We characterized the HSV-1 pUL7:pUL51 complex by solution scattering and chemical crosslinking, revealing a 1:2 complex that can form higher-order oligomers in solution, and we solved the crystal structure of the core pUL7:pUL51 heterodimer. While pUL7 adopts a previously-unseen compact fold, the helix-turn-helix conformation of pUL51 resembles the cellular endosomal complex required for transport (ESCRT)-III component CHMP4B and pUL51 forms ESCRT-III–like filaments, suggesting a direct role for pUL51 in promoting membrane scission during virus assembly. Our results provide a structural framework for understanding the role of the conserved pUL7:pUL51 complex in herpesvirus assembly.Herpesviruses acquire their membrane envelopes in the cytoplasm of infected cells via a molecular mechanism that remains unclear. Herpes simplex virus (HSV)−1 proteins pUL7 and pUL51 form a complex required for efficient virus envelopment. We show that interaction between homologues of pUL7 and pUL51 is conserved across human herpesviruses, as is their association with  Most people suffer from occasional cold sores, which are caused by the herpes simplex virus. This virus causes infections that last your entire life, but for the most part it lies dormant in your cells and reactivates only at times of stress. When it reactivates, the virus manipulates host cells to make new virus particles that may spread the infection to other people. Like many other viruses, herpes simplex viruses also steal jelly-like structures known as membranes from their host cells to form protective coats around new virus particles.In cells from humans and other animals, proteins belonging to a molecular machine known as ESCRT form filaments that bend and break membranes as the cells require. Many viruses hijack the ESCRT machinery to wrap membranes around new virus particles. However, herpes simplex viruses do not follow the usual rules for activating this machine. Instead, they rely on two viral proteins called pUL7 and pUL51 to hot-wire the ESCRT machinery. Previous studies have shown that these two proteins bind to each other, but it remained unclear how they work.Butt et al. used a combination of biochemical and biophysical techniques to solve the three-dimensional structures of pUL7 and pUL51 when bound to each other. The experiments determined that the structure of pUL51 resembles the structures of different components in the ESCRT machinery. Like the ESCRT proteins, pUL51 formed filaments, suggesting that pUL51 bends membranes in cells and that pUL7 blocks it from doing so until the time is right. Further experiments showed that the equivalents of pUL7 and pUL51 in other members of the herpes virus family also bind to each other in a similar way.These findings reveal that herpes simplex viruses and their close relatives have evolved a different strategy than many other viruses to steal membranes from host cells. Interfering with this mechanism may provide new avenues for designing drugs or improving vaccines against these viruses. The pUL7 and pUL51 proteins may also inspire new tools in biotechnology that could precisely control the shapes of biological membranes. Herpesvtrans-Golgi markers in infected cells -III component . We charEscherichia coli, the palmitoylation site of pUL51 (Cys9) having been mutated to serine to avoid aberrant disulfide bond formation (g) of 4.3 and 4.8 nm, with maximum particle dimensions (Dmax) of ~18 nm and 20 nm, respectively  complex visually resembles the folded core of the full-length complex  complex was crystallized and its structure was solved by four-wavelength anomalous dispersion analysis of a mercury acetate derivative. The structure of native pUL7:pUL51(8–142) was refined to 1.83 Å resolution with residuals ran plot –Table S2Strikingly, the molecules of pUL7 and pUL51 in the structure were arranged as a hetero-octamer, with single β-strands from each pUL7 and pUL51 molecule in the asymmetric unit forming a central β-barrel . Closer L7(Seven) Tegument Assembly/Release Domain (CUSTARD) fold. pUL51 comprises a hydrophobic loop region followed by a helix-turn-helix. The interaction with pUL7 is extensive and largely hydrophobic in nature (The core heterodimeric complex formed by pUL7 residues 11–296 and pUL51 residues 41–125 is shown in n nature : The hydn nature .2 < 1.26). These models showed the second copy of pUL51 to have the same general orientation relative to pUL7, binding near pUL7 helices α1, α2, α6, α7, and the loop between helices α7 and α8  293 T cells. In all cases, pUL51-mCherry or the relevant homologue could be readily co-precipitated with the GFP-pUL7 homologue, whereas pUL51-mCherry homologues were not efficiently co-precipitated by GFP alone . The assGiven the large evolutionary distance between α-, β- and γ-herpesvirus pUL7 and pUL51 homologues, and consequent sequence divergence, it was unclear whether the molecular details of the interaction between these proteins would be conserved. GFP-tagged pUL7 was thus co-transfected with mCherry-tagged pUL51 from HSV-1 or with mCherry-tagged homologues from VZV, HCMV or KSHV. Co-precipitation was observed for HSV-1 pUL51 and for pORF7 from VZV, an α-herpesvirus, but not for the homologues from HCMV or KSHV . This su6 data sets where interacting sites were paired at random. True pairings showed more correlated change than 94% of the randomized pairings and the results were little changed when different subsets of the data, including fewer strains and more interactions, were analyzed  was generated by manual inspection of the binding interface. The amino acids carried at these sites across an alignment of pUL7 and pUL51 homologues from 199 strains of α-herpesvirus were tested for correlated changes. Initially, 35 of the 63 interacting-residue pairs where homology could be confidently assigned were analyzed, results being compared to a null distribution determined from 10analyzed –Table S4In addition to interacting with pUL7, it has previously been shown that HSV-1 pUL51 is able to interact with HSV-1 pUL14  and thattrans-Golgi marker, and with paxillin and zyxin at the cell periphery, markers of focal adhesions . Bacteria were cultured in 2 × TY medium, recombinant proteins being expressed overnight at 25°C following addition of 0.4 mM isopropyl β-d-1-thiogalactopyranoside. The complex of HSV-1 strain KOS proteins pUL7 (UniProt ID A0A110B4Q7) and pUL51, or truncations thereof, were co-expressed in the E. coli strain T7 express qlysY/I (New England BioLabs) using the polycistronic vector pOPC −1 strain KOS protein pUL51 (UniProt ID D3YPL0), either with the wild-type sequence or where residue Cys9  had been substituted with serine, was expressed with an N-terminal MetAlaHistor pOPC . The nuc2, 1.4 mM β-mercaptoethanol, 0.05% Tween-20) supplemented with 400 U bovine pancreas DNase I (Merck) and 200 μL EDTA-free protease inhibitor cocktail (Merck) at 4°C. Cells were lysed using a TS series cell disruptor (Constant Systems) at 24 kPSI and the lysate was cleared by centrifugation at 40,000 × g for 30 min at 4°C. For soluble proteins, cleared lysate was incubated with glutathione sepharose 4B resin  equilibrated in GST wash buffer ) for 1 hr at 4°C before being applied to a column and washed with >10 column volumes (c.v.) of GST wash buffer. To remove contaminating nucleic acids, pUL7:pUL51 complexes were resuspended in 25 mM sodium phosphate pH 7.5, 150 mM NaCl, 0.5 mM DTT, 1 mM MgCl2 and incubated with 2000 U of benzonase (Merck) for 1 hr at room temperature before being applied to a column, washed with 8 c.v. of 50 mM sodium phosphate pH 7.5, 1M NaCl, and then washed with 4 c.v. of GST wash buffer. Bound protein was eluted using GST wash buffer supplemented with 25 mM reduced l-glutathione, concentrated, and further purified by size-exclusion chromatography (SEC) using an S200 16/600 column  equilibrated in 20 mM tris pH 7.5, 200 mM NaCl, 1 mM DTT. The GST tag was removed by supplementing the pooled SEC fractions containing pUL7:pUL51 complex with 0.5 mM EDTA and then incubating with 40 μg of GST-tagged human rhinovirus 3C protease. Free GST and uncleaved GST-tagged pUL7 were captured using glutathione sepharose resin and the cleaved complex was again subjected to SEC using S200 16/600 or 10/300 columns  equilibrated in 20 mM tris pH 7.5, 200 mM NaCl, 1 mM DTT, 3% (v/v) glycerol. Purified pUL7:pUL51 was concentrated, snap-frozen in liquid nitrogen as small aliquots, and stored at −80°C. Protein concentrations were estimated from absorbance at 280 nm using calculated extinction coefficients  EDTA-free protease inhibitor cocktail (Merck)) that was briskly stirred for 2 hr at 4°C. Refolded pUL51(1-170) was buffer-exchanged into 20 mM phosphate buffer pH 7.5 or 20 mM HEPES pH 7.5 using a Sephadex PD-10 gravity column  or into 20 mM tris pH 8.5 by exhaustive dialysis overnight at 4°C.pUL51(1-170) was purified from inclusion bodies and refolded by rapid dilution as described previously for vaccinia virus CrmE . BrieflyMulti-angle light scattering (MALS) experiments were performed immediately following SEC (SEC-MALS) by inline measurement of static light scattering , differential refractive index , and UV absorbance . Samples (100 μL) were injected onto an S200 Increase 10/300 column  equilibrated in in 20 mM tris pH 7.5, 200 mM NaCl, 3% (v/v) glycerol, 0.25 mM tris(2-carboxyethyl)phosphine (TCEP) at 0.4 mL/min. Molecular masses were calculated using ASTRA 6 (Wyatt Technology) and figures were prepared using Prism 7 (GraphPad).−1, 2θ is the scattering angle, and λ is the X-ray wavelength, 0.124 nm) were recorded using a Pilatus 6M detector (Dectris) with 1 s sample exposure times for a total of 3600 data frames spanning the entire course of the SEC separation. 