Two-dimensional (2D) materials and their composites have gained significant importance as the functional layer of various environmental sensors and nanoelectronics owing to their unique properties. This work reports for the first time a highly sensitive, fast, and stable humidity sensor based on the bi-layered active sensing area composed of graphene flower (GF) and poly  PVA thin films for multifunctional applications. The GF/PVA humidity sensor exhibited stable impedance response over 15 days, for a relative humidity (RH) range of (40–90% RH) under ambient operating conditions. The proposed bi-layered humidity sensor also exhibited an ultra-high capacitive sensitivity response of the 29 nF/%RH at 10 kHz and fast transient response of 2 s and 3.5 s, respectively. Furthermore, the reported sensor also showed a good response towards multi-functional applications such as non-contact skin humidity and mouth breathing detection. Thin film-based capacitive and impedimetric sensors offer numerous benefits over optical and chemical sensors in vapor detection applications, namely low energy consumption, higher sensitivity response and selectivity, good stability, and fast response/recovery time ,2. In inCarbon-based materials, such as graphene and carbon nanotubes (CNTs), have sparked a great deal of interest in chemical sensing because of their distinctive structural and electronic properties, such as a large surface area, compact size, high electronic mobility, and high sensitivity response to electrical interruptions from water or other gaseous molecules ,20,21,22Polymers are another class of material with a high sensitivity to many environmental factors. Low cost, simple synthesis, high stability, and good mechanical characteristics for flexible electronics are just a few of the major advantages of utilizing these materials ,32,33,342/g, transparent nature, remarkable conductivity, and highly crystalline structure [Herein, for the first time, we demonstrate a unique sorption layer (multi-layered graphene nanostructures known as graphene flower deposited over PVA film), used to fabricate a fast, highly responsive, and extremely sensitive humidity sensor for future applications in printed electronics. GF material has attractive properties such as a large surface area of 500–2500 mtructure ,45,46,473, and metal content ~70 ± 2 wt% from InkTec, Ansan-si, Gyeonggi-do, Korea. We purchased the 100-µm-thick PET substrate from AgIC paper. The PVA solution was formed by mixing PVA powder in the DI Water to 1:10 mass ratio and kept at room temperature for 24 h to swell. The solution was then magnetically stirred by keeping it on the hot plate for 12 h at 343 K to dissolve PVA completely as seen in GF solution in propylene glycol monomethyl ether acetate (PGMEA) (99.9 wt%) was purchased from InALA . PVA polymer was purchased from Sigma-Aldric . For the electrode’s preparation, we purchased screen-printing Ag ink with viscosity 155,000 ± 15,000 cps, density ~2.8 ± 0.2 g/cmThe scanning electron microscope TESCAN MIRA 3  was used to analyze the surface morphology of the deposited active material, while dispersive X-ray (EDS) spectroscopy was used to investigate the elemental composition. The crystal structure of GF and its composite with PVA was studied using an X-ray diffractometer . Fourier transform infrared (FTIR) spectroscopy was performed by using Fourier Transform infrared spectrometer ALPHA II . Additionally, Raman spectroscopy was performed using Micro Raman Spectrometer System LABRAM HR EV .rms source alternating voltage signal. When the RH of the chamber changed the capacitance and impedance spectra incorporating the impedance and capacitance responses were acquired by interfacing the LCR meter with a personal computer. The proposed sensor’s humidity responses were recorded over a wide humidity range, and the temperature was kept constant at 22 °C during the experiment. The test frequency was maintained constant at 1 kHz and 10 kHz while examining the sensor’s electrical response. The humidification level of the chamber was gradually increased while recording the measurement to get the maximum number of data points.We studied the electrical response of the sensor by exposing it to varying levels of RH over a wide range. θ = 27°), in θ = 25.8° disappears in the composites as the graphene flower is dispersed into the PVA matrix, implying graphene disorder and loss of structural regularity and intercalation of PVA into inter-layers of graphene. As a result, the graphene nanostructures are thought to have been disseminated into the PVA matrix on a molecular level. In the composites, similar results were reported for studies performed previously, by adding graphene nanostructures in the PVA with different volume ratios. To determine the purity of the materials and study their probable interaction, Fourier transform infrared spectroscopy (FTIR) was used to investigate the functional groups in active layer materials, as shown in −1 originating from C–H stretching vibrations. From the FTIR spectrum of PVA, peaks at 1450 cm−1 (C–H bending) and1100 cm−1 (C–O–C stretching) can be observed, confirming the presence of pure PVA in the composite. Small peaks positioned at 1376 cm−1 can be attributed to C–H bonds, while at 1260 is specific to C–O bonds, and a small peak at 839 cm−1 is attributed to C–C stretching vibration. Chemical composition of the sensor film was determined using Raman spectroscopy (LabRAM HR Raman). −1, 1575 cm−1, and 2696 cm−1, respectively. The 3D Nano Profiling System (NV2200) was used to find out the surface roughness of sensing active material. From this 3D view, the thickness of PVA/GF, and GF was estimated to be 4.7 µm, and 2.1 µm respectively as shown in To examine the surface morphology of the proposed humidity sensor’s PVA/GF composite thin film, a scanning electron microscope image was taken of the active layer, as shown in We measured the impedance and capacitance responses under different humidity levels at test frequencies of 1 kHz, and 10 kHz to explore electrical performance of the proposed humidity sensor. RHC and RHoC are the capacitances at %RH and 40% RH, respectively. + ions will rise under these conditions, and they will be able to hop between water vapor molecules (Grotthuss mechanism). When the amount of water vapor in the atmosphere increases, more H+ ions migrate, causing the PVA/GF conductivity to rise. Protons are the primary carriers responsible for electrical conductivity when exposed to moisture.The capacity of PVA to sense moisture is due to an OH group, which can be seen in the PVA structure. The capacity of GF to identify water molecules is dependent mainly upon the high specific surface area percentages and hydrophilic functional groups, including such hydroxyl and carboxyl groups, linked to its surface. Additional adsorption sites, such as voids and flaws, are created when PVA is mixed into GF layers. They can be formed at the interface between the two nanomaterials, which helps to increase humidity sensing. Moreover, GF has a low electrical resistance and a high concentration of charge carrier. It can act as an anchor to enhance electron transport in metallic nanoparticles, which helps to modify PVA. When the sensor is exposed to varying humidity levels in this study, it easily interacts with the water vapor, causing the dielectric constant to fluctuate. As per the findings of the research, GF nanostructures without PVA have a lack of stability and reproducibility, which is a stumbling barrier for sensor applications. PVA will absorb or desorb water molecules in a humid atmosphere, resulting in a change in the dielectric constant. The dielectric constant will increase as the humidity level rises, electrical response, i.e., capacitance and impedance, will change as the dielectric constant changes. When humidity rises, there will be more hydrogen bonding between water vapor and the OH groups in PVA. The quantity of HThe suggested sensor was evaluated for various applications depending on its outstanding sensing capability, including non-contact humidity sensing of the fingertip. Furthermore, we put our sample to the test for human healthcare monitoring applications by introducing it to human respiration to detect water vapors. Our sensor quickly responded to and detected minor %RH fluctuations. In summary, this paper proposed a highly sensitive, fast, and stable humidity sensor based on 2D graphene nanoflakes, with PVA/GF nanocomposite film as the sensing layer. The spray-coated GF solution has a high electrical conductivity and homogeneous dispersion. The suggested humidity detecting device showed a humidity sensing range of 40–90% RH, with the response and recovery periods of 2 s and 3.5 s, respectively, at room temperature. The developed humidity sensor exhibited a capacitance sensitivity response of 29 nF/%RH at 10 kHz with a good linearity and reproducibility. Our proposed device also exhibited different applications, such as contactless skin humidity monitoring, due to its superior sensing performance characteristics. Similarly, we tested our sensor in an open environment and found it to be stable. The humidity detecting performance of the constructed sensor was outstanding. Based on these findings, we anticipate that humidity sensors based on PVA/GF nanocomposite will have a bright future in the field of real-time humidity detecting electronic devices.


