The presence of trigonal GeO2 under graphene was identified by Raman and XRD measurements. The oxidation of Ge leads to the formation of blisters under the graphene layer. It is suggested that oxidation of Ge is connected with the dissociation of water molecules and penetration of OH molecules or O to the Ge surface. It has also been found that the formation of blisters of GeO2 leads to a dramatic increase in the intensity of the graphene Raman spectrum. The increase in the Raman signal intensity is most likely due to the screening of graphene by GeO2 from the Ge(001) surface.The problem of graphene protection of Ge surfaces against oxidation is investigated. Raman, X-Ray diffraction (XRD), atomic force microscopy (AFM) and scanning electron microscopy (SEM) measurements of graphene epitaxially grown on Ge(001)/Si(001) substrates are presented. It is shown that the penetration of water vapor through graphene defects on Gr/Ge(001)/Si(001) samples leads to the oxidation of germanium, forming GeO Due to the tight graphene lattice, it may be expected that it should protect surfaces against oxidation well. In this work protection of germanium surfaces by graphene was investigated. The CVD growth of graphene on Ge001)/Si(001) substrates has been used for this purpose. This method of growth was proposed as a promising method for obtaining graphene layers on large area substrates [/Si(001) 2 . TherefoThe graphene growth on Ge(001)/Si(001) substrates is complicated due to the formation of germanium nano-facets underneath the graphene ,14. The Graphene films were synthesized in a 6-inch Aixtron Black Magic system using the CVD method on Ge(001)/Si(001) wafers which contained 3 μm thick Ge film. During the graphene deposition a pressure of 850 mbar was sustained, and the temperature was 900 °C. Methane gas mixed with Ar in the ratio of 1:200 was used as a carbon precursor. The CVD growth was performed on 2 inch wafers preceded by annealing the substrate under pure hydrogen to reduce native oxides.A scanning electron microscope (SEM)  equipped with a secondary-electron detector was used to observe the influence of water vapor on the graphene morphology.Room temperature Raman measurements were done using a Renishaw Via Raman micro-scope system with a 532 nm Nd:YAG laser as an excitation source, with a laser spot diameter of approximately 0.5 μm. The laser was focused on the sample using a ×100 objective and a numerical aperture NA = 0.9 with the average power of the laser being about 5 mW. Spatial Raman maps were performed in the area of 10 μm × 10 μm (15) with a distance of 1 micron between collected spectra. The Raman peaks were fitted with a single Lorentzian/Gaussian shape using the Renishaw WiRE.4.0 program .The atomic force microscopy (AFM) was measured on a Veeco Dimension V SPM with a NanoScope V controller. The surface images were flattened and plain fitted using Nanoscope analysis software. OTESPA Al coated probes with a constant force of ~42 N/m with a typical nominal tip radius of 7 nm were used for the AFM imaging. All the AFM measurements were performed in a normal atmosphere at room temperature.The measurement of the XRD curve was performed using a SmartLab diffractometer equipped with a 9-kV rotating Cu-anode.8 cm−2. The AFM image of one group of blisters is shown in After growth the 1 cm × 1 cm samples were subjected to a humid atmosphere (about 90%) at 50 °C for about 3 h or about 150 h. The samples were placed in a heated container filled with water upon the water surface. The temperature and humidity were measured in the simplest way using a thermometer and a hygrometer. After 3 h of water vapor treatment it was possible to observe regular spots of the size of a few micrometers on the surface of the Gr/Ge/Si(001) sample on the SEM. The SEM image of this surface is shown in The longer water vapor treatment, up to 150 h, dramatically changed the morphology of the surface of the Gr/Ge/Si(001). The SEM image of the surface is shown in −1, 166 cm−1 and 123 cm−1 shown in 2 [2 Raman modes appears approximately proportional to the thickness of the blisters. The fact that GeO2 blisters already appear after 3 h of water vapor treatment indicates that the influence of water vapor strongly speeds up the oxidation process of Ge. The samples kept in the “drawer” (room temperature and low humidity atmosphere) have not shown any occurrence of GeO2. Additionally, there is no sign in the spectra of Raman Si mode (520 cm−1), which indicates that the Ge layer is not completely dissolved under the blisters. As can be seen from the lower part of We wondered what the origin of the blisters on the surface of the Gr/Ge/Si(001) was. The Raman measurements indicate that in the blister locations, for both samples after 3 and 150 h of water vapor treatment, in addition to the intense Ge mode, the spectrum characteristic for germanium oxide occurs. The observed lines at 444 cmown in 2 . This spThe intensities of the Raman spectra of graphene are quite different in the region of blisters versus further away from them. The corresponding spectra are shown in 12 cm−2 before, to about 1.5 × 1012 cm−2 after, the water vapor treatment. The ratio of the D/D’ intensity provides information about the nature of the defects created by the water vapor. The ratio of D/D’ modes is about eleven for the untreated sample (virgin) (measured only in a few points because of a very long collection time) and changes to about seven for the sample after 150 h of water vapor treatment, as shown in the histogram in 3 defects in the virgin sample changes to domination by point defects in the water vapor treated sample. Some experimental points around a value of three to four of the D/D’ ratio /Si(001) sample for 150 h in the humid atmosphere results in a shift of a group of points in ent with . This ch2 blisters on the surface of graphene/Ge(001)/Si(001) surface indicate the presence of graphene defects which are responsible for process water vapor penetration and oxidation of the germanium surface. However, it is puzzling why the oxidation of germanium under graphene takes place so effectively. Exposing pure germanium without graphene to the same oxidation conditions does not lead to formation of GeO2 on the surface. The question arises as to why presence of graphene on the surface of germanium speeds up the process of oxidation. The answer can be found in previous experiments connected with the investigation of the interaction of water with the graphene monolayer. Electron energy loss spectroscopy measurements on water adsorption on quasi-freestanding monolayer graphene on Pt(111) have shown that water molecules at room temperature dissociate, giving rise to C–H vibrational bands [2O --- C–H + OH, or 2C + H2O --- 2(C–H) + O. Most likely, O atoms or OH molecules may go through graphene defects and will interact with the germanium surface, leading to faster oxidation of Ge by O or by OH according to reaction:Formation of GeOal bands . This in2 underneath the graphene identified by Raman measurements, proves that oxidation of Ge in these places takes place. Most likely, extended defects connected with poor bonding between graphene sheets may be responsible for sites of dissociation of water molecules and places of penetration of OH molecules to the Ge surface.A similar process was reported in the case of oxidation of Cu surfaces ,26. It w2 compared to Ge(001), is strongly enhanced. This increase is similar to the increase in the Raman signal by surface enhanced Raman spectroscopy (SERS) by metallic nanoparticles, which is a well-known effect [2 blisters as being highly conductive. The Raman signal from the area of the blisters is about one or two orders of magnitude stronger than the one outside of this area. This increase in the graphene-related Raman bands has already been reported recently when water penetrates underneath the graphene layer and detaches it from the Ge substrate [2 seems to be connected with the isolation of the graphene by the GeO2 from the Ge(001) surface. The 2D vs. G diagram shown in 2 is predominantly under tensile strain, which is in qualitative agreement with the AFM results shown in The intensity of the Raman spectra obtained from the graphene that is on GeOn effect ,31,32,33ubstrate . The low3 defects. Apparently, the hill-and-valley nano-structures which are positioned at 90° to each other and run along the <100> directions on the Ge(001) surface create the conditions for the formation of sp3 bonds between some Ge surface atoms and carbon atoms from the graphene lattice. The presence of water vapor may break these sp3 bonds and change them into graphene point defects. The histogram presented in 12 cm−2 before, to about 1.5 × 1012 cm−2 after, the water vapor treatment. These point defects may be vacancies and may have the character of five-fold and seven-fold rings called Stole-Wales defects [8 cm−2 which is several orders of magnitude lower than the density of point defects established from the Raman D/G ratio. That clearly proves that the defects responsible for the oxidation of germanium are not point defects. This is in agreement with the histogram presented in The Raman spectrum of graphene outside and inside the blister is characterized by a large D peak. The ratio of the intensity of the D to G peak being 1.5 for the virgin sample, changes to 1.9 for the water vapor treated sample as shown in  defects . However2. It is proposed that graphene defects are sites of dissociation of water molecules and penetration of OH molecules or O to the Ge surface and become the origin of oxidation processes. The formation of GeO2 under graphene, dramatically increases the intensity of the graphene Raman spectrum. It is believed that the increase is due to the screening of graphene by GeO2 from the Ge(001) surface.To conclude it has been shown that water vapor penetrates through defects in the graphene/Ge(001)/Si(001) layer and oxidizes underneath the germanium forming blisters of GeO