90 μL of purified pUL7:pUL51 (8 mg/mL) or pUL7:pUL51(8–142) (4.5 mg/mL) was injected at 0.5 mL/min onto an S200 Increase 10/300 column  equilibrated in 20 mM HEPES pH 7.5, 200 mM NaCl, 3% (v/v) glycerol, 1 mM DTT (pUL7:pUL51) or 20 mM tris pH 7.5, 200 mM NaCl, 3% (v/v) glycerol, 0.25 mM TCEP (pUL7:pUL51(8–142)). Data presented in g through the elution profile   are shown in Continuous flow small-angle X-ray scattering (SAXS) experiments were performed immediately following SEC with in-line MALS and dynamic light scattering (SEC-SAXS-MALS-DLS), at EMBL-P12 bioSAXS beam line  . Scatterimation)  were scaimation) . Moleculimation) , datmow imation) , datvc (imation)  and Bayeimation)  of the Aimation) , from whMBIMETER . SAXS dag GASBOR  and DAMMg GASBOR . For pULg GASBOR . Dummy-ag GASBOR , average5 AGC target and 50 ms maximum injection time. MS2 used quadrupole isolation at an isolation width of m/z 1.6 and CID fragmentation (25% NCE). Fragment ions were scanned in the Orbitrap with 5 × 104 AGC target and 100 ms maximum injection time. MS3 was triggered by a targeted mass difference of 25.979 for DSBU and 31.9721 for DSSO with HCD fragmentation (30% NCE) and fragment ions scanned in the ion trap with an AGC target of 2.0 × 104.Purified pUL7:pUL51(8–142) at 1 mg/mL (16.4 μM) in HEPES SAXS buffer was incubated with 20- to 100-fold molar excess of disuccinimidyl sulfoxide  or disuccinimidyl dibutyric urea  dissolved in DMSO, or with DMSO carrier alone  in all cases). Reactions were incubated at room temperature for 30 min before quenching by addition of 1 M tris pH 7.5 to a final tris concentration of 20 mM. Samples were separated by SDS-PAGE using a 4–12% Bolt Bis-Tris gel (ThermoFisher) in MOPS running buffer and stained with InstantBlue Coomassie Protein Stain (Expedeon) according to the manufacturers’ instructions. Cross-linked samples corresponding to pUL7:pUL51(8–142) heterodimers (1:1) or heterotrimers (1:2) were excised, reduced, alkylated and digested in-gel using trypsin. The resulting peptides were analyzed using an Orbitrap Fusion Lumos coupled to an Ultimate 3000 RSLC nano UHPLC equipped with a 100 µm ID × 2 cm Acclaim PepMap Precolumn and a 75 µm ID × 50 cm, 2 µm particle Acclaim PepMap RSLC analytical column (ThermoFisher Scientific). Loading solvent was 0.1% formic acid (FA) with analytical solvents A: 0.1% FA and B: 80% (v/v) acetonitrile (MeCN) + 0.1% FA. Samples were loaded at 5 µL/min, loading solvent for 5 min before beginning the analytical gradient. The analytical gradient was 3% to 40% B over 42 min, rising to 95% B by 45 min, followed by a 4 min wash at 95% B, and finally equilibration at 3% solvent B for 10 min. Columns were held at 40°C. Data were acquired in a DDA fashion with MS3 triggered by a targeted mass difference. MS1 was acquired from 375 to 1500 Th at 60,000 resolution, 4 × 10E. coli proteome (downloaded 06.09.2019 with OPGE removed) and 246 common contaminants; full trypsin digestion; carbamidomethyl static modification of cysteines; oxidation variable modification of methionines; 1% FDR using XlinkX validator Percolator. Workflow 2: spectra filtered for either MS2 or MS3 scans with each set searched separately using Mascot against a database containing an E. coli proteome (downloaded 06.09.2019 with OPGE removed) with 246 common contaminants, and HSV-1 proteome (downloaded 04.04.2016); PSM validator Max. Delta Cn = 0.05. Statistical validation of identified cross-link peptides from both workflows was carried out by a joint consensus workflow.Raw files were process using XLinkX 2.2 in Proteome Discoverer 2.2.0.388 (ThermoFisher). MS2 or MS3 spectra were selected based on the identification of either DSSO (K +158.004 Da) or DSBU (K +196.085 Da) and then processed in two workflows in parallel with the following parameters. Workflow 1: XlinkX Search against a database containing an HSV-1 proteome (downloaded 04.04.2016), −1). The final distribution of target function (F) values was clearly bimodal: models from the cluster with higher F values were unable to simultaneously satisfy the provided cross-link restraints and the SAXS data, and were thus discarded. Remaining models were assessed for fit to the SAXS profile (χ2) using CRYSOL.Pseudo-atomic modelling of the pUL7:pUL51(8–142) heterotrimer was performed using CORAL . The corpUL7:pUL51(8–142) was crystallized in sitting or hanging drops by mixing 1 µL of 5.3 mg/mL protein with 0.5 µL of 0.5 M benzamidine hydrochloride and 1 µL of reservoir solution containing 0.15 M sodium citrate pH 5.5, 12% (v/v) 2-methyl-2,4-pentanediol, 0.1 M NaCl and equilibrating against 200 µL reservoirs at 16°C for at least one week. Crystals of pUL7:pUL51(8–142) were cryoprotected by brief immersion in reservoir solution supplemented with 20% (v/v) glycerol before flash cryo-cooling by plunging into liquid nitrogen. For multiple-wavelength anomalous dispersion (MAD) phasing experiments, 1 μL of 1 mM mercury(II) acetate in reservoir solution was added to the mother liquor and incubated at 16°C for 4 hr before cryoprotection and cryo-cooling as described above. Diffraction data were recorded at 100 K on a Pilatus3 6M detector (Dectris) at Diamond Light Source beamline I03. Images were processed using DIALS , either P 4 21 2) was performed using CRANK2 (P 21) using MolRep .Four-wavelength anomalous dispersion analysis of the mercury derivative  in 20 mM phosphate buffer, pH 7.5. A total of 20 spectra were recorded per sample at 50 nm/min with 1 nm bandwidth between 260–190 nm. Spectra were converted to mean residue ellipticity, averaged, and smoothed  using Prism 7 (GraphPad). Spectra were decomposed using CDSSTR  as imple–/A2 across 1 s exposures.Copper grids (300 mesh) coated with formvar and continuous carbon (EM Systems Support) were glow discharged in air for 20 s. Three microlitres of 10–100 μM pUL51(1-170) in 20 mM HEPES pH 7.5 or 20 mM tris pH 8.5 was applied to the grid and allowed to adsorb (30 s to 2 min) before wicking away excess solvent with filter paper (Whatman). Grids were sequentially applied to two 30 µL drops of 2% (w/v) uranyl acetate for approximately 3 s and then 30 s, respectively. Excess stain was wicked away using filter paper (Whatman) and grids were allowed to air dry. Images were obtained using a Tecnai Spirit transmission electron microscope (FEI) operating at 120 kV, equipped with an Ultrascan 1000 CCD camera (Gatan). Images were acquired at 30,000–120,000 × magnification with −1 μm defocus and a total electron dose of 20–40 eProtein sequences of pUL7 and pUL51 homologues from representative α-, β- and γ-herpesviruses that infect humans were as follows (Uniprot ID): HSV-1 pUL7 (A0A110B4Q7) and pUL51 (D3YPL0), VZV pORF53 (P09301) and pORF7 (P09271), HCMV pUL103 (D3YS25) and pUL71 (D3YRZ9), human herpesvirus 7 (HHV-7) U75 (P52458) and U44 (P52474), KSHV pORF42 (F5HAI6) and pORF55 (F5H9W9), Epstein-Barr virus (EBV) BBRF2 (P29882) and BSRF1 (P0CK49). Secondary structure prediction was performed using the NetSurfP-1.1 server , disordeAlphaherpesvirinae  or across all sub-families . Seed alignments were used to generate hidden Markov models (HMMs) using the HMMER (Alphaherpesvirinae) or using the HMMER web server (Herpesviridae). We mapped the proteins thus identified to the source virus genomes, discarding any protein sequences from partial genome sequences where pUL7 or pUL51 were absent. Our initial alignments comprised distinct pairs of pUL7 and pUL51 sequences from 205 Alphaherpesvirinae, 147 Betaherpesvirinae and 78 Gammaherpesvirinae, and the alignments for homologues in each subfamily were improved by manual correction.Clustal Omega  was usedhe HMMER  program he HMMER  program b server   that are predicted to interact. For each pair of interacting sites, we calculated the strength of the correlation between its amino acid states across the alignment. For this purpose, we followed Zaykin and colleagues . This led to an alignment containing 199 strains, for which the amino acids of 35/63 interacting sites could be confidently aligned across all strains. These 35 interactions involved 21 sites from pUL51 and 19 sites from pUL7 , or because we could not be confident in the alignment of some sites. There was thus an inherent trade-off between maximizing the number of interactions and maximizing the number of strains in the test. We initially examined alignments across the Data set 2; Data set 3; Data set 4; R code for performing the analysis is available as file Because so many interactions were missing from this analysis, we next excluded two further pUL7 homologue sequences  where many of the interacting sites could not be confidently aligned. This led to an alignment of 197 strains, for which 54/63 interactions could be tested . Despite the increase in the size of the data set, results were little changed (l-glutamine (ThermoFisher). Cells were maintained at 37°C in a humidified 5% CO2 atmosphere.Mycoplasma-free human embryonic kidney (HEK) 293 T and HeLa cells were maintained in Dulbecco’s modified Eagle’s medium  supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) and 2 mM Plasmids for GFP-pUL7  and pUL51-mCherry  were as used in 6 HEK 293 T cells were transfected by adding 1 µg total DNA (split evenly by mass between the plasmids indicated) and 1.5 µg of branched polyethylenimine  that had been diluted in Opti-MEM (ThermoFisher) and incubated together for 20 min before addition to cells.For co-precipitation experiments, 5 × 104 HeLa cells/well were seeded in six-well plates containing four sterile no. 1.5 coverslips/well and grown overnight before being transfected by addition of 625 ng total DNA (split evenly by mass between the plasmids indicated) and 6 µL/well TransIT-LT1 (Mirus) that had been diluted in Opti-MEM and incubated together for 20 min before addition to cells.For immunocytochemistry, 7.5 × 102HPO4, 1.8 mM KH2PO4), and washed twice in PBS. Cell pellets were resuspended in lysis buffer , 1% (v/v) EDTA-free protease inhibitor cocktail (Merck)) and incubated at 4°C for 30 min before clarification by centrifugation at 20,000 × g, 4°C for 10 min. The protein concentration in each lysate was normalized after assessment using the bicinchoninic acid assay (ThermoFisher) according to the manufacturer’s instructions. Normalised lysates were incubated for 1 hr at 4°C with GFP-Trap or RFP-Trap bead slurry (Chromotek) that had been pre-equilibrated in wash buffer . Following incubation, beads were washed three times, the supernatant was completely removed, beads were resuspended in SDS-PAGE loading buffer and the samples were heated at 95°C for 5 min to liberate bound proteins before removal of the beads by centrifugation. Samples were separated by SDS-PAGE using 12% or 15% polyacrylamide gels and transferred to Protran nitrocellulose membranes (Perkin Elmer) using the Mini-PROTEAN and Mini-Trans-Blot systems (BioRad) following the manufacturer’s protocol. After blocking in PBS with 5% (w/v) non-fat milk powder, membranes were incubated with primary antibody overnight at 4°C and then secondary antibody for 1 hr at room temperature. Dried blots were visualized on an Odyssey CLx infrared scanner (LI-COR).Cells were harvested 24 hr post-transfection by scraping in phosphate buffered saline  FBS, 0.1% saponin in PBS) for 30 min. Primary antibodies (below) were diluted in blocking buffer and incubated with coverslips for 2 hr. Coverslips were washed five times with blocking buffer before incubation for 1 hr with the relevant secondary antibodies (below) diluted in blocking buffer. Coverslips were washed five times with blocking buffer, three times with 0.1% saponin in PBS, three times with PBS, and finally with ultrapure water. Coverslips were mounted using Mowiol 4–88 (Merck) containing 200 nM 4′,6-diamidino-2-phenylindole (DAPI) and allowed to set overnight. Images were acquired using a Zeiss LSM780 confocal laser scanning microscopy system mounted on an AxioObserver.Z1 inverted microscope using a 64× Plan Apochromat objective (NA 1.4). Images were processed using Fiji  electron microscopy-grade formaldehyde  for 5 min on ice before being incubated with 250 mM HEPES pH 7.5, 8% (v/v) PFA at room temperature for 10 