Microplastics (MP), as novel substrata for microbial colonization within aquatic ecosystems, are a matter of growing concern due to their potential to propagate foreign or invasive species across different environments. MP are known to harbour a diversity of microorganisms, yet little is understood of the dynamics of their biofilms and their capacity to successfully displace these microorganisms across different aquatic ecosystems typically marked by steep salinity gradients. To address this, we performed an in situ sequential incubation experiment to simulate MP transport from riverine to coastal seawaters using synthetic  and natural (Wood) substrata. Bacterial communities on incubated particles were compared to each other as well as to those in surrounding waters, and their dynamics along the gradient investigated. All communities differed significantly from each other in their overall structure along the salinity gradient and were shaped by different ecological processes. While HDPE communities were governed by environmental selection, those on TW and Wood were dominated by stochastic events of dispersal and drift. Upon transfer into coastal seawaters, an almost complete turnover was observed among HDPE and TW communities. While synthetic particles displaced a minor proportion of communities across the salinity gradient, some of these comprised putatively pathogenic and resistant taxa. Our findings present an extensive assessment of MP biofilms and their dynamics upon displacement across different aquatic systems, presenting new insights into the role of MP as transport vectors. Since the advent of microplastic (MP) research, scientists have endeavoured to draw a clearer picture of the distribution and impact of these synthetic items throughout their largest sink—the ocean . The intMP have been posited through several studies to act as a novel niche within aquatic environments, with MP-associated communities found to be distinct from those in surrounding waters –14. AddiWhile the threat of a MP-mediated propagation of microorganisms throughout aquatic ecosystems remains a central concern, still very little is understood of the processes that drive the formation of these biofilms and the capacity of MP to successfully displace these communities across the strong salinity gradients that mark these different systems . As riveA sequential incubation experiment was conducted from July to September 2018 along a salinity gradient that spanned the transect from the Weser River to the offshore island of Helgoland (North Sea) in Germany Fig. .Fig. 1ThSynthetic particles of approximately 4 mm, consisting of high-density polyethylene  and tyre wear , were enclosed within stainless steel mesh capsules along with natural wood particles of similar diameters , and contained within two metal cages . One cage (Transfer), comprising 36 capsules , was sequentially incubated along four sites , for predetermined periods of time reflective of modelled retention times at each site. Particles were first exposed to freshwater conditions (daily mean conductivity = 1.16–1.37 mS/cm) upstream of the weir in Bremen for 33 days, with no tidal influence due to the presence of the weir. The cage was then transferred to Brake, located roughly 40 km from the river mouth, where particles were incubated for 14 days. These waters, comprising the tidal freshwater reaches of the estuary, generally receive a well-mixed influence of marine and freshwater (daily mean conductivity = 2.32–3.72) and are characterized by oligohaline conditions. The third site was situated by the river mouth in Bremerhaven and marks the border of the reach. In these brackish waters, particles were exposed to strong salinity shifts due to incoming tides (daily mean conductivity = 26.93–29.03 mS/cm) over a period of 13 days. Lastly, at the final site located off the island of Helgoland, particles were incubated for 23 days in coastal seawaters characterized by highly saline conditions (daily mean conductivity = 41.20–44.67 mS/cm) and year-round mixing. Concurrently, the other cage (Stationary), comprising 9 capsules , was incubated in the final offshore site (Helgoland) throughout the entire duration of the experiment as a marine control.For the transferred cage, three capsules of each type were collected following incubation at each site and surrounding waters were sampled at the beginning and end of each incubation period to provide a comparison against source communities. Abiotic conditions were similarly measured before and after each incubation using a multi-probe . Additionally, data on the conductivity and temperature at each site throughout the incubation experiment were obtained from the responsible agencies and displayed in Fig. Total biofilm DNA was extracted from incubated particles using the DNeasy PowerBiofilm kit  according to the manufacturer’s instructions. Waterborne communities were analysed by serial filtration through sterile 3.0 µm and 0.2 µm polycarbonate filters  to assess both the particle-associated  and free-living  fractions, respectively. DNA of waterborne communities were isolated using the DNeasy PowerWater kit  as per the manufacturer’s instructions. Details on starting sample material and DNA concentrations are provided in Table Library preparation and amplicon sequencing were performed by LGC Genomics GmbH . Bacterial 16S rRNA genes were amplified using the primer pair 341F (5′-CCTACGGGNGGCWGCAG-3′) and 785R (5′-GACTACHVGGGTATCTAAKCC-3′) , targetiReads were processed on the QIIME 2™ platform (v.2020.8 – 2021.2) . QualitySRS package [Analyses of processed reads were performed in R (v.4.0.3 onwards) . Reads w package  and a spvegan package [picante package [stats and FSA packages [Both the taxonomic and phylogenetic diversity of communities were investigated in this study. Shannon and Gini-Simpson indices were calculated using the  package  to estim package . The stapackages .metacoder package [UpSetR package [Significant compositional differences between sample types were explored through differential abundance testing using the  package . To eluc package , where t package  to assesvegan package, weighted and unweighted taxonomic dissimilarities were computed based on Bray–Curtis and Jaccard indices, respectively, while weighted and unweighted UniFrac distances were calculated using the phyloseq package [Dissimilarities in the taxonomic and phylogenetic structure between and within sample types were investigated. Using the  package  to asses package  performebetapart package [ape [phyloseq packages, respectively [Dissimilarities were partitioned based on the Jaccard index into components of turnover and nestedness using the  package  to invesage [ape  and phylectively .picante package and custom scripts [bray). Deviations from the null expectation indicate dispersal limitation (RCbray > 0.95) or homogenizing dispersal (RCbray < -0.95), while values that fall within the null distribution (−0.95 < RCbray < 0.95) suggest a turnover undominated by selection or dispersal, otherwise referred to as ecological drift.A null model framework  was impl scripts , 44 to i scripts  based onhttps://github.com/jessicaxsong/TheTravellingParticles.16S amplicon sequences generated in this study have been deposited in the European Nucleotide Archive at EMBL-EBI under accession number PRJEB47707. Processing scripts are made available at Proteobacteria  while TW and particle-associated waterborne communities (Water 3) were predominated by Gammaproteobacteria. A significantly greater abundance of Gammaproteobacteria and Alphaproteobacteria were detected on TW and Wood, respectively, each constituting a mean relative abundance of more than 50% of their overall communities  and Cyanobacteria (2%), along with others that made up less than 1%.Bacterial communities were profiled through 16S amplicon sequencing, from which 13 320 unique ASVs were inferred and classified into 8 phyla and 10 classes with a mean relative abundance greater than 1%. Overall communities detected for all sample types consisted predominantly of dia Fig. . Alphapries Fig. . TW-assoles Fig. , with siOnly a minor proportion of the total ASVs detected on the incubated particles were also found in surrounding waters Fig. . HDPE shThe taxonomic and phylogenetic diversity of HDPE communities were the highest of all sample types across all sites Fig. . While sThe effects of Sample Type and Sampling Site on structural dissimilarities were tested through PERMANOVA using a multifactorial design. Taxonomic and phylogenetic dissimilarities were shaped by both Sample Type and Sampling Site, with a significant interaction between both factors Table . OverallCommunities on incubated particles adhered to different patterns of variation along the salinity gradient. The overall structure of HDPE and TW communities did not change significantly across riverine sites, except in abundances between certain sites Table . Signifi). Wood communities differed by an average of 51% and were slightly more nested, with a respective 10% and 15% of the taxonomic and phylogenetic dissimilarities observed along this stretch of the gradient attributed to species loss/gain. For HDPE communities, displacement across these transitional waters resulted in a high compositional turnover due to a shift in selective pressures (variable selection). The phylogenetic turnover of TW and Wood communities, conversely, did not deviate from the null expectation, rather a significant turnover was observed in their taxonomy as a product of low dispersal rates .Between riverine sites, the compositional turnover of HDPE communities was lower than expected by chance due to the selective pressures imposed by homogenous environmental conditions (homogenous selection, Fig. S11Alphaproteobacteria contributed the most to differences within HDPE communities along the gradient (Table Rhodobacteraceae. The recruitment of Thiotrichaceae and Flavobacteriaceae upon transfer into coastal seawaters additionally contributed to the large compositional differences observed along this stretch of the gradient. Variations within Wood communities were similarly driven by an increase in Rhodobacteraceae and Sphingomonadaceae across riverine sites but with a near two-fold decline in their counts upon transfer into coastal seawaters. A large increase in Flavobacteriaceae was also observed upon the transfer of Wood from freshwater to brackish and coastal seawaters. Compositional differences within TW communities along the gradient were attributed chiefly to Gammaproteobacteria, represented mostly by changes in the SS1-B-06-26 family. Members of SS1-B-06-26 increased gradually across riverine sites but declined upon transfer into coastal seawaters, where the recruitment of Flavobacteriaceae and Pseudohongiellaceae was observed instead.nt Table , namely Rhodobacteraceae and Thiotrichaceae detected on transferred particles. Transferred and stationary TW particles were distinguished predominantly by lower counts of SS1-B-06-26 detected on the former which instead harboured a greater abundance of Pseudohongiellaceae and Flavobacteriaceae. Only 14% and 15% of the total ASVs detected on transferred HDPE and TW at the final site, respectively, were displaced from riverine sites and absent from their stationary equivalents. ASVs transferred on HDPE consisted mostly of Anaerolineae and Deinococci variants, along with classes that were entirely absent from their stationary counterparts such as Nitrospira and Acidobacteria