Caenorhabditis elegans, ablation of germline stem cells (GSCs) and activity changes of the conserved signaling mediators unc-43/CaMKII  and egl-8/PLCβ (phospholipase Cβ) also increase lifespan. Like IIS, these pathways depend on the conserved transcription factor daf-16/FOXO for lifespan extension, but how they functionally interact is unknown. Here, we show that altered unc-43/egl-8 activity further increases the lifespan of long-lived GSC-deficient worms, but not of worms that are long-lived due to a strong reduction-of-function mutation in the insulin/IGF1-like receptor daf-2. Additionally, we provide evidence for unc-43 and, to a lesser extent, egl-8 modulating the expression of certain collagen genes, which were reported to be dispensable for longevity of these particular daf-2 mutant worms, but not for other forms of longevity. Together, these results provide new insights into the conditions and potential mechanisms by which CaMKII- and PLCβ-signals modulate C. elegans lifespan.Reduction of insulin/insulin-like growth factor 1 (IGF1) signaling (IIS) promotes longevity across species. In the nematode  Caenorhabditis elegans and the fruit fly Drosophila melanogaster, provided key insights into the biology of lifespan regulation that apparently are also applicable to human aging [C. elegans to cause a dramatic, ~two-fold increase in lifespan and, subsequently, was confirmed to promote longevity in mammals [C. elegans, lifespan extension by reduced IIS, e.g., by reduction-of-function mutations in the daf-2 gene , is dependent on daf-16  [FOXO3 have been repeatedly and robustly associated with lifespan in humans [Studies in invertebrate model organisms, such as the roundworm an aging . A proto mammals . In C. edaf-2 mutation, additional mechanisms for lifespan extension have been described in C. elegans, including two pathways that can even further extend the extraordinary long lifespan of daf-2 mutant worms: the absence of germline stem cells (GSCs) [unc-43 [daf-2 lifespan raises the possibility that the germline- and the unc-43 signaling pathways are at least in part mechanistically different from the IIS/daf-2 pathway. Interestingly, both the germline- and the unc-43 pathway, just as the daf-2 pathway, depend on daf-16 for lifespan extension [daf-2 deficient animals, such as hlh-30 (TFEB), hsf-1 (HSF1), and skn-1 (NRF2) [unc-43 pathway has not yet been investigated.Beyond s (GSCs) ,4, and a [unc-43 . This fuxtension . Whether1 (NRF2) ,8,9, areGSC(−) and daf-2(−) C. elegans. For example, only GSC(−) worms require the conserved adaptor protein KRI-1 and the translation elongation factor TCER-1 for lifespan extension [GSC(−) longevity, it acts in the intestine and neurons to ensure daf-2(−) longevity [2+) levels by binding to Ca2+-calmodulin complexes [2+-signaling are conserved across metazoans and present in C. elegans, including multiple types of sarcoplasmic/endoplasmic reticulum or plasma membrane channels, G-protein coupled receptors (GPCRs), phospholipase C (PLC) enzymes, and Ca2+-pumps [egl-30, the γ-aminobutyric acid type B (GABAB) receptor gbb-1, and the PLCβ-ortholog egl-8, through their function in neurons, also contribute to intestinal daf-16 activation and longevity [Several lines of evidence indicate that activation and function of the FOXO-transcription factor DAF-16 differ between xtension ,11. Moreongevity . DAF-16/ongevity , includiongevity . CaMKII,omplexes . Many ad2+-pumps . Among tongevity ,18.unc-43 gain-of-function mutation, activate DAF-16 and further extend daf-2(−) lifespan, we hypothesized that the two pathways are largely overlapping. We tested this hypothesis in the present study and provide evidence for a novel longevity-promoting mechanism triggered by unc-43(gf) and also, albeit more weakly, by unc-43 and egl-8 loss, which may explain their different effects on various lifespan-extending pathways in C. elegans.Given that both, germline-ablation and E. coli OP50 [glp-1(e2144ts) mutation served as a genetic model for germline ablation [glp-1(ts) strains (referred to as GSC(−) in Results/Discussion), and corresponding glp-1(+) ) control strains were incubated at 25 °C for the first 24 h of postembryonic development and subsequently shifted to 20 °C for the remainder of the experiment. daf-2(e1370) worms and corresponding daf-2(+) control worms were continuously cultured at 20 °C. For experiments, all strains were routinely cultured on plates containing 20 µM 5-Fluoro-2′-deoxyuridine  from L4 onward to avoid progeny development and internal hatching in fertile strains carrying the unc-43(gf) mutation .Strains used in this study are listed in oli OP50 . Worms c[glp-1e214ts mutatablation . To elimWorms were synchronized by hypochlorite treatment, cultured under the appropriate temperature regimen (cf. above), and scored for survival every other day starting on days 8–10 of adulthood. From the late L4 stage onward, animals were maintained on 6 cm plates at a density of 40 worms/plate and every 10 days, they were transferred to fresh OP50-seeded FUDR-containing NG agar plates to prevent progeny development and desiccation, respectively. Worms were considered dead if they did not respond to gentle touching with a worm pick. Animals that showed a protruding vulva or had ruptured, died from internal progeny hatching (bagging), or escaped from the plate were censored. tert-butyl hydroperoxide (TBHP) and were prepared 12 h before starting the experiment.Worms were synchronized by hypochlorite treatment, cultured under the appropriate temperature regimen (cf. above), and transferred to assay plates on day 2 of adulthood . Survival was scored every 1–2 h. Oxidative stress assay plates contained 15.4 mM E. coli OP50 (for RNA-seq: 1600 worms/2 plates) and cultured at the required temperatures (cf. above). At the L4-stage, 500 worms per strain were manually transferred to two E. coli OP50-seeded 6 cm NG agar plates supplemented with 20 µM FUDR to inhibit germ cell proliferation and progeny production (for RNA-seq: 1500 worms to two 10 cm plates). At day 1 of adulthood, worms were harvested by washing them off their plates with M9 buffer. After additional washing with M9 and RNAse-free water, worms were suspended in 1 mL Trizol, snap-frozen in liquid nitrogen, and stored at −80 °C until RNA extraction. To obtain synchronized populations, gravid adults were treated with hypochlorite, and eggs were allowed to hatch in M9 overnight. ~700 L1 larvae per strain were plated on 10 cm NG agar plates seeded with concentrated RNA was extracted using Trizol and cleaned up with the Monarch Total RNA Miniprep Kit or RNA Cleanup Kit  according to the manufacturer’s instructions. Equal amounts of RNA extracted from three biological replicates were pooled for RNA-sequencing to mitigate batch effects.6 reads  were obtained, resulting in at least 15.7× genome coverage . A minimum of 94.5% of reads  could be mapped to the C. elegans reference genome (cf. below).RNA quality control, library preparation, and 50-bp paired-end RNA-sequencing on the Illumina HiSeq4000 platform were performed at Eurofins Genomics, Ebersberg, Germany. For each sample, a minimum of 31.6 × 10https://usegalaxy.eu (accessed on 30 May 2020) were used for the initial analysis steps: FastQC (v0.72) and MultiQC (v1.7) for initial quality control; Trimmomatic  for gentle trimming of reads; STAR (v2.7.2b) for alignment to the C. elegans reference genome (WS276 release); and featureCounts (v1.6.4) for read summarization [NOIseq  were defined as having a probability of differential expression >95%. Only DEGs that were detected in 20 repetitions of replicate simulation and that displayed an expression-fold chance ≥1.5 with each of the two normalization methods in each of the three repetitions of the entire NOIseq-analysis were considered further. PCA-analysis of CPM-filtered, TMM/UQUA-normalized RNA-seq count data was performed using the NOIseq PCA function. Heatmaps of filtered, normalized, log-transformed count data or of sample-to-sample distances were generated using the R package pheatmap (v1.0.12). Complete NOIseq-data is provided in NOIseq M and probability values for all DEGs, along with gene ontology (GO) term information.The following tools provided by the European Galaxy Server , at rization ,22,23. Arization ) and cusv2.34.0; ,26). Lowgenome WS6 releasephyper function in R. Representation factors were calculated as the number of overlapping genes divided by the expected number of overlapping genes in the two lists . For all calculations, the number of genes in the genome was set to 10,602, i.e., the number of genes in Wormbase WS276 that passed CPM-filtering during NOIseq-analysis. DEG-lists from published studies were, if necessary, converted to WBGene-IDs using WormMine  and adjusted to genes in WS276 using custom R-scripts and manual curation. The statistical significance of overlaps between gene lists was calculated as the hypergeometric probability of detecting at least as many common genes as observed in the two lists using the https://wormbase.org/tools/enrichment/tea/tea.cgi; accessed on 27 May 2021) [http://revigo.irb.hr/; accessed on 27 May 2021). For WormCat analysis [www.wormcat.com (accessed on 9 January 2022). Collagen and matrisome classification [http://CeColDB.permalink.cc/ (accessed on 3 January 2022) and http://ce-matrisome-annotator.permalink.cc/ (accessed on 4 January 2022) and custom R scripts.GO term enrichment analysis was performed using the enrichment tool provided by Wormbase (ay 2021) . The q-vay 2021) ; http://analysis , the resfication  was perfDaf-2(−) strains, which all formed 100% dauers when incubated at 25 °C, were allowed to recover from dauer by incubation at 15 °C for 120 h before classification. Experiments in daf-2(+) strains were either conducted in parallel with experiments in daf-2(−) strains or included daf-2(−) control plates to provide a reference for the typical dauer morphology.Synchronized (hypochlorite treatment) L1 larvae were plated at a density of ~50 larvae/6 cm plate (2 plates per strain), incubated at 20 °C or 25 °C, and inspected for the presence of dauers every 12 h. At the 60 h (20 °C) or 48 h (25 °C) timepoint, when wildtype worms reached adulthood, worms were morphologically classified as “dauer” or “non-dauer”. cdc-42, tba-1, and Y45F10D.4 expression [A total of 1 µg total RNA was reverse transcribed using LunaScript RT SuperMix . qPCR-reactions were performed in duplicates or triplicates in a 20 µL reaction volume on an CFX Connect Real-Time PCR Detection System  with iTaq Universal SYBR Green Supermix (Bio-Rad Laboratories). The thermal cycling protocol comprised one activation step at 95 °C for 3 min, followed by 40 cycles of denaturation at 95 °C for 10 s and combined annealing/extension at 60 °C for 30 s. Melting curve analysis was performed from 65 °C to 95 °C with 0.5 °C increments at 5 s per step. Data were analyzed by the ΔΔCt method, and target gene expression levels were normalized to the geometric means of pression ,33. PrimStatistical analysis was performed using Prism 5 or 9 . Details on the particular tests used are specified in the figure legends.C. elegans lifespan at least in part by activating unc-43/CaMKII, we generated a set of relevant double mutants and measured their lifespans. Thereby, we took advantage of a widely used genetic model for germline stem cell deficiency, glp-1(e2144ts) (hereafter referred to as GSC(−); [unc-43 alleles n498 and n498n1186 (referred to as unc-43(gf) and unc-43(−), respectively). All experiments were conducted in the presence of FUDR , to avoid internal progeny hatching in fertile unc-43(gf) worms and to facilitate comparison with similar previous studies that also took advantage of FUDR to prevent progeny development in fertile strains [GSC(+), i.e., otherwise wildtype worms, unc-43(gf) robustly extended lifespan  also consistently produced a small to moderate lifespan increase in otherwise wildtype worms  worms, unc-43(gf) substantially extended lifespan (67–92%), while unc-43(−) had a more modest effect  and unc-43(−) both trigger longevity-promoting mechanisms. Moreover, these mechanisms apparently are not, or are not maximally, triggered by germline ablation.To test the hypothesis that germline ablation extends  GSC(−);  unc-43 a strains ,18,34. I 36–87%; , consists 6–21%; . Similarunc-43 alleles, we investigated how loss of egl-8/PLCβ influences the lifespan of GSC(−) worms. As unc-43(−), egl-8(md1971), a putative loss-of-function allele, extended the lifespan of otherwise wildtype worms  further extended lifespan in GSC(−) worms  (egl-8(−), similar to unc-43(−) and unc-43(gf) appears to induce lifespan-extending programs that are not, or not fully, active in GSC(−) worms.In parallel with the  13–38%; , in agre 30–43%, . Similar8(e2917) . Thus, eunc-43(gf), unc-43(−) and egl-8(−) in intact and in GSC-deficient animals, we examined the effect of these alleles on the lifespan of worms harboring a strong reduction-of-function mutation in the insulin/IGF-1 like receptor daf-2  [daf-2(+)) animals, unc-43(gf) substantially and unc-43(−) more modestly extended lifespan  worms, however, unexpectedly in the light of published work by others [unc-43(gf) did not further extend but rather slightly decrease lifespan  did not significantly decrease