TDP-43 pathology is a disease hallmark that characterizes both amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP). TDP-43 undergoes several posttranslational modifications that can change its biological activities and its aggregative propensity, which is a common hallmark of different neurodegenerative conditions. New evidence is provided by the current study pointing at TDP-43 acetylation in ALS cellular models. Using both in vitro and in vivo approaches, we demonstrate that TDP-43 interacts with histone deacetylase 1 (HDAC1) via RRM1 and RRM2 domains, that are known to contain the two major TDP-43 acetylation sites, K142 and K192. Moreover, we show that TDP-43 is a direct transcriptional activator of CHOP promoter and this activity is regulated by acetylation. Finally and most importantly, we observe both in cell culture and in Drosophila that a HDCA1 reduced level (genomic inactivation or siRNA) or treatment with pan-HDAC inhibitors exert a protective role against WT or pathological mutant TDP-43 toxicity, suggesting TDP-43 acetylation as a new potential therapeutic target. HDAC inhibition efficacy in neurodegeneration has long been debated, but future investigations are warranted in this area. Selection of more specific HDAC inhibitors is still a promising option for neuronal protection especially as HDAC1 appears as a downstream target of both TDP- 43 and FUS, another ALS-related gene. TDP-43 is a predominantly nuclear RNA/DNA-binding protein essential for the development of the central nervous system (CNS) from the earliest stages of embryonic life to adulthood2. In the nucleus, TDP-43 is involved in transcriptional regulation, splicing and miRNA biogenesis, but upon stressing conditions it is partially relocated in the cytoplasm, becoming involved in mRNA stability control, translation, and nucleocytoplasmic transport by forming stress granules3. TDP-43 undergoes several posttranslational modifications that can change its structure, localization, overall functions, its aggregative propensity and, importantly, TDP-43-positive aggregates are a common hallmark of different neurodegenerative conditions4. Ubiquitinated, phosphorylated, and acetylated-TDP-43 aggregates were, in fact, identified in 95–97% of patients affected by amyotrophic lateral sclerosis (ALS) and in about 50% of Frontotemporal Lobar Degeneration (FTLD)6. Moreover, the direct role of TDP-43 in disease pathogenesis is underscored by the identification of more than 40 ALS-associated dominant missense mutations in the TDP-43 gene (TARDBP) in both familial and sporadic patients7. Several different pathological mutants display a propensity to form both nuclear and cytoplasmic aggregates indicating that loss of TDP-43 homeostasis and aggregation play a critical role in pathogenesis. In particular it has been demonstrated that acetylation modifies both TDP-43 function and localization, by decreasing its ability to interact with nucleic acids8. According to Choen et al.9 TDP-43 can be acetylated mainly on K154 and K192 by the HAT (histone acetyl transferase) activity of CBP (CREB binding-protein). Once acetylated, TDP-43 can be relocated in the cytoplasm where it can be deacetylated by the histone deacetylase 6 (HDAC6)10. Given this experimental evidence, it is plausible that modulating TDP-43 acetylation might be a good strategy to prevent TDP-43 aggregation and cell damage.TDP-43 or TARDBP (TAR-DNA binding protein-43) is a 43 kDa ribonucleoprotein, originally identified as transcriptional repressor of HIV1 TAR-DNA, associated during the past decades with a spectrum of neurological diseases, namely TDP-43 proteinopathy11. HDAC substrates, which are implicated in neuronal development and survival, are not restricted to histones. As demonstrated, several different physiological functions are regulated by deacteylation14. Among the different HDACs functionally associated with ALS onset and progression we focused our attention on HDAC1. HDAC1 is a Zn+-dependent deacetylase of 482 amino acids, that can be found in large transcriptional repression complexes consisting of SIN3A, NuRD, and Co-REST15, which inactivate the expression of neuronal genes in nonnervous tissues16. The role of HDAC1 in regulating neuronal viability is quite controversial ranging from neuronal protection from death to an increase in neurodegeneration and axonal death12. The involvement of class I HDACs in the onset of ALS was first shown by Janssen et al.17. Different studies also suggest that some of these HDACs regulate vitality and mortality of nerve cells; first, HDAC1 assumes neurotoxic or neuroprotective function as it interacts with HDAC3 or HDAC918. In fact, the role of HDAC1 in regulating neuronal vitality is quite controversial, since some studies show that such deacetylase is able to protect neurons from death, while other studies demonstrate that HDAC1 induces neurodegeneration and axonal death19. Moreover, recent studies indicate that in degenerative neurons, HDAC1 moves to cytoplasm where it becomes implicated in axonal alteration and degeneration of the cell19.HDACs are a complex family of proteins involved in many different cellular functions in CNS, including chromatin shaping to adjust transcriptional profiles during neuronal development and neuronal response to injury21. Notably, ALS patients show increased levels of ROS but also of 8-hydroxy-2 ‘-deoxyguanosine (OH 8dG), a marker of DNA damage. In this context, the interaction between FUS  and HDAC1 could have an important role in preserving DNA stability and cell survival, and the alteration of this interaction in ALS patients could lead to an imbalance in the delicate equilibrium between cell survival and cell death. Recently also TDP-43 has been shown to play a key role in DNA damage response (DDR), since its loss of function results in a faulty repair of DNA damage associated with a stopping in transcription and an inhibition of recruitment of critical components of the NHEJ repair system23.Most relevant to ALS, HDAC1 interacts with FUS, another ALS-causative gene, on DNA double strand breaks and this interaction seems to be important for chromatin integrity; as a matter of fact, many FUS pathological mutations impair this interaction and lead to impaired DNA break repair24, as well as the observation that acetylation appears to promote aggregation and diminish TDP-43 functionality10, prompted us to investigate a possible interaction between HDAC1 and TDP-43. Since epigenetic drugs are at present one of the most promising strategies for ALS treatment, information on the physical, and functional interaction between TDP-43 and HDAC1 will provide the rationale for using the HDAC inhibitor (HDACi) in ALS therapy25.The striking functional and structural similarities between TDP-43 and FUS20 showing a more prominent binding of HDAC1 to TDP-43 compared with FUS. Since HDAC1 and HDAC2 exhibit redundancies in various systems, a co-immunoprecipitation experiment was performed demonstrating no interaction between HDAC2 and TDP-43  or acetylation-mimic (KK-QQ) or acetylation-null (KK-AA) mutations were generated to assess whether they can affect the interaction with HDAC1. They were transiently expressed in SH-SY5Y cells and their localization was analysed by immunofluorescence  responsible for the interaction with HDAC1 a series of Myc-tagged TDP-43 fragments was generated, lacking the various putative functional domains of the protein . Since the N-terminal deletion causes the complete cytoplasmic re-localization of TDP-43 Fig. , while Hnce Fig.  demonstrInterestingly, in the co-immunoprecipitation experiments performed the acetylation-mimic mutant (KK-QQ) displays a significant decrease in HDAC1 binding Fig. .To study the interaction between TDP-43 and HDAC1, first their subcellular localization in SH-SY5Y cells was analysed. By immunofluorescence staining it was demonstrated that cellular stress induced by the overexpression of different TDP-43 mutants does not significantly affect HDAC1 localization Fig. .9. In order to investigate if HDAC1 alters TDP-43 acetylation, we performed a 2D gel-analysis of immunoprecipitated TDP-43 from SH-SY5Y cells transfected with TDP-43 alone or in combination with HDAC1. After immunoprecipitation TDP-43 was separated by isoelectrofocusing and SDS/PAGE. The level of TDP-43 acetylation was evaluated using anti-acetyl lysine antibody  promoter31. TDP-43 has been also shown to induce a transcriptional upregulation of C/EBP-homologous protein (CHOP) promoter and CHOP gene disruption markedly attenuates TDP-43-induced cell death32. Moreover, TDP-43-induced upregulation of CHOP expression is mediated by both reduction of CHOP degradation and by the increase of CHOP mRNA level. Thus, we decided to use CHOP promoter (from −954 to +91) to drive the expression of luciferase reporter and to test the ability of TDP-43 in regulating it. As shown in Fig. TDP-43 was originally described as transcription factor for TAR DNA of HIV133. As shown in Fig. This experimental evidence indicates a direct interaction between TDP-43 and CHOP promoter, which was confirmed by the ChIP approach. In a first attempt, the CHOP promoter activation was tested in response to sodium arsenite using the dual modification of histone H3, which is phosphorylated at serine 10 and acetylated at lysine 14 (H3-PS10/AcK14), as a marker of transcriptional activation. Accordingly, we observed that sodium arsenite treatment induces CHOP transcriptional activation demonstrated by histone H3 phosho-acetylation detection Fig. . Subsequ10. Starting from this observation the effects of double acetylatyion mimic or acetylation-null mutations on TDP-43 toxicity were assessed by measuring TDP-43 nuclear loss and cell death of SH-SY5Y cells34. In particular, SH-SY5Y transduced with TDP-43 WT or bearing different mutations, were exposed to sodium arsenite, a classical agent used to induce stress granules formation, or UV-C to induce DDR36. Although both treatments induce the re-localization of WT or acetylation-null mutations in the cytoplasm , 4-phenylbutyrate (SPB), trichostatin A (TSA), and fatty acid derivatives, which inhibit most class I and II HDACs, have been used in SOD1-G93A mouse model. In fact, SPB treatment extends survival and motor performance in SOD1-G93A mice models and it has been demonstrated to be safe and well tolerated42 in a phase 2 clinical trial. TSA induces a modest improvement in motor function and survival as well as protection against motor neuron death43. Furthermore, butyrate and valproic acid are also known to readily cross the BBB44. Finally, the therapeutic potential of NaB has been also demonstrated in other neurodegenerative diseases such as Alzheimer disease, Parkinson’s and Huntington disease40. Thus, the effects of these four HDACis on TDP-43-mediated cell death were assessed.HDAC inhibition has been shown to be protective in a wide range of pathological conditionsFirst, cellular toxicity of the different HDACis was evaluated by testing their dose-response effect on cell viability Fig.  and two Then HDAC1 expression level was manipulated to assess its effect on TDP-43 toxicity. The CRISPR/Cas9 genome editing technique was used in SH-SY5Y cells to generate stable cell lines in which the expression of HDAC1 was ablated. SH-SY5Y were transfected with a plasmid coding for the humanized Cas9 and different gRNA targeting the second exon of