Carbon nanostructures have played an important role in creating a new field of materials based on carbon. Chemical modification of carbon nanostructures through grafting has been a successful step to improve dispersion and compatibility in solvents, with biomolecules and polymers to form nanocomposites. In this sense carbohydrates such as chitosan are extremely valuable because their functional groups play an important role in diversifying the applications of carbon nanomaterials. This paper reports the covalent attachment of chitosan onto graphene oxide, taking advantage of this carbohydrate at the nanometric level. Grafting is an innovative route to modify properties of graphene, a two-dimensional nanometric arrangement, which is one of the most novel and promising nanostructures. Chitosan grafting was achieved by redox reaction using different temperature conditions that impact on the morphology and features of graphene oxide sheets. Transmission Electron Microscopy, Fourier Transform Infrared, Raman and Energy Dispersive spectroscopies were used to study the surface of chitosan-grafted-graphene oxide. Results show a successful modification indicated by the functional groups found in the grafted material. Dispersions of chitosan-grafted-graphene oxide samples in water and hexane revealed different behavior due to the chemical groups attached to the graphene oxide sheet. Chitosan has been extensively investigated for several decades due to its biocompatibility, biodegradability, solubility in aqueous medium, presence of multiple functional groups and non-toxicity, which can be applied in molecular separation, food packaging film, artificial skin, bone substitutes, water treatment, electrochemical sensors and biosensors among others [Chitosan, a polysaccharide composed of β-,4-2-aming others ,4,5. Cheg others ,7. Speciβ-,4-2-aming others .−1 K−1, and consideration as a zero-bandgap semiconductor [On the other hand, today, graphene attracts as much attention as carbon nanotubes did in the last decade. So, this material, a single layer of carbon atoms in a closely packed honeycomb two-dimensional lattice, has engaged considerable interest from both the experimental and theoretical scientific communities ,9,10,11.onductor . Each gronductor ; graphenonductor ,16, in wonductor  and in eonductor ,19. Alsoonductor ,21.etc. Its importance is reflected in the great number of researches focused toward this aim, for instance: Stable dispersions of graphene sheets have been achieved using as dispersing agent amphiphilic polymers, alkylamines and hydrophilic carboxyl groups, among others [One disadvantage of graphene is the characteristic poor dispersion observed also in other carbon based nanomaterials: nanotubes and fullerenes. Dispersion of carbon materials depends on different factors, such as polarity of the solvent, chemical group attached to carbonaceous structure, graphene sheets stack, nature of interactions between graphitic layers and the solvent, g others . Thus, fg others ,23,24,25g others . For insg others , Poly(vig others  and (2) g others  have resg others ,9. Severg others ,9,23, isg others , 1-pyreng others  and diffg others ,28,29,30g others ; with teg others  and withg others ,34 for ng others ; magnetig others ; covaleng others ,38, and g others . These rg others ,41,42,43Next the results obtained with the characterization of grafted graphene oxide with chitosan are presented together with the samples used as references. A summary of the nomenclature for all samples synthesized is shown in The graphene oxide sheet morphology (GEO) is observed in et al. [et al. . AFM ima−1, v(C=O) of carboxyl groups; 1620 cm−1, v(C=C); and 1065 cm−1, v(C–O). These signals provide evidence of the presence of oxygen-containing groups, caused by the chemical reaction. These moieties have also been reported by different authors [Different functional groups were observed in all graphene samples through FTIR analysis . Pure gr authors ,27.3, this last from remaining acetylated parts of chitosan. At 1535 cm−1 a new band is observed in CGEO spectra (−1 is attributed to va(C–O–C) and the band at 899 cm−1 corresponds also to C–O–C, in both cases related to the glycosidic linkage [−1) in CGEO spectra. This indicates that C–O–C bond vibrations are also modified due to grafting treatment; when C–O– bonds of chitosan interact with GEO in CGEO hybrids. Thus, not only amine groups can take part in grafting, also the OH, of the chitosan structure can play an important role.The effect of chemical modification due to grafting chitosan onto graphene oxide is observed in CGEO1, CGEO2 and CGEO3 spectra ( spectra d–f. It c linkage . These s−1 (G band), a line around 1350 cm−1 (D band) and 2D band in the region of 2700 cm−1 [2 atoms, it reveals the presence of certain defects, whereas 2D is the second order of this vibrational mode [2 sites affect positions, intensities and widths of the D and G bands [2 nanocarbons [Raman spectra of carbon allotropes, such as graphite, show distinctive signals: A strong band close to 1580 cm700 cm−1 ,48,49,50700 cm−1 ,48,49,50nal mode ,52,53. I G bands ,25. In tocarbons ,55,56. Cocarbons .et al. [Similar behavior is observed for chitosan-grafted graphene samples CGEO1 f, D and et al. . Howeveret al. ,55,56 an2SO4 used in the redox reaction. Also, chitosan analysis obtained by Salam et al. of similar samples to that used in this research is added in the table as reference for the nitrogen, carbon and oxygen content of this natural polymer [Chemical modification is also revealed by the presence of oxygen and nitrogen in GEO and grafted samples, which was verified by Energy dispersive X-ray spectroscopy (EDS).  polymer . It is iFinally, our dispersion study provides results that reflect a strong affinity between water and the different groups attached to graphene sheets A. Disper4) was acquired from Merck. Sulfuric acid (H2SO4) (98%), hydrogen peroxide (H2O2) (30%), acetone (99%) and hexane (99%) were purchased from Baker and malic acid (99%) was supplied by Fluka.Chitosan (CS) (degree of deacetylation >85%) was purchased from Sigma-Aldrich. Graphite rods, spectro-grade (ash content <2 ppm) were obtained from Electron Microscopy Science. Potassium permanganate (KMnO2SO4 (46 mL) was added into the reaction flask maintaining 0 °C (±2°) with an ice bath, graphite (2 g) and KMnO4 (6 g) were added slowly, then the temperature was increased to 35 °C (±2°) and the mixture was stirred with a magnetic stirring bar for 2 h. Later, excess water was incorporated and H2O2 (10 mL) was added until there was no gas production. A glass filter was used to wash with water and a brown powder was obtained (GO), which was dried at 65 °C for 12 h. Then, a solution containing 100 mg of dried GO in 10 mL of H2O was produced, this solution was sonicated for 3 h at room temperature with the aid of an ultrasonic bath (Branson 1510R-MTH with frequency of 50–60 Hz), thus producing graphene oxide sheet solution (GEO), which was conserved for its subsequent characterization.Graphite oxide (GO) was obtained from graphite by a modified Hummers method , only qu4, 50 mg of malic acid and 0.75 mL of H2SO4, were added. The mixture reacts for 3 h at fixed temperature. In order to observe the temperature effects on chitosan graftings, three ranges were applied: 55 to 60 °C, 75 to 80 °C and 95 to 100 °C . Finally the reaction products were washed with acetone and dried for 24 h at room temperature. A scheme of this reaction taking as basis previous grafting works [The solution obtained in the last procedure was constantly stirred in a reaction flask whereby 125 mg of chitosan, 37.5 mg of KMnOng works ,59 is sh−1. Fourier transform infrared spectroscopy (FTIR) was recorded in a FT-IR Avatar Model 360 by Nicolet spectrometer, at a resolution of 4 cm−1 and by the attenuated total reflectance technique (ATR). Finally energy dispersive X-ray spectroscopy (EDS) was done in a JEOL JSM-6060LV SEM using an Oxford Inca X-sight system. All samples for EDS were prepared previously as solid disks, which were fastened to a double-sided adhesive tape attached to aluminum stubs.Transmission Electron Microscopy (TEM) micrographs were obtained using a JEOL TEM-1010 microscope operating at 80 kV. The AFM images were recorded using an Atomic Force Microscope, AA5000 Multifunction SPM Systems. In the case of chitosan samples, the scans were performed in contact mode, operating at a frequency of 5 kHz. For graphene oxide and grafted samples AFM Digital Instrument CP-II was used, the scans were performed in non-contact mode, operating at a frequency of 200 kHz and the scanning speed was 0.5 Hz. Micro Raman  measurements were carried out at 368 nm wavelength incident laser light with a spectral resolution of 1 cmet al. [Dispersion measurements were performed maintaining the carbon material-solvent relation as 2:10, the same as proposed by Chattopadhyay et al. . GraphitTo complement the dispersion study UV-Vis spectra were obtained in UV Vis DR 5000 HACH equipment in scan mode of 190 nm to 800 nm for the samples dispersed in water (after sonication and 24 h after settling), only water dispersions were run, inasmuch as, dispersions in hexane were very poor.etc., taking advantage of the potential associated with this nanoscale materialResults show successful grafting indicated by the functional groups of graphene oxide and chitosan moieties, since carboxyl, carbonyl, and hydroxyl groups are found in GO, GEO and grafted samples. The presence of amide bands in FTIR of grafted samples is significant as this reveals the chemical interaction between the amine group from the polysaccharide and the oxygen-containing groups of GEO. The TEM and AFM images demonstrate important changes in the morphology of GEO sheets in comparison to the chitosan-grafted samples. Also grafted conditions produce important changes in the grafted graphene oxide structure as was shown by Raman spectroscopy, where G, D and 2D bands present important shifts related to the linked chitosan. These changes in the graphene structure are also corroborated by microscopy images. In spite of morphologic variations dispersion in water is not affected significantly as hydroxyl and carbonyl groups remain attached to the graphene sheets. For all samples the most suitable solvent was water, since polarity and hydrogen bonds play an important role in dispersion behavior. The temperature range used in the grafting reactions plays an important role in the morphology of grafted sheets and therefore for solubility properties. Thus, chitosan grafting through redox reaction is an appropriate route to improve dispersion and provides an interesting umbrella for bio-related applications, such as nanocomposites, biocomposites, biosensors, biomedical materials,


