Bilayer graphene is a contender of interest for functional electronic applications because of its variable band gap due to interlayer interactions. Graphene growth on Cu is self-limiting, thus despite the fact that chemical vapor deposition (CVD) has made substantial strides in the production of monolayer and single-crystal graphene on Cu substrates, the direct synthesizing of high-quality, large-area bilayer graphene remains an enormous challenge. In order to tackle this issue, we present a simple technique using typical CVD graphene growth followed by a repetitive wrinkling-etching-regrowth procedure. The key element of our approach is the rapid cooling process that causes high-density wrinkles to form in the monolayer area rather than the bilayer area. Next, wrinkled sites are selectively etched with hydrogen, exposing a significant portion of the active Cu surface, and leaving the remaining bilayer areas, which enhance the nucleation and growth of the second graphene layer. A fully covered graphene with 78 ± 2.8% bilayer coverage and a bilayer transmittance of 95.6% at room temperature can be achieved by modifying the process settings. Bilayer graphene samples are examined using optical microscopy (OM), scanning electron microscopy (SEM), Raman spectroscopy, and an atomic force microscope (AFM) during this process. The outcomes of our research are beneficial in clarifying the growth processes and future commercial applications of bilayer graphene. Monolayer graphene is a desirable material for future electrical and photonic devices due to its extremely high carrier mobility and distinct one-atom-thick layered structure . However2O-assisted CVD technique. In order to facilitate the nucleation and growth of the bilayer beneath, the H2O strategically etches the edge of the top graphene layer, slowing the growth of the top layer ..2/Si subWe devised a G-rW-rE-rG method to produce large-area bilayer graphene films, which exhibit bilayer coverage of 78 ± 2.8% and a transmittance of 95.6% at room temperature. The formation of large-area bilayer graphene can be attributed to edge attachment and diffusion-based growth mechanisms. Following wrinkling and selective hydrogen etching, the coverage of the top monolayer graphene decreases while a portion of the bilayer area remains, and this facilitates the nucleation and growth of the bilayers during the regrowth process. By combining the G-rW-rE-rG strategy with other methods to induce the formation of bilayer graphene with a specific stacking order in the initial growth step, it is anticipated that substantially larger or even wafer-sized homogeneous-stacked bilayer graphene may be produced. We are certain that our work will help in understanding the growth mechanisms of CVD-derived bilayer and multilayer graphene on Cu, as well as clear the way for the mass production of bilayer graphene for industrial applications.


Poly(A) binding protein interacting protein 1 (PAIP1) is a translation regulator and also regulate the decay of mRNA. PAIP1 has also been reported to be a marker of increased invasive potential of liver cancer. However, the roles and underlying molecular mechanism of PAIP1 in liver cancer is still unclear. Here, cell viability and the gene expression profile of liver cancer line HepG2 transfected with PAIP1 siRNA was compared with cells transfected with non-targeting control siRNA. The results showed that PAIP1 knockdown inhibited cell viability, and extensively affects expression of 893 genes at transcriptional level in HepG2 cells. Gene function analysis showed that a large number of PAIP1 up-regulated genes were enriched in term of DNA-dependent transcription and the down-regulated genes were enriched in some pathways including immune response and inflammatory response. qPCR confirmed that PAIP1 knockdown positively regulated the expression of selected immune and inflammatory factor genes in HepG2 cells. Expression analysis of TCGA revealed that PAIP1 had positive correlations with two immune associated genes IL1R2 and PTAFR in liver tumor tissue. Taken together, our results demonstrated that PAIP1 was not only a translation regulator, but also a transcription regulator in liver cancer. Moreover, PAIP1 could function as a regulatory factor of immune and inflammatory genes in liver cancer. Thus, our study provides important cues for further study on the regulatory mechanism of PAIP1 in liver cancer. Liver cancer is one of the most common cancers, 75%–85% of which is hepatocellular carcinoma (HCC) . HepatitRNA binding proteins (RBPs) are proteins that function by binding to RNA, which could regulate transcription and post-transcriptional alternative splicing, modification, transport, translation and degradation metabolism of RNA . Abnormaβ signaling pathway (Poly(A) binding protein interacting protein 1 (PAIP1) is a translation regulator that interacts with poly(A) tail binding protein (PABP) and the translation initiation factor eIF4G, which facilitate translation initiation process  and can  pathway . PAIP1 k pathway . PAIP1 p pathway . Results pathway . HoweverHere, to further explore the roles and regulated targets of PAIP1, we knocked down PAIP1 in HepG2 cells that was isolated from liver cancer tissue. Then, comprehensive gene expression profiles of PAIP1-knockdown cells and controls were detected by high-throughput RNA sequencing (RNA-Seq) to identify genome-wide targets regulated by PAIP1. The results showed that PAIP1 knockdown could extensively affect the expression of a large number of genes. In particular, PAIP1 knockdown inhibited expression of immune genes at mRNA level in HepG2 cells. Moreover, there were significant correlation between expression of PAIP1 and PAIP1-regulated immune genes in liver cancer tissues. These results indicated that PAIP1 cloud function as a regulatory factor of transcription, which affect expression of immune and inflammatory genes in liver cancer. Our study provides an important basis and data platform to further clarify the role of PAIP1 in mediating progress of liver cancer.The human liver cancer cell line HepG2 and Huh7 was purchased from Procell Life Science & Technology Co., Ltd. . HepG2 cells line were cultured at 37 °C with 5% CO2 in MEM with 10% fetal bovine serum (FBS), 100 µg/mL streptomycin, 100 U/mL penicillin.To knockdown PAIP1, three different siRNA duplexes and non-targeting control siRNA were purchased from Gemma . The sequence of these siRNA were present in −ΔΔCT.The total RNA of HepG2 cells transfected with different vectors was isolated using TRIzol reagent . Then, cDNA was synthesized. qPCR was performed using Bestar SYBR Green RT-PCR Master Mix  on the Bio-Rad S1000. GAPDH  was used as a reference gene for assessing the efficiency of PAIP1 knockdown. The primers used for qPCR are listed in HepG2 cells were lysed in ice-cold wash buffer  supplemented with a protease inhibitor cocktail  and incubate on ice for 30 min. Samples were boiled for 10 min in boiling water with 1X SDS sample buffer and separated on 10% SDS-PAGE. With TBST buffer  containing 5% non-fat milk power for 1 h at room temperature, membranes were incubated with primary antibody: PAIP1 antibody , GAPDH  and then with HRP-conjugated secondary antibody. Bound secondary antibody was detected using the enhanced chemiluminescence (ECL) reagent .A MTT assay was used to evaluate cell viability according to a previous study . CultureThe total RNA of HepG2 cells was extracted with TRIZOL  and purified with two phenol-chloroform. Then, the purified total RNA was treated with RQ1 DNase  to remove DNA. Further purified RNA was detected for the absorbance at 260 nm/280 nm (A260/A280) using Smartspec Plus  to assess the quality and quantity. Agarose gel electrophoresis (1.5%) was performed to assess the integrity of the purified RNA.For RNA-seq library preparation, 1 µg RNA was used for each sample. Oligo(dT)-conjugated magnetic beads  were used to purify and concentrate polyA mRNAs from the total RNA sample. The purified and concentrated RNA sample was fragmented. End-repair and 5′  adaptor ligation of fragmented RNA were performed. Then, the RNA were reversely transcribed using primers harboring 3′  adaptor sequence and randomized hexamer for cDNAs. The cDNAs were further amplified and purified. The purified cDNAs were stored at −80 °C until sequencing.According to the manufacturer’s instructions, high-throughput sequencing libraries were prepared. Pair-end Sequencing (151-bp) were conducted by Illumina HiSeq4000 system in a commercial company .First, raw sequencing reads with more than 2-N bases were discarded. Then, FASTX-Toolkit (Version 0.0.13) was used to trim the adaptors and low-quality bases from raw reads. The resulting reads less than 16nt were further discarded. Tophat2 were used to align the clean reads to the GRch38 genome allowing 4 mismatches . Then, uFPKM was used to evaluate the expression level of genes. edgeR software was used to identify the differentially expressed genes (DEGs)  with a tp-value < 0.05 or FDR < 0.05).Overrepresentation analysis (ORA) were performed for the upregulated or downregulated DEGs to detect gene set enrichment by Gene Ontology (GO) and reactome pathway analysis was used to predict the gene function and calculate the functional category distribution frequency . ReactomQuantitative real-time PCR (qPCR) of selected DEGs was conducted to elucidate the validity of the RNA-seq data. GAPDH was used as a reference gene. Primers for qPCR was presented in https://www.cancer.gov/). Expression of PAIP1 and PAIP1-reguated DEGs in liver cancer sample were analyzed.The RNA-seq data of liver cancer were downloaded from TCGA database . Pearson’s correlation test was used to assess the relationship between expression of PAIP1 and PAIP1-regulated DEGs in liver cancer tissues from TCGA at P values <0.05.Statistical analysis was performed using R (v3.1.3). qPCR data of the two groups were compared by an unpaired two-tailed To verify whether PAIP1 was overexpressed in liver cancer tissues, we detected the expression of PAIP1 in liver cancer. We analyzed the expression of PAIP1 in liver cancer sample of the TCGA and the Human Protein Atlas database, the results showed that the expression of PAIP1 was obviously up-regulated in liver cancer tissues compared to normal tissues at both transcript and protein level  and 1B. To explore the regulated targets of PAIP1 in liver cancer, the expression of PAIP1 was knocked-down in HepG2 cells by transfecting vector with three different PAIP1 siRNAs  . CompareThen, RNA-seq was used to detect the gene expression profiles of PAIP1 knockdown with siRNA3 and control HepG2 cells. Both siPAIP1 and control HepG2 cells had three biological replicates. There were six RNA-seq samples . After cleaning raw sequence data, at least 38.8 million pair-end reads were obtained for each sample. After mapping those clean reads to the human genome, more than 66.8 million uniquely mapped reads of each sample were used for gene expression further gene expression analysis .FPKM were calculated to represent the expression level of each gene. The results showed that there were unique 30,705 genes with FPKM >0 and unique 12,862 genes with FPKM >1 in at least one sample  and S5. Then, DEGs between the siPAIP1 and control cells were identified by the edgeR to further compare the gene expression profile. A total of 633 upregulated and 260 downregulated DEGs were identified between