min. Coverslips were washed with PBS before quenching of residual PFA by addition of 25 mM NHing Fiji .Primary antibodies used for immunoblotting were rabbit anti-GFP , rat anti-RFP , or mouse anti-GAPDH . Secondary antibodies for immunoblotting were LI-COR IRDye 680T conjugated goat anti-rat (926–68029), donkey anti-rabbit (926–68023) or goat anti-mouse (926–68020), or LI-COR IRDye 800CW conjugated donkey anti-rabbit (926–32213) or goat anti-mouse (926-32210). Primary antibodies used for immunocytochemistry were anti-TGN46 , mouse anti-Paxillin , rabbit anti-Zyxin , and secondary antibodies were Alexa Fluor 647 conjugated donkey anti-sheep , goat anti-mouse  or goat anti-rabbit .www.pdb.org (accession code 6T5A), and raw diffraction images have been deposited in the University of Cambridge Apollo repository (https://doi.org/10.17863/CAM.44914). SAXS data, ab initio models and pseudo-atomic models have been deposited into the Small-Angle Scattering Biological Data Bank (SASBDB)   under th(SASBDB)  partner  In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:Graham and colleagues describe the structural characterization of the Herpes virus tegument protein complex pUL7-pUL51, which is important for membrane envelopment of the capsid in the cytosol and is conserved across herpesviruses. The most exciting observation that truncated pUL51 is structurally similar to the helical hairpin formed by CHMP4B, the most abundant subunit of the ESCRT-III membrane fission complex. The authors therefore propose that pUL51 may function in viral envelopment and budding by mimicking (or recruiting) the cellular ESCRT-III complex, which is used by many other enveloped viruses to bud from cells. Although this hypothesis remains to be tested functionally, the authors provide strong support by demonstrated that truncated pUL51 proteins can form helical filaments that resemble those formed by ESCRT-III proteins.Decision letter after peer review:eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Wes Sundquist, Reviewing Editor and Cynthia Wolberger, Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Insights into herpesvirus assembly from the structure of the pUL7:pUL51 complex" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:The manuscript by Graham and colleagues describes the structural characterization of the Herpes virus tegument protein complex pUL7-pUL51, which is important for membrane envelopment of the capsid in the cytosol. The complex is conserved across herpesviruses.The major findings are:1) SAXS analysis indicates that pUL7:pUL51 forms a 1:2 complex with an elongated shape likely due to an unstructured C-terminal region of pUL51.2) Deletion of the pUL51 C-terminus resulted still in a 2:1 pUL7-pUL51(1-170) complex, which was characterized by SAXS and shown to be more compact.3) An even smaller pUL7-pUL51(8-142) complex forms a 1:1 complex and its structure was determined at high resolution by X-ray crystallography.4) The pUL7 structure adopts a novel, predominantly helical fold that the authors term a "Conserved UL7(Seven) Tegument Assembly/Release Domain (CUSTARD)" fold5) The structure of the truncated pUL51 construct reveals a surprising feature that could provide an essential clue for understanding its critical function in viral replication because there is striking similarity between pUL51 and the helical hairpin formed by CHMP4B, a subunit of the ESCRT-III membrane fission complex. The authors therefore propose that pUL51 may function in viral envelopment and budding by mimicking (or recruiting) the cellular ESCRT-III complex.5) Based on the heterodimer data, chemical cross linking, Mass spec and the SAXS data a model of the pUL7-pUL51 complex was reconstructed to position the missing C-terminal region of pUL51.6) Part of the interface of the pUL7-pUL51 complex is conserved across herpesviruses, and bioinformatics analysis suggest co-evolution of interacting residue pairs.7) pUL51 does not interact with pUL14 as had been suggested previously.8) Co-localization of pUL7-pUL51 with focal adhesion markers paxillin and zyxin as previously suggested, was not observed.Essential revisions:Overall, this is likely to be an important piece of work for understanding the mechanism of envelopment of Herpesviruses. The manuscript is well written, the data are of high technical quality, and the structure is novel and presents a new fold of pUL7. However, the study currently lacks experimentally-based insights into how the pUL7-pUL51 complex functions in membrane envelopment. In particular, the hypothesis that pUL51 functions by mimicking CHMP4B needs to be supported by functional evidence because this is the most important claim in the paper. Given the strong similarity between pUL51 and CHMP4B, the modeling of key pUL51 residues predicted to function in membrane binding and remodeling should be straightforward.To substantiate the ESCRT-III hypothesis, the authors need to provide convincing functional data on pUL51. Relevent experiments might include testing membrane binding and polymerization in vitro, testing Golgi membrane binding in cells using structure-based mutagenesis, and evaluating relevant mutations in the context of viral replication. The key questions that need to be answered are:1) Does pUL51 bind to membranes without palmitoylation and, more importantly, is the binding mode consistent with the ESCRT-III model? It is not obvious from the figures that the complex can be positioned on a membrane via the helical hairpin in the complex.2) Does pUL51 form ESCRT-III-like filaments. The authors report that pUL51 forms large aggregates in vitro. Are these aggregates ESCRT-III-like filaments?3) How does pUL7 affect pUL51 polymerization and membrane binding?eLife.If the authors are able to provide these types of convincing functional studies, then the manuscript will become a conclusive work that advances the field significantly and should be acceptable for publication in  Essential revisions:Overall, this is likely to be an important piece of work for understanding the mechanism of envelopment of Herpesviruses. The manuscript is well written, the data are of high technical quality, and the structure is novel and presents a new fold of pUL7. However, the study currently lacks experimentally-based insights into how the pUL7-pUL51 complex functions in membrane envelopment. In particular, the hypothesis that pUL51 functions by mimicking CHMP4B needs to be supported by functional evidence because this is the most important claim in the paper. Given the strong similarity between pUL51 and CHMP4B, the modeling of key pUL51 residues predicted to function in membrane binding and remodeling should be straightforward.To substantiate the ESCRT-III hypothesis, the authors need to provide convincing functional data on pUL51. Relevent experiments might include testing membrane binding and polymerization in vitro, testing Golgi membrane binding in cells using structure-based mutagenesis, and evaluating relevant mutations in the context of viral replication. The key questions that need to be answered are:1) Does pUL51 bind to membranes without palmitoylation and, more importantly, is the binding mode consistent with the ESCRT-III model? It is not obvious from the figures that the complex can be positioned on a membrane via the helical hairpin in the complex.It has been published previously that pUL51 does not readily associate with membranes when its palmitoylation is blocked by mutation of the acceptor cysteine residue to serine or alanine . The palmitoylation site of pUL51 (residue 9) is located well before the first residue of pUL51 in the pUL7:pUL51 core heterodimeric complex . While it is likely that helix 1 of pUL51 is more extended in the polymeric form of the protein , there would still be approximately 16 amino acids between this palmitoylated Cys residue and the predicted start of the core ESCRT-III–like helical hairpin. We posit that the N-terminal palmitoylation of pUL51 and homologues is akin to the ANCHR motif of Snf7 and the myristylation of the CHMP4B nucleating protein CHMP6, and that pUL51 therefore fulfils the functions of both CHMP6 and CHMP4B in infected cells. This hypothesis is consistent with observations that ‘canonical’ ESCRT-III initiation signals, mediated by ESCRT-I, ESCRT-II or the Bro1-domain containing proteins, are dispensable for HSV-1 membrane envelopment .We have included a schematic diagram in Figure 5 and have updated the Discussion to clarify our proposal for pUL51 polymerisation and its role in virus assembly. We have also clarified our proposal that pUL7 is a negative regul,ator of pUL51 polymerisation, blocking the pUL51 self-association interface akin to the regulation of Shrub polymerisation by Lgd  or CHMP4B by CC2D1A .2) Does pUL51 form ESCRT-III-like filaments. The authors report that pUL51 forms large aggregates in vitro. Are these aggregates ESCRT-III-like filaments?E. coli inclusion bodies. As shown in Figure 5—figure supplement 1, this refolded protein is pure and largely helical, and as shown in Figure 5 this protein forms ESCRT-III like filaments.We have now shown that a truncated form of pUL51, including the N-terminal regions predicted to be helical (residues 1-170), forms ESCRT-III like filaments. We initially concentrated our efforts on assaying the polymerisation of the full-length pUL51 protein shown in Figure 1—figure supplement 1. However, the aggregation-prone nature of this protein made it exceedingly challenging to work with – the yields were very low and it would irreversibly aggregate before we could apply it to EM grids. We therefore designed a new purification strategy by refolding pUL51(1-170) from 3) How does pUL7 affect pUL51 polymerization and membrane binding?eLife at a later date.Our previously-published work shows that pUL51 recruits pUL7 to membranes . Based on the similar orientations of Lgd DM14-3 relative to Shrub and pUL7 relative to pUL51, we predict that pUL7 will inhibit pUL51 polymerisation. We planned to test this hypothesis using a ‘poisoning’ method, by adding increasing concentrations of pUL7:pUL51(8-142) to pure pUL51(1-170) and monitoring the effect upon pUL51(1-170) polymerisation in vitro. However, given the challenges in producing sufficient pure pUL51, these polymerisation assays were optimised only days before our laboratory was closed due to the COVID-19 outbreak. We were therefore unable to perform these poisoning experiments. We hope to perform these experiments as soon as we are back in the lab, in addition to probing pUL51 mutants that were designed to block polymerisation based on the structural homology to cellular ESCRT-III components. We humbly suggest that these experiments may be suitable for publication as a Research Advance in