E. coli clinical isolate, catalyzes methylation of 30S at A1408 of the 16S rRNA and confers aminoglycoside resistance. Using sucrose cushion centrifugation and isothermal titration calorimetry, we first confirmed the binding between NpmA and 30S. Next, we performed amide Hydrogen/Deuterium Exchange Mass Spectrometry (HDXMS) of apo NpmA and in the presence and absence of SAM/SAH. We observed that ligand binding resulted in time-dependent differences in deuterium exchange not only at the ligand-binding pocket (D25–D55 and A86–E112) but also in distal regions (F62-F82 and Y113-S144) of NpmA. These results provide insights into methylation group donor cofactor-mediated allostery in NpmA in the ligand-bound states, which could not be observed in the static endpoint crystal structures. We predict that the two distal sites in NpmA form part of the allosteric sites that importantly are part of the main 16S rRNA binding interface. Thus HDXMS helped uncover allosteric communication relays that couple SAM/SAH binding sites with the ribosome-binding site. This highlights how HDXMS together with X-ray crystallography can provide important allosteric insights in protein-ligand complexes.Aminoglycosides are broad-spectrum antibiotics that bind to the 30S ribosomal subunit (30S) of bacteria and disrupt protein translation. NpmA, a structurally well-characterized methyltransferase identified in an  Serious infectious diseases caused by Gram-negative and Gram-positive bacteria are commonly treated with aminoglycosides345Excessive use of aminoglycoside antibiotics, however, has resulted in selection and the rampant progression and propagation of bacterial strains with resistance to these antibiotics. Specifically, methylation of the target site in bacteria hinders the binding of aminoglycosides to the ribosome. This methylation is carried out by methyltransferases (MTases) belonging to the Arm (aminoglycoside resistance MTase) and Kam (kanamycin-apramycin MTase) families1A1408 MTase (NpmA) belongs to the Kam family and was identified in an E. coli clinical isolate1 position of A1408apo and ligand-bound NpmA (NpmA-SAM and NpmA-SAH) were solved by our group10The novel plasmid-mediated 16S rRNA mE. coliAs a continuation of our efforts to understand the mechanism of NpmA-mediated antibiotic resistance, we purified the 30S ribosomal subunit from 6 tag and were expressed and purified from E. coli strain JE28. This strain was obtained from Dr. Suparna Sanyal from Uppsala University, Sweden. The expression and purification was carried out as described earlier600 nm of 1.0 and then cooled to 4 °C to accumulate the “run-off” ribosomes. The 1 L culture was then centrifuged at 3500× g for 10 min at 4 °C to harvest the cells. The pellet was stored at −80 °C or re-suspended in lysis buffer comprising 20 mM Tris–HCl pH 7.6, 10 mM MgCl2, 150 mM KCl, 30 mM NH4Cl, protease inhibitor (Sigma-Aldrich), lysozyme (0.5 mg/ml) and DNAse I (10 μg/ml) for purification. After sonication, the cell lysate was centrifuged twice at 20,000 × g for 10 min at 4 °C.The ribosomes used in this study have a tetra-(His)TM Chelating HP column  connected to an AKTA prime chromatography system . The system was equilibrated with lysis buffer and the column washed until a baseline level was reached. The column was then washed with wash buffer  and the 70S ribosome was eluted using an elution buffer containing a high concentration of imidazole .The lysate was injected into a HiTrap2+ . The HisTrapTM HP column was then equilibrated with SUB buffer, the sample injected, and the flow-through collected. As the tetra (His)6 tag is present only on the 50S subunit, it was trapped in the column whereas the free 30S ribosomal subunit was eluted in the flow-through. The eluted 30S ribosomal subunit was then concentrated using sucrose-cushion centrifugation or 30-kDa cut-off Amicon concentrators (Millipore) to a final concentration of 70 μM, flash frozen and stored in aliquots at −80 °C.To separate the ribosomal subunits, the 70S ribosome eluent was dialyzed 4 times for 10 min with a ‘SUB’ buffer that comprised less Mg2, 150 mM KCl, 30 mM NH4Cl, was added to the sucrose cushion solution  in a ratio of 2:1. The sample was then centrifuged in thick-walled polycarbonate tubes at 51,000 rpm in a TLA 100.4 rotor on a TLX-120 Beckmann ultracentrifuge for 12 h. The ribosome pellet was then resuspended to obtain the desired concentrationThe ribosome sample, in a buffer consisting of 20 mM Tris–HCl pH 7.6, 1 mM MgClFor ITC experiments, NpmA was first incubated with an excess of SAM/SAH  in a 1:12 molar ratio. Two ml of 30S ribosomal subunit (1.75–2.5 μM) in the sample cell was then titrated with 25–50 μM NpmA-SAM/SAH that was loaded into the injector of a VP-ITC calorimeter  at 20 °C. The samples were degassed and centrifuged before the experiment. The interval between successive injections was 4 min and a volume of 10 μl was dispensed per injection. The ITC data was analyzed using Origin 7.0 (OriginLab Corp.) software.apo-NpmA and SAH/SAM-bound NpmA. Deuterium exchange was initiated by dilution of 2 μl of each sample with 18 μl of equivalent buffer reconstituted in D2O (pH 7.6) to achieve a final concentration of 90% D2O. All samples were kept at 20 °C and the exchange reaction was quenched after 0.5, 1, 2, 5 and 10 min using 40 μl ice-cold buffer containing 0.1% TFA and 2.5 M guanidinium hydrochloride; this addition brought the final pH read of the quenched sample to 2.5. Besides the shorter deuterium labeling times, maximum time of 10 min chosen was suitable as substantial methylation levels have been shown previously at 10-minutes in an in vitro methylation assayi HDXMS mass spectrometer  in the MSE mode, a non-biased, non-selective CID method181920NpmA was concentrated to 60 μM and the SAH or SAM cofactor was added at a ratio of 1:17 (NpmA: SAH/SAM). Finally, each of the following complexes was used: 60 μM E based sequence identification of peptides from non-deuterated samples was carried out with ProteinLynx Global Server 2.4 software (Waters)21MSE. coli JE28 strain (MG1655 (rplL-his6):kan:rpoB). The eluted 70S (tetra (His)6 tagged) ribosome from the nickel affinity column was dialyzed against a buffer containing 1 mM Mg2+ to separate the ribosomal subunits , sucrose cushion centrifugation was performed with 10–60 μM NpmA and 0.6–1.8 μM 30S ribosomal subunits. Sucrose cushion centrifugation was carried out with 30S lane  but not printing . These fd) for NpmA-SAH:30S and NpmA-SAM:30S was estimated to be 5.1 μM (N = 1.1) and 12.5 μM (N = 0.94), respectively  in the peptide spanning residues F62-F82 is shown in The sequence coverage of the pepsin proteolyzed NpmA peptides obtained in our HDXMS experiment was 92% . Examinaapo-NpmA and the SAM/SAH-bound NpmA complexes showed a decrease in deuterium exchange in peptides in the SAM-  with the same ligand-binding residues but HDXMS results show an enhanced initial reduction in deuteration in the region F62-F82 in the NpmA-SAM complex as compared with the NpmA-SAH complex 926. Due 9apo state. The magnitude of decrease in deuterium exchange was greater in the SAM-bound state than in the SAH-bound state, even though the residues in both SAM and SAH are identical. Interestingly, this decrease in deuteration was also observed in distal regions that do not interact with SAM/SAH, suggesting that ligand binding to NpmA facilitates allosteric effects that are not discerned from the crystal structures.X-ray crystallographic studies of NpmA bound to its cofactors, SAH and SAM have provided atomic resolution insights into the cofactor-binding site. HDXMS provides a comprehensive view of conformational changes associated with protein-ligand interactions that encompass both binding and allosteric changes. The HDXMS experiments revealed an overall decrease in deuteration in ligand binding (orthosteric sites) and distal regions of NpmA in the SAM/SAH-bound state compared with its apo-NpmA in solution detected by HDXMS, offers a strong support for Conformational SelectionImportantly, a bimodal distribution of deuterium exchange kinetics was observed in one of the distal regions (F62-F82)Our foot printing and computational docking studies further suggested a base-flipping mechanism for the A1408 nucleotide of 16S rRNA to facilitate its methylation9How to cite this article: Husain, N. et al. Ligand-mediated changes in conformational dynamics of NpmA: implications for ribosomal interactions. Sci. Rep.6, 37061; doi: 10.1038/srep37061 (2016).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


In vitro studies revealed that SC-43 promoted HSC apoptosis, increased the SHP-1 activity and inhibited phospho-STAT3. The enhanced SHP-1 activity in HSCs significantly inhibited HSC proliferation, whereas SHP-1 inhibition rescued SC-43-induced HSC apoptosis. Furthermore, SC-43 interacted with the N-SH2 domain of SHP-1 to enhance the activity of SHP-1 as its antifibrotic mechanism. In conclusion, the SHP-1–STAT3 pathway is crucial in fibrogenesis. SC-43 significantly ameliorates liver fibrosis through SHP-1 upregulation. A SHP-1-targeted antifibrotic therapy may represent a druggable strategy for antifibrotic drug discovery.This study aimed to investigate the role of src-homology protein tyrosine phosphatase-1 (SHP-1)–signal transducer and activator of transcription 3 (STAT3) pathway in liver fibrogenesis and the anti-fibrotic effect of SHP-1 agonist. The antifibrotic activity of SC-43, a sorafenib derivative with an enhanced SHP-1 activity, was evaluated in two fibrosis mouse models by carbon tetrachloride induction and bile duct ligation. Rat, human, and primary mouse hepatic stellate cells (HSCs) were used for mechanistic investigations. The results showed that SHP-1 protein primarily localized in fibrotic areas of human and mouse livers. SC-43 treatment reduced the activated HSCs and thus effectively prevented and regressed liver fibrosis in both fibrosis mouse models and improved mouse survival.  