Introns are ubiquitous in eukaryotic genomes and have long been considered as ‘junk RNA’ but the huge energy expenditure in their transcription, removal, and degradation indicate that they may have functional significance and can offer evolutionary advantages. In fungi, plants and algae introns make a significant contribution to the size of the organellar genomes. Organellar introns are classified as catalytic self-splicing introns that can be categorized as either Group I or Group II introns. There are some biases, with Group I introns being more frequently encountered in fungal mitochondrial genomes, whereas among plants Group II introns dominate within the mitochondrial and chloroplast genomes. Organellar introns can encode a variety of proteins, such as maturases, homing endonucleases, reverse transcriptases, and, in some cases, ribosomal proteins, along with other novel open reading frames. Although organellar introns are viewed to be ribozymes, they do interact with various intron- or nuclear genome-encoded protein factors that assist in the intron RNA to fold into competent splicing structures, or facilitate the turn-over of intron RNAs to prevent reverse splicing. Organellar introns are also known to be involved in non-canonical splicing, such as backsplicing and trans-splicing which can result in novel splicing products or, in some instances, compensate for the fragmentation of genes by recombination events. In organellar genomes, Group I and II introns may exist in nested intronic arrangements, such as introns within introns, referred to as twintrons, where splicing of the external intron may be dependent on splicing of the internal intron. These nested or complex introns, with two or three-component intron modules, are being explored as platforms for alternative splicing and their possible function as molecular switches for modulating gene expression which could be potentially applied towards heterologous gene expression. This review explores recent findings on organellar Group I and II introns, focusing on splicing and mobility mechanisms aided by associated intron/nuclear encoded proteins and their potential roles in organellar gene expression and cross talk between nuclear and organellar genomes. Potential application for these types of elements in biotechnology are also discussed. Introns, or intervening sequences, are segments that are transcribed but removed from a transcript before translation can proceed. There are several categories of introns, such as tRNA introns, nuclear spliceosomal introns, and Group I and Group II introns . IntronsOrganellar genomes can be quite variable in size due to the absence and presence of introns, however, little is known about the mechanisms that promote intron gain and loss and why in some instances, the organellar genomes are devoid of introns, or in extreme examples where introns comprise most of the nucleotides that makeup the genome ,9,10. OrOrganellar introns are mainly classified as self-splicing introns that are either Group I and II type introns, based on differences in their splicing pathways and RNA secondary structural features. Sequence conservation among Group I or Group II introns is very low, but conservation among these elements can be found at the structural level ,17,18,19Group I and II introns at the RNA level assume RNA folds that generate the active sites needed for the splicing reactions to be initiated A,B. Grou2+ ions that permits the 2′OH Group on the budged adenine to destabilize the phosphodiester bond (PDE) that links the upstream exon with the intron . Ad. Ad16]. ) intron ,258. “Zoox3 gene . With thox3 gene ,259.Ophiostoma ips revealed two examples of complex introns: cytochrome b (cob I4) and the cytochrome oxidase III (cox3 I2) introns. The cox3 I2 intron is composed of two Group IA1 type intron modules in a possible tandem arrangement, and most likely is excised as a composite intron (‘zombie’ twintron). The complex cob I4 intron is the first report of a trintron, which is a complex intron composed of three intron modules. Two Group I introns are in tandem arrangement with the first intron module being interrupted by a Group II intron module. Based on in silico predictions this complex intron could splice by a ratchet-type mechanisms, whereby the upstream Group I intron module splices first followed by the splicing of the downstream Group I intron module. The Group II intron module interrupts the ORF of the first intron module and the Group II module is assumed to splice independently of the other intron modules [Characterization of the mitogenome for the fungus  modules .Monomorphina aenigmatica [Atypical Group II introns and twintrons have been identified in the plastid genome of the freshwater euglenoid igmatica . Throughigmatica . To be aChaetomium thermophilum var. thermophilum, in which the splicing of the internal Group II intron reconstitutes a HE ORF of the external (resident) Group I intron, facilitating its expression which, in turn, contributes to the mobility of the twintron to cognate intron-less rns genes [E. coli; within E. coli, the expressions of the intron encoded protein could be manipulated by modulation the splicing of the internal Group II intron module by manipulating Mg2+ concentrations in the media [E. coli.Twintron/complex introns offer new ribozyme scaffolds that could be engineered where the expression of intron encoded ORFs can be regulated by the splicing of nested ribozyme type components . An examns genes . It was he media . EssentiThe study of complex introns is interesting from an evolutionary point of view, with these configurations evolving independently multiple times involving different categories of introns, to generate complexity. These complex ribozymes my evolve into platform of alternative splicing or as regulatory elements that can modulate the expression of the host gene ,263. TheExploring organellar genomes and their intron complements is important as these introns can impact genome sizes, genome architecture, and organellar genome diversity. Organellar introns have the potential to serve as regulatory elements that can impact gene expression and, in some instances, introns are associated with a phenotype ,269. MutTetrahymena thermophila can function in RNA repair by replacing mutated regions of mRNA with corrected sequences and, thus, contribute to therapeutic strategies [Polymorphism in mitochondrial Group I introns have been linked to drug susceptibility in fungal pathogens and are being explored as important markers in clinical research . Trans-srategies . Group Irategies ). Organerategies ,272. Forrategies ,274. In rategies ,274; revrategies ,276), corategies ,277,278.Group I and II introns, complex introns, and trans-splicing introns could be considered examples of constructive neutral evolution where complex systems evolved by non-adaptive mechanisms  ,280. Pos


Alzheimer’s disease (AD) is a neurodegenerative disorder for which there is currently no effective treatment. Despite advances in the molecular pathology of the characteristic histopathological markers of the disease (tau protein and β-amyloid), their translation to the clinic has not provided the expected results. Increasing evidences have demonstrated the presence of aggregates of TDP-43 (TAR DNA binding protein 43) in the postmortem brains of patients diagnosed with AD. The present research is focused on of the study of the pathological role of TDP-43 in AD. For this purpose, immortalized lymphocytes samples from patients diagnosed with different severity of sporadic AD were used and the TDP-43 pathology was analyzed against controls, looking for differences in their fragmentation, phosphorylation and cellular location using Western blot and immunocytochemical techniques. The results revealed an increase in TDP-43 fragmentation, as well as increased phosphorylation and aberrant localization of TDP-43 in the cytosolic compartment of lymphocytes of patients diagnosed with severe AD. Moreover, a fragment of approximately 25 KD was found in the extracellular medium of cells derived from severe AD individuals that seem to have prion-like characteristics. We conclude that TDP-43 plays a key role in AD pathogenesis and its cell to cell propagation. Alzheimer’s Disease (AD) is a neurodegenerative disorder marked by progressive impairment of cognitive ability, generally in later life, affecting more than 50 million people worldwide . Age is The disease begins with a decline in cognition followed by a number of other changes in brain functioning, including impairments in language and visual-spatial skills, and disorientation. This impairment in cognitive functions is due to anatomical atrophy of AD brains, which correlates with severe neuronal loss . In partamyloid precursor protein (APP), presenilin-1 (PSEN-1), presenilin-2 (PSEN-2) [Based on age of onset, AD can be divided into early onset AD, and the more common, late-onset AD, in people over 65 years old, accounting for more than 90% of all AD cases . Most ca(PSEN-2) . On the AD is considered a multifactorial disease associated with a number of risk factors other than advanced age and genetic factors, such as environmental factors, diet, cardiovascular diseases, and diabetes, head injuries, and obesity among others ,11,12. RAD is characterized by two main neuropathological hallmarks: extracellular amyloid-β (Aβ) deposition in senile plaques and intracellular accumulations of hyperphosphorylated microtubule-associated tau, called neurofibrillary tangles (NFT) ,15,16.Based in the density of NFTs in various brain regions, AD cases have been classified in three different subtypes hippocampal-sparing, with lower NFT in hippocampus, limbic-predominant with lower cortical NFT, and the more frequent typical AD forms . In addiTDP-43 was originally found to associate with frontal temporal lobar degeneration with ubiquitin inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS) . These nThe manner in which TDP-43 pathology induces neuron degeneration is not yet completely understood, and the role of TDP-43 in cognition remains elusive, since conflicting results had been reported ,29. AppaTDP-43 is a predominantly nuclear protein, although it can shuttle between the nucleus and cytosol. It plays a variety of roles in RNA metabolism, including transcription, splicing, mRNA transport, mRNA stability through recruitment into stress granules (SGs), and microRNA biosynthesis ,32. UndeThe present work was undertaken to better understand the role and significance of TDP-43 in AD. To accomplish this goal, we assessed and compared pathological characteristics of TDP-43 of control and AD-derived immortalized lymphocytes. These lymphoblastoid cells lines had been extensively characterized in our laboratory, demonstrating that they display disease specific signature molecules ,37,38, aThe results presented here indicate an increase in TDP-43 fragmentation, as well as increased phosphorylation and aberrant localization of TDP-43 in the cytosolic