HDAC1 gene. After puromycin selection, single stable clones were isolated and analysed for HDAC1 expression leading to the identification of two lines in which HDAC1 protein expression was absent as demonstrated by immunofluorescence experiment Fig.  and sequThese HDAC1-KO lines were used in cellular vitality tests, after being transduced with TDP-43 expressing adenoviruses. Interestingly, the ablation of HDAC1 expression significantly ameliorates TDP-43-mediated cell death Fig. .To confirm the evidence coming from HDCA1 genetic ablation, a commercial HDAC1 small interfering RNA (siRNA) to downregulate HDAC1 was used. Thus, we observed that the reduction of HDAC1 protein level by 70%, which causes a statistically significant decrease of TDP-43-induced cell toxicity, compared with the random sequence control Fig. .Conversely, TDP-43 toxicity was exacerbated Fig.  when HDA37 were exploited.The present study demonstrated that in three different experimental paradigms  the reduction of HDAC1 activity significantly reduces TDP-43-mediated cell death. To confirm these results through an in vivo approach, Drosophila ALS models45, which is associated to a strong cell death phenotype characterized by depigmentation, roughness, and dark spots. When these flies were crossed with a line in which Rpd3, the HDAC1 and HDAC2 drosophila ortholog, is inactivated by the expression of an RNAi specific construct47, a reduction in retinal degeneration can be clearly observed. These eyes exhibit a significant reduction of the dark apoptotic areas  loses its ability to induce CHOP transcription, indicating that TDP-43 acetylation can affect TDP-43-mediated pathways downstream ER stress, as well as further promote the nuclear retention of TDP-43 upon exposition to stressful signals.By using cellular and animal models we were able to describe a specific interaction between HDAC1 and TDP-43, via RRM1 and RRM2 domains. Interestingly, our experimental results suggest that HDAC1 may play a pivotal role on the deacetylation of TDP-43 in the nucleus. In addition, we observed as the impairment of TDP-43 deacetylation, by TDP-43 mutagenesis or by genetic/pharmacological HDAC1 inhibition, has a positive effect on TDP-43-induced cell death. Notably, through ChIP analysis and luciferase assays, evidence is provided that TDP-43 is a direct activator of CHOP (C/EBP‐homologous protein) transcription, expanding what previously reported49.The phenotype described is in line with the multiple crossroads between epigenome, epigenetic machinery, and ALS, described in the past 10 years33, that can affect TDP-43-induced cell toxicity at multiple levels, since changes in acetylation of both histone and nonhistone proteins have been reported to affect cell physiology spanning from transcription, to DNA repair signal transduction, and protein aggregation50.We had previously demonstrated that TDP-43 M337V expression induces a decrease in global histone H3 phosho-acetylation13. In fact, the levels of HDAC1, HDAC2, and sirtuins (a family of class III HDACs) are impaired in post-mortem ALS tissues17. In yeast Set-3, a component of the histone deacetylation complex, is a modulator of TDP-43 toxicity51. HDAC1, like most HDCAs, is a nuclear enzyme that can be re-localized in the cytosol in damaged axons of demyelinating models, such as in patients affected by multiple sclerosis and in cultured neurons exposed to glutamate or TNF-α53. More recently, during development of the Xenopus brain, HDAC1 was observed in the mitochondria of developing neurons54. In addition, in a FUS knock-in mouse model HDAC1 is mislocalized to the cytoplasm55, probably following dephosphorylation on serine 421 and 42353. Despite this evidence we were not able to highlight HDAC1 re-localization in our cellular models.HDACs are key modulators of the acetyloma and they have been demonstrated to be deregulated in ALS experimental models and patients40. At present more than 350 clinical trials involving HDACi have been carried out or are on-going not only as single therapeutic but also in combination with other targeted agents against various human diseases, including neurodegenerative diseases. VPA was approved by FDA in 1978 as an anticonvulsant drug for the treatment of seizure disorders, even if the molecular target of this drug it is not known yet. The possibility of using HDACi for neurodegenerative treatment originates in 2008, when Hahnen identified two major HDACi neuroprotective mechanisms, including the transcriptional activation of disease-modifying genes and the rectification of destabilization in histone acetylation homeostasis56. Several pan-HDACis reduced ALS development in mice58. SPB was shown to extend survival and motor performance in transgenic ALS SOD1 animal model, and these effects were attributed to an upregulation in the expression of nuclear factor κB (NF-κB) and bcl-2 proteins41, although the genetic inhibition of NF-κB in SOD1 mice does not ameliorate disease onset and progression59. Unfortunately, even if VPA and SPB are safe, tolerable, and efficient in improving histone acetylation levels, they failed to ameliorate clinical parameters in ALS patients60. In line with this observation the work by Pigna et al. identified HDAC4, a class IIa HDAC, as having a crucial role in preserving the innervations and skeletal muscle in SOD1 ALS mouse model61. HDAC4 genetic ablation in skeletal muscle accelerates ALS pathological features, indicating a possible risk for using HDACs pan-inhibitors in ALS treatment. On the other hand, HDAC6 inhibition in motor neuron cultures derived from iPSCs, originated from fibroblasts of ALS patients carrying different FUS mutations, reverses axonal transport defects62. Treatment of FUS transgenic mice with ACY-738, a potent class I HDAC brain penetrable inhibitor, largely restores global histone acetylation, and metabolic gene expression in the spinal cord63. ACY-738 inhibits HDAC6 with low nanomolar potency and a selectivity of 60- to 1500-fold over class I HDACs, but its effect in FUS mouse model is independent from HDAC6 itself, indicating that other members of the family, including HDAC1, can be the key element mediating the observed therapeutic effects63. Moreover HDAC1 appears as a downstream target of both FUS and TDP-43 related ALS in mediating double strand-breaks repair65.HDACis were originally applied to cancer therapy and some of them, such as panobinostat, have been approved from FDA for multiple myeloma treatment, while others, like VPA and SPB, are in phase II or III clinical trials, respectively, for hematological and solid malignanciesAlthough HDACi translational failure underlies ALS complexity and can be related to the lack of selectivity for different HDACs, more specific drugs would be very useful. Particularly, evidence provided indicates that HDAC1 inhibition can be a precious therapeutic option in ALS therapy.The following primary antibodies were used in this study: Myc monoclonal antibody , β-actin , Flag , HDAC1 , TARDBP , Acetylated-Lysine antibody , GFP , caspase-3 , PARP , LC3B , anti-rabbit peroxidase-conjugated secondary antibody (AP132P EMD Millipore) and anti-mouse peroxidase-conjugated secondary antibody (AP124P EMD Millipore); anti-rabbit, anti-mouse Alexa 488  or 647-conjugated secondary antibody . All antibodies were used at the dilution recommended by the manufacturer’s instructions.The following HDACis were used in this study: Sodium phenil butyrate , Trichostatin A , Sodium butyrate , Valproic acid sodium salt .Istituto Zooprofilattico della Sardegna . All animal procedures have been performed according to the European Guidelines for the use of animals in research (86/609/CEE) and the requirements of Italian laws . The ethical procedure has been approved by the Animal welfare office, Department of Public Health and Veterinary, Nutrition and Food Safety, General Management of Animal Care and Veterinary Drugs of the Italian Ministry of Health . Authorized investigators performed all the experiments. Dissected tissue was immediately frozen in liquid nitrogen and stored at −80 °C.Mice tissues were dissected from BALB31c mice housed at the 66 under UAS promoter during fly eye development, driven by GMR-Gal4, were crossed at 25 °C with flies expressing a unique dsRNA that targets Drosophila HDAC1. In particular, GMR-GAL4 on X-chromosome was originally placed in trans to generate a GMR-GAL4;gl-TDP-165 43CO/CyO transgenic fly, which was obtained from Bloomington Stock Center67. RNAi line to target HDAC1 (v30599) , which was previously utilized to downregulated Rpd3 according to47, was obtained by Vienna Drosophila Research Center. Eye neurodegeneration was evaluated according to68. A minimum of 60 flies were randomly choosed, with investigators blind to the genotypes, during the analysis.Flies expressing insect codon-optimized version of human wild-type TDP-43 (gl-TDP-43CO)Sequence coding for human TDP-43 (NM_007375.3) or human HDAC1 (NM_004964.2) were cloned in different expression vectors  and used for site-directed mutagenesis . Mutants were obtained by mutagenesis starting from hTDP-43, by site-directed mutagenesis. Positive clones were screened by sequencing. Human HDAC6 (NC_000023.11) and HDAC2 (NM_001527) were cloned in expression vector pCMV-3xFlag.pAdenoX-hTDP-43WT/Q331K/M337V/A382T/ΔRRM1-2/K145A-K192A/K145Q-K192Q and pAdenoX-hHDAC1) were generated using the Adeno-X Expression System 1 (Clontech) and partially described. All constructions were verified by automated sequencing. Adenoviral particles were produced and titrated using the Adenoviral-X Expression System 1 (Clontech) according to manufacturer’s instruction. Cells were transduced by adenoviral particles (5–30 pfu/cell) in DMEM–F12 and incubated at 37°C for 1 h. The transduced cells  were analysed 48 h transduction.All adenoviral vectors  at 37 °C. The plasmid pcDNA3 containing cDNA coding for 3 × Flag-HDAC1 was transfected using Lipofectamine® LTX Reagent (Life Technologies) according to the manufacturer’s protocol. The different SH-SY5Y clones were maintained under selection by 400 μg/mL of G418. Individual clones were picked after 14 days of selection, moved in a 96 well plate, and maintained under selective medium until confluence growth. Different individual clones were analysed for HDAC1 expression by western blot and immunofluorescence.Human neuroblastoma SH-SY5Y cells  and SH-SY5Y-CFP-DEVD-YFP2). After microirradiation, cells were incubated for 4 h at 37 °C in a humidified atmosphere containing 5% CO2. All slides were processed by assaying the previously mentioned immunofluorescence protocol and analysed by confocal microscopy.To expose cells to stressful conditions after transduction with the indicated adenoviral particle, cells were either exposed to NaArs 50 µM for 16 h or for UV irradiation: cells were treated with UV-C (254 nm) using a low pressure mercury lamp, and the cells were subjected to global  was used to package and propagate the recombinant adenoviral- based vectors produced with the BD Adeno-X Expression System.Adeno-X 293 cell line were grown in DMEM, 10% FBS at 37 °C. Transient expression of each vector  was obtained with Lipofectamine Plus reagent (ThermoFisher) according to manufacturer’s instructions. After an incubation of 4 h with transfection reagents, the cells were cultured in normal growth medium for 24 or 48 h. Trasduction with adenoviral particle