siPAIP1 and control cells . All theIn addition, the expression of PAIP1 was knocked-down in another liver cancer cell Huh7 by transfecting vector with same three different PAIP1 siRNAs  . CompareTo reveal the potential roles of DEGs, GO analysis was performed to annotate 633 upregulated DEGs. GO analysis was divided into three categories: biological process (GO-P), molecular function (GO-F) and cellular component (GO-C). The results revealed 170 upregulated genes annotated with 45 GO-P, terms . The topTo further reveal the roles of downregulated DEGs, GO analysis was performed to annotate those DEGs. The results revealed 124 downregulated genes annotated with 33 GO-P terms . The topTo further explore the potential function of PAIP1-regulated genes, reactome pathway analysis was performed. The results showed that the upregulated DEGs were enriched in terms such as HDACs deacetylate histones, DNA methylation, and PRC2 methylates histones and DNA . The dowTherefore, we selected some genes related to immune response and inflammatory response in DEGs and did hierarchical clustering of standardized FPKM values in HepG2 cells. The results indicated that there was a significant separation between the PAIP1-knockdown sample and the control sample, and the three replicate data sets were highly consistent . To veriP <  0.05) with that of APOC3, CCL14, IL1RN, SERPINA3, and HAMP in liver cancer tissues (P < 0.05) between PAIP1 expression and PTAFR and IL1R2  were significantly positively correlated with PAIP1 expression in liver tumor samples from TCGA, which is same to regulation direction of PAIP1 on these genes in HepG2 cells. We speculated that tumor tissues were highly cellular heterogeneous, and a gene may had different expression level in tumor cell from tumor microenvironment (TME) cells. In fact, single-cell RNA sequencing of liver tumor tissue has reveal the heterogeneity of malignant cells and immune cells in hepatocellular carcinoma . At leasIn conclusion, this study demonstrated that PAIP1 was high expressed in liver cancer tissue, and PAIP1 knockdown inhibit cell viability and extensively regulate the expression of a number of genes in HepG2 cells. This result indicated that PAIP1 was not only a translation regulator, but also a transcription regulator in liver cancer. In particular, we first revealed that PAIP1 promote expression of many genes associated with immune and inflammatory response in HepG2 cells. Thus, PAIP1 may play a role as immune regulator in liver cancer. Our study provides important cues for further study on the regulatory mechanism of PAIP1 in liver cancer.10.7717/peerj.15070/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.15070/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.15070/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.15070/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.15070/supp-5Supplemental Information 5Click here for additional data file.10.7717/peerj.15070/supp-6Supplemental Information 6Click here for additional data file.10.7717/peerj.15070/supp-7Supplemental Information 7Click here for additional data file.10.7717/peerj.15070/supp-8Supplemental Information 8Click here for additional data file.10.7717/peerj.15070/supp-9Supplemental Information 9Click here for additional data file.10.7717/peerj.15070/supp-10Supplemental Information 10Click here for additional data file.10.7717/peerj.15070/supp-11Supplemental Information 11Click here for additional data file.10.7717/peerj.15070/supp-12Supplemental Information 12Click here for additional data file.


TAR DNA binding protein 43 kDa (TDP-43) plays an important role in several essential cell functions. However, TDP-43 dysfunction has been implicated in the development of various brain diseases including amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), and limbic predominant age-related TDP-43 encephalopathy (LATE). Recent investigations into the individual components of TDP-43 pathology show how broader TDP-43 dysfunction may precede these disease end states, and therefore could help to explain why TDP-43 dysfunction continues to be implicated in a rapidly expanding category of neurodegenerative diseases. The literature reviewed in this article suggests that dysregulation of TDP-43 initiated by some environmental and/or genetic insults can lead to a snowballing dysfunction across the cell, involving impaired gene expression, mRNA stability, as well as the function and coordination of those pathways directly regulated by TDP-43. Furthermore, the hallmarks of TDP-43 pathology, such as hyperphosphorylation and insoluble cytoplasmic accumulation of the protein may actually be artifacts of an upstream impairment in TDP-43’s normal function. Overall, the present article summarizes current knowledge regarding TDP-43’s normal and pathological cell functions and sheds light on possible mechanisms that underlie its causal role in neurodegeneration. TAR DNA binding protein 43 kDa (TDP-43) is a highly conserved ubiquitously expressed nuclear protein that plays an important role in several essential cell functions, including transcriptional repression, RNA splicing, mRNA transport, microRNA maturation, translational regulation, and the formation of stress granules . Under nOn the other hand, pathological TDP-43 has been implicated in a wide range of neurodegenerative conditions. For example, hyperphosphorylated and ubiquitinated TDP-43 inclusions in the cytoplasm of cells in the nervous system represent a major pathological feature of amyotrophic lateral sclerosis (ALS), and frontotemporal lobar degeneration (FTLD) . TDP-43 TDP-43 pathology is usually characterized by insoluble, hyperphosphorylated and ubiquinated aggregates of TDP-43 in the cytoplasm, nucleus and cell processes of neurons and glia . MislocaIn the pathological condition, post-translational modifications are also typically made to the TDP-43 protein, especially hyperphosphorylation and/or fragmentation . AlthougWhile hyperphosphorylated TDP-43 is associated with a variety of neurodegenerative diseases, regulated phosphorylation is necessary for TDP-43’s normal function . There aCaenorhabditis elegans ALS models and human cell cultures could reduce TDP-43 phosphorylation, improve behavioral phenotypes, and reduce neurotoxicity. The authors also found that C. elegans with the phosphatase calcinuerin knocked out, had significantly worsened motor control phenotypes, along with phosphorylated TDP-43 aggregates, and neurodegeneration  mRNAs, and a 30% decrease in ATG4B protein levels; changes that are consistent with a loss of TDP-43 function. Due to TDP-43’s direct binding and splicing of ATG4B mRNA, TDP-43 is considered necessary for autophagy  and autophagy . AdditioOther studies have identified potential disease mechanisms in TDP-43 pathology by investigating the direct transcription regulation and mRNA splicing targets of TDP-43, and how its dysfunction affects systems in the cell downstream. For example, The literature investigating TDP-43’s phosphorylation, prion like seeding activity, over expression, aberrant splicing function, and role in processes like autophagy, axonal regrowth, and neurite branching paints a complex picture of TDP-43 pathogenesis. Interference at each of these components has been shown to ameliorate the accumulation of TDP-43, prevent neurodegeneration, and improve behavioral symptoms in some models. However, these studies suggest that dysfunction in TDP-43 could present itself in many ways outside of the traditional characterization of TDP-43 pathology; it is likely a mosaic of events with several discreet yet interrelated steps. The dysregulation and dysfunction of TDP-43 seems to feedback on itself and further exacerbate its own pathogenesis. Consequently, development of effective therapies for TDP-43-associated diseases may need to focus on interfering with the mechanisms that initiate this dysregulation. Eliminating only one element, for example hyperphosphorylation, may be insufficient or even harmful to disease outcomes, and could fail to overcome the adverse involvement of TDP-43 pathology in full-blown neurodegenerative diseases. These studies, taken together, suggest that the hallmarks we consider to be characteristic or even causative of TDP-43 pathology may only be the end state of a complex cascade of events with likely both environmental and genetic components. Further investigations into the factors that trigger TDP-43 dysregulation and subsequent dysfunction may elucidate more effective therapeutic targets, and enrich our understanding of the mechanisms of pathogenesis in the expanding category of diseases associated with TDP-43.BIN drafted the current manuscript, with writing and editing contributions from HFU. All authors contributed to the article and approved the submitted version.Funding for this article was provided by the National Institutes of Health through grants AG062220, AG066518, and OD011092.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The development of cryogenic semiconductor electronics and superconducting quantum computing requires composite materials that can provide both thermal conduction and thermal insulation. We demonstrated that at cryogenic temperatures, the thermal conductivity of graphene composites can be both higher and lower than that of the reference pristine epoxy, depending on the graphene filler loading and temperature. There exists a well-defined cross-over temperature—above it, the thermal conductivity of composites increases with the addition of graphene; below it, the thermal conductivity decreases with the addition of graphene. The counter-intuitive trend was explained by the specificity of heat conduction at low temperatures: graphene fillers can serve as, both, the scattering centers for phonons in the matrix material and as the conduits of heat. We offer a physical model that explains the experimental trends by the increasing effect of the thermal boundary resistance at cryogenic temperatures and the anomalous thermal percolation threshold, which becomes temperature dependent. The obtained results suggest the possibility of using graphene composites for, both, removing the heat and thermally insulating components at cryogenic temperatures—a capability important for quantum computing and cryogenically cooled conventional electronics. Graphene composites can serve both as efficient thermal insulators at low temperatures and thermal conductors at high temperatures. Here, the authors report the evolution of thermal conductivity of composites with graphene fillers from cryogenic to room temperature. The main progress with quantum computing technologies is associated with superconducting qubits, which require cryogenic temperatures3. Space exploration needs electronics operating in harsh low-temperature environments. Cryogenic thermal management usually relies on both thermal conductors and thermal insulators6. The former is the polymer-based thermal interface materials (TIMs) with fillers that conduct heat well, facilitating heat removal, and the latter are polymer materials, which have low thermal conductivity and can act as thermal insulators between electronic components operating at different temperatures. Examples of low-temperature thermal management applications include protective coatings of superconductive power cables7, adhesives in cryogenic low-noise amplifiers for radio-astronomy and space communication systems8, optical mounts