compartment of lymphoblasts of patients diagnosed with severe AD. Moreover, a fragment of approximately 25 KDa was found in the extracellular medium of cells derived from AD individuals, that appears to be transported by extracellular vesicles (EVs) inducing TDP-43 pathological features and cytoskeletal changes in control cells. The data may suggest a role of these EVs containing fragmented TDP-43 in contributing to the propagation of AD disease in a prion-like manner.RPMI 1640 culture medium , DMEM culture medium , Fetal Bovine Serum (FBS) , penicillin/ streptomycin , polyvinylidene difluoride (PVDF) membranes for Western blots , enhanced chemiluminescence (ECL) system , Pierce BCA Protein Assay kit , protease inhibitor complete mini mixture .Antibodies used in this study were from Santa Cruz Biotechnologies , Cell Signal , Thermo Fisher , Molecular Probes (Thermo Fisher), Bio-Rad  or Proteintech,  and are listed in Healthy controls and patients were recruited from Hospital Doce de Octubre, Madrid Spain. The clinical diagnosis of probable AD was based on the criteria of the National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer’s Disease and Related Disorders Association (NINCDS-ADRDA) , and diaBlood samples (approximately 8 mL) were obtained through antecubital vein puncture in EDTA-treated tubes. Peripheral blood mononuclear cells (PBMCs) were isolated on Lymphoprep™ density-gradient centrifugation according to the instructions of the manufacturer .Establishment of lymphoblastoid cell lines (LCLs) was performed in our laboratory, by infecting peripheral blood lymphocytes with the Epstein Barr virus (EBV) .v/v) fetal bovine serum (FBS), and 1% penicillin/ streptomycin. U2OS cells were obtained from the American Type Culture Collection and grown in DMEM media, supplemented with 10% (v/v) fetal bovine serum and 1% penicillin/streptomycin. All cell lines were grown at 37 °C in a humidified 5% CO2 atmosphere.Lymphoblastoid cells were grown in suspension in T flasks in an upright position, in approximately 10 mL RPMI 1640 medium that contained 10%  and the associated software using the manufacturer’s recommended conditions. Each reaction was performed in biological triplicates with 20 ng of cDNA by using FastStart Essential DNA Green Master . The sequences of the forward and reverse primers used are the following: for hTDP-43 FW: 5′-GAGAAGTTCTTATGGTGCAG-3′and RV: 5′-TGGCTTTGCTTAGAATTAGG-3′, for hRPS17 FW: 5′-CCATTATCCCCAGCAAAAAG-3′ and RV: 5′-GAGACCTCAGGA ACATAATTG-3′. Data analysis was based on the ΔΔCT method with normalization of the raw data to hRPS17 housekeeping gene.Total RNA extraction, cDNA synthesis and quantitative polymerase chain reaction (PCR) was done as detailed in the previous article . Quantit6 cells × mL−1 and 24 h later, cells were harvested, washed in PBS and then lysed in ice-cold RIPA lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 15 mM MgCl2, 0.5% (vol/vol) sodium deoxycholate, 0.5% (vol/vol) NP-40 and 0.1% (vol/vol) SDS), containing 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonylfluride (PMSF), 1 mM sodium pyrophosphate and protease inhibitor mixture. The protein content of the extracts was determined by de Pierce BCA Protein Assay kit.To prepare whole cell extracts the lymphoblasts were seeded at initial density of 1 × 1050-µg of protein from cell extracts or 35 µL of extracellular medium were fractionated on an SDS polyacrylamide gel, transferred to PVDF membrane and blocked with 5% non-fat milk. The antibodies used are listed in 2B4O7·10H2O 15 mM, pH 8.4) overnight at 37 °C.Immunofluorescence analysis was performed on cells grown on coverslips. For the attachment of lymphoblastoid cells, the coverslips were previously coated with a solution of 0.025% Gelatin  for 30 min at room temperature followed by a solution of 1 mg/mL poly-L-lysine  diluted 1:50 in Borax buffer  following manufacturers’ recommendations. EV-containing pellet was resuspended in phosphate-buffered saline and stored at −20 °C until further analyses.To generate conditioned medium for extracellular vesicles isolation, lymphoblasts were grown in RPMI medium with 10% of exosome-depleted FBS , instead of normal FBS. After 72 h, conditioned medium was recollected by centrifugation at 2000× EV characterization was performed by nanoparticle tracking analysis (NTA) and Western Blot. Size distribution and quantification of EVs preparations were analyzed by measuring the rate of Brownian motion with a NanoSight LM10 system with a 630 nm laser , equipped with fast video capture and particle-tracking software. EVs samples were diluted 1:1000 in Hank’s balanced salt solution (HBSS), disaggregated and injected in the sample cubicle of the NanoSight. The mean of the number of particles acquired per milliliter was compared between AD and control cells and the measurements of EVs concentration (particles/mL) were normalized with the cell number from which conditioned medium containing EVs was recollected. For Western Blot analysis 20 µL of EVs were lysed with Laemmli buffer and denaturalized at 95 °C for 5 min. Western Blots were performed in parallel in EVs samples and cell lysates and the antibodies used are described in t-test for statistical comparisons between groups. A “p-value < 0.05” was considered statistically significant.Statistical analyses were performed using Graph Pad Prism software version 6 . Data are presented as means ± standard deviation (SD) of the mean. Statistical significance was estimated using one-way ANOVA followed by the Bonferroni test for multiple comparations, or, two-tailed Student’s To examine TDP-43 pathology in human lymphoblasts, we first analyzed the expression of TDP-43 mRNA and protein levels in immortalized lymphocytes from AD patients as a function of disease severity. Interestingly, a longer exposure of TDP-43 blots revealed the presence of a TDP-43 fragment of approximately 25–30 KDa perhaps due to TDP-43 cleavage, in most of the AD cases but not in control samples B.We then, analyzed the phosphorylation status of TDP-43 in control an AD  patients, using a phospho-specific antibody by Western blotting and confocal laser microscopy. As shown in Increased phosphorylation of TDP-43 had been associated with mislocalization of the protein in TDP-43 proteinopathies such as ALS and FTD . For thiMoreover, immunofluorescence staining for filamentous actin (F-actin) with Alexa Fluor-568 phalloidin, revealed cytoskeletal abnormalities in AD lymphoblasts A such asIt has been documented that full-length and fragmented TDP-43 may be released in extracellular vesicles or exosomes in different neurological diseases. For this reason, we first preformed Western blot analysis looking for TDP-43 protein in the extracellular medium of control and AD lymphoblasts. Furthermore, CM from AD lymphoblasts induces also TDP-43 pathological changes, mislocalization and generation of TDP-43 aggregates in osteosarcoma U2OS cell line .Finally, we isolated extracellular vesicles (EVs) from CM of control and AD cells, and NTA analysis was used to identify their size and concentration. As shown in Over the past decade, TDP-43 deposition has been associated with an increasing number of neurodegenerative diseases  being thIn this study we have examined some TDP-43 features in peripheral cells from AD patients as a function of disease severity. For this purpose, a comparative study on TDP-43 expression levels as well as TDP-43 phosphorylation pattern, protein fragmentation and subcellular localization was performed in lymphoblasts derived from control subjects and mild, moderate and severe AD patients.The results herein presented show the presence of pathological features of TDP-43 in these easily accessible patient-derived cell lines, that could be considered systemic manifestations of the disease. Increased TDP-43 phosphorylation, protein cleavage, and cytosolic TDP-43 accumulation are more evident in lymphoblasts from severe AD patients. Moreover, higher expression of TDP-43 mRNA was observed in moderate and severe AD cases, which may be the result of increased self-regulation of TDP-43 expression, in order to maintain homeostasis in late stages of AD . These oTDP-43 pathological changes observed in AD lymphoblasts are similar to those found in lymphoblastoid cell lines derived from FTLD-TDP and ALS patients ,40, despNowadays, there is an expanding field of research trying to elucidate how TDP-43 pathology may be mechanistically related with AD, especially with the limbic-predominant subtype, in which TDP-43 deposition is more frequent . It is nTDP-43 may participate in various pathogenic mechanisms underlying AD, such as amyloid β deposition, tau hyperphosphorylation, mitochondrial dysfunction, and neuroinflammation . TDP-43 TDP-43 pathological features, such as increased phosphorylation, cleavage, aggregation and cytoplasmic accumulation are believed to impaired neuronal function . It is sSince TDP-43 protein contains a prion-like domain in its C-terminus, it was hypothesized that TDP-43 toxic species could be transmitted intercellularly . TherefoFinally, it is worth highlighting that fragmented TDP-43 (25 KDa) and, in less amount, full-length TDP-43 were detected in EVs isolated from AD lymphoblasts. EVs have been shown to participate in the communication and transfer of cargo among different cell types within the CNS , modulatWe confirmed that TDP-43 pathology is present in peripheral cells from AD patients, being exacerbated with disease severity. Thus, protein kinase inhibitors able to modulate TDP-43 emerge as promising agents for AD including those patients in more advanced stages. Furthermore, the addition of these treatments to those able to decrease amyloid-β may open new hopes for AD therapy. A prion-like disease propagation of TDP-43 pathology via actin protrusions and secretion of fragmented TDP-43 in EVs is in line with the finding that conditioned medium from AD cells provoked TDP-43 phosphorylation and cytoplasmic accumulation in control cells. Although further work is warranted for a better understanding of mechanisms involved in disease dissemination and to elucidate the possible interactions of pathological TDP-43 with other AD-associated molecules, amyloid-β and tau, for patient stratification and development of novel therapeutic strategies, this last discovery support TDP-43 potential therapies as disease-modifying agents able to modulate the neurodegeneration.The results of this study are somehow limited by the scarce number of patients included in the analysis. The patient-sample set should be increased to validate these results. We believe that the analysis of the complex interactions among TDP-43, amyloid-β, and tau may open new areas of therapeutic interest for the treatment of this devastating disease, mainly based in multitarget approaches. Research aiming to study the influence of protein kinase inhibitors able to recover TDP-43 homeostasis, in pathogenic mechanisms underlying AD pathology and spreading is ongoing in our laboratory.


