymes increased steadily in embryos during desiccation at 100% relative humidity, whereas they decreased at lower humidity. The total content of free PAs in the embryos gradually decreased throughout desiccation. The increase in free putrescine (Put) and perchloric acid-insoluble Put conjugates was observed in embryos desiccated at lower humidity. These changes were accompanied to some extent by the transcription of the genes for the PA biosynthesis enzymes. Desiccation at 100% relative humidity increased the activity of the cell wall-modifying enzymes β-1,3-glucanases and chitinases; the activities of these enzymes were also significantly suppressed at reduced humidity. The same pattern was observed in the transcription of some β-1,3-glucanase and chitinase genes. Desiccation treatments triggered metabolic processes that responded to water availability, suggesting an active response of the embryo to the reduction in humidity. A positive effect was demonstrated only for desiccation at high relative humidity. Some of the physiological characteristics described can be used as markers of inappropriate relative humidity during somatic embryo desiccation. Plants are commonly exposed to various environmental stresses  during their development. Optimal and efficient stress responses at different stages of plant development are prerequisites for a good plant survival strategy . In coniin vitro plant propagation, including conifers , which is required for the formation of the aminopropyl group in Spd and Spm , which are potentially harmful to all cellular components and negatively affect cellular metabolic processes . Consequvice versa). β-1,3-glucanases (EC 3.2.1.39) catalyze the cleavage of 1,3-β-D-glucoside bonds in β-1,3-glucans, a major structural component of fungal cell walls  is routinely used in conifer SE . RecentlP. abies (L.) Karst]. Embryogenic cultures were induced in our laboratory in August 2020 from immature zygotic embryos. Induction, proliferation, and maturation proceeded under conditions described in °C.In the first series of experiments, we investigated the importance of the desiccation step for the development of emblings from 12 embryogenic lines of Norway spruce ornithine  or 7.4 kBq L-[U-14C]arginine , for ODC and ADC, respectively, in the presence of 2 mM unlabeled substrate during a 1.5-h incubation at 37°C. 14CO2 was entrapped in hyamine hydroxide and the radioactivity was counted on liquid scintillation analyzer, Tri-Carb 2900TR, Packard. To determine SAMDC activity samples were homogenized in three volumes of 0.1M phosphate buffer, pH 7.6 containing 2 mM β-mercaptoethanol and 1 mM EDTA, and centrifuged at 20,000 × g for 30 min at 4°C. The supernatant and resuspended pellet  were incubated separately with 3.7 kBq [1-14C]S-adenosylmethionine  in the presence of 2.8 mM unlabeled substrate and 3 mM Put. 14CO2 evolution was measured for 1 h at 37°C. The radioactivity was counted on liquid scintillation analyzer, Tri-Carb 2900TR, Packard.The activities of ODC (EC 4.1.1.17), ADC (EC 4.1.1.19), and SAMDC (EC 4.1.1.50) were determined using the radiochemical method developed by Total protein content of the samples was measured by the Bradford method using bovine serum albumin as a standard .°C with 1 ml of 5% (w/v) perchloric acid (PCA) per 100 mg fresh mass of tissue. 1,7-Diaminoheptane was added as an internal standard, and the extracts were centrifuged at 21,000 g for 15 min. PCA-soluble free PAs were determined in one half of the supernatant. The remaining supernatant and the pellet were acid hydrolyzed in 6N HCl for 18 h at 110°C to obtain PCA-soluble and PCA-insoluble PA fractions. PA standards  and extracted free, PCA-soluble and insoluble PAs were benzoylated according to the method of °C until analysis. Detection and quantification of benzoylamines were carried out using an HPLC-MS system consisting of an HTS-Pal auto-sampler with cooled sample stack , Rheos 2200 quaternary HPLC pump , Delta Chrom CTC 100 Column oven , and TSQ Quantum Ultra AM triple-quad high-resolution mass spectrometer  equipped with an electrospray interface. The dried extract was re-dissolved in 5 ml of 80% (v/v) methanol, and 0.5 ml was taken for further analysis and filtered using a 0.2 μm Micro-Spin centrifugal filter . An aliquot of 5 μl was injected onto a Kinetex 2.6 μm C18 100 Å HPLC column, 50 × 2.1 mm  and analyzed by elution with a binary gradient (water/acetonitrile) starting at 20% (v/v) and ending at 50% (v/v) acetonitrile. The residual sample was removed from the column by increasing the acetonitrile content to 90% (v/v) for 6 min, and then the column was equilibrated at 20% (v/v) acetonitrile for 6 min before the next injection. The mass spectrometer was operated in the positive SRM (single reaction monitoring) mode and 2–4 transitions were monitored for each compound. The most intensive ion was used for quantification, the others for identity confirmation. PAs were quantified using a multilevel calibration graph with diaminoheptane as the internal standard.Embryos were ground in liquid nitrogen and extracted for 1 h at 4°C. After electrophoresis, proteins were renatured overnight in 50 mM sodium acetate buffer (pH 5.0), 1% (v/v) Triton X-100. Chitinase fractions were stained with 0.01% (w/v) Fluorescent Brightener 28 (Sigma) in 250 mM Tris–HCl (pH 8.9) for 15 min, and detected by illumination with UV light. Fractions with β-1,3-glucanase activity were visualized as red bands by boiling the gels in 200 ml of 1M NaOH containing 0.3 g of 2,3,5-triphenyltetrazolium chloride (Sigma) in a water bath for 5–10 min. After photography , gel images were processed using Scion Image Software1 (N × (mean-background) where N is the number of pixels in the selected area and the background is the modal gray value (pixels). The sizes of individual isoforms were determined based on the co-separated molecular standard.Total proteins were extracted from embryos according to oftware1 . BackgroP. abies (GenBank: X57980.1) was used as a reference. Primers for the genes of interest were designed based on either gene homology: β-1,3–glucanase (GenBank: L49179.1), putative class I chitinase (GenBank: AY450922.1), class IV Chia4-Pa chitinase (GenBank: AY270016.1), or literature data – ADC, SAMDC, spermidine synthase (SPDS), and SPMS ; PCR products were purified using QIAquick PCR Purification kit (Qiagen) and sequenced (Eurofins Genomics). Sequences were then compared with database data (NCBI). Relative transcript levels were analyzed by the modified 2CT–ΔΔ method using individual amplification efficiency for each gene  and treated with DNaseI (Thermo Scientific). cDNA was prepared using Revert Aid First Strand cDNA Synthesis Kit (Thermo Scientific). Quantitative RT-PCR was performed in 12 μl PCR mix containing 6 μl of PCR MasterMix (Generi Biotech), 3.5 μl of nuclease-free water, 0.5 μl of mixture of forward and reverse primers  and 2 μl of cDNA. The alpha-tubulin gene from and SPMS . Primer ach gene  and compP-values less than 0.05 were considered significant. The statistical package SigmaPlot 14.0 was used for all analyses.Three or four independent experiments were performed, each with at least two biological replicates. Results are presented as means ± standard deviations. The data were analyzed by one-way analysis of variance (ANOVA). The means were compared using the Tukey test, and differences between means with In the first series of experiments, the effect of treatment with 100% relative humidity was tested in 12 embryogenic lines. Germination of a total of 400 emblings derived from treated and untreated embryos was compared based on the percentage of emblings with developed primary root and/or terminal bud and on the number of malformed emblings . The resIn the second series of experiments, the 3-week-old emblings derived from control embryos , had green cotyledons, an elongated hypocotyl, and a primary root, just as in the first series of experiments. Water deprivation during the first 11 days of desiccation treatment affected the development of emblings. Hypocotyls often thickened and did not elongate, and cotyledons were poorly developed . The degRoot formation was less affected than shoot growth; 33% of G95 emblings and 77% of G90 emblings were able to form roots compared to 53% in G100, and root length remained comparable to the G100 control emblings .In control embryos exposed to 100% relative humidity, DW remained unchanged (20–23%) throughout the desiccation process , ED100. In embryos exposed to high relative humidity (D100), the level of ROS production (measured by the amount of MDA present) increased slightly compared to mature embryos M; . HoweverThe content of ABA and its metabolites was also measured in desiccated embryos and preliminary results showed the expected decrease in ABA levels . TreatmeIn the course of desiccation of embryos, a significant decrease in the total content of PAs was observed. Mature somatic embryos (M) were characterized by higher Spd than Spm content, while Put content represented the lowest proportion of the sum of free, soluble, and insoluble conjugates of PAs . In embrThe contents of PA conjugates were lower than those of free PAs. The significantly higher contents of Spd conjugates determined in D95 and D90 were the only obvious differences