of cryogenic refractive optics, and cryosorption pumps10.There is a rapidly emerging need for thermal management at cryogenic temperatures. It is driven by several trends and developments. There is a strong motivation to run conventional semiconductor electronics at low temperatures to implement “cold computing,” which allows one to increase computational and energy efficiency while reducing power consumption12. Polymers are used in TIMs as the base, i.e., matrix material, that fills the air gaps between two adjacent solid interfaces and provides adhesive functionality when used in curing composites. The common strategy to increase the thermal conductivity of polymers is to add micrometer- and nanometer-scale fillers with a higher intrinsic thermal conductivity that can couple well with the base polymer. A mixture of the single-layer and few-layer graphene (FLG) flakes, termed “graphene” in the thermal context, has proven to be an efficient filler material for a variety of TIMs, including non-curing mineral oil-based thermal pastes13, and curing epoxies15. Graphene for thermal management applications can be mass-produced via liquid-phase exfoliation, graphene oxide reduction, or other techniques17. Graphene TIMs with thermal conductivity above ~15. The excellent performance of graphene TIMs near RT originates in the extraordinarily high intrinsic thermal conductivity of graphene and few-layer graphene19, strong coupling to the matrix, good dispersion, and appropriate viscosity range of the resulting composites20. The enhancement of thermal conductivity is achieved both below and above the thermal percolation threshold—a loading fraction at which the graphene fillers start to form continuous thermally conductive paths22. The thermal percolation threshold can be identified when the dependence of the thermal conductivity on filler loading becomes super-linear23. One should note that the thermal properties of graphene composites have only been studied at RT and above—the temperature range of interest for conventional electronics. We are not aware of any report on the cryogenic thermal characteristics of graphene composites. In general, the data for the thermal properties of any polymer composites at cryogenic temperatures are scarce. The understanding of heat propagation in amorphous polymers at low temperatures is far from complete, even if one does not consider the solid inclusions, i.e., fillers25.Polymers are poor heat conductors with thermal conductivity in the range from ~0.2 to conceptual change for thermal management, which typically rely on different materials for heat conduction and isolation.Here, we investigated the thermal properties of epoxy–graphene composites at temperatures from 2 K to RT. Epoxy is a practically important material and is often used as a reference material to compare the effect of different types of fillers on its thermal conductivity. We found that at cryogenic temperatures, the thermal conductivity of graphene composites can be both higher and lower than that of the reference pristine epoxy, depending on the graphene filler loading and temperature. This is drastically different from what is observed near RT. Moreover, there exists a well-defined cross-over temperature that above it, the thermal conductivity increases with the addition of graphene, whereas, below it, the thermal conductivity decreases with the addition of graphene. Graphene composites are unique in a way that they can provide both the strongest enhancement in thermal conductivity and the strongest suppression. We offer a physical model explanation of the counter-intuitive trends and provide numerical simulation data which agree with the measurements. The obtained results suggest the possibility of using composites with the same constituent materials for, both, removing the heat and thermally insulating electronic components at cryogenic temperatures. The latter constitutes a 26. To maximize the thermal conductivity of the composite, one normally wants to keep the lateral dimensions above the gray phonon mean free path (MFP) in graphene, which is ~1 μm near RT27. In contrast, to improve the insulating properties of composites, fillers with smaller lateral dimensions are preferred28.The polymer matrix is a thermoset epoxy set consisting of a base resin  and a hardening agent . We used few-layer graphene with the specified average lateral dimension of 25 µm, an average thickness of 15 nm, and an average surface area of 50 to 29. The percolation results in significant enhancements in both the electrical and thermal characteristics of the composites29.Several composite samples were prepared by mixing precalculated quantities of the resin, the hardener, and the FLG fillers to hit a targeted filler loading level. First, a certain amount of FLG fillers was distributed into the epoxy resin using a high-speed shear mixer . The hardener was then added to the mixture in the mass ratio of 12:100 with respect to the base epoxy resin’s weight. The blend was vacuumed several times to eliminate any air bubbles that might have been trapped during the composite preparation process. The mixture was poured into special molds to cure and solidify  was carried out under laser excitation with the wavelength of λ = 633 nm in the conventional backscattering configuration. In all experiments, the laser power was kept at ~3 mW. Raman spectra of several samples at random spots are presented in Fig. −1 and ~2700 cm−133, confirming an even distribution of graphene throughout the samples. The intensity of the 2D peak is much lower than the G-peak indicating the presence of a mixture of single-layer and few-layer graphene in the composite samples. The Brillouin spectroscopy allows probing low-energy acoustic phonons near the Brillouin-zone (BZ) center with energies in the range of 2 GHz to 900 GHz36. In bulk polymers, the Brillouin spectrum is dominated by the inelastic scattering of light from bulk phonons, i.e., elastic vibrations, through the opto-elastic effect37. In the conventional backscattering geometry, the phonon wavevector is 40. In our experiments, we used an excitation laser with 38. These observations provide additional support to the conclusion of well-dispersed and randomly oriented fillers in the composites. The data for the composites show more scatter owing to increased light absorption and decreased intensity of the scattered light. Similar trends in Brillouin signatures for the acoustic phonons in the pristine epoxy and epoxy with graphene indicate that the addition of the fillers has not negatively affected the mechanical properties of the material.The composites were further characterized using Raman and Brillouin—Mandelstam light scattering spectroscopy, also referred to as Brillouin light scattering spectroscopy41. The details of the error analyses are described in the Supplementary Information. The results for the neat epoxy agree well with the literature42. The variation of the specific heat with temperature resembles that in other amorphous polymers44. In all samples, We start with the investigation of the specific heat and illustrate its typical temperature trends in disordered amorphous materials. Figure ies Fig. . One can46. Below 46. This behavior is explained by the tunneling model of two-level systems for amorphous materials at low temperatures47. This anomalous quasi-linear trend is followed by a hump in the 49. The “boson peak” shown in Fig. 49. The temperature at which the boson peak occurs decreases slightly with increasing the filler loading. The “boson peak” eventually fades away with increasing the filler content as the material acquires more “crystalline” characteristics owing to the graphene content. The overall value of One can notice from Fig. T~80 K at which point a second plateau extending up to T~175 K occurs. The existence of the first plateau in T dependence is universal for amorphous materials, and it occurs almost in the same temperature range. It starts at the temperature where the “boson peak” appears in the specific heat , Tc = 11 K, and RT. The systematic errors for the data points at low temperatures were smaller than the size of the symbols and not shown for clarity. At T = 2 K, the thermal conductivity of the epoxy falls approximately two times with the addition of only 2.6 vol% of graphene fillers. The sharp decrease in thermal conductivity suggests that FLG fillers contribute significantly to the phonon scattering processes. At this temperature, as the concentration of the filler increases, the thermal conductivity declines linearly. At RT, however, the trend is the opposite. The addition of graphene fillers improves the thermal conductivity monotonically in line with many prior reports23. The variation of Figure 64. The ratio of the thermal conductivity of the fillers to the matrix, 21. The wavelength of the thermal phonons, which make the dominant contribution to heat conduction, is 45. For a typical semiconductor material 65. This means that for the thermal percolation to occur at RT, the fillers should be in physical contact or close to each other, i.e., on the order of ~1 nm. At low temperatures, 66. If we accept this picture of the process, then the dependence of the thermal conductivity at 2 K and 300 K are those below and above thermal percolation, correspondingly  with a molecular weight of 700 , the hardener, triethylenetetramine , and few-layer graphene (FLG) fillers  to hit a targeted filler loading level. The average lateral dimension and surface area of the FLG fillers were 25 Raman experiments were conducted in the conventional backscattering configuration using a red laser with an excitation wavelength of 633 nm. All experiments were conducted at room temperature. The Brillouin–Mandelstam light scattering experiments were conducted in the backscattering configuration using a continuous-wave solid-state diode-pumped laser with an excitation wavelength of 532 nm. Samples were launched in a specially designed helium-cooled stage. The temperature of the sample can be varied from 4 K up to 300 K. The laser light was focused using a ×10 objective with NA = 0.28. The laser power on the sample was kept low to avoid any possible laser-induced heating effects. This is important, especially for experiments at cryogenic temperatures. The scattered light was collected by the same objective and directed to the high-resolution 3 + 3 tandem Fabry-Perot interferometer , detector, and spectrum analyzer.3 dimensions. The measurements were conducted using the steady-state 4-probe continuous mode. The heating rate was adjusted at 0.3 K min−1. The details of the heat capacity and thermal conductivity measurements as well as comprehensive error and uncertainty analyses are described in the Supplementary Information.The heat capacity and thermal conductivity of the epoxy and composite samples were measured in the temperature range of 2 K to 300 K using a Quantum Design Physical Property Measurement System (PPMS). For thermal conductivity measurements, samples were cut into rectangular bars with 1 × 1 × 10 mmSupplementary InformationPeer Review File