GAP-43 is a vertebrate neuron-specific protein and that is strongly related to axon growth and regeneration; thus, this protein has been utilized as a classical molecular marker of these events and growth cones. Although GAP-43 was biochemically characterized more than a quarter century ago, how this protein is related to these events is still not clear. Recently, we identified many phosphorylation sites in the growth cone membrane proteins of rodent brains. Two phosphorylation sites of GAP-43, S96 and T172, were found within the top 10 hit sites among all proteins. S96 has already been characterized , and here, phosphorylation of T172 was characterized. In vitro (cultured neurons) and in vivo, an antibody specific to phosphorylated T172 (pT172 antibody) specifically recognized cultured growth cones and growing axons in developing mouse neurons, respectively. Immunoblotting showed that pT172 antigens were more rapidly downregulated throughout development than those of pS96 antibody. From the primary structure, this phosphorylation site was predicted to be conserved in a wide range of animals including primates. In the developing marmoset brainstem and in differentiated neurons derived from human induced pluripotent stem cells, immunoreactivity with pT172 antibody revealed patterns similar to those in mice. pT172 antibody also labeled regenerating axons following sciatic nerve injury. Taken together, the T172 residue is widely conserved in a wide range of mammals including primates, and pT172 is a new candidate molecular marker for growing axons. Growth-associated protein of 43-kDa  is a neuron-specific protein in vertebrates and is related to axon growth and regeneration –4. Thus,2+-independent manner , 5 μM of SB203580 (Sigma-Aldrich) as a p38 inhibitor, or 5 μM of U0126  as a MEK1/2 inhibitor that at this concentration works as an ERK inhibitor ) for reducing background staining for 10 min. They were then permeabilized with 0.1% Triton X-100  in CSB for 10 min, were blocked with 3% bovine serum albumin  for 30 min, and reacted with primary and secondary Abs in 3% BSA. To visualize F-actin, the cells were incubated with rhodamine-phalloidin . Quantitative distribution of the region of interest (ROI) of the growth cone area was measured by fluorescence intensity along a line plot from the filopodial tip to display a two-dimensional graph of pixel intensity for pT172 and F-actin images using ImageJ software Fiji (http://rsb.info.nih.gov/ij). The x-axis represents the distance along the line, and the y-axis is the normalized gray value.Mouse E15 cortical or hippocampal neurons were differentiated into stage 3, when each neuron has an axon , and immFluorescent images were acquired with a confocal laser scanning microscope  as previously described . For linThe ratio of the fluorescence values of GAP-43 and pT172 in the longest neurite defined as an axon and the second longest neurite defined as a dendrite was obtained using Fiji. The average fluorescence intensity of each ROI was measured.Human induced pluripotent stem cells (1210B2), which were established from peripheral blood mononuclear cells  at the Center for iPS Cell Research and Application, Kyoto University  by an integration-free reprogramming method, were induced into neural progenitor cells using the dual SMAD-inhibition method with dorsomorphin plus SB431542 as previously described , 66. hiPThe shotgun proteomics method was described previously , 13. BriThe gel slices were reduced with 10 mM dithiothreitol, carbamide methylated with 55 mM iodoacetamide, and subjected to in-gel trypsin digestion to improve recovery of the digested peptides. The peptides from each sample were finally dissolved in 15 μl of 0.3% trifluoroacetic acid. A 50-kDa-level gel sample (3 μl of 1/5 dilute solution) and a 10-kDa-level gel sample (4 μl) were injected into a nano-flow-LC  coupled with a tandem MS .Analysis was conducted in triplicate for each sample under direct injection mode with an analytical column of 75 μm × 150 mm . Mobile phases A and B were 0.1% formic acid and 0.1% formic acid in acetonitrile, respectively. Peptides were eluted using 40 (10 kDa) or 60 (50 kDa)-min gradients from 2 to 32% B at 300 nl/min. MS spectra (250 ms) followed by 10 MS/MS spectra (100 ms each) were acquired in data-dependent mode. The dynamic exclusion time was set at 5 s. Autocalibration using 50 fmol of tryptic peptides of bovine serum albumin was performed. Protein identification was carried out using Mascot version 2.6.0 (Matrix Science) as a search engine  protein database was used to search for amino acid sequences in human, cynomolgus monkey, marmoset, ferret, rat, and mouse GAP-43. The GAP-43 protein sequence of ferret was that of the predicted one. Multiple sequence alignment analysis was displayed using these search results by Snap gene software .t test and one-way analysis of variance (ANOVA) were used for statistical analysis, as appropriate, and a p-value < 0.05 was considered statistically significant. Significance testing using one-way ANOVA was further performed followed by a post hoc test using Bonferroni methods. All statistical analyses were performed using GraphPad Prism 8 software.All data are shown as the means ± standard deviation (SD). Paired Student’s Additional file 1: Table 1. Antibodies used for immunological detection in this paper.Additional file 2: Figure S1. Intracellular localization of phosphorylated GAP-43 T172 and its responsible kinase. (a) As well as pan-GAP-43 Ab (left), the immunoreactivity of pT172 Ab on full-size membranes (right), showing a wider range of molecular masses, indicated that this Ab mostly recognizes a single band of mouse GAP-43 (mGAP-43) in the total proteins derived from the lysate of cultured mouse cortical neurons (DIV3). (b) In vitro phosphorylation of GAP-43 by activated JNK. Myc-GAP-43 was immunoprecipitaed using anti-myc and used as the JNK substrate. Phosphorylation experiments were performed following the manufacturer’s manual of “JNK1, Acitve” . (c) Immunostaining of cultured hippocampal neurons using pT172 Ab (blue) with rhodamine-phalloidin (red) and Tuj-1 Ab (green). Note that pT172Ab labeled the tubulin-positive area of the axon more intensely than the actin-positive area. Scale bar: 20 μm. (d) pT172Ab immunoreactivity in the growth cone was colocalized with GAP-43  at ROI (white box). Scale bar: 10 μm.Additional file 3: Figure S2. pT172 and GAP-43 are regulated in mouse developing brain. (a) The distribution of GAP-43, pS96, and pT172 persists throughout life . GAPDH: positive control. The frontal cortical lysates of a C57BL6N mouse were used. (b) Densitometric quantitation of (a). Values are represented as the means ± SD for three experiments. Although some GAP-43 expression was maintained throughout life, the immunoreactivity of pT172 and pS96 decreased rapidly with development. One-way ANOVA followed by a post hoc test using the Bonferroni method. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. (c) Immunoblot for pT172Ab on full-size membranes. Note that this Ab mostly recognizes a single band of GAP-43 in the total protein of cortical lysate.Additional file 4: Figure S3.. Expression pattern of pT172 in the developing mouse brain. (a) Negative control immunostaining of E15 thalamocortical pathway. pT172 pAb (a-1), secondary Ab alone , and nonspecific IgG . Each concentration of proteins was 0.26 µg/ml. Scale bar: 200 µm. (b) Microscopic images of sagittal sections derived from various brain regions were DAB-stained using pT172. Boxes in (b) represent the regions enlarged in (c-g). pT172 succeeded in labeling nerve fibers under a growing state. Scale bar: 500 µm in (b), 200 µm in .  Z-stack maximum-intensity projection images of basal nuclei (h) and OB (i). pT172 was more closely colocalized in the developing axons labeled by L1 than pan-GAP-43 in OC, LOT, and STR. GAP-43 and pT172 were detected in neurons of MCL and ONL (i). CP: cortical plate; LOT: longitudinal olfactory tract; MCL: mitral cell layer; OB: olfactory bulb; OCh: optic chiasm; ONL: olfactory nerve layer; Teg: longitudinal tegmental tracts; STR: striatum; PON: pons; EGL: external granular layer, PK: Purkinje cell layer.Additional file 5: Figure S4. pT172Ab immunoreactivity in regenerating axons after neural tissue injury in young adult mice.  pT172Ab immunoreactivity detected in injured sciatic nerve (day 1 (a) and day 3 (b) after crush injury). Black-arrowhead: damaged point; white-arrowhead: the farthest point of immunoreactivity. (c) pT172Ab immunoreactivity was not present in uninjured sciatic nerves, while GAP-43 reactivity itself was detected there. Scale bar: 500 μm. (a-c). See also Fig. 3.Additional file 6: Figure S5. Amino acid sequences of GAP-43 surrounding the residue corresponding to S96 of rodents, in various mammalian species. S96 position of rodent GAP-43 is shown in red. See Discussion.Additional file 7: Figure S6. Phospho-specificity of GAP-43 pT181 in the P1 marmoset. (a) Negative control immunostaining of P1 Marmoset visual cortex. pT172 pAb (a-1), secondary Ab alone , and nonspecific IgG . See also Fig. S3. Scale bar; 500 µm. (b) Immunoblot reactivity of pT172 Ab on a full-sized blot membrane. These results show the specificity of pT172Ab. (c) Phosphoproteomics procedure for the mid-brain including pons of P1 marmoset. The band corresponding to GAP-43 was cut out after SDS-PAGE of the sample, trypsinized in-gel, and subjected to MS analysis. *, molecular mass marker. Note that the GAP-43 immunoreactivity band (arrow in red) did not consist of the GAP-43 amino acid sequence by LC/MS/MS analysis, suggesting that it may be a non-specific reaction. The analyzed results are shown in Fig. 5p.Additional file 8: Figure S7. Stress-dependent JNK activation phosphorylates human GAP-43 T181 and is recognized by pT172Ab. Extracts from the transfected U251G cells were prepared at 30 min after osmotic stress (0.5 M NaCl) for JNK activation and immunoblotted using pT172, GAP-43, GFP, pJNK, JNK, and GAPDH Abs by the methods of Kawasaki et al. (2018).