with a MOI of 5–10 pfu/cell was performed according to69).SH-SY5Y cells (ATCC number CRL-2266) were grown in DMEM–F12, 10% FBS at 37 °C whereas, Staphylococcus aureus (Sigma-Aldrich P3391). Subsequently, immunocomplexes were analysed by means of western blotting, using specific antibodies.Briefly, cultured cells were lysed with lysis buffer , containing protease inhibitors (SIGMA P 8340). Cell lysates were immunoprecipitated overnight at 4 °C with specific antibodies; immunocomplexes were then captured by incubating for 16 h at 4 °C with continuous gentle shaking, with protein-A sepharose from Protein content was determined using Bradford protein assay (27813 SIGMA). Equal amounts of protein extracts were resolved by standard SDS/PAGE. Samples were then electroblotted onto Protan nitrocellulose membranes . Afterwards, membranes were incubated in 3% low-fat milk, diluted in 1 × PBS-Tween 0.05% solution with the indicated antibody for 16 h at 4 °C. Anti-Rabbit IgG (whole molecule)- and Anti-Mouse IgG (whole molecule)-peroxidase antibody (EMD Millipore) were used to reveal immunocomplexes by enhanced chemioluminescence (ThermoFischer). The apparent molecular weight of proteins was determined by calibrating the blots with prestained molecular weight markers . Where indicated, the relative signal intensity acquired by using the ChemiDoc XRS+  was quantified using QuantityOne Software.Two-dimensional electrophoresis (2-DE) was used to separate proteins according to their isoelectric point (1st dimension) and, orthogonally, to their molecular weight (2nd dimension).71. Briefly, samples were applied to 70 mm IPG strips , by overnight rehydration loading at 20 °C, and then isoelectrofocused at 50 μA/IPG strip for 22 kVh at 20 °C. Once isoelectric focusing was completed, proteins were in-gel reduced by incubating IPG strips with 50 mM tris buffer containing 6 M urea, 30% glycerol v/v, 3% SDS w/v, and 1% DTT w/v, followed by in-gel alkylation with the same solution containing 2.5% iodoacetamide w/v, in place of DTT. Each step was performed keeping strips under continuous shaking for 15 min. IPG strips were then sealed with 0.5% low melting point agarose w/v, in SDS running buffer, at top of second dimension gels (8 × 7 cm × 0.1 cm). SDS-PAGE was carried out using 15% T, 3% C polyacrylamide gels at the following conditions: 50 V for 15 min and subsequently at 150 V until the Bromophenol dye front reached the lower limit of the gel, in a Mini-Protean Tetra Cell . Later gels were subjected to western blot analysis, as described above.2-DE was performed as reported elsewhere®488 goat anti-mouse IgG (Life Technologies), diluted 1∶1000 in blocking solution, for 1 h at the room temperature. Cells were then analysed with a Leica TCS SP5 confocal microscopy, with LAS lite 170 image software.Cells were grown on a cover-glass, were washed twice with PBS 1X and then fixed with 4% paraformaldehyde in 1 × PBS and permeabilized with 0.2% Triton X-100 in 1 × PBS. After a blocking step for 1 h in 5% BSA, diluted in 1 × PBS-0.05% Tween-20, cells were incubated with the primary antibody mouse anti-Myc (Sigma-Aldrich), diluted 1∶10000 in blocking solution, overnight at 4 °C, and then incubated with a secondary antibody Alexa Fluor6) were plated 24 h before transduction, and infected by using viruses encoding for TDP WT at a multiplicity of 30 pfu/cell. After 24 h, cells were harvested and chromatin immunoprecipitation was performed using EZ-Magna ChIP™ (Millipore), according to the manufacturer’s protocol.SH-SY5Y cells  and Normal Rabbit Ig (reagent supplied) as negative control. Purified chromatin was eluted and DNA fragments were used for qPCR (S2). The results were normalized using the Fold Enrichment Method . **p < 0,01 Student’s t test.Each immunoprecipitated (IP) reaction was performed using about 1 × 1072) promoter from −954 to +91 was cloned between the XhoI and HindIII sites in the pGLE-Basic Vector. All constructions were verified by automated sequencing. SH-SY5Y cells were seeded in 24-well plates and cultured for 16 h. Cells were then transfected by wild-type or mutant of TDP-43, HDAC1 and luciferase constructs, in addition to a Renilla vector, used as an internal control for luciferase activity; transfected cells were further cultured for 48 h. Luciferase assays were conducted using dual luciferase assay system (Promega). Each experiment was performed in triplicate.The DNA-damage-inducible transcript 3  were seeded 24 h before the first transfection with the siRNA oligonucleotide specific for HDAC1 gene. Lipofectamine 3000 reagent  was combined with Optimem medium (Promega) (reaction 1); meanwhile, in a different tube, 148 pmol of HDAC1 siRNA and 500 ng of TDP, WT or mutant, were mixed with Optimem (reaction 2). Both reactions were mixed and left for 5 min at the room temperature. Afterwards, the mixture was added to the cells and incubated at 37 °C and 5% of CO2. After 48 h the transfection procedure was repeated only for HDAC1 siRNA, and cells were incubated at 37 °C and 5% of CO2 for other 24 h. After 72 h a MTS assay was performed, and cells were finally washed two times with PBS; subsequently, cell lysates were subjected to western blot analysis with anti-HDAC1 antibodies to evaluate expression level.SH-SY5Y cells (1 × 10http://tools.genome-engineering.org) was used to develop gRNAs. Off-target activity was evaluated additionally with Blastn (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The pSpCas9(BB)-2A-Puro (Addgene # 48139) that expresses the Streptococcus pyogenes Cas9 (including an NLS and a FLAG tag) from a CAG promoter and has a U6 promoter driven gRNA, was used as cloning backbone according to73. Briefly, phosphorylation and annealing were performed with the three pairs of oligos, mentioned above, harboring a BbsI overhang. Afterwards, BbsI  mediated digestion and T4 DNA ligase  directed ligation in the linearized pSpCas9(BB)-2A-Puro was performed. After the transformation, cloning was verified with a control PCR with the primers in the table. Plasmids were purified and sequenced. After transfection of the indicated combinations of pSpCas9(BB)-2A-Puro-gRNAs (Addgene #62988)), positive cells were selected using puromycin (2 μg mL−1) for 5 days prior to clonal expansion. Empty pSpCas9(BB)-2A-Puro was used as negative control.The web interface of CRISPR Design Tool -5-(3-carboxymethoxyphenyl)-2-(4- sulfophenyl)-2H-tetrazolium (MTS) assay , according to manufacturer’s instructions. Absorbance at 490 nm was measured in a multilabel counter  72 h post trasnduction.n ≥ 3 independent experiments. Statistical evaluation was conducted by one-way or two-way ANOVA and Bonferroni post test. Values significantly different from the relative control are indicated with a symbol: *p < 0,05; **p < 0.01; ***p < 0.001 or §p < 0.05; §§p < 0,01; §§§p < 0.001.The results are presented as means ± S.D. of Supplementary Figure legends-clean copyFigure S1-revFigure S2Figure S3Figure S4-revFigure S5Figure S6-revFigure S7-rev


Zablocki Veterans Affairs Medical Center. The medical center contains a fully-equipped state-of-the-art simulation center with simulation trainer models and ultrasound machines. This allows us to provide teaching sessions on ultrasound-guided vein cannulation to trainees and advanced practice providers.After months of attending-only taught sessions on ultrasound-guided vein cannulation at the simulation center, a curriculum was created for the Pulmonary and Critical Care Medicine fellows to become the teachers of the sessions. The curriculum included a formal in-service program for the fellows to learn their roles and responsibilities as educators. During the in-service program, the fellows learned how to assess the trainees' needs and how to adapt their coaching to help to close any knowledge gaps. After the in-service was given, we established the fellow-taught sessions, and they were integrated into the monthly academic schedule. The fellow-taught sessions eventually substituted the attending-taught sessions. A faculty member observed the fellow-taught sessions and provided feedback to improve the fellows' teaching competencies. The Veterans Affairs Medical Center Senior IRB Analyst approved this project. In this letter, we report our experience with implementing the curriculum of fellows as teachers of ultrasound-guided vein cannulation. We also provide details about the trainees' overall experience during the teaching sessions.We conducted the ultrasound-guided vein cannulation teaching sessions 1-2 times a month, at the Clement J. Zablocki Veterans Affairs Medical Center simulation center. During the sessions, the focus was on teaching ultrasound basics, image acquisition, identification of major veins, review of key anatomical structures, and advancing the needle into the center of the vein. The sessions’ participants were internal medicine residents and interns, medical students, and advanced practice providers who filled out an eight-point total pretest before the session, and an eight-point total posttest afterwards. The participating trainees also filled out an anonymous and voluntary evaluation. The pretest and posttest were similar and were created to explore three specific domains. The domains were: (i) recognition of the vessels and surrounding structures under ultrasound; (ii) identification of the ultrasound views that will assure a safe and successful central vein cannulation; (iii) the proper use of knobology to assure successful peripheral vein cannulation. The test contained two multiple-choice questions, one true or false, and one question asking for the identification of the structures in an ultrasound image.Seventy-four trainees in total participated in the sessions. Fifty-four (73%) were residents or interns, 17(23%) Medical Students, 2(3%) Advanced Practice Providers, and 1(1%) did not answer. Twenty-four (32%) trainees participated in the fellow-taught sessions and fifty (68%) in the attending-taught sessions. There was a higher proportion of medical students in the fellow-taught sessions (38%) compared to the attending-taught ones (16%). There was a trend to higher trainees' posttest scoring in both groups. However, the improvement in the posttest scoring was more pronounced and significant in the fellow-taught group. In the fellow-taught group, nineteen out of twenty-four participant trainees (79%) completed the session's evaluation.  All the trainees subjectively felt that fellows' teaching was effective, and 89% of them thought the fellows' teaching positively influenced their education.Although there was a higher proportion of medical students in the fellow-taught group, the group had a more significant improvement in the posttest scores. This finding attests to fellows' ability to deliver high-quality education to learners at different levels of training. As Woodfield and colleagues,Thus far, our experience with the implementation of this curriculum has been very positive. The curriculum is giving the fellows additional exposure to clinical education in an environment other than inpatient consults. Most of the fellows' teaching opportunities that have been published in medical literature occur in the inpatient consultation setting.,The lack of opportunities for fellows to develop as teachers is at odds with the multitude of residents-as-teachers' programs available, and their role as teachers may be underrecognized.