Sarcoidosis and uveitis are chronic inflammatory conditions with potentially debilitating effects on quality of life. Steroids form the mainstay standard therapy in both conditions. Biologic agents are considered to be appropriate alternatives for treatment in steroid-refractory sarcoidosis and uveitis due to the role of tumor necrosis factor (TNF) in mediating the inflammatory cascade seen in both conditions. We performed a thorough literature search using PubMed to compare the extent of use, efficacy, and safety profile of individual anti-TNF agents in the management of these conditions. Our review consists of two systematic reviews with meta-analysis, thirteen observational studies, and fifteen case series/reports. Infliximab had the widest range of organ-system usage in extra-pulmonary sarcoidosis but is equivalent to adalimumab in terms of efficacy. In uveitis, adalimumab was found to be the most efficacious agent for maintaining disease remission in adults and children with chronic non-infectious uveitis. Etanercept was neither used widely, nor was it efficacious in the management of either condition. In terms of safety profile, biologic agents were found to be well tolerated and have a similar safety profile. More randomized clinical trials are needed to inform evidence-based use of biologic agents in these conditions. Sarcoidosis is a systemic inflammatory disorder wherein the central feature is the formation and propagation of granulomas in affected organs. It has a prevalence of 10 per 100,000 people in the United States, with the incidence and prevalence being higher in African American populations compared to Asians, Hispanics, and Caucasians . Organ-s Uveitis refers to intraocular inflammation, which, in a strict sense, may involve the iris, ciliary body, or choroid . This inTumour Necrosis Factor (TNF) is a participatory cytokine in many inflammatory disorders and plays a role in the formation & propagation of sarcoid granulomas . The rolCorticosteroids are considered to be the mainstay and standard treatment in both sarcoidosis and chronic uveitis , howeverThe purpose for this article is to review the literature to highlight the role of the common anti-TNF agents, infliximab, adalimumab, and etanercept, in the management of extra-pulmonary sarcoidosis and non-infectious uveitis, summarize key articles comparing the efficacy and safety profile of these agents as well as discuss possible areas for future research.MethodsA thorough literature search was conducted using PubMed with infliximab, adalimumab, etanercept as keywords. These were used in combination with sarcoidosis and chronic uveitis. Search filters applied include the availability of full text and publishing within the last ten years. This initial search yielded 248 articles, but only 50 papers were shortlisted for abstract review after careful screening of articles based on relevance to the research question. Duplicate studies in review articles were excluded. Inclusion and exclusion criteria were applied during in-text screening, and 30 papers were chosen for the final review. Inclusion criteria helped include articles relevant to the research question with full text available published in English. Articles focused on pulmonary sarcoidosis were screened out using the exclusion criteria.Additional supportive references were included in the introduction and discussion sections. PRISMA diagram describing the selection of data is shown in Figure ResultsThirty articles included for the final review are composed of two systematic reviews with meta-analysis ,12, fiftDiscussionMechanism of Action of Anti-TNF AgentsAdalimumab and infliximab have similar mechanisms of action by binding to soluble TNF-α, thereby rendering it biologically inactive. Adalimumab also causes lysis of inflammatory cells bound to TNF by interacting with the TNF-cell receptor complex . InflixiInfliximab and adalimumab are associated with the formation of neutralizing antibodies after prolonged usage; etanercept is not associated with the formation of neutralizing antibodies with prolonged use .Use of Biologic Agents in Extra-pulmonary SarcoidosisSeveral recent observational studies have described the use and efficacy of anti-TNF agents in extra-pulmonary sarcoidosis in the organ systems Table .We summarize the organ-systems affected in the patients with extra-pulmonary sarcoidosis (including case series/reports) alongside with the anti-TNF agent used for treatment in Figure Our selected studies describe the use of infliximab in neural, cutaneous, gastrointestinal, articular, renal, and bone sarcoidosis. Adalimumab was used predominantly in cardiac, cutaneous, articular, muscle, and ocular sarcoidosis. Etanercept was used only in cutaneous and articular sarcoidosis.Methods by which studies assessed for the efficacy of individual anti-TNF agents include the extent of regression of lesions on imaging, steroid-sparing property, or improvements on clinical evaluation. The studies, however, fall short of making direct head to head comparisons in efficacy between the three agents.InfliximabThe reported cases, case series, and observational studies on extra-pulmonary sarcoidosis wherein infliximab were used suggest that infliximab is effective. Gelfand et al. performed a retrospective chart review on 66 patients demonstrating that patients with neurosarcoidosis refractory to steroids on infliximab therapy had significant improvement in clinical and imaging evaluations . Two casAdalimumabThere is literature describing the use of adalimumab in extra-pulmonary sarcoidosis refractory to steroids. These include studies by Rosenthal et al., a retrospective study involving 28 patients with cardiac sarcoidosis wherein adalimumab was successfully used in combination with methotrexate to achieve notable suppression in myocardial inflammation . The stuEtanerceptThere is insufficient evidence supporting the use of etanercept as an effective steroid-sparing agent in sarcoidosis. Marques et al. describe a case of peripheral neuropathy successfully treated with etanercept . The patIn summary, medium level literature evidence pooled from observational studies, and case series suggest that infliximab, adalimumab, and etanercept are widely used either as monotherapy or in combination with methotrexate in extra-pulmonary sarcoidosis refractory to steroids with infliximab having the broadest range of organ system use. Although there are no head-to-head comparisons of their efficacy, the studies show that infliximab and adalimumab have been used extensively with positive outcomes in various multi-organ systems except in articular sarcoidosis. Infliximab has even been noted to give better results in extra-pulmonary locations compared to patients with predominantly pulmonary sarcoidosis ,36. EtanUse of Biologic Agents in Adult Non-infective UveitisNon-infective uveitis (NIU) is a sight-threatening condition frequently associated with autoimmune disorders. Although the inciting antigen is often not known, TNF plays a key role in the mediation of the inflammatory response. Causative autoimmune disorders include Behcet's disease, ankylosing spondyloarthropathy, sarcoidosis, and juvenile idiopathic arthritis.There is high-quality evidence supporting the use of adalimumab in treating adult NIU such as Leal et al. , a systeWhat Is the Comparative Efficacy of Infliximab and Adalimumab in Refractory Adult Non-infectious Uveitis?A study to compare infliximab and adalimumab in terms of efficacy and safety profile in uveitis was published in 2016 and conducted by the French uveitis network . It was In summary, even though only adalimumab is approved for use in treating adult NIU, infliximab still has extensive off-label usage, and a study has shown that both are equally efficacious . A randoRole of Biologic Agents in Pediatric NIUPediatric uveitis is a potential cause of blindness in children. It is commonly caused by juvenile idiopathic arthritis (JIA) and typically occurs anteriorly (chronic anterior uveitis- CAU). Steroids are the standard first-line treatment for this condition . The corThere are no recently published randomized clinical trials to guide the timing of biologic agents, but there is data from recent observational studies and case reports to support its use ,30,33-35A study published in 2019 performed on a cohort of children with CAU concludes by supporting the use of biologic agents in severe uveitis or where patients do not tolerate methotrexate . In thisThere were two observational studies comparing infliximab and adalimumab in terms of efficacy in pediatric CAU ,33. BothTo summarize, several recent observational studies and a systematic review support the use of biologic agents in severe pediatric uveitis to preserve vision or when methotrexate is not used due to side effects. Adalimumab is more effective than infliximab at achieving uveitis remission in the long term. Etanercept is not an effective agent in chronic pediatric uveitis.The Safety Profile of Biologic AgentsBiologic agents are well tolerated as monotherapy or add-on therapy in sarcoidosis or uveitis. The most common adverse events seem to be localized administration site reactions, infections , and hypersensitivity reactions ,33,37,41There is, however, a black box warning for reactivation of latent tuberculosis and invasive mycotic infections . These aLimitationsThere were no randomized clinical trials within the period of study that compared the efficacy of anti-TNF agents to placebo or each other in refractory extra-pulmonary sarcoidosis.Extra-pulmonary sarcoidosis and chronic uveitis are inflammatory conditions that both have tumor necrosis factor as a common denominator in their pathogenesis. Our review was focused primarily on determining the extent of the use of biologic agents in these conditions. The secondary aim of the review was to compare the efficacy and safety profile of the common biologic agents used for their management. Our findings in both conditions show that infliximab and adalimumab are used as alternatives to steroids. In sarcoidosis, compared to adalimumab, infliximab has been used successfully in more organ-systems. Infliximab has been used mostly for neural, gastrointestinal, renal, and bone sarcoidosis, while adalimumab has principally been used for treatment in cardiac, muscle, and ocular sarcoidosis. Both agents have been used for cutaneous and albeit unsuccessfully, in articular sarcoidosis. In terms of efficacy of use, both agents are similar and are superior to etanercept, which has limited use. In chronic pediatric uveitis, adalimumab is superior at maintaining long term disease remission. In terms of safety profile, biologic agents are mostly well tolerated but require surveillance due to the potential for life-threatening systemic infections and reactivation of latent tuberculosis that may occur. Due to the rarity of extra-pulmonary sarcoidosis in the population, there were no randomized clinical trials within the period of study to make direct comparisons between agents. More randomized clinical trials are required to inform better evidence-based use of these agents by clinicians in the future.