Amyotrophic lateral sclerosis (ALS), also known as motor neuron disease (MND), is a progressive neurological disorder, characterised by the death of upper and lower motor neurons. The aetiology of ALS remains unknown, and treatment options are limited. Endogenous retroviruses (ERVs), specifically human endogenous retrovirus type K (HERV-K), have been proposed to be involved in the propagation of neurodegeneration in ALS. ERVs are genomic remnants of ancient viral infection events, with most being inactive and not retaining the capacity to encode a fully infectious virus. However, some ERVs retain the ability to be activated and transcribed, and ERV transcripts have been found to be elevated within the brain tissue of MND patients. A hallmark of ALS pathology is altered localisation of the transactive response (TAR) DNA binding protein 43 kDa (TDP-43), which is normally found within the nucleus of neuronal and glial cells and is involved in RNA regulation. In ALS, TDP-43 aggregates within the cytoplasm and facilitates neurodegeneration. The involvement of ERVs in ALS pathology is thought to occur through TDP-43 and neuroinflammatory mediators. In this review, the proposed involvement of TDP-43, HERV-K and immune regulators on the onset and progression of ALS will be discussed. Furthermore, the evidence supporting a therapy based on targeting ERVs in ALS will be reviewed. Amyotrophic lateral sclerosis (ALS), also known as motor neuron disease (MND), is a progressive and fatal neurological disorder, characterised by the death of both upper and lower motor neurons in the primary motor cortex and spinal cord , 2. DeatTARDBP, 95% of ALS cases have a cellular TDP-43 pathology. Cytoplasmic TDP-43 aggregation is present in both sporadic and familial ALS with the exception of those with familial ALS caused by SOD1 mutations [A hallmark of ALS pathology is cytoplasmic TDP-43 aggregation. While only 4% of familial ALS cases can be attributed to mutations in utations . In healutations , 11. Cytutations , 13.Both central and peripheral inflammation have been well established in ALS, with this pathology encompassing increased levels of inflammatory cytokines including tumour necrosis factor α (TNF-α) and interleukins  the involvement of non-neuronal cells including activation of microglia and astrocyte dysfunction, and T lymphocyte infiltration into the central nervous system (CNS) –17.Expression of endogenous retroviruses (ERVs) has also been implicated in neurodegenerative mechanisms in ALS . ERVs arC9orf72; 40%) [SOD1; 12%) [TDP-43; 4%) [FUS; 4%) [TBK1), NIMA-related kinase 1 (NEK1) and additional yet-to-be identified genes [Inherited genetic causes, known as familial ALS, account for 10% of cases, while the remaining 90% are regarded as sporadic . Interes72; 40%) , 28, misD1; 12%) , point mFUS; 4%) , 31. Theed genes , 32.SOD1 and TARDBP [The identification of genes involved in ALS pathology has helped elucidate some of the mechanisms underlying neurodegeneration including protein accumulation from d TARDBP , 33, 34.d TARDBP , impaired TARDBP , axonal d TARDBP , mitochod TARDBP , neuroind TARDBP  and TDP-d TARDBP .ACRV with the promotor region containing TDP-43 binding sites [. Lalmansingh et al. [ACRV, localising the repressor activity to the RRM1 region of TDP-43. Mutations in TDP-43 causing dysfunctional RRM1 mitigated the repressor activity of TDP-43. In addition, TDP-43 was also found to be involved in the alternative splicing of human cystic fibrosis transmembrane conductance regulator (CFTR) exon 9 [SMN2), a gene associated with sporadic ALS [TDP-43 contains a nuclear localisation sequence, a nuclear export signal, two highly conserved RNA recognition motifs (RRM1 and RRM2) and a glycine-rich C-terminal domain . The nucng sites . Lalmansh et al.  experime) exon 9  and humaadic ALS . Furtheradic ALS –54.In 2006, the initial link between TDP-43 and familial ALS was identified through the presence of ubiquitinated and hyper-phosphorylated TDP-43 inclusions in histological sections of the cortex and spinal cord, which are now considered a hallmark pathology of ALS , 55. SinAnother potential mechanism contributing to the formation of TDP-43 protein aggregates is via the disruption to the autoregulatory activity of TDP-43. Under normal conditions, TDP-43 self-regulates its own expression through binding to 3′UTR sequences in its own mRNA, promoting degradation to decrease TDP-43 levels . HoweverOne hypothesis for TDP-43 linked neurodegeneration is a loss-of-function of nuclear TDP-43. DNA damage and an impaired DNA repair system have been proposed as a cause of neurodegeneration from nuclear loss of TDP-43 , 63. In In addition to loss-of-function TDP-43-associated neurodegeneration, a gain of toxicity from the cytoplasmic TDP-43 inclusions may also induce neurodegeneration. Barmada et al.  used ratAs mentioned above, a gain-of-function toxicity from cytoplasmic TDP-43 can also influence mitochondrial function. Using transgenic mice expressing wild-type hTDP-43 under a mouse prion promoter, Xu et al.  identifiIn conclusion, the combined effects of both loss of nuclear TDP-43 and the gain of toxic cytoplasmic TDP-43 aggregates should not be ruled out as the cause of TDP-43-related neurodegeneration. Knock-down of endogenous TDP-43 by siRNA in the murine spinal cord-x neuroblastoma hybrid cell line (NSC-34) was used to measure cell viability in the absence of cytoplasmic TDP-43 aggregates. Neuronal toxicity was indicated by both a significant reduction in cell viability and an increase of caspase-3 activity was found, suggestive that loss-of-function toxicity can occur without the need for TDP-43 aggregation. However, a similar result was found when TDP-43 inclusion bodies were intracellularly delivered via a plasmid expressing human TDP-43 into the NSC-34 cell line to mimic cytoplasmic aggregation. The relative contributions of loss-of-function and gain-of-function toxicity were calculated and determined to equally contribute to neuronal toxicity .It has been proposed that another mechanism of TDP-43-associated neurodegeneration involves the expression of ERVs, which were previously thought to remain dormant within the genome. This mechanism is discussed further below.SOD1, C9orf72 and TARDBP also have dysregulated inflammatory processes, as seen in human ALS [C9orf72 mutation had increased inflammatory cytokine expression within plasma and reduced survival rates compared to wild-type controls [Q331K mouse model was used to investigate the inflammatory processes with the transgenic TDP-43 mice showing increased microglial activation that correlated with motor deficits and subsequent increased progression of neurodegeneration compared to WT mice [SOD1G93A mice and increase the expression of interferon-γ (IFN-γ) [OPTN, SQSTM1, VCP and TBK1 are also associated with inflammation [The impact of inflammation has been well established in ALS with dysregulation of inflammatory cytokines in ALS patients, involvement of astrocytes and microglia, and T lymphocyte infiltration into the CNS linked to ALS disease progression . Animal uman ALS , 75, 76.controls . Further WT mice . Modulat (IFN-γ) . Removalammation , 80. In ammation –83. Otheammation .C9orf72−/− mice, there is an upregulation of type 1 IFNs resulting in systemic CNS inflammation due to increased cGAS-STING pathway signalling [−/− in a neurodegenerative disease model, Nazmi et al. [The cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS) and stimulator of interferon genes (STING) pathway (cGAS/STING) pathway has been implicated in neuroinflammation-mediated neurodegeneration , 87. cGAgnalling . Throughi et al.  proposedi et al. . In conti et al.  in ALS pi et al. , providiTo further outline the role of the immune response in neurodegeneration, TBK1 mutations have also been linked to ALS in a small number of familial ALS cases , 97. TBKNeuroinflammation in ALS includes the activation of microglia and the polarisation of microglia into two different phenotypes, either pro-inflammatory, M1, or anti-inflammatory, M2 . In earlG93A mice was shown to reduce the neuroprotective state of microglia and resulted in faster disease progression [Astrocyte-mediated neurotoxicity has been proposed to be caused by protein aggregation such as mutant SOD1 and TDP-43 , 111. Fugression . Moreovegression . In the gression , 119.In conclusion, while the exact mechanism of neuroinflammation-mediated neurodegeneration remains unknown, it is proposed to occur through a perpetual cycle of motor neuron death and sustained microglia and astrocyte activation with neurotoxic pro-inflammatory cytokine increases Fig. . Cell-toRetroviruses are enveloped, positive-sense single stranded RNA viruses . Retrovigag, pol and env coding regions, flanked by long terminal repeats (LTRs). Gag (group-specific antigen) encodes structural proteins including the capsid, matrix and nucleocapsid; pol encodes the enzymatic functions of the virus, viral protease, RT and integrase; and the env encodes the surface and transmembrane glycoproteins, gp120 and gp140 [tat within the HIV genome, encoding a transcriptional activator. Each LTR consists of a unique 3′ region (U3), a repeat (R) and unique 5′ region (U5). The U3 region of the LTRs serves as the viral promoter regions controlling gene expression. The R region contains the trans-activation response element (TAR), which interacts with viral tat protein during transcription and recruits cellular factors to enhance viral gene transcription [The proviral DNA genome consists of nd gp140 . Complexcription .ERVs are a type of transposable element which is a type of mobile genetic element that can move to other locations in the genome. Transposable elements are classified as DNA transposons or RNA transposons. Based on the presence of LTRs, retrotransposons are further classified into non-LTRs including short interspersed nuclear elements (SINE) and long interspersed nuclear elements (LINE) or ERVs with LTRs. LTR retrotransposons can be transcribed from the host cell genome into ERV RNA, then, in the same cell, reverse transcribed back into double-stranded DNA and re-integrated into another site of the host genome , 132. ThWhile exogenous retroviruses are capable of horizontal transmission from person to person and produce infectious virions, ERVs, in contrast, are traditionally not thought to produce infectious virions and are not horizontally transmitted. Instead, ERV’s have integrated into the host genome and are vertically transmitted through the germ lines , 135. ThMost human endogenous retroviruses (HERVs) are not transcribed and contain deletions and mutations resulting in a lack of functional protein production and lack the components required for a functional virus . RecentlThus, while some HERVs have physiological importance, dysfunctional expression of these HERVs can have detrimental effects on the host. Other HERVs have been correlated with a variety of diseases: HERV-K, HERV-E, and HERV-W are associated with cancers such as ovarian cancer and breast cancer –158, HERThe evolutionarily youngest ERV to enter the human genome is HERV type K (HERV-K), which is predicted to have endogenized into the human genome approximately 700,000 years ago . HERV-K env and encode accessory protein, Np9 while type II proviruses encode accessory protein, Rec [. Rec is similar to HIV-1 Rev accessory gene, a protein that is involved in RNA splicing. While the biological role of these proteins is still unclear, mRNA transcripts for Rec and Np9 from multiple HERV-K loci have been found in many human tissue types [Two LTR regions are also present on a portion of other HERV families , 166. HEein, Rec . Rec is ue types .Many of the HERVs that exist in the human genome are in the form of