Subgroup 6. Variants of the SS1-B-06-26 family made up the majority of the transferred ASVs on TW, along with Alteromonadales and Pseudomonadales, which were undetected on their stationary equivalents.The structure of transferred communities upon reaching the final offshore site differed significantly from that of their stationary equivalents which remained offshore throughout the experiment Tables  and S13.Rhodobacteraceae and Sphingomonadaceae detected on stationary Wood particles. Transferred Wood particles instead harboured greater counts of Flavobacteriaceae. Wood, however, displaced the greatest proportion of ASVs along the gradient relative to synthetic substrata, with 26% of their total ASVs detected in the offshore site having originated from riverine sites. These consisted predominantly of variants of Rhodobacteraceae, Sphingomonadaceae, and Flavobacteriaceae.Relative to the synthetic substrata, differences between transferred and stationary Wood particles were the greatest. At the final site, both transferred and stationary Wood communities differed in their composition by an average of 93%, due predominantly to greater proportions of The potential of MP to raft bacteria across different aquatic systems was investigated in this study through a sequential incubation experiment performed along a salinity gradient. Bacterial biofilms detected on synthetic and natural substrata were analysed and compared to each other as well as to surrounding waterborne communities. In our study, HDPE communities were significantly more diverse than other sample types except particle-associated waterborne communities, to which their overall diversity was largely comparable. These findings are consistent with those previously reported of plastic sampled from coastal seawaters , 15. OthGammaproteobacteria consistently made up a dominant proportion of TW communities, comprising significantly greater counts of hydrocarbon-degrading families of the Oceanospirillales order, such as Oleiphilaceae, Pseudohongiellaceae, and SS1-B-06-26 than other sample types [Gammaproteobacteria, such as Porticoccaceae, Immundisolibacterales, and Methylophilaceae [Oceanospirillales have previously been reported to occur on MP of different polymer types [Gammaproteobacteria. Aquabacterium, a genus of Betaproteobacteria reportedly capable of hydrocarbon utilization, was also detected. The pronounced concentration of hydrocarbonoclastic bacteria consistently detected along the salinity gradient in our study, with the collective capability to degrade linear, branched, or aromatic hydrocarbons, suggests that TW act as a carbon source for a selective consortium of core bacteria. While further investigations are required to substantiate this thesis, the detection of Oleiphilus and Porticoccaceae strongly support our hypothesis as these taxa have been categorized as obligate hydrocarbon degraders and rely on aliphatic hydrocarbons or mono- and polycyclic aromatics as their primary sources of energy [Given the hydrophobicity of both HDPE and TW surfaces , 48, simle types –56. Signhilaceae –59 were er types –63. To tf energy , 57, 64.Alphaproteobacteria and Gammaproteobacteria were the two most dominant classes detected of all sample types [Alphaproteobacteria were significantly more abundant on Wood and were composed predominantly of Rhodobacteraceae and Sphingomonadaceae. These families are among the most dominant and well-known surface colonizers reported of aquatic environments [Consistent with published studies, le types , 15, 65.ronments  and as sronments , where mAlong the salinity gradient, all sample types differed significantly from each other in their structure. The most pronounced differences were observed between communities on incubated particles and those within surrounding waters, which differed consistently from each other along the gradient. While dominated by similar bacterial classes, only a minor proportion of the total ASVs on incubated particles were also found in surrounding waters. These findings, though consistent with published studies , 12, conGammaproteobacteria and Alphaproteobacteria, comprised more than 50% of their respective overall populations.Distinct patterns of variation were also observed within each community along the salinity gradient. For HDPE, these changes were driven predominantly by environmental selection. While seemingly susceptible to environmental filtering on a local scale, TW and Wood communities were governed more by stochastic events on a regional scale. The opposing local and regional patterns observed among TW and Wood communities likely indicate that, while community assembly is shaped by deterministic factors, their turnover is more strongly influenced by stochastic drift or dispersal events . Upon trFlavobacteriaceae and Thiotrichaceae, which have been reported within estuarine environments and described as marine bacterioplankton with an adaptive tolerance to lower salinities [A gradual convergence in the overall structure of HDPE and TW communities was observed along the salinity gradient. This might first be explained by the coalescing effect that confluent water bodies of differing salinities reportedly have on microbial communities. In a study by Rocca et al. , communilinities . A compolinities , who obslinities . Wood colinities , 74. ConNitrospira and Acidobacteria Subgroup 6 on HDPE from freshwater to coastal seawaters is not surprising given their reportedly ubiquitous nature across aquatic environments [Pseudomonadales and Alteromonadales on TW, which were respectively recruited from freshwater and brackish waters. These orders consisted mostly of an unclassified genus of Moraxellaceae and Aestuariibacter, both of which have been detected within riverine and coastal environments [Aestuariibacter are known hydrocarbon-degrading bacteria [Moraxellaceae family, however, bear particular clinical importance. In addition to several species having been identified as causal agents for a range of human and animal infections, Moraxellaceae are often reported to harbour antibiotic resistance genes [This was also evident when considering the transport potential of the different substrata. Wood successfully displaced a greater proportion of their total ASVs along the salinity gradient than HDPE or TW. ASVs transported on Wood, however, consisted of variants of the most dominant taxa detected of Wood communities whereas the synthetic substrata additionally rafted taxa completely undetected on their stationary counterparts on the class and order level. The recruitment and successful displacement of ronments , 76. Morronments , 77. As bacteria , their dce genes –80. ThesThis study offers a first look into the dynamics of bacterial communities on the surfaces of synthetic and natural substrata along a salinity gradient. In addition to providing a deeper understanding of MP biofilms, we show that the displacement of these substrata from riverine to coastal seawaters elicits a high turnover of communities, suggesting an unlikely translocation of species across these aquatic environments on a substantial scale. Nonetheless, HDPE and TW were found capable of rafting certain freshwater taxa into seawater environments, some of which may be harmful. Putatively, we also demonstrate the specificity of TW as a substrate for bacteria and present exciting new avenues for further research into the functionality of these biofilms to more fully assess the role of MP as transport vectors for bacteria and their potential threats.Supplementary Material GuideSupplementary MaterialSupplementary Table 1Supplementary Table 2Supplementary Table 3Supplementary Table 8Supplementary Table 9Supplementary Table 10Supplementary Table 11Supplementary Table 13


Pseudomonas aeruginosa (PA) infections, and 2) examine whether antimicrobial resistance in PA infections is associated with worsening functional status and higher health care resource utilization (HCRU).The aims of the study were to 1) evaluate the prevalence of multidrug resistant (MDR) /Extensively drug resistant (XDR) strains among hospitalized adults with PA as confirmed by local evaluation of microbiological results. MDR PA was defined as acquired non-susceptibility to at least one agent in three or more antimicrobial categories. XDR PA was defined as acquired non-susceptibility to at least one agent in all but two or fewer antimicrobial categories. HCRU metrics evaluated included hospital length of stay (LOS) and intensive care unit (ICU) LOS.This multicenter prospective study was conducted in 9 large Italian teaching hospitals between June 2018-February 2020. We included patients aged ≥18 years with a diagnosis of nosocomial pneumonia (NP), complicated urinary tract infections (cUTI) or complicated intra-abdominal infections (cIAI) due to PA were enrolled. The main baseline characteristics of overall patients were reported in Table 1. Almost one-third of patients (28.4%) reported either MDR or XDR PA infection, with more patients experiencing MDR (Table 1). Health care resource use stratified by patients with and without MDR/XDR status are reported in Table 2. Overall, in our study population, median hospital LOS and ICU LOS were 42.0 (IQR=39.0) and 15.5 (IQR=37.0) days, respectively. There was a statistically significant longer median hospital LOS for patients with MDR/XDR infections compared to non MDR/XDR PA infections . ICU LOS also trended towards being longer for patients with MDR/XDR infections compared to those with non-MDR/XDR infections .A total of 95 patients with a nosocomial infection due to PA, particularly in those with cIAI. Overall, the present study may suggest a positive correlation between having MDR-XDR PA nosocomial infections  and increased HCRU that require further attention from a disease management perspective.MDR/XDR isolates were prevalent among patients with nosocomial infections due to Matteo Bassetti, PhD, Angelini: Advisor/Consultant|Astellas: Grant/Research Support|Bayer: Advisor/Consultant|Bayer: Honoraria|BioMe ´ rieux: Advisor/Consultant|BioMe ´ rieux: Honoraria|Cidara: Advisor/Consultant|Cidara: Honoraria|Cipla: Advisor/Consultant|Cipla: Honoraria|Gilead: Advisor/Consultant|Gilead: Honoraria|Menarini: Advisor/Consultant|Menarini: Honoraria|MSD: Advisor/Consultant|MSD: Honoraria|Nabriva: Advisor/Consultant|Pfizer: Advisor/Consultant|Pfizer: Board Member|Pfizer: Grant/Research Support|Pfizer: Honoraria|Shionogi: Advisor/Consultant|Shionogi: Honoraria|Tetraphase: Advisor/Consultant Francesco Menichetti, n/a, Aneglini: Advisor/Consultant|Aneglini: Board Member|Aneglini: Grant/Research Support|Aneglini: Honoraria|Astellas: Advisor/Consultant|Astellas: Honoraria|Becton: Advisor/Consultant|Becton: Honoraria|bioMérieux: Advisor/Consultant|bioMérieux: Honoraria|Biotest: Advisor/Consultant|Biotest: Board Member|Biotest: Honoraria|Bristol-Myers Squibb: Advisor/Consultant|Bristol-Myers Squibb: Honoraria|Correvio: Advisor/Consultant|Correvio: Speaker honoraria|Dickinson: Advisor/Consultant|Dickinson: Honoraria|Gilead: Advisor/Consultant|Gilead: Grant/Research Support|Janssen: Advisor/Consultant|Janssen: Honoraria|MSD: Advisor/Consultant|MSD: Speaker honoraria|Nordic pharma: Board Member|Nordic pharma: Honoraria|Pfizer: Advisor/Consultant|Pfizer: Honoraria|Shionogi: Advisor/Consultant|Shionogi: Honoraria|ViiV: Advisor/Consultant|ViiV: Honoraria Nicola Petrosillo, n/a, Becton & Dickinson,: Honoraria|MSD: Honoraria|ohnson & Johnson: Honoraria|Pfizer: Honoraria|Shionogi: Honoraria.