Upon binding, Q22 unfolds and elongates on both grapheme and MoS2 surfaces, regardless of its initial conformation, with graphene showing slightly stronger effect. In contrast, initially collapsed Q46 remains mostly collapsed within our simulation time on both nanosheets even though they do provide some “stretching” to Q46 as well. Further analyses indicate that the hydrophobic nature of graphene/MoS2 promotes the stretching of polyQ on nanosheets. However, there is strong competition with the intra-polyQ interactions  leading to the disparate folding/binding behaviors of Q22 and Q46. Our results present distinct Q-length specific behavior of the polyQ domain upon binding to two types of 2D-nanomaterials which holds clinical relevance for Huntington's disease.There is a strong negative correlation between the polyglutamine (polyQ) domain length (Q-length) in the intrinsically disordered Huntingtin protein (Htt) exon-1 and the age of onset of Huntington's disease (HD). PolyQ of Q-length longer than 40 has the propensity of forming very compact aggregate structures, leading to HD at full penetrance. Recent advances in nanobiotechnology provided a new platform for the development of novel diagnosis and therapeutics. Here, we explore the possibility of utilizing 2D-nanomaterials to inhibit the formation of supercompact polyQ structures through the so-called “folding-upon-binding” where the protein structure is dependent on the binding substrate. Using molecular dynamics simulations, we characterize two polyQ peptides with Q-length of 22  and 46  binding to both graphene and molybdenum disulfide (MoS Neurodegenerative Huntington's disease (HD) is caused by expansion of the trinucleotide CAG repeats in exon-1 of the HD gene, the mutation that encodes an extended polyglutamine (polyQ) tract within the N-terminal exon-1 of the Huntingtin protein (Htt)  and C38 , are absent and the Q-length is 20 or greater, polyQ collapses into a disordered but compact globule , offering a biomimetic environment for protein binding when interfaced with water  nanomaterials with unique compositional, structural and physicochemical features, such as graphene and MoS2 nanosheet. We investigate two poly glutamine-lengths, 22  and 46  to determine if the binding mechanism of the polyQ on these nanosheets is Q-length dependent. We find that Q22 exhibits a similar binding mode on both graphene and MoS2 surfaces (with graphene showing a slightly stronger effect) regardless of its initial configuration - the final conformation of Q22 is fully extended. On the other hand, the initially collapsed Q46 remains mostly collapsed when adsorbing onto graphene or MoS2 nanosheet, indicating that neither graphene nor MoS2 is sufficient to fully stretch the supercompact Q46 structure (again with graphene showing slightly stronger effect). Further energetic and hydrogen bonding analyses indicate that the difference in Q22 vs. Q46 binding conformations is determined by the competition between polyQ internal interactions (mainly intra-hydrogen-bonds) and polyQ-nanosheet interactions. Insights derived from our simulations of polyQ interaction with these novel 2D-nanomaterials may lead to future potential applications in diagnoses and therapeutics for Huntington's disease (HD).Herein, we use all-atom molecular dynamics (MD) simulations to study Htt-polyQ adsorption onto graphene and MoS2 nanosheets. 2 surface are shown in 2 nanosheet by 0.75 nm to prevent large initial interactions. The OPLS-AA force field  and 46 (Q46) adsorbing onto graphene and MoSAll MD simulations were performed with GROMACS  and root-mean-square deviation (RMSD) of Q22 and Q46 as a function of time , again indicating graphene has slightly stronger denaturing capability toward Q46 than MoS2. The Rg of the initially fully extended Q46 is 1.94 nm on graphene and 2.67 nm on MoS2. The smaller Rg of the initially fully extended Q46 on graphene is a result of the extended peptide “winding back” toward itself, which should not be over-read in terms of significance, but rather as an indication of non-convergence in our simulations for the fully extended case. The collapsed case, on the other hand, should be more indicative. Collectively, these results indicate that the 2D-nanosheets can induce unfolding of the Q22 structure, but not Q46, with graphene showing slightly stronger capacity, which is consistent with previous studies where graphene displays stronger interaction with biomolecules than MoS2 , while the rest of the systems fluctuates mostly randomly. The exception is when the initially fully extended Q22 and Q46 systems bind to graphene, their RMSDs fluctuate more widely when compared to the control systems, indicating large configurational diversity.2 surfaces. Both the initially fully stretched and collapsed Q22 adsorbed onto the hydrophobic graphene and MoS2 nanosheets in a similar stretched manner, while the initially collapsed Q46 did not stretch out on either of the nanosheets like the collapsed Q22 did. For the graphene system, the collapsed Q22 starts to unfold and spread out on the surface of the graphene after 236 ns  and the COM distance of the initially fully extended Q22 show that Q22 quickly binds the nanosheets (<20 ns). The ACN and COM distance of the initially collapsed Q22 reveal more gradual, detailed binding and unfolding behavior. The initially collapsed Q22 systems exhibit two distinct binding stages: (i) In the early binding stage , a portion of the Q22 atoms are in direct contact with the graphene and MoS2 nanosheets. The binding is not strong enough to restrain the Q22 motion, so the Q22 still diffuses along the graphene/MoS2 surface, while the number of contacting atoms slowly increases. (ii) This is the stable binding stage  where the ACN reaches the maximum, which is very close to the stable binding stage of the fully extended Q22. For the Q46 system, the final ACN of the initially collapsed Q46 are far less than the initially fully extended Q46, and the COM distances with the graphene (0.67 nm) and MoS2 (0.92 nm) surface are far away from the initially fully extended Q46 (0.42 nm on graphene and 0.33 nm on MoS2), indicating different nanosheet binding structures of Q46 based on different initial conformations or non-convergence in simulations .In order to further characterize the structure of polyQ on the surface of nanosheets, we analyzed the contacts between the polyQ peptides and the nanosheets. This involved: (1) calculating the number of heavy atoms in contact with graphene and MoSnosheets , (2) com interaction energies between polyQ and graphene/MoS2 nanosheets . Although glutamine is normally a hydrophilic residue, isolated polyQ in water shows strong hydrophobic protein properties, including the propensity of forming a supercompact structure and self-aggregation. Therefore, polyQ adsorption onto the graphene/MoSanosheet . On the 2 simulations. The polyQ sidechain-nanosheet interaction is sufficiently strong in the Q22 systems to not only prevent the collapse of Q22 but also to unfold collapsed Q22, reducing the number of intra-chain hydrogen bonds. However, the polyQ sidechain-nanosheet interaction is not strong enough to unfold the collapsed Q46 though it can prevent the collapse of initially extended Q46 on graphene and MoS2. Therefore, from our work, we find that the two-dimensional nanomaterial graphene and MoS2 have the potential to inhibit the collapse of polyQ, but not for Q-lengths up to Q46.PolyQ peptides are known to form hydrogen bonds using both the glutamine backbone and sidechain which is the main cause of the formation of polyQ supercompact structures . We believe our current work reveals important polyQ length-dependent folding/binding behavior upon binding to novel 2D-nanomaterials, which might have implications for future potential clinical applications to Huntington's disease.To summarize, we have investigated polyQ peptides of two lengths (22 and 46) binding to both graphene and MoSAll datasets generated for this study are included in the article/MF: job design, MD simulation, data collection, draft paper, and approve final paper for publication. DB and WZ: make important changes to the paper and approve final paper for publication. ZW: data collection and approve final paper for publication.DB was employed by the company IBM Thomas J. Watson Research Center. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling Editor declared a shared affiliation, though no other collaboration, with one of the authors DB.


Die durch das Coronavirus verursachte Pandemie stellt ein beachtliches gesundheitliches Risiko dar. Zur Bewältigung dieser Krise müssen in erheblichem Umfang wirtschaftliche Ressourcen für das Gesundheitssystem eingesetzt werden. Angesichts der gesundheitlichen Risiken sind die hierfür aufzubringenden Kosten gegenwärtig nicht relevant. Wenn der Lockdown endet, ist jedoch mit erheblichen Anpassungsproblemen und Stockungen auf dem Weg zu einer wieder friktionsfrei arbeits-, risiko- und wissensteiligen Volkswirtschaft zu rechnen.


The past decades have seen growing research interest in developing efficient fabrication techniques for preparing bioinspired graphene surfaces with superwettability. Among the various fabrication methods, laser fabrication stands out as a prominent one to achieve this end and has demonstrated unique merits in the development of graphene surfaces with superwettability. In this paper, we reviewed the recent advances in this field. The unique advantages of laser fabricated graphene surfaces have been summarized. Typical graphene surfaces with superwettability achieved by laser fabrication, including superhydrophobic graphene surfaces, oil/ water separation, fog collection, antibacterial surfaces, surface enhanced Raman scattering (SERS), and desalination, have been introduced. In addition, current challenges and future perspectives in this field have been discussed. With the rapid progress of novel laser physical/ chemical fabrication schemes, graphene surfaces with superwettability prepared by laser fabrication may undergo sustained development and thus contribute greatly to the scientific research and our daily life. Inspired by the intriguing superwetting phenomena existed in nature, such as lotus leaf, rice leaf, butterfly wing, and water strider legs, considerable efforts have been devoted to the development of bioinspired surfaces with superwettability including superhydrophilic (water contact angle (WCA) close to 0°), superhydrophobic (WCA > 150°), superoleophilic (oil contact angle (OCA) close to 0°), and superoleophobic (OCA > 150°) surfaces , may have particular merits in superwetting surfaces , and desalination. In addition, the challenges and outlook of this field have been discussed briefly.Up to now, various laser sources have been adopted for the fabrication of graphene surfaces. Generally, they can be classified into two categories: CW lasers and pulsed lasers. CW lasers can provide continuous output of light, which interacts with graphene or its related materials as a consequence of photothermal effect. While pulsed lasers  can interact with graphene-related materials in an ultrafast process, which can reduce the heat-affected zone and thus result in a high resolution  have been extensively studied due to their promising applications in water droplet transportation (Wu et al., In addition to direct processing of graphene surfaces, laser structured templates-supported graphene films serve as an alternative approach to creating superhydrophobic surfaces. Jiang et al.  employed2 plasma etching and laser etching, thus facilitating unidirectional transport of oil or water. Besides, the surfaces showed reversible adhesion. More importantly, the graphene surfaces showed prominent self-healing capability after flame treatment, holding great promise for high-efficiency and reusable oil/ water separation surfaces.Currently, oil spillage, industrial wastewater discharge, and emission of edible oil have become a non-negligible problem that seriously threatens the ecological environment and people's health. Therefore, considerable research efforts have been devoted to the development of efficient oil/ water separation surfaces. Liu et al.  employedSpider nets and desert beetles have demonstrated their unique superiority in fog harvesting, which is significant for their survival in harsh environment. Inspired by this property, Song et al.  successfAntibacterial surfaces are quite important for eliminating antibiotic-resistant bacteria since they can be coated onto various desired substrates. To achieve this goal, Jiang et al.  employedIn addition to the above-mentioned functions, laser fabricated graphene surfaces with superwettability also demonstrate promising applications in other fields, such as SERS and desalination. Yan et al.  fabricatIn conclusion, featuring designable patterning, hierarchical micro/nanostructuring, and flexible tuning capability of chemical composition, laser fabrication holds great promise for the fabrication of bioinspired graphene surfaces with superwettability. Typical laser fabricated superwetting graphene surfaces and their applications are summarized in this paper, including superhydrophobic graphene surfaces, oil/ water separation, fog collection, antibacterial surfaces, SERS, and desalination. However, at present, a major challenge lies in that the micro/nanostructures on graphene surfaces may be scratched or even damaged after long term use. In this regard, developing wear-resistant and self-healing graphene surfaces might be a possible solution. In addition, the cost of laser fabrication is relatively high, thus restricting its further applications in industrial manufacturing. Nevertheless, with the rapid development of various laser sources, laser fabrication of superwetting graphene surfaces will find much broader applications in our daily life.