daf-2(−) lifespan. Consistent with our previous observations (egl-8(−) alleles extended lifespan in daf-2(+) worms , similar to unc-43(−), had essentially no effect on daf-2(−) lifespan (unc-43(gf), unc-43(−) and egl-8(−) all trigger longevity-promoting pathways that are already highly active, or that are dispensable for longevity, in daf-2(−) worms.In addition to our lifespan analyses on e e1370) . As mentn 9–13%; . Moreovervations A, both e 27–39%; . Finallylifespan . In summunc-43 and egl-8 affect the resistance of GSC(−), daf-2(−) and otherwise wildtype worms to selected forms of physical stress. Specifically, we examined heat and TBHP-induced oxidative stress. Only in daf-2(−) background, unc-43(gf) slightly increased heat stress resistance, while only in otherwise wildtype worms, unc-43(−) conferred heat sensitivity (unc-43(gf) and unc-43(−) both consistently caused at least trends towards sensitivity in most or all backgrounds, with only the GSC(−); unc-43(gf) strains being clearly not sensitive (egl-8(−) strains consistently tended to be or were more resistant than controls to oxidative stress but more sensitive to heat stress, with the exception of daf-2(−); egl-8(−) strains, which did not consistently show heat sensitivity (unc-43(gf), unc-43(−) and egl-8(−) did not confer broad stress resistance but rather sensitivity to at least one of the two physical stressors examined.Longevity frequently correlates with stress resistance . Therefositivity . On the ensitive . All eglsitivity . In summunc-43(−) and egl-8(−) extended C. elegans lifespan more modestly, but in the same genetic background-dependent manner as unc-43(gf). To identify novel factors and pathways that may mediate unc-43′s and/or egl-8′s lifespan-regulatory functions in addition to daf-16, we performed an exploratory RNA-sequencing (RNA-seq) study in daf-2(−) and otherwise wildtype (daf-2(+)) worms with normal or altered unc-43/egl-8 activity. GSC(−) worms were omitted from this study since unc-43/egl-8 mutations changed their lifespan in essentially the same way as the lifespan of otherwise wildtype worms. Out of the two egl-8 loss-of-function alleles, md1971 was chosen, and hereafter, egl-8(−) refers to md1971 unless otherwise stated. Initial comparison of gene expression between daf-2(−) and wildtype worms revealed highly significant overlaps of differentially expressed genes (DEGs) from our study with published work [daf-2(−) control strains from each other, and strikingly, egl-8(−) and especially unc-43(gf) single mutant worms appeared closer to daf-2(−) in these analyses (unc-43(gf) and egl-8(−) in otherwise wildtype worms significantly overlapped with genes induced by daf-2(−) in our and in published studies (unc-43(gf)/egl-8(−)- and most to all lists of daf-2(−) repressed genes significantly overlapped with each other. Interestingly, in otherwise wildtype worms, unc-43(−) induced and repressed genes also significantly overlapped with most to all lists of daf-2(−) induced and -repressed genes (daf-2(−) worms, unc-43(gf), unc-43(−) and egl-8(−) seemed to support some gene expression changes already induced by daf-2(−), while opposing others (unc-43(gf), unc-43(−) and egl-8(−)-regulated DEGs to genes regulated by daf-2(−) in a daf-16 dependent manner [unc-43(gf) and egl-8(−) globally shift gene expression towards daf-2(−), and they are consistent with previously reported roles of unc-43(gf) and egl-8(−) as positive regulators of daf-16 [In our hands, hed work ,37, thusanalyses A–C. In l studies . Similared genes . In daf-g others . Similart manner ,39,40,41f daf-16 ,16,17,18unc-43(gf), unc-43(−) and egl-8(−) DEGs in otherwise wildtype and in daf-2(−) worms, we performed gene ontology (GO) term enrichment analysis [unc-43 and egl-8 in neurons, muscle, and immunity, respectively [unc-43(gf), unc-43(−) or egl-8(−) strains were also enriched among DEGs between daf-2(−) and wildtype worms, consistent with the previously observed overlaps between the respective DEG-lists (daf-2(+) strains was “structural component of cuticle”, followed by additional GO terms containing many collagen genes , and by multiple GO terms related to pathogen defense. “Structural component of cuticle” and pathogen defense-related GO terms were also enriched among upregulated DEGs of most to all daf-2(−) strains. On the other hand, unc-43(gf), unc-43(−) and egl-8(−) downregulated DEGs were most significantly and commonly enriched for pathogen defense related GO terms in all daf-2(+)- and in the daf-2(−); egl-8(−) strain, and for organic acid-metabolism-related GO terms in daf-2(−); unc-43(gf) and daf-2(−); unc-43(−) worms. Enrichment of similar functional categories was detected by a different analysis tool, WormCat [unc-43(gf), unc-43(−) and egl-8(−)-induced genes, “extracellular material: collagen”  allele analyzed by us, daf-2(e1370), at least not at the standard culture temperature of 20 °C at which we conducted our study [Unc-43(gf), unc-43(−), and egl-8(−) do not sensitize C. elegans to dauer entry at 20 °C or 25 °C (daf-2(−) collagens” ([unc-43(−), egl-8(−) and especially unc-43(gf) upregulated DEGs from daf-2(+), as well as among unc-43(gf) upregulated DEGs from daf-2(−) worms  appeared to up- and downregulate significant numbers of genes that are up- or downregulated by skn-1 under normal conditions (unc-43(gf), unc-43(−) and egl-8(−), especially in daf-2(+) backgrounds, revealed strong overrepresentation of cuticular collagens, as also seen for skn-1 upregulated (non-dauer longevity-associated) collagens ([unc-43(gf), unc-43(−), egl-8(−) and skn-1(−) induced DEG-sets, all of which showed particular enrichment of nematode-specific core matrisome genes  relative to otherwise wildtype worms, while induction in unc-43(−) and egl-8(−) was less consistent and/or weaker (GSC(−) and daf-2(−) worms, col-120, col-141, and col-176 upregulation by unc-43(gf) was repeatedly observed, while unc-43(−) did not trigger strong alterations (in daf-2(−) worms, egl-8(−) appeared to at least mildly suppress all four collagen genes examined (GSC(−) and daf-2(−) worms displayed at least trends towards elevated mRNA levels for most of the collagen genes examined (unc-43(gf), and to a smaller extent, unc-43(−) and egl-8(−), promote the expression of non-dauer longevity-associated collagens. Moreover, the effects of the three alleles appear strongest in GSC(+)/daf-2(+), and weakest, or even lost, in daf-2(−) background.Certain cuticle-forming collagens have been shown to be required for longevity, specifically under conditions that do not induce processes related to dulthood . Expressdulthood . Non-dauur study . Unc-43  at 20 °C) worms. This result may be explained by at least two, not mutually exclusive, models. First, unc-43(gf), unc-43(−) and egl-8(−) all may trigger processes that are already maximally induced by daf-2(−), but not by GSC(−) and wildtype worms. This model is supported by the fact that all three mutations induce similar gene expression changes than daf-2(−). Alternatively, processes triggered by unc-43(gf), unc-43(−) and egl-8(−) may not be critical to daf-2(e1370) longevity. Consistent with this possibility, unc-43(gf) and, to a lesser extent, unc-43(−) and egl-8(−), induce the expression of certain collagen genes, which are required for longevity under conditions that do not induce dauer-like traits in adults. These conditions include germline deficiency but not the daf-2(e1370) allele under our culture conditions of 20 °C [daf-16, by which in particular unc-43(gf), but also unc-43(−) and egl-8(−) modulate lifespan. Of note, overexpression of a single key collagen, such as col-120, also examined by us by qPCR, is sufficient for lifespan extension [daf-2(e1370), mild induction of collagen expression, which is still observed, at least in response to unc-43(gf), may not be functionally relevant. Clearly, it will be extremely interesting to directly examine the role of non-dauer longevity-associated collagens in unc-43(gf), unc-43(−) and egl-8(−) dependent C. elegans lifespan regulation and, eventually, to define the mechanisms that link unc-43/egl-8 to expression of these collagens. Although we have not yet experimentally confirmed or excluded the possibility that the known mediator of unc-43(gf)/egl-8(−) longevity, daf-16, regulates non-dauer longevity-associated collagen expression in the specific context of unc-43/egl-8 mutation, even though it does not do so in other contexts, an alternative candidate is emerging from the literature: skn-1 [unc-43(gf), unc-43(−) and egl-8(−) modulate the expression of similar gene sets, including similar collagens, than skn-1. A key finding of our study consists in the observation that altered of 20 °C . Thus, ixtension . Converse: skn-1 . In suppunc-43(−) and unc-43(gf) differ from each other in several details despite the use of the same alleles. In the case of unc-43(−), these discrepancies may be explained by different experimental conditions, i.e., different temperatures, and by the use of FUDR, both of which have been shown to affect lifespan in certain genetic backgrounds [unc-43(−) and unc-43(gf) alleles within the same experiments, which required the addition of FUDR not just to unc-43(gf) and corresponding control [unc-43(−) strains. Importantly, FUDR does not affect wildtype lifespan nor daf-2(−)’s and GSC(−)’s basic ability to promote longevity [Yet, our study and a previous report  on the lkgrounds . Specifikgrounds  and anal control , but alsongevity ,34,43.daf-2(e1370), rather than daf-2 RNAi [daf-2 activity may account for unc-43(gf) in our hands not extending the lifespan of worms with reduced IIS. Differential interactions of “strong”  and “weak”  daf-2 inhibition with other lifespan-regulatory mechanisms are not unprecedented in the literature, e.g., with somatic gonad signaling and collagen expression [daf-2 RNAi does not efficiently reduce daf-2 activity in neurons [daf-2(−)- and unc-43(gf) lifespan-extending factors daf-16 in neurons accounts for a fraction of daf-2(−) longevity [unc-43(gf) may trigger daf-16 regulated processes in neurons that are already active in daf-2(e1370), but not in daf-2 RNAi worms, which may explain why it further extends daf-2 RNAi- but not daf-2(e1370) lifespan. Beyond different assay temperatures, our choice of f-2 RNAi  to inhibdaf-2e170, rathepression ,45. More neurons ,46,47. Yongevity . Thus, uunc-43(−) and unc-43(gf) extend lifespan, at least in certain genetic backgrounds under certain experimental conditions, appears paradoxical at the first glance. On the other hand, the moderate to strong longevity increase of unc-43(−) and unc-43(gf) worms can be expected, based on the nature of the—relatively moderate to relatively large—changes in gene expression triggered by these two mutations. Indeed, as discussed above, unc-43(−) and unc-43(gf) regulated gene sets are similar to each other and to daf-2- and daf-16-regulated gene sets. In addition, both mutations, although to different extents, induce non-dauer longevity-associated collagens. How unc-43(−) and unc-43(gf) affect collagen expression and/or its indirect regulator skn-1 is currently unclear [daf-16 targets upon unc-43(−) loss, as observed in our RNA-seq study, represents additional, indirect effects of unc-43 on daf-16. Moreover, UNC-43′s direct contribution to DAF-16 activity appears to be dispensable for the maintenance of a normal/normally long lifespan in all genetic backgrounds tested, as unc-43(−), GSC(−); unc-43(−) and daf-2(−); unc-43(−) worms are long-lived, in contrast to daf-16(-), GSC(−); daf-16(-) and daf-2(−); daf-16(-) worms, which are short- to normal-lived [unc-43 loss may “overpower” (direct) DAF-16 regulation by UNC-43.The observation that both,  unclear . Previou unclear  suggesteal-lived . As propal-lived , GSC losegl-8 further extends wildtype lifespan. However, in contrast to previous work [egl-8 loss. Again, this difference may be explained by different experimental conditions, such as different egl-8(−) alleles (md1971 vs. n488), different assay temperatures (20 °C vs. 25 °C) and treatment with different chemicals to impose oxidative stress (TBHP vs. arsenite). Indeed, TBHP and arsenite have already been shown to trigger different transcriptional responses [egl-8 modulates lifespan cell non-autonomously through its function in neurons: egl-8 loss reduces neuronal secretion of insulin-like peptides, which in turn increases daf-16 activity in the intestine; in addition, evidence suggests that this pathway is further mediated by the EGL-8-generated second messenger diacylglycerol (DAG) and the DAG-dependent kinase dkf-2 [egl-8(−) mutation, while GSC(−) worms may display further intestinal daf-16 activation and lifespan extension. Results from our lifespan analyses are in line with this prediction. Conceptually, egl-8 loss also decreases the levels of another second messenger, inositol-1, 4, 5-trisphosphate and subsequently, intracellular Ca2+ levels, and CaMKII/UNC-43 activity [egl-8(−) and unc-43(−) interact to modulate daf-16 activity and C. elegans lifespan.In agreement with published studies ,17,18, wous work , we did esponses . In a cuse dkf-2 . This moactivity . Yet, adunc-43 and egl-8 can/cannot modulate lifespan in C. elegans and suggests collagen expression as a second lifespan-regulatory mechanism functioning downstream of unc-43 and egl-8, in addition to the known regulator daf-16.In summary, our study identified new genetic backgrounds in which


Brain-derived neurotrophic factor (BDNF) induces activation of the TrkB receptor and several downstream pathways , leading to neuronal survival, growth, and plasticity. It has been well established that TrkB signaling regulation is required for neurite formation and dendritic arborization, but the specific mechanism is not fully understood. The non-receptor tyrosine kinase c-Abl is a possible candidate regulator of this process, as it has been implicated in tyrosine kinase receptors’ signaling and trafficking, as well as regulation of neuronal morphogenesis. To assess the role of c-Abl in BDNF-induced dendritic arborization, wild-type and c-Abl-KO neurons were stimulated with BDNF, and diverse strategies were employed to probe the function of c-Abl, including the use of pharmacological inhibitors, an allosteric c-Abl activator, and shRNA to downregulates c-Abl expression. Surprisingly, BDNF promoted c-Abl activation and interaction with TrkB receptors. Furthermore, pharmacological c-Abl inhibition and genetic ablation abolished BDNF-induced dendritic arborization and increased the availability of TrkB in the cell membrane. Interestingly, inhibition or genetic ablation of c-Abl had no effect on the classic TrkB downstream pathways. Together, our results suggest that BDNF/TrkB-dependent c-Abl activation is a novel and essential mechanism in TrkB signaling. Regulation of dendritic arborization is essential for brain function since it determines the receptive field of neurons . ExtrinsThe process of dendritic growth and branching requires active reorganization of the actin cytoskeleton. Neurite outgrowth has been proposed to be directly regulated by lamellipodia and filopodia formation, which in turn are dependent on actin cytoskeleton dynamics mediated by Rho GTPases such as Rac1, Cdc42, and RhoA ,14. InteHere, we show that BDNF-induced dendritic branching requires c-Abl expression and activity. Interestingly, BDNF increases c-Abl activity and c-Abl-TrkB interaction. Although this process requires TrkB activity, it does not require the activation of MAPK, PI3K, and PLC-γ signaling pathways. These results suggest that c-Abl activation is a key downstream mechanism required for BDNF-TrkB-induced dendritic growth, and its mechanism of action does not involve the regulation of the classical signaling pathways regulated by BDNF/TrkB. To obtain further insight into the mechanism of action of c-Abl activity in the context of BDNF/TrkB signaling and function, we investigated the role of c-Abl in the regulation of BDNF/TrkB internalization and endocytic trafficking. We found that c-Abl activity was not required for BDNF-induced endocytosis or degradation of the TrkB receptor. However, we found that c-Abl was required for basal endocytosis of TrkB and that its activation increased the speed of TrkB retrograde traffic.Altogether, our results show that c-Abl activation downstream of BDNF/TrkB signaling mediates a cellular process leading to dendritic growth that does not involve the activation of classical signaling pathways triggered by BDNF/TrkB but is required in BDNF/TrkB-dependent dendritic growth.BDNF induces activation of TrkB, promoting dendritic growth ,15,33,34As shown in previous studies, neurons treated with BDNF showed increased dendritic branching compared to control neurons A. We assTo confirm the requirement of c-Abl expression in the dendritic arborization increase induced by BDNF, we used hippocampal neurons derived from c-Abl KO embryos. Seven DIV c-Abl KO and WT hippocampal neurons were treated with BDNF for 48 h, and primary neurites and branching points were evaluated. Consistent with the results obtained with c-Abl inhibitors, the absence of c-Abl expression in neurons reduced BDNF-induced dendritic growth to basal levels F. The Shabl/arg double-null mice exhibit defective neural tube morphology and gross alterations in neuroepithelial actin cytoskeleton structures, suggesting that c-Abl knockout neurons could have alterations in dendritic tree development -2,5-diphenyltetrazolium bromide (MTT) assay. Then, the MTT reagent (5 mg/mL) was added and incubated for 4 hrs. The medium was then removed and replaced with lysis buffer (50% Dimethylformamide and 20% SDS) overnight. Subsequently, the absorbance of the sample was measured at 620 and 570 nm.t-test or one-way ANOVA followed by appropriate multiple comparisons test depending on the number of groups used in each experiment. Details of the specific tests used, level of significance, and number of replicates are indicated in each figure legend. Statistical analyses were performed using GraphPad Prism 8 (Scientific Software). The significance level was p < 0.05 for all treatments.All data are presented as the mean ± Standard Error of the Mean (SEM). Sholl’s analysis curves were compared with two-way repeated measures ANOVA, followed by Bonferroni’s multiple comparisons. Statistical analyses were performed using a Student’s


This paper presents a rapid assessment of coronavirus disease 2019 (COVID-19) pandemic plans and explores the representation of culturally and linguistically diverse (CALD) communities in such plans. Four levels of pandemic plans were reviewed: regional, state, national and international.Discussions with representatives from four CALD communities informed the development of search and selection criteria for the COVID-19 plans, which were gathered and assessed using a CALD lens. Six COVID-19 pandemic plans that met the inclusion criteria were critically assessed.The reviewed plans did not report any CALD community voices, views or consultations with community groups in the development phase, nor did they acknowledge the diversity of CALD populations. A few plans noted the vulnerability of CALD communities, but none discussed the challenges CALD communities face in accessing health information or health services during the pandemic, or other structural barriers .Our analysis revealed major gaps in all pandemic plans in terms of engaging with immigrant or CALD communities. Policies and plans that address and consider the complex needs and challenges of CALD communities are essential. Collaboration between public health services, multicultural services and policy-makers is vital for the inclusion of this higher-risk population. In Australia, as in other developed nations, the coronavirus disease 2019 (COVID-19) pandemic has disproportionately affected culturally and linguistically diverse (CALD) communities. In many countries, immigrant communities have experienced higher rates of COVID-19 infection, hospitalization, severity of disease and death.  conducting the study. It explores whether the needs, expectations and challenges of CALD communities are represented in these plans. The terms “immigrant” and “CALD communities” are used interchangeably here. The study did not include a review of all relevant literature or research papers; rather, the focus was on pandemic plans and policies at multiple settings and levels .Pandemic plans at regional, state, national and international levels were selected, accessed and then critically assessed through a “CALD lens.”The selection criteria included plans that were:available online at the time of assessment;updated or published within the past 5 years; andapplicable to the region or state of the PHU undertaking the review, to a neighbouring state or to international plans published by the World Health Organization (WHO).As part of using a CALD lens, discussions with representatives from four CALD communities informed the development of the search and selection criteria. These key informants were e-mailed a series of questions. Four consumer representatives of local multicultural health services then discussed the e-mailed questions with the informants to finalize the assessment questions. The final questions were as follows:Does the plan describe a governance structure that includes CALD community representatives?Does the plan describe any consultation with CALD communities before or during the development of the plan?Does the plan outline how it reflects and embraces the diversity of CALD communities?Does the plan reference the challenges CALD communities encounter in accessing health systems?Does the plan describe how CALD communities would be involved in the oversight, implementation or review of the plan when it is operationalized?Six plans met the inclusion criteria and were critically reviewed . However, none of the documents discussed CALD community challenges in accessing health information or health services during the pandemic or other structural barriers such as social determinants of health. Also, the documents did not mention factors such as unemployment, crowded housing, visa status, low health literacy, racism and cultural beliefs. A few references were made to challenges in communication between health organizations and immigrant communities, but the plans tended to fall short of addressing solutions for overcoming barriers to reduce risk. Only two plans  talked about the necessity of providing translated information.In general, Australian policies and plans do not engage with CALD communities, and there is little data regarding the needs of these communities and the challenges they face in accessing health-care systems. This may be an indication of structural racism in the system. (Despite the health inequities faced by people from CALD communities, their voices and needs were not reflected in the pandemic plans assessed in this study. The plans failed to address embedded inequities, which are particularly important in health emergencies. It is recommended that CALD communities be included in the development and implementation of pandemic plans. Further research should be undertaken with diverse communities to enable effective public health actions for COVID-19 and future pandemics.