We show by UV-Vis reflectance investigations that the local surface plasmon resonance (LSPR) of Ag NPs partially covered with graphene is preserved for at least three months, i.e., three times longer than the LSPR of bare Ag NPs. The gradual loss of LSPR is due to the spontaneous sulfurization of non-covered Ag NPs, as revealed by scanning electron microscopy and energy-dispersive X-ray spectroscopy. We show that the Ag NPs completely sandwiched between graphene and HOPG do not sulfurize, even after one year.Silver nanoparticles (Ag NPs) play important roles in the development of plasmonic applications. Combining these nanoparticles with graphene can yield hybrid materials with enhanced light–matter interaction. Here, we report a simple method for the synthesis of graphene–silver nanoparticle hybrids on highly oriented pyrolytic graphite (HOPG) substrates. We demonstrate by scanning tunneling microscopy and local tunneling spectroscopy measurements the electrostatic  This leads to morphological changes of the NPs and significant diminishing of the optical properties [Noble metallic nanoparticles (NPs) are extensively applied for chemical and biological sensing due to their local surface plasmon resonance (LSPR) ,2 and suoperties . Preservoperties  for a reoperties , since ioperties ,23. Neveoperties .The synthesis of Ag NPs can be realized over a wide range of strategies , dependi−7 mbar and rate of 0.1 nm s−1. During the process, the substrate remained at room temperature. Immediately after silver deposition and opening of the vacuum chamber, the thin silver films were covered with CVD graphene, which was synthesized as described in a previous paper [2 aqueous solution (20%) and HCl (37%) in a 4:1 volume ratio. The tape holding the graphene was rinsed in distilled water, dried, and pressed onto the deposited silver thin film. Graphene-covered silver thin films were obtained by lifting the tape with tweezers. In order to drive the surface diffusion of deposited silver and to form the Ag nanoparticles, subsequent annealing of both bare and graphene-covered thin silver films was performed at 400 °C under inert gas (Ar) atmosphere for 90 min. We used these annealing parameters, which worked earlier for the preparation of gold nanoparticles, as reported recently [Bulk silver of 99.99% purity  was placed in an electrically heated tungsten boat for evaporation. Thin silver films of 7 nm nominal thickness were evaporated onto HOPG substrates at a background pressure of 5 × 10us paper . For therecently .The Ag NPs and graphene–Ag NP hybrid structures were investigated by tapping mode AFM measurements performed on a MultiMode 8 , along with STM and STS measurements using a DI Nanoscope E  operating under ambient conditions. The optical reflectance properties of the samples were measured in the wavelength range of 200 to 1000 nm using an Avantes AvaSpec-HS1024 × 122TEC fibre optic spectrometer . We used a bifurcated probe for illumination and detection with 200 μm core diameters. The reflectance spectra of the samples were recorded by collecting the specular reflected light under normal incidence illumination with an Avantes AvaLight DH-S-BAL balanced UV-Vis light source . Scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX) investigations were performed with Thermo Scientific Scios2  and Oxford X-max 20  instruments, respectively.The surface of 7 nm Ag deposited onto HOPG is shown in We also prepared graphene-covered samples by transferring the two-dimensional carbon sheet onto the as-deposited silver thin film. Typical AFM images of the graphene–Ag hybrid nanostructures obtained after a similar annealing procedure are shown in p-doped graphene on HOPG (Dirac point around 70 mV) and slightly n-doped graphene on the Ag NP (Dirac point around –10 mV). This local probe measurement is in agreement with previous results, where electron transfer from Ag NPs to graphene was demonstrated by Raman spectroscopy [Graphene–Ag NPs were also investigated by STM and STS in order to study the effect of silver contact on the density of electronic states of graphene. troscopy .Next, we discuss the optical properties of the prepared samples. The reflectance spectra of the as-deposited Ag thin film a, bare A through :(1)ωLSPRcoupling  between coupling , can alscoupling .Further, we investigated how the optical properties of Ag NPs and graphene–Ag NPs kept under ambient conditions vary in time. We performed the same reflectance measurements on the same samples one month and three months after preparation. The corresponding spectra are shown in For a more detailed study of spontaneous sulfurization, we investigated the graphene-covered Ag NPs by SEM and EDX. The SEM image of freshly prepared and partially covered Ag NPs is shown in Importantly, no sulfur is observed on graphene-encapsulated Ag NPs d, black,n-doping of graphene. We demonstrated by optical reflectance investigations that a graphene overlayer preserves the local surface plasmon resonance properties of Ag NPs for at least three months, although the LSPR is gradually redshifted. We showed by SEM and EDX that graphene can protect Ag NPs from ambient sulfur for more than one year. Nevertheless, with the applied transfer process, only 40–50% of Ag NPs are coated and the observed loss of plasmonic properties is primarily attributed to the spontaneous sulfurization of non-covered NPs. The long-term stability of LSPR could be significantly improved by increasing the total graphene coverage. Such protection of Ag NPs by an atomically thin cover layer can be very useful, for example in LSPR shift-based sensor applications, photocatalysis, or the preparation of advanced substrates for surface-enhanced Raman spectroscopy.We reported a simple method for the synthesis of graphene–silver nanoparticle hybrids on HOPG substrates. We showed that in the case of a graphene overlayer, the Ag NPs tend to coalesce and to form larger nanoparticles. STM and STS measurements performed on graphene–Ag NPs revealed charge transfer from silver resulting in the


The graphene paper microsieves can be applied in the filtration of biological fluids or separation of solid particles from exploitation fluids. To produce graphene paper microsieves for specific applications, good control over fabrication should be achieved. In this study, a laser ablation method using a picosecond laser was applied to fabricate graphene paper microsieves. Holes in the microsieves were drilled using pulsed laser radiation with a pulse energy from 5 to 100 µJ, a duration of 60 ps, a wavelength of 355 nm, and a repetition rate of 1 kHz. The impact method was applied using 10 to 100 pulses to drill one hole. To produce holes of a proper diameter which could separate biological particles of a certain size (≥10 µm), optimum parameters of graphene paper laser ablation were defined using the MATLAB software taking into account laser pulse energy, repetition rate, and a desired hole diameter. A series of structural tests were carried out to determine the quality of an edge and a hole shape. Experimental results and Laguerre–Gauss calculations in MATLAB were then compared to perform the analysis of the distribution of diffraction fringes. Optimum experimental parameters were determined for which good susceptibility of the graphene paper to laser processing was observed. Graphene is a 2D crystal composed of a single layer of carbon atoms arranged in a hexagonal mesh. The development of efficient fabrication methods and the ability to determine its functional characteristics have resulted in an increasing interest in the application of graphene and its derivatives in many areas of technology and medicine ,2,3,4,5.2 substrate were patterned using 532 nm laser irradiation under a fluence of 356 mJ·cm−2, only the selective ablation of graphene was observed. However, when the fluence was increased to above 1030 mJ·cm−2, damage to substrate was observed instead of graphene ablation [The available literature shows published studies in which a laser ablation process was carried out on the graphene deposited mainly by the chemical vapor deposition (CVD) method on various substrates ,13,14, iablation .In the case of graphene paper composed of many layers of graphene and not deposited on the substrate, the substrate does not disturb its structure during laser ablation. However, the applied laser ablation parameters have influence on the shape and topography of the area of laser beam interaction. This is a very important factor when particular sizes and shapes of the holes are required for specific applications. Such a situation occurs, for instance, in the case of the microsieves made of graphene paper. During the production of microsieves from graphene paper, which can be applied in the filtration of biological fluids as well as separation of solid particles from exploitation fluids, selection of parameters for laser processing is an important issue ,4,20. AtIn the literature, there are several reports on laser-assisted synthesis, reduction, and micro-patterning of graphene or graphene oxide ,13, inclThe studies were conducted on graphene paper of the thickness in a range of 80–100 μm produced at the Department of Chemical Synthesis and Flake Graphene, Łukasiewicz Research Network—Institute of Electronic Materials Technology in Poland, according to patented technology . A graphThe morphology of graphene paper before and after laser ablation was investigated by scanning electron microscopy (SEM) using a Quanta 250 FEG SEM, FEI, Hillsboro, OR, USA. SEM images were acquired using a backscattered detector  with an accelerating voltage of 10 kV. The analysis of the SEM images allowed us to determine the kind and extent of damage arising as a result of laser beam interaction with graphene paper and optimization of size and shape of microholes generated during ablation.Graphene paper laser ablation was conducted with the use of a PL2210/SH/TH/FH picosecond laser produced by the company EKSPLA, Vilnius, Lithuania. A diagram of the used laser setup is presented in The laser beam pulse energy of the first harmonic (wavelength—1064 nm) was 1.3 mJ, on the third one (355 nm) it was 0.45 mJ, and on the fourth one (266 nm) it was 0.25 mJ. The duration of the pulse of the first harmonic was equal to 70 ps, whereas the frequency of repetition was 1 kHz. The energy of the pulses was regulated by the use of a 990-0070-355 polarizing damper produced by the company EKSPLA, Vilnius, Lithuania.A galvanometric scanner, produced by the company RAYLASE , with a telecentric lens of focal length equal to 100 mm, which focuses radiation, was used on the surface of the processed material. Microsieves were produced using laser beam radiation on the third harmonic (355 nm).Laser pulse energy and laser pulse numbers of 10, 20, 50 and 100 µJ and 10 to 80 pulses, respectively, during graphene paper laser ablation were selected