solitary LTRs , 20. HowThe link between ALS and retrovirus activity was first identified in 1975, through discovery of RT activity in brain tissue of two ALS patients . FurtherExperimental studies further supported the proposed association between ALS and ERVs. Hadlock et al.  evaluateRecently, an antibody response to specific epitopes of HERV-K (HML-2) env has also been demonstrated with a greater antibody response in ALS patients compared to age- and sex-matched controls .gag, pol and env compared to control brain tissue [env gene in human neuronal cultures, derived from iPSCs, has demonstrated the toxicity env, with reduced viable neuronal cell number after env transfection [Furthermore, HERV-K pol transcripts, measured through quantitative real-time PCR, in the brain tissue from the prefrontal cortex, sensory cortex and occipital cortex of 28 ALS patients were compared to levels in brain tissue from people who succumbed to other diseases. These HERV-K pol transcripts were found to be significantly higher in ALS patients than age-matched controls . Post-mon tissue . Transfesfection . Similarsfection  found anThe association between HERV-K and ALS pathology is proposed to occur through an interaction with TDP-43. Importantly, chromatin immunoprecipitation identified 5 binding sites for TDP-43 on the consensus sequence of HERV-K LTR . This suenv throughout the genome resulted in a decrease in TDP-43 mRNA and protein levels in human prostate adenocarcinoma cells. While previous findings have identified TDP-43-dependent increases in HERV-K expression [HERV-K has also been shown to influence TDP-43 expression and aggregation , 184. Ibpression , the abopression , 186. Chpression  establisgag and pol determined by q-RT-PCR and increased RT activity in an astrocytic cell line [As outlined above, there is increased activity of transcription factors that drive inflammatory mediator production including IRF-1, IRF-3 and NF-κB in ALS , 188. Thell line . Additioell line , 192. Geell line .In support of the relationship between inflammation and ERVs, NF-κB is also thought to induce HERV-K expression. Manghera et al.  demonstr50 and IC90 concentrations of the drug. Triumeq is an example of combination ART that is widely used for treatment of HIV which consists of two NRTIs, abacavir and lamivudine and an integrase inhibitor, dolutegravir, all of which are capable of penetrating the CNS [As described, HERV-K has been implicated in the causation and perpetuation of the signals that drive neurodegeneration in ALS. This and the early clinical anecdotal findings of improved ALS-like symptoms in HIV patients on ART led to the proposal that targeting ERVs could be used as a treatment for ALS, through ART designed to target HIV , 195. Tw the CNS –200.Theoretically, the two reverse transcriptase inhibitors within Triumeq, abacavir and lamivudine could inhibit the formation of HERV-K double-stranded DNA inside cells where HERV-K has been activated. cGAS/STING is a cell sensor that detects dsDNA in the cytoplasm as a danger signal, and an activation of the cGAS/STING pathway has been suggested to occur in ALS . Thus, NRecently, the de-repression of HERV-K was proposed to be involved in ageing, with increased HERV-K gag, pol and env transcript levels and protein levels in senescent human mesenchymal progenitor cells (hMPCs) compared to phenotypically young cells . The incECD, neurofilament-light and phosphorylated neurofilament heavy. Levels of serum HERV-K were also measured through droplet digital PCR.A phase IIa clinical trial for Triumeq as a treatment for ALS has recently been completed . This clThe results of the study showed patients on Triumeq treatment had a slower clinical decline as measured by the ALSFRS-R compared to pre-treatment. HERV-K DNA serum levels were significantly decreased over the treatment course , 205. ThPrevious studies in mice and drosophila have identified the regulation of ERVs from TDP-43 expression suggesting a similar mechanism of TDP-43 binding to ERVs as is seen with HERVs and TDP-43 , 208. FuThe causation and progression of ALS are elusive, and the current approved therapeutics for ALS have limited effect . The inv


Objective Scaffolds provided a surface on which cells could attach, proliferate, and differentiate. Nowadays, bone tissue engineering offers hope for treating bone cancer. Poly(e-caprolactone) (PCL)/graphene have capability as an osteogenic and regenerative therapy. It could be used to produce bone tissue engineering scaffolds. The purpose of this study was to investigate the ability of PCL/graphene to enhance the osteoinductive mechanism.Materials and Methods The PCL/graphene scaffold was developed utilizing a particulate-leaching process and cultured with osteoblast-like cells MG63 at 0.5, 1.5, and 2.5 wt% of graphene. We evaluated the porosity, pore size, migratory cells, and cell attachment of the scaffold.Statistical Analysis Data was expressed as the mean ± standard error of the mean and statistical analyses were performed using one-way analysis of variance and Tukey's post hoc at a level ofp-value < 0.05.Results Porosity of scaffold with various percentage of graphene was nonsignificant (p > 0.05). There were differences in the acceleration of cell migration following wound closure between groups at 24 hours (p < 0.01) and 48 hours (p < 0.00). Adding the graphene on the scaffolds enhanced migration of osteoblast cells culture and possibility to attach. Graphene on 2.5 wt% exhibited good characteristics over other concentrations.Conclusion This finding suggests that PCL/graphene composites may have potential applications in bone tissue engineering. Typically, bone reconstruction or regeneration requires the use of a biocompatibility scaffold with a porous structure. The scaffold should possess sufficient strength to support the injured bone in place. A scaffold is generally able to control the proliferation of cells that have migrated from surrounding tissue or been seeded inside the porous surface of the scaffold. Therefore, the scaffold's pore and pore interconnectivity contribute to cell adhesion and proliferation, as well as to the transport of nutrients and oxygen throughout the three-dimensional (3D) constructs.Previous studies have reported that scaffolds for bone repair must be biodegradable, biocompatible, porous, and have strong interconnectivity between pores for the cells to adhesion, proliferation, and differentiation.Poly (e – caprolactone) (PCL) is one of the materials meeting these characteristics. In recent years, PCL has been utilized as a biomedical scaffold due to its biodegradable and biocompatible properties. However, this material's poor mechanical qualities and pore size, limit its use in bone engineering.in vitroexperiments have demonstrated graphene's capacity to promote osteoblast proliferation and enhance their differentiation into mature osteoblasts. Thus, graphene is a promising material for enhancing the bioviability and bioactivity of synthetic scaffolds, especially in cooperation with polymer, such as PCL.Graphene, a new allotrope of carbon, is characterized as a two-dimensional honeycomb lattice composed of carbon atoms in monolayers. Graphene and its derivatives have garnered considerable attention in the field of materials research since graphene has no visible toxicity and exhibits high biocompatibility. Indeed,In vitrocell migration can be studied using the wound healing test (scratch assay), which is a straightforward approach.Not only are physical properties an important thing for a good scaffold, but the ability of cells to migrate is important too in a variety of physiological and pathological processes.The purpose of this study was to disclose the physicochemical behavior of PCL and graphene at varied concentrations  on osteoblast-like cells MG-63 in determining which concentrations to enhance the osteoinductive mechanism.n80,000) from Sigma-Aldrich  was used as the matrix material and natrium chloride (NaCl) as the porogen (Sigma-Aldrich). Graphene was produced by heating a graphite intercalation compound to 700°C in a common furnace, positioned in front of a fume closet to avoid inhalation of the nanoparticles, and leaving it there for 60 seconds. These layers grew by ultrasonication process, resulting in graphene dispersion in the solvent. For 12 hours at room temperature, PCL was dissolved in chloroform in a 1:10 w/v ratio. The PCL solution was then added to the NaCl and graphene solutions and stirred for 2 hours with a magnetic stirrer. The blended solution was poured into a mold and allowed to dry at room temperature for 1 day. Chloroform residues were eliminated during a 24-hour period in a vacuum oven set to 37°C. The PCL/graphene scaffolds were immersed in deionized (DI) water for 24 hours to eliminate the porogen, with the DI water being replaced every 2 hours throughout this time period. The PCL/graphene scaffold was then dried in a vacuum oven set to 50°C for 12 hours. Finally, a porous PCL/graphene-blended scaffold was developed.3format  was used to observe samples of 3D porous scaffolds. Gold was sputtered onto samples using a sputter coater in a vacuum chamber and then observed. Then, using Image J software, the SEM picture was analyzed to determine the pore size.4cells were seeded into each well and allowed to reach 90% confluency. Using a 200-μL tip, the cell monolayers were scratched and rinsed with phosphate-buffered saline (PBS) to remove detached cells and other debris. Three representative images from each of the scratched areas were photographed to estimate the relative migration of cells. The migration cell (scratch assay) was analyzed using inverted microscope , in 100× magnification, and has been processed using Gen 5.0 software. The distance between the two edges of the wound sites was detected at 24 and 48 hours and analyzed by Image J software. Wound closure was calculated using the equatioonThe osteoblast-like cells MG-63 were cultured in 12-well plates. Around 3 × 102)in a cell culture incubator, and the media was replenished every 2 to 3 days. Each sample used in the cell culture was 10 × 10 × 2 mm3in size. Sterilization of samples was accomplished by soaking them overnight in 95% ethanol and then washing them twice with PBS (Gibco) to eliminate residual ethanol. After that, samples were transferred to 24-well plates. Cells were detached using 0.25% trypsin-ethylenediaminetetraacetic acid (Gibco) and each sample was seeded with 0.5 mL of a cell suspension at a concentration of 104cells/mL in 24-well tissue culture plates. For 21 days, the 24-well plates were incubated in a cell culture incubator. Throughout this period, the medium was renewed every 2 to 3 days. On days 7 and 21, samples were withdrawn to observe the results of cell culture. A SEM was used to determine the cell morphology.Osteoblast-like cells MG-63 were seeded onto PCL/graphene scaffolds to examine cell adherence and growth characteristics at various graphene weight ratios. First, osteoblast-like cells MG-63 were grown in Dulbecco's Modified Eagle Medium (Gibco) with 10% fetal bovine serum  and 1% penicillin . Cells were cultured in T75 flasks . Significant differences between groups were determined at a level ofPhysical properties, like as porosity and pore size are critical in determining the scaffold's quality. In this study, the porosity of PCL incorporated with various concentration of graphene had been similar