Three-dimensional graphene-based aerogels (3D GAs), combining the intrinsic properties of graphene and 3D porous structure, have attracted increasing research interest in varied fields with potential application. Some related reviews focusing on applications in photoredox catalysis, biomedicine, energy storage, supercapacitor or other single aspect have provided valuable insights into the current status of Gas. However, systematic reviews concentrating on the diverse applications of 3D GAs are still scarce. Herein, we intend to afford a comprehensive summary to the recent progress in the preparation method (template-free and template-directed method) summarized in Preparation Strategies and the application fields  illustrated in Application of 3D GAs with varied morphologies, structures, and properties. Meanwhile, some unsettled issues, existing challenges, and potential opportunities have also been proposed in Future Perspectives to spur further research interest into synthesizing finer 3D GAs and exploring wider and closer practical applications. Aerogel, generated by the replacement of liquid inside a gel with gas by freeze-drying or supercritical drying technique, was first presented by S. Kistler in the 1930s ,3,4. The2/g), chemical stability, and high electron mobility, as well as excellent mechanical and optical properties, which has great application potential in many fields [As the most important component of two-dimensional (2D) carbon-based material, graphene possesses superior thermal conductivity (~5000 W/m·K), high specific surface area , hydrazine vapor, Fe (II), sodium hydrogen sulfite (NaHSO3), sodium sulfide (Na2S), and vitamin C and hydrogen iodide (HI) [3O4 aerogel via in situ self-assembly of graphene with NaHSO3 as the reducing agent in the presence of Fe3O4 nanoparticles. The capture of nanoparticles was similar to a fishing process, wherein nanoparticles acted as the fish and graphene/GO nanosheets served as the fishing net that was formed through the self-assembly of GO during reduction. Similarly, Li et al. [ide (HI) ,41. The ide (HI) ,42, Yan ide (HI)  preparedi et al.  proposedi et al. , moving i et al.  developeHydrothermal reduction is another facile and effective strategy to prepare graphene from GO. The reduction degree is determined by reaction temperature and time. Generally speaking, the higher the temperature, the higher the reduction degree. However, there is no consensus on the required time and temperature of the heat-treatment for complete reduction of GO [2-graphene aerogel (SGA), which involved the synchronous processes of hydrothermal-induced reduction of GO, self-assembly of graphene sheets, and in suit growth of SnO2 nanoparticles, presented in on of GO ,48. Moston of GO . Li and on of GO  proposedElectrochemical reduction is an appealing strategy used to form 3D GA. Unlike other self-assembly methods that rely on the unique shape of graphene sheets and features of the second components and whose materials are bulky monoliths and unsuitable for electrode modification, electrochemical reduction-induced self-assembly approach is believed to be the most promising means to fabricate a 3D structure applied in electrode material. Chen et al. [n et al.  proposedThe abundant oxygen-containing functional groups on their basal planes and edges make GO sheets sensitive to the experimental conditions, and only assemble into 3D microstructure under certain conditions . The obtPhysical cross-linking refers to linking GO sheets and a crosslinker mainly by hydrogen bonding, as illustrated in 3O4 nanoparticles and GO nano-sheets).Chemical cross-linking refers to linking GO sheets and a crosslinker by covalent bonding, which has the characteristics of high uniformity and stabilization. Ye et al. [e et al.  developeDuring the synthesis process, hydrogen bonding and/or covalent bonding between crosslinker and GO sheets accounts for forming the cross-linking sites, which contribute to the generation of hydrogel with a 3D network. Such aerogels usually show significantly improved mechanical properties. However, the introduction of cross-linkers inevitably sacrifices the electrical conductivity of GAs to a certain extent. So, there is a fundamental tradeoff to grasp. Utilizing conductive polymer as a crosslinker to fabricate highly compressible and electrically conductive 3DGAs might be a new attempt.The template-directed strategy, including the emulsion technique, breath figure method, and ink-printing technique, is one of the mainstream approaches employed in the formation of 3D GA with ordered and hierarchical structures . It usesHard templating strategy, using solid nanoparticles, porous metal foams, and specific movable nozzles as a guide is conceptually the simplest method to synthesize 3D graphene aerogel with the desired microstructure and performance . In geneCompared with the hard template approach, the soft template method with the emulsion droplet, vesicle/micelle, and gas bubble as templates is easier due to the unnecessity of removing the templates . AlthougThe carbon aerogels  prepared by various methods generally have lower density than the graphene/GO-based hybrid (other nanoparticle or polymers) aerogels. Moreover, for the same GO aerogels obtained by the two-stage preparation procedure involving subsequent microwave irradiation (MWI) or thermal treatment, which further eliminates functional groups after freeze-drying, are ultralight and their density came down to a lower scale compared with those prepared via one-step. However, in any case, all the as-prepared GA possess an interaction network and hierarchical pores ranging from micropores to mesopores and macropores, which can be characterized by SEM and the nitrogen adsorption/desorption isotherm . The porGraphene aerogel, with characteristics of low density, high surface area and porosity, and good electrical and thermal conductivity, has attracted the attention of researchers and flourished in the recent decades . They poWater contamination caused by harmful chemicals, particularly oils and soluble dyes and phosphate, has become an issue of serious global concern. Various technologies including chemical precipitation ,78, bioln-decane adsorption as an example, 8.5 mg of aerogel can completely adsorb 1.4 g of oil within 6 s. The average absorption rate (27 g/g·s) is much faster than that of pure graphene (0.57 g/g·s). The capacity for absorption is up to 120–250 g/g, depending on the liquid densitY.S.milarly, Gao et al. [n-hexane, crude oil, toluene, motor oil, vegetable oil, ionic liquid, and phenixin)  d, fallin3), high surface area (214.75 m2/g), paramagnetic property (26.59 emu/g), excellent adsorbent efficiency (93%) for cationic malachite green (MG) dye through the Yoshida H-bonding, dipole-dipole H-bonding, π-π interaction, n-π interaction, electrostatic attraction, and physical adsorption, as shown in 4), once adhered to the graphene or GO, will show great application potential in capturing phosphates in water, Losic et al. [3O4 nanoparticles, respectively. The prepared aerogels showed superior capacity of 350 mg/g at an initial phosphate concentration of 200 mg/L under acidic conditions and the adsorption process followed the second order model and Freundlich isotherm  with high energy density and voltage, which can store and supply electricity, have a wide range of applications with the development of modern electronic devices. Currently, the biggest challenge is to develop durable, nontoxic, and inexpensive materials for electrodes.3O4 [3O4 [3O4 [2 [2O4 [3O4 aerogel via in situ self-assembly of graphene in the presence of Fe3O4 nanoparticles. The graphene/Fe3O4 composite aerogel showed a high capacity and cyclic stability (a remaining capacity of 1100 mA h/g after 50 cycles of charge and discharge), meaning it can work well as an anode for LIBs, as shown in 3O4 nanoparticles working as spacer and graphene nanosheets, as both sides can adsorb lithium ions [2-graphene aerogel (SGA), which displays a well-defined and interconnected network porous microstructure and possesses higher capacity retention and discharge capacity (602 mA h/g after 60 cycles) compared to other SG composites , with mechanical stability under dynamic tension and compression deformations, can find potential applications in strain sensors, stretchable components, and lightweight mechanical devices.Another approach is to enhance the interconnection of aerogels to produce a hierarchical structure by adopting freeze-shaping, 3D ink-printing, and other synergistic assembly techniques ,99. Guo Based on the major advances made in the design of flexible 3D graphene aerogels, the trend is to push their structure towards a smarter and more well-controlled system , in ordeThe existing commercial fire-warning equipment, including temperature, smoke, and infrared flame detectors, is usually unsatisfactory , as theyWith the increase of temperature, the electrical resistance of GO decreases dramatically, which endows it an attractive application prospect in fire-warning materials ,104,105.The GO modified aerogel with excellent flame retardancy, thermal isolation, and intrinsic fire warning performance broadens its application territory to cover the drawbacks in delayed response and restricted application scenarios of traditional fire detectors. Therefore, the aerogel that can be triggered in the precombustion stage to offer favorable opportunities for firefighting and emergency rescue is endowed with enticing prospects in chemical industries, pipeline transportation, and high-rise buildings.2 reduction [A 3D network structure provides multidimensional electron transport pathways and large accessible surface area, which is conducive to improve the separation efficiency of photogenerated electron-hole pairs and the adsorption of reactants. Such intrinsic hierarchical porous structure characteristics and properties make GAs endowed with potential as promising and efficient photocatalysts for practical applications in solar energy conversion , such aseduction , and cheeduction .VI, showing that after visible light irradiation for 8 min, the MO was completely degraded by AgBr/GAs compared with the 65% of degradation rate by AgBr  and reducing CO2 to CH4 via acquiring 8-electrons process (CO2 + 8H+ + 8e–→CH4 + 2H2O) [2, with higher catalytic efficiency compared to other semiconductors. Tong and co-workers [3N4/GO aerogel (CNGA) by the hydrothermal induced self-assembly method, in which g-C3N4 acted as the efficient photocatalyst, and GO was responsible for supporting the 3D framework. The as-prepared GAs could reduce CO2 into CO with a high yield of 23 mmol/g (within 6 h), which was about 2.3-fold increment compared to pure g-C3N4  . The 3D -workers  designede g-C3N4 . That isThe 3D GAs also play an important role in catalyzing some chemical reactions, such as the selective oxidation of alcohol to carbonyl, the reduction of nitroaromatic compound to amino compound, and the synthesis of ammonia . Yang etThere is no doubt that considerable advances have been achieved in the fabrication of 3D GA materials in the past few decades. Various preparation methods involving a series of template-free and template-guided methods are proposed to fabricate 3D GAs with integrated properties of graphene and other unique characters. The excellent properties make them attractive and promising in potential applications in varied fields, which brings us closer to practical applications. In this review, we summarized the advancement of literature, historical progress of synthesis of 3D GAs, and introduced the main application in absorption, anode material, mechanical device, fire-warning material, and catalysis aspects.p-conjugation compared to pure graphene, which causes the improvement of electrical correlation performance of 3D GAs to often be restricted. Therefore, rational utilization of graphene with superior electrical conductivity to advance the 3D GAs still needs continuous efforts. Secondly, the widely used drying methods of preparation of aerogel are mainly freeze-drying and supercritical drying [Currently, although dramatic progress has been realized, the performance and application filed of 3D GAs has not been fully exploited. There are still many challenges toward the preparation and mechanism elaboration of highly efficient 3D GAs. Firstly, most of the 3D GAs are derived from the reduction of GO, so their electrical conductivity and charge carrier mobility are remarkably decreased due to the disruption of l drying . Freeze-l drying . Therefol drying . TherefoWith the output of constant efforts, we believe that substantial breakthroughs for the finer structural control and practical applications of 3D GAs would be expected in the near future.