Adult adrenocortical carcinoma (ACC) is a rare and aggressive tumor in adults, usually associated with excessive steroid secretion. It is highly metastatic and has few therapeutic options and a poor prognosis. Here, we explore the hallmarks of ACC influenced by transcription factors and their target genes (regulons) to provide a prognostic overview of ACC biology. Using an in silico clinical data analysis approach, we assessed human transcriptomic data from publicly available datasets. We found four distinct clusters of regulons associated with good and worse prognoses associated with cell proliferation and/or immunologic activity. Some findings require further bench analyses, primarily focusing on worse prognostic regulons and their targets.NR5A1  and CENPA (Centromeric Protein A), that were consistently associated with overall survival for further downstream analyses. The CENPA regulon was the primary regulator of MKI-67 (a marker of proliferation KI-67), while the NR5A1 regulon is a well-described transcription factor (TF) in ACC tumorigenesis. We also found that the ZBTB4 (Zinc finger and BTB domain-containing protein 4) regulon, which is negatively associated with CENPA in our transcriptional regulatory network, is also a druggable anti-tumorigenic TF. We anticipate that the ACC regulons may be used as a reference for further investigations concerning the complex molecular interactions in ACC tumors.We reconstructed a transcriptional regulatory network for adrenocortical carcinoma (ACC) using transcriptomic and clinical data from The Cancer Genome Atlas (TCGA)-ACC cohort. We investigated the association of transcriptional regulatory units (regulons) with overall survival, molecular phenotypes, and immune signatures. We annotated the ACC regulons with cancer hallmarks and assessed single sample regulon activities in the European Network for the Study of Adrenal Tumors (ENSAT) cohort. We found 369 regulons associated with overall survival and subdivided them into four clusters: RC1 and RC2, associated with good prognosis, and RC3 and RC4, associated with worse outcomes. The RC1 and RC3 regulons were highly correlated with the ‘Steroid Phenotype,’ while the RC2 and RC4 regulons were highly correlated with a molecular proliferation signature. We selected two regulons,  TP53 mutation, and an upregulation of the WNT/β-Catenin pathway , were the regulons with the most associations   and Wound Healing signatures , and a significant negative correlation with leukocyte fraction , Macrophage Regulation , and Lymphocyte Infiltration Signature Score   . Interes × 10−5) .CENPA was the main regulator of the MKI-67 gene (MI = 0.77), the most common histopathologic marker of proliferation, followed by FOXM1 (MI = 0.59), MXD3 (MI = 0.48), and DNMT1 (MI = 0.34)  . MXD3 anPA Duals , and FOXCENPA was significantly associated with both the Steroid IP and the Proliferation IS  and clustered together with RC3  and in OS , in addition to the ENSAT cohort for OS   . In the CENPA’s activity were THRB (Thyroid Hormone Receptor Beta), STAT5B , and ZBTB4 (Zinc Finger And BTB Domain Containing 4) . All of them from RC1 related to good prognosis in the TCGA-ACC and ENSAT cohorts .In the TCGA-ACC cohort, the three regulons with higher negative Spearman correlation with  cohorts  and presThe TCGA-ACC cohort is the largest ACC cohort, thus being the most suitable for regulatory network inference. The inferred network had a good balance between positive and negative targets , with 61Here we present 369 regulons associated with OS, where 188 are related to a good prognosis and 181 to a worse ACC prognosis. Within these regulons, we identified subgroups with specific relationships for each set of molecular characteristics and functional annotations, resulting in four RCs. Regulon cluster 1 and RC3 are more related to the Steroid Phenotype, while RC2 and RC4 are more related to the molecular Proliferation Score as defined by Zheng et al. (2016) . As the RC1 and RC3 are related to Steroid IP, with RC1 associated with LSP and RC3 with HSP. These two clusters presented opposite profiles, both in activity and functional annotations. These clusters were at high activity in the immune and proliferation pathways, as previously shown for the Steroid Phenotype . The strWe also observed that RC2 and RC4 follow opposite patterns in terms of activity and characteristics. RC2, with a good prognosis, is related to low scores in the Proliferation IS, while RC4, with a poor prognosis, is related to high scores in this phenotype. We identified from the Hallmarks enrichment  that theTP53 gene, may alter the P53 pathway and its DNA repair activity [Our functional analysis using the MSigDb Hallmarks presented an overall panorama, providing insights into the biology and function of each group of inferred regulons. However, the MSigDb Hallmarks represent themes of gene sets rather than pathways , which mactivity ,64. The FLI1 (Friend Leukemia Virus Integration 1), STAT5A , and IKZF1 (Ikaros Family Zinc Finger Protein 1). For example, in our results, IKZF1 appeared related to good prognosis , and with the strongest association with the LSP  and the highest positive correlation with leukocyte fraction , Macrophage Regulation , and Lymphocyte Infiltration Signature Score  among the 369 regulons  , shows tregulons . FindingNR5A1 TF, an important player in adrenal development and ACC tumorigenesis [NR5A1 regulon correlated with low OS in the multivariate Cox and Kaplan-Meier analyses in the TCGA-ACC cohort, which was also confirmed in the ENSAT cohort , a common prognostic and proliferation marker widely used in cancer histopathology [Remarkably, we identified ormation . In thisathology .CENPA was described as relating to proliferation and prognosis in ovarian cancer [CENPA overexpression was also associated with proliferation and metastasis in kidney carcinoma by activating the WNT/β-Catenin signaling pathway [CENPA appeared positively enriched in this pathway , but moe cancer  showed tFOXM1 is assigned as a prognostic marker in ACC by Yuan et al. (2018) [It should be noted that our study had some limitations. Our selection strategy may have removed some interesting regulons, which are not listed in the 369 prognostic regulons, thereby excluding their possible role in the ACC regulatory scenario. For example, . (2018) . This TFIn conclusion, we have generated a regulatory network for ACC, evaluated the regulons inferred in concern to OS, clustered them in four RCs, and investigated how they relate to characteristics associated with worse outcomes like the steroid phenotype and the immune and proliferation pathways. The list of prognostic regulons, and their characterization, may open new research avenues and relevant questions to be answered in this hard-to-treat and aggressive malignancy.


The reason for this is that the stronger thermal vibration reduces the load stress difference near the crack tip and, therefore, the crack tip can pass through the sp link. A crack propagates more easily along the zigzag direction than along the armchair direction. The out-of-plane tearing is more suitable than the in-plane tearing for graphene patterning. The path of crack propagation can be adjusted by changing the loading direction, e.g., a rectangular graphene ribbon can be produced by oblique tearing. This new understanding will benefit the application of graphene patterning via the tearing approach.The path of crack propagation in a graphene sheet is significant for graphene patterning via the tearing approach. In this study, we evaluate the fracture properties of pre-cracked graphene during the tearing process, with consideration of the effects of the aspect ratio, loading speed, loading direction, and ambient temperatures on the crack propagation in the monolayer sheet. Some remarkable conclusions are drawn based on the molecular dynamic simulation results, i.e., a higher loading speed may result in a complicated path of crack propagation, and the propagation of an armchair crack may be accompanied by  Cai et al. [Graphene  is a typectronic , thermalectronic , and mecectronic ,6,7 propectronic ,15,16,17ectronic  fabricatectronic  preparedectronic ,14 invesectronic ,19 develi et al.  introduci et al. ,22. Songi et al. ,24 modeli et al. ,26 builti et al.  also sug1/2. Xu et al. [1/2 for zigzag and 3.71 MPa·m1/2 for armchair cracks. Moura and Marder [2, which is about twice that of pristine graphene. Budarapu et al. [In preparing the graphene-based components in a nanodevice or nanostructure, several types of patterning techniques have been developed, e.g., lithographic techniques ,28,29,30u et al.  reportedd Marder  studied d Marder  estimated Marder  designedu et al.  studied u et al.  investigu et al.  also shoIn the above studies, which have provided a deep understanding of graphene fractures, much attention has been paid to functionalization , types oL1, has three options. The length of the moving area, i.e., L2, has four options, and determines the value of the aspect ratio (AR = L1/L2). Correspondingly, the three pairs of models are referred to as AC1, ZZ1; AC2, ZZ2; and AC3, ZZ3, with the values of AR listed in v, is constant in the tearing process. The moving direction of the moving area determines the tearing mode, e.g., in-plane , P(−30°), and P(30°)—marked in The tearing displacement is defined as the product of v) on the crack propagation in the graphene sheets with initial cracks as shown in sp2 carbon sheet, its bending deformation also leads to increasing potential energy, while an overlap of two parts of the sheet via van der Waals interaction reduces the potential energy of the system. For monitoring the state of the sheet during a tearing process, here we adopted a concept named variation of potential energy (VPE), which can be calculated by subtracting the initial potential energy (PE(0)) of the system from the current potential energy (PE(t)), i.e.,First, we studied the effect of loading speed , the cuL1 and L2 , e.g., In-AC1  at first, and then changes to P(−30°)  . Hence, L1 = 30.8 Å  before the sheet breaks in half. When observing the cracking processes, we found that the crack always propagated along with the path P(0°) perpendicular to the loading direction , the sheet still does not split off. For the sheet with an armchair crack, its path of crack propagation is mainly in the “SC” style. Conversely, the propagation path of a zigzag crack is mainly split off, with smooth edges along the initial direction of the crack, which benefits the graphene patterning by tearing.In the above discussion, the dynamic response of the crack in torn graphene is collected at an extremely low temperature, i.e., 1 K, which is difficult, but can be achieved in an experiment. It is known that the ambient temperature of the graphene sheet indicates the intensity of the thermal vibration of atoms. In general, a stronger thermal vibration of atoms leads to a lower strength of the sheet . At 1 K,sp carbon link appeared at the crack edge , which was obviously different from that at 1 K. The reason for this is that the stress distributes randomly in the sheet at higher temperatures . The top five rows of atoms at the zigzag edge were confined as the fixed area or the moving area for applying the tearing load. Five cases of oblique tearing together with the in-plane and out-of-plane tearing results were collected for comparison. For example, in According to the tearing direction shown in θ =75° or 90°, the crack always propagates along with the path P(0°)  for about 300 ps, and then changes to be aligned with P(−90°)  b. When θIn this study, the tearing process of graphene sheets is simulated using the molecular dynamics approach, and conducted on