In conclusion, SAHA relieved axonal damage and neurological dysfunction after SAH via the HSP70 acetylation-induced degradation of TDP-43, highlighting a novel therapeutic target for SAH.Increased focus has been placed on the role of histone deacetylase inhibitors as crucial players in subarachnoid hemorrhage (SAH) progression. Therefore, this study was designed to expand the understanding of SAH by exploring the downstream mechanism of the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) in SAH. The expression of TDP-43 in patients with SAH and rat models of SAH was measured. Then, western blot analysis, immunofluorescence staining, and transmission electron microscope were used to investigate the in vitro effect of TDP-43 on a neuronal cell model of SAH established by oxyhemoglobin treatment. Immunofluorescence staining and coimmunoprecipitation assays were conducted to explore the relationship among histone deacetylase 1 (HDAC1), heat shock protein 70 (HSP70), and TDP-43. Furthermore, the in vivo effect of HDAC1 on SAH was investigated in rat models of SAH established by endovascular perforation. High expression of TDP-43 in the cerebrospinal fluid of patients with SAH and brain tissues of rat models of SAH was observed, and TDP-43 accumulation in the cytoplasm and the formation of inclusion bodies were responsible for axonal damage, abnormal nuclear membrane morphology, and apoptosis in neurons. TDP-43 degradation was promoted by the HDAC1 inhibitor SAHA  Analysis of the molecular processes involved in the progression of hemorrhage-induced brain damage has yielded a potential therapeutic target. Fei Liu and Aihua Liu at the Central South University in Changsha, China, and co-workers examined mechanisms behind the extensive brain damage that can be caused by subarachnoid hemorrhage, bleeding in a space between the brain and its surrounding membranes. In experiments on human samples and rat models, the researchers showed that the TDP-43 protein was over-expressed in cerebrospinal fluid after hemorrhage, and that the build-up of this protein aggravated nerve cell damage and dysfunction. TDP-43 is regulated by a pathway involving a potent enzyme involved in regulation of gene expression called HDAC1. Treatment of rat models with an HDAC1 inhibitor degraded TDP-43 and limited the progression of brain damage. SAH, which is mainly induced by aneurysmal rupture and peri-mesencephalic bleeding, is a dangerous disease that is commonly characterized by acute headache, nausea/vomiting and neck pain, with a mortality rate within 24 h of 25% and an overall mortality rate of 50%3. Accumulating evidence has suggested that SAH-induced early brain injury is a key factor that affects the prognosis of SAH patients4. Although great achievements have been made in understanding the pathogenesis and pathophysiology of SAH, as well as in the treatment and management of SAH, its underlying molecular, cellular, and circulatory dynamics remain largely unclear, and its prognosis is still not satisfactory6. Thus, in-depth exploration of SAH is needed to improve management.Subarachnoid hemorrhage (SAH) is a lethal and devastating intracranial hemorrhage that is frequently misdiagnosed in the initial stage and increases the risk of mortality and disability8. It was shown that SAHA exerted great clinical utility as a therapeutic intervention after intracerebral hemorrhage9. Moreover, HDACs are potent enzymes that posttranslationally modify both histone and nonhistone acetylation sites, thereby affecting many cellular processes, such as the cell cycle and apoptosis10. Notably, the exacerbating effects of HDAC4 on SAH can be inhibited by SAHA11. HDAC1 inactivation was shown to protect against neuronal death and brain injury12. Moreover, it was identified that heat shock protein 70 (HSP70) was a cytosolic substrate of HDAC5 and could be hyperacetylated by HDAC5, which was further associated with the proliferation of hypoxic tumor cells13. HSP70 is a well-known therapeutic target for SAH14. In addition, HSP70 was shown to inhibit the cytoplasmic accumulation of TDP-43, which is responsible for the formation of insoluble inclusion bodies and is a hallmark of neurodegenerative diseases16. Moreover, TDP-43 was identified as a prognostic biomarker for SAH17. Herein, SAHA is hypothesized to be involved in the development of SAH by interacting with the HDAC1/HSP70/TDP-43 axis.Suberoylanilide hydroxamic acid (SAHA), also called vorinostat, belongs to the hydroxamic acid class of histone deacetylase (HDAC) inhibitorsThe current study was approved by the IRB of The Third Xiangya Hospital of Central South University and was performed in accordance with the Helsinki Declaration. Written informed consent was obtained from each participant. Animal experiments were approved by the Animal Ethics Committee of The Third Xiangya Hospital of Central South University and Central South University and were conducted according to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health.P < 0.05.Aneurysmal SAH-related microarray data (GSE54083) were downloaded from the Gene Expression Omnibus (GEO) database. Because the occurrence of SAH was induced by rupture of an intracranial aneurysm, eight cases of intracranial aneurysm rupture samples and ten normal control samples in GSE54083 were selected, and the differential analysis was conducted using the “limma” package of R language. Differentially expressed genes were identified with a threshold of g for 15 min at 4 °C. The supernatant of the cerebrospinal fluid, which was free from blood cell components, was collected, subpackaged in 1.5 mL centrifuge tubes in equal amounts, and stored at −80 °C for subsequent experiments. Control cerebrospinal fluid and blood samples were collected from age-matched patients who underwent elective surgery that was not associated with the central nervous system.A total of 15 patients with aneurysmal SAH and 10 patients without SAH (non-SAH patients) from The Third Xiangya Hospital of Central South University were selected for cerebrospinal fluid collection. Cerebrospinal fluid (5 mL) was collected every 6 h for 8 days and then immediately centrifuged in a 10 mL sterile centrifuge tube at 12,000 × https://www.rndsystems.com/cn).ELISA was performed to measure the levels of TDP-43 in cerebrospinal fluid based on the instructions of the kit , and 15 μL (μg/μL) of lentivirus  were injected into the rat lateral ventricle  within 10 min at a constant rate (5 μL/min). The needle was maintained for 10 min to prevent blood reflux and then slowly pulled out, after which the scalp was sutured. The success rate of the model was ~85% . Rats were subjected to sham operation or SAH modeling. After successful modeling, the rats were treated with oe-NC, sh-NC, oe-TDP-43, sh-HDAC1, dimethyl sulfoxide , or SAHA  or without any injection (n = 6 each). No rats died during viral infection or drug treatment.A Wistar rat endovascular puncture model of SAH was constructed according to previous research methodsl-glutamine, 50 U/mL penicillin, and 50 U/mL streptomycin . Half of the medium was renewed every 2 days. An in vitro cell model of SAH was established by stimulating neurons with oxyhemoglobin (OxyHb). The neurons were incubated with OxyHb (20 μM) at 37 °C with 5% CO2 for 6 h. Next, the medium was removed, and the neurons were washed with PBS (three times) for subsequent experiments.Primary neurons were isolated from the brain tissues of fetal rats (embryos aged 16–18 d). The meninges and blood vessels were removed, and the brain tissues were digested with 0.25% trypsin (containing ethylenediaminetetraacetic acid (EDTA)) at 37 °C for 5 min. Then, the tissues were washed three times with phosphate-buffered saline (PBS) to stop the trypsinization. Next, the cells were resuspended on neural basal medium supplemented with 2% B27, 2 mM 2.Human embryonic kidney (293T) cells (American Type Culture Collection) were cultured in Dulbecco’s Modified Eagles Medium (Gibco) with 10% (v/v) fetal bovine serum. Primary neurons were cultured in neural basal medium (Gibco) in a cell incubator at 37 °C with 5% CO5 cells/mL cell suspension, seeded in a six-well plate in 2 mL per well, and cultured overnight.293T cells were cotransfected with the packaging virus and the target vector with LV5-green fluorescent protein (GFP)  and pSIH  for 48 h, followed by supernatant collection. After filtration and centrifugation, viral particles in the supernatant were obtained, and the viral titer was determined. Viruses in the logarithmic growth phase were collected. Cells were treated with OxyHb (treated with 20 μM OxyHb for 24 h), OxyHb + sh-NC (treated with 20 μM OxyHb for 24 h after being transfected with sh-NC for 48 h), OxyHb + sh-HDAC1 (treated with 20 μM OxyHb for 24 h after being transfected with sh-HDAC1 for 48 h), OxyHb + sh-TDP-43 (treated with 20 μM OxyHb for 24 h after being transfected with sh-HDAC1 for 48 h), OxyHb + DMSO (treated with 20 μM OxyHb for 24 h after being transfected with 5% DMSO for 48 h), and OxyHb + SAHA (treated with 20 μM OxyHb for 24 h after being transfected with SAHA for 48 h). sh-NC, sh-HDAC1, oe-NC, and oe-TDP-43 were all synthesized by GenePharma Co., Ltd. . Cells were prepared into a 4 × 10Neurological status was evaluated 72 h after SAH modeling. The modified Garcia test included vibrissae touch, trunk touch, spontaneous movement, spontaneous movement of limbs, forelimb extension, and climbing ability (maximum score = 18). Neurological evaluations were performed by an investigator who was blinded to the experimental conditions.20. All rats were trained 3 d before modeling and were tested 1 d before SAH modeling and 1, 3, 7, and 14 d after SAH modeling.This test was used to evaluate the exercise ability of rats by rotating a rod cylinder 21. Starting from the 18th d after modeling, all rats were trained for 4 d, three times per day. Each training session lasted for 1 min, with 5 min intervals between every two sessions. The test was performed from the 22nd to the 26th d after SAH modeling.The Morris water maze test was carried out as previously described in a circular tank with a diameter of 180 cm and a depth of 50 cm22. In short, rats were deeply anesthetized with pentobarbital sodium 72 h after SAH modeling and euthanized. An electronic analytical balance  was used to weigh each part.Brain water content was measured by the wet/dry method2O2 for 10 min. After being blocked with 5% bovine serum albumin (BSA) for 20 min, the sections were incubated with primary antibodies against amyloid precursor protein  overnight at 4 °C and biotinylated goat anti-mouse immunoglobulin G (IgG) secondary antibodies  for 20 min. Finally, the sections were stained with 3,3-diaminobenzidine and counterstained with hematoxylin. Images were obtained with a microscope . Two independent pathologists evaluated the sections. The rate of positive cells for each sample was counted according to the APP staining intensity.Brain section (4 μm in thickness) were prepared and treated with an EDTA buffer solution , followed by antigen retrieval. Endogenous peroxidase activity was inactivated with 0.3% Hn = 6). ImageJ  was used to count the total number of TUNEL and NeuN double-positive cells in five areas near the injury area.Sections were washed in PBS and blocked in 10% BSA at 25 °C for 1 h. Then, the sections were incubated with the following primary antibodies  at 4 °C for 12–16 h: rabbit anti-HDAC1 , mouse anti-HSP70 , rabbit anti-TDP-43 , rabbit anti-Iba1 , rabbit anti-GFAP , and rabbit anti-NeuN . Then, the sections were incubated with Alexa Fluor 647 donkey anti-goat IgG , Alexa Fluor 488 donkey anti-mouse IgG (Thermo Fisher Scientific), and CY3 donkey anti-rabbit IgG  at 25 °C for 1 h. Next, the sections were stained with 4’,6-diamidino-2-phenylindole  at 25 °C for 15 min. Transferase-mediated TUNEL  costaining with rabbit anti-NeuN  and neuronal apoptosis were assayed. The sections were observed with an Olympus BX51 fluorescence microscope  or a laser scanning confocal microscope . The image control parameters were set by the control group, and all other parameters remained the same for image capture. Five hematoma border regions were selected in each part for analysis, and four parts were selected for each animal , neurofilament light chain (NFL) , ApoE , TDP-43 , Acetyl-K , glyceraldehyde-3-phosphate dehydrogenase , and Lamin A , as well as horseradish peroxidase-labeled goat anti-rabbit IgG . Then, a developing solution  was used for development. ImageJ 1.48 u software  was used to perform quantitative protein analysis .