HT), assisting in enhancing the electronic conductivity coupled with the mechanical robustness of VNBs. From electron microscopy, surface sensitive spectroscopy and other complementary structural characterization, hydrothermally-produced rGO nanosheets/nanoribbons are decorated with and inserted within the VNBs’ layered crystal structure, which further confirmed the enhanced electronic conductivity of VNBs. Following the electrochemical properties of GVNBs being investigated, the specific capacitance Csp is determined from cyclic voltammetry (CV) with a varying scan rate and galvanostatic charging-discharging (V–t) profiles with varying current density. The rGO-rich composite V1G3  showed superior specific capacitance followed by VO-rich composite V3G1 (VO/GO = 3:1), as compared to V1G1 (VO/GO = 1:1) composite, besides the constituents, i.e., rGO, rGOHT and VNBs. Composites V1G3 and V3G1 also showed excellent cyclic stability and a capacitance retention of >80% after 500 cycles at the highest specific current density. Furthermore, by performing extensive simulations and modeling of electrochemical impedance spectroscopy data, we determined various circuit parameters, including charge transfer and solution resistance, double layer and low frequency capacitance, Warburg impedance and the constant phase element. The detailed analyses provided greater insights into physical-chemical processes occurring at the electrode-electrolyte interface and highlighted the comparative performance of thin heterogeneous composite electrodes. We attribute the superior performance to the open graphene topological network being beneficial to available ion diffusion sites and the faster transport kinetics having a larger accessible geometric surface area and synergistic integration with optimal nanostructured VO loading. Computational simulations via periodic density functional theory (DFT) with and without V2O5 adatoms on graphene sheets are also performed. These calculations determine the total and partial electronic density of state (DOS) in the vicinity of the Fermi level , in turn complementing the experimental results toward surface/interfacial charge transfer on heterogeneous electrodes.Graphene nanosheets and graphene nanoribbons, G combined with vanadium pentoxide (VO) nanobelts (VNBs) and VNBs forming GVNB composites with varying compositions were synthesized via a one-step low temperature facile hydrothermal decomposition method as high-performance electrochemical pseudocapacitive electrodes. VNBs from vanadium pentoxides (VO) are formed in the presence of graphene oxide (GO), a mild oxidant, which transforms into reduced GO (rGO Likewise, graphene derivatives, including graphene oxide (GO), with various surface functional groups, and its reduced form (rGO), prepared using different reduction methods, are equally promising for various technologies. In general, epoxide (–O–) and carboxyl (–COOH) groups located on the basal plane of the GO nanosheet are major components; carbonyl (–C=O) and hydroxyl (–OH) groups distributed at the edges of GO sheets are minor components. Preferential reduction of basal plane moieties leaving residual and random edge functional sites are produced using chemical (rGO), electrochemical (ErGO) and thermal (rGOHT) reduction methods [x)/hydroxides [x, MnxOy, CoxOy, VxOy, FeOx, NiO, Ni(OH)2 and Co(OH)2, exhibited attractive performance for pseudocapacitor applications. In spite of extensive exploitation of diverse nanocarbons and transition metal oxides as electrochemical electrodes, they are limited due to insufficient electrochemical activity, cycle lifetime and high cost. The continued interest in the strategic combination of TMeOx and low-dimensional carbons is relevant, since it generates synergistic and complementary effects, yielding versatile properties to fit new functions for applied electrochemistry  [Intense research activity on sustainable renewable energy is stimulated by the increasingly global demand of electric energy. Electrochemical energy storage systems (EES), namely electrochemical super- (SCs) and pseudo-capacitors (PCs) and rechargeable secondary batteries, represent the most efficient and environmentally-benign technologies. The need for next-generation stable, high-performance, cost-effective electrode materials (higher energy and power density with longer cycle life) and novel architectures is the driving force for addressing the scarcity of energy availability worldwide while mitigating environmental pollution. While energy is stored in supercapacitors by forming an electrochemical double layer by the adsorption of