In addition to HCC, liver cirrhosis is associated with variceal bleeding, spontaneous bacterial peritonitis, hepatic encephalopathy, and hepatorenal syndrome. Liver cirrhosis is a major health issue; however, an effective therapy against liver fibrosis is presently unavailable. An antifibrotic treatment is a critical unmet clinical requirement2.Chronic hepatitis caused repeated liver inflammation and gradually evolved into liver cirrhosis and hepatocellular carcinoma (HCC). Patients with chronic hepatitis B (CHB) related liver cirrhosis harbor a 12-fold increased risk of HCC3. The signal transducer and activator of transcription 3 (STAT3) pathway in hepatic stellate cells (HSCs) regulate their survival, proliferation, and activation4. Adiponectin was recently reported to have an antifibrotic activity in the liver by inhibiting the Janus kinase (JAK)–STAT pathway5. According to the aforementioned studies, STAT3 may play a major role in hepatic fibrogenesis.Discovering the genetic regulation of fibrosis and the liver-specific feature of fibrogenesis is crucial for identifying a mechanism-based antifibrotic therapy6, inhibit the proliferation of HCC cell lines through STAT3 inactivation8. Furthermore, both sorafenib and SC-1 ameliorated liver fibrosis in vivo and promoted HSC apoptosis in vitro through STAT3 pathway inhibition9. We confirmed that STAT3 is crucial in fibrogenesis and suggested that STAT3 is a potential target for antifibrotic therapy in patients with CHB related liver fibrosis9. Furthermore, a novel sorafenib derivative, SC-43, 43, Fig.  was deveOur hypothesis is that the SHP-1–STAT3 pathway is involved in liver fibrogenesis. In this study, we investigated the role of SHP-1 in fibrogenesis. The antifibrotic activity of the SHP-1 agonist, SC-43, and its antifibrotic mechanism were further explored.4 induction for 4 weeks, SHP-1 primarily located in areas with α-SMA-expressed activated HSCs and significant fibrosis  and sorafenib (10 mg/kg) were concurrently administered with CCl4 in the following 6 weeks. The SC-43 and sorafenib treatment significantly ameliorated liver fibrosis, as observed through sirius red staining  was achieved after CCling Fig. . A dose–ing Fig. . Furthering Fig. . We chos4 induction for 8 weeks. SC-43 (10 mg/kg) and sorafenib (10 mg/kg) were administered for the following 4 weeks without CCl4. At sacrifice, we observed that hepatic fibrosis regressed even in the control group  developed through CCloup Fig. , indicatoup Fig. .We further investigated the antifibrotic activity of SC-43 in the BDL model. In the fibrosis prevention model, the vehicle and SC-43 (10 mg/kg) were administered from day 1 until sacrifice on day 14 of BDL. The SC-43 treatment significantly reduced the collagen-positive area Fig. . In the These animal study results suggested the antifibrotic activity of SC-43 in the prevention, treatment, and regression of liver fibrosis.in vitro. The SC-43 treatment had dose-dependent effects to reduce the viability of HSC-T6 and LX2 cells, more significant than sorafenib , followed by a catalytic protein tyrosine phosphatase (PTPase) domain and C-terminal tail. In its inactive form, the D61 site at the N-SH2 domain interacts with the WPD site on the PTPase domain and hinders its PTPase activity. The SHP-1 activity constitutively increased in dN1 and D61A mutants, mimicking an open conformation10 Fig. .Figure 7To investigate the role of SHP-1 in HSC apoptosis, we observed that SHP-1 overexpression significantly reduced cell viability since day 2 of transfection Fig. . The SC-These results suggest that SHP-1 activation reduced HSC proliferation. The SC-43 treatment increased the SHP-1 activity and downregulated p-STAT3 to promote HSC apoptosis, whereas SHP-1 inhibition counteracted the effects of SC-43.The associations of the ectopic expression of wild-type SHP-1, dN1, and D61A with HSC proliferation were further examined using a colony formation assay. As shown in Fig. Next, we examined the effects of SC-43-induced SHP-1 activity on the ectopic expression of different SHP-1 mutants. The SC-43 treatment significantly increased the SHP-1 activity in the ectopic expression of the vector and wild-type SHP-1 but not in the ectopic expression of dN1 and D61A Fig. . BecauseThese results suggested that the ectopic expression of SHP-1 and dN1 and D61A significantly inhibit cell proliferation. The D61 site of the inhibitory N-SH2 domain is crucial for SC-43-induced SHP-1 upregulation. The overexpression of the dN1 and D61A mutants abolished the SC-43-induced SHP-1 activation, cell apoptosis, and p-STAT3 downregulation. The antifibrotic mechanism of SC-43 in the SHP-1–STAT3 pathway is summarized in Fig. 11. SHP-1 is primarily expressed in hematopoietic cells and functions as a key regulator of intracellular phosphotyrosine levels in lymphocytes12. SHP-1 functions as an antagonist to the growth-promoting and oncogenic potentials of tyrosine kinase; therefore, it was proposed as a tumor suppressor gene in lymphoma, leukemia, and other cancers12. Furthermore, SHP-1 acts as a negative regulator of several receptor tyrosine kinases, including PDGFR14, the insulin receptor15, the epidermal growth factor (EGF) receptor, and vascular EGF receptor type 216. Therefore, SHP-1 acts as a suppressor of the PDGFR signaling pathway and may serve as a molecular target to prevent HSC proliferation and the fibrogenic effects following HSC activation14. SHP-1 downregulates and subsequently blocks p-STAT3 dimerization through a direct interaction and dephosphorylation of p-STAT310. By contrast, SHP-2 is a positive mediator of the Ras–extracellular signal-regulated kinase 1/2 and Akt signaling pathways14. In addition, SHP-1 and SHP-2 modulate cellular signals involving PI3K, Akt, JAK2, STAT, mitogen-activating protein kinases, extracellular signal-related kinases, c-Jun-amino terminal kinases, and nuclear factor-kB11. Overall, SHP-1 and SHP-2 modulate progenitor cell development, cellular growth, tissue inflammation, cellular chemotaxis, and cell survival16.The role of SHP-1 in liver fibrosis remains unclear. The SHP family contains a nonreceptor protein tyrosine phosphatase comprising two members, SHP-1 and SHP-2, which exert contrasting biological functions17. Moreover, we demonstrated that SHP-1 was predominately distributed in fibrotic areas. The phosphatase of these SHP-1 protein may remain in inactive states, which do not have antifibrotic effect. Only activated SHP-1 phosphatase has antifibrotic activity. Therefore, SHP-1 agonist might be used as a molecular target and tissue-specific regimen for antifibrotic therapy.In this study, we found SHP-1 inhibition by sodium stibogluconate increased fibrogenesis, suggesting SHP-1 plays important role in fibrogenesis. SHP-1 was recently reported to be abundant in the liver, with tissue specificity19. The interaction between N-SH2 and PTP domains results in an intramolecular inhibition, which is stabilized by a salt bridge between Asp61 (D61) and Lys36220, and further blocks the entrance of phosphopeptide activators. Our results suggested that SC-43 affects SHP-1 by triggering a conformational switch, thus relieving its autoinhibition. However, the dN1 and D61A mutants lacked this autoinhibition; therefore, SC-43 could not further increase their SHP-1 activity. The docking model indicates that the trifluoromethyl group of SC-43 may dock into the pocket between the N-SH2 domain and form a hydrogen bond with Q529, which subsequently releases the active PTP domain of SHP-120. The phenylcyanyl group of SC-43 is shorter than the pyridine–methylamide group of sorafenib, and the phenyl ring between urea and the phenylcyanyl moiety in SC-43 reduced its total length. These two conformational factors contribute to a more efficient fit in the N-SH2 pocket and reduce the steric-hindering effect of the N-SH2 domain, resulting in a more potent SHP-1 activation in SC-43 than in sorafenib10.The crystal structure of the ligand-free SHP-1 protein has an autoinhibition conformation depending on the inhibitory effect of the N-SH2 domain on the catalytic PTP domain4. This finding is consistent with previous studies reporting that fibrosis may reverse after withdrawing the toxic agent21 and similar to a recent clinical finding that sustained viral suppression by antiviral therapy can regress cirrhosis and related complications in patients with CHB23. Even in the process of fibrosis regression, the SC-43 treatment significantly reduced the fibrosis, suggesting that HSC apoptosis during the fibrosis resolution stage might be crucial. Whether SC-43 treatment triggers other fibrinolytic mechanisms in this stage warrants further investigation. Sorafenib-induced multikinase inhibition causes various adverse reactions through off-target effects and preclude its use as an antifibrotic agent. The SC-43 lacks the Raf kinase activity, and theoretically it should have lower adverse effects. Future studies are needed to compare the in vivo antifibrotic activity of SC-43 with sorafenib in other animal models and investigate the adverse effects of SC-43 in preclinical studies.The antifibrotic activity of SC-43 was well demonstrated in both hepatotoxic and cholestatic animal models. In addition, compared with the control, the SC-43 treatment improved the survival of fibrotic mice. Fibrogenesis involves multiple signaling pathways. SC-43 downregulates p-Akt and p-STAT3 through PDGFR dependent and independent mechanisms might be another advantage of this compound for antifibrotic therapy. Notably, we observed cirrhosis regression after withdrawing the causative agent CClIn conclusion, our results suggest the relevance of the SHP-1–STAT3 pathway in fibrogenesis. SC-43 activates SHP-1 through the direct interaction of the inhibitory N-SH2 domain and promotes HSC apoptosis as the antifibrotic mechanism. Furthermore, SC-43 reduces liver fibrosis in both hepatotoxic and cholestatic fibrosis mouse models, indicating that the SHP-1 agonist is a potential target for antifibrotic drug discovery.SC-43, or 1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(3-(4-cyanophenoxy)phenyl) urea, was synthesized by Dr. Chung-Wai Shiau at National Yang-Ming University. Sorafenib (Nexavar) was kindly provided by Bayer HealthCare AG . Antibodies for alpha-smooth muscle actin (α-SMA), P-STAT3 (Tyr705), STAT3, cyclin D1, glyceraldehyde-3-phosphate dehydrogenase, P-Smad2 (Ser465/467), P-Smad3 (Ser423/425), Smad2, Smad3, poly (ADP-ribose) polymerase (PARP), platelet-derived growth factor receptor (PDGFR)-β, P-PDGFR-β (Tyr857), and P-Akt (Ser473) were purchased from Cell Signaling . Akt was purchased from Santa Cruz Biotechnology . Furthermore, sodium vanadate was purchased from Cayman Chemical . PTP inhibitor III was purchased from Calbiochem . Cremaphor and sodium stibogluconate were obtained from Sigma .We enrolled 25 patients with CHB with different degrees of liver fibrosis . Their biopsied liver tissues were stained with SHP-1. The study conformed to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the Institutional Review Board of the National Taiwan University Hospital. Written informed consent was obtained from all patients at enrollment.4 (1 µL/g) was administered through a biweekly intraperitoneal injection to the C57BL/6JNarl mice for 4 or 8 weeks. A vehicle (cremaphor), SC-43 , or sorafenib (10 mg/kg) were administered through oral gavage for 5 days a week during a specified schedule until sacrifice. The concentrations, route and frequency of SC-43 and sorafenib were selected according to previous published results10. Sorafenib served as the positive control because of its proven antifibrotic activity9.Eight-week-old male C57BL/6JNarl mice or Balb/C mice (20–25 g) were obtained from the National Laboratory Animal Center, Taiwan. In the hepatotoxic model, CClIn the BDL model, the common bile duct was double ligated, followed by resection in the Balb/C mice. A vehicle or SC-43 were administered through oral gavage daily from day 1 or 8 until sacrifice on day 14.24, were administered daily  in the