® Premix Ex TaqTM II kit  was used to perform fluorescent qPCR. The samples were subjected to RT–qPCR in a real-time fluorescent qPCR instrument . The 2−ΔΔCT method was used to quantify expression, and GAPDH was used as the internal reference. The primers are shown in Supplementary Table Cells were lysed using a TRIzol kit , and total RNA was extracted from cell and tissue samples. RNA quality and concentration were measured by UV–Vis spectrophotometry . For mRNA analysis, an RT kit  was used to perform RT to obtain cDNA. Subsequently, cDNA was used as a template, and the SYBRhttps://www.thermofisher.cn/) was used to separate the nucleus and cytoplasm in each group of cells, as well as to separate nuclear and cytoplasmic proteins. Then, western blot analysis was performed to measure the expression of TDP-43 . GAPDH  was used as the internal reference for cytoplasmic proteins, and Lamin A  was used for nucleoproteins. The ratio of the gray value of TDP-43 to the gray value of each control protein was used for quantitative analysis by ImageJ 1.48 u software .A NE-PER™ Nuclear and Cytoplasm Extraction Kit . SOD activity was measured by the WST-1 method.Brain sections were deparaffinized, dehydrated, incubated in a 0.06% potassium permanganate solution (Sigma–Aldrich) for 10 min, and reacted in FJC working solution (0.1% acetic acid) for 20 min. A fluorescence microscope (Olympus) was used to observe FJC-positive cells, and the number was counted by technicians who were blinded to experimental conditions.23. The ventral side of the left hemisphere was examined with the help of a laser scanning confocal microscope . The representative image was obtained from a Section 2 mm posterior to the bregma.DHE staining was carried out to measure superoxide anions, which indicate the level of oxidative stress in tissuesEqual amounts of cell lysates (1500 µg) were immunoprecipitated with 1 μg of HSP70, HDAC1, TDP-43, or acetyl-K antibodies and 50 μL protein A/G agarose. The immunoprecipitate was washed twice with 10 mM HEPES (pH 7.9), 1 mM EDTA, 150 mM NaCl, and 1% Nonidet P-40 and boiled for 10 min. Then, the precipitated proteins were eluted with 30 μL of sodium dodecyl sulfate (SDS)–PAGE buffer. The eluted proteins were separated by 8% SDS–PAGE, transferred to a nitrocellulose membrane, and examined with the corresponding antibodies.6 cells were resuspended in 100 μL of dye solution, incubated at room temperature for 15 min, and 1 mL HEPES buffer solution was added , followed by cell apoptosis detection.After 48 h of transfection, the cells were detached with EDTA-free 0.25% trypsin , collected in a flow tube, and centrifuged, and the supernatant was discarded. According to the directions of the Annexin-V-fluorescein isothiocyanate (Annexin-V-FITC) cell apoptosis detection kit , the Annexin-V-FITC/PI dye solution was prepared. Then, 1 × 10ImageJ software was used to quantify neurite length. Images of cells labeled with anti-GFP and anti-neurofilament antibodies were captured by an Axiovert camera (images were processed using Volocity software). The Neuron J ImageJ plug-in was used to measure the lengths of neurites. Approximately 150 cells under each experimental condition were analyzed by three independent researchers.The grid was checked at 80 KV on a Philips CM120 electron microscope with a low-magnification image  of the white matter area containing the axon tract, as well as a high-magnification image (X15000 or X20000) of axons and other subcellular elements that were captured. From the examined samples, axons with myelin sheaths and axon organelles conformed to the ultrastructural standards. TEM images were captured with a charge-coupled device camera  and processed with Digital Micrograph software (Gatan).t tests, while data comparisons among multiple groups were performed using one-way analysis of variance (ANOVA). Comparisons among data at different time points were performed by repeated-measures ANOVA with Tukey’s post-hoc test. A value of P < 0.05 was considered statistically significant.SPSS 21.0 statistical software  was used for statistical analysis. Measurement data are displayed as the mean ± standard deviation. First, the normality and homogeneity of variance were tested. Data with a normal distribution and even variance between two groups were analyzed by unpaired To explore the mechanism of TDP-43 in SAH, cerebrospinal fluid was collected from 10 non-SAH patients and 15 patients with aneurysmal SAH. We found that the expression of TDP-43 in the cerebrospinal fluid of SAH patients was much higher than that in the CSF of non-SAH patients Fig. .Fig. 1TDThen, a rat model of SAH were constructed by the endovascular puncture. In model rats, we found severe cerebral hemorrhage, increased brain water content -TARDBP (TDP-43) was a regulatory pathway Fig. . The colThe SAH-related microarray GSE54083 was obtained from the GEO database and further analyzed, and the results revealed that HDAC1 was highly expressed in SAH Fig. . KEGG enNeurons were treated with the HDAC1 inhibitor SAHA (1 μM) for 30 min, and the acetylation level of HSP70 was measured. SAHA treatment notably promoted HSP70 acetylation in 293T cells , frontotemporal lobar degeneration (FTLD), Parkinson’s disease and Huntington’s disease30. Specifically, TDP-43 acetylation increased the accumulation of insoluble and hyperphosphorylated TDP-43 inclusions, which contributed to the pathogenesis of ALS and FTLD31. Another study showed that TDP-43 promoted Alzheimer’s disease development32. In summary, these lines of evidence validated the exacerbating role of TDP-43 in SAH progression.We discovered that TDP-43 was highly expressed in the cerebrospinal fluid of SAH patients and in the brain tissues of a rat model of SAH. Consistent with our findings, a previous study also showed upregulated expression of TDP-43 in the cerebrospinal fluid of patients with aneurysmal SAH and the brain tissues of a rat model of SAH33. Moreover, the deacetylation of HSP70 was promoted by HDAC1, and HDAC1 silencing promoted TDP-43 degradation, thereby alleviating neuronal and axonal damage, abnormal nuclear membrane morphology, and apoptosis in a neuronal cell model of SAH. A prior study showed that protein deacetylation, which is the reverse process of acetylation , was mediated by deacetylases34. Moreover, the inhibition of HDACs was reported to exert a protective effect against acute lung injury by acetylating and suppressing HSP90 activity35. Moreover, HDAC silencing was shown to reduce white matter damage after intracerebral hemorrhage36. Furthermore, this study showed that the HDAC inhibitor SAHA alleviated axonal injury and cognitive impairment in rats with SAH by elevating TDP-43 degradation by enhancing the acetylation of HSP70 by inhibiting HDAC1 enzymatic activity. SAHA is a well-recognized HDAC inhibitor that has also been confirmed to alleviate intracerebral hemorrhage9 In addition, a recent study revealed that SAHA attenuated hemorrhagic shock and resuscitation-induced lung injury by repressing histone acetylation37. Hence, these findings support the role of SAHA in ameliorating SAH development by regulating the HDAC1/HSP70/TDP-43 axis.Then, TDP-43 protein degradation was inhibited by HSP70 deacetylation, which induced cytoplasmic aggregation and further promoted inclusion body formation. Similar to our findings, another study showed that the pathological accumulation of insoluble TDP-43 could be reduced by HSPs and attenuate amyotrophic lateral sclerosisTaken together, the findings of this study confirmed that SAHA plays an inhibitory role in SAH progression by promoting the degradation of TDP-43 by enhancing the acetylation of HSP through the suppression of HDAC1 activity Fig. . HoweverSupplementary information


As global use of surgical robotic systems is steadily increasing, surgical simulation can be an excellent way for robotic surgeons to acquire and retain their skills in a safe environment. To address the need for training in less wealthy parts of the world, an affordable surgical robot simulator (PoLaRS) was designed.n = 18) performing tasks on PoLaRS and dVSS, and the control group (n = 20) only performing tasks on the dVSS. The performance parameters were Time, Path length, and the number of collisions. Afterwards, the test group participants filled in a questionnaire regarding both systems.The aim of this pilot study is to compare learning curve data of the PoLaRS prototype with those of Intuitive Surgical’s da Vinci Skills Simulator (dVSS) and to establish face- and construct validity. Medical students were divided into two groups; the test group (P ≤ 0.028). No differences was found between the test group and the control group in the dVSS performances during the post-test phase. Learning curves showed similar shapes between both systems, and between both groups. Participants recognized the potential benefits of simulation training on the PoLaRS system.A total of 528 trials executed by 38 participants were measured and included for analyses. The test group significantly improved in Time, Path Length and Collisions during the PoLaRS test phase (Robotic surgical skills improved during training with PoLaRS. This shows the potential of PoLaRS to become an affordable alternative to current surgical robot simulators. Validation with similar tasks and different expert levels is needed before implementing the training system into robotic training curricula.The online version contains supplementary material available at 10.1007/s00464-021-08906-z. The utilization of robotic systems for common surgical procedures is rapidly increasing –4. TheseSimulation training allows surgical trainees to train in a low-stakes environment that enables deliberate practice , withoutTo cater for the demand for affordable and efficient ways to train robotic surgeons, a Virtual Reality (VR) training system called ‘Portable Laparoscopic Robotic Surgery’ (PoLaRS) was developed in collaboration with the Delft University of Technology . It is designed to train fundamental robotic skills on a remote basis at an affordable cost of less than 25 k Euro per system. The technical skills that are acquired on the PoLaRS system could potentially be transferred to any currently available surgical robotic system to continue the learning curve and to hone these skills. Therefore, the availability of affordable simulators can be helpful in supporting localized training opportunities, thereby greatly improving the acquisition and retention of skills. This study aims to compare performance parameters of the PoLaRS system and the da Vinci Skills Simulator (dVSS) by establishing learning curves, as well as to assess validity evidence. It is hypothesized that construct validity can be indicated by similarity in learning curves of both systems, and prior training on the PoLaRS device will result in a shorter learning curve compared to the da Vinci system.https://www.randomizer.org/, using block randomization and a block size of four. Thereafter, participants of the test group completed two exercises on the PoLaRS prototype, performing each exercise three times . The control group participants viewed an instructional video from the company giving an overview of the da Vinci system instead. Afterwards, both groups performed two exercises on the da Vinci system, both performing each exercise three times. A questionnaire was sent to test group participants asking for feedback regarding face and construct validity of the PoLaRS and da Vinci systems. Due to the nature of this study and only passively controlled interface movements of both training systems, IRB approval and written consent are not required .Medical interns at the Leiden University Medical Center  were recruited for voluntary participation in this initial validation study. Participant demographics and baseline characteristics  were obtained by a pre-course survey . All parLaparoscopic box trainer with an endoscopic camera, Maryland graspers and ForceSense measuring system;PoLaRS prototype;da Vinci console with Skills Simulator add-on.In this study, the following equipment was used:The ForceSense laparoscopic system  consists of two TrEndo instrument tracking sensors, one ForceTRAP tissue interaction force sensor, an endoscopic video camera, and a Windows tablet with pre-installed ForceSense.NET software . These items were installed in a T5 boxtrainer . Two endoscopic Maryland graspers  were inserted into the TrEndo instrument tracking sensors .The Portable Laparoscopic Robotic Surgery system (PoLaRS) Fig.  is a proThe dVSS  is a VR system that is added to the console of a da Vinci Xi surgical robot Fig.  