as independent variables. A hole diameter of ≤10 µm was used as a criterion for a quality assessment of laser ablation. The optimum laser ablation parameter values were determined using the MATLAB program  taking into consideration Equations (1) and (2).The morphology of the graphene paper used in these studies is shown in During selection of the laser ablation parameters of the graphene paper a degradation of ablated material was observed when laser energy was increased to 100 μJ with a laser pulse number above 30, as is shown in The results of SEM imaging of the structural changes occurring in graphene paper upon laser ablation are presented in In In the MATLAB software, the obtained results of the graphene paper laser ablation were used to develop a function determining a range of optimum parameters. For this purpose, value 1 was assigned to sets of holes with diameters in a range of 6–10 µm, whereas value 0 was assigned to the diameters which did not satisfy this condition. Based on the assumptions, a function of the selection of graphene paper laser ablation parameters was developed .Analysis of the conducted tests proved that holes can be fabricated only at certain parameters of laser processing with diameters in a range of 6–10 µm, which enable the separation of circulating tumor cells. It should be noted that once a hole is created, increasing the number of pulses does not significantly influence any increase in diameter. However, this may cause greater development of the edges e(r)=The Equation (1) was obtained assuming that a beam incident on the diaphragm has Gaussian distribution of energy density. The cut off Gaussian beam underwent diffraction. The calculations of the distributions were carried out in MATLAB by the Laguerre–Gauss method for the initial distribution described in Equation (1) . The axi−2 as a threshold energy density for engraving, were carried out. The comparison of the experimental and theoretical results is shown in A focused 355 nm laser beam causes microdrilling in graphene paper leading to holes with diameters depending on the pulse energy and distance of the processed surfaces from the focal plane. Measurements of the diameters of the craters engraved by laser pulses of different energies were conducted and calculations of the diameters of those craters, assuming 3.5 J·cmA good compliance of the results of calculations and measurements occurs at low energies of the pulse. When the energies of the pulse are higher, resulting from interference of thermal conductivity and shock waves, compliance decreases, particularly in the area of the focal plane  revealed multi-layered petals of graphene arranged in parallel. Typical lines in Raman spectra, i.e., D, G, D’, D + G, 2D, proved the presence of graphene oxide in the graphene paper;Calculated and measured distributions of the laser beam fluence show a good compliance. The tests conducted on the graphene paper laser ablation process demonstrated that the energy of a laser pulse is a crucial parameter decisive for the result of the processing. The lower the pulse energy, the smaller the diameter of the produced holes and the better the quality of their edges. Increasing the pulse energy decreases the number of the shots required to produce the hole. However, it decreases the quality of the processing and, moreover, causes uncontrolled failure of the processed material due to thermal conductivity and shock wave effects;The results of the test performed also show that after producing the holes, increasing the number of pulses does not significantly affect their diameter any longer;The analyses conducted proved that in order to successfully produce a microsieve from graphene paper, 50 to 70 pulses for one hole must be applied at energy levels of 20 µJ or 30 to 50 pulses at energy levels of 50 µJ.


Rsh) is also observed across transferred graphene using this improved process.In this work, an improved rosin transfer process is initiated. An anisole coating is introduced based on the rosin transfer process to reduce the residue particles on the surface of transferred graphene. Rosin/graphene and anisole/rosin/graphene samples are handled without baking and with baking at different temperatures, i.e., 100 °C, 150 °C, and 200 °C. Atomic force microscopy (AFM) and Raman spectroscopy are employed to characterize the surface properties of transferred graphene. The removal of the protective rosin layer and anisole/rosin layers without baking is found to be more effective and beneficial compared to the conventional PMMA transfer process. Furthermore, better results in terms of reduced surface roughness and residue particles are accomplished by introducing anisole in the improved rosin transfer process. Uniform and low sheet resistance ( Based on aforementioned rare properties, graphene has become one of the most promising alternatives for Si. All of these features make graphene to step into the new generation of technologies beyond the limitations of conventional semiconductor materials [The isolated two-dimensional 2D) nature of graphene has attracted tremendous interest due to its exceptional properties. However, these excellent properties are attributed to the isolated single-layer graphene. Such unique properties include mechanical breaking strength of ~ 130 GPa D nature  comparedaterials –8.The properties described above are mostly related to intrinsic graphene. In reality, to achieve these complex properties, large area growth of graphene is required. For the growth of graphene, chemical vapor deposition (CVD) method is an efficient and inexpensive process for producing large area single-layer graphene . HoweverE.ad), good support strength, and good solubility in solvents and the last guarantees the easy removal of this protective layer after graphene transfer. Recently, rosin (C19H29COOH), a small natural organic molecule, was reported to provide a good protective layer with low Ead (1.04 eV) compared to popularly used PMMA (Ead > 1.45 eV), a good supporting strength, and, more importantly, an easy removal in solvents due to the intrinsic property as a small molecule [To meet these requirements, the first need is to study the reason for impairments on the surface of graphene. The impairments mainly result from the removal of the protective polymer layer in solvents. The purpose of this polymer protective layer is to protect the graphene from folding, tearing, and cracks. A good protective layer should have low adsorption energy  by low pressure chemical vapor deposition (LPCVD) in a quartz tube furnace [4:H2 = 0.5:300 sccm) was flowed in the furnace at the same temperature/pressure for 50 min to grow thin crystalline film of graphene. After the synthesis, graphene samples were cooled down to room temperature (the flow of methane was stopped at 600 °C). However, the carbon dissolves in metal up to a few atomic percent; the use of non-carbide forming metals, e.g., Cu, Ni, and Pt, is preferred [The graphene samples employed here were grown on a 25-μm-thick copper (Cu) foil  dissolved in ethyl lactate was used because of high viscosity and good film forming ability. Note that the employment of rosin with concentration less than 50 wt. % usually leads to less viscous, smother, and low film forming ability which cannot offer sufficient support for graphene. The rosin/graphene/Cu samples were then placed in cleansing solution (HCl:H2O2:H2O = 1:1:1) for 50 s to remove the dust and the residues attached on the back side of Cu during the spin coating. The accessible graphene-copper face was then etched by immersing in a marble solution HCl (50 ml):H2O (50 ml):CuSO4·5H2O (10 g) for 1.5 h, leaving behind a pliant membrane of rosin/graphene suspended in the solution. The suspended membrane was transferred to DI water for 5 times to rinse residual etching solution. The floating flexible and fragile membrane was transferred on the SiO2 substrate with care and precision. A modified rosin transfer process was proposed to further reduce the polymer residues and to improve the quality of transferred graphene, where rosin/graphene/SiO2 samples were spin-coated with anisole at 500 rpm for 10 s and at 1200 rpm for 30 s. All samples were categorized into not baked  and baked at 100 °C, 150 °C, and 200 °C for 30 min. The supporting rosin layer is removed by acetone bath, while anisole is used in the improved rosin-enabled transfer process which was also then removed by acetone bath. All transferred graphene was characterized using the Raman spectroscopy at 532 nm excitation wavelength in air using × 100 objective to determine the quality of pristine and as-transferred graphene layer using the improved rosin-enabled transfer process. The AFM characterization is done in tapping mode using the Bruker Dimension Icon model at standard temperature and atmosphere conditions. The four-point measurement (Kelvin technique) is performed to measure the sheet resistance at random points on the 2 × 2 cm2 area of samples.Figure Rq) values of as-transferred graphene are also collected by scanning surface area of 10 μm × 10 μm. Compared to Rq values of 0.889 nm, 0.97 nm, and 0.992 nm for graphene baked at 100, 150, and 200 °C, the lowest Rq value of 0.668 nm occurs for the graphene without baking. This, however, points out that baking is not beneficial in achieving a low Rq value which is also desired for practical device application of graphene. This Rq value can be especially used as the quantification of surface morphology of transferred graphene. The water molecules trapped between pliant graphene membrane and SiO2 during pickup from DI water would rupture the graphene, hence producing cracks within the graphene. As a result, the Rq value increases with increasing baking temperature. It is, therefore, not recommended to bake graphene at high temperatures even if baking is good at reducing residue particles.Figure Rq values for graphene without baking and with baking at 100, 150, and 200 °C are 0.523 nm, 0.887 nm, 0.95 nm, and 0.98 nm, respectively. A relaxation to as-transferred graphene with the introduction of anisole may help in achieving the lower Rq value of 0.523 nm in the improved rosin transfer process than that of 0.668 nm in the rosin transfer process, while the lowest value for Rq in case of conventional transfer method using PMMA is 1.03 nm. In this improved rosin transfer process, it is again proved that the baking is not beneficial in achieving a low Rq value because of similar reason, i.e., cracks produced during the baking at high temperature. Note that compared to the Rq value of 1.03 nm in the PMMA transfer process, both the rosin and improved rosin transfer process show much smaller Rq values, which manifests the superiority of adopted graphene transfer processes in this work. Compared with Rq roughness, the maximum height of large residual particles (Rmax) is also an important parameter in the application