from those determined in the control . The bouAs desiccation progressed, changes in the individual PAs resulted in changes in the ratios of Spd/Put, Spm/Put, and Spd/Spm . The decThe activities of the three enzymes responsible for the biosynthesis of the major PAs were measured in the soluble and particulate fractions; the results are presented by the sum of the activities of both fractions . In embrA and D). The activity of all four basic enzyme fractions was pronounced during the second half of desiccation (Gel separation based on protein size (SDS-containing gels) and subsequent enzyme detection identified a single fraction of ∼37 kDa β-1,3-glucanases in mature (not shown) somatic embryos. Under control conditions, their activity remained unaffected, whereas desiccation at reduced relative humidity markedly suppressed their activity. However, when embryos were subsequently transferred to a 100% relative humidity environment, strong activation of the enzyme was observed in embryos exposed to 95 and/or 90% relative humidity during the first half of desiccation . This paiccation .A and D). Most of the chitinase activity in the embryos likely came from the basic isoforms  comprised four different isoforms of ∼140, 37, 28, and 20 kDa . Their aisoforms ; unfortuThe expression levels of four polyamine biosynthetic enzyme genes were followed by qPCR analysis during the desiccation of embryos at different humidity levels. The expression level of the ADC gene  increaseThe expression of the β-1,3-glucanase gene studied was affected as a function of humidity . TreatmeAs for chitinases, the expression of a single class I chitinase gene and a single class IV chitinase gene were analyzed. The expression of the class I chitinase gene was not affected by any desiccation treatment , in contP. abies  as well as the expression of a single β-1,3-glucanase gene  in Norway spruce somatic embryos . Previoucaribaea  and moredormancy .While chitinases likely alter cell wall elasticity  and prevDesiccation at high relative humidity promoted germination of Norway spruce somatic embryos, increased the activities of ADC and ODC, the activities of β-1,3-glucanases and transcription of β-1,3-glucanase and class IV chitinase genes. Exposure of matured somatic embryos to decreased humidity levels resulted in stress-induced changes in PA homeostasis as well as the content of ABA and its metabolites. The adaptation of embryos desiccated at reduced relative humidity resulted in changes in the content of free, conjugated, and bound forms of PAs. A high rate of embryo dehydration elicited a significant increase in the content of free Put, although the activities of ADC and ODC decreased, which together with a sharp decrease in the content of free Spm indicated reconversion of free Spm. The increase in bound PAs was triggered by adaptation to a severe water deficit (90% relative humidity), confirming the role of PAs in strengthening the cell wall under environmental stress conditions. Low-humidity desiccation also markedly suppressed the activities of cell wall-modifying enzymes β-1,3-glucanases and chitinases, as well as the expression of β-1,3-glucanase and class IV chitinase genes. Most of the monitored enzyme activities were temporarily attenuated by low relative humidity, but were restored after the water deficit conditions passed away. Desiccation at lower relative humidity appears to impede the processes leading to physiological maturity and subsequent ability of the embryos to develop into emblings.The original contributions presented in the study are included in the article/MC, IM, and ZV had the main responsibility of planning and experimental set up for the study. MC and LG were responsible for MDA analysis, polyamine content and biosynthetic enzyme activity experiments. JMo and ZG did in gel analysis of β-1,3-glucanase and chitinase activities. JMa and VM conducted LC-MS analysis of polyamines and ABA. JK and JP carried out RT-PCR analysis. LF and ZV were responsible for experimental material, obtained micro-morphological data, had main responsibility for data analysis, and for writing of the manuscript draft. KE, IM, ZV, LF, MC, and VM writing—review and editing. MC, IM, and LG funding acquisition. All authors contributed to the article and approved the submitted version.


Over the past decade, a new family of ceramic matrix composites has been developed from the incorporation of homogeneously dispersed graphene-based fillers  into the ceramic matrices. These composites have shown a significant increment of their fracture toughness accompanied by other electrical and thermal functionalities, which make them potentially attractive for a wide range of applications. Here, the main methods for testing the fracture toughness of these composites are described, then the principal observations on the reinforcing mechanisms responsible for this improvement are briefly reviewed, and we discuss the relation with graphene platelets type, morphology and alignment.This article is part of the theme issue 'Nanocracks in nature and industry'. This has been the preferential route for obtaining dense bulk materials, and also produces, as a consequence of the uniaxial pressure applied, anisotropic composites in which graphene platelets ab-plane is aligned perpendicular to the pressing direction (a).The common composites processing method has been mixing either the GNP or GO/rGO platelets with fine ceramic matrix powders, using powder mixing methods in different media , and morphologies , have been reported in most cases as homogeneously dispersed but start to agglomerate as the filler content increases. For these reasons, more complex routes for mixing graphene with the matrix, avoiding agglomerate formation, have been effectively used. For example, e growth ,48, CVD e growth  and the e growth .b), functionally graded materials [c), bioinspired structures [d), porous [e).The excellent results in the reinforcement of basic bulk composites, alongside the interest in replicating natural materials structures and using novel fabrication methods like additive manufacturing, have motivated the study of the mechanical response in other designs of ceramics containing graphene fillers. Under these premises, fracture resistance has been studied also in laminated materials ,45,51 fb, functiaterials ,52, coataterials ,53  is the most frequently used due to its accessibility, small sample size required and rapidity. It has become an essential technique for the observation of crack patterns and for localizing the GNP and GO sheets within them with the aid of scanning electron microscopy (SEM), which often reveals a toughening mechanism. This has also allowed us to establish relationships between the dispersion, morphology and orientation of the platelets and the crack-stopping mechanisms. IFT however, though a highly used method for determination of l cracks . Nonethe5 vol.-% , which rKIC. CB is a good alternative for obtaining stable fracture tests but is limited by having special tools for cutting the Chevron notch. On the contrary, SCF presents the advantage of introducing an easy artificial flaw by indentation but post-fracture identification of the pre-crack is complicated and could be affected by high filler contents. These bending test configurations are especially convenient for composites containing highly exfoliated graphene sheets. Yihua et al. [An effort to perform more reliable studies has been made through the application of single edge notched beam (SENB)  or singla et al.  comparedin-situ crack propagation on Si3N4/rGO samples inside SEM [Complementarily, the inspection of fracture surfaces from the two halves of the fractured beams has provided interesting information on the tortuosity of the crack propagation paths ,33 and tside SEM  has perm. 4ab-plane with a weak bonding in the c-axis could generate additional non-conventional mechanisms.When compared to one-dimensional fillers, e.g. CNT, the use of platelets as reinforcing phase in composite materials presents various advantages associated with a reduced production cost, easier mixing methods and large available surface for effective stress transfer and increased interfacial energy available. Therefore, graphene (and its derivatives) was considered from the beginning as the two-dimensional variant of CNT from which higher relative enhancement could be expected. Moreover, it was also predicted that the particular layered microstructure of the platelets combining the extraordinary stiffness of the a shows the tendency of the maximum increments in fracture toughness both achieved by GNP and rGO for materials studied before 2017 [KIC could be achieved by the incorporation of rGO. Such platelets, as has been summarized in Figure 2ore 2017 . It is nb–d depicts toughness increment for individual materials within the same family of ceramics. Besides, all the results have been included in figure 2e for a direct comparison between families. Oxide-based materials are characterized by the preferential use of GO sheets that, as it was indicated above, are suitable for colloidal processing and possess a defective surface which improves interfacial strength. For nitrides, both types of graphene platelets have been used, achieving the highest increments with rGO, and also using