Right. IThe ForceSense system makes use of pre-installed ForceSense.NET software, which records the real-time video feed from the endoscopic video camera and the force parameters and raw data collected by the ForceTRAP sensor. All data collected during the performance of a task are stored on the ForceSense.NET server, which is accessible with a personal user account.In trainer mode, the PoLaRS makes use of a VR environment in which several exercises can be performed, similar to the da Vinci Skills Simulator. On request, visual and tactile force feedback is provided in case of instrument-environment collisions. In the PoLaRS software, when a collision is detected, a 3-s ‘cool-down’ period is applied, after which another collision can be detected. This ‘cool-down’ period is effective in a 10 mm radius around the center of the active collision. This means that longer collisions and/or larger movements cause multiple collision counts compared to a single hit as registered by the dVSS. From all parameters, the time to completion (s), left and right instrument path length (mm) and the number of collisions between instruments and non-movable environmental objects were used in this study. All performance metrics are saved locally in a designated folder according to the date and time of the trial.The dVSS software provides training exercises for handling the da Vinci Xi system controls. The software includes training statistics, and every trial is scored according to technical performance. Exercises available on the dVSS system can be made by either Intuitive Surgical itself, or by Mimic Simulation . Both provided a performance score after each trial. Time to completion (s), Path length for both left and right instruments  (cm), and number of instrument collisions were recorded. Intuitive Surgical records a single Instrument collisions metric, while Mimic-developed exercises record different types of collisions separately, namely Instrument-Endoscope collisions, Endoscope-Environment collisions, and Instrument-Instrument collisions. To provide comparability between them, Mimic’s collision metrics were summarized into a single Collisions parameter. All scoresheets are saved consecutively and locally and can be accessed by viewing the exercise history.The main performance parameters that were used in this study were: time taken to complete the task; instrument path length left and right; and the number of collisions Table . There whttps://www.randomizer.org/, using block randomization with a block size of four. After randomization, the test group participants were instructed to complete two exercises on the PoLaRS system. In the first exercise , participants were tasked with sorting marbles according to their color. They were instructed to sort the green marbles with their left hand and the blue marbles with their right hand to ensure bimanual performance. The second task  consisted of picking up marbles and feeding them through circular cut-outs in walls, making use of depth perception. Three trials of each exercise were performed. The PoLaRS system was set up the skills lab Reinier de Graaf Gasthuis teaching hospital, which was not accessible to the control group participants. Instead, they were asked to view a short introductory video on the da Vinci system, showing the different elements of the system, but not showing how the controls relate to the movement of the instruments. A link to this video can be found in Supplemental File 3.Participants first completed a pre-test on the ForceSense laparoscopic system Fig. . The preThe post-test was performed on a dVSS unit, connected to the da Vinci Xi console  in the OR at the Reinier de Graaf Gasthuis teaching hospital. All participants completed two exercises, with three trials each. Contrary to the PoLaRS system, the dVSS has many different exercises to choose from. Thus, exercises were selected beforehand by AK and BB to reflect the PoLaRS exercises, which are similar in nature, without the use of foot pedals, clutch, or camera handling. ‘Pick & Place’ and ‘Sea Spikes 1’ were selected for this study, as can be seen in Supplemental Files 10 and 11.Participants in the test group were asked to fill out a questionnaire  in which they could provide feedback on the PoLaRS and da Vinci systems after completion of all test sessions. This questionnaire contained both open and closed questions. Closed questions regarded face- and content validity and were answered using a 1–10 Likert scale. Open questions regarded the benefits and possible points of improvement for both the PoLaRS prototype and the DaVinci system. Responses were grouped into categories and interpreted by AK.p < 0.05. Baseline performance was compared using the third trial of each exercise on the ForceSense system. Normality tests using Shapiro–Wilks tests were performed for all collected parameters. For normal distribution, unpaired t-tests were used to compare the data. In the case of non-normal distribution, the Mann–Whitney U test was performed. Parameter data were compared in each group between the first and last trials of the test and post-test phases to identify learning effects, and between the two groups in the post-test phase to identify the effects of prior PoLaRS training on performance. Questionnaire data were used to indicate whether face and construct validity were reached based on an average score above six on a 1–10 Likert scale.The data from all systems were collected and analyzed using IBM SPSS . Differences were considered significant at A total of 528 trials executed by 38 enrolled participants were measured and included for analyses. All participants completed the pre-test, after which 18 were randomly placed in the test group, and 20 in the control group. Both groups had similar baseline characteristics , and both groups performed similarly on the ForceSense system (Table p = 0.018). For the other parameters, no significant differences were found. Figure Figure The test group participants were sent a questionnaire after the testing sessions, asking for their feedback. Questions were either answered with a score of 1–10 on a Likert scale Fig.  or a wriThe questionnaire also contained several open questions, where participants could provide feedback on the benefits and points of improvement of the PoLaRS prototype and the da Vinci system. According to the participants, the main benefit of the PoLaRS prototype was the fact that one can practice robotic surgery in a very low-stakes, accessible way (11 mentions). Further benefits were a large range of movement (two mentions), a good feeling of precision (one mention), and similarity of exercises to DaVinci (one mention). Points of improvement varied: suboptimal depth perception (five mentions), movement not as smooth as da Vinci (five mentions), lack of resistance in controllers (three mentions), ergonomics (three mentions), coordination feels counterintuitive (two mentions), less precision in small movements (one mention), controllers could be more like DaVinci (one mention), exercise realism/medically oriented exercises (one mention).The benefits of the DaVinci system, according to participants, are the precision of movements (seven mentions), comfortable ergonomics (seven mentions), smooth movements (five mentions), ease of use (four mentions), three-dimensional view (three mentions), good resistance on controllers (one mention). Points of improvement mentioned for the DaVinci system include lack of tactile feedback (four mentions), controllers sometimes colliding with each other (two mentions), exercise realism/medically oriented exercises (two mentions), limited instrument reach (one mention), and suboptimal depth perception (one mention). In this study, we examined whether the PoLaRS prototype would provide a noticeable benefit in performance on the dVSS system. Although no significant differences were found, prior training on the PoLaRS prototype did not negatively impact performance on the da Vinci Skills Simulator and the questionnaire indicated that prior training with PoLaRS was helpful in performing on the dVSS system. Furthermore, performances in the test group seemed to have fewer outliers than the control group.Although we expected some effect from training on the PoLaRS prototype prior to the dVSS, the individual learning curves reveal that it is most likely not the system but the training task that has a dominant influence on performance. In fact, for both systems, similar learning curve shapes and similar reductions in parameter data variation were observed for all tasks. Comparing the final trial on the first task and the first trial on the following task in the PoLaRS parameter data, large differences were found for each parameter. This same differences are found when switching from the first task to the second task on the dVSS. Moreover, when switching from PoLaRS to dVSS, a similar difference was found for all parameters without any disturbances. This indicates construct validity, as the data suggest that both systems are intuitive enough to be used as an effective extension of the human arm. To determine if the PoLaRS can be used as a training device for complex surgical tasks on a specific robotic platform, it is advised to validate similar training tasks for both systems with multiple experts . In contIt is evident that the complexity of the training system and training tasks have an influence on the performance , 17. In Although the questionnaire scores were lower for the PoLaRS system compared to the DaVinci Skills Simulator, an average score of 6.9 (SD 1.2) for the questions about the performance of PoLaRS indicates that face validity was established. A possible explanation for the lower scores is that the questionnaire was completed after all testing sessions were completed. Therefore, participants compared the PoLaRS system directly to the DaVinci system, rather than rate it on its own merit. In future studies it might be wise to have participants fill out these questions after each testing session. An interesting observation can be made from the questionnaire: when asked whether the exercises on the PoLaRS system made them perform better on the DaVinci system, participants varied greatly in their responses. Some participants did not feel like the PoLaRS system contributed to their performance on dVSS, while others did. Interestingly, there does not seem to be a correlation between those that performed well on the PoLaRS system and those that felt that the PoLaRS system contributed to their dVSS performance; the two highest raters (both rating 9/10) performed similarly to the two lowest raters (rating 2/10 and 3/10). This indicates that it may be difficult for participants to relate objective performance to factors of influence.Concerning the questionnaire’s open questions, the three most important points of improvement were smoothness of movement, depth perception, and resistance in the controllers. However, the majority of them saw the benefits of using the PoLaRS system as an accessible and low-stakes means to develop their skills in operating surgical robots. Many participants noticed the lack of tactile feedback in both PoLaRS and dVSS systems, a feature of which implementation is currently being researched –20, whicOn two separate occasions, the dVSS computer experienced a freeze-up, where the participant could not finish their current task. It was believed to be an overheating issue; therefore, the computer was switched off completely and restarted after approximately five minutes. On one of the testing days, the computer froze two more times after restarting although the computer’s workload was markedly lower compared to other testing days where it functioned without issue. Although the influence of this kind of events on the learning curve during training seems minimal, it did raised the question whether a “freeze up” can happen during surgery and what the consequences can be.Many companies are working on surgical robots that are expected to enter the market in the coming years –23. FromRobotic surgical skills improved during training with PoLaRS. Although skills transfer between training tasks and systems was not demonstrated yet, this study shows the potential of PoLaRS to become an affordable alternative to current surgical robot simulators. Full construct validity needs to be established for different experience levels and training tasks before implementing this system into curricula.Supplementary file1 (PNG 180 kb)Supplementary file2 (PNG 242 kb)Supplementary file3 (DOCX 14 kb)Supplementary file4 (DOCX 18 kb)Supplementary file5 (DOCX 13 kb)Supplementary file6 (DOCX 15 kb)Supplementary file7 (DOCX 14 kb)Supplementary file8 (MP4 18086 kb)Supplementary file9 (PDF 426 kb)Supplementary file10 (PNG 448 kb)Supplementary file11 (PNG 374 kb)Supplementary file12 (PNG 396 kb)Supplementary file13 (PNG 221 kb)Supplementary file14 (PNG 211 kb)Below is the link to the electronic supplementary material.