electrolyte ions on the electrode surface, the energy storage in pseudocapacitors is through actual electron transportation, unlike rechargeable secondary batteries (Faradaic redox reactions) via the conversion between chemical energy into electricity. However, it is noteworthy that in practical electrochemical electrodes, the two energy storage mechanisms often work concomitantly. Nanotechnology has accelerated the innovation of novel electrode formulations to replace conventional carbon-based nanomaterials as supercapacitive cathodes and transition metal oxides as pseudocapacitive cathodes by themselves, which calls for hybrids or composites. Hybrid composites specifically comprising metal-graphene interfaces are generally developed for several electronic, electrochemical, electroanalytical and biological applications. Graphene, which is a seamless sheet of sp methods . Transitdroxides , such assensing) ,6,7,8. Asensing) ,10,11,12sensing) ,17,18,192O5; VO) has been widely investigated as a potential candidate material, because of being a low-cost, abundant resource, having a layered structure, high energy density and wide potential window arising from its multivalent oxidation states (V3+/V4+/V5+) [xOy, augmenting electrochemical (re)activity and, thus, energy storage. Although several studies have shown progress [Among known pseudocapacitive materials offering higher energy density, vanadium pentoxide (VV4+/V5+) ,22,23,24V4+/V5+) ,26,27. Iprogress ,29,30,312O5, chemically-reduced graphene oxide (rGO), hydrothermally-processed rGO (rGOHT) and composites of rGOHT (from GO) mixed with VO micro-particles at VO/GO ratios of 1:1, 1:3 and 3:1 were synthesized. The rGOHT and composites (VO/GO) in the form of powders were prepared using a one-step hydrothermal process, where dispersions of mixed materials (1 mg/mL GO and 0.1 mg/mL VO) with DI water were placed in a Teflon-lined autoclave and placed in a box furnace at constant temperature for a certain deposition time  as a binder and 10 wt % carbon black was formed by mixing these components with NMP (N-methyl-2-pyrrolidone) in a mortar and pestle. The slurry was applied to cleaned substrates using a blade knife, and they were left to dry at room temperature for at least 24 h. After the electrodes had dried, they were placed into a quartz tube furnace in Ar atmosphere for annealing at 120 °C for 4 h.Graphene oxide (GO) was prepared by the modified Hummer’s method followed by chemical reduction using hydrazine monohydrate, producing reduced GO (rGO) . The hydVacc) of 10 kV and a constant current of 45 μA in secondary electron imaging (SEI) mode collected with an in-lens detector equipped with an X-ray ISIS EDS system providing surface morphology. Samples for TEM (transmission electron microscopy) and SAED (selected area electron diffraction) were prepared by placing one to two drops of individual component and of hybrids on commercial lacey carbon-coated Cu mesh grids  and allowed to air dry. They were taken using a JEOL instrument  operating in cryo-EM and SAED modes at 200 kV or 100 kV and 1 nA with a Be specimen holder, a Gresham SiLi detector with a Moxtek AP3.3 window. For SAED patterns, we used a 0.20-μm aperture, producing a small spot size, and spread the beam to increase the electron coherence length at the samples. TEM and SAED measurements provided intrinsic microstructure and nanoscale morphology and determined interplanar spacing. X-ray diffraction (XRD) patterns were obtained with the Siemens Model D5000 instrument  in Bragg–Brentano θ-2θ geometry ranging 2θ from 8° to 60° using a Cu Kα X-ray source (λ = 1.5405 Å) operating at a voltage of 45 kV and a current of 40 mA. Samples were run at a scan rate of 0.04°/s or to improve the signal-to-noise ratio; a scan rate of 0.02°/s was used. Raman spectra were measured to determine the lattice vibration at various regions of interest on the samples. The spectra were recorded using a micro-Raman spectrometer  equipped with a laser providing an excitation wavelength of 633 nm (EL = 1.92 eV) and an ~4–6-mW power incident on the sample, with the edge filter cutting at ~100 cm−1. The scattered light from the sample is collected in backscattering geometry transmitted by a beam splitter and detected by a CCD camera. The reflected light is filtered using an edge filter to remove the laser excitation and sent to a spectrometer. An objective lens of 50× was used providing a spot size of ~1–2 μm, and extreme care was taken to avoid sample damage or laser-induced thermal degradation. Either 5% or 10% light intensity was used to accurately obtain spectra while avoiding thermal