GNP of high aspect ratio obtained after specific milling conditions [As the types of matrices used and the number of publications have increased over time, presently there is enough data to study this tendency in the different families of structural ceramics. Figure 2nditions . NitrideKIC in the range of approximately 7–10 MPam1/2. However, as can be observed in figure 2f,g, higher values can be achieved with complex bioinspired or layered designs that allow better control of anisotropy, larger layers and reduction of agglomerates, enhancing the activation of mechanisms for energy dissipation, and giving values above the average of bulk materials.The selection of ceramic matrices has also changed from one-component matrices to systems that include other ceramic phases in the form of dispersed particles or whiskers which enhance hardness, high temperature properties and oxidation resistance. For instance, the fracture toughness of a high entropy ceramic composed of four materials  has been also successfully enhanced by 70% with the use of 0.15 wt-% of GNP . The max. 5KIC measurements, confirm that both GNP and rGO act as suitable reinforcements in many types of ceramic matrices, clearly developing two types of mechanisms: (i) principal extrinsic mechanisms of conventional CMC reinforced by ligaments, which implicate the interaction between the matrix and the secondary phase, specifically crack deflection, bridging and pull-out (a–e) and (ii) mechanisms which involve how the graphene layers internally split and slide during fracture, namely the splitting of graphene individual layers, the bonding of external layers to the matrix allowing sliding of internal sheets during pull-out, and the kinking, bending and stretching of platelets (e–g).The static and dynamic observations of crack propagation paths and fracture surfaces, accompanied by the results of pull-out a–e and . Moreover, brick-mortar biomimetic designs take advantage of this effect, forcing crack to propagate through the weak phase, achieving higher stability of the fracture process and more tortuous paths [a,b). It is worth mentioning that interfaces with varied fracture properties could be formed between matrix and graphene due to the direct bonding between the layers and the ceramic grains or with the grain boundary phase composed by the sintering additives. Moreover, two situations seem to occur in the light of the type of platelets, (i) GNP platelets form weak bonds with the matrix allowing debonding at interface, activating deflection, and short bridging and pull-out. (ii) rGO platelets form strong bonding with the matrix which leads to improved stress transfer, debonding in between graphene planes, higher closure stresses and large pull-out distances.Crack deflection occurs when a propagating crack tilts and twists, surrounding the platelets at the brittle interface formed with the matrix. Ovid'ko and Sheinerman have calculated that for a crack propagating at grain boundaries and encountering randomly oriented platelets p to 50% , dependius paths . Other tus paths  , which would produce tensile stresses perpendicular to the graphene plane; on the contrary, other conditions are expected at the edges of the platelets, affected by a lower in-plane CTE.The factors mentioned act in combination with residual stresses from thermal expansion mismatch and with the final structure of the platelets once the composite is sintered, as in many cases full exfoliation could not be achieved and big stacks formed by weakly bonded layers constitute the reinforcing ligament. In fact, Liu l layers . In regil layers  and furt3N4 composites with aligned rGO and GNP platelets, estimated a value for graphene fillers strength in the order of 20–40 GPa [Crack wake bridging is the mechanism that contributes the most to fracture toughness enhancement by the generation of closing forces from the resistance of partially debonded graphene fillers and the mechanical interlocking with the matrix. An analysis using the model for fibre reinforced ceramics, taking experimental data from Si0–40 GPa , while t0–40 GPa , also ta0–40 GPa . Recentl0–40 GPa .It is important to note that in addition to the differences observed in the relative increment of toughness related to the processing routes, it is commonly reported that toughness increment reaches a maximum at a specific filler volume and then drops gradually. The effect of higher graphene contents could lead to agglomeration and the formation of pores surrounding the platelets (or in between the agglomerate) which can contribute to diminishing the fracture toughness. Increased filler volume also augments the contact between platelets with different alignments which can contribute to rapid crack propagation creating easy paths between the weakly bonded planes.. 6et al. were the first to report rising R-curve behaviour in Al2O3/rGO composites obtaining 1.6 times higher steady-state toughness than initial K0 [a). The results confirmed that the highest reinforcements are obtained for crack fronts facing platelets planes and the materials showed rapid increment in RK at short crack extensions when compared with other reported composites. Closure stresses of 50 MPa and a length of the bridged zone of 800 nm were calculated. A similar analysis of R-curve behaviour for two different orientations of the graphene platelets against crack propagation path, and for two morphologies of graphene platelets was performed in Al2O3/rGO materials by measurement of crack-opening displacements. The calculated maximum bridging stresses for thin and thick rGO sheets perpendicular to crack growth direction were 137 and 183 MPa, respectively [2/SiC/rGO composites fabricated following different Bouligand patterns which exhibit RK/K0 ratios up to 2 (for the fibres with 45° rotation angle at crack lengths of 0.5 mm) [Fewer works have been published addressing the study of R-curve behaviour, due to inconveniences related to the varied raw materials, the reproducibility of GNP and rGO dispersions, and the methods for performing stable crack propagation tests. Centeno itial K0 . The achitial K0  allowed 2 substrate and in bulk TaC/GNP ceramics has been measured under quasi-static loading and low-frequency dynamic loading demonstrating an effective increase in damping and the possibility of reducing flaw formation by shock waves and vibrations through intrinsic energy dissipating mechanisms as rippling and flattening, bending, kinking and sliding [3N4/polyethylene naphthalate substrate subjected to cyclic bending stress showed a fatigue limit two orders of magnitude higher than ITO deposited on the same substrate [et al. reported for the first time the observation of bridging rGO platelets crinkling and recovering flat morphology without apparent damage under the loading–unloading–reloading operations of a controlled crack propagation test, which was caused by mechanisms associated with weak Van der Waals forces, strain gradients and electromechanical coupling [c).Two interesting characteristics associated with cracks formation and propagation in graphene/ceramic composites that have been scarcely investigated are the behaviour of materials under cyclic stresses and the effect of temperature on fracture resistance. Fatigue in some monolithic ceramics may be difficult to assess as crack growth can be promoted by ambient conditions, and catastrophic failure may also occur instantly once the crack starts propagating, however, it has been studied in reinforced ceramics which exhibit more stable crack growth. For graphene/ceramic materials, the research is still at an early stage but a few works have shown important results. The damping behaviour of graphene deposited on Si/SiO sliding ,87. Grapubstrate . In a recoupling   the improvement of fracture resistance for making more damage-tolerant materials, intended to be used in parts subjected to high tensile stress, impact stress and wear, and (ii) the simultaneous improvement of the mechanical properties and the electrical or thermal functionalities of the composites which allow increasing the efficiency of the components in electronic devices or machining through alternative methods. The former includes, for example, Al. 8Huge progress has been made in the last decade taking advantage of graphene's extraordinary mechanical properties in crack initiation and arrest. The increased availability of graphene in larger quantities at lower cost has brought a wide variety of fillers with differences in size and surface functionalization, complicating the task of optimizing the conditions for structural reinforcement. The main achievements have been the demonstration of the reinforcing capability in different types of matrices and the identification of the mechanisms that reduce fracture energy. There is no unique approach to achieving a significant increment of fracture toughness, rather the characteristics of the matrix and the nature and distribution of the graphene sheets must also be considered. Additionally, in-depth studies on the role of defects and residual stresses are still needed for designing proper interfaces. The interesting results obtained from reinforcing ceramics with graphene have opened new fields of study focused on the use of other two-dimensional nanoreinforcements.