the open-source code LAMMPS . Each siStep 1: Build the graphene sheet with an initial crack;Step 2: Reshape the sheet by minimizing its potential energy;Step 3: Before tearing, relax the system in an NVT ensemble for 100 ps, using the Nose–Hoover thermostat ,56 to coStep 4: Fix the atoms in the fixed area, and apply a constant speed on the atoms in the moving area along a direction for 200 ps;Step 5: Collect the essential data for post-processing.In a simulation, the interactions between the carbon atoms in the graphene sheet are determined by the adaptive intermolecular reactive bond-order (AIREBO) potential , with a In this study, we estimated the fracture properties of pre-cracked graphene during the tearing process with constant velocity. The effects of the aspect ratio, loading speed, loading direction, and ambient temperatures on the crack propagation in the monolayer graphene sheet were investigated using the molecular dynamics simulation approach. Some remarkable conclusions can be drawn based on the numerical results for preparing a graphene ribbon by tearing; for example:Firstly, in an in-plane tearing process at an extremely low temperature, a high loading speed on the graphene sheet with an initial armchair crack may produce a complicated path of crack propagation. The influence can be reduced when the two loading areas  in the sheet contain more atoms. If the sheet has an initial zigzag crack, the crack propagates straight forward, i.e., along P(0°).sp carbon link may appear soon after the initial crack propagates through the whole graphene sheet. These appear more frequently in the graphene sheets with armchair cracks than in the sheets with zigzag cracks.Secondly, when the two loading areas contain fewer atoms, a new crack may occur at the edge of the shorter area—especially at a higher loading speed, e.g., >0.4 Å/ps. An sp carbon link may appear on the path of crack propagation, i.e., the crack tip passes through the carbon link due to a slight stress difference near the crack tip when the atoms have strong thermal vibration. Fortunately, the sp carbon link seldom appears in the graphenes sheet with initial zigzag cracks.Thirdly, in an in-plane tearing of a graphene sheet with an initial armchair crack, temperature influences the crack propagation. For example, at a temperature higher than 200 K, an Fourthly, in an out-of-plane tearing process, if the loading speed is less than 1 Å/ps, the armchair crack propagates along P(30°), while the zigzag crack propagates along P(0°), despite the length difference between the two loading areas and the difference in ambient temperature. Hence, out-of-plane tearing is more suitable for graphene patterning than in-plane tearing.Finally, when an oblique tearing process is applied to a larger sheet with an initial zigzag crack, the path of propagation depends on the loading direction.sp carbon links on the path of the armchair crack propagation should be avoided, and the two loading areas should have sufficient length to counteract the local stress concentration.From above, for preparing a graphene ribbon from a sheet by tearing, the


Conventional fabrication methods to produce graphene are cumbersome, expensive, and not ecologically friendly. This is due to the fact that the processing of a large volume of raw materials requires large amounts of acids and alkalis which, in turn, require special disposal. Therefore, it is necessary to develop new technologies or to refine existing ones for the production of graphene—and to create new, ecologically-safe and effective methods. Here, we utilized physical sonication to extract graphene films from natural mineral shungite rock. From our study of the structure of shungite by Raman spectrometry and X-ray phase analysis, we found that shungite is characterized by graphite-like mineral structures. Transmission electron microscopy images obtained from the processed material revealed graphene films—with surfaces as small as 200 nanometers long and several layers wide. Our green method of fabicating graphene can be widely used in a variety of fields, from electro-optics to ecology, to list a few. At the moment, graphene is a material that is being actively researched as it finds wide application in various industries ,2,3,4,5.Graphene films are produced by the following methods ,26,27: tEach of the presented methods has its pros and cons; during chemical processing, there is a need to wash the resulting graphene of the substances with which the material reacted, which increases the time to obtain graphene. During the mechanical processing (exfoliation) of graphite, large sheets of graphene are obtained; however, in this case, a large amount of tape is needed, which can also increase the amount of side debris, and the loss of some graphene on the tape. When grown on a substrate, graphene layers have a smaller film size than when machined. High energy consumption is required during plasma treatment. We chose ultrasound treatment because when using mechanical methods, films with large surfaces are formed. This method includes only two stages and is easy-to-use. In addition, it is environmentally friendly since it uses an aqueous medium without the addition of surfactants.Here, we used shungite in the sonication which consists of hybrid structures of sp1+sp2+sp3 forms of hybridized carbon atoms . Mostly, sp2 + sp3 forms structures are present in a small fraction of sp1 . It is kHere, we report the production of graphene from shungite rock, as illustrated in Shungite sample from the Zazhoginsky field were retrieved during geological processes. We investigated the surface of the shungite, the presence of mineral inclusions, and the composition of the elements. The studies were carried out using a scanning electron microscope (SEM) with a low vacuum and a Quanta 200 SEM tungsten electron source. The Quanta SEM electron microscope was also equipped with an energy-dispersive X-ray spectroscopy (EDS) system for elemental analysis. For the analysis of shungite, we also used the X-ray method. The studies were carried out using a PANalytical Empyrean multipurpose diffractometer for the analysis of powder and solid substances, nanomaterials, thin films, and suspensions.To test the technique, we used shungite from the Zazhoginsky deposit of group I (group I—more than 96% carbon). Before producing graphene, we broke up the shungite samples. A FRITSCH grinding mill 6 was used to grind shungite samples. Grinding time is 10 min at a room temperature of 25 We checked the shungite surface using a scanning electron microscope (SEM) to determine the type of shungite. We observed the heterogeneous and stepped surface of shungite; the surface-shell spalls characteristic of shungite, as can be seen from After detecting inclusions of various shapes on the surface, we investigated the chemical composition of shungite using energy-dispersive X-ray spectroscopy (EDS). We selected random regions in the shungite and selected several areas in each region with the following dimensions: shown in a,d. The We investigated two areas in each chosen region, as shown in Shungite is also characterized by graphite and graphene shapes, respectively, which have specific absorption peaks at specific wavelengths.Using the method of X-ray spectrometry and Raman spectroscopy, we studied how carbon binds to other elements. The data obtained are shown in graphite . The ban G bands a, it can G bands a, represTo determine the numbers of sp2 and sp3 carbon bonds, we studied the samples using X-ray photoelectron spectrometry. Data are presented in The XPS spectra of shungite after deconvolution is shown in After examining the shungite samples, we processed the samples via a mechanical method—sonication. Since large volumes of samples can be used with this method, there is no secondary contamination and there is no need to carry out subsequent sample processing.The XPS spectra of sonicated shungite after deconvolution is shown in ure data ,65, to aWe considered the production of graphene films from natural mineral shungite using a mechanical method—low-temperature  and optics to biotechnology , energy , and ecology .


Fasciola gigantica excretory-secretory products (FgESP) shows better diagnostic value than the FgESP, but its immunomodulatory function and potential as a molecular vaccine candidate are unknown. Thus, the effect of F22 on the mitogen-induced proliferation of buffalo peripheral blood mononuclear cells (PBMCs) in the innate immune response was preliminarily studied, and its components were also explored. The results indicated that F22 significantly decreased the proliferation of PBMCs stimulated with mitogen. Two hundred and sixteen were identified as components of the F22, and these included eighty-six proteins present in more than one pathway and some with robust immunomodulatory ability. Further studies should be performed to investigate the immunomodulatory function of F22 in the adaptive immune response, and the components of F22 can be further studied as potential vaccine candidate molecules.The 22nd chromatography component (F22) of the Fasciola gigantica excretory-secretory products (FgESP) shows better diagnostic value than the FgESP, and diagnostic methods based on F22 have also been established. Thus, exploring its immunomodulatory function and potential as a molecular vaccine candidate is attractive. In the present study, the effect of F22 on the mitogen-induced proliferation of buffalo peripheral blood mononuclear cells (PBMCs) in the innate immune response was preliminarily studied using the FgESP as a control. PBMCs were incubated with concanavalin A (ConA) and phytohemagglutinin (PHA) at optimal (1 µg/well) or suboptimal (0.25 µg/well) doses coupled with FgESP and F22 at different doses (1–16 µg/well). Cell proliferation was then assessed by microenzyme reaction colorimetry -2,5-diphenyl tetrazolium (MTT) assay). In addition, the components of F22 were also explored by mass spectrometry and then subjected to Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis to infer their functions. The results indicated that FgESP decreased the proliferation of PBMCs stimulated with ConA and PHA at specific doses, whereas F22 significantly decreased the proliferation of PBMCs stimulated with ConA and PHA at both optimal and suboptimal doses (p < 0.05). Two hundred and sixteen proteins were identified in F22, and these included 86 proteins that could be assigned to more than one pathway and some with robust immunomodulatory ability. Further studies should be performed to investigate the immunomodulatory function of F22 in the adaptive immune response, and the components of F22 can be further studied as potential vaccine candidate molecules.The 22nd chromatography component (F22) of the  Fasciola hepatica, Fasciola gigantica, and their intermediate form that causes great global losses annually _15305.fasta) was selected for library comparison of F22. Proteins for which the number of unique peptides ≥ 2 were selected for subsequent analysis to detect the number of proteins related to Fasciola spp. Proteome Discoverer 1.3 (Thermo Scientific) software was used to match and score the original map files (raw files) from the peptide identification with the Q Exactive chromatographic mass spectrometer through SEQUEST software. According to the criterion FDR < 0.01, the peptides were screened based on the confidence, and the presence of at least two unique peptides was used for identification to obtain highly reliable qualitative results.FgESP and F22 were collected as previously described [FgESP obtained by gel filtration chromatography showed four higher peaks of UV280 , and F22 was located under the P1 peak (not shown). F22 obtained after