CCl4 model for 2 weeks and in the BDL model for 1 week to investigate the effect of SHP-1 inhibition on fibrogenesis. All experimental animal procedures were in accordance with protocols approved by the Institutional Laboratory Animal Care and Use Committee of National Taiwan University.Sodium stibogluconate, a specific SHP-1 inhibitorMouse liver specimens were preserved in 10% formaldehyde, dehydrated in a graded alcohol series, embedded in paraffin blocks, sectioned to 3-μm thickness, placed on glass slides, and stained using a Picrosirius Red Stain kit , according to the manufacturer’s instructions. The liver fibrosis severity was graded according to the Ishak fibrosis scoring system. The collagen-positive area was evaluated through densitometry by using ImageJ software. The liver hydroxyproline concentration was measured using a hydroxyproline assay kit , according to the manufacturer’s instructions. Immunohistochemical staining was performed using the Leica BOND-MAX autostainer (Leica Biosystems), according to the manufacturer’s protocol. The TUNEL assay  was performed according to the instructions.9. HSC-T6 cells were cultured in Waymouth medium and LX2 cells and primary mouse HSCs in Dulbecco’s modified Eagle medium . All media were supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin G, 100 μg/mL streptomycin sulfate, and 2 mM L-glutamine. All cells were cultured in a 37 °C humidified incubator under 5% CO2 in air. The quiescent HSCs were activated following FBS supplementation. For in vitro studies, various concentrations of SC-43 and sorafenib were dissolved in dimethyl sulfoxide and subsequently added to the cells in 5% FBS for the scheduled incubation duration.HSC-T6 and LX2 cells were immortalized rat and human HSC lines, respectively, and were kindly provided by Prof. Scott Friedman from Mount Sinai Hospital, New York. Primary mouse HSCs were isolated according to previously described proceduresHSC-T6 or LX2 cells or primary mouse HSCs were seeded in 96-well, flat-bottomed plates with 5000 cells per well with 10% FBS for 24 h. The cells were exposed to various concentrations of SC-43 or sorafenib for 24 h and 48 h. The effects of the individual agents on cell viability were evaluated using a CellTiter 96 AQueous one solution cell proliferation assay containing 3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), an inner salt  in triplicate, according to the manufacturer’s protocol. After SC-43 or sorafenib treatment for 24 h, the percentage of apoptotic cells were determined through Annexin V and propidium iodide (PI) double staining after pooling both early (Annexin V+/PI−) and late (Annexin V+/PI+) apoptotic cells on a BD FACS Verse flow cytometer . The cell cycles were evaluated by PI staining.Whole cell extracts were obtained using a radioimmunoprecipitation assay (RIPA) buffer , and the protein concentrations were quantified using a BCA protein assay kit . Twenty-five micrograms of a sample protein was loaded onto sodium dodecyl sulfate–acrylamide gels of various percentages, electrophoresed, and transferred to polyvinylidene fluoride membranes. After treatment with 5% bovine serum albumin in Tris-buffered saline and Tween 20 for 1 h, the membranes were subsequently incubated overnight with the appropriate primary antibodies. Following extensive washing, the membranes were incubated for 1 h with a blocking buffer containing horseradish peroxidase (HRP)-conjugated secondary antibodies. The proteins were detected using the Immobilon Western Chemiluminescent HRP substrate  or the ECL detection system .For transforming growth factor (TGF)-β induction, the HSCs were serum deprived for 4 h, subsequently treated with 10 μM SC-43 for 4 h, and followed by stimulation with 10 ng/mL recombinant human TGF-β1  for 20 min.For PDGF-BB induction, the HSCs were serum deprived for 4 h, treated with 10 μM SC-43 for 4 h, followed by induction with 50 ng/mL recombinant human or rat PDGF-BB (R&D Systems) for 10 min on LX2 or HSC-T6 cells, respectively.For interleukin (IL)-6 stimulation, the HSCs were serum deprived for 4 h, treated with 10 μM SC-43 for 4 h, followed by induction with 100 ng/mL IL-6 (R&D Systems) for 30 min.5 HSC-T6 cells, which were seeded on a 6-cm dish; the rat STAT3 stable overexpression clone was generated.Rat STAT3  was constructed into a pLVX–AcGFP-N1 expression vector , which was later cotransfected into 293FT cells in addition to lentiviral packaging and expression vectors (P8.91 and VSV-G) by using the Lipofectamine 2000 transfection reagent (Invitrogen). The lentiviral supernatant was harvested 48 h post transfection and used to infect 5 × 10Plasmids encoding the human wild-type SHP-1 and SHP-1 mutant, in which the N-SH2 domain was truncated (dN1) or one aspartic acid at site 61 was changed into an alanine residue (D61A) were cloned into the pCMV6-entry vector with myc-tag. These mutants were confirmed through DNA sequencing. Smart-pool siRNA, including the control (D-001810-10), and SHP-1  were obtained from Dharmacon Inc . For transient expression, SHP-1 plasmids or siRNA  with the Lipofectamine 2000 transfection reagent were pretransfected into the LX2 cells for 24 h, according to the manufacturer’s instructions.After 24 h of SC43 treatment with 5% FBS in DMEM in HSCs, protein extracts were incubated overnight with an anti-SHP-1 antibody in the RIPA lysis buffer. Protein G-Sepharose 4 Fast Flow  was added into each sample, followed by incubation for 3 h at 4 °C, with rotation. The RediPlate 96 EnzChek Tyrosine Phosphatase Assay Kit  was used for determining the SHP-1 activity, according to the manufacturer’s protocol.LX2 cells were plated in 10-cm dishes (1500–5000 cells per dish) and cultured in DMEM for 2 weeks. The cells were subsequently fixed with 4% formaldehyde and stained with 0.1% crystal violet.25, or serum alanine aminotransferase (ALT). The mouse survival rate was estimated using the Kaplan–Meier method. The log-rank test was used to determine the statistical differences in survival of the different experimental groups. Statistical analysis was performed using STATA . All tests were two-sided, and P < 0.05 was considered significant.Continuous variables are presented as mean (standard error), and categorical data are presented as number (percentage), as appropriate. Differences between subgroups were evaluated using the Student t test. Spearman’s correlation was used for the association between SHP-1 and aspartate aminotransferase (AST) to platelet ratio index , a biomarker for liver fibrosisSupplementary Data


MAPT), progranulin (GRN) and chromosome 9 open reading frame 72 (C9orf72) being the most common genetic causes. No other risk factors for FTD had been identified until a genome-wide association study of patients with the most common pathological form of FTD, frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP), showed an association with single nucleotide polymorphisms (SNPs) in a region encoding transmembrane protein 106b (TMEM106b).GRN mutations (who have FTLD-TDP pathology).GRN cohorts, in those with C9orf72 expansions  and in one undifferentiated clinical FTD cohort.GRN cohort.Frontotemporal dementia (FTD) is a neurodegenerative disease associated with impaired behaviour, language and motor function. Around a third of FTD is familial, with mutations in microtubule-associated protein tau (GRN (n=10), C9orf72 (n=17), dual GRN/C9orf72 (n=1), TBK1 (n=1) and SQSTM1 (n=2) mutations) and those with clinical syndromes associated with FTLD-TDP (FTD-MND and svPPA). Genotyping of the rs1990622 polymorphism in the total FTD cohort showed a frequency of AA 77 , AG 90  and GG 31 , and in the TDP-43 subset of AA 33 (43.4%), AG 34 (44.7%) and GG 9 (11.8%). The frequency within a UK control population of 5020 people from the Wellcome Trust Case Control Consortium was AA 1637 (32.6%), AG 2485 (49.5%) and GG 898 (17.9%). Using a homozygous protective allele model, there was no difference in the TDP-43 subset  or the total FTD cohort  compared with the control group, nor did allelic tests show significant differences between patients and controls. However, a homozygous risk allele model in the TDP-43 subset gave Fisher’s exact test, p=0.049 compared with the control group, and in the total FTD cohort gave Fisher’s exact test, p=0.076 compared with the control group.The UCL FTD DNA cohort database was searched for those with volumetric MRI. Data were available from 198 patients with an FTD spectrum disorder: 87 with behavioural variant FTD (bvFTD), 46 with non-fluent variant primary progressive aphasia (nfvPPA), 43 with semantic variant PPA (svPPA), 6 with a primary progressive aphasia (PPA) syndrome not meeting criteria for any of the other PPA syndromes , 5 with FTD associated with motor neurone disease (FTD-MND), 7 with corticobasal syndrome (CBS) and 4 with progressive supranuclear palsy (PSP). Within this cohort, 76 individuals had definite or likely TDP-43 pathology: this group included cases with genetic mutations known to cause TDP-43 pathology  for the insula, cingulate, frontal, temporal, parietal and occipital lobes in each hemisphere were generated from volumetric T1 MRI using a previously described methodology.In the total FTD cohort, significantly lower volumes were present in the AA  group compared with the AG/GG group in the frontal (p=0.009), temporal (p=0.029), cingulate (p=0.014) and insula (p=0.018) cortices .As previous studies have shown an association of the rs1990622 polymorphism with decreased volumes in the left hemisphere,In the TDP-43 subset, although similar or larger absolute differences in volume were seen in the AA group compared with the AG/GG group, a significant difference was only seen between the groups in the frontal lobe (p=0.044), with a trend to a difference in the cingulate (p=0.085) and insula (p=0.084) cortices. As with the clinical cohort, this was driven by an effect within the left hemisphere, with significant differences seen between groups in the frontal (p=0.013) and parietal (p=0.037) cortices, and a trend to a difference in the cingulate (p=0.082) and insula (p=0.072) .In a cohort of patients clinically diagnosed with FTD and also in a subset of patients with likely TDP-43 pathology, we show that homozygosity for the TMEM106B risk allele is associated with reduced grey matter volume in key cortical regions implicated in FTD. Consistent with previous research showing an effect of the TMEM106B risk allele on left hemisphere structures within the general population, decreased grey matter volumes were found in the left but not right hemisphere in both the total cohort and the TDP-43 subset.GRN or C9orf72, as well as those without.Despite some studies showing no effect, overall, previous studies have shown that the effects of TMEM106B variants are seen in cases of FTD with FTLD-TDP (but not FTLD-tau), including those with mutations in TMEM106B is a glycoprotein critical for normal lysosomal function.