with each other. PCL has porosity 88% ±1.5). The porosities of the PCL/graphene scaffolds with 0.5, 1.5, and 2.5 wt% were 88 (±1.2), 87 (±0.9), and 89 (±2.1) respectively . This f. The porPore distribution was determined by a SEM . It shoTo evaluate the osteoblast-like cells MG63 migration response, the cells were exposed to different graphene concentrations  and allowed to migrate for 24 and 48 hours. Using a wound healing assay, we observed a graphene's concentration-dependent effect on osteoblast-like cells MG63 migration. The microscopic figure has to be analyzed to obtain information about the migration characteristics of the cultured cells. This could be accomplished manually by measuring the surface area and gap distance using image processing software such as Image J . The daThe condition of 100% cell confluence was utilized to get the signal that the scratch defect had been completely closed. When treated with 2.5 wt% graphene, osteoblast-like cells MG63 showed nearly full closure of the scratch site within 48 hours, compared with the other treatments. As a result, a statistically significant increase in responsiveness was detected at 2.5 wt% graphene concentrations when compared with the others concentration and control .Cells cultured on PCL and PCL/graphene scaffolds were obtained by SEM on days 7 and 14 . It demThe total porosity of the scaffolds was measured in this study using the liquid displacement method. The constructions were more than 85% porous in total. When compared with cancellous bone (which have an average porosity of 79.3%), the porosity of the scaffolds is optimal since it promotes bone ingrowth without reducing mechanical qualities such as strength.A variety of scaffolds made from different biomaterials and constructed utilizing a variety of fabrication techniques were used. Biocompatibility, biodegradability, mechanical qualities, scaffold architecture, and manufacturing technique are all key factors to consider when creating or establishing the appropriateness of a scaffold for use in tissue engineering. In a successful tissue engineering technique, these properties are deciding criteria when selecting a biomaterial.Pore size, along with porosity, is an essential physical property for cell adhesion, gas diffusion, and the delivery of nutrients and wastes. In general, it has been noted that while the scaffold's large pore size or porosity promotes effective nutrition supply, gas transport, and metabolic waste disposal, it results in low cell attachment and intracellular signaling. While a small pore size or porosity may have the opposite effect, the optimal pore size is in the diameter range of 100 to 500 μm, which is thought to stimulate osteogenesis and angiogenesis due to the size of osteoblasts, which is approximately 10 to 50 μm.Cell migration and proliferation are required for a range of physiological and pathological processes, including wound healing,Because of their osteoinductive properties and antibacterial activity, graphene-based materials (GMs) have a bright future in bone tissue engineering. The GMs trigger osteogenic differentiation via a variety of mechanisms and routes. To begin with, mechanical stimulation from the porous folds of graphene or graphene oxide can launch a cascade of processes that enhance osteogenic development without the need of chemical inducers. In addition, GMs regulate osteogenesis through the ECM, macrophage polarization modulation, the oncostatin M signaling route, the mitogen–activated protein kinase signaling network, the bone morphogenetic protein signaling pathway, the Wnt/-catenin signaling pathway, and other pathways.SEM images demonstrate cell attachment, proliferation, and differentiation. It demonstrated that osteoblast-like cells could adhere to, proliferate, and differentiate on the scaffold surface.The rapidly expanding science of tissue engineering for dental tissues will undoubtedly result in a dramatic shift in the availability of novel items for practitioners to employ on a daily basis. When implanted on the side of an injury, a scaffold material should strive for endogenous cell repopulation as well as recipient remodeling.PCL/graphene composites may have potential applications as novel bone tissue engineering. The porosity and pore size were suitable to induce osteogenesis and angiogenesis, and also stimulated osteoblast migration by adding graphene start from 24 hours.


Flexible piezocapacitivesensors utilizing nanomaterial–polymercomposite-based nanofibrous membranes offer an attractive alternativeto more traditional piezoelectric and piezoresistive wearable sensorsowing to their ultralow powered nature, fast response, low hysteresis,and insensitivity to temperature change. In this work, we proposea facile method of fabricating electrospun graphene-dispersed PVAcnanofibrous membrane-based piezocapacitive sensors for applicationsin IoT-enabled wearables and human physiological function monitoring.A series of electrical and material characterization experiments wereconducted on both the pristine and graphene-dispersed PVAc nanofibersto understand the effect of graphene addition on nanofiber morphology,dielectric response, and pressure sensing performance. Dynamic uniaxialpressure sensing performance evaluation tests were conducted on thepristine and graphene-loaded PVAc nanofibrous membrane-based sensorsfor understanding the effect of two-dimensional (2D) nanofiller additionon pressure sensing performance. A marked increase in the dielectricconstant and pressure sensing performance was observed for graphene-loadedspin coated membrane and nanofiber webs respectively, and subsequentlythe micro dipole formation model was invoked to explain the nanofiller-induceddielectric constant enhancement. The robustness and reliability ofthe sensor have been underscored by conducting accelerated lifetimeassessment experiments entailing at least 3000 cycles of periodictactile force loading. A series of tests involving human physiologicalparameter monitoring were conducted to underscore the applicabilityof the proposed sensor for IoT-enabled personalized health care, softrobotics, and next-generation prosthetic devices. Finally, the easydegradability of the sensing elements is demonstrated to emphasizetheir suitability for transient electronics applications. The last two decades have witnessed asignificant growth in thewearable-focused consumer electronics industry, and the rise in theconsumption of smart watches and accessories is a testament to thatfact. Especially, with the rapid incremental progress being made everyday, the field of wearable electronics is hovering around the brinkof science fiction. The Internet of Things (IoT) revolution in thelast decade has pushed wearable sensors to the forefront of researchinterest across the globe. Today, the demands for seamless integrationof wearable sensors which can monitor vital physiological parametershave skyrocketed, and this dramatic rise in demand has been partiallyfueled by the onslaught of the COVID-19 pandemic since late 2019.The need of the hour is reliable, facile, low-powered, and inexpensivesensors fit for seamless integration into various wearable apparelthat will enable nonintrusive acquisition of vital health parameterswith the aim of improving the quality of life of the user. However,for any wearable sensor technology to be considered seriously in thehealthcare domain, parameters such as sensitivity, ease of use, costeffectiveness, long term-reliability, and, most importantly, a lowpower budget, are of paramount importance. In addition to applicationsin human physiological function monitoring, flexible sensors are alsoof great importance in developing artificial skin for next-generationprostheses, soft human–machine interfaces, robotics-assistedmedical facilities, and other similar areas.7 piezoelectric,13 and capacitive.17 Other mechanisms utilizing transistors for pressure sensing havealso been reported in the literature.20 More recently, the triboelectricsensing mechanism has also been employed in the development of pressureand strain sensors.22 Though the piezoelectric sensingmechanism offers the advantage of self-powered sensing, their inabilityto sense static pressure places a major hindrance on their applicationsin tasks requiring both static and dynamic pressure sensing. In contrast,piezoresistive sensing offers functionality in a wide range of pressuresensing conditions, including static, quasi-static, and dynamic pressure/strainsensing. However, resistive sensors have the inherent problems ofthermal drift, high hysteresis, and a relatively higher power budget.Finally, the fundamental problem with transistor-based sensors liesin their complex fabrication process and associated circuitry. Piezocapacitivesensing offers an attractive alternative to more traditional piezoelectricand piezoresistive sensing owing to its ultralow-powered nature dueto the lack of flow of DC current. Furthermore, piezocapacitive sensorsoffer added advantages, such as superior stability, temperature independence,low hysteresis, and fast response time.Historically, strainand pressure sensing devices have relied ondifferent sensing mechanisms, namely piezo-resistive,d) between the electrodes, more recently, methods are beinginvestigated to achieve pressure-induced dielectric constant (εr) changes to achieve enhanced pressure sensitivity.24 Furthermore, it is desirable that the dielectric layer has a lowcompressive modulus, which will further contribute toward enhancingthe pressure sensitivity. In the past, elastomeric materials withmicrostructured air voids have been employed for enhancing the sensingperformance of capacitive sensors.25 For instance,Mannsfeld et al. demonstrated a microstructured polydimethylsiloxane(PDMS)-based large-area flexible capacitive sensor array with fastresponse.23 More recently, researchershave employed ionic hydrogels for developing capacitive sensors forpressure sensing, humidity sensing, and human physiological parametersmonitoring.27 Ding et al. reported a polyvinylalcohol-cellulose nanofiber-based biocompatible, self-healing, breathable,stretchable, and degradable organohydrogel ionic skin with superiorhumidity responsiveness.26Traditional parallel-plateelectrode-based capacitive pressuresensors comprise two conductive electrodes separated by a dielectricmedium. Upon application of external pressure, the distance betweenthe two electrodes changes, thereby leading to a change in capacitancethat can be measured with an external circuit. The structural propertyof the dielectric layer between the two electrodes has a profoundimpact on the sensing performance of the sensor. Though traditionalcapacitive pressure sensors rely on the changing distance (d) between the electrodes reduces, squeezing the sandwicheddielectric layer and replacing the low permittivity air voids withhigh permittivity dielectric material in the process. The change inthe dielectric constant with applied pressure contributes to the enhancedsensing performance. Though dielectric elastomeric structures withmicropatterned air voids offer an attractive proposition, the inherentcomplexity of fabrication involving multistep patterning processesand reliance on expensive machinery place constraints on small andmedium-sized enterprises to develop and commercialize them.In thecase of capacitive sensors employing dielectric layers withair voids, the initial effective dielectric constant