of large area thin film devices, because it determines whether short circuit may occur in devices. Figure Rmax at room temperature, 100 °C, 150 °C, and 200 °C. The minimum value for the Rmax, i.e., 15 nm, is achieved at RT for anisole/rosin/graphene. This also confirms the advantage of improved rosin transfer process at RT.Figure Rq values and Rmax, the quality of as-transferred graphene deserves to be evaluated. In Fig. −1 (G), a primary in-plane vibrational mode, and 2676 cm−1, a second-order overtone of a different in-plane vibration (2D), are found. These peaks are adduced from a 532-nm excitation laser. The position and shape of these two peaks are prominent, clearly defining the material to be graphene. Also, the ratios of 2D band to G band intensities (I2D/IG) are 1.61 to 1.65, indicating the single layer of as-transferred graphene. The absence of D peaks in the Raman spectra for as-transferred graphene with baking at different temperatures confirms that the disorder is unlikely to appear using both the rosin and improved rosin transfer process. Also, no rosin- and anisole-related peaks are detected for all transferred graphene. The assumption no rosin- or anisole-related peaks was made on the fact that the Raman spectra appeared to be same after transfer process as those observed compared to the Raman spectra of pristine graphene grown on Cu. The appearance of D peak after transfer process in the baked sample shows the induced defects during the removal of rosin. Furthermore, the rosin residues after transfer process are very low. Therefore, rosin-related peaks are unlikely to appear in the Raman spectra of as-transferred graphene.Despite that the improved rosin transfer process is obviously advantageous in terms of residue particles and −1 demonstrates the doping of rosin-enabled transfer process; the described phenomenon has been reported previously in the literature [−1 which is the smallest compared to those with baking at high temperatures. These results mean that room temperature is favorable for achieving high-quality graphene during the rosin transfer process.Shifts in both the G and 2D Raman peaks of graphene are usually produced by a combination of strain and doping due to the interaction with the substrate or support layer during transfer process. It is known that the blue shift of both the G band and 2D band positions indicated p-doping of graphene. The entailed 2D peak upshift of ~ 6 cmterature , 25. The−1 which is a little bit lower than that in Fig. In Fig. Rsh) data are collected and illustrated in Fig. Rsh is measured at 5 points on each sample. The size of the sample is about 2 × 2 cm2 in order to get reliable results. In Fig. Rsh data for as-transferred graphene using the rosin transfer process at random spots are presented. As seen, for all graphene, scattered Rsh values in the range of 500–700 Ω/□ are found across the surface of as-transferred graphene. The lowest value of Rsh occurs for the graphene without baking which is also in good agreement with the observations from the Raman spectra. In Fig. Rsh values for as-transferred graphene using the improved rosin transfer process are shown. As seen, compared to Fig. Rsh is much better and the range of Rsh values is significantly narrower, i.e., 500–600 Ω/□. More importantly, the Rsh values in the improved rosin transfer process are generally lower than those in the rosin transfer process and the lowest Rsh value of ~ 500 Ω/□ also happens for the graphene without baking. Figure Figure −1, which is obviously lower than 38.18 cm−1 for transfer graphene using the rosin transfer process. In addition, as-transferred graphene using the improved rosin transfer process shows generally lower Rsh values of 500–600 Ω/□ than those of 500–700 Ω/□ using the rosin transfer process. The baking at high temperatures is found to exert marginal effects on residue particles and quality for as-transferred graphene which is thus not recommended. Achieved results in this work are ought to be helpful in advancing clean graphene transfer process in order to realize graphene-based devices of high performance in the future.In this work, an improved rosin transfer process is proposed for the purpose to reduce residue particles further on the basis of the rosin transfer process. The established improved transfer process is compared with the conventional PMMA transfer process. It is found that this improved rosin transfer process by the introduction of anisole is indeed advantageous in terms of significantly reduced residue particles as well as good quality of transferred graphene. This remarkable reduction of residue particles would rather be attributed to the capability of anisole as a strong solvent in collaboration with acetone. Anisole/rosin dissolves more easily than bare rosin in acetone which leads to cleaner graphene in this improved rosin transfer process. The FWHM value of 2D band for as-transferred graphene using the improved rosin transfer process is 35.79 cm


In this study, the effect of the argon, nitrogen, and hydrogen gases on the final properties of the reduced graphene oxide- hydroxyapatite nanocomposites synthesized by gas injected hydrothermal method was investigated. Four samples were synthesized, which in the first sample the pressure was controlled by volume change at a constant concentration. In subsequent samples, the pressure inside the autoclave was adjusted by the injecting gases. The initial pressure of the injected gases was 10 bar and the final pressure considered was 25 bar. The synthesized powders were consolidated at 950 °C and 2 MPa by spark plasma sintering method. The final samples were subjected to Vickers indentation analysis. The findings of this study indicate that the injection of argon, hydrogen, and nitrogen gases improved the mechanical properties of the nanocomposites. Injection of gases increased the crystallinity and particle size of hydroxyapatite, and this increase was greater for nitrogen gas than for others. Injection of these gases increased the rate of graphene oxide reduction and in this case the effect of nitrogen gas was greater than the others.  But still, it has poor mechanical properties such as intrinsic brittleness, low fracture toughness (fracture toughness of 0.28–1.08 MPa.m0.5), poor tensile strength, and weak wear resistance which have limited the scope of its applications [Calcium phosphates have been widely used in the medical field. Members of this family include hydroxyapatite (HA), Tetracalcium phosphate (TeCP), α- Tricalcium phosphate (α-TCP), β- Tricalcium phosphate (β-TCP), dicalcium phosphate dehydrate (DCPD), dicalcium phosphate anhydrous (DCPA), and octacalcium phosphate (OCP). Among these bioceramics, HA is less soluble in the biological environment and therefore suitable for orthopedic applications as an implant , 2. HA iications –31. Reseications –49.Different methods have been applied to synthesize hydroxyapatite and graphene nanocomposites so far. Among these methods, the hydrothermal process has received much attention because of its lack of calcination stage, excellent control and the ability to synthesize in situ. In this method, precursors containing calcium and phosphate ions are used as suppliers of the elements required for hydroxyapatite and graphene oxide as the graphene precursor. The high-pressure hydrothermal process results in the synthesis of hydroxyapatite and the reduction of graphene oxide . During the process, calcium ions first bind to the graphene oxide surface agents by ionic bond, and finally, in reaction with phosphate ions, hydroxyapatite is synthesized in situ on the surface of the graphene sheets. The synthesized powders are consolidated in various ways such as spark plasma sintering (SPS) and hot pressing (HP). Recently, gas injection into hydrothermal autoclave has been used to increase the rate of graphene oxide reduction. Increasing hydrothermal pressure by injecting gas also improves the mechanical properties of hydroxyapatite. Increasing the hydrothermal pressure in this method increases the crystallinity and crystallite size of HA. But the type of gas injected will affect the final mechanical properties obtained. Previously, researchers have shown that nitrogen gas reduces graphene oxide more than hydrogen gas –63.To investigate the effect of the type of injected gas on the final mechanical properties, three types of gas including argon, nitrogen, and hydrogen are considered in this study and the pressure of the gases was assumed to be constant. The synthesized powders were then consolidated by SPS and examined for mechanical properties. Characterization methods for synthesized powders and sintered samples include X-ray diffraction (XRD), Raman spectroscopy, Fourier transform infrared spectroscopy (FTIR), field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), high angle annular dark field (HAADF), energy-dispersive X-ray spectroscopy (EDS) and Vickers indentation technique. The results obtained in this study are expected to be very useful for similar researches.The chemicals used in this study include calcium nitrate tetrahydrate , diammonium hydrogenphosphate , ammonium solution , and GO . Figure For sintering of the powders, SPS method was used (Dr Sinter SPS-1050-CE). Details of the sintering operation have already been described in detail , 65.Table Eq. (112/300) and I300 are the intensity of the hollow between diffraction peaks of HA in the planes (300) and (112) and the intensity of the peak of HA in the plane (300), respectively. Eq. where υ11/300 and In this equation, d, θ, and λ are grain size, Bragg diffraction angle, and wave length of used X-ray (Cu), respectively. β and ε are full width at half height (FWHM), and crystalline lattice strain, respectively. Diamond 3.2, ImageJ, and Origin pro 2016 were used in this study for drawing shapes and graphs.Figure Scheme Figure Figure Figure Figure −1 is related to the HA structure (Fig. Figure ure Fig. b. Accordure Fig. c, d. TheFigure Figure The findings of this study indicate that injection of argon, hydrogen and nitrogen gases improved the mechanical properties of rGO-HA nanocomposites. Injection of gases increased crystallinity and particle size of HA, and this increase was greater for nitrogen gas than for others. Injection of these gases increased the rate of GO reduction and in this case the effect of nitrogen gas was greater than the others. The powders synthesized in this study included graphene sheets decorated with HA nanorods. The addition of graphene sheets made the mechanical properties of HA more uniform. Powder characterizations showed that injection of gases increased the HA-related peak intensities in XRD analysis, decreased the GO-peak intensities in FTIR analysis, and increased the ID/IG ratio in Raman spectroscopy. The results of this study could be useful for gas injection applications in hydrothermal autoclave.