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Obesity has become a worldwide epidemic, and obesity-related problems are becoming more severe in public health. Increasing brown adipose tissue (BAT) mass or/and activity in mice and humans has been demonstrated to help lose weight and improve whole-body metabolism. Studies on the conversion of white adipose tissue (WAT) to BAT under certain conditions have provided new possibilities for treating obesity and the related disorders. It has been established that long non-coding RNAs (lncRNAs) play an important role in the regulation of mouse adipocyte differentiation and thermogenic programs; however, the function and potential mechanism of lncRNA in the process of human white adipocyte browning remains unclear. In the present study, we identified a lncRNA called Forkhead Box C2 antisense RNA 1 (FOXC2-AS1), which was first identified in osteosarcoma, and it was highly expressed in human adipocytes but decreased during the white adipocyte differentiation program. FOXC2-AS1 expression was also induced by the thermogenic agent forskolin. Lentivirus-mediated overexpression of FOXC2-AS1 in human white adipocytes did not affect lipid drop accumulation, but significantly promoted the browning phenotype, as revealed by the increased respiratory capacity and the enhanced protein expression levels of brown adipocyte-specific markers. In contrast, inhibiting FOXC2-AS1 with small interfering RNA led to attenuated thermogenic capacity in human white adipocytes. RNA-sequencing analysis and western blot were used to identify a possible regulatory role of the autophagy signaling pathway in FOXC2-AS1 to mediate white-to-brown adipocyte conversion. The autophagy inhibitor 3-methyladenine restored the reduced UCP1 protein level and thermogenic capacity caused by inhibiting FOXC2-AS1. Overall, the present study characterized the potential role of FOXC2-AS1 and further identified a lncRNA-mediated mechanism for inducing browning of human white adipocytes and maintaining thermogenesis, further providing a potential strategy for treating obesity and related disorder. Obesity and its related complications such as type 2 diabetes mellitus, coronary heart disease and obstructive sleep apnea, have been considered significant health problems . AlthougThe two main types of adipose tissue are white adipose tissue (WAT) and brown adipose tissue (BAT) . WAT priLong non-coding RNAs (lncRNAs) are RNA molecules whose transcripts are longer than 200 nt in length and they do not encode a protein . MountinIn this study, we determined the role and possible mechanism of FOXC2-AS1 during white adipocyte differentiation and browning. The results showed that overexpressing FOXC2-AS1 did not affect adipogenesis of human preadipocytes but promoted the browning phenotype, whereas knockdown of FOXC2-AS1 inhibited the process.FOXC2-AS1 may participate in the autophagy signaling pathway to mediate white-to-brown adipocyte conversion. These observations further deepen our understanding of the role and mechanism of lncRNAs during the browning process of white adipocytes and provide a potential molecular target to the treat of obesity and related metabolic disorders.Subcutaneous WAT samples were obtained from the patients undergoing abdominal liposuction at Nanjing Maternal and Child Health Hospital. Signed informed consent for the tissue was obtained. These patients did not have any malignancies, endocrine diseases or severe systemic illness. The details of the participants such as gender, age, body mass index, and health status are listed in Primary human preadipocytes were isolated from adult subcutaneous fat as described previously . The segHuman preadipocytes from subcutaneous adipose tissue before passage 5 were thawed and maintained in growth medium  supplemented with 5% fetal bovine serum (ScienCell Research Laboratories), 1% growth supplement (ScienCell Research Laboratories), and 1% penicillin/streptomycin solution (ScienCell Research Laboratories). After reaching confluence and waiting another 1–2 days, the preadipocytes were exposed to serum-free induction medium containing 0.5 mM 3-isobutyl-L-methylxanthine (Sigma-Aldrich), 1 μM dexamethasone (Sigma-Aldrich), 100 nM insulin (Sigma-Aldrich), 1 μM rosiglitazone (Sigma-Aldrich), and 1% penicillin/streptomycin solution for 4 days. Subsequently, the medium was replaced with the maintenance medium including DMEM/F-12 (Gibco), 100 nM insulin (Sigma-Aldrich), and 1% penicillin/streptomycin solution (Gibco) until the adipocytes were fully mature with and accumulated lipid droplet. The differentiated adipocytes were collected on days 4 and 8 for further detection.To access FOXC2-AS1 expression level stimulated by forskolin (Fsk), differentiated adipocytes on days 4 and 8 were incubated in serum-free DMEM/F-12 medium for 6 h followed by stimulation with Fsk at a final concentration of 10 µM in DMEM/F-12 medium for additional 4–6 h to induce the thermogenic process in the adipocytes.6 TU/mL) as well as Polybrene (5 μg/ml). The cells were recovered in PAM growth medium 16–18 h after transfection, and grown to confluence. FOXC2-AS1 expression was knocked down by the small interfering RNA (siRNA) method. Interfering sequences were specifically synthesized and purchased from Invitrogen Co.  as follows: Si-1 CCGTTCAAGGTTTCCTTGCACCCTT;Si-2:CGGCTGCGTATTCGATTCTCAGCAA; Si-3: GGGCGTGCCACTTATTTCCAATAAA. A negative control sequence (moderate GC content) was also purchased from Invitrogen. When the preadipocytes reached 50%–60% confluence, they were transfected with siRNAs (Si-FOXC2-AS1) and the negative control (Si-NC) at 100 nM using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions. Upon reaching confluence and after 1–2 days, FOXC2-AS1 overexpressed- or knockdown-cells were induced to differentiate until they contained large lipid droplets and were harvested respectively on days 4 and 8 for the functional evaluation. The primary adipocyte sample size was three biologically independent samples from participants.Overexpression of the full-length FOXC2-AS1 sequence was mediated by a lentivirus purchased from GenePharma Co. . The procedures were as follows. Once reaching 30%–40% confluence, the medium was exchanged with growth medium supplemented with FOXC2-AS1 overexpressing (FOXC2-AS1) or negative control (NC) lentivirus, respectively . The RNeasy Mini Kit  was used to extract and purify total RNA according to the manufacturer’s instructions. RNA  from each sample was reverse transcribed into cDNA using the Scientific™ RevertAid Fist Strand cDNA Synthesis kit (Thermo Fisher Scientific). Quantitative RT-PCR was carried out using the QuantStudio™ 7 Flex Real-Time PCR System  and the SYBR Green method  according to the manufacturer’s instructions. The results were presented by comparing the relative expression of genes to the internal reference PPIA by the 2via pipetting to a new 96-well plate (200 µl/well), and the optical density was detected spectrophotometrically at 510 nM with the Synergy H4 Hybird microplate reader .Lipid accumulation was detected by Oil Red O staining on days 4 and 8 of differentiation program. The adipocytes were washed twice with PBS and then fixed for 30 min with 4% paraformaldehyde . The cells were rinsed twice with PBS, stained with 0.2% (m/v) Oil Red O (Sigma-Aldrich) dissolved in isopropanol  for 30 min at 37°C. The lipid droplets were evaluated by the Imager A2 fluorescence microscope  and representative figures were shown. To determine lipid content in the differentiated adipocytes on days 4 and 8, isopropanol (0.5ml/well of a 12-well plate) was added to the plates as reported previously , 34. AftTotal protein was isolated from cells using RIPA lysis buffer  containing 2% proteinase inhibitor cocktail . The protein samples were prepared according to the protein concentration determined by a BCA assay kit (Thermo Fisher Scientific). The candidate proteins were separated by 8%, 10%, or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis based on molecular weight, transferred to a PVDF membrane , and immunoblotted with specific primary antibodies as follows: anti-PRDM16 , anti-PGC1α , anti-UCP1 , anti-CEBPα , anti-PPARγ , anti-ATGL , anti-FOXC2 , anti-LC3 , and β-actin . The second antibodies were: goat anti-mouse IgG HRP  and goat anti-rabbit IgG HRP . The gray values were scanned and calculated with ImageJ software .5 per well) were seeded in a specific 24-well plate  coated with 0.1% galectin (Sigma-Aldrich). The cells were induced into mature adipocytes as described above and the oxygen consumption rate (OCR) was determined on days 4 and 8 using the XF Cell Mito Stress Test Kit (Agilent Technologies) on an XF Extracellular Flux analyzer (Agilent Technologies) according to the manufacturer’s instructions. After detecting the basal OCR, the adipocytes were exposed to the following mitochondrial complex inhibitors: oligomycin , FCCP , and a rotenone/antimycin mixture . Basal respiration, maximal respiration, ATP production, protein leak, and spare respiration were calculated according to the manufacturer’s instructions.The preadipocytes (5 × 10https://www.ncbi.nlm.nih.gov/), UCSC (http://genome.ucsc.edu/), ENSEMBL (http://asia.ensembl.org/index.html), and Hisat2 software (v2.0.1). An expression analysis was performed by HTSeq (v0.6.1), and the differentially expressed genes were analyzed using the DESeq2 Bioconductor package with |fold change| ≥ 1.5 and a q-value < 0.05. A Gene Ontology analysis was performed using GOSeq (v1.34.1) and the KEGG pathway analysis was performed at the website (http://en.wikipedia.org/wiki/KEGG).Total RNA of the NC and FOXC2-AS1 groups from differentiated adipocytes (day 4) was extracted with TRIzol reagent. The Agilent 2100 Bioanalyzer (Agilent Technologies) was used to quantify and qualify total RNA in each sample. One μg of total RNA with a RIN > 7 was used in subsequent steps. Then, the RNAClean XP Kit  and the RNase-Free DNase Set  were used to purify high quality total RNA. Next, the purified samples were subjected to RNA-seq analysis with the Agilent 2100 Bioanalyzer according to the Illumina User Guide. High quality clean data were filtered using Cutadapt (V1.9.1). The following data were processed using websites, such as NCBI  were treated by acute stimulation with the 3-MA (1 and 5 mM)(Sigma-Aldrich) or vehicle (DMSO) (Sigma-Aldrich) for additional 4 h. The treated cells were used for subsequent experiments at the indicated times.t-test, and a P-value < 0.05 was considered significant.The data analysis was performed using GraphPad Prism 7  and expressed as mean ± standard deviation. Comparisons were made using the unpaired two-tailed . FOXC2-AS1 is 319 bp in length and predicted to lack the ability to encode proteins according to the Coding Potential Assessment Tool (http://lilab.research.bcm.edu/cpat/index.php) . To study the effect of FOXC2-AS1 on adipogenesis, we analyzed its expression by quantitative RT-PCR during subcutaneous white adipocyte differentiation program. Compared to the undifferentiated preadipocytes (Day 0), the relative FOXC2-AS1 expression level decreased significantly with the adipogenic time . Fsk is a thermogenic agent that activates cAMP from ATP, thereby promoting fatty acid release from adipose tissue , is located on chromosome 16 (16q24.1) and transcribed from the negative strand of forkhead box protein C2 (FOXC2) . FOXC2-A. FOXC2-Ae tissue . To vali. FOXC2-Avia lentivirus infection or knocked down via siRNA transfection respectively in human subcutaneous white adipocytes. Quantitative RT-PCR