We synthesized NiO layers on Ni foam through a thermal oxidation process; subsequently, we grew graphene layers directly on the surface of NiO after a hydrogen-assisted reduction process. The uniform graphene coating renders high electrical conductivity, structural flexibility and high elastic modulus at atomic thickness. The graphene-coated 3D NiO–Ni structure delivered a high areal density of ∼23 mg cm−2. It also exhibits a high areal capacity of 1.2 mA h cm−2 at 0.1 mA cm−2 for its Li-ion battery performance. The high capacity is attributed to the high surface area of the 3D structure and the unique properties of the graphene layers on the NiO anode. Since the entire process is carried out in one CVD system, the fabrication of such a graphene-coated 3D NiO–Ni anode is simple and scalable for practical applications.The high theoretical specific capacity of nickel oxide (NiO) makes it attractive as a high-efficiency electrode material for electrochemical energy storage. However, its application is limited due to its inferior electrochemical performance and complicated electrode fabrication process. Here, we developed an  The processing of graphene coated NiO–Ni anode using one CVD system delivered high Li-ion battery performance. However, such a high specific capacity has never been translated into increased areal capacity (capacity per footprint area), which is crucial for practical application in LIBs. The low areal capacity has been considered a critical drawback in most nanomaterial-based anodes. Nevertheless, no attention has been paid to enhancing areal capacity of NiO–graphene anode for large-scale, advanced LIB.The increasing demand for high-efficiency, large-scale electrochemical energy storages  method, in which graphene layers were grown directly on a NiO–Ni structure. In this structure, porous 3D Ni substrate offered high surface area to accommodate large loading of NiO; in addition, the porous structure facilitated lithium ion diffusion within NiO.8,9,15,16 The in situ graphene growth on NiO was achieved by a simple CVD process right after reduction of the NiO process in the same CVD chamber;23–32 the process was effective, yet facile, to produce a highly stable graphene network throughout the 3D NiO structure. The graphene-coated 3D NiO–Ni anode delivered improved areal density (∼23 mg cm−2) and higher areal capacity (1.2 mA h cm−2 at 0.1 mA cm−2) than previously reported NiO-based anodes;8–10,12–15,18,19 such values are critical for practical applications. The excellent properties and novel design of the graphene coated 3D NiO–Ni anode would expand the development of large-scale LIBs.Our previous experiment demonstrated how 3-dimensional (3D) structures could enhance the areal capacity of electrodes in LIBs.2.2.1.2 (125 sccm) mixed gas environment (4 : 1 volume ratio), to a temperature of 1000 °C for 2 hours. At the final stage, the as-grown NiO on Ni foam was naturally cooled to room temperature within the tube under inert Ar gas atmosphere. After the growth process, the gray Ni foam was transformed into a greenish stoichiometric NiO structure. Subsequently, the CVD tube was rapidly heated to 700 °C within the Ar gas environment. Once temperature was reached, the mixture of CH4, H2, and Ar gases was introduced at flow rates of 50, 100 and 500 sccm (1 : 2 : 10 volume ratio), respectively. During the graphene growth process of 1 minute, the thermally decomposed carbon from the precursor CH4 gas was absorbed onto the reduced Ni from NiO (the NiO reduction process simultaneously occurred by H2 gas). Consequently, a graphene-coated NiO–Ni nanocomposite structure was synthesized. Porous Ni foam, with nominal cell size of 450 μm and porosity of 85% (Alantum), was used as a pristine substrate for NiO growth. The Ni foam was cut and then inserted into a quartz tube of a thermal chemical vapor deposition (CVD) system (Atomate). For NiO growth, the substrate was rapidly heated, in an Ar (500 sccm) and O2.2.θ) with step size 0.01° and with a Raman spectroscope  using Ar+ laser with λ = 514 nm and 0.5 mW power.The morphologies of the NiO–Ni foam and graphene-coated NiO–Ni foam structures were identified with a field emission scanning electron microscope (FESEM) . Elemental analysis for both structures was carried out using an energy dispersive spectroscope (EDS) (FEI Helios 650). For cross-sectional SEM-EDS analysis, the samples were frozen in liquid nitrogen (77 K) and then cut into two pieces. The structural property of the samples was also characterized with an X-ray diffractometer (XRD)  using Cu K-alpha radiation in the range of 10–90° . 1.0 M LiPF6 in ethylene carbonate–dimethylene carbonate–diethylene carbonate (EC–DMC–DEC) (1 : 1 : 1 in volume) and a typical polypropylene (PP) based membrane  served as an electrolyte and a separator, respectively. The complete cell assembly was conducted in an argon-filled glovebox that maintained oxygen and humidity levels less than 0.5 ppm. The charge–discharge cycling behaviors of the cell were characterized with a multi-channel battery tester (MACCOR-series 4000) in galvanostatic mode (constant current). In this study, charge and discharge processes were related to the oxidation and reduction (conversion) reactions as NiO + 2Li+ + 2e− ↔ Ni + Li2O, respectively. The cells were cycled in the voltage range of 0.01–3.0 V at different current densities. Cyclic voltammetry (CV) measurements for the 3D graphene–NiO–Ni and 3D NiO–Ni anode samples were conducted using a multi-channel potentiostat  in the voltage range of 0.01 to 3.0 V (vs. Li+/Li) at a scan rate of 0.1 mV s−1.A CR2032 coin cell (Wellcos Ltd.) was assembled with as-grown 3D graphene–NiO–Ni as a working electrode and lithium foil as both counter and reference electrodes. No current collector or additive was incorporated into the assembled anode; this is advantageous for enhanced energy and power density of an LIB cell  pattern confirms the evolution of NiO phase grown on Ni by thermal CVD oxidation , (2 0 0) and (2 2 0) for Ni, respectively (JCPDF card 4-850). Note that the presence of the Ni phase has the advantage of enhancing electrical conductivity of NiO and catalytic activity that facilitates decomposition of Li2O and formation of the solid electrolyte interphase (SEI) layer during the charging process.4 No other peaks relevant to the impurities were identified in the XRD patterns. The Raman spectra further corroborate the results from the XRD analysis (−1 (LO mode), three two-phonon peaks at ∼730 cm−1 (2TO mode), ∼906 cm−1 (TO + LO mode) and ∼1090 cm−1 (2LO mode), and one strong two-magnon peak at ∼1490 cm−1 (2M mode) were observed; the peaks were consistent with previous results from NiO.41 In contrast, no Raman peak for Ni indicates the lack of active vibrational Raman mode in Ni.9 In −2. As evident in Fig. S1,8–10,12–19 The average areal density is obtained by the formula mNiO = Δm × 149.38/32 for the reaction of 2Ni + O2 = 2NiO, where mNiO is the weight of NiO, Δm (the weight of O) is the real weight difference between NiO and Ni after NiO growth, and 149.38 and 32 g mol−1 are the molecular weights of 2NiO and O2, respectively.11 Therefore, we could confirm that our exerted oxidation condition is more intense than other reported ones that processed the Ni–NiO structures.8–10,12–19,28FESEM image of porous Ni foam showed the average pore size was 150 μm and the width was approximately 40 μm  with smo Ni foam . The groxidation . The stranalysis . A typic3.2.e.g., Cu and Ni); additionally, it allows excellent physico-chemical properties of graphene.22,23,31 In principle, graphene is not directly grown on NiO due to insolubility of carbon into NiO.42 Thus, the H2 reduction process is required to transform NiO to Ni for graphene growth. The amount of O vacancies in the NiO structure increases with elevated temperature during the reduction process and catalyzes cleavage of the hydrogen bond (H–H); thus, the reaction produces H2O gas and leaves behind a Ni structure.43 Graphene growth proceeded right after the NiO reduction process by using the same CVD system.44 The process steps are summarized as follows: (1) 3D NiO–Ni structure was annealed in Ar environment at up to 700 °C. (2) CH4/H2 (1 : 2 volume ratio) gas mixture was introduced into the reactor. (3) The structure was cooled to room temperature in Ar environment. In step (2), hydrocarbon  is thermally decomposed; subsequently, the resultant carbon atoms dissolved into Ni after NiO is transformed into Ni by H2 reduction process. During step (3), carbon atoms are segregated and then precipitated on the Ni surface; thus, graphene layers are grown.4546 The presence of the as-grown graphene is also confirmed by the typical D peak at 1355 cm−1, G peak at 1581 cm−1 and 2D peak at 2706 cm−1 in the Raman spectra (47ID/IG peak ratio (∼0.2) and I2D/IG (∼0.5) are indicative of high-quality and multi-layered graphene. Note that peaks corresponding to NiO structure are not observed due to the screening effect caused by graphene on the graphene/NiO surface. This is evidenced by the Raman spectra with wavenumber ranging from 300 to 1000 cm−1, where no NiO peaks are observed  during graphene synthesis; thus, LO-mode peak for NiO at ∼490 cm−1 is observed in the Raman spectra . The areal density of graphene (∼0.17 mg cm−2) was measured by the weight difference of the sample before and after CVD growth.Thermal CVD is more facile for direct growth of high-quality graphene on metal substrates  was observed between 0.25 V and 0.5 V. The plateau region is ascribed to the formation of solid electrolyte interphase (SEI) on the surface of the 3D graphene–NiO–Ni through electrolyte decomposition; it is also due to the reduction from Ni2+ to Ni0 by Li+ ion uptake into NiO based on NiO + 2Li+ ↔ Li2O + Ni, forming Li2O.4,23 It should be noted that the voltage fluctuation at the plateau region might come from the large amount of SEI formation at the graphene–electrolyte interface. Nanostructured materials are commonly susceptible to their unstable or irreversible capacity loss that occurs in the first cycle.48 The gradual increase of the first charge profile up to 2.0 V shows the plateau region of 2.0–2.1 V, in which oxidation reaction from Ni0 to Ni2+ occurred, forming NiO.23,30 Notice that coulombic efficiency (the ratio of charge (1.2 mA h cm−2) to discharge (2.2 mA h cm−2) capacities) for the first cycle at 0.1 mA cm−2 is measured as ∼55%  to discharge (1.18 mA h cm−2) capacities from the second cycle. The gradual decrease in voltage profile for the discharge process was observed in 1–1.5 V; the profile is related to the reduction reaction from Ni2+ to Ni0.4,23 The second discharge curve exhibited the voltage plateau region at ∼1.5 V; the higher voltage over