Electrical performances of the fabricated graphene interconnects were evaluated, and the impact of temperature and size on the current density and reliability was investigated. The maximum current density of 1.18 ×108 A/cm2 was observed for 0.3 μm graphene interconnect on SiO2/Si substrate, which is about two orders and one order higher than that of conventionally used copper interconnects and CVD grown graphene respectively, thus demonstrating huge potential in outperforming copper wires for on-chip clocking. The drop in current at 473 K as compared to room temperature was found to be nearly 30%, indicating a positive temperature coefficient of resistivity (TCR). TCR for all cases were studied and it was found that with decrease in width, the sensitivity of temperature also reduces. The effect of resistivity on the breakdown current density was analysed on the experimental data using Matlab and found to follow the power-law equations. The breakdown current density was found to have a reciprocal relationship to graphene interconnect resistivity suggesting Joule heating as the likely mechanism of breakdown.Graphene interconnects have been projected to out-perform Copper interconnects in the next generation Magnetic Quantum-dot Cellular Automata (MQCA) based nano-electronic applications. In this paper a simple two-step lithography process for patterning CVD monolayer graphene on SiO Copper being the state-of-art interconnect material is facing severe challenges while being scaled down to nano dimensions due to its increased resistivity that is mainly because of its surface and grain boundary scatterings4 and also susceptibility to electromigration effect5. Moreover, copper as an on-chip clocking material requires high currents and large dimensions6 in order to generate the external field in MQCA. To illustrate, copper interconnects (CI) used in MQCA occupies more than 2000 nm in order to generate the required external field for data propagation between the nano-magnets7. Therefore CI impose limitations on the emerging nanoelectronic applications leading to extensive research in finding an alternate material that can replace the CI.Interconnects are going to play an important role in the next generation Magnetic Quantum-dot Cellular automata (MQCA) based nano-electronics14 can be envisaged as a potential interconnect which could replace copper. Moreover, graphene is projected to be an excellent candidate material for interconnects due to its high carrier mobility (2/V-s)15, ballistic transport16, high current carrying capacity17 and high thermal conductivity18. On the other hand, the length of the nanomagnets used in typical MQCA devices are more than 100 nm and therefore the underline interconnects should be in size ranging from more than 100 nm and less than the CI which are currently used.Thus in this context, graphene20 resulting in the maximum current density of 4 ×107A/cm2 and 1.2×107 A/cm2 on SiO2 substrate respectively. This is an order of magnitude less than the state-of-the-art Graphene Nanoribbon where the size is typically much less than 100 nm hence, it is not suitable as interconnects in MQCA22. Thus in an attempt to increase the current density in the CVD grown graphene, several hybrid structures have been reported27 with an additional overhead of significant increase in the fabrication complexity. To address these issues we introduce here a simple fabrication procedure of patterning graphene interconnects in the context of next-generation MQCA, still retaining an order of magnitude higher current density as compared to the present CVD graphene interconnects.CVD based multilayer and monolayer graphene interconnects have been investigated in2/Si substrate. Thus in this paper we report-a simple two-step lithography process for patterning CVD monolayer graphene interconnects resulting in one order of magnitude higher current density andeffect of temperature and size on the breakdown current density of CVD-monolayer graphene interconnects.Furthermore, in order to analyse the problem of integrating graphene interconnects in next-generation MQCA based nano-electronics, it is necessary to understand the effect of temperature and size impact on the electrical parameters. Graphene being projected as a potential alternative for interconnect material, the assessment of temperature and size would be of utmost importance. However, published literature lacks data on symbiotic effect of temperature and size on the electrical conductivity of graphene. Hence an organised attempt has been made here to understand the combined effect of temperature and size on the electrical behaviour of CVD grown monolayer graphene on SiOThe rest of the paper is organised as follows: Section 2 describes the experimental setup, section 3 describes the results followed by its discussions and the section 4 draws the conclusion.20. This results in the usage of different polymer resist that leaves behind the resist residues in each step of the lithographic process thus reducing the current carrying capacity of the graphene interconnects.State of the art, patterning of CVD based graphene interconnects involves a minimum of five lithographic processes i.e. two times optical and three times electron beam lithographyIn this paper, we report a novel two-step lithography process for CVD monolayer graphene patterning. A detailed description with pictorial representation has been given in Fig. 2 (300 nm thickness)/Si substrate using wet transfer process that was procured from M/s. Graphenea Inc. USA. The grain size was approximately 10 μm for monolayer graphene with greater than 95% coverage and with small multilayer islands.Initially, we started with graphene monolayer (10 mm × 10 mm) produced by CVD on copper catalyst and transferred to a SiO2. Figure 2 drying. Subsequently metal deposition was carried out at a pressure of 10−6 Torr and then lift off was done in acetone for 4 hours.The first lithography step involved patterning the metal contacts and the alignments marks. The alignment marks and the metal contacts Cr/Au (10 nm/70 nm) were fabricated by electron-beam lithography (EBL). Bilayer EBL resist, has been used in the reported experiment in order to achieve better liftoff of Gold (Au). PMMA - a high-resolution e-beam resist and Copolymer EL9 is selected as the bilayer resist material for the EBL. The sample is exposed to an electron beam of acceleration voltage 16 kV and a dose of 180 μC/cm2. The resist was developed in Microposit MIF319 developer and Tetra-methyl ammonium hydroxide (TMAH 2.3%) for 1 sec. While using HSQ an overlying dielectric layer is formed on the graphene interconnects. A low power oxygen plasma etch for 1 min 30 sec and at 50 Watt power was used, but still the fine line remained. After the plasma etch the HSQ resist pattern was etched into the graphene flake.The second lithography step involves patterning the graphene interconnects with width (W) in the range of 300 nm to 1500 nm, length of 1 μm and thickness of 0.335 nm. HSQ (Hydrogen Silsesquioxane) was used for patterning the graphene channels at 2000 rpm for 45 sec and exposing it to acceleration voltage of 18 kV and a dose of 190 s μC/CmThe Current-voltage (I-V) characteristics of the device were measured using Proxima (Fast IV Measurement/B1500) and Keithley 4200 Semiconductor Characterization System with two-probe configuration. The (I-V) characterisation was done by sweeping voltage in steps of 0–0.5 V, 0–1 V followed by 0 to breakdown voltage. Due to increasing current density in the graphene interconnects, there was a voltage at which the break down occurred in the graphene interconnect, resulting in a visible drop in current. The device testing was stopped at this point. This way of measurement helps in suppressing the effect of trapping centres due to resist residuals. Breakdown voltage for all cases was nearly found at 2 V. The temperature measurements were carried out at 298–473 K respectively. Temperature vs I-V characterisation results are discussed in the following section.22 that HSQ coating does not degrade the carrier mobility or rather they sometimes improve the mobility. Thus the reported two-step process of fabricating graphene interconnect not only reduces the rigour of fabrication but also reduces the residual trapping leading to higher current capacities of the graphene interconnects that is discussed in the subsequent section.However, it has been reported inSEM and Raman spectrum studies were conducted at several locations in order to confirm the uniformity of the monolayer graphene film.−1, respectively. This ensures I(G)/I(2D) = 0.55, which indicates that it is a monolayer graphene sample33. Figure 2 (300 nm/Si substrate).Figure 38. Therefore we have used CVD Monolayer graphene film on SiO2/Si substrate procured from M/s. Graphenea Inc, USA, with a current density of 102 A/cm2, having width of 1 Cm × 1 Cm × 0.35 nm as the starting material for patterning graphene interconnects.In spite of graphene devices that are produced from highly oriented pyrolytic graphite (HOPG), laser ablation, spin coating, CVD graphene is widely known method for device fabrication since it is by far the most popular way for producing graphene and also results in relatively high quality graphene, potentially on a large scale20 have also used CVD graphene for electrical characterization of the patterned samples and the maximum current densities reported are up to 4×107 A/cm2 and 1.2×107A/cm2 respectively which is about an order lesser than that of our fabricated graphene interconnects with the maximum current density of 1.18 ×108 A/cm2, which is attributed by the proposed simplistic two-step procedure.Besides, study reported in20.The major bottleneck for graphene application is, while patterning due to multistep process - the electronic transport properties gets degraded due to the resist residual trapped in the grain boundaries and on the surface of the CVD graphene that acts as scattering sites, limiting the transport of charge carriers resulting in the reduction of the electron mobility and also increase in the sheet resistance of the CVD graphene sheet20.Thus in this paper, a simple two-step lithography process has been reported for patterning graphene. As the number of lithography steps has been reduced, the effect of resist residuals and contaminants on the graphene surface also gets reduced resulting in one order higher current density as compared to other patterned CVD graphene current densities reported inThe results obtained has been divided into four main parts3 39, hence in order to place the easy axis of the nanomagnets on the graphene interconnect, the minimum width of the graphene interconnect was selected in the range of equal to or more than 300 nm and less than the copper interconnect, so as to maintain the keep-out zone .The variation of current with width 0.3 to 1.5 µm) of monolayer graphene interconnect is given in Table .5 µm of It was observed that with an increase in width of the graphene interconnects, the current also increased. It was found that the current (at breakdown voltage) increases at an increasing rate, though the breakdown current density decreases as shown in Fig. 40.The results obtained indicate that as the width increases the influence of the interconnect boundaries (rough area) decreases leading to lesser scattering and increased conductivity & current8 A/cm2, was observed which is an order more than state-of-the-art20, whereas the minimum was found to be 3.68 ×107 A/cm2 for 1.5 μm interconnect at room temperature.Breakdown current densities for all the cases were calculated and a maximum current density of 1.18×1021. The voltage and current were found to follow ohm’s law, i.e. V α I.This may be attributed by the reduction in the number of lithography steps as compared to the state of the patterning of CVD Graphene interconnects. Figure Patterning CVD monolayer graphene samples is quite an exhaustive, complicated and expensive process and thus we have formulated a mathematical expression for calculating the current from the width of the interconnects. The relationship between the current and width has been derived from Fig. The best fit for the obtained values of the intercept were found to be a = 143.93, b = −177.47 and c = 288.44. The graphene interconnects between the two metal electrodes on an insulating oxide layer was electrically characterised in the temperature range of T = 298 K to 473 K. The typical I-V characteristics of the CVD monolayer graphene interconnect of a 0.6 µm is given in Fig. ρ = ρ0 + AT (A