Pseudomonas phage Phi6. According to the results, concentration-dependent antiviral activity was observed, and the strongest viral inhibition graphene oxide-loaded nanofibers were 33.08 ± 1.21% at the end of 24 h.As the world has experienced in the Coronavirus Disease 2019 pandemic, viral infections have devastating effects on public health. Personal protective equipment with high antiviral features has become popular among healthcare staff, researchers, immunocompromised people and more to minimize this effect. Graphene and its derivatives have been included in many antimicrobial studies due to their exceptional physicochemical properties. However, scientific studies on antiviral graphene are much more limited than antibacterial and antifungal studies. The aim of this study was to produce nanocomposite fibers with high antiviral properties that can be used for personal protective equipment and biomedical devices. In this work, 10 wt% polycaprolactone-based fibers were prepared with different concentrations  of porous graphene, graphene oxide and graphene foam in acetone by using electrospinning. SEM, FTIR and XRD characterizations were applied to understand the structure of fibers and the presence of materials. According to SEM results, the mean diameters of the porous graphene, graphene oxide and graphene foam nanofibers formed were around 390, 470, and 520 nm, respectively. FTIR and XRD characterization results for 2 w/w% concentration nanofibers demonstrated the presence of graphene oxide, porous graphene and graphene foam nanomaterials in the fiber. The antiviral properties of the formed fibers were tested against  Cystoviridae family and infects Pseudomonas bacteria. Similar to SARS-CoV-2 it is enveloped by a lipid membrane, has spike proteins, and is of similar size (80–100 nm), thus making it a good surrogate for studying RNA viruses , etc.) play a crucial role in the development of PPE, wound dressing films and filters to reduce the level of microbial contamination and bioburden  honeycomb shape lattice of carbon atoms that was initially prepared by micromechanical cleavage of bulk graphite , 17. Graioburden –32.The antimicrobial activity of graphene and its derivatives is due to a combination of physicochemical properties. These can be listed as oxidative-stress mediated, layer number, lateral size effect, tailored surface, time and concentration dependency –36. OxidEscherichia coli (E. coli) (E. coli and Staphylococcus aureus (S. aureus) bacteria decreased the at most at the highest concentration level (10 wt%). Since this study was conducted for water purification application, it is not a concern of toxic effects on the human body, but it is essential for graphene-based materials for biomedical research.Many studies have been reported on graphene and its derivatives and their nanocomposites discussing antibacterial, antifungal and antiviral activities , 40–42. E. coli) , 20. AccE. coli) . It was GO, porous graphene (PG) and graphene-based foam (GF) are among the graphene derivatives  sought aIn this study, the antiviral activity of electrospun PG, GO and GF nanocomposite fibers were assessed against a double-stranded RNA virus. Electrospinning is one of the most common fiber production methods used to obtain nano-sized polymeric fibers from various polymer solutions applying high voltage. PCL was used as the carrier polymer as it is a biocompatible, easily processable polymer, and when it is dissolved in acetone, a non-toxic environmentally friendly solution is obtained when used in the human body. Previous antimicrobial studies of graphene were mostly focused on the antibacterial and viral inhibition was not included in much research. At the same time, the antiviral activity of PG and GF are investigated for the first time in this study. These comparative antiviral fibrous structures enable us to find the optimum material for PPE for future antiviral filtration systems.PCL , GO (average number of layers 15–20) and acetone were purchased from Sigma Aldrich . PG (pore size around 3–5 nm) and GF (sized about 4 μm with folded area and number of layers varied from 2 – 3 to 9 – 15) were synthesized as reported by Tabish et al. in the previous research , 48, 50.PG, GO, GF powders and PCL polymer (10 wt%) were weighted on precision scales for 5 different concentrations  determined as indicated in Surface tension measurement of 15 different nanocomposite solutions was performed using Kruss Tensiometer . During this measurement using the Du Nouy ring method, approximately 10 mL of solution was taken from the glass bottle and a platinum-iridium ring was dipped into it and slowly withdrawn. In order to ensure correct results, the ring was first calibrated with distilled water and then used for solutions. The maximum surface tensions obtained during extraction were recorded. This process was performed three times for each solution and the averages were recorded. The ambient temperature was recorded as approximately 23°C.A Brookfield Viscometer DV-III was used to determine the viscosity of the solutions . For each of the solutions, approximately 6 ml was poured into the viscometer and the values measured with a small-sample spinner were recorded. Viscosity measurements of nanocomposite solutions were carried out at ambient conditions (23°C) and were performed three times to get an average result.PG, GO, and GF incorporated fibers were collected using a grounded metal flat plate collector . The neeThe fibers were gathered after manufacture, mounted on aluminum studs, and gold sputter coated for 90 s . After that, these samples were examined using a scanning electron microscope (SEM)  with a 5 kV working voltage. The morphology of the nanocomposite fiber mats were determined using SEM at about 10 different areas of a sample. A total of 100 fibers were randomly measured using Image J software, and the mean diameter and standard deviation were computed using Excel. OriginPro software was used to create the histogram graphs of the frequency distribution of fiber diameters.