is a combinationof the dielectric constants of air and the porous/microstructuredmaterial. Upon application of an external pressure, the gap  and grapheneand MXene nanoflakes, has proven to enhance the dielectric performanceof the polymeric materials.36 Furthermore, the addition of nanomaterial fillers in low volumefractions has the potential to enhance the dielectric property ofthe polymeric materials without compromising their flexibility ormechanical properties. Yang et al. reported CNT-polyvinylidene fluoride(PVDF) nanofibrous membrane-based flexible piezocapacitive tactilesensors for wearable applications.36 However,due to the one-dimensional (1-D) nature of CNTs, they have a lessersurface area in comparison to 2D materials with a larger specificsurface area and tend to form agglomerates that lead to poor dispersionin nanofibers and can subsequently reduce the mechanical performanceof the membranes. Incorporation of 2-D materials like Graphene andMXene (Ti3C2Tx)can potentially alleviate the problems associated with lower-dimensionnanofillers and lead to the fabrication of better performing dielectriclayers. Previously, Sharma et al. reported that MXene dispersed polyvinylidenefluoride-trifluoroethylene (PVDF-TrFe) nanofibers for developing highperformance flexible and wearable capacitive sensors.33 The harsh acid etching used for synthesizing MXene sheetsled to defect sites in the flakes that formed centers of oxidation,leading to the loss of conductivity.38 Furthermore,the presence of hydroxyl groups on the MXene surface (to achieve betterdispersion in water) resulted in a reaction with dissolved oxygenand water molecules, thereby causing oxidation and a subsequent lossof conductive properties.38 In contrast,graphene can prove to be an attractive alternative owing to its superiorstability coupled with excellent electrical and mechanical properties.Piezocapacitive sensors utilizing polymeric nanofibers withnanomaterialfillers are relatively new and must be explored further to assesstheir suitability for applications in wearables. Although polymericnanofibers enable the facile fabrication of flexible and pliant structureswith low compression modulus, their low dielectric permittivity limitstheir usage for applications in supercapacitors and piezocapacitivesensors. Incorporation of fillers in polymeric materials near thepercolation threshold has proven to enhance the dielectric propertiesof the composites.40 polymer having the formula (C4H6O2)n,which makes it a promising candidate for usage in sensors targetedfor human physiological monitoring applications.In contrast to the previous works utilizing materials like PVDFand PVDF-TrFe, in this research, we have employed PVAc, an inexpensiveand degradable polymeric material ,for synthesizing the nanofibrous membranes. PVAc is a rubbery, thermoplasticsynthetic nontoxic−1(∼2.73–56.06 kPa)and 0.00653 kPa−1(∼75.76–318.18 kPa).To stress the robustness and reliability of the sensors for real-lifeapplications, a series of accelerated lifetime assessment tests involvingat least 3000 cycles of tactile force loading are demonstrated. The 0.25 wt % graphene-dispersed PVAc nanofibrousmembrane-based piezocapacitive sensors are chosen for the subsequentapplications demonstrated in this work. The suitability of the sensorsfor applications in wearables and human physiological monitoring hasbeen validated by conducting a series of demonstration experimentsinvolving muscle movement detection, respiratory rate monitoring,sensorized smart shoe insoles for gait detection, and a posture sensingsmart chair for encouraging pressure-relieving maneuvers. The readinessof the proposed piezocapacitive sensors for IoT integration has beenunderscored through custom-written embedded software programs (implementedon Arduino Uno and Nano platforms) and subsequent applications inwireless gait monitoring smart insoles and posture sensing smart chairs.Finally, the degradability and subsequent recyclability of the sensorhave been demonstrated to emphasize the relevance of the sensor forthe circular electronics initiative theme. The facile sensor reportedin this work would inspire a future telemedicine ecosystem comprisingof low-powered, highly sensitive piezocapacitive sensor networks coupledwith IoT hardware for continuous monitoring of human physiologicalvitals, thereby reducing the load on existing healthcare infrastructure,as conceptually shown by the schematic in In this work,we propose a facile method for fabricating graphene-dispersedpolyvinyl acetate (PVAc) electrospun nanofiber-based piezocapacitivesensors for flexible and wearable electronics applications. A methodentailing the sandwiching of graphene-dispersed PVAc nanofibrous membranesbetween two layers of flexible fabric electrodes for fabricating afully flexible and apparel-integrable sensor is demonstrated. Dielectricresponse assessment tests are conducted on both the pristine PVAcand graphene-dispersed PVAc membranes (fabricated by spin coating)to understand the effect of the addition of graphene nanofillers onthe dielectric constant enhancement. The microcapacitor network theoryand microscopic dipole formation model have been invoked to explain the dielectric response enhancement.Material and morphological characterization experiments are conductedon both the pristine and graphene-loaded PVAc nanofibers to understandthe effect of graphene dispersion on nanofiber properties and morphologies.Electromechanical characterization experiments are conducted on thepristine and graphene-loaded PVAc nanofibrous membrane-based piezocapacitivesensors for assessing the effect of graphene nanofiller addition onpressure sensing performance. The 0.25 wt % graphene-loaded PVAc nanofibrousmembrane-based piezocapacitive sensor demonstrated superior performancewith two distinct linear regimes and pressure sensitivity figuresof 0.01355 kPaIn this work, electrospinning was employed for fabricating pristinePVAc nanofibers. To understand the effect of nanomaterial fillerson the dielectric constant/permittivity, commercially acquired graphenenanoflake dispersion was added to the pristine PVAc polymer solutionto achieve 0.1, 0.25, and 0.50% weight of graphene nanofillers inPVAc. Electrospinning was subsequently conducted to synthesize thegraphene-loaded PVAc nanofibers. More details of the polymer solutionpreparation and subsequent electrospinning process are furnished inthe The as-spunnanofibers on aluminum foil substrate are transferred from the rotatingmandrel collector and diced into 2 cm × 2 cm sections. To developall flexible, wearable, and apparel-integrable sensors, the dicednanofibrous membranes were sandwiched between two layers of circularsilver-plated nylon fabric bonded to single-strand copper wires forsubsequent electrical connections. A complete encapsulation was achievedby employing two layers of optically clear adhesive plate seals. Video SV1). The micrographs in –1) that is triggered by the defectsand a G band (at 1596 cm–1) for the characteristicof graphite/graphene. The intensity ratio of the D band to the G bandis 1.10, indicating that graphene contains many defects.In orderto understand the effect of the addition of graphene nanofillers onthe dielectric characteristics of the polymer composite, pristinePVAc and graphene-loaded PVAc nanofibers were synthesized by electrospinning.Graphene was added in varying concentrations  to a PVAc polymer solution, and electrospinning was conductedto achieve graphene-loaded PVAc nanofibers. 41 However, the graphene fillers used in this workare intended to enhance the dielectric constant of the compositesby harnessing the phenomenon of interfacial polarization. As such,eventual changes in the conductivity of the graphene nanofillers ascompared to the defect-free case do not significantly impact the pressuresensing performance of the resulting sensors. Furthermore, the relativeintensity of the peaks improved progressively with increasing thegraphene loadings, as observed from the plots in Lattice defects are inevitably introduced duringthe production/synthesisprocess of graphene, which lead to deviations from its theoreticalmechanical and electrical properties. The defects introduced duringthe synthesis process lead to changes in the bond length of the interatomicvalence bonds and potentially to changes in their conductivity.36 From the perspective of percolation theory, in the case of conductivefillers dispersed in nonconductive polymers, the dielectric constant(κ) of nanomaterial-polymer composites follows the relation:κ p|f–fc|s–, where κp represents the dielectricconstant of the insulating polymer matrix, s is anexponent with a value of ∼1, f representsthe fractional volume of the nanofiller in the polymer matrix, and fc represents the critical fractional volumeat percolation threshold.42 A number oftheories have been hypothesized to explain the phenomenon of dielectricconstant enhancement in the case of insulating polymeric materialsloaded with nanomaterial fillers.45 In particular, the microcapacitornetwork formation theory has been invoked by several researchers toexplain the giant enhancement of dielectric constant near percolationthreshold.46 The sketch in fc),several microcapacitors are formed by neighboring conductive nanofillersseparated by very thin layers of the polymer insulator. Each of theseindividual microcapacitors demonstrate a very high capacitance figureand subsequently contribute to the overall enhancement of the dielectricconstant of the composite.43 Another mechanism thatalso contributes to the dielectric constant enhancement is the microscopicdipole formation model.47 In this case, applicationof an external electric field to the nanomaterial polymer compositeleads to charge accumulation at the interface between the nanomaterialfillers and the polymer matrix, which subsequently leads to interfacialpolarization (Maxwell–Wagner-Sillars polarization) as depictedby the schematic in Researchworks conducted in the past have established that the incorporationof nanomaterial fillers in polymeric materials generally improvestheir dielectric performance.34 The microscopicdipole model, as discussed earlier, is likely to be the contributingfactor behind the dielectric constant enhancement observed in thiswork.The plot in The piezocapacitivesensors proposed in this work utilize graphene–PVAc nanofibrousmembranes as the dielectric layer. For a nanofibrous membrane-basedcapacitor with air voids, the initial effective dielectric constantis a combination of the dielectric constants of air and the nanofibrousmaterial. Upon application of an external pressure, the gap betweenthe electrodes reduces, thereby squeezing the sandwiched nanofibrousmembrane and replacing the low permittivity air voids with high permittivityof the nanofibrous material in the process. This pressure-induceddensification process increases the effective dielectric constantof the capacitor. The phenomenon of pressure-induced dielectric constantenhancement coupled with the usual thickness change significantlyenhances the sensitivity of the sensor. 