elution was collected for concentration determination of the concentration, and F22 was then adjusted to specific concentration for further use.escribed . The chrFgESP at multiple concentrations (1~8 µg/well) had no effect on the proliferation of PBMCs stimulated with ConA at either dose, whereas FgESP at 16 µg/well decreased the proliferation of PBMCs stimulated with ConA at a suboptimal dose (p < 0.05) ( < 0.05) a.FgESP had no effect on the proliferation of PBMCs stimulated with PHA at the optimal dose. With the suboptimal dose of PHA, FgESP at 2 and 4 µg/well significantly decreased proliferation (p < 0.05) (FgESP is shown in  < 0.05) b. The pep < 0.01). With a suboptimal dose of ConA, F22 at 1~16 µg/well decreased PBMC proliferation (p < 0.01).The ConA-induced proliferation of PBMCs was decreased by F22 (2~16 µg/well) a. With ap < 0.01). With a suboptimal dose of PHA, F22 at 4~16 µg/well clearly decreased PBMC proliferation (p < 0.01), whereas at lower doses (1 and 2 µg/well), F22 had no regulatory effect on PBMC proliferation. The percent inhibition induced by F22 is shown in The PHA-induced proliferation of PBMCs was also decreased by F22 (4~16 µg/well) b. With aFgESP and F22 were subjected to mass spectrometry analysis (with a Q Exactive combined mass spectrometer), and proteins with ≥2 unique peptides were selected for analysis. FgESP contains 548 proteins, whereas F22 contained 216 proteins (FgESP score: 323.31), calcium-binding protein (CaBP) (FgESP score: 323.31), and heat-shock protein (HSP) (FgESP score: 323.31), were present in FgESP. Cathepsin L (CatL) (F22 score: 143.76), thioredoxin peroxidase (TPx) (F22 Score 103.37), and fibronectin type II domain (F22 Score 100.63) were present in F22 with high scores  of proteins), whereas “Digestive system” had the highest number of proteins within “Organismal Systems” (21.60% (27 of proteins) . MoreoveFgESP functions poly-directionally in F. gigantica—buffalo interaction and attracts a great deal of attention. However, its complex composition hindered relevant research to some extent. Encouragingly, the chromatography component of FgESP, F22, shows substantial diagnostic value and immunosuppressive ability, which indicates that chromatography may act as a tool in complex, mixed-component identification and can be applied to immunomodulatory molecular screening.As is well known, F. gigantica Ras-related protein Rab10 (rFgRab10) enhances the apoptosis and migration of PBMCs, promotes the phagocytic ability of monocytes, and significantly inhibits cell proliferation [Filarial nematodes (Onchocerca volvulus and Acanthocheilonema viteae) cystatins suppress the proliferation of human PBMCs and murine spleen cells [Haemonchus contortus (Hc-AK) decreased the proliferation and increases the apoptosis of goat PBMCs [H. contortus excretory-secretory 15-kDa protein induced decreased migration ability of PBMCs and stimulated PBMCs to increase NO production [ConA and PHA are two mitogenic substances that can stimulate the proliferation and activation of T lymphocytes. These substances can activate different T-cell subpopulations, and activated T lymphocytes then function in the cellular immune response. In a previous study , ConA stferation . Filariaen cells . Argininat PBMCs . Recombioduction .FgESP decreased mitogen-stimulated PBMC proliferation at specific doses, which is consistent with the findings reported by Zhang [FgESP, it can be inferred that FgESP may consist of inhibitory and activator components with immunomodulatory functions. Specific molecules in FgESP, such as Fg14-3-3e and FgTPx, have been identified, and relevant research is underway [FgESP components in the immunomodulatory activity of FgESP. F22 decreased mitogen-induced PBMC proliferation stimulated by both ConA and PHA. As the results indicate, it can be inferred that the components of F22 may affect different T-cell subpopulations, and identifying the correlation between specific molecules in F22 and their target subpopulations will deepen our understanding of specific immunomodulatory processes.In the present study, by Zhang . Howeverunderway ,21. FurtFgESP Score: 323.31) and CaM (FgESP Score: 263.37) have been preliminarily explored. For CaBP2 and CaM2, a previous study showed that calmodulin-targeted drugs can impede the growth of Schistosoma mansoni, Plasmodium falciparum, and malarial parasites [Fg-CaBP4 protein can actively bind to the surface of goat PBMCs and regulate the immune response of PBMCs [FgCaBP2 and FgCaM2 may need to be explored further.As components of FgESP, the immunomodulatory roles of CaBP  proliferation in vitro; then, FhESP was fractionated, and a proliferation assay showed that the suppressive activity of FhESP is shown to correlate with the presence of Cat L proteases [FhCat L1 and FhCat L2 show a protective range of 33% to 79% against F. hepatica infection [TPx and cathepsin L, which exhibited high scores in the mass spectrometry analysis of both us study  showed troteases . Recombinfection .FgESP, which is helpful for the screening of potential vaccine candidate molecules. FND (F22 Score: 100.63), β-TUB (F22 Score: 26.29), and HDM-1 (F22 Score: 20.95) were identified in F22. Fibronectin type III domain-containing 5 (FNDC5) is a transmembrane glycoprotein that can be hydrolyzed and lysed to form irisin, which has many benefits in preventing human diseases [F22 exhibits stronger immunosuppressive ability than the diseases . β-TUB idiseases . HDM-1 cSchistosoma mansoni Hsp70 may be associated with regulated cecal invasion as well as cecal transformation [Fg14-3-3e protein, which inhibits the proliferation and migration of goat PBMCs, inhibits phagocytosis of monocytes and may play an important role in the interaction of F. gigantica with goat PBMCs [KEGG analyses indicated that 86 of the proteins in F22 are active in more than one pathway. Heat-shock protein 70 (Hsp70) is a very conserved and prevalent protein , and theormation ,33. The ormation ,36,37,38at PBMCs . Glutathat PBMCs , and subIn the present study, F22 decreased mitogen-induced naïve buffalo PBMC proliferation, indicating its strong immunosuppressive effect on the initial buffalo immune response. Further studies on F22 and its specific component-mediated immunomodulation under the adaptive immune state may facilitate the screening of potential vaccine candidate molecules for the prevention of fascioliasis.


Scanning near-field optical microscopy was used to image hybrid polaritons in graphene/α-MoO3. We demonstrate the topological transition and characterize hybrid modes, which can be tuned from surface waves to bulk waveguide modes, traversing an exceptional point arising from the anisotropic plasmon-phonon coupling. Graphene/α-MoO3 heterostructures offer the possibility to explore dynamical topological transitions and directional coupling that could inspire new nanophotonic and quantum devices.Polaritons in hyperbolic van der Waals materials—where principal axes have permittivities of opposite signs—are light-matter modes with unique properties and promising applications. Isofrequency contours of hyperbolic polaritons may undergo topological transitions from open hyperbolas to closed ellipse-like curves, prompting an abrupt change in physical properties. Electronically-tunable topological transitions are especially desirable for future integrated technologies but have yet to be demonstrated. In this work, we present a doping-induced topological transition effected by plasmon-phonon hybridization in graphene/α-MoO Hyperbolic phonon polaritons – mixed states of photons and anisotropic lattice vibrations – offer appealing properties for nanophotonic applications. Here, the authors show that the plasmon-phonon hybridization upon electronic doping in graphene/α-MoO3 heterostructures can induce topological transitions of the polariton wavefront. The term “hyperbolic” refers to the shape of the isofrequency surface, which is hyperboloidal rather than ellipsoidal like in common anisotropic media, because of the opposite signs of the real dielectric permittivities of metallic and dielectric media. Hyperbolic isofrequency surfaces give rise to an increased photonic density of states, which can help engineer spontaneous2 and thermal3 emission. Furthermore, the unique properties of hyperbolic media have allowed for the development of subwavelength imaging4, polarization converters5, and negative refraction6, among many other technologies.Hyperbolic optics first rose to prominence with the development of hyperbolic metamaterials constructed by periodically alternating metallic and dielectric media9 and α-phase molybdenum trioxide (α-MoO3)11 have been found to exhibit hyperbolicity around anisotropic phonon resonances that drive permittivity to negative values along one crystal axis or plane14. Van der Waals crystals display relatively weak phonon damping, enabling propagating phonon polaritons that behave like waveguide modes with deeply subdiffractional wavelengths15. Phonon polaritons in the two reststrahlen bands of uniaxial h-BN are out-of-plane hyperbolic, meaning that the out-of-plane optic axis and its normal plane have opposite-signed permittivities. h-BN polaritons can propagate along any in-plane direction9. On the other hand, α-MoO3 is biaxial with three infrared reststrahlen bands. The upper band hosts out-of-plane hyperbolic modes 3) is rooted in crystal structure. Heterostructuring and hybridizing hyperbolic modes with other modes can modify their properties22, but adjusting tuning parameters typically requires altering device design. With α-MoO3, for example, in-plane hyperbolic phonon polaritons can be modified by fabricating double-layer structures with a predefined twist angle as the tuning parameter19. The isofrequency contour (IFC) characterizing the propagation-direction-dependent momenta of in-plane hyperbolic modes can be tuned through a topological transition23 from an open hyperbola to a closed curve by sweeping twist angle. On the other hand, dynamical tuning of h-BN phonon polaritons has been demonstrated by means of hybridization with graphene surface plasmons20. Surface plasmon-phonon polariton (SP3) and hyperbolic plasmon-phonon polariton (HP3) hybrid modes have gate-tunable dispersions enabled by the carrier-density-dependent Fermi energy 24. However, graphene/h-BN admits only modest electronic tunability without topological transitions.Integrated nanophotonics requires dynamical tuning of hyperbolic polaritons, but this is challenging since hyperbolicity in the two most widely studied systems  where the topological transition occurs. Finite difference time domain simulations (Methods) show that, without doping, graphene is devoid of plasmonic response and the heterostructure is optically equivalent to bare α-MoO3 . In the cleavage plane of the [100] (x) and [001] (y) crystal axes  phonon frequency 25 has recognized that the transition occurs at an exceptional point (EP), above which plasmons experience Rabi splitting from strong coupling to phonons. Below the EP, there is no mode splitting: plasmons and phonons are weakly coupled in a manner akin to electromagnetically-induced transparency.Along the [001] direction in the middle reststrahlen band, the α-MoO3 with high-frequency permittivities set to unity. The total dielectric function of Drude-like graphene with electron coupling to in-plane hyperbolic phonons modulated by the quantity 3 to SP3 in the finite slab.We now show quantitatively that tuning mentclass2pt{minimely Fig. . \docume}Γ− Fig. . Splittix) to dope graphene26 by a charge transfer process (Methods). Doping graphene using thin high-work-function materials like WOx or α-RuCl3 has proven to be nondetrimental to s-SNOM imaging28. Furthermore, by