–1 was applied to pure PCL and 2w/w% PG-PCL, GO-PCL, GF-PCL nanofibers. Nanofiber samples were adjusted to be approximately 2 mm thick and 5 mm in diameter and placed in the spectrometer. X-ray diffraction (XRD) (at 40 kV and –40 mA) characterization was used for 4 different samples at the same concentration.Fourier transform infrared spectroscopy (FTIR)  characterization with a wavelength range of 4,000–500 cmPseudomonas Phi6 bacteriophage was used to model double-stranded RNA viruses. Pseudomonas syringae (P. syringae) and Pseudomonas Phi6 bacteriophage were sourced from DSMZ . The received microorganisms were cultured following the manufacturer’s instructions. Stock cultures of P. syringae were stored in a Microbank™ at −20°C, whilst the Phi6 bacteriophage was stored in a cool dark place. Antiviral activity was assessed against this microorganism as it is a safe, easy to work with and well-studied model surrogate for SARS-CoV-2.In this work, P. syringae were prepared by incubating a single colony in 30 ml of tryptone soya broth for 24 h at 25°C and 150 rpm.Actively growing broth cultures of P. syringae culture and 3 ml of molten semi-solid agar (0.5% agar) and poured onto agar plates. The plates were incubated for 24 h at 37°C and the number of plaques was counted. The viral reduction was calculated by comparing the number of virions at 24–0 h. Antiviral activity was statistically analyzed and compared to the control samples using unpaired t-tests. The difference was considered significant when p < 0.05.Bacteriophage suspensions containing the made fibers in PBS were prepared. A total of 100 μl of the suspension at 0 and 24 h was added to 300 μl of the overnight Graphene suspensions were prepared in different concentrations  as PCL-PG, PCL-GO and PCL-GF. The fibers obtained from these suspensions were formed by the electrospinning method, which is affected by the surface tension and viscosity values of the solution.In The viscosity effect of graphene materials added to the PCL solution is shown in 0.1, 0.5, 1, 2 and 4 w/w% PCL-PG, PCL-GO and PCL-GF nanofibers were prepared using electrospinning. The prepared fibers were imaged using SEM to understand fiber morphology. A histogram was created with fiber diameter distribution by measuring the diameter of 100 sample fibers. The formation of some large beads was observed due to the rapid evaporation of the solvent acetone used during the electrospinning process. Due to the nature of the electrospinning method, the fibers dispersed with the aid of high voltage are not uniformly aligned. However, it is observed that adding graphene nanoparticles, which are affected by high voltage with increasing concentrations, increases the entanglement of fibers. While the applied high voltage is necessary to overcome the surface tension, the electrical conductivity of the nanomaterial used is affected by this high voltage, causing asymmetric fiber formation .As seen in SEM characterization of PCL-GO nanofibers and fiber diameter distribution is shown in The PCL-GF nanofibers shown in –1 . PCL fibers loaded with GF and PG at the same concentration, showed similar but slightly lower antiviral activity, with reductions of 31.2 ± 1.11 and 30.5 ± 2.0%, respectively. Fibers containing the lowest nanoparticle concentration (0.1 w/w%) exhibited viral reductions of 13.4 ± 1.9, 15.4 ± 4.9, and 14.01 ± 1.8%, for GO, GF and PG, respectively . At a concentration of 0.5 w/w% of GF or PG, fibers showed a statistically significant viral reduction when compared to the control. Whereas GO fibers only showed a statistically significant reduction at a concentration of 1.0 w/w% or more. This indicates that porous-like structures are more effective at lower concentrations. Whereas at higher concentrations, solid structures are more effective, likely due to their increased exposed surface area.As seen in Overall, the results shown in It has been explained in previous studies that graphene and its derivatives inhibit bacteria by many different mechanisms –60. The In antibacterial nanofiber studies, GO was mostly preferred because of its oxidative stress physicochemical properties and high biocompatibility. However, in general, GO polymer nanocomposites, which exhibit dose-dependent and time-dependent antibacterial activities , are morThe antiviral activity of GO has been investigated in previous studies , and durSince PG, GO and GF nanomaterials are not completely soluble in acetone, they are dispersed and form a suspension. If the graphene derivatives in the formed polymer nanocomposite suspensions are not sufficiently dispersed, they may undergo agglomeration , 62. AggFinally, electrospinning is a system that provides fiber formation from polymer solutions in the high electric field created by a high voltage power supply. At the same time, graphene and its derivatives have a high electroconductivity. Graphene-based materials in nanocomposite solution may be affected by the high voltage applied during nanofiber production and may not adhere to the fiber surface. In such cases, as the concentration may decrease, the antimicrobial activity will also decrease. At the same time, using the entire solution in the syringe in fiber formation is another important point. Since graphene is not completely dissolved in solution, it may collapse and remain in the syringe. This can likewise affect the amount of concentration. Therefore, it should be ensured that all the solution in the syringe and tube is used.In this work, morphology, chemical analysis and the virus inhibitory properties of 0.1, 0.5,1, 2, 4 w/w% concentration PG, GO and GF-loaded fibers were compared. SEM, FTIR and XRD characterizations were applied to the nanofibers. According to the results, the ultrafine fibers obtained mostly have porous surface properties and the mean diameter of all fibers was measured at around 460 nm. It has been observed mostly that the general trend is an increase in nanofiber diameters as the nanomaterial concentration increases. PG, GO and GF nanofibers showed antiviral activity against the SARS-CoV-2 surrogate and the highest inhibition was recorded as 33.08 ± 1.21% after 24 h. In this present study, PG and GF antiviral properties were investigated for the first time. The data showed that overall graphene-based nanomaterials are promising for biomedical applications such as PPE against the ongoing COVID-19 pandemic.The original contributions presented in this study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.SGE performed conceptualization, resources, visualization, analysis, and wrote the original draft. TAT provided resources and characterization, also reviewed the original draft. ME was involved in the conceptualization and overall supervision, reviewed and edited the manuscripts. RKM wrote the original draft and was involved in the conceptualization and overall supervision, reviewed, and edited the manuscripts. All authors contributed to the article and approved the submitted version.