This not only thoroughly fixes the issue of dissolution of zinc electrode but also increases the specific surface areas of zinc and promotes chemical reaction rate of the charge-discharge processes. By performing experimental measurements, we found that the discharge capacity of the new designed Ni-Zn batteries can be as high as 2603 mAh/gZno, and the superior electrochemical performance can be kept in 10,000 test cycles, suggesting that the new developed in-situ cutting technique is very useful in electrochemical fields.Rechargeable Ni–Zn batteries, with high safety, low cost and nontoxicity, can be expected to compete with lithium-ion batteries for market share. However, the issue of dissolution of zinc electrode largely limit the battery cycle life and remains unsolved. We designed a kind of graphene-ZnO hybrid electrode in which  Among all existing energy storage devices, lithium-ion batteries are becoming more and more popular due to their high energy density, high specific power, and long cycle life5. However, high cost due to low abundance of lithium and safety issues due to flammable organic electrolyte become the two major bottlenecks for wide promotion of lithium-ion batteries.Energy storage has been considered as an important element in modern energy supply chain, which can enhance stability of grids, promote penetration of renewable energy resources, improve energy efficiencies, reduce consumption of fossil energy resources and environmental impacts of energy generation6. Furthermore, natural abundance of zinc largely reduces the cost of Ni–Zn batteries. And compared to toxicity of heavy metals in Ni-Cd batteries and lead-acid batteries, nontoxicity of electrode materials makes Ni–Zn batteries to be environmentally friendly7. Moreover, rechargeable Ni–Zn batteries, with high safety and nontoxicity, can be expected to compete with lithium-ion batteries for electric bus. All the above advantages make rechargeable Ni–Zn batteries to be one of promising energy storage systems6. However, the low cycle life becomes the obstacle to limit the widespread commercialization of Ni–Zn batteries, which is mainly due to the shape change, dendrite growth, passivation and self-discharge of the zinc electrode14. In essence, all the above issues affecting the cycle life of Ni–Zn batteries are associated with dissolution of the zinc electrode into solution during charge or discharge processes16. A variety of attempts have been made to fix the above issues, including adding additives to the zinc electrode or electrolytes24, changing separators27, and designing three-dimensional zinc sponge anodes2, etc. However, the issue of dissolution of zinc electrode remains unsolved.Rechargeable Ni–Zn batteries, using aqueous nonflammable solutions as electrolytes, greatly reduces the risk of fire and explosion when batteries are charged or used30, however, it has been rarely reported to directly cut graphene into short nanoribbons and smaller sized graphene can help to largely improve the mass and specific activity of the oxygen reduction reaction31. Here we developed a kind of in-situ cutting technique which can directly cut graphene into short nanoribbons, and the strong interatomic interactions between graphene and zinc can help to anchor plenty of zinc atoms onto graphene surfaces. On the one hand, anchoring of zinc atoms onto graphene surfaces can completely solve the issue of dissolution of zinc electrode in alkaline solution during discharge process and avoid dendrite formation; on the other hand, the specific surface areas of zinc can be greatly increased and the chemical reaction rate can be promoted. Furthermore, the superior electrical conductivity of graphene is helpful to largely improve the speed of charge-discharge processes. We performed a series of experiments and molecular dynamics simulations to investigate the electrochemical performance of the new designed rechargeable Ni/Zn secondary battery and the mechanisms behind.Cutting and unzipping technique has been widely used to cut carbon nanotubes into graphene nanoribbons for many applicationsin-situ cutting of graphene. To further confirm that the short nanosheets are not water-soluble materials e.g. KOH, the test electrode  were washed 12 times with deionized water, and Fig. SEM is used to examine the microstructures of the new designed graphene-ZnO hybrid electrode. The microstructures of the test electrode before cycling are captured by SEM as shown in Fig. in-situ cutting of graphene, we further performed TEM experiments to investigate the microstructures of the graphene-ZnO electrode. The TEM images of the electrode are shown in Fig. in-situ cutting of graphene is confirmed.To achieve a higher resolution to confirm the Energy dispersive X-ray spectroscopy (EDS) analysis was performed to determine the elemental composition of the SEM microstructures in the graphene-ZnO hybrid electrode (80.7 wt% ZnO) after 1,000 cycles and the analysis results are shown in Fig. 32 with a timestep of 0.1 fs. The reactive force-field (ReaxFF) interatomic potential34, capable of describing the dynamics of chemical reactions at the atomic scale and widely used to model chemical reactions in batteries36, was used to describe all the interatomic interactions in the model system. To confirm the key role of graphene to anchor the Zn atoms and avoid their dissolution, we constructed two model systems. One model system consists of 527 OH− ions and 4175 water molecules, corresponding to about 7 mol/L OH- solution which is consistent with our experimental condition, and a single layer of 572 Zn atoms are placed below the OH− solution, and a graphene layer with 1196 C atoms are placed beneath the Zn layer. The size of the whole model system is about 6 nm × 6 nm × 7 nm. To compare with it, the other system with OH− solution and Zn layer the same to the first model system but without graphene layer is constructed. NPT ensemble  was used to control the temperature of the model system to be 300 K and the pressure in z direction to be 1 atm. To ensure that the model system achieved the stable status, we set the total running time to be 1,000,000 steps (100 ps). The initial and final structures of the model system of the graphene-Zn composite with OH− solution are shown in Fig. − solution are shown in Fig. − solution are shown in Fig. − solution are shown in Fig. − solution which is consistent with experimental observations. By comparison, we can conclude that graphene can play a key role in anchoring Zn atoms and avoid them to be dissolved. Furthermore, from Fig. 37. From Figs To further confirm that graphene can anchor zinc atoms onto its surface, we performed a series of molecular dynamics simulations. All molecular dynamics simulations were performed using the Large-scale Atomic/Molecular Massively Parallel Simulator (LAMMPS) package/discharge of voltage variation of the battery is shown in Fig. To evaluate the cycle life and the stability of the new designed Ni-Zn batteries, we conducted a series of electrochemical measurements in as high as 10,000 test cycles. The temporal changein-situ cutting was finished. According to the information shown above, the midpoint charge/discharge voltage is very stable, and the capacity decay rate is only 0.0011% . From the results we can conclude that the electrochemical performance of the new designed Ni-Zn batteries is very stable in 10,000 test cycles and the battery cycle life is far larger than 10,000 cycles. For comparison, we also show the midpoint discharge capacity with the ratio of graphene to zinc oxide of 1:4.19 (80.7% ZnO)  in Fig. As shown in Figs f 1:4.19 0.7% ZnO ZnO, which is far larger than the theoretical specific capacity of Zn electrode of 987 mAh/gZnO38. This is far better than the discharge capacity of other ZnO based composite anode materials, in which the discharge capacity cannot maintain beyond 1000 mAh/gZnO. The ZnO–Ni3ZnC0.7–C composite for lithium ion batteries exhibited an initial discharge capacity of 1120 mAh/gZnO and retained a discharge capacity of 372 mAh/gZnO at the 50th cycle39. Even though the initial discharge capacity of ZnO-carbon black nanocomposite anode material for lithium ion batteries was up to 2096 mAh/gZnO, which fell quickly to only 1026 mAh/gZnO after 500 cycles38. Reference40 constructed a kind of high-power micro-supercapacitor with enhanced capacity that is close to our capacity results by high packing density unidirectional arrays of vertically aligned graphene, so we believe that the superior performance achieved here should be due to the supercapacitor effect of graphene. Furthermore, the properties of the battery, such as cycle life, irreversible capacity loss, self-discharge rate, electrode corrosion and safety are also highly dependent on the thickness of the solid electrolyte interface (SEI) which is a hierarchical structure formed in the transition zone between the electrode and the electrolyte41.The temporal change of voltage during deep discharge process of the battery is shown in Fig. in-situ cut into short nanoribbons that effectively anchor plenty of zinc atoms onto it surface, possessing largely enhanced cycle life and discharge capacity compared to conventional Ni/Zn secondary battery yet the fabrication method is very easy to operate. The issue of dissolution of zinc electrode is thoroughly solved and the strong interatomic interactions between graphene and zinc atoms and the increased specific surface areas of zinc are the two main mechanisms for the enhanced electrochemical performance.A kind of graphene-ZnO hybrid electrode in rechargeable Ni/Zn secondary battery is designed in which graphene can be 2) electrode is served as the cathode. The ratio of designed capacity for the β-Ni(OH)2 cathode and ZnO anode was about 5:1, so the capacity obtained in this present work just reflected the performance of ZnO electrode. A solution of 7 M KOH and 0.1 M LiOH, was used as the electrolyte, and a polyolefin microporous membrane is used as the separator. The ZnO anode and β-Ni(OH)2 cathode were assembled into a cell. The microstructure and morphology of the electrodes were characterized by a scanning electron microscope  and a transmission electron microscope .We synthesized graphene-ZnO hybrid electrode in an easy method: A mixture of graphene and zinc oxide with the mass ratio of graphene to zinc oxide of 1:4.19 (80.7% ZnO) and 31:19 (38% ZnO). The as-prepared ZnO-graphene mixtures were smeared into a brass net (2 cm × 2 cm) to fabricate the anode. Afterwards, the pasted electrodes were dried at 70 °C and pressed at a pressure of 10 MPa. A pasted nickel hydroxide (β-Ni(OH)in-situ cutting was finished.The galvanostatic charge-discharge tests were conducted on a BS9300 battery program-control test system at room temperature. The cells were charged at 0.1 C for 5 min, and then discharged at 0.1 C down to 0.8 V cut-off. After 1000 test cycles, the


This method paves the way for the environmentally friendly and cost-effective production of graphene-based materials.The unique properties of graphene are highly desired for printing electronics, coatings, energy storage, separation membranes, biomedicine, and composites. However, the high efficiency exfoliation of graphene into single- or few-layered nanoplates remains a grand challenge and becomes the bottleneck in essential studies and applications of graphene. Here, we report a scalable and green method to exfoliate graphene nanoplatelets (GNPs) from nature graphite in pure water without using any chemicals or surfactants. The essence of this strategy lies in the facile liquid exfoliation route with the assistance of vapor pretreatment for the preparation of edge hydroxylated graphene. The produced graphene consisted primarily of fewer than ten atomic layers. Such the water soluble graphene can be stored in the form of dispersion (~0.55 g L However, the production of true (single-layer) graphene in bulk quantity in liquid form graphite dispersions has been an unreachable goal for materials scientists in the past decade5. It was known that several routes have been explored to produce graphene which include micromechanical cleavage, topological growth on metal and non-metal substrate, and chemical oxidation of graphite7. It is also evident that the three approaches suffer from scalability, cost effectiveness and quality issues. Liquid-phase exfoliation is a promising approach to realize scalable production of high-quality graphene or other layered materials8. Generally, graphite can be exfoliated in aqueous or organic solutions with a surfactant by using mechanical cleavage approach 10. During the processes for purification and dispersion, the exfoliation method suffers from the need for large quantities of solvents, which are usually expensive and need special care when handling. Despite having many impressive features, to date, graphene nanoplatelets can be dispersed in pure water