stacking either monolayer or bilayer tungsten diselenide (WSe2) prior to oxidation, we can tune the 3 between ~0.60 eV and 0.45 eV, respectively. Doping levels were determined by fitting  was used to image polaritons and evaluate their energy-momentum  onto SiO2/Si substrates. A monolayer or bilayer WSe2 on graphene heterostructure is then transferred onto a predetermined α-MoO3 flake. Finally, we treat samples in a UV-ozone generator (Jelight UVO-cleaner) at 300 K to oxidize the topmost WSe2 monolayer into WOx37.High-quality WO38.A Neaspec neaSNOM near-field microscope was used with a DRS Daylight Solutions Hedgehog tunable quantum cascade laser TLS-SK-41112-HHG (λ=10.86–11.67 µm). Standard PtIr-coated Arrow tips with 75 kHz resonance frequencies were used with tapping amplitudes of about 40 nm in contact. The signal localized under the apex of the tip is isolated in the backscattered signal by demodulation at the 1st−5th tip tapping harmonics in a pseudoheterodyne detection scheme3 heterostructures were performed with the Ansys Lumerical finite difference time domain (FDTD) solver for Maxwell’s equations . We used a point dipole source positioned 20 nm above the top surface of the graphene/MoO3 structure and polarized along the z direction. Electric field monitors were placed on the top surface. The boundary conditions were set to the perfectly matched layer option. The NumPy fast Fourier transform library was used to compute isofrequency contours from FDTD wavefronts. In particular, we used the fft2 function followed by the fftshift function to set the zero-frequency component to the center.Simulations of the electric field distributions on graphene/MoOTo extract polariton momenta −1.Raman measurements were conducted on a commercial Bruker Senterra system using an excitation wavelength of 532 nm at 2 mW with a 60 second integration time. A grating of 1200 line/mm was used to attain an energy resolution of 3–5 cmSupplementary Information


The tunneling current–bias voltage (It − bV) properties of GWMHs can be tuned by 5 × 106 times in magnitude for current increasing from 0.2 nA to 1 mA with applied bias voltage increasing from 10 mV to 2 V. Moreover, the transfer properties of GWMHs exhibit n-type conduction at bV = 0.1 V and bipolar conduction at bV = 2 V; these findings are explained well by direct tunneling (DT) and Fowler–Nordheim tunneling (FNT), respectively. The results show the great potential of GWMHs for high-power field-effect transistors (FETs) and next-generation logic electronic devices.Van der Waals heterojunctions, formed by stacking two-dimensional materials with various structural and electronic properties, opens a new way to design new functional devices for future applications and provides an ideal research platform for exploring novel physical phenomena. In this work, bilayer graphene/WS Graphene FETs have the advantages of high carrier mobility and large current density ,3,4,5,6,Among the device applications of Van der Waals heterojunctions, tunneling transistors based on the carrier tunneling effect have particular significance due to their advantage of a large on/off ratio and the associated promising prospect of low power consumption. To achieve a sufficiently high on-state current density and a very low off-state current density in graphene-based tunneling transistors, a suitable tunneling layer should be delicately designed. Among 2D materials, hexagonal boron nitride (h-BN) is widely used as the tunneling layer ,30 becau2) is an n-type semiconductor [2 is 4.2 eV [2, taking advantage of graphene’s gate-voltage controllability. Hence, WS2 is an ideal material for investigating the tunneling mechanism and improving the performance of graphene-based tunneling devices [Tungsten disulfide  were fabricated via a controlled dry transfer method (see details in Methods) [2 substrate on the monolayer graphene. Moreover, Cr/Au was selected as the top metal because Cr’s work function (~4.6 eV) [2 barrier and the Cr/Au electrode with a relatively low barrier, resulting in a high on-state current density (4 × 107 A m−2) and a high on/off ratio (5 × 106) by sweeping the bias voltages. More interestingly, the GWMHs device exhibits n-type conduction at 0.1 V bias voltage and bipolar conduction at 2 V bias voltage, which is unusual in semiconducting devices but can be explained well by direct tunneling (DT) and Fowler–Nordheim tunneling (FNT), respectively. The results of this study not only pave the way to polarity-controllable high-performance 2D transistors but also provide an ideal paradigm for designing next-generation logic electronic devices.In this study, bilayer graphene–WSMethods) . Bilayer~4.6 eV)  is closeDevice fabrication. Few-layer bottom graphene and a WS2 tunneling barrier layer were mechanically exfoliated from bulk graphite and WS2 single crystals from the HQ Graphene company  [2/Si substrate, and the exfoliated WS2 layer was transferred onto a polydimethylsiloxane (PDMS) membrane. Then, the vertical graphene–WS2 Van der Waals heterostructures were carefully assembled with the dry transfer method [2 layers were deposited with Cr/Au electrodes using standard e-beam lithography and an e-beam evaporation process. The deposited film thicknesses of Cr and Au electrodes were 5 nm and 50 nm, respectively.erlands) . After er method  using a Structure characterization. The Raman spectra of graphene and WS2 layers were measured by a confocal Raman spectrometer  at room temperature in ambient conditions. The excitation wavelength was 532 nm. The laser spot was about 1 μm in diameter and had a power of 1 mW. The thickness of the WS2 layer was determined by atomic force microscopy using the tapping mode .Electrical transport measurements. The tunneling I–V characteristics of our devices were measured by a low-temperature probe station equipped with source meters and lock-in amplifiers. The samples were placed in a vacuum chamber (<10−6 Torr) with a variable temperature from 300 to 5 K.2 layers were mechanically exfoliated from high-quality bulk crystals and then vertically stacked layer-by-layer via a dry transfer method to form the heterostructures. The bottom graphene layer was electrically attached to Cr/Au metal electrodes, and the whole heterostructure device was placed on an Si substrate with a 290 nm SiO2 layer on top . 2. The thicknesses of the graphene bottom layer and the WS2 middle layer were measured by atomic force microscopy (AFM) and were about 0.89 and 6.4 nm  at one edge of the device, as shown in the −1, integration width: 20 cm−1). The shapes of the graphene and WS2 layers are clearly visible, correlating well with the optical image. Raman spectroscopy was used to characterize the number of layers of WS2 and graphene, as shown in 2 exhibits two characteristic peaks—the E1g2 peak at 350 cm−1 and the A1g peak at 420 cm−1—which are consistent with the previous Raman studies of few-layer WS2 [Both the graphene and WSayer WS2 . The numayer WS2 ,38,39.jt − bV) characteristics were performed in the GWMHs devices. By applying a bias voltage (bV) between the bottom graphene layer and the top Cr/Au electrode, the tunneling current density (jt) through the multilayer WS2 could be detected. jt − bV curves under various gate voltages (Vg) at 300 K, and jt as a function of the bias voltage bV and the gate voltage Vg at 300 K. jt − bV curves and the jt map at 5 K, respectively. As illustrated in jt) of GWMHs can be tuned by a factor of 5 × 106, e.g., from 8 A m−2 to 4 × 107 A m−2, with the bias voltage changing from 10 mV to 2 V at 5 K. Under a very small bias at 10 mV, current leakage still exists with magnitude 8 A m−2, resulting from host point defects in the WS2 layer [6 to 6 × 104 because of the increased current leakage at higher temperatures [6 A m−2 at 25 V bias) [7 A m−2 at 2 V bias).After structural characterization, electrical measurements of the tunneling current density–bias voltage  , our GWMjt−Vg) exhibit typical n-type conduction at a low bias (bV = 0.1 V) but bipolar conduction at a high bias (bV = 2 V). By changing the gate voltage, the Fermi level of bottom graphene can be tuned. 2, but the Fermi level of the top metal electrodes cannot be tuned because of their high DOS, as shown in 2, lowering the tunneling barrier and increasing the tunneling current, as shown in As depicted in 2–metal heterojunction device, it is possible to tune the Fermi level of the bottom graphene and tailor the shape of the tunneling barrier, providing a platform for exploring the tunneling mechanism under different bias voltages. At low bias voltages, direct tunneling occurs, and the tunneling barrier width is equal to the WS2 barrier thickness, which is unaffected by the bias and gate voltages. The direct tunneling current density DTj can be approximated by the following equation [In the graphene–WSequation ,42:(1)jD2 barrier can be further shaped by higher bias voltages, converting it from a trapezoid to a triangle. In this circumstance, the tunneling barrier width is no longer equal to the WS2 barrier thickness, which is reduced as bV is increased. Therefore, the tunneling probability is markedly increased, and the resistance of bottom graphene dominates the total resistance. Fowler–Nordheim tunneling (FN tunneling) occurs, which can be described by the following equation [The WSequation ,42:(2)jFm and m* are the masses of free and effective electrons, respectively. d are the effective barrier height and width, respectively, determined by the difference between graphene’s Fermi level and the bottom of WS2’s conduction band at the graphene–WS2 interface, respectively. h is Planck’s constant, and e is the unit charge of the electron.In Equations (1) and (2), jt − bV characteristics under different back-gate voltages at a temperature of 5 K. The Equation (2) can be transformed into [Because of the high electric field generated during the FN tunneling process, the Joule heating effect frequently causes a large thermionic emission current over the triangle-shaped barrier. To suppress the thermionic current and determine the effective barrier height med into ,42:(3)lnjt − bV curves and establish a reference point for defining the tunneling barrier height. tj|/bV2) as a function of 1/bV in the GWMHs tunneling device at different Vg values at 5 K. At a higher bV, the larger negative slope indicates that the FN tunneling dominated the carrier transport and current flow in this region, while at lower bV, direct tunneling of charge carriers through the ultrathin WS2 layer dominated. If the effective mass value of the electron (m*) is set at 0.3 m [Equation (3) helps investigate the at 0.3 m , then thtion (4) ,42:(4)k=Vg changes from zero to negative values (hole doping). The gate-tunability of the carrier tunneling regions and the barrier height in our GWMHs devices opens a new road to control the characteristics of vertical field-effect tunneling transistors. In addition, with the continuous progress of lithography technology, lateral tunneling heterojunctions have also attracted much attention [ttention ,45,46. H2–metal heterojunctions (GWMHs) was studied in this work. The tunneling current density (jt) of GWMHs changes by a factor of 5 × 106, with the applied bias voltages changing from 10 mV to 2 V, and a high on-state current density (4 × 107 A m−2) was achieved. Moreover, the transfer properties of GWMHs exhibit n-type conduction at a 0.1 V bias and bipolar conduction at 2 V, which are well-explained by direct tunneling (DT) and Fowler–Nordheim tunneling (FNT), respectively. Our results show great potential for GWMHs in high-power FET devices and next-generation logic electronic devices.In summary, a tunable field-effect tunneling transistor based on graphene–WS