The ability to identify frail older adults using a self-reported version of the physical frailty phenotype (PFP) that has been validated with the standard PFP could facilitate physical frailty detection in clinical settings.N = 182), ages 65 years and older, in an aging research registry in Baltimore, Maryland. Measurements included: standard PFP ; and self-reported questions about walking and handgrip strength. We compared objectively-measured gait speed and grip strength to self-reported questions using Cohen’s Kappa and diagnostic accuracy tests. We used these measures to compare the standard PFP with self-reported versions of the PFP, focusing on a dichotomized identification of frail versus pre- or non-frail participants.We collected data from volunteers (Self-reported slowness had fair-to-moderate agreement (Kappa(k) = 0.34–0.56) with measured slowness; self-reported and objective weakness had slight-to-borderline-fair agreement (k = 0.10–0.21). Combining three self-reported slowness questions had highest sensitivity (81%) and negative predictive value . For weakness, three questions combined had highest sensitivity (72%), while all combinations had comparable NPV. Follow-up questions on level of difficulty led to minimal changes in agreement and decreased sensitivity. Substituting subjective for objective measures in our PFP model dichotomized by frail versus non/pre-frail, we found substantial (k = 0.76–0.78) agreement between standard and self-reported PFPs. We found highest sensitivity (86.4%) and NPV (98.7%) when comparing the dichotomized standard PFP to a self-reported version combining all slowness and weakness questions. Substitutions in a three-level model  resulted in fair-to-moderate agreement (k = 0.33–0.50) with the standard PFP.Our results show potential utility as well as challenges of using certain self-reported questions in a modified frailty phenotype. A self-reported PFP with high agreement to the standard phenotype could be a valuable frailty screening assessment in clinical settings.The online version contains supplementary material available at 10.1186/s12877-022-03376-x. Frailty is a recognized health state of elevated susceptibility, hypothesized to emerge from physiologic declines, to adverse health outcomes when encountering a stressor event , 2. It iThe physical frailty phenotype (PFP) is a commonly used frailty assessment  developePrevious studies of self-reported versions of the PFP have often used single questions about current physical function / functional difficulty to replace each of the objective measures –14. One In this cross-sectional study, we examined the agreement between the standard PFP and versions of a self-reported phenotype where objective measures were replaced by subjective questions. Given that the frailty syndrome is conceptually distinct from disability and related to declines in physiologic reserve , 3, we hStudy participants were recruited to the “Registry of Older Adults Who May Be Willing to Participate in Research” (IRB# NA_00013162) on the Johns Hopkins Bayview Medical Campus. It is comprised of community-dwelling adults, aged 65 years or older, living in the Baltimore Metropolitan area and recruited from a Johns Hopkins outpatient clinic, off-site educational events, or responses to newsprint and online advertisements. Participants must not have an advanced illness with < 6-month life expectancy. Once consented, demographic and health-related information is collected and participants undergo a frailty assessment protocol by trained personnel. A set of self-reported measures related to walking speed and grip strength (described below) was collected on all registry participants between December 2016 and July 2019. The Johns Hopkins Medicine Institutional Review Board approved this study and analyses.1) they walk very slow or are unable to walk in comparison to the walking speed of their peers; 2) walking a quarter mile is difficult; or 3) they have more difficulty crossing an intersection in a timely manner compared to age 60. Question 3 served as our dynamic, change-based question for self-reported walking speed.For walking speed, the participants met the slowness criterion if they responded that: 4) carrying groceries is difficult; 5) opening jars is difficult; or 6) lifting an object once with slight difficulty soon becomes strenuous if they attempt to lift it repeatedly. Question 6 served as our dynamic, change-based question for self-reported grip strength.For grip strength, the participants met the weakness criterion if they responded that: We used three subjective questions on slowness –20 and tWe examined the agreement between these individual questions  with their corresponding objective performance measures.Additionally, we examined agreement when applying the severity of difficulty  as a cutoff for meeting the criteria. Questions 2–5 included follow-up questions regarding level of difficulty. Only those who reported moderate or extreme difficulty met the criteria in this scenario.A standard PFP assessment was performed , 7. In bA self-reported PFP that included solely “static” questions – meaning questions about current function or functional difficulty; if the response was met for questions 1 or 2 for slowness or for questions 4 or 5 for weakness, the participant was frail for that criterion.A self-reported PFP that included solely “dynamic” questions – meaning questions about changes in functional ability over time; if the response was met for question 3 for slowness or for question 6 for weakness, the participant was frail for that criterion.A self-reported PFP that sought to maximize agreement as well as diagnostic accuracy, specifically sensitivity and negative predictive value (NPV), with the objective measure, to be described in the results.We developed three self-reported PFPs by using self-reported questions described above in place of the objective walking speed and grip strength tests:Demographic information  and health-related information  were obtained using a standardized questionnaire. Height and weight were measured to calculate BMI.We calculated percent agreement by summing the number of values in agreement between objective and self-report measures and dividing by the study N. Inter-rater reliability measured by Cohen’s Kappa coefficient was used to determine the agreement, accounting for chance, between objective and self-reported measures, and between standard and self-reported phenotype instruments , 25. KapTo assess the validity of self-reported measures in comparison with corresponding objective tests, we calculated sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). Sensitivity refers to a screening test’s ability to accurately identify a condition among those who actually have the condition as determined by a reference standard) . SpecifiOne-hundred-eighty-two registry participants completed the standard frailty phenotype assessment and a self-report questionnaire of walking and grip related questions. As shown in Table The agreement between measured walking speed and the individual self-reported slowness questions was fair for Question 1 (Kappa(k) = 0.34), and moderate for Question 2 (k = 0.54) and Question 3 (k = 0.47). By combining questions, including using all three questions, we found concordance still within the moderate range (k = 0.51–0.56). Highest sensitivity and NPV were found when comparing walking speed with a combination of all questions; highest specificity and PPV were found using Question 1. See Table The agreement between measured grip strength and the self-reported weakness questions was slight for Question 4 (k = 0.16) and Question 5 k = 0.13), and fair for Question 6 k = 0.21). By combining questions, including using all questions, we found slight agreement (k = 0.10–0.13). The highest sensitivity was found when comparing grip strength with a combination of all questions (72%), and the highest specificity (85%) and PPV (57%) with Question 4. Comparable values for NPV were found using individual or combined questions (65–68%). See Table , and fai1. By comWe also explored agreement and accuracy when including measures of severity  in the self-reported slowness and weakness questions. The findings were similar to the results without accounting for the severity of difficulty  standard PFP to the self-reported PFP, substantial agreement was found across the different self-reported PFPs (k = 0.76–0.78), with confidence intervals remaining in the substantial range. Highest sensitivity 86.4%) was found in the version using all self-reported questions for slowness and weakness criteria. All versions of the dichotomized self-reported PFP had high specificity (96.3–98.8%) and high NPV (96.3–98.7%). The greatest PPV (88.9%) was found in the version using only dynamic self-reported questions. See Table % was fouComparing agreement between the three-level (frail vs pre-frail vs non-frail) standard PFP and the self-reported PFP – where only the static self-report questions for walking speed and grip strength were combined to determine slowness and weakness, respectively – we found fair agreement (k = 0.33). See Table When compared to the dichotomized (frail vs pre/non-frail) standard PFP, we defined a self-reported PFP with substantial agreement (k = 0.78), high sensitivity (84.6%), and high NPV (98.7%). This self-reported PFP combined all self-reported questions per the slowness and weakness criteria. However, in comparison to the three-level (frail vs pre-frail vs non-frail) standard PFP, the three-level self-reported versions in this study had only fair to moderate agreement (k = 0.33–0.50). We found that including more self-report items led to increased sensitivity for frailty (vs. non-frail) detection while maintaining similar levels of NPV. The importance of identifying and treating frailty early in its course, as well as the relative ease of performing more objective tests for confirmation, if needed, without harm to patients even if frailty is not present, makes reducing the false negatives imperative .In comparison to objective measures of slowness and weakness in the standard PFP, self-reported walking questions had fair to moderate agreement with objective slowness, while self-reported grip/hand strength questions had slight to borderline fair agreement with objective weakness. This discrepancy may be due to the specific self-reported questions included in this study. The combination of multiple self-reported questions led to slightly improved Kappa agreement over the individual questions for slowness only. However, including multiple self-reported items had a marked impact on improving sensitivity relative to NPV. Static self-reported questions commonly used to determine difficulties in physical function  had fair to moderate agreement with objective slowness but only slight agreement with objective weakness. Change-based dynamic questions showed moderate agreement for walking speed but only fair agreement for grip strength. Using self-reported questions that included severity, we found minimal change in agreement scores, but both sensitivity and NPV decreased. Therefore, self-reported measures of slowness and weakness that include degrees of difficulty do not appear to improve efforts to minimize false negatives (high sensitivity) and to maximize true negatives (high NPV) .Our work provides novel information about agreement and diagnostic accuracy between standard and self-reported frailty phenotypes, and expands upon on previous studies that have examined agreement between subjective and objective measurements for weakness and slowness. A 2018 study reported observed agreement of 71.1% and k = 0.55 between a self-reported (including four static slowness questions and two weakness questions) and standard three-level frailty phenotype , which aA critical consideration is whether self-reported information can fully equate with performance measures , 29. SevWe tested agreement between our new self-reported frailty assessment and the PFP. We focused on this comparison due to the physiological basis of frailty assessment using the PFP and the potential avenues for prevention and treatment this basis offers . We furtN = 166 participants with available data, out of 182 participants total). We found fair agreement (0.36) between the standard PFP and the FRAIL scale when assessing frail vs non-frail status, which is consistent with previous studies. In these same subset analyses, we found substantial agreement (k = 0.76) for the self-reported PFP, as well as greater sensitivity, positive predictive value, and negative predictive value for the self-reported PFP. See Appendix Regarding existing self-report measures, we did not include the FRAIL Scale in our main study for two main reasons: 1) we view it as conceptually distinct from physical frailty due the inclusion of multimorbidity as one of its criteria; 2) data to calculate the FRAIL scale was available only for a subset of our study population. However, we did analyze the agreement between the standard PFP and the self-reported version of the PFP, and the agreement between the standard PFP and the FRAIL scale, in a subset of our study population  and objective PFP vs FRAIL scale in a subset (N=166) of the study population.