33 The relationbetween the external pressure (p) and capacitancechange (ΔC) is given by the following expressionC0 is theinitial capacitance of the sensor and E0 is the Young’s modulus of the dielectric layer.24 For low pressure regimes, the applied pressurebeing less than the Young’s modulus (p ≪ E0), the quadratic term can be neglected, andthe normalized capacitance change is expected to demonstrate a linearbehaviorIn the past, Kim et al. and Sharma et al. independentlyproposed analytical models to explain the dependence of capacitancechange on applied external pressure.24 From the above discussion, it is evident thatnanofibrous membranes with air voids are more likely to be suitablefor the development of wearable piezocapacitive sensors.Kim et al. argued that, atlow pressureregimes, the normalizedcapacitance change for nanofibrous piezocapacitive sensors is doublethat of their bulk dielectric material counterpart (without air voids).Figure S1). The mismatch between the response and recovery times can be attributedto the hysteresis of the graphene–PVAc nanofibrous membrane.To further understand the effect of graphene nanofiller addition,the pressure sensitivity of the pristine PVAc nanofiber-based sensorwas compared with that of the graphene-dispersed nanofiber sensors.The normalized capacitance change versus the applied uniaxial pressurewas plotted, as shown in –1 (in the pressure range 9.85–80.30 kPa)and 0.00135 kPa–1 (in the pressure range 95.45–275kPa) with R-square values of 0.9873 and 0.9833, respectively.Similar uniaxial pressure sensitivity assessment tests were conductedon the graphene-dispersed PVAc nanofiber-based sensors, as shown bythe plots in 33 The dynamic range, sensitivity,response/recovery times, and degradability of the sensor reportedin this work are compared with recent similar piezocapacitive sensorsin the literature and are presented in Toassess the pressure sensing performance of the graphene–PVAcflexible pressure sensor, a series of tests involving tactile pressuresensing were performed. Figure S2a). The plot in 51Owing to its maximum pressure sensitivity,the 0.25wt % graphene–PVAcnanofiber-based sensor was chosen for further mechanical and lifetimeassessment analyses. To assess the compressibility and hysteresisbehavior, the sensor assembly was subjected to a series of 10 cycliccompressive loading and unloading. Further details of the test setupare furnished in the Supporting Information section S2a. The Figure S2b). The plotin st and 10thcycles with the average stress–strain characteristics (averagedover 10 cycles). The average tensile modulus of the nanofiber membranewas determined as 212.69 MPa with an ultimate tensile strength of4.61 MPa at 15.10% strain (elongation at break).Furthermore, the tensile modulus and ultimate tensilestrengthof the 0.25 wt % graphene-loaded PVAc nanofiber membrane were assessedby subjecting it to a series of cyclic tensile loading and unloading.Further details of the test setup are furnished in the SupportingInformation section S2b. The To demonstratethe robustness and reliability of the sensor forlong term applications, accelerated lifetime assessment experimentsinvolving a combined total of 3000 cycles of tactile force loadingat three different force levels  were conducted.The photograph in To demonstrate the applicability of ourpiezocapacitive sensorsfor physiological function monitoring applications, the 0.25 wt %graphene-dispersed nanofiber-based sensors were chosen owing to theirsuperior pressure sensing performance, as demonstrated previously.The plot in N = 107).52 Peak plantar pressures werereported to lie in the range 53–139 kPa. In our previous research,we have demonstrated utilizing graphene–PDMS squeezable sensorsfor developing smart sensorized shoe soles for gait monitoring applications.53 As seen previously, the sensors presented inthis work have been calibrated for pressure sensitivities in the rangeof ∼2–320 kPa that is the regime of interest for plantarpressure sensing. Here, we have utilized the graphene–PVAcnanofiber sensors for demonstrating IoT-enabled gait monitoring. Asdiscussed previously, capacitive sensors are more suitable due totheir low hysteresis, fast response, and low power budget. Three identicalsensors were secured on the heel, lateral midfoot, and toe ball regionsof a soft shoe insole and placed inside a sport sneaker. For dataacquisition, embedded systems software utilizing the Arduino Nanoplatform was employed. The details of the connections are furnishedin the experimental section. For wireless data acquisition, an ArduinoHC05 Bluetooth module utilizing the serial communication protocolwas used. The IoT enabled system utilizing the Arduino nanoplatformis schematically represented in –1 (determined by multiplying the step length by cadence).The sensorized shoe insole demonstrated in this work can potentiallybe used by physicians to determine problems with gait and determineif further diagnostic tests are warranted to find the underlying condition.In the past,Cavanagh et al. have reported standing plantar pressure distributionunder human feet for a heterogeneous sample of feet . The solution was stirred using a hot platemagnetic stirrer at 60 °C to accelerate the decomposition process.The large specific surface area of the nanofibrous membrane providesmore area for the water–ethanol solution to penetrate, therebyfacilitating the decomposition process. At the end of 30 min, cleargraphene dispersion in the solution was observed. The solution wasremoved from the stirrer and allowed to cool to room temperature.By the end of 60 min, graphene precipitation was observed, and thesolution turned whitish, indicating a homogeneous solution of PVAcin the water–ethanol mixture. The photographs in With the growing burden of electronicswaste lately and their detrimental impact on the environment and publichealth, there is a surge in demand for transient electronics.33 can be used in conjunction with flexible andbiodegradable packaging to circumvent the recyclability issue oftenassociated with deposited metal-based thin film electrodes.Though the PVAc-graphene nanofiber membrane isdegradable, thelack of suitable degradable packaging restricts the applicabilityof the reported sensor for applications demanding true, fully degradablesensors. To make the sensor fully degradable yet robust and reliable,water-soluble packaging materials like polyvinyl alcohol (PVA), polyethyleneglycol (PEG), and water-soluble hydrogels can be considered in thefuture. Furthermore, conductive polymer-based flexible electrodes−1 (∼2.73–56.06 kPa)and 0.00653 kPa–1 (∼75.76–318.18 kPa).A series of accelerated lifetime assessment tests involving at least3000 cycles of tactile force loading at three different forces wereconducted to demonstrate the robustness and reliability of the sensor.Finally, the 0.25 wt % graphene-dispersed PVAc nanofibrous membrane-basedsensors were chosen for demonstration experiments. A series of humanphysiological parameter monitoring experiments comprising muscle movementdetection, respiratory rate detection, and gait monitoring were conducted.To further emphasize the readiness of the sensors for IoT applications,a wireless smart gait monitoring shoe insole and a posture sensingsmart chair, coupled with a custom embedded program implemented onArduino Uno and nanoplatforms were demonstrated. Finally, the suitabilityof the proposed sensing element for the circular electronics initiativetheme has been demonstrated through the simple decomposition of thesensor in a water–ethanol solution.In conclusion, this work established afacile method of utilizinggraphene-dispersed PVAc nanofibrous membranes for IoT-enabled wearablepiezocapacitive sensing applications. A series of electrical and materialcharacterization tests involving dielectric response assessment, SEMand TEM observation, and Raman spectral analyses were conducted onthe nanofibers to understand the effect of graphene dispersion onnanofiber morphology, and electrical properties. Microcapacitor networkand microdipole formation mechanisms were invoked to explain the observednanofiller-induced dielectric constant enhancement. A series of dynamicuniaxial pressure sensing performance tests were conducted on thepristine and graphene-dispersed PVAc nanofibrous membrane-based piezocapacitivesensors to observe and understand the effect of graphene nanofilleraddition on pressure sensing performance. The nanofibrous membranewith 0.25 wt % graphene loading showed superior pressure sensing performancewith two distinct linear regimes and pressure sensitivity figuresof 0.01355 kPaMW 500,000 g mol–1), N,N-dimethylformamide (DMF),and electrochemically exfoliated graphene ink (1 mg mL–1 in DMF) purchased from Sigma-Aldrich, the Netherlands, were used.For the pristine PVAc nanofibers, 1.5 g of PVAc was dissolved in 10mL of DMF and stirred at room temperature using a magnetic stirrerto achieve 15% (w/v) solution. For the graphene–PVAc nanofibers,the commercially acquired graphene dispersion was added to the PVAcpolymer solution and subsequently sonicated employing an ultrasonicbath sonicator to achieve 0.1, 0.25, and 0.50% weight of graphenenanofillers in PVAc, while keeping the overall concentration of thesolution fixed at 15% (w/v). An electrospinning setup (NanoSpinnerNE300) from Inovenso electrospinner fitted with a stainless-steelrotating mandrel collector was used for electrospinning the nanofibers.Inovenso IPS-14RS syringe pump fitted with a 10 mL syringe was usedto feed the polymer solution through a needle (20 G) at a constantflow rate of 1 mL h–1. The electrospinning was conductedfor the pristine PVAc solution and different concentrations of thegraphene-loaded solutions by applying a potential difference of 15kV between the tip of the needle and the rotating mandrel collector(rotating at 2200 rpm) separated by 15 cm. The electrospinning processwas conducted for 3 h to obtain a film of uniform nanofibers depositedon the aluminum foil substrate.For electrospinning of pristine PVAc and graphene–PVAc nanofibers,PVAc beads  was employed. Samples havingdimensions of 1 cm × 1 cm were sputtered with gold and placedon appropriate SEM stubs for subsequent imaging. For conducting theimaging experiments, an acceleration voltage of 7 kV and a spot sizeof 3.0 were employed.To confirmthe dispersion ofthe graphene nanoflake fillers within the PVAc nanofibers, Raman spectroscopicanalyses and TEM imaging were conducted. Raman spectra analysis wascarried out on a PerkinElmer Raman station by using 633 nm laser excitation.Raman analyses were conducted on both the pristine and graphene-dispersedPVAc nanofibers. For the TEM measurements, the specimens were preparedby directly transferring the nanofibers from the aluminum foil tocopper grids. Bright-field imaging was performed with a JEOL 2010at an accelerating voltage of 200 kV.Thefabrication flow for developing the pristine PVAc and graphene–PVAcnanofiber pressure sensors is schematically represented in 55 was employed for exporting the data from theArduino serial output to CSV format. For all the experiments involvinghuman motion monitoring and the smart chair, unamplified signal outputswere presented in the article.For demonstrating the capability of the sensorin IoT-enabled humanphysiological monitoring applications, a custom-made capacitance meterutilizing the Arduino Uno and Arduino Nano platforms was developed.The embedded system setup utilizes an open-source Arduino capacitorlibrary that enables the measurement of capacitance in the range 0.2pF–100 μF. For measurement and sensing experiments, eachindividual sensor was connected to an analog pin and a digital pinof the Arduino Uno/Nano board. For data acquisition using the Arduinoplatform, an open-source plug-in code (Ardu Spreadsheet 1.0)