only with a concentration less than 0.01 g L−1 and there is only slightly improved by adding dispersants like surfactants, ionic liquids11, superacids12, even long time sonication13. On the other hand, long time storage or reducing the amount of solvent destabilizes the dispersion and leads to the re-stacking of nanoplatelets. This means that an enormous amount of solvent is needed and only a small amount of graphene is produced, which is economically infeasible and environmentally unfriendly18.Following the discovery of graphene-based materials, graphene has been applied vigorously in fields ranging from electronics to medicine recent years because of its attractive electrical, mechanical, optical and chemical propertieset al.19 found a novel ball-milling exfoliation procedure with the addition of modifier of DY50 during the milling process to produce graphene nanoplatelets with high production yield (~100%). Salvatierra et al.20 reported a simple and novel route, based on a reaction between the aqueous acid solution and the magnesium to provide a large amount of hydrogen gas and heat to enhance production yield of graphene. According to these results, high-speed ball-milling generates kinetic energy sufficient to break C-C bonds, as well as π-π bonds between the graphite layers. However, these methods are commonly time-consuming, comprising multiple steps in hazardous conditions. Also, the proposed approaches were mostly resulted in the functionalized multi-layered GNPs that need further treatment to reach the pure few layered graphene22. Dong et al.23 present an fully-chemical-free method for pretreatment, on mild oxidation of graphite exfoliation and dispersion of graphene in water, which can be stored in the form of a concentrated slurry (50 g L−1) or filter cake for months without the risk of re-stacking. Bepete et al.4 achieved single-layer graphene stable dispersion in degassed pure water with a concentration of 0.16 g L−1.Some new approaches for scalable production of graphene nanoplatelets (GNPs) were recently reported, which include ball-milling graphite in the presence of some chemical reactants. For example, Zhang −1. This process is environmentally friendly since thermal energy is readily available and water is a nontoxic medium, leaving no impurities in the product.In this paper, we present a facile and eco-friendly method to exfoliate graphite in a layer-by-layer way to produce GNPs in pure water. In contrast to the conventional exfoliations in a solvent dispersed system, we strive for an approach in which GNPs are produced and stored as flocculated aqueous solution with a high concentration. In this typical process, natural graphite powder was added to distilled water at a mass fraction of 10%, followed by sonication the mixture into a homogeneous form. The exfoliation process involves the following sequences: high temperature vapor leads to the curling and delamination of the graphite, insertion of water molecule along the fissure, cutting by the reaction of graphite edge and water molecule, or direct peeling off from the parent materials. The yield of ultrathin graphene is about 23.5% by weight, and the concentration of graphene in pure water is as high as 0.55 g LNatural graphite powder  and ethanol (95%) were purchased from Sigma-Aldrich. Distilled water was subjected to agitation with a rotating Teflon stirrer under a reduced pressure of 0.1 mbar for 40 min to obtain degassed water. All the other reagents were used as received.The exfoliation of graphite was following by two steps. Firstly, 5 g of graphite powder was added into 50 mL degassed water. The mixture was subjected to sonication at ~500 W  for 30 min by using a tip sonicator (SJIA 650 W), resulting in a black graphite slurry. The mixture was further transferred to a stainless steel autoclave and kept at 150 °C for 2 h with a 1500 rpm mechanical stirring. After cooling down to room temperature, the black slurry was collectedfrom the autoclave and diluted by adding 500 mL distilled water. Subsequently, the solution was sonicated for 20 min at ~500 W using a tip sonicator, and centrifuged at 3000 rpm for 30 min to remove other unreacted materials. The as-obtained sample was dried at 60 °C in a vacuum oven for 24 h.2 substrate with the graphene ethanol suspension deposited on it. X-ray photoelectron spectroscopy (XPS) (AXIS ULTRA DLD) was applied to the confirmation of the chemical composition. Fourier transform infrared spectrometer  was used to confirm the structure of graphene. Raman spectroscopy was performed to confirm the quality of the graphene nanosheets exfoliated in water and compare them with the raw materials. The thermal behavior was performed with a TG-209 F3 instrument  at a heating rate of 10 °C min−1 from room temperature to 800 °C under nitrogen atmosphere. Contact angle measurements were carried out by a contact angle goniometer  and the results were presented as the average of three measurements at different locations. UV-vis absorption spectra were recorded on a UV-2550 spectrophotometer with the wavelength ranging from 200 nm to 800 nm. Electrical conductivity was measured from current-voltage curves under small currents that do not generate significant self-heating.The following equipment was used: Scanning electron microscope (SEM) images were acquired using a FEI QUANTUM 250FEG field emission scanning electron microscope. The SEM samples were prepared by placing a small amount of as-obtained solid on the conductive carbon adhesive, which was stuck to an aluminum sample holder. Transmission electron microscope (TEM) images were observed on a high-resolution JEOL JEM-2100 transmission electron microscope instruments at an acceleration voltage of 200 kV. Atomic force microscope (AFM) images were obtained measurements with a Digital Instruments Dimension 3100 instrument using Si/SiO−1. Although the dispersibility of graphene can be improved in the present of surfactant or special ionic liquids in water, higher viscosity and limited exfoliation efficiency make the large-scale production of GNPs impractical. In contrast, our graphene can be dispersed in pure water with a concentration of 0.55 g L−1 with remaining stable more than months in room temperature, which is much higher than most previous reports 24. As the result, this affords opportunities for a high concentration and low-cost as well as massive scale production of GNPs.In this research, we used bulk-layered graphite material to obtain atomically thin graphene platelets in pure water Fig. 27. In addition, it can be seen from Fig. et al.7. This phenomenon demonstrates that the flexibility of platelets is high and the thickness of the peeled-off sheets is small. Due to the novel scroll topology, we believe these platelets may have different properties from common carbon nanoscrolls28.Once graphene could be exfoliated, the morphology of GNPs was an important parameter for further application or characterization. In order to study the morphology change after the exfoliation, scanning electron microscopy (SEM) images of the parent initial graphite powder and GNPs were observed and the results were shown in Fig. 31. GNPs were further analyzed through high-resolution TEM (HR-TEM) and these images were listed in Fig. The morphology of GNPs was further analyzed by transmission electron microscopy (TEM). Samples for TEM were prepared by drying a droplet of the graphene suspension on a mica substrate. TEM images were obtained using a JEM-2100F high-resolution transmission electron microscope at an acceleration voltage of 200 kV. Figure The thickness and fine structure of GNPs were further measured on an Atomic force microscope (AFM). The samples used for AFM tests were prepared by depositing the corresponding dispersions on Lacey carbon grid and dried under vacuum at 50 °C for 12 h. As shown in Fig. 33. We have carried out detailed FTIR investigation to monitor the evolution of the functional groups in graphene samples during the exfoliation process. Figure −1 are assigned to stretching vibrations of C-O in hydroxyl, and the sharp absorption peak at 1255 cm−1 belongs to stretching vibrations of C-O in carboxyl groups, while they were not observed in the FTIR spectra of initial graphite sample. Raman spectroscopy has been used as a powerful tool to study graphene-based materials, to determine the number of layers, the stacking sequence in the case of multiple layers, doping, and the amount and nature of defects. Raman spectroscopy provides a non-destructive method to determine whether these functional groups of graphene are present on the surface, as in graphene oxide, or on the edges34. Generally, graphene possesses three main features in Raman spectroscopy, G-peak, D-peak and 2D-peak and every peak has different physical origins. The G-peak at about 1580 cm−1 and the D-peak at about 1350 cm−1 are attributed to the sp2-hybridiaed carbon bonds in the graphene lattice and the edge or defects in the lattice, respectively18. From Fig. −1 and the larger intensity ratio of the 2D-peak to the G-peak (I2D/IG) imply the few layer feature of graphene18. The amount of –OH and –COOH groups was also evaluated from the thermogravimetric analysis (TGA), which suggests ~2.26 wt% content of hydrophilic groups in the functionalized graphene edges , Fourier transform infrared spectroscopy (FTIR), and Raman spectroscopy. XPS spectrum of powder samples is shown in Fig. −1and centrifuged at 2000 rpm for 30 min, and the supernatant was collected as stable graphene dispersion. The concentration of aqueous GNPs dispersion was calculated to be ~0.62 g L−1 , which is the highest at present report based on dispersion in pure water. Although stability seems to decrease over time in the case of room temperature storage, we observed that precipitation ceases completely after 30 days and that the solutions maintain their same colour for longer than six months. For example, the aqueous dispersions with different concentrations about 0.015~0.5 g L−1 were unchanged after stored for six months at room temperature  with a PTFE membrane with size of 0.22 μm. These films can be easily peeled from the filter membrane. They tend to be shiny on the side which faced the membrane but matt black on the other side. It is well known that carriers must cross many inter-sheet junctions as they pass through the graphene films, however, which has been confirmed that such junctions limit the conductivity of graphene films. Thus, the graphene film was sandwiched between two thin and smooth polyimide films and pressed under a pressure of 30 MPa for 20 min to get a highly densed compress graphene film with densities of 1.75–1.85 g cm−3 (and so porosities of 20–25%). We expect that electrical conductivity of graphene film can be significantly improved by mechanical compression. As displayed in Fig. 4 S m−1 before annealing and can be further improved to 4.8 × 104 S m−1 after annealing at 500 °C for 2 h, which is much higher than the most of graphene films prepared by similar solution exfoliation36. Because the films were produced through a slow process, the annealing would be more ordered and decrease the content of oxygen-containing groups on graphene surface, which would result in the electrical conductivity enhancement. As a result, this level of conductivity is quite encouraging that the graphene films would be used as an electrode for electric devices.With the rapid development of the electronics industry, conductive paper has become increasingly critical in enhancing the performance and reliability of the devices. GNPs have been used as fillers in polymer composites to considerably enhance the electrical conductivity, which is particularly important for applications in electronic packaging. For the purpose of developing a conductive material, we propose to construct an ultrathin conductive film made of GNPs only. Therefore, the as-prepared GNPs were used as building blocks for the fabrication of a free-standing conductive paper by a simple filtration process. Graphene paper was prepared by filtrating 0.1 g L−1 with a shelf life of more than months at room temperature. This approach has several advantages including simple-step, low-cost, even without any use of expensive or sophisticated equipment. This remarkable feat results from the graphene-based layered materials. We have achieved graphene conductive film with water dispersed graphene material, which thus have significant potential for real-life applications.In summary, we present an effective and green method for large-scale synthesis of few-layered. This ability to use pure water to exfoliate and disperse 2D materials will enable not only cost-effective industrial and commercial applications but also facilitate research that requires water-based experiments. The results of our experiments imply that water vapor exfoliation at a high temperature results in the synthesis of graphene with good dispersion stabilities in water. As-prepared graphene can be highly dispersed in water, without any additives, at a concentration of 0.55 g LSupplementary Information