Ea of ∼8 meV for the cold region which is 375 percent lower than the hot region (∼30 meV). The dual TCR behaviour is of great importance for application as a stable resistor up to room temperature, and as a thermistor above room temperature.Printing of electronic devices on a paper substrate using 2D graphene-based ink is an opening gate to innovative applications, where devices would be biodegradable, eco-friendly and can be disposed of with negligible impact on the environment. A resistor is a key element of electronic devices and their application area depends upon its power rating and temperature coefficient of resistance (TCR). In this work, in house developed graphene ink is successfully utilized to fabricate a paper-based resistor using a bar coating technique. Dimensional patterning with precise known values of resistance is achieved using a laser with freedom of shape and size which has been explored for the first time on a paper substrate. The resistor has potential to handle ∼7 W power at room temperature with capacity to withstand up to 200 V which is the highest among reported printed resistors. A dual, low and high TCR is observed, correspondingly in cold (173 K to 300 K) and hot (300 K to 373 K) temperature regions with an activation energy  Printing of electronic devices on a paper substrate using 2D graphene-based ink is an opening gate to innovative applications, where devices would be biodegradable, eco-friendly and can be disposed of with negligible impact on the environment. Printing technologies like inkjet, screen, and flexography are widely in use for the production of functional structures on an arbitrary substrate such as polymers and paper.3 But their utilization is restricted due to the inconsistent range of ink parameters such as rheology, particle size, surface tension5etc. and optimization of these parameters is a very tedious job as a small change in one deviates others significantly. Comparatively, the bar coating technique can be successfully implemented for a broader range of ink parameters. Various nanoparticles such as copper, silver, and carbon nanotubes are utilized to formulate functional inks and used to fabricate flexible printed devices. Graphene has advantages over the traditional nanoparticles, due to its tremendous properties like higher mechanical strength, excellent flexibility, transparency, and higher conductivity. The exceptional flexible properties of graphene are by virtue of its crystalline structure in which sp2 hybridized carbon atoms connect with each other and the high electrical conductivity is due to the formation of π-bonds. These properties made graphene a strong contender for the fabrication of flexible devices using printing technology.6–12 Graphene has also been explored for the fabrication of various active and passive flexible devices like Field Effect Transistor (FET),13 antenna,14 super-capacitor,15 resistor,16 sensor17etc.In recent years, the demand for small, lightweight, flexible and thin film devices for applications such as consumer electronics, defense, health monitoring, aerospace engineering and many more is increasing day by day which motivates researchers to work in the field of flexible and printable electronics.18 vacuum filtration & cutting,19 chemical vapor deposition,20 and ink jet printing3,16etc. are a few techniques used to fabricate resistors. Ink formulation is a customized job which is defined by the printing process, substrate, layer thickness, drying mechanism etc. Therefore, the in-house developed graphene ink for the fabrication of resistors for flexible electronics systems is explored. The temperature coefficient of resistance (TCR) is an important parameter of a resistor, to be used as a temperature sensor or as a stable passive component. The TCR value and its nature are two defining parameters of a device which decides whether it can be used as a temperature sensor or as a stable resistor in high-reliability circuits and systems.21 More emphatically, in the case of temperature sensing applications, the device is supposed to show high TCR, on the other hand a resistor for reliable circuits must have a very low TCR.22 The TCR of the printed resistor mainly depends upon the temperature coefficients of the ingredients of the ink along with the humidity and pressure of the surrounding environment. It has been reported that humidity, temperature and other gases present in the environment affect the properties of graphene and consequently affect the resistance and TCR.23–27 There are numerous studies on the TCR of graphene-based resistors on various substrates. However, the nature of the TCR reported in the literature is contradictory i.e. in some cases the value of the TCR reported is near zero or very low and other cases very high.16,28–31 This makes it necessary to explore the value of the TCR in graphene-based resistors in detail. In this work, resistors were fabricated on a paper substrate using graphene ink with bar coating film deposition technique and laser cutting to result in a predetermined resistance value. Further detailed study to understand the mechanism for the observed high/low value of the TCRs is presented. In this work, the dual TCR nature of the graphene-based resistor on paper substrate is presented for the first time to the best of our knowledge.On the basis of the resistor electrical resistance property, this two terminal passive electrical component is ubiquitous in various electronic circuits. Presently, resistors are being fabricated using various novel methods as compared to their earlier fabrication procedures. Now a days screen printing,2.32 Graphene was purchased for the ink formulation and in order to analyze the quality, Raman spectroscopy of the printed resistor from formulated graphene ink was carried out , ESI−1 respectively. The G band originates from the stretching of the covalent bond in the carbon lattice whereas the D band gives a signature of the present disorder in the structure. Further, the 2D band observed at 2729 cm−1 is an overtone of the D band signifying the formation of the multilayer structure of the graphene ink printed resistor. For better comparison, the Raman spectrum of the purchased graphene powder was also done at the same excitation wavelength and laser power , ESI33 Such broadening may be attributed to the stack formation during printing of the ink. However, the ratio of the intensity of the D band w.r.t. that of the G band depicts the quality of the graphene-like structure. For powdered graphene the ratio ID/IG has been found to be 0.16 which increases to 0.23 in the printed sample; still this ratio is quite low thus confirming the formation of a good quality graphene ink printed resistor.34,35Homogeneously dispersed graphene ink was formulated with the help of a vibroshaker using a combination of organic solvents to get proper wettability, stability and a fissure-free homogenous printed layer . The combination of solvents enhances dispersion of the solid components and substrate wettability along with smoothness in the printed layer. Solvents get evaporated after thermal treatment and therefore do not impact the conductivity. Other additives such as resin, sticking agent, wetting and dispersion modifier which remain in the coated layer contribute to an increase in resistance due to their insulating nature.V(I) characteristics of the resistors were recorded and follow Ohm’s law. Variation in the curve’s slope indicates that the current decreases apropos of voltage with an increase in length and increases with an increase in width as shown in 36 Through selecting these dimensional parameters graphene film resistors with customized resistance values can be fabricated roll-to-roll using a laser source.In the present work, graphene ink was primarily coated using a wire wound bar coater on the paper substrate . Predefi−1 when the sample was annealed for 10 min and drops to 13 Ω min−1 for the annealing time 60 min (Table S1−1), more than the sample annealed at 373 K for 10 min. After that it nearly stabilized with much less change (4 Ω min−1) as compared to the first 10 min. Solvents used in the ink boiled around 373 K and most of the volatile organic components (VOCs) were evaporated