detection confirmed the FOXC2-AS1 expression level increased significantly in the overexpression group (FOXC2-AS1) compared to the NC group . Additionally, the interference efficiency of the Si-2 sequence on FOXC2-AS1 was the most significant among the three siRNAs’  and adipose triacylglyceride lipase (ATGL) . However, C/EBPα and ATGL mRNA expression levels tended to be comparable between the two groups at the terminal stage of the differentiation program on day 8 . Knockdown of FOXC2-AS1 consistently did not alter the mRNA levels of any of the adipogenic-related genes . Furthermore, the PPARγ, C/EBPα, and ATGL protein levels did not change significantly after FOXC2-AS1 overexpression  or knockdown . Additionally, phosphorylation levels of ATGL and AMPK were detected after FOXC2-AS1 overexpression or knockdown to verify their lipolytic activity and increased p-ATGL/ATGL and p-AMPK/AMPK ratios were only observed in adipocytes after FOXC2-AS1 overexpression on day 4 . Overall, our results demonstrate that FOXC2-AS1 is not required for adipogenic processing but promotes lipolytic activity of human subcutaneous white adipocytes.To further reveal the possible role of FOXC2-AS1 in adipocyte differentiation, FOXC2-AS1 was overexpressed NC group . AdditioNC group . AdditioNC group . AdditioNC group . AdditioNC group . AdditioNC group . AdditioNC group . AdditioNC group . AdditioNC group . Additio. Cellular metabolic parameters, including basal respiration, maximal respiratory respiration, ATP production, protein leak, and spare respiration increased after FOXC2-AS1 overexpression . However, at the terminal stage of the differentiation program on day 8, OCR rates and the above parameters became equal between the NC and FOXC2-AS1 overexpressing groups . When FOXC2-AS1 was knocked down, a downregulated trend in cellular metabolic parameters, including basal respiration, protein leak, ATP production, and spare respiration was observed on days 4 and 8 . Accordingly, the protein levels of brown adipocyte-specific markers, such as UCP1, PGC1α, and positive regulatory domain containing 16 (PRDM16), were upregulated , while these mRNA expression levels did not change in human subcutaneous white adipocytes after FOXC2-AS1 overexpression . FOXC2-AS1 knockdown presented a dramatic decrease in the PRDM16 mRNA expression level  and resulted in the decreased protein levels in UCP1, PGC1α, and PRDM16 protein expression levels . These observations indicate that FOXC2-AS1 may promote the white-to-brown adipocyte conversion by affecting thermogenic capacity and mitochondrial content.To determine whether FOXC2-AS1 is involved in regulating of human white adipocyte browning, we detected the browning phenotype after FOXC2-AS1 overexpression or knockdown. FOXC2-AS1-overexpressing cells had a significantly higher thermogenic capacity compared to the NC group, as measured by the OCR on day 4 of the differentiation program . Cellula. Cellula. Cellula. Cellula. Cellula. Cellula. Cellula, showing a consistent FOXC2-AS1expression trend . Next, we tried to determine whether FOXC2-AS1 affected FOXC2 expression. However, overexpression or knockdown of FOXC2-AS1 did not alter FOXC2 mRNA  or protein levels . We employed RNA-sequencing to search for a possible regulatory mechanism and reveal the mechanistic insight underlying the white-to-brown adipocyte conversion by FOXC2-AS1. Considering the browning phenotype on day 4 after FOXC2-AS1 overexpression, the differentially expressed genes (DEGs) between FOXC2-AS1-overexpressed and NC white adipocytes were profiled. A total of 147 DEGs were detected  as shown in . By querying these DEGs in the KEGG pathway analysis, these genes were mainly enriched in ubiquitin-mediated proteolysis, ribosome biogenesis, glutathione metabolism, the autophagy signaling pathway, and the AGE-RAGE signaling pathway (and). Notably, mounting evidence has demonstrated that inhibiting the autophagy signaling pathway promotes the conversion of white to brown adipocytes  after FOXC2-AS1 overexpression or knockdown by western blot. As results, the LC3 II/LC3 I ratio decreased significantly after FOXC2-AS1 overexpression  but increased after FOXC2-AS1 knockdown  on days 4 and 8 of adipogenic differentiation. To explore if FOXC2-AS1-regulated white adipocyte browning was mediated by affecting the autophagy signaling pathway, adipocytes were acutely stimulated with the autophagy inhibitor 3-MA  when FOXC2-AS1 expression was inhibited. As a result, 3-MA (5 mM) significantly inhibited autophagy . Acute stimulation with 3-MA did not affect lipid accumulation . Additionally, 3-MA restored the reduced UCP1 protein level and thermogenic capacity caused by inhibiting FOXC2-AS1 . The inhibition of autophagy in adipocytes attenuated the inhibitory effects of FOXC2-AS1 on adipocyte browning, indicating that FOXC2-AS1 may mediate the adipocyte thermogenic program by regulating the adipocyte autophagy level.Several studies have reported that antisense lncRNAs can affect regulatory function by affecting their neighboring protein-encoding gene expression . Thus, w, showing, showing, showing, showing, showing, showing, showingipocytes –39. To v, showing, showing, showing, showing, showingAccumulating evidence has demonstrated that lncRNAs are involved in regulation of adipocyte differentiation and functions . The funIn combination with our results, FOXC2-AS1 expression continued to decline during the differentiation program, suggesting a potential role of FOXC2-AS1 in regulating adipocyte differentiation program as shown in . This inconsistent finding may be explained that the increased levels of browning proteins were insufficient for the changed phenotype. Stimulation with the thermogenic agent Fsk may be a more useful approach to explore the role of FOXC2-AS1 in the white-to-brown conversion in future research. Accordingly, knockdown of FOXC2-AS1 also did not affect lipid accumulation or adipogenic marker genes expression as presented in . However, this effect was not observed after inhibiting FOXC2-AS1, further demonstrating that this lncRNA is not required to regulate lipolytic activity in adipocytes.The change in FOXC2-AS1 during differentiation and the thermogenic program persuaded us to confirm its possible role in white adipocytes. From our results, gain-of-FOXC2-AS1 was sufficient to promote the browning program of white adipocytes in . This in. This in. This inOverall, our results demonstrate that FOXC2-AS1 is not required for adipogenic processing. Thus, our observations suggest that FOXC2-AS1 is not required for adipocyte differentiation and no additional coactivators are needed to achieve its functional effect. More importantly, some authors have reported the importance and the specific regulation of protein factors merely in the thermogenic program or the browning process. For example, KDM1 lysine (K)-specific demethylase 6B mediated Ucp1 and Cidea expression but not for adipogenesis, and facilitates browning of white adipocytes . Another), while FOXC2-AS1 knockdown increased this ratio . The reduction of UCP1 was suppressed and thermogenic capacity was inhibited when the autophagy inhibitor 3-MA was administered while inhibiting FOXC2-AS1 . This finding further elucidates that FOXC2-AS1 regulates adipocytes browning by affecting autophagy. 3-MA has also been used to explore the relationship between novel adipokine CTRP5 and adipocytes browning . Signed informed consent for the tissue was obtained.YW and SH performed experiments and interpreted results of experiments. XWC and YC prepared the figures. JW analyzed the data. XC and CJ participated in the discussion. LY and LP conceived and designed experiments, provided main funding to regents, and approved the final version of the manuscript. All authors contributed to the article and approved the submitted version.This study was supported by the National Natural Science Foundation of China , the Jiangsu Province Natural Science Foundation (Grant No. BK20191126 and BK20180146), the Jiangsu Provincial Medical Innovation Team Program (Grant No. CXTDA 2017001), the Nanjing Medical Science and Technique Development Foundation (Grant No. QRX17160), the Science and Technology Development Foundation Item of Nanjing Medical University (Grant No.2017NJMUZD074), and the “six talent peak” High-level Talents Training Project of Jiangsu Province (Grant No. C-YY-081).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Graphene and phospholipids are widely used in biosensing and drug delivery. This paper studies the mechanical and electronic properties of a composite based on two graphene flakes and dipalmitoylphosphatidylcholine (DPPC) phospholipid molecules located between them via combination of various mathematical modeling methods. Molecular dynamics simulation showed that an adhesion between bilayer graphene and DPCC increases during nanoindentation of the composite by a carbon nanotube (CNT). Herewith, the DPPC molecule located under a nanotip takes the form of graphene and is not destroyed. By the Mulliken procedure, it was shown that the phospholipid molecules act as a “buffer” of charge between two graphene sheets and CNT. The highest values of electron transfer in the graphene/DPPC system were observed at the lower indentation point, when the deflection reached its maximum value. Graphene is a two-dimensional allotropic modification of carbon with a thickness of one atom . High biSimulation of graphene-based composites’ nanoindentation by molecular dynamic methods allows to study local characteristics of the considered objects, as it makes it possible to place a nanotip strictly above the surface of interest. Huang et al. MD research showed that the strength and hardness of bio-inspired nanocomposites decreased with the increasing length of graphene layers, while increasing the length of the graphene layer avoids the generation of dislocations at the edge of the graphene sheet . NanoindThe object of this study is a composite based on two graphene flakes and dipalmitoylphosphatidylcholine (DPPC) phospholipid molecules located between them. Previously, the authors found the configurations of such composites that provide optimal current–voltage characteristics and electron transfer values . The aimx × Ly = 25.5 Å × 35.51 Å. This composite structure acted as the supercell with translation vectors Lx, Ly   with a closed edge was placed at a distance of 4.1 Å from the surface of the upper graphene sheet a. The leThe dependence of the system’s total energy on the CNT shift is shown in To assess the strength of graphene sheets during deflection, we calculated the map of the local stresses’ distribution by atoms at each step a–h. As tAs the calculation of atoms’ Mulliken charges by the SCC DFTB 2 method is resource consuming, the central part was cut out from the graphene/DPPC composite. This fragment containing two isolated DPPC molecules directly contacted the CNT and was responsible for charge transfer in the system a.The charge distributions between the atoms at the initial moment of time, at the lower point of the forward stroke F, and at the end of the reverse stroke I are shown in The search for the ground state of the graphene/DPPC composite film, as well as the study of its atomic structure changes during deflection by a nanotip, was performed by the AMBER empirical method  implemenThe adhesion energy between the phospholipids and graphene sheets at various steps was calculated by the following formula (Equation (1)):iσ on each atom is calculated by the formula r = 1.7 Å).To estimate the strength of graphene sheets during deflection, the previously developed original method for calculating local stresses of the atomic grid was used . AccordiA: The study of changes in the electronic structure during indentation and electronic transfer between phospholipid molecules and graphene was performed by Mulliken population analysis . Accordi) method  in the d) method  in the 3The nanoindentation of the composite on the base of bilayer graphene and 16 DPPC phospholipid molecules was simulated by the molecular dynamics method. It was noted that, during indentation, the adhesion between graphene flakes and DPPC molecules increased and reached a maximum at the end of the reverse stroke. The maximum values of local stresses (in the region of 2.53 GPA) were observed on the upper graphene layer at the lower indentation point. At this moment, the DPPC molecule located under the nanotip took the form of curved graphene, while chemical bonds of the phospholipid molecules were not destroyed. The pressure of the CNT tip led to the growth of adhesion energy between graphene sheets and DPPC molecules. It is known that, getting into blood vessels, drug carriers sense abnormally high shear stresses ,35, so t