is a positive constant). Therefore, it can be observed from the above figure, that the resistance dependence of temperature can be modelled by the equation.It was observed that with the increase in temperature, the current decreased. The resistivity was found to follow the law Temperature Coefficient of Resistance (TCR), defined as:The sensitivity to temperature was also calculated for this case by using the −1. Thus it was found that TCR was positive and therefore with increase in temperature, the current decreased.The temperature coefficient of resistance at 298 K for 0.6 µm was found to be 0.00248 KTCR for all other cases of the study was also calculated and is listed below:-i.e. the sensitivity of the resistivity to temperature variations decreases. The TCR for 1 µm was found to be about 38% larger compared to 300 nm wide interconnects.From Table The variation of current as a function of a) width of graphene interconnects, and b) temperature ranging from 298 K to 473 K are shown in Fig. We observed that interconnect of width 0.3 μm could not withstand the temperature of 473 K and rather failed after 423 K. The drop in current at 423 K for 0.3 μm width as compared to room temperature was noted to be 17% whereas, for all other cases, the drop in current at 473 K as compared to room temperature was more than 30%. The maximum drop in current was observed for 1.5 μm as 51%. Thus temperature was found to play a significant role in the current-carrying capacity of the graphene interconnect thus leading to decreased current densities.et al.41, that Cr/Au forms a good Ohmic contact with graphene and hence the contact resistance (Rc) due to the Cr/Au pads were found to be negligible as compared with the sample resistance. Similarly, as reported by Wei et al.42 the contact resistance versus contacts inter-spacing plot for CVD grown graphene with Cr/Au contact pads deposited on top of graphene channel was found to be 20 ohms for the contact inter-spacing of 1 µm. The similar observation on the contact resistance was also reported by Cai et al.43, where Cr/Au metal contact pads were fabricated on Single-layer graphene yielding 33.5 Ω which was found to be compared favourably with the study reported in42. We have adopted the procedures followed in the previously reported studies43, with a similar kind of setup for planning our experiments. The results shown in43 can also be correlated to the experimentally fabricated monolayer graphene interconnect used in our study.It has been reported in the literature by Withers 43, the contact resistance is within the range of 20–34 ohm. The total resistance of the proposed fabricated graphene interconnects falls in the range of 3.5–18 KΩ, and thus the impact of the contact resistance (in the order of 20–34 ohm) on the total resistance can be considered as negligible which is also aligned with the findings reported in17 by Murali et al.Moreover from Fig. 2 substrate on which graphene interconnect is directly lying.Figure 2, C and b are constants while 8 and b = −0.64 with ρ having the units of μΩ-cm. The effect of resistivity on the breakdown current density was analysed on the experimental data using Matlab. From the plot obtained Fig. , it was The exponent term of 0.64 represents the breakdown of the graphene interconnects with increasing resistivity, indicating that higher resistivity is responsible for the degradation of breakdown current density. This higher resistivity can be attributed to the defect morphologies such as the lattice imperfections, in-plane defects and voids, thus resulting in - jJoule’s heating that finally results in the electrical breakdown.x) along a conductor is governed by the heat equation –g is the thermal conductance of the GNR, and a is the area of cross-section. Inserting the values of k, g from ref. 17, the maximum temperature was found to be 1073 K (800 °C) at x = l/2, where l is the length of the graphene interconnect.The maximum temperature point of a suspended longitudinal dc biased graphene interconnect was found at the middle point. In steady-state conditions, the distribution of temperature θ, where l is the length of the graphene interconnect resulting thermal oxidation of graphite45 and maybe intimately connected with self-heating of the graphene interconnects.Thus the mechanism of breakdown reported here is believed to be Joule’s heating. Similar phenomenon has been reported by Collins Furthermore, in order to explain the constant breakdown voltage with the increment in width, the Power dissipation (Joule’s heating) for all cases was calculated as shown below:-It can be observed from Table Thus from the experimental observation, it can be found that:-It can be noted that, although the product of I and R, i.e., breakdown voltage (V) remains the same, for various widths, the quadratic component I play the dominant role in contributing to the self-heating phenomenon which is referred here as the Joule’s heating and the same can be represented using the inequality below and is also tabulated in Table 2R is causing the breakdown in the graphene interconnects which is the jJoule’s heating. Also it can be noted that with the wider graphene patterns, both the heat dissipation and heat absorption capability increases due to increase in the surface area. Since the radiative heat transfer is directly proportional to the surface area, increase in Joule’s heating due to the quadratic component of current (I2) is therefore compensated by the higher heat dissipation resulting in averaging out the effect of increase in the temperature of the patterned graphene sample and hence the breakdown voltage doesn’t get lowered down even for the wider graphene patterns.Thus, the quadratic component of the current I is not same throughout and therefore the Power dissipation, P = IHence from the experimental observations and the theoretical calculations, the authors intuitively conclude that the mechanism of breakdown is attributed primarily by the self-heating referred to as Joule’s heating.A simple two-step lithography for patterning CVD monolayer graphene has been used, thus resulting in lower complexity, cost-effective solution and higher current density as compared to the state-of-the-art fabrication methods for CVD graphene.8 A/cm2 (for 0.3 μm width) that is 100 times (106 A/cm2) more than the copper, resulting in outperforming copper-based interconnects.The fabricated graphene interconnects were found to have a current density of 1.18 ×10Based on our experimental observation, we formulated a generalised equation between the current and width of the CVD monolayer graphene-based interconnect that enables to calculate the current or width values without doing fabrication.The current at the breakdown point was found to decrease significantly with the increase in temperature, indicating a positive temperature coefficient of resistance. Further, as width of interconnects increases from 0.3 to 1.5 μm, the current at breakdown voltage was found to increase, at an increasing rate. This is due to decrease in scattering because of lesser contribution of boundaries, as the width of interconnects increases.It was observed that the graphene interconnects failed at the mid-segment of the interconnects due to incipient fusion at the breakdown voltage.The failure mechanism of the graphene interconnects was analysed. We found that the current density and the resistivity follows a power-law relationship signifying that higher resistivity of the interconnects were also responsible in the degradation of breakdown current density. This higher resistivity can be attributed to the defect morphologies such as the lattice imperfections, in-plane defects and voids, thus resulting in - joule’s heating that finally results in the electrical breakdown.2 substrate obeyed Ohm’s law.Monolayer graphene interconnects on SiOThe drop in current at 473 K for nearly all other cases as compared to room temperature was more than 30%.The temperature was found to have a profound effect on the graphene interconnects. Results show that while working with lower widths of the monolayer graphene interconnects at high temperatures pose challenges.The effect of width and temperature on the electrical properties of CVD monolayer graphene has been investigated experimentally. The outcomes of the study are summarised below:8 A/cm2 for 0.3 μm graphene interconnect on SiO2/Si substrate, i.e. two orders higher than that of conventionally used copper interconnects, Graphene has an enormous potential in outperforming copper wires.Thus with the maximum current density being 1.18 ×10


The majority of cases of dyspepsia are functional, with a very rare cause of organic dyspepsia being gastric sarcoidosis. Although gastric sarcoidosis is the most common gastrointestinal manifestation of sarcoidosis, it is asymptomatic in >99% of cases. This is a case of a 48-year-old with known pulmonary sarcoidosis who was diagnosed with gastric sarcoidosis after presenting with dyspepsia. Dyspepsia is one of the most common gastrointestinal problems encountered in the outpatient setting, affecting 7%-45% of the population worldwide . The majOur patient was a 48-year-old African American female with a history of sarcoidosis, depression, and vitamin D deficiency. Her sarcoidosis was initially diagnosed by bronchoscopy and endobronchial ultrasound-guided biopsy after the patient was found to have incidental pulmonary nodules about six years prior to this presentation. She had been treated with systemic steroids and methotrexate, and had been stable off medications for one year prior to presentation. During a routine healthcare visit, she reported new onset of nausea and heartburn for one month. She was started on pantoprazole 40 mg daily, which only slightly improved her symptoms. In view of her systemic disease and advanced age, she was referred for an esophagogastroduodenoscopy, which showed Los Angeles classification grade A esophagitis of the gastroesophageal junction Figure  and erosBiopsies of gastric antrum and body showed severe active chronic gastritis with non-necrotizing granulomatous inflammation Figure , with neThe patient resumed treatment with steroids and improved symptomatically, and she is now being followed outpatient.Dyspepsia is a constellation of upper gastrointestinal (GI) symptoms, including the presence of upper abdominal pain or discomfort with or without other symptoms such as nausea, belching, and vomiting. Less than 30% of cases of dyspepsia are found to have an organic cause. Most cases of dyspepsia are functional, as defined by the Rome IV criteria . These iSarcoidosis is a multisystemic disease characterized by the histological presence of non-caseating granulomas, which most commonly manifests in the lungs. Gastrointestinal sarcoidosis is rare and usually asymptomatic, with only 0.1%-0.9% of all cases displaying symptoms . The mosRegarding diagnostic approach to GI sarcoidosis, a high degree of suspicion is required as the diagnosis is made histologically with upper endoscopy and biopsy. The endoscopic appearance varies depending on the type of involvement, but can range from normal appearing mucosa to erosive, friable mucosa with nodularity and prominent folds, ulceration, or polyps. A deep mucosal or full thickness biopsy is recommended, as superficial biopsies may be non-specific. Tissue biopsy typically demonstrates the characteristic histologic findings of sarcoidosis: non-caseating granulomatous lesions with multinucleated giant cells. It is imperative to rule out infections and other etiologies of granulomas with appropriate staining. Management consists of immunosuppressive and symptomatic therapy in patients with symptoms; asymptomatic patients are usually not treated . The maiPatients with dyspepsia can have significantly decreased quality of life, although their life expectancy is normal. A large number of cases of dyspepsia remain uninvestigated, with their overall pooled prevalence estimated at 21% . Althoughttps://insights.ovid.com/crossref?an=00000434-201910001-02739) [This article has been presented as an abstract (1-02739) .A rare organic cause of dyspepsia is gastric sarcoidosis, which can have a variety of presentations and endoscopic appearances. It can be diagnosed by its characteristic biopsy appearance, and treated with systemic steroids when symptomatic. Our case highlights a rare and likely underdiagnosed cause of dyspepsia, the importance of maintaining a broad differential diagnosis, and appropriate use of upper endoscopy for evaluation of patients with new-onset dyspepsia.