In both neuronal types, the cell nuclei forfeit their normal (non-phosphorylated) expression of the 43-kDa transactive response DNA-binding protein (TDP-43). Here, we present initial evidence that in α-motoneurons the loss of normal nuclear TDP-43 expression is followed by the formation of phosphorylated TDP-43 aggregates (pTDP-43) within the cytoplasm, whereas in Betz cells, by contrast, the loss of normal nuclear TDP-43 expression remains mostly unaccompanied by the development of cytoplasmic aggregations. We discuss some implications of this phenomenon of nuclear clearing in the absence of cytoplasmic inclusions, namely, abnormal but soluble  cytoplasmic TDP-43 could enter the axoplasm of Betz cells, and following its transmission to the corresponding α-motoneurons in the lower brainstem and spinal cord, possibly contribute in recipient neurons to the dysregulation of the normal nuclear protein. Because the cellular mechanisms that possibly inhibit the aggregation of TDP-43 in the cytoplasm of involved Betz cells are unknown, insight into such mechanisms could disclose a pathway by which the development of aggregates in this cell population could be accelerated, thereby opening an avenue for a causally based therapy.Two nerve cells types, Betz cells in layer Vb of the primary motor neocortex and  Selecteposition , 71; (3)2O2 and 80 % Tris. Following pretreatment with 100 % formic acid for 3 min to facilitate the immunoreactions, blocking with bovine serum albumin was performed to inhibit endogenous peroxidase and to prevent nonspecific binding. Subsequently, each of the sets of free-floating 100 µm sections was incubated for 18 h at 20 °C using the primary antibodies. Subsequent to processing with a corresponding secondary biotinylated antibody  for 1.5 h, all immunoreactions were visualized with the avidin–biotin complex  for 2 h and the chromogen 3,3′-diaminobenzidine tetrahydrochloride . Omission of the primary antiserum resulted in non-staining. Positive as well as negative control sections were included. The tissue sections were cleared, mounted, and cover-slipped in a medium with a refraction index of 1.58 . All sections were viewed and neuropathological staging was performed with an Olympus BX61 microscope . A plan apochromat 40:1 lens was utilized to evaluate and photograph individual Betz cells. Digital micrographs were taken with an Olympus XC50 camera using the analysis® Soft Imaging System .Tissue sections for immunohistochemistry were treated for 30 min in a mixture of 10 % methanol plus 10 % concentrated (30 %) HTo investigate what proportion of Betz cells in a single section displayed (A) a normal nuclear TDP-43 or (B) a reduced nuclear TDP-43 immunoreactivity in comparison to the proportion of those (C) showing complete loss of nuclear TDP-43 immunoreactivity with an empty somatic cytoplasm or with traces of aggregates, and to determine (D) how many Betz cell had been lost (the presence of lipofuscin pigment remnants in the immediately surrounding neuropil was used as a marker for cell loss), one observer (HB) examined a single slide from two sALS cases at neuropathological stage 1 Table , A–D. Coα-motoneurons, a TDP-43 immunonegative cell nucleus was consistently accompanied by pTDP-43-immunopositive inclusions in the cytoplasm of the cell body  In Betz pyramidal cells, a TDP-43 immunonegative cell nucleus Fig. a–i or byIn midline sections through the primary motor neocortex of sALS brains, not only a large number of uninvolved Betz giant pyramidal cells were observed in layer Vb between stages 1 and 4 but also noticeable fluctuations in the degree of their intranuclear TDP-43 immunoreactivity Table . Betz ceNotably, most Betz cells in which the intranuclear immunostaining was either extremely weak or absent also failed to display TDP-43-immunopositive cytoplasmic inclusions  sites becomes involved first. Nevertheless, it is remarkable that both of the sites that become involved very early, although spatially distant from one another, are directly connected by axonal contacts.In the cohort examined, evidence during stage 1 of TDP-43 immunonegative nuclei in Betz cells that occurred in the absence of bulbar and spinal pTDP-43-immunopositive During subsequent sALS stages, additional nerve cell types are drawn into the pathological process: Parvocellular projection neurons of the red nucleus that receive cortico-rubral afferents become involved in stage 2 and consistently display pTDP-43 immunopositive cytoplasmatic inclusions . Given tα-motoneurons [The question also arises of how much importance should be assigned to the monosynaptic contacts between the cortical Betz cells and bulbar/spinal oneurons , 45, 56.oneurons , 50, 69.The existence of even the most subtle cytoplasmic inclusions in isolated Betz cells Fig. g could mα-synuclein [α-motoneurons in the spinal cord and lower brainstem of the cases examined  contacts to bulbar and spinal oneurons –46, 63. l tracts , 20. By l tracts , 58, 68.l tracts , 72. Witl tracts .α-motoneurons may be induced via corticofugal axons and via complete synapses that directly contact these neurons. These secondarily involved subcortical nerve cells could convert the pTDP-43 in their cytoplasm into insoluble inclusions and thereby prevent further propagation of the pathology. If these assumptions were to prove accurate and pending additional studies, it would be important to identify the cellular mechanisms that might cause TDP-43 in the cytoplasm of susceptible neocortical projection neurons to persevere there in a soluble state [The considerations above are related to the idea that sALS may be primarily a disorder of the human neocortex , 20. Thile state . Of simile state , 59, 69.α-motoneurons [α-motoneurons would be taken up by or transferred to Betz cell axons and transported retrogradely to their somata, where physiological TDP-43 function could become perturbed without TDP-43 phosphorylation or with only minimal phosphorylation, thereby leading to a dysmetabolism of the Betz cells in the complete absence, or virtual absence, of somatic cytoplasmic aggregates. Convincing morphological evidence for a retrograde degenerative process does not currently exist. Although additional studies, including experimental models, are required to test both hypotheses [In contrast to the hypothesis of anterograde degeneration discussed above, a ‘dying-back’ theory has been proposed, according to which the pathological process underlying sALS could originate either in spinal cord oneurons , 18, 65,oneurons , or in moneurons . This wopotheses , the cha


CTDSPL and CTDSPL2 as common integration sites in ALV-induced B-cell lymphomas, suggesting a potential role in driving oncogenesis. We show that in tumors with integrations in these genes, the viral promoter is driving the expression of a truncated fusion transcript. Overexpression in cultured chick embryo fibroblasts reveals that CTDSPL and CTDSPL2 have oncogenic properties, including promoting cell migration. We also show that CTDSPL2 has a previously uncharacterized role in protecting cells from apoptosis induced by oxidative stress. Further, the truncated viral fusion transcripts of both CTDSPL and CTDSPL2 promote immortalization in primary cell culture.Avian leukosis virus induces tumors in chickens by integrating into the genome and altering expression of nearby genes. Thus, ALV can be used as an insertional mutagenesis tool to identify novel genes involved in tumorigenesis. Deep sequencing analysis of viral integration sites has identified  MYC, MYB, TERT, mir-155, MET and EGFR, have been seen in past screens [Avian leukosis virus (ALV) is a simple retrovirus that induces tumorigenesis by integrating into the host genome and altering expression of cellular genes . ALV typ screens .CTDSPL  and CTDSPL2  as common integration sites in ALV-induced B-cell lymphomas [in vitro [Our lab has previously identified ymphomas . The recymphomas . The CTDin vitro , 12. Thrin vitro , 12. CTDin vitro .CTDSP1/2/L genes have all been found to contain an intronic microRNA that belongs to the miR-26 family. These miRNAs have been shown to act synergistically with the CTDSP1 and CTDSPL proteins to dephosphorylate, and thus activate, pRb and block the G1/S cell cycle transition [The CTDSP proteins are able to act on additional targets as well. For instance, CTDSP1/2/L proteins have been shown to induce TGF-β signaling and attenuate BMP signaling , 16. CTDansition . CTDSP2 ansition .CTDSPL has been characterized as a tumor suppressor gene that is frequently deleted or mutated in many major epithelial cancers such as lung, renal cell and breast carcinoma [Due to involvement in these pathways, it comes as no surprise that the CTDSP1/2/L and the miR-26 family have been implicated in tumorigenesis. arcinoma , 21–23. arcinoma . Comparaarcinoma , 25. HowCTDSPL2 as a novel gene involved in oncogenesis and further characterize the role of CTDSPL. Specifically, we investigate the function of viral induced truncations of both genes in cancer. Overexpression of CTDSPL and CTDSPL2 leads to changes in expression of ribosomal genes and genes involved in cellular migration and metabolism. We show that overexpression of both CTDSPL and CTDPSL2 causes accelerated cellular migration in primary cell culture. Interestingly, expression of CTDSPL2, but not CTDSPL, protects cells from apoptosis induced by oxidative stress, indicating that the two genes may not be redundant. Importantly, the truncated viral fusion transcripts of both CTDSPL and CTDSPL2 promote immortalization when overexpressed in primary cell culture.In this work, we characterize High throughput sequencing was used to identify retroviral integration sites in ALV-induced B-cell lymphomas . IntegraCTDSPL and CTDSPL2 were identified to be common integration sites previously [CTDSPL in 12 tumors from 7 birds. All expanded integrations are in the same transcriptional orientation as CTDSPL and fall upstream of exon 4  and secondary (liver and kidney) tumors found in the same bird  were infected with retroviral vectors (RCAS(A)) carrying either the truncated or full-length transcript of either CTDSPL or CTDSPL2. Transcripts were overexpressed approximately 100-fold relative to wild type CEF expression.To better characterize the role of CTDSPL or CTDSPL2. Cufflinks was used to detect genes differentially expressed in cells carrying a CTDSPL or CTDSPL2 construct relative to cells infected with an empty retroviral construct.Both CTDSPL and CTDSPL2 are believed to act on the CTD of RNA polymerase II to regulate gene expression . We reasCTDSPL or CTDSPL2 had the most similar changes in gene expression profiles with approximately 1/3 of the deregulated genes overlapping between the two conditions, suggesting that they may play partially redundant roles.We observed between 4 and 30 genes differentially expressed in each condition Figure . There wCTDSPL or CTDSPL2 genes, we performed a GO analysis of genes differentially expressed between the full length and truncated form of both CTDSPL and CTDSPL2 separately . The truncated form of CTDSPL2 had a more modest effect with 18% closure observed on average  signaling through dephosphorylation of Smad proteins [CTDSPL and CTDSPL2 transcripts.We show that viral integrations in proteins . This haproteins . Furtherproteins . This roproteins . The proCTDSPL and CTDSPL2 promotes immortalization of primary cells in culture. This is a feature unique to the truncated transcripts, as overexpression of full-length forms of both genes did not significantly improve proliferation rates at times past the normal lifespan of CEFs. We believe that this role in immortalization is likely the reason that integrations promoting the expression of truncated forms of both genes are selected for in ALV-induced B-cell lymphomas. This role in immortalization for CTDSPL and CTDPSL2 is interesting to note due to the co-occurrence of CTDSPL and CTDSPL2 integrations with integrations into TERT, which has previously been reported to promote immortalization [The overexpression of truncated viral fusion transcripts of both lization .CTDSP1/2/L are fairly well characterized genes that have been repeatedly shown to play partially redundant roles. CTDSPL2 seems to be fairly similar to the other members of the CTDSP family in many regards. Some functions are known to overlap, such as regulation of BMP signaling. Here we show that CTDSPL2 promotes metastasis similar to CTDSPL. These overlapping functions, in addition to the observation that despite integrations in both genes, only one is overexpressed in individual tumors, would suggest that CTDSPL and CTDSPL2 might be redundant. However, we observed that expression of CTDSPL2, and not CTDSPL, can protect cells from apoptosis induced