around it. Thermogravimetric analysis (TGA) of the sample also discloses this and after that there is a nearly stable mass of ink up to 650 K  have been selected to examine their effect on device performance. The measured resistance reduced from 27 kΩ to 23.6, 21.8, 21.5 for successive increases in annealing time and reaches the lowest of 18.5 kΩ for annealing time 120 min at 373 K . The red Table S1. At 423  Table S1. The sloK Fig. S2.As the annealing time increases, it impacts less because most of the solvent was evaporated in the first 10 min and nothing much has been left to fly away. TGA analysis of the graphene ink coated paper also supports this conviction Fig. S3. HoweverThe temperature–time profile of the samples on annealing demonstrated that the annealing time has less effect on device resistance than the annealing temperature. Therefore, more annealing time can be traded for less annealing time, which requires the annealing temperature must increase to drive-off excessive remnant VOCs. Reduction in annealing time is advantageous to expand the portfolio of materials that would otherwise be excluded in roll-to-roll device fabrication on flexible substrates. Longer annealing time is not acceptable in roll-to-roll printing of electronic devices which significantly affects the process’s throughput. The annealing temperature is still low and easily acceptable for polymer substrates in flexible and printable electronics with the added advantage of low annealing time.37–39 The operational power of the printed devices was explored with diverse annealing times and temperatures to examine their power operational capabilities as well as the best parameters which can deliver maximum power. SEM analysis of the as fabricated resistor reveals the uniformity in micro-structure of the material. While a lower resistance value with high temperature bearing capability leads the design of a resistive, high power structure on paper substrate and exhibited high performance characteristics which will be described here in detail. High power resistors have applications as resistance heaters, in current sensing, snubber applications, bleeder resistor, inrush current limiting, ballast resistor, electronic dissipater and so on.l × w = 5 × 4 mm) for that purpose was taken into account which hopes to convey the highest power by keeping in mind that power is inversely proportional to the resistance. The sample was then annealed for 120 min at 423 K. The potential difference up to 200 V was applied and the power delivered by the device measured was 6.5, 6.6 and 8.4 W at temperature 290 K, 300 K and 353 K respectively  characteristics  thermal imaging and it was found that there was no increase in temperature after operating the device for 60 seconds at 200 V  in the eristics  clearly V Fig. S5. Paper aR–T measurements of the devices were carried out from 173 to 373 K and their TCR, thermal index and activation energy were calculated. The changes in resistance have been studied in cold and hot regions taking room temperature (300 K) as the reference temperature. For the measurement of the TCR the following equation is usedα is the temperature coefficient of resistance of the device, ΔR is the change in resistance w.r.t. change in temperature (ΔT) and R300 is the resistance of the device at 300 K. −1 or PPM K−1 by multiplying α by 106. Previously reported graphene resistors have shown a high TCR and have been used as temperature sensors while some have shown a near zero TCR which can be used as a stable resistor.28–30 It is the intrinsic behaviour of the synthesized graphene material and defects occurring during synthesis that are the crucial factors which are responsible for low or high observed TCR.28 Here dual TCR behavior of the device is being reported which can be used as a stable resistor along with as a temperature sensor, as shown in 40 Therefore, to verify any change in phase, differential scanning calorimetry (DSC) of the graphene, resin and ink was carried out, but no evidence of phase transition was found  and activation energy (Ea) of the fabricated resistor by using the Arrhenius equation mentioned below:R is the resistance at temperature T, R0 is the resistance at infinite temperature (T) = ∞, B is the thermal index  of the cold and hot zones for all studied conditions (Table S2R2) is from 0.86 to 0.99, indicating a reasonable linear fitting of experimental data. The measurement conditions played an important role and therefore, above room temperature measurements were done in both vacuum and a normal environment at relative humidity of about 50%. Humidity has a direct influence on graphene resistance and it has been confirmed by researchers previously.25,45 Along with this, the paper substrate has a hygroscopic nature therefore temperature and humidity both are responsible for the high value of TCR in the normal environment  for a graphene-based device, however, these devices are on a SiO2/Si substrate and are not considered for flexible electronic devices. Similarly various low TCR resistors have been reported by other researchers, but most of them are on rigid substrates and can’t be considered for roll-to-roll fabrication of devices.31–45Al-Mumen 3.n-propyl acetate (5 grams). All constituents were pre-mixed in an SS container using an electric stirrer  for 5 minutes. The graphene ink was then shaken by the vibroshaker using 100 grams of zirconium oxide beads (0.5 mm diameter) to act as grinding media for 2 hours in four intervals of half-hour each having a half-hour resting period between two successive shakings to avoid over heating of the ink. After shaking, the ink was separated from the zirconium oxide beads and stored in an air-tight glass bottle. The detailed characterization of the as formulated ink will be reported elsewhere.Graphene powder was purchased from United Nanotech, India and other materials were borrowed from CYMK inks LLP, India. A vibroshaker  was used for graphene ink formulation with the following composition: graphene powder (10 grams), resin solution (40 grams), ANTI-TERA-U (0.5 grams), MEK (34.5 grams), methyl isobutyl ketone (10 grams), cyclohexene (5 grams), Thick graphene film of approximate thickness 20 μm was deposited using a wire wound bar coater  of thread size 20 micron on a paper substrate  of thickness 71 ± 8 μm with surface roughness of 0.9 ± 0.4 μm.The resistors were first designed in Inkscape software (Open Source Vector Graphics Editor). The graphene coated film was patterned by laser  to fabricate the resistor.Electrical contacts of the resistor to the external electrical measurement system were made by silver paste . A Keithley 2450 source meter was used to take the electrical measurements in vacuum with the help of a Cryostat. Low temperature measurements (173 to 300 K) were carried out in cryogenic conditions using liquid nitrogen as the coolant.2) and a heating rate of 10 K per minute was maintained during the characterization. Differential Scanning Calorimetry (DSC) measurements were carried out using an SDT Q600 analyzer (TA Instruments) in the presence of nitrogen gas (N2) and a heating rate of 2 K per minute was maintained during the characterization.Surface morphological characterization was carried out using a scanning electron microscope (SEM) on a Zeiss (EVO-18). Thermogravimetric analysis (TGA) was performed using a HITACHI STA 7200 in the presence of nitrogen gas (N4.A high power and dual TCR device was successfully bar coated using stable graphene ink. Characterization of the film by electrical measurements showed that the optimum annealing temperature and time are 423 K and 10 min, respectively, which makes it suitable for roll-to-roll device fabrication. Laser patterned bar coated devices have been successfully fabricated and demonstrated high power handling capacity up to 7 W along with high voltage bearing capability of 200 V. The devices showed dual TCR behaviour in cold and hot regions separately which is believed to be the first report amongst previously studied 2D materials. It can be suitably used as a stable resistor in the low temperature region and as a thermistor at above room temperature.All authors do not have any conflict of interest.RA-009-C8RA10246E-s001