Considering the emerging use of internet-based psychotherapy during the COVID-19 pandemic, information on videoconference-based EMDR (eEMDR) would be beneficial for many therapists. In this study, 23 therapists from the EMDR-Institute in Germany provided information about their experiences with eEMDR in a questionnaire-based survey. Information on the effectiveness and the course of 102 eEMDR sessions was recorded. Results showed the potential of eEMDR as an effective and viable method. The decrease in the subjective unit of disturbance (SUD), which is an important indicator of treatment outcome, was found to be at a similar level compared to that of previous EMDR studies that were not administered in eEMDR format. The most important predictor of the SUD decrease was the type of bilateral stimulation used in eEMDR sessions. Eye movements resulted in significantly greater SUD reductions than tapping. Perceived disadvantages and impediments for the implementation of eEMDR were mainly of bureaucratic and technical concerns. In addition, about one-third of the therapists stated that some patients were not willing to engage in eEMDR. In our study, eEMDR proved to be a practically applicable therapy method and therefore, therapists can consider using eEMDR. These findings will hopefully encourage EMDR therapists and their patients to use eEMDR due to its effectiveness and viability as an online treatment approach.Research on the effectiveness and applicability of eye movement desensitization and reprocessing (EMDR)  Mental health consequences of the COVID-19 pandemic suggest a high demand for psychological support during this unprecedented situation e.g., . At the Eye movement desensitization and reprocessing (EMDR) as well as trauma-focused Cognitive-behavioral therapy (CBT) are the primary choice for treatment of PTSD . EMDR isvia videoconference is feasible and appropriate ; however, they do not provide general conclusions about the effectiveness of eEMDR in an outpatient treatment setting. In times of COVID-19 and beyond, improvements in the provision of eEMDR and information on its effectiveness in treating PTSD especially in routine outpatient care are strongly required.A recently published review showed a lack of available evidence on effects of internet-based EMDR . Furtherropriate . As of nropriate . In the nce SUD; , signifiTo address this research gap, we studied the effectiveness of eEMDR on patients with PTSD and other diagnoses within the framework of an explorative approach. For the study purpose, we consulted therapists from the EMDR-Institute in Germany regarding their experiences with eEMDR sessions during the COVID-19 pandemic. Our study focused on obtaining information regarding the effectiveness of eEMDR sessions in a standard outpatient routine treatment, i.e., ratings of the EMDR process and the SUD decrease in patients reported during an eEMDR session. Notably, the SUD decrease can be seen as an important tool for therapists to assess the treatment process . Potentivia email, and 32 (8%) of them responded and returned filled-in questionnaires. Therapists were mainly female (81.3%) and more than half of them (65.6%) were aged between 51 and 65years. They had a mean professional experience of 19.77years  and a mean EMDR experience of 10.72years . Therapists were mainly trained in administering CBT (56.3%) and depth psychology (40.6%). Some therapists (12.5%) indicated they have already had experience with online-based psychotherapy before the COVID-19 pandemic.Four hundred EMDR-therapists were addressed M=41, SD=12.09). On average, patients had received 4.84 eEMDR sessions . Regarding their mental health, roughly 50% of the patient cohort fulfilled criteria of one diagnosis, while the other half of the patients had multiple comorbid diagnosis  and aged between 18 and 68years  and eventually, 102 protocols were available for evaluation. SD=19.75, range=30–100) minutes.Therapists were requested to return eEMDR session protocols of at least three different patients. Nine therapists did not return protocols because they had not yet conducted eEMDR. However, they provided information on generic questions like perceived barriers to online therapy, alternatives to eEMDR in COVID-19 pandemic, and experience with online therapy in general. The remaining 23 therapists on average returned 4.44 eEMDR session protocols , their experiences with online-based psychotherapy  and provided description of the technical as well as organizational measures they implemented to administer eEMDR .Therapists reported sociodemographic data, information about their professional background , technical aspects of the eEMDR session , and ratings concerning the quality of the eEMDR session . Moreover, on qualitative items, the therapists reported their opinion on advantages and disadvantages of eEMDR as well as adjustments and improvements necessary for the further use of eEMDR.via e-mail to therapists from the EMDR network in Germany. The e-mail informed therapists about the purpose and all procedures of the study as well as data protection issues. Most of the questionnaires were returned via postal mail; only a few were returned via e-mail. All study procedures were approved by the ethics committee of Ulm University.Questionnaires were sent Data were prepared and analyzed with IBM SPSS statistics (version 26.0.0.0) and R (version 1.3.1093). Our aim was to relate patient and therapist characteristics to quantified session effectiveness. Decrease in SUD ratings was analyzed for associations with potentially influential variables using Spearman’s rank correlation coefficients and Pearson’s correlation coefficients where applicable. Group comparisons with potential predictors of SUD decrease were computed with Kruskal-Wallis tests. Bonferroni-Holm corrections were conducted to correct the results for multiple testing. A multiple linear regression analysis was computed to examine the combined relevance of various predictors of SUD decrease. Qualitative statements about therapists’ attitudes towards eEMDR were collected as free text. The statements were reviewed, and frequency statistics were derived based on superior categories. This classification was crosschecked by one of the other authors (VT).In most eEMDR sessions, patients used computers or laptops (86.8%) followed by smartphones (18.4%). Likewise, therapists conducted the sessions mainly on computers or laptops (95.7%), followed by smartphones (8.7%), and tablets (8.7%). Some therapists also switched between different technical devices. Occasionally, they used additional equipment, i.e., headsets (30.4%), telephones (17.4%), and additional cameras (8.7%).Eye movement desensitization and reprocessing is a treatment algorithm developed by Shapiro that uses 8 phases and 3 temporal focusing steps to process stressful material. EMD is a modified EMDR protocol that focuses on a single high charge memory. EMDr is another slightly differently modified EMDR protocol that focuses on a single memory and allows only a limited range of associations in the processing phase . The stavia eye movements and in 36.3% of the sessions via tapping. In 7.8% of the sessions, both modes of bilateral stimulation were combined. When using eye movements for bilateral stimulation, supportive tools  were often (51.9%) used to evoke eye movements. Less often (31.5%), therapists guided the patients’ eyes with their fingers, while acoustical guidance (9.3%) and instructing the patients to switch their gaze between room corners (5.6%) were rarely used.In 52.9% of the sessions, bilateral stimulation was administered In most of the sessions, patient and therapist agreed on raising the hand as a stop signal (43.8%), while in 20.8% of the sessions, patient and therapist agreed on saying “stop,” and in 15.6% of the sessions, both signals were used concomitantly. Rarely, other stop gestures  were agreed.good or very good (Mdn=5 very good), and no session has been considered very bad as compared to face-to-face EMDR.In roughly 90% of the sessions, the therapists rated the overall impression of eEMDR sessions as very good . Similarly, when asked more specifically about the impression of the eEMDR process, therapists also submitted very good ratings. M=73.1% from the beginning to the end of an eEMDR session =8.82, p=0.116. The SUD decrease was lower in sessions with patients who have already had more eEMDR sessions, r=−0.23, p=0.049.Correlational analyses indicated significant associations neither between the therapists’ general professional experience and the relative SUD decrease, χ2(2)=14.11, p<0.001. As displayed in post hoc tests showed, eye movements have been more effective in reducing the SUD (Mdn =1) than tapping (Mdn =0.68).The relative SUD decrease differed significantly depending on the mode of bilateral stimulation, F=3.93, p=0.006, R2=18.1%. The analyses corroborated the relevance of the mode of bilateral stimulation as a significant predictor of the relative SUD decrease. However, the therapists’ general work experience and EMDR experience were not of relevance , while there was no difference between eye movements and combined eye movements and tapping (pHolm=0.166) and tapping and combined eye movements and tapping (pHolm=0.649).Linear Therapists most frequently reported that unstable internet connection either on patient or therapist side to be a barrier to implement eEMDR (37.5%). Several therapists also indicated that their patients refused online-based therapy (31.3%). Some therapists considered lack of personal contact (28.1%) and missing withdrawal options for the patients in their flat (21.9%) as barriers. 6.3% of the therapists considered their own media and technology skills or their patients’ cognitive or sensory limitations as barriers. Interestingly, no therapist indicated high impulsivity of patients as a barrier for online therapy.Furthermore, therapists were asked to report advantages and disadvantages of eEMDR sessions compared to face-to-face EMDR sessions as free text. Qualitative statements about advantages and disadvantages were reviewed and combined to superior categories. Results and frequencies of reported disadvantages can be seen in Difficulties with internet connection and dissociating patients were frequent answers concerning perceived disadvantages of eEMDR sessions. We therefore took a closer look at the actual connectivity issues and occurrence of dissociation during eEMDR sessions. Results showed that in 13.7% of the sessions, the connection was interrupted. Nevertheless, a SUD decrease of 68.3% could be accomplished in those sessions, and half of those sessions could be ended completely. Dissociations occurred in patients in 11.8% of the sessions. In those sessions, the mean relative SUD decrease was 53%, and one-third of those sessions could be ended as complete sessions.not to offer eEMDR post pandemic. Qualitative statements indicated, the reasons against and in favor of continuing to offer eEMDR coincided with its perceived disadvantages and advantages. Besides, some therapists suggested eEMDR could be inappropriate for certain patients, including patients with dissociation and severe mental disease status. With appropriate patient groups, however, therapists stated they consider continuing to offer eEMDR beyond the COVID-19 pandemic.Vast majority (87%) of the therapists stated, they would continue offering eEMDR beyond the COVID-19 pandemic. Conversely, 13% of the therapists considered In roughly half of the session protocols, the therapists provided information about their ideas and wishes for adjustments and improvements with respect to the future use of eEMDR beyond the COVID-19 pandemic. Answers were clustered into four categories as shown in good or very good. In this study, the most important indicator of effectiveness was the extent to which eEMDR enabled to reduce the patient-rated SUD from beginning to end of the session. According to Responses from therapists and the patients’ relative SUD decrease suggest eEMDR sessions can be an efficient and practically applicable alternative for face-to-face EMDR. Throughout eEMDR sessions examined in this study, therapist ratings of adherence, confrontation, grounding, and process were consistently positive, and the quality of more than 90% of the sessions was rated as Importantly, the SUD decrease was independent from the therapists’ professional experience or their EMDR experience. The question whether therapists’ experience influences treatment outcome has been debated since the origins of psychotherapy  and findvia eye movements enabled significantly stronger decrease in the patients’ SUD than the use of tapping or a combination of eye movements and tapping. These results were also corroborated in a regression analysis when controlling the therapists’ professional and EMDR experience. With regard to the debate on the effectiveness of different modes of bilateral stimulation . However, technology and connectivity issues are concerns that can be resolved through further network expansion throughout the country  and also by providing adequate technical equipment. According to the therapists, one of the advantages of eEMDR compared to face-to-face EMDR is that the patients would better focus on and engage in the therapy process. Some therapists also observed positive implications of the patients’ domestic surroundings . The barriers mentioned by therapists were not of therapeutic concerns, but rather related to the bureaucratic and technical framework of eEMDR. Importantly, more than 80% of the therapists participating in our study indicated they consider offering eEMDR beyond the COVID-19 pandemic, and this underscores the potential of eEMDR.The present study is of explorative nature and it focused on the outcome from a single eEMDR session instead of an entire EMDR therapy. In addition, there was no comparison group to compare the effects of eEMDR to a waitlist-control group, face-to-face EMDR, or other internet-delivered therapy methods. Although this was not the intention of our exploratory study, randomized clinical trials are clearly warranted to ascertain the effectiveness of eEMDR as compared to its face-to-face version and other therapy approaches.via videoconference but also to a lack of time and capacity considering the strenuous workload for therapists due to the pandemic situation.Notably, only 8% of the contacted therapists decided to participate, thus representing a selective and potentially biased subgroup of all therapists. The low response rate may be attributed to the restraint to offer EMDR The questionnaire asked therapists regarding their attitude and experiences with eEMDR. There could be therapist bias due to social desirability, especially regarding effectiveness measures as they may have chosen not to report on those eEMDR sessions which were assessed as ineffective. However, in the sessions where the standard protocol was applied, nearly half of the sessions ended incompletely, thus dispelling the presumption that therapists selectively returned protocols of only effective sessions. Furthermore, patient-rated SUD values might be also biased by social desirability.Future research could assess the experiences and compliance of patients participating in eEMDR, as this will provide additional insights for improving effectiveness of eEMDR. Furthermore, studies could investigate which patients benefit most from eEMDR formats and conversely, which diagnoses, or patient characteristics contraindicate the use of eEMDR.via videoconference (This study was one of the first to address the effectiveness of eEMDR by asking EMDR-therapists about their experiences with this treatment format. Our results show eEMDR as an effective and viable alternative to face-to-face EMDR. Especially the high SUD decrease in eEMDR sessions, an important indicator of treatment outcome was very promising. Thus, the results help to dispel doubts regarding the feasibility and appropriateness of EMDR nference .Therapists can consider conducting eEMDR even beyond the COVID-19 pandemic situation, as perceived impediments and disadvantages were mainly related to the bureaucratic and technical framework of eEMDR. However, with certain adjustments to the framework it is feasible to overcome these barriers. Thus, eEMDR has the potential to not only be a temporary solution during the pandemic but to become an integral part of everyday therapy.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent from the patients/ participants or patients/participants legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements.VT conceptualized the study, obtained ethical approval, and recruited therapists. CM wrote the manuscript under supervision of VT and AB, and performed statisical analysis together with LM under supervision of AB. AH provided expertise and feedback and recruited therapists. ML provided data. SV and RR edited the manuscript. I-TK supervised the study, revised and edited the manuscript. All authors contributed to the article and approved the submitted version.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.


E. The charge density distribution in the vicinity of the graphene monolayer that is caused by E was determined using the pseudopotential density-functional theory approach. Different geometries were considered. The centroid of this extra density induced by an external electric field was determined as E, and its dependence on E has been obtained. The thus determined We present a detailed first-principles investigation of the response of a free-standing graphene sheet to an external perpendicular static electric field  The numerous properties of graphene have been intensively investigated after its experimental realization. Thousands of papers on this material were published. However, there still remains a simple unanswered question regarding the way in which the induced charge density is distributed in the vicinity of a graphene monolayer when an external electric field is applied to the graphene sheet. This topic was addressed, to some degree, by considering the problem of screening of the electric field induced by point charges in graphite ,2,3,4,5.z axis as pointing in the direction perpendicular to the carbon atoms basal plane) that is a “real position” of a solid surface for many phenomena occurring there. It determines a “physical” position of a metal surface when an external perturbation is applied. This problem was widely studied in the case of metal surfaces. In general, this “real” surface position is different from the spatial localization of the top atomic layer or a geometrical crystal edge, staying towards the vacuum side [The perpendicular charge distribution was studied by considering two- and multi-layer graphene films ,12,13, tuum side ,16,17,18It is usually assumed for a quasi two-dimensional (2D) system that the excess charge is confined within an infinitesimally thin 2D layer ,19. CertKnowledge of the position of the center of mass of the induced charge density is important in many fields of surface science. Thus, it determines the reference plane for the image-potential felt by an external charge placed in front of a surface. If this charge is an excited electron with energy below the vacuum level, it can be trapped by this image potential in a state belonging to an infinite Rydberg -like series ,24. The In the previous work devoted to the IPSs in graphene, it was assumed ,26 that redicted  to existUp to now, IPSs for a free-standing graphene were not studied experimentally. On the other hand, numerous measurements were performed on the graphene supported on various metallic or semiconducting substrates. Usually, the interface distance between the graphene sheet and the surface atomic layer is such that the conventional single Rydberg series of a whole system is observed. Thus, in the graphene/metal systems where the graphene atomic layer is placed closer to the substrate, only a single series of IPSs was observed ,34,35,36For a description of the IPSs in the graphene/substrate systems, a number of potentials have been developed. Indeed, an accurate description of IPSs is a challenge since the conventional density-functional theory (DFT) calculations do not accurately account for a correct long-range interaction in front of solid surfaces. One of the approaches consists of constructing the nonlocal van der Waals functional . Althougthe Ref. , the fitications ,26.Our goal in this work is to determine the The rest of this paper is organized as follows. In The band structure of a graphene monolayer in the absence and presence of an external electric field of varying intensity was obtained within the LDA by solving the Kohn-Sham equations employing a home-made band structure computer code . We usedx-y plane as shown in z-dependence is defined by a Gaussian with a decay length of 1 a.u. This extra charge was placed at a distance of 10 Å from the graphene plane. In order to ensure the neutrality of the system, the charge +z variation of the extra potential added to the system is schematically shown in The self-consistent procedure was realized by considering a repeated-slab geometry with the lateral lattice constant of 2.424 Å. The external electric field applied in the direction perpendicular to the graphene plane has no translation symmetry. In order to implement it in the repeated-slab geometry, we added to the Hamiltonian a term corresponding to the extra charge In other sets of calculations, we considered a geometry when the external electric field is applied from both sides of the graphene sheet as shown in The electronic structure of graphene around the Fermi level at zero external electric field is presented in E. x-y plane for the values of E ranging from −0.4 to 0.5 V/Å. In order to perform a comparison, E. One can see that its shape deviates qualitatively from the total valence density depicted by the green solid line. This can be understood, since the total density is dominated by the E. In general, we observe that at larger E, the shapes of E amplitude, the variations in E, defined asE. In the former case, E, the size of the Fermi surface shrinks and the possible calculation oscillations increase. For comparison, in the insert of E in a free-standing Al(111) monolayer. Since, for Al, the Fermi surface is large because there are three valence electrons, the deviation from the linear behavior is small.We have examined the way in which the induced charge density profile onolayer . A similonolayer . We expeWhen we apply an external electric field to the graphene sheet from both sides according to the scheme depicted in Clearly, in the symmetric case b, the cagen atom .Es reported in In the case of the geometry described in In our numerical calculations devoted to IPSs, the perpendicular lattice constant was increased up to 80 Å which allowed us to obtain convergent energies for the six lowest-energy members of the series. As it was mentioned previously, IPSs cannot be properly described with the use of conventional DFT calculations since the long-ranged image-potential on the vacuum side is not reproduced correctly. Additionally, the tight-binding methods are not inherently desired for its description ,50. Index and y coordinates, since The parameters x-y plane constructed with z larger than z only. For comparison, in z larger than n is increased this difference is gradually reduced. With In the Ref. . One canthe Ref.  to 1.58 z and z is large, let us account for the interaction between the charge A with real charges Az is large, for a point charge A, it can be considered as a point charge located at n. However, it may not be good for The approach described above for construction of a hybrid “LDA+image−tail” potential is an adoption of the conventional image-potential picture employed for solid surfaces . It can ocalized  around 2Based on this picture, we constructed a hybrid “LDA+image−tail” potential for which ce state ,54 obtaice state . HoweverWe believe, the measurements of energies of free-standing graphene will provide important information on its screening properties. It may contribute to establishing a detailed picture of what is going on there due to an external perturbation. So far, all the experiments on IPSs were performed on supported graphene. Sensitivity of the image-potential states to the environment where graphene was kept was significant. Our findings point out that they can also provide important information about free-standing graphene screening properties.In this theoretical study we have reported the detailed charge density distribution produced in free-standing graphene by external static electric fields with three geometries. The image-plane position was established. Surprisingly, it is rather large, located at 1.048 Å outside the carbon atom plane. Using this information, we constructed a new potential felt by an electron excited to the image-potential states. We checked several kinds of such a potential, demonstrating sensitivity of the energies of the lowest image-potential states to the details of this potential. It would be of interest to obtain the experimental information, such as from the photoemission spectroscopy, on the image-potential state energies for free-standing graphene. We believe that the experimentally determined image-potential energies will be extremely helpful for development of a more detailed picture for the graphene potential and how it reacts to the external perturbation on the atomic scale.Our data on  systems . Such pographene ,57,58,59graphene ,65,66,67graphene , borophegraphene , MXenes graphene ,71,72, agraphene .