the first discharge is closely related to the large variation of NiO microstructure and texture involved in the irreversible formation of Li2O and the decomposition of SEI layer formed during the first cycle.4 For the second charge, the voltage profile was similar to that of the first charge except with a slightly higher plateau voltage range of 2.1–2.2 V due to increased anodic polarization in a cell.30 The overall voltage–capacity curves showed similar profiles to the previously reported NiO-based anodes.4,15,17,23,30 Our prepared 3D NiO–Ni anode before graphene growth demonstrates different voltage profiles with negligibly low areal capacities  curves for the 3D graphene–NiO–Ni and 3D NiO–Ni anode samples are displayed in Fig. S4.vs. capacity profiles of The electrochemical properties of the 3D graphene–NiO–Ni working electrode were tested using a CR2032 coin-type half-cell. −2 at 0.2, 0.5, 1, 2, 5 and 10 mA cm−2, respectively; the values are higher than those from other reported NiO anodes.18,19 The average areal capacity of the anode (0.6 mA h cm−2), after running through 10 mA cm−2, recovered to the former value of 2 mA cm−2 (higher than 99% capacity retention); this confirmed high structural integrity and rate capability of our proposed 3D graphene–NiO–Ni anode. The subsequent cycling performance at 1 mA cm−2 resulted in an average areal capacity of 0.75 mA h cm−2, which is ∼140% higher than the previous reports on nanoscale NiO anodes;18,19 thus, the resulting capacity retention (∼90%) indicated excellent cell stability. Furthermore, cycling stability of the anode at 1 mA cm−2 for 100 cycles is presented in e.g., carbon black) for anode fabrication. Such materials will act as inefficient deadweight constituents.33 Although 3D graphene–NiO–Ni anode demonstrated improved areal capacity, gravimetric specific capacity could not be acquired due to the difficulty in determining weight fraction among the three components of the anode  after the CVD processing. Nevertheless, as mentioned earlier, no one has yet applied the current CVD approach to enhance the areal capacity of NiO and LIB efficiency with graphene. Thus, the importance of our results from the CVD approach would be realized by its implementation into practical applications for large-scale LIBs and other energy storage systems.Moreover, 4.via a simple two-step thermal CVD method to increase its areal density up to ∼23 mg cm−2. Such value provides a higher amount of active materials for LIBs which is important for large-scale practical applications. The in situ graphene grown on the reduced 3D NiO–Ni exhibits wrinkled, high-quality, and multi-layered structures. Such growth is a simple and highly effective method for large-scale coating of graphene onto a nano-porous electrode. While graphene layer grown on NiO is effective as an electrically conducting and structurally buffering for the nano-porous NiO, the 3D graphene–NiO–Ni anode exhibits a high rate capability with an areal capacity of 1.2 mA h cm−2 at 0.1 mA cm−2. Our 3D graphene–NiO–Ni anode could be applied to ever-expanding development of large-scale, advanced LIBs.We have fabricated a novel 3D graphene–NiO–Ni anode There are no conflicts of interest to declare.RA-008-C7RA10987C-s001


The treatment of sarcoidosis remains uncertain, despite 70 years of study. The conventional approach is to initiate corticosteroids in individuals who require treatment. The position of more aggressive regimes is unknown.Recent recognition that many patients will require prolonged therapy, and the observation that corticosteroids lead to overt and insidious toxicities, have led to suggestions that steroid-sparing medications be used earlier in the management of sarcoidosis. Individuals with poor prognostic features, designated as ‘high-risk’ sarcoidosis may, especially benefit from a broader palette of therapeutic options in the initial treatment regimen. An even more aggressive approach, known as ‘top-down’ or ‘hit-hard and early’ therapy has emerged in the fields of gastroenterology and rheumatology in the past 15 years, on the premise that highly effective early control of inflammation leads to better outcomes. These regimens typically involve early initiation of biologic therapies.For certain subpopulations of sarcoidosis patients, ‘top-down’ therapy could be helpful. Severe pulmonary sarcoidosis, neurosarcoidosis, cardiac sarcoidosis and multiorgan sarcoidosis are phenotypes that may be most relevant for revised therapeutic algorithms. Precision medicine approaches and randomized trials will be necessary to confirm a role for top-down therapy in the routine management of sarcoidosis. It should be noted that these opinions are based not on controlled data but rather on the absence of head-to-head comparisons between corticosteroids and other agents.Corticosteroids have been used for sarcoidosis for over 70 years. Even now, they remain the most commonly used medication for systemic management ▪. Cortic3Although sarcoidosis experts typically advocate for earlier inclusion of steroid-sparing agents in treatment algorithms ▪, in praSarcoidosis is an extremely heterogeneous disease – whereas many patients enjoy spontaneous resolution, a quantitatively important subset experience severe morbidity, loss of quality of life, or even death as a result. Most of these patients with poor outcomes exhibit chronic, nonself-limiting sarcoidosis, ongoing granulomatous inflammation and sometimes fibrosis. Uncontrolled inflammation is believed to be the instigator of fibrotic sarcoidosis, perhaps in the context of profibrotic genetic features and immune responses . For pulCompared with the general population, sarcoidosis leads to increased mortality –19. A la▪▪]. Patients in definite need for treatment are those with severe organ dysfunction such as severe pulmonary sarcoidosis, significant hypercalcemia, clinically important cardiac sarcoidosis or clinically important neurosarcoidosis .As mentioned previously, the initial need for treatment is a predictor for mortality and prolonged treatment duration . However3Immunomodulatory drugs such as methotrexate, azathioprine and leflunomide can be used as steroid sparing options in sarcoidosis, with methotrexate being used most often ▪,33,34. Significantly, a recent cohort study compared pulmonary sarcoidosis patients treated with first-line methylprednisone or methotrexate . Patient▪▪]. The ERS guideline provides a conditional recommendation with low-quality evidence for their use [▪▪]. High disease activity on 18-fluorodeoxyglucose (FDG)-PET correlates with effectiveness of TNFi [Biologics such as tumor necrosis factor inhibitors (TNFi) drugs have mostly been reserved for severe refractory sarcoidosis cases, as outlined in the ERS Guideline ▪▪. The Eheir use ▪▪. High  of TNFi , but rou of TNFi ▪. Whethe of TNFi ,41. Howe of TNFi ▪. A high of TNFi ▪.In the current guidelines, biologics are reserved for cases proven refractory to first-line and second-line therapy which often leads to a potential suspension of months to years until patients receive biologic treatment. In case of high-risk sarcoidosis it is unknown whether this is harmful. Two cohort studies of infliximab and inflectra treated patients showed a decrease in lung function while on first-line or second-line treatment in the 6 months prior to biologic therapy ,44. For In Crohn's disease, a granulomatous inflammatory bowel disease (IBD), the use of top-down versus step-up treatment has been a subject of investigation in the last decade with promising results for a combination therapy of immunomodulators and biologicals for severe disease . A largeSimilar to IBD, current management philosophy in rheumatoid arthritis also emphasizes earlier disease control. The European League Against Rheumatism endorses the immediate initiation of methotrexate or another immunomodulator (with or without corticosteroids) to diminish the chance of permanent joint damage at the time of diagnosis . After 3Sarcoidosis is already an orphan disease, with a treatment-requiring proportion of approximately 50%. The category of patients truly refractory to standard first/second-line treatment is even smaller, but ‘refractory’ is an arbitrary term that may overestimate how many patients can be managed with tolerable or minimally toxic doses of corticosteroids. It is estimated that 10–30% of patients develop progressive pulmonary disease . Due to A highlight related to this particular article is that two extant trials are currently investigating the role of methotrexate as a substitute for initial treatment of sarcoidosis. The PREDMETH trial is a multicenter RCT currently enrolling patients with pulmonary sarcoidosis and impaired lung function in the Netherlands ▪. This sBesides RCTs, the use of  registries with well phenotyped patients is a critical first step to systematically study high-risk or severe sarcoidosis and the optimal treatment regiments. The marginal benefit of registries compared with RCTs is the option to include a more heterogeneous (and more representative) sarcoidosis population, gain large numbers and also gain insight in the use of off-label biologics.With the recent publication of the ERS and BTS guidelines on sarcoidosis management the gap of knowledge and paucity of rigorous evidence in this field becomes strikingly clear. However, these guidelines do provide consistency of direction for physicians treating sarcoidosis patients. A step-up approach with initial corticosteroid monotherapy is favored by the ERS/BTS guideline, although some other reviews or expert opinions leave room for initiation of upfront second-line treatment (e.g. methotrexate) together with steroids in severely endangered or ‘High risk’ sarcoidosis patients.Patient selection will always pose a significant challenge, because not all patients need systemic treatment and all available treatment options have potential side effects. The optimal treatment strategy for these severe, sometimes even life threatening, sarcoidosis cases is unknown. These cases include severe pulmonary, cardiac and neurosarcoidosis. Potentially, extrapolating literature from other disciplines, a ‘hit-hard and early’ approach could benefit severe life or organ threatening sarcoidosis for which a prolonged treatment regimen is anticipated. Evidence from ongoing and future trials in sarcoidosis treatment are needed.None.The current work was not supported or sponsored.A.D.M.V. received consulting fees from Boehringer Ingelheim. D.A.C. received grant support from Boehringer-Ingelheim, Mallinkrodt, AI therapeutics, aTyr Pharma and Janssen. Personal consulting fees, steering committee or travel fees from aTyr Pharma, Krevant, Roche, Boehringer-Ingelheim, Fibrogen and Pliant.