by oxidative stress.CTDSP1/2/L genes have an intronic microRNA from the miR-26 family. No intronic microRNA has been reported in CTDSPL2. The CTDSPL2 protein, at 53 kDa, is significantly larger in size than CTDSP1/2/L proteins, which all weigh in at around 32 kDa on average. Each protein in the family contains a C-terminal phosphatase domain, but CTDSPL2 has significantly more N-terminal sequence of unknown function. The N-terminal region that is truncated in the viral fusion transcript is predicted to be intrinsically disordered . Fusion transcripts were detected by performing PCR with a forward primer in gag immediately before the viral splice donor (TCAAGCATGGAAGCCGTCATAAAG) and a reverse primer within the gene of interest .RNA was extracted using RNA-Bee reagent . cDNA was prepared using Maxima H reverse transcriptase with an oligo(dT)Protein order was predicted using PONDR  VL-XT . StretchCTDSPL  or CTDSPL2 . A housekeeping gene, GAPDH, was used as an internal reference [t method (ΔΔCt)qPCR to quantify transcript abundance was performed using PowerUp SYBR Green Mastermix (ThermoFisher Scientific) according to the manufacturer’s protocol on a BioRad C1000 thermocycler / CFX96 Real-Time System. Expression was measured using primers in either 2 in 199 media supplemented with 1% chick serum, 1% calf serum, and 2% tryptose phosphate. Overexpression constructs were generated by cloning either full length or a truncated transcript into the RCAS(A) viral expression vector [CTDSPL truncated construct begins at exon 4 (nt 281 from transcription start site in cDNA); CTDSPL2 truncated construct begins at exon 3 (nt 348 from transcription start site in cDNA). Virus was generated via electroporation of constructs into CEFs with subsequent collection of the viral supernatant.Chicken embryonic fibroblasts (CEFs) were maintained at 39°C, 5% CO6 cells in a 10 cm dish at time 0. To induce apoptosis, cells were treated with 50 μM H2O2 as described [Cells were seeded at 0.8 × 10escribed . After 4A scratch assay was used to detect differences in cell migration as described previously . Briefly


In 2016 and 2017, monoclonal antibodies targeting PD-L1, including atezolizumab, durvalumab, and avelumab, were approved by the FDA for the treatment of multiple advanced cancers. And many other anti-PD-L1 antibodies are under clinical trials. Recently, the crystal structures of PD-L1 in complex with BMS-936559 and avelumab have been determined, revealing details of the antigen-antibody interactions. However, it is still unknown how atezolizumab and durvalumab specifically recognize PD-L1, although this is important for investigating novel binding sites on PD-L1 targeted by other therapeutic antibodies for the design and improvement of anti-PD-L1 agents. Here, we report the crystal structures of PD-L1 in complex with atezolizumab and durvalumab to elucidate the precise epitopes involved and the structural basis for PD-1/PD-L1 blockade by these antibodies. A comprehensive comparison of PD-L1 interactions with anti-PD-L1 antibodies provides a better understanding of the mechanism of PD-L1 blockade as well as new insights into the rational design of improved anti-PD-L1 therapeutics. PD-1 is a type I membrane protein with a single extracellular immunoglobulin superfamily (IgSF) V-set domain that is expressed on the surface of activated T cells in peripheral tissues5. PD-L1 and PD-L2 are commonly expressed on dendritic cells and macrophages, and their ectodomains are composed of a membrane distal IgSF V-set and a membrane proximal IgSF C-set domains7. Ligation of PD-1 with its two ligands initiates co-inhibitory signaling through the cytoplasmic domain of PD-1, containing an immunoreceptor tyrosine-based inhibitory motif and an immunoreceptor tyrosine-based switch motif, thus leading to activation of SHP phosphatases that downregulate TCR signaling by dephosphorylating effector molecules involved in the signaling9. As a result, PD-1 signaling prevents excessive or harmful inflammation and maintains immune tolerance to self-antigens under normal conditions10.Programmed death 1 (PD-1) and its ligands PD-L1 and PD-L2 are key co-inhibitory molecules in the modulation of T-cell mediated immune responses11. Thus, blocking the PD-1/PD-L1 interaction can restore T-cell activation and antitumor responses14. The success of antibody-based PD-1/PD-L1 blockade therapies has provided a major breakthrough in the fight against human cancers, especially for solid tumors. The FDA approved the first anti-PD-L1 drug, atezolizumab (Tecentriq®), for the treatment of advanced urothelial carcinoma in May 2016 and metastatic non-small cell lung cancer (NSCLC) in October 201618. In 2017, avelumab (Bavencio®) and durvalumab (Imfinzi®) were also approved by the FDA for Merkel-cell carcinoma and advanced bladder cancer, respectively  or complement-dependent cytotoxicity (CDC), thereby preventing the depletion of T cells expressing PD-L133.Combinations of anti-PD-1/PD-L1 antibodies with other therapies are being explored as potentially synergistic therapeutic strategies. Among these, the combination of anti-PD-1/PD-L1 and anti-CTLA-4 antibodies has been the most successful, probably because PD-1/PD-L1 and CTLA-4/B7 interactions play different roles in T-cell immunity37. Recently, the crystal structures of PD-L1 in complex with the Fab fragment of BMS-936559 and the single-chain Fv fragment (scFv) of avelumab have been determined, revealing details of the antigen-antibody interactions39. However, the epitopes and the PD-L1 blocking mechanism of atezolizumab and durvalumab remain unclear. It is also unknown whether there are novel binding sites on PD-L1 for the design and improvement of anti-PD-L1 agents and whether atezolizumab and durvalumab utilize a similar or distinct competitive binding mode to PD-L1 compared with other anti-PD-L1 antibodies for the development of better combination therapies.The structures of murine PD-1 in complex with human PD-L1, murine PD-1 in complex with murine PD-L2, and human PD-1 in complex with human PD-L1 have established the structural foundations of the interaction of PD-1 with its ligands, PD-L1 and PD-L2In the present study, we report the crystal structures of the N-terminal IgSF V-set domain of PD-L1 in complex with the Fab fragments of atezolizumab and durvalumab, thereby elucidating the structural basis for the blockade of the PD-1/PD-L1 interaction by these therapeutic antibodies. In addition, a comprehensive analysis of the PD-L1 interactions with the receptor PD-1 and anti-PD-L1 antibodies, including atezolizumab, durvalumab, BMS-963559, and avelumab, provides new insights into the development of future anti-PD-L1 therapeutics, including small-molecule modulators and next-generation therapeutic antibodies.E. coli and refolded in vitro to obtain a soluble form. The Fab fragments of the anti-PD-L1 antibodies were produced by periplasmic expression in E. coli. Gel filtration analysis confirmed that that the 1:1 complexes of PD-L1 with each antibody exist as monomers in solution. The crystal structure of the IgSF V-set domain of human PD-L1 in complex with the atezolizumab Fab fragment was determined at a resolution of 3.10 Å with R/Rfree = 0.208/0.256 . Most of the buried solvent accessible area is contributed by the heavy chain (67%)  Fig. . The ate7%) Fig. . In tota7%) Fig. . Of couries Fig. 34, 38, 32, which is smaller than the PD-1/PD-L1 interface by 346 Å2. The binding interface is almost equally contributed by both the heavy and light chains  for the binding of avelumab-scFv to PD-L1 has been reported to be 42.1 pM by surface plasmon resonance (SPR) and other antibodies are also known to have Kd values less than 1 nM, whereas the binding affinity between PD-1 and PD-L1 was reported as 8.2 μM by SPR42. The buried surface areas of PD-L1 in complex with PD-1, atezolizumab, durvalumab, BMS-963559, and avelumab are 1970, 2106, 1624, 1349, and 1865 Å2, respectively  competitive antagonist of PD-1 showing superior tumor penetration43. There are also several small-molecule immunomodulators targeting the PD-1/PD-L1 axis in preclinical or clinical investigations46. Structural studies on the PD-L1 interaction with therapeutic antibodies can provide insight into the design of small molecules targeting PD-L1, as their potency can be enhanced by mimicking the diverse interactions of these antibodies, including the involvement of the BC, CC′, C′C″, and FG loops for binding to atezolizumab; the CC′ loop and N-terminal region for durvalumab; the BC, C′C″, and FG loops for BMS-963559; and the CC′ loop for avelumab. We also believe that the accumulation of such structural studies will provide invaluable information for developing next-generation therapeutic antibodies, such as antibody drug conjugates (ADCs) and bi-specific antibodies, and for coping with any possible antigen mutational escape of PD-L1 in future.Cancer immunotherapy via antibody-based PD-1/PD-L1 blockade has provided a major breakthrough for the treatment of multiple advanced and metastatic cancers since the approval of the monoclonal antibodies targeting the PD-1/PD-L1 axis. Despite the great achievement of the therapeutic antibodies blocking the PD-1/PD-L1 interaction, they have specific shortcomings as therapeutics. Poor tissue/tumor penetrance of antibody drugs due to their large size can be problematic, especially when targeting PD-1/PD-L1 signaling as PD-1-expressing T cells are found infiltrated within the solid tissue of PD-L1-expressing tumors47. PD-L2-deficient mice exhibit increased airway hyperactivity and lung inflammation48. While PD-1 inhibition by anti-PD-1 antibodies disrupts both PD-L1 and PD-L2 pathways, anti-PD-L1 antibodies, including atezolizumab, durvalumab, BMS-963559, and avelumab, are known to target only PD-L1 to inhibit the PD-1/PD-L1 interaction while preserving the PD-1/PD-L2 interaction, thereby avoiding the immune-related toxicity associated with PD-L2 blockade24. The complex structures of PD-L1 with these antibodies explain their lack of binding to PD-L2  as inclusion bodies. The cells were grown at 37 °C in LB medium supplemented with 50 μg mL−1 ampicillin until OD600 reached 0.6–1.0, and the protein expression was induced with 1 mM IPTG and incubated for 4 h at 37 °C. The cells were harvested by centrifugation, re-suspended in lysis buffer  and lysed by sonication on ice. Inclusion bodies were recovered by centrifugation  and solubilized in 8 M urea, 20 mM Tris, pH 8.0, 200 mM NaCl by stirring overnight. After removing undissolved residue by centrifugation , solubilized fraction was applied to HisTrap HP column  and washed with five column volumes of wash buffer . The protein was then eluted with elution buffer . The eluted protein was refolded by dialysis 3 times against 20 mM Tris, pH 8.0, 200 mM NaCl and purified further by gel filtration chromatography using a HiLoad 16/60 Superdex 200 pg column . The protein purity was evaluated by reducing and nonreducing SDS-PAGE.Genes encoding the IgSF V-set domain of human PD-L1 (aa 18-134) were subcloned into pET-21a (Novagen). The protein was expressed in E. coli . The sequences for heavy chain and light chain were cloned into a modified pBAD vector, containing the STII signal sequence in each chain for periplasmic secretion and a C-terminal 6His-tag in heavy chain38. The plasmid pBAD-Fab was transformed into E. coli Top10F (Invitrogen). The cells were grown at 37 °C in LB medium supplemented with 50 μg mL−1 ampicillin. At an OD600 of 1.0, the protein expression was induced with 0.2% arabinose and cells were grown at 30 °C for 15 h. The cells were harvested by centrifugation, re-suspended in lysis buffer  and lysed by sonication on ice. After removing cell debris by centrifugation , the supernatant containing soluble protein was applied to HisTrap HP column  and washed with five column volumes of wash buffer . The protein was then eluted with elution buffer . The eluted protein was concentrated for gel filtration chromatography using a HiLoad 16/60 Superdex 200 pg column . The column had previously been equilibrated with gel filtration buffer . The elution profile of the protein showed a single major peak and the protein quality was evaluated by reducing and nonreducing SDS-PAGE.The DNA sequences for the Fab fragments of atezolizumab and durvalumab were synthesized after codon-optimization for expression in Details of the crystallization, X-ray data collection, structure determination, and refinement of the PD-L1/atezolizumab Fab and PD-L1/durvalumab Fab complexes are described in Supplementary Information. Data collection and refinement statistics are summarized in Table http://www.rcsb.org) under the accession codes 5X8 L and 5X8 M, respectively.The atomic coordinates and structure factors for the structures of PD-L1 in complex with the atezolizumab and durvalumab Fab have been deposited into Protein Data Bank (Supplementary Information