Understanding the structural behavior of graphene flake, which is the structural unit of bulk crumpled graphene, is of high importance, especially when it is in contact with the other types of atoms. In the present work, crumpled graphene is considered as storage media for two types of nanoclusters—nickel and hydrogen. Crumpled graphene consists of crumpled graphene flakes bonded by weak van der Waals forces and can be considered an excellent container for different atoms. Molecular dynamics simulation is used to study the behavior of the graphene flake filled with the nickel nanocluster or hydrogen molecules. The simulation results reveal that graphene flake can be considered a perfect container for metal nanocluster since graphene can easily cover it. Hydrogen molecules can be stored on graphene flake at 77 K, however, the amount of hydrogen is low. Thus, additional treatment is required to increase the amount of stored hydrogen. Remarkably, the size dependence of the structural behavior of the graphene flake filled with both nickel and hydrogen atoms is found. The size of the filling cluster should be chosen in comparison with the specific surface area of graphene flake. The transformation of nanoscale structural elements into three-dimensional 3D) complex architecture is currently an important task of materials science. Since the discovery of fullerenes in 1985, a lot of new carbon structures have been proposed. Carbon polymorphs can be used to obtain nanostructures with unique mechanical and physical properties applicable in nanoelectronics, energy storage devices, sensors, supercapacitors, Li-ion batteries, etc. D complex,5,6,7.Graphene is capable of enhancing the performance, functionality as well as durability of many applications. The one-atom thin structure can serve as the platform for other materials especially since the graphene layer can be bent or crumpled. Extensive studies have been carried out in recent decades to investigate the crumpling behavior of thin sheets like graphene, by both theoretical and experimental methods ,17,18,19It is known that coupling metal nanoclusters with carbon materials can efficiently promote catalysis and electrocatalytic activities ,24 or prThe other important advantages of such structures are a large specific surface area (SSA) and, respectively, a high rate of gas adsorption, which makes it possible to predict their use in hydrogen storage ,51,52,53In the present work, two types of nanofillers for crumpled graphene flake were considered—Ni and hydrogen nanoclusters. Thus, the idea of using crumpled graphene flake for the storage of different atoms using Ni and hydrogen clusters as the example was realized. It is important to understand the dynamics of interaction between single graphene flake and a metal or non-metal nanocluster before the study of storage in three-dimensional crumpled graphene. The behavior of the hybrid structure is studied by atomistic simulation at different temperatures. For the carbon–nickel system, room temperature (300 K) and temperature close to the melting point of Ni (1000 K) were chosen. However, for the carbon–hydrogen system, the temperature of liquid nitrogen (77 K) and room temperature were chosen. The dynamics of the structure were studied during exposure for 20 ps.N = 21; 38; 47; 66; 78) inside the flake was considered  it is called “flake” rather than “nanoribbon”. This GF was filled with nanoclusters of two types: (i) nickel nanocluster and (ii) hydrogen nanocluster.To study the interaction between graphene flake (GF) and atomic nanocluster, an initial structure composed of the GF of one size . Equations of motion for the atoms were integrated numerically using the fourth-order Verlet method with the time step of 0.2 fs. The Nose–Hoover thermostat was used to control the system temperature. The periodic boundary conditions were applied in all directions, however, the simulation box is much bigger than the size of GF filled with nanocluster. The adaptive intermolecular reactive empirical bond order potential (AIREBO)  was usedGraphene flake with the nanocluster was exposed at 300 K and 1000 K for 20 ps to study the dynamics of nanocluster inside graphene flake. Previously , it was ab-initio simulation [To describe the interatomic interaction between Ni–Ni and Ni–C, Morse interatomic potential was used with the parameters or Ni–Ni ; and De mulation ,60.Graphene flake with hydrogen nanocluster was exposed at 77 K and 300 K for 20 ps. The temperature of 77 K was chosen since it was previously shown that better sorption of hydrogen can be found at 77 K . The temTo describe the interatomic interaction between C and H, AIREBO interatomic potential was used.It should be mentioned that the initially obtained structure contains atomic hydrogen, however, H atoms transform to Hngle see b. As it to 3.2 Å . HydrogeAt first, the evolution of the potential energy of the system during the crumpling process for graphene flake filled within spherical the Ni nanocluster was analyzed. In a is two times higher than for the biggest one which means that the time required to attach the nanocluster by the graphene flake is longer. For nanocluster NiThe longest time of stabilization at 300 K was found for NiAt higher temperature (1000 K), again a strong correlation between equilibration time and the size of the nanocluster was found. For GF with NiIn ioned in . Ni atomt = 1.5 ps one covalent bond appeared between two edges. Nanocluster disturbed by the temperature and several atoms attach to the graphene plane (state II). This leads to the spreading of the Ni atoms over the graphene plane (state III). Simultaneously, such a small nanocluster allowed the graphene flake to easily bend and the structural unit transforms to the bi-layer graphene with Ni atoms spread inside (state IV). Several more covalent bonds appeared on the side edges of GF. At 300 K, not many covalent bonds between the edges of GF can appear, since the temperature is far from the melting temperature of graphene [At first, consider GF with Nigraphene ,63.Increase in the exposure temperature to 1000 K facilitates the transformation. At 300 K, equilibration time is equal to 5 ps, while at 1000 K, it is equal to 3.5 ps. Subsquently, the structure came to a stable state and no further changes were observed despite slight thermal fluctuations. At 1000 K, more carbon atoms on the edges of GF found neighbors since the structure is disturbed by thermal fluctuations.For structures with NiA graphene flake with NiOne of the important applications of such structural elements is the fabrication of composites. In eratures ,42,43,44In At 77 K a, the eqAt 77 K, the time of equilibration was the longest (3 ps) for hydrogen clusters consist of 77, 66, and 38 atoms, and the shortest (1.5 ps) is for 21 and 47 atoms. At that temperature, the size of the cluster plays quite an important role which is connected with the number of sites for hydrogen on the side of GF. All hydrogen molecules and atoms can easily find sites on graphene for an initial number of atoms less than 47, while for bigger clusters, the number of sites is not enough and some molecules and atoms will move outside GF or seek better sits near GF.At 300 K, the main point is the process of dehydrogenation which is quite quick at such a high temperature, and for clusters with 38–78 atoms, about 1.5 ps is enough for all molecules to detach the side of GF and move outside. The long time of equilibration for cluster HTo understand the dynamics of the hydrogen sorption/desorption, snapshots of the structural units altogether with their potential energy are presented in At 300 K, hydrogen molecules and atoms have no chance to form even a weak bond to graphene. At 300 K, all hydrogen atoms move outside GF which is quite understandable. Numerous theoretical and experimental works confirm the sorption of molecular hydrogen on carbon nanostructures ,64,65,66A large number of hydrogen atoms almost completely fills the cavity of the GF. At 77 K, hydrogen atoms are easily converted into hydrogen molecules and under the action of chemical attraction or van der Waals forces, attach to the walls of the graphene flake. Mainly, hydrogen, which is located in the center of the flake under the influence of temperature, tends to leave the graphene cavity. Therefore, in structures with 66 and 78 hydrogen atoms, the stabilization of the structure takes longer (3 ps), in contrast to small hydrogen clusters, such as 21 and 47 (1.5 ps).For a bigger nanocluster, energy curves almost coincide for two temperatures, since hydrogen molecules at 77 K can find the places for sorption just after the first steps. In this case, hydrogen atoms are placed near graphene and link it as far as the van der Waals radius of the molecule reaches. Thus, the hydrogen adsorption capacities depend considerably on the initial size of the hydrogen cluster. The SSA of the GF should be big enough for a chosen number of H atoms and HIn As well as for a metal nanocluster, the three-dimensional structure of crumpled graphene filled with hydrogen is presented in Molecular dynamics simulation is used to study the dynamics of graphene flakes filled with the nanoclusters of two different types: metal and non-metal. The obtained results can shed the light on understanding the possibility of using crumpled graphene as a storage media from the point of single flake behavior.It was found that cavities of crumpled graphene (the structure consists of crumpled graphene flakes) can be used as containers for metal nanoclusters—for instance, for Ni. A nanocluster consisting of 21 to 78 Ni atoms was considered. A graphene flake can easily cover nanocluster, however, the dynamics of the interaction strongly depend on the nanocluster size. Small nanoclusters can be easily bent by rigid graphene flake, while the biggest conserve the shape. Such a structure, composed of Ni nanoclusters and graphene flakes, can be further used to obtain composite material with improved mechanical properties.The problem of hydrogen storage has been of high importance for decades and the application of carbon nanostructures from this point of view also looks promising. Crumpled graphene has a high specific surface area, light weight and a lot of pores and cavities which can be filled with hydrogen. It was shown that at 77 K, hydrogen molecules and atoms can be absorbed by both sides of graphene flakes. However, a single flake cannot store enough hydrogen for practical application. However, when graphene flakes composed in another structure and with additional treatment like hydrostatic compression ,47, it c