It was the prevalent cause of the significant decrease in the reverse current and short-circuit current. No photovoltaic effect dependence on the graphene roughness and work function could be observed.Graphene was synthesized directly on Si(100) substrates by microwave plasma-enhanced chemical vapor deposition (MW-PECVD). The effects of the graphene structure on the electrical and photovoltaic properties of graphene/n-Si(100) were studied. The samples were investigated using Raman spectroscopy, atomic force microscopy, and by measuring current–voltage (I-V) graphs. The temperature of the hydrogen plasma annealing prior to graphene synthesis was an essential parameter regarding the graphene/Si contact I-V characteristics and photovoltaic parameters. Graphene n-type self-doping was found to occur due to the native SiO In most studies, graphene is synthesized by chemical vapor deposition (CVD) on copper foil and then transferred to the silicon substrate ,13,14,15The abovementioned problems can be solved using graphene directly grown on silicon by plasma-enhanced chemical vapor deposition , althougTherefore, the present study investigates the effects of the directly synthesized graphene structure on current–voltage characteristics and photovoltaic properties of the graphene/n-Si photovoltaic devices. Various synthesis conditions were used to grow graphene samples of different structures and surface morphologies. Only a small influence or no influence of the graphene thickness, defect density, surface morphology, and work function was found. The impact of substrate-induced self-doping and silicon surface pretreatment on the graphene/n-Si device’s current-voltage and photovoltaics characteristics was revealed.Samples were produced using a microwave PECVD system Cyrannus (Innovative Plasma Systems (Iplas) GmbH, Troisdorf, Germany). Monocrystalline, double-side polished, n-type Si(100) , with a resistivity of 1–10 Ω·cm, was used as a substrate. A precursor gas mixture of hydrogen and methane was used for graphene synthesis. Before the growth of graphene, hydrogen plasma was ignited, and methane gas was only introduced when the target temperature was reached. In some cases, the silicon substrates were plasma pre-annealed at higher temperatures than the temperature of the subsequent graphene synthesis. A special enclosure was used to protect from direct plasma that results in high etching rates of the sample . Synthes4OH + H2O2 + H2O), impurity removal (1:50 solution of HF + H2O), and RCA 2 (1:1:6 solution of HCl + H2O2 + H2O) treatments [2 [After graphene synthesis was carried out, diode fabrication began with Al back contact formation (on the uncoated Si side) using e-beam technology. The DMF + acetone boiling and RCA 1  being at 1620 cm−1. The Lorentzian function was used for the best peak fit, considering Merlen et al. [D/IG ratio was used to reveal the defectiveness of our produced samples [2D/IG ratio contributed to the graphene thickness evaluation [Thickness and defect characterization was carried out via Raman scattering spectroscopy using a Raman spectrometer, InVia . The measurement was done just after the graphene synthesis and before the graphene/Si(100) diode fabrication. We acquired Raman spectra at several different places on each sample, considering possible differences in the graphene structure across the specimen. The beam power was set to 1.5 mW, and the excitation wavelength was 532 nm. Several peaks were analyzed for in-depth characterization and defect estimation . The G peak was separated into two components, with the actual G peak being at 1600 cmn et al.  who made samples . In contaluation   was employed to detect any structural peculiarities of the graphene surface. The surface morphology was investigated at room temperature and ambient air conditions using a NanoWizard III atomic force microscope . The measurements were done in tapping mode. The silicon probes  with a thin layer (25 ± 5 nm) of Pt/Ir coating on both re-ex and tip sides of the probes were used. The probe parameters were as follows: spring constant 2.7 N/m; 60 kHz frequency; 30 nm tip ROC; pyramidal shape. Images of 2 µm × 2 µm size were acquired from the measured data using JPKSPM Data Processing software . Kelvin probe measurements were carried out using the same instrumental setup to evaluate graphene work function.R(0.3 V)), forward current vs. reverse current at ±0.1V (IR(0.1 V)/IF(0.1 V)), forward current vs. reverse current at ±0.3 V (IR(0.3 V)/IF(0.3 V))). The photovoltaic parameters (short-circuit current (ISC) and open-circuit voltage (UOC)) were derived from current–voltage characteristics measured under illumination. The I-V characteristic’s dependence on temperature was measured using a similar setup to the photovoltaic parameter measurements. The same Keithley 6487 picoampere meter/voltage source was employed, with thermal operational conditions being changed by a custom-made Peltier element configuration. The temperature varied from −20 to 40 °C. Each measurement was made after the temperature value had settled down.The current-voltage (I-V) characteristics were measured using a Keithley 6487 picoampere meter/voltage source. The measurements were done at several points on the sample to evaluate the possible dispersion of the characteristics. Characteristics were investigated in three different regimes to study the photovoltaic properties of the fabricated devices. These were dark mode (sample was not illuminated), UV mode (when the sample was illuminated by 406 nm wavelength light-emitting diode (LED)), and IR mode (when the sample was illuminated by 800 nm wavelength light-emitting diode). In all instances, the voltage range was from −2 to +2 V. To ensure the same optical power (5.2 mW) between different measurement modes, currents supplied to the LEDs were selected accordingly. The measurements were done at several different places on the samples to evaluate the dispersion of the results. Diode behavior was studied by examining the I-V characteristic parameters in the dark  characteristics of the produced photodiodes can be seen in 2D/IG ratio values indicated the presence of few-layer graphene. The thickness of the one graphene layer was ~0.4 nm [The graphene AFM images and topography parameters were studied to supplement our Raman spectroscopy findings , Table 2 ~0.4 nm ,58. Thus ~0.4 nm . Sample In most cases, graphene Raman scattering spectra were investigated for defect-free or few-defect graphene (no Raman D peak). However, directly synthesized graphene usually contains a significant number of defects . This ca2D/IG ratio with ID/IG ratio was found  [2D decrease with the I2D/IG ratio increase in 2D values can be found for graphene samples of the same thickness. Thus, FWHM2D depends on some other factors. The Pos2D, in our case, was upshifted with the I2D/IG ratio  . One canIG ratio c. In con samples ,63,64,65G is related to the ID/IG ratio of defective graphene [G dependence on ID/IG ratio was found in our case  pre-annealed at 900 °C. For the samples synthesized on the silicon pre-annealed at 700 °C temperature, the tendency of the FWHM2D to decrease with increased FWHMG was found  characteristics of the graphene/Si(100) heterojunctions and graphene structure were studied. The initial surface preparation significantly influences the Schottky and ohmic contact I-V characteristics ,79,80,81R(0.3 V), IR(0.1 V)/IF(0.1 V), IR(0.3 V)/IF(0.3 V)) on the main Raman peak ratios was found , when the Si(100) substrate was hydrogen plasma pre-annealed at 700 °C /IF(0.3 V) ratio, it is seen that values of the reverse current and IR(0.3 V)/IF(0.3 V) of the samples grown after annealing in 700 °C gradually decrease when FWHM2D decreases  decrease (IR(0.3V)/IF(0.3 V) increase) with increasing surface roughness can be observed, although the plot’s strange “branching out” is seen. Due to this, it is impossible to conclude whether this magnitude of roughness impacts device performance.Inclusions of the non-planar graphene, such as wrinkles, can significantly influence charge transport properties ,89. HoweTypical I-V curves of produced photovoltaic devices under illumination can be seen in SC and UOC were investigated concerning Raman parameters   samples, Irameters . Figure creases) a,b. The function . In the function ,30. In ofunction ,27,28,38d FWHM2D c when sarelation d, with sSC correlation with Pos2D and PosG   , and datrameters . It shouain size  after anoughness . The mosterlayer . At the terlayer .R(0.3V) was analyzed. An increase in the ISC following a rise in IR(0.3V) can be seen at least in two groups of samples (SC can be seen when IR(0.1 V)/IF(0.1 V) increases (R(0.3 V)/IF(0.3 V) is taken into account  plot was employed to show that UOC tends to increase with a short-circuit and reverse current ratio increase, although it branches out when the ratio reaches a value of ~1 (SC/IR(0.3V), which tends to be minimal (<0.183 V) when annealing was carried out at 900 °C, different illumination regimes show that IR irradiation yields higher UOC and ISC. As in many discussed relations, samples that were annealed at 700 °C also had the most significant spread of ISC/IR(0.3 V), with values situated in a range of 0.336–3.375. UOC in samples that had been annealed at 800 °C before graphene growth tended to increase with ISC/IR(0.3V), although moderate dispersion of values was observed.The photovoltaic parameter’s relation with I samples a. Curve ncreases b, with o account c, the dispersive . The UOCue of ~1 d. While 2 surface [The electron transfer from the n-Si(100) to the graphene should result in decreased graphene/Si contact barrier and, hence, increased reverse current ,96. In o surface ,98.2 interface results in a dipole formation, and charge redistribution imposes n doping in the graphene [2 interface [2 can also be n-type doped [The charge exchange at the graphene/SiOgraphene , althougnterface . The grape doped .2D  with graphene dopant concentration is significantly suppressed by increasing the graphene layer number [2 to the graphene and subsequent charge redistribution within the graphene [Graphene work-function shift with doping significantly decreased when the graphene layer number increased . The mair number . This isgraphene . The chagraphene . Numerougraphene .2D should downshift and the FWHM2D should increase with an increase in doping and, hence, increased native oxide surface positive charge density. Thus, the reverse current and IR/IF ratios decrease with the graphene substrate-induced self-doping seen in SC increase with IR/IF ratio and with IR can be explained by the flow of the tunneling photocurrent similarly to the quantum dot and superlattice solar cells where the tunneling effect was used to raise the short-circuit current [The analysis of samples’ I-V characteristics measured at different temperatures revealed the flow of the tunneling and thermionic emission currents . At lowe current ,106. It  current . In addi current ,108.It was revealed that the silicon substrate hydrogen plasma pre-annealing was a very important technological parameter regarding the photovoltaic parameters. An increase in the annealing temperature to 900 °C resulted in suppression of the photovoltaic effect. The AFM study revealed no clear morphology and phase changes due to the silicon surface treatment by hydrogen plasma at both 700 and 900 °C . Si(100)OC did not depend on the ISC and increased with ISC/IR ratio for ratios up to 1–1.5  photovoltaic devices properties were revealed.2 interlayer to the graphene was the main reason for the notable reverse current (IR) and short-circuit current (ISC) decrease. Due to the tunneling photocurrent flow, the UOC increased with a short-circuit current, and the reverse current ratio increased. Significant hydrogen plasma pre-treatment effects on the current-voltage characteristics and photovoltaic parameters were observed, revealing the importance of the graphene/silicon interface.The graphene n-type self-doping due to the charge transfer from the native SiOIt was found that the graphene samples’ work functions were nearly the same (4.820–4.826 eV), even though the graphene structure and properties of the photovoltaic devices varied dramatically. No effects of graphene surface morphology and defects on the electrical and photovoltaic characteristics were found. The short-circuit current and open-circuit voltage only slightly increased with graphene layer number.Thus, directly synthesized graphene/n-Si solar cells can be improved by preventing n-type self-doping and optimizing the graphene/silicon interface, whereas graphene defects, layer number, work function, and morphology are much less critical.