Kelvin probe force microscopy (KPFM) verifies that the WF-tuned regions, where the WF increases by the order of ~250 meV, coexist with the regions of intrinsic WF within a single graphene flake. By combining KPFM with lateral force microscopy (LFM), it is demonstrated that the WF-tuned area can be manipulated by pressing it with an atomic force microscopy (AFM) tip and the tuned WF returns to the intrinsic WF of graphene. A highly plausible mechanism for the WF tuning is suggested, in which the increased graphene-substrate distance by excess H2 gases may cause the WF increase within a single graphene flake. This novel WF tuning method via a simple CVD growth control provides a new direction to manipulate the WF of various 2-dimensional nanosheets as well as graphene.Besides its unprecedented physical and chemical characteristics, graphene is also well known for its formidable potential of being a next-generation device material. Work function (WF) of graphene is a crucial factor in the fabrication of graphene-based electronic devices because it determines the energy band alignment and whether the contact in the interface is Ohmic or Schottky. Tuning of graphene WF, therefore, is strongly demanded in many types of electronic and optoelectronic devices. Whereas study on work function tuning induced by doping or chemical functionalization has been widely conducted, attempt to tune the WF of graphene by controlling chemical vapor deposition (CVD) condition is not sufficient in spite of its simplicity. Here we report the successful WF tuning method for graphene grown on a Cu foil with a novel CVD growth recipe, in which the CH For applications such as transparent electrodes, tuning the work function (WF) of graphene is essential since the mismatch of the energy levels between the graphene electrode and materials in the active layers would degrade the performance of devices4. In particular, when graphene makes a contact with other semiconductors or metals in electronic or optoelectronic devices, the WF difference between them determines the band structure alignment in the interface and the contact characteristics, i.e. whether it is Ohmic or Schottky8. Therefore, it is significant to control the WF of graphene in graphene-based devices.Since the first exfoliation of a single graphene layer from highly oriented pyrolytic graphite (HOPG), graphene has been considered as one of the most promising materials for next generation of electronic devices, due to its outstanding optical, electrical, and mechanical properties15. A major approach to tune the graphene WF is surface engineering, particularly by depositing a very thin layer (about one layer) of alkali metal, such as Cs, Li, Sr, or Ba, that are sometimes combined with a proper amount of oxygen12. Another approach is by the doping of graphene due to a lack of dangling bonds and surface states15. Although a great deal of meaningful results of graphene WF tuning have been reported by doping or surface engineering, this approach has a limitation because basically it needs an additional process in the device fabrication, which increases the fabrication cost and time. Thus, a simple WF tuning method for large-area graphene film without additional fabrication process is highly desirable.Lots of efforts have been made for tuning the WF of graphene4/H2 gas ratio is simply controlled. This method does not add any additional process to a routine CVD graphene growth process. Raman and X-ray photoelectron spectroscopy (XPS) confirmed that the graphene grown with the CVD recipe of high H2 gas portion has considerably increased WF, while its chemical structure is almost same as that of a conventional graphene. Moreover, Kelvin probe force microscopy (KPFM) verifies that the WF-tuned regions, in which WF increases by the order of ~250 meV, coexist with the regions of intrinsic WF within a single graphene flake. It is also demonstrated that the WF-tuned area can be manipulated by pressing it with an atomic force microscopy (AFM) tip and the tuned WF returns to the intrinsic WF of graphene. This novel WF tuning method via a simple CVD growth control provides a new direction to manipulate the electronic properties of graphene and various two-dimensional analogues.Here we report the WF tuning method for graphene grown on a Cu foil with a controlled CVD growth recipe, in which the CH4 to that of H2 from 40:10 to 40:40. The growth process was conducted for 20 to 40 minutes to ensure enough coverage of graphene. The quality of graphene on Cu foils was identified by Raman spectroscopy . XPS experiments were conducted to investigate the chemical structure and the work function of the graphene samples by using an ultrahigh vacuum (UHV) XPS instrument  with a Ar gas cluster ion beam (GCIB) sputtering equipment.Two kinds of graphene samples were synthesized through low-pressure chemical vapor deposition (LPCVD) on a Cu foil, by varying a volumetric velocity ratio of CHTo obtain the spatial variation of WF for the two kinds of graphene on Cu, we also performed KPFM, which is an AFM based technique to acquire WF information via measuring contact potential difference (CPD) for every pixel. A map of CPD, the relative surface potential (SP) of an AFM tip to that of a sample, is simultaneously obtained with a topographic image of the sample surface. The AFM tip for KPFM is generally metallic or metal-coated since measurement of CPD requires at least one conductive component. In addition, manipulation on graphene surfaces and graphene-copper interfaces was done by lateral force microscopy (LFM), a contact mode AFM technique which can characterize or modify tribological properties of the sample surface. All AFM-based measurements were conducted with a commercial atomic force microscope . KPFM measurements involved electrically grounding graphene samples, through which SP of any sample may have the same zero point.4 to H2 ratio in a LPCVD method. The conventional graphene was grown with the CH4:H2 ratio of 40:10 and the ratio was changed to 40:40 for WF-tuning by the higher H2 content. Figure 4:H2 = 40:40). In the Raman spectrum, the characteristic G and 2D peaks of graphene are clearly measured at 1575 and 2673 cm−1, respectively. The most common method to determine the layer number of graphene is to calculate the intensity ratio of G and 2D peaks in Raman spectrum19. For the graphene sample grown with the new recipe, the intensity ratio of 2D/G was ~1.6, which corresponds to monolayer or bilayer graphene, thinner than trilayer19. Other commonly used method to estimate the layer number of graphene is to measure the full width at half maximum (FWHM) of 2D peak. The FWHM of the 2D peak in Fig. −1 and it corresponds to the monolayer or bilayer graphene, which is in a good agreement with the estimation by 2D/G calculations. Based on the Raman spectroscopy analysis, it was confirmed that the graphene grown by the new CVD recipe is same as a conventional CVD-grown graphene on a Cu foil, in terms of its intrinsic properties.As shown in Fig. 2 content, successfully tunes the WF of graphene. In addition, to check the chemical structure of the WF-tuned graphene by the new CVD recipe, we performed XPS depth profiling measurements by using a mild Ar GCIB sputtering technique, which is known as the most powerful technique to sputter organic sample surfaces without a severe chemical damage21. Figure To investigate the WF properties of the graphene grown on a Cu foil with the new CVD recipe, we performed XPS, a representative surface analysis technique to explore the electronic structure including WF. Figure i.e. ~100 meV lower WF, than that of Cu. On the contrary, besides the graphene area is still clearly distinguishable from a Cu surface by the difference of SP in Fig. Although XPS is a reliable method to measure WF of graphene, it has a limit because its lateral resolution of ~10 μm is not sufficient to examine the spatial variation of WF on a graphene surface. For this purpose, we performed KPFM that is a useful AFM-based technique to image the spatial variation of WF on surfaces of various materials at the nanoscale. Figure 2 gas portion, show the increased WF by the order of ~250 meV and this increment is significantly high in terms of WF tuning. However, although the graphene WF tuning by our new CVD recipe is indubitable, its mechanism is still ambiguous. Here, we examine four possible cases of graphene WF variation. The first one is that the WF tuning may originate from the formation of a bilayer structure within a single graphene flake. It is known that, at a high H2 pressure, the (second) adlayer graphene is grown beneath the (first) top layer, i.e. between the first layer and the Cu surface and variation in the number of layers can change the WF of graphene23. However, the formation of bilayer structure cannot explain our WF increase of ~250 meV, because WF tends to decrease as the number of graphene layers increases24. The second possible origin of our WF-tuning is the local curvature induced by the ripples within a single domain graphene flake, which may be formed by excessive H2 gases in the new CVD process. This hypothesis also cannot elucidate the WF increase in our WF-tuned graphene, because it was reported that the curvature increase on graphene ripples leads to the gradual decrease of WF, although its tendency is not monotonous24. The third possibility is that the doping of graphene, in particular, hydroxylation, happens in the new CVD process. In the literature, the functionalization of graphene by hydroxyl (-OH) groups significantly increases the WF of graphene13. However, the difference in hydroxylation between our conventional and WF-tuned graphene samples is not severe in the XPS results 25. According to the literature, as the distance between graphene and a Cu (111) surface increases from the equilibrium distance of ~3.3 Å, the WF of graphene also monotonously increases by ~ 380 meV up to the distance of ~4.0 Å and it is almost saturated for longer distances25. This monotonous increase and saturation of WF with increasing graphene-substrate distance can explain the feature of our WF-tuned graphene samples.From our XPS and KPFM results, the WF-tuned graphene samples, synthesized with the new CVD recipe of a higher Hlts Fig. . In addi27. Because the LFM is operated in a contact mode AFM, it can be used to modify the distances between two-dimensional materials and substrates. Figure i.e. dark regions), are indicated by a green arrow and a green dotted square. Figure i.e. the WF-tuning originates from the increase of graphene-substrate distance within a single graphene domain, is highly plausible. The detailed process for our suggested WF-tuning mechanism is depicted in Fig. 2 gases are encapsulated beneath a graphene in the CVD process and the graphene-substrate distance slightly increases on the H2 encapsulated area. Then, the H2 gases are released through graphene defects or the side gap between graphene and Cu substrate after the CVD process. Finally, the increased graphene-substrate distance which increases the WF maintains.For the experimental verification of the mechanism of our WF-tuning method, we performed LFM on the WF-tuned graphene sample in Fig. 4:H2 gas ratio in a CVD growth. While the WF-tuned graphene does not have any significant difference in a chemical structure comparing with a conventional graphene in Raman spectroscopy and XPS measurements, it shows the considerably increased WF. In addition, it was demonstrated that the WF-tuned regions, in which the WF increases by the order of ~250 meV, coexist with the regions of the intrinsic WF within a single graphene flake in Kelvin probe force microscopy (KPFM). In particular, it was possible to manipulate the WF-tuned graphene area by pressing it with an AFM tip and return the WF to its intrinsic value by the combination of KPFM and LFM. Furthermore, we suggest a highly plausible WF tuning mechanism for the new CVD process, based on the combined KPFM and LFM experiment. In the suggested WF tuning mechanism, the graphene-substrate distance increases by encapsulation of excess H2 gases beneath a graphene flake and it gives rise to the increased graphene WF. This study provides an experimental work on the modulation of graphene WF and the WF-tuning method by controlling the graphene-substrate interface could be applied to alternative two-dimensional analogues. This novel WF tuning technique suggests a new concept in the modulation of electronic properties of various two-dimensional analogues as well as graphene.In this study, we developed a novel WF tuning method for graphene grown on a Cu foil simply by changing the CHSupplementary Information.