degradation. Spectra were acquired at time intervals of 30–60 s, although increased to a few minutes to maximize signal throughput. The Raman shift ranged from 150 to 3200 cm−1 for hybrid composites with a spectral resolution of 1 cm−1. Allowing more details and understanding the interaction between rGOHT and VNB, we measured room temperature electrical (I–V) properties’ in-plane configurations. We made electrical contacts with colloidal silver paste and attached a Cu wire for connection with the Keithley 2400 source meter . We measured in-plane two-point contact resistance and determined room temperature electrical conductivity (σdc) for all of the samples studied. The hybrids apparently displayed semiconducting  behavior, as anticipated. Quantitatively, the electrical conductivity measured for these hybrids ranged from 0.01 to 2.1 s/cm  images were taken with an instrument  operating at primary electron acceleration voltage . CV was conducted in the potential window of 0.0–0.8 V at scan rates between 2.5 and 200 mV/s in 0.5 M sodium sulfate (Na2SO4) electrolyte using a Ag/AgCl reference electrode (RE), Pt wire as the counter electrode (CE) and the working electrode (WE) as prepared samples. Electrochemical impedance spectroscopy (ac EIS) was performed at open circuit voltage and +0.2 V with superimposed 5 mV ac signal within a frequency range from 0.01 Hz to 98 kHz. Galvanostatic charging-discharging tests were conducted using cathode and anode currents ranging between 2.5 and 150 mA (or 0.5 A·g−1 and 30 A·g−1). Each test allowed the cell to charge for 10 s or until its voltage reached ~1 V, at which point, the cell began discharging, and this cycle was repeated >500 times.Electrochemical tests were performed on those samples prepared on nickel foam substrates using an electrochemical bi-potentiostat workstation  including cyclic voltammetry (CV), galvanostatic charging-discharging EF) and the density matrix calculations (Gilat grid) [−9 Hartrees for clean graphene substrates and the adatom-graphene systems to 10−7 Hartrees, suggesting covalent and electrostatic bonding.Density functional theory is employed to determine the density of state s and charge transfer between graphene and VO. Briefly, a monolayer of graphene is modeled as a two-dimensional hexagonal lattice with a calculated lattice constant of 2.45 Å, which is slightly lower than the corresponding experimentally-measured value for graphite reported at 2.46 Å . The adaRYSTAL09  program RYSTAL09 ,38 was eRYSTAL09 , which aRYSTAL09 ,41  ,44. The HT, crystalline VNB and their composites along with the EDS image and elemental spectra  and pressure (greater than a few atmospheres) in an autoclave, GO with oxygenated surface functionalities acts as a mild oxidizing agent to promote the synthesis of nanostructured VO  in DI water. They reveal a relatively uniform surface morphology by themselves and as hybrid composites, wherein the interconnected network and crumpled silk-liked structured graphene nanosheets (GNS) as larger nanoribbons are uniformly blended with VNBs with a lateral size range of 50–100 nm, which could prevent the commonly-observed restacking of GNS. To investigate the structural information of the components and hybrids at the nanoscale, TEM combined with SAED results are presented in dhkl)  u revealsHT, VO and GVNBs are investigated by X-ray diffraction (XRD), shown in HT is somewhere in between the GO precursor and the traditional graphene, and that of VO matched the corresponding bulk pattern (JCPDS Card No. 89-0612). Qualitatively, the relatively sharper peaks are reflective of the medium range order of the nanocrystalline grain size distribution from Debye–Scherrer’s equation [3G1, V1G1 and V1G3) of GVNBs contain peaks of GO, rGOHT and VNBs. The peaks at 15.2°, 22.1°, 25.9°, 28.5°, 32.0° and 41.7° correspond to the (200), (101), (110), (111) and (002) planes of VNBs, respectively [101) was determined to be 0.584 nm [HT [HT. Interestingly, the GVNBs show sharp characteristic peaks associated with both VO and rGO. Toward further gaining insights into the chemical structure of hybrids, we used nondestructive Raman spectroscopy (RS) to probe chemical structural bonding. −1 for all of the samples studied. For a realistic comparison, the spectra are normalized to the intense spectral band at a wavenumber of ~1595 cm−1. Raman spectra show characteristic crystalline VO peaks (marked) appearing at 218, 267, 310, 360, 486, 529, 586, 768 and 982 cm−1 ascribed to the A1g, B1g and B3u normal modes of VxOy (see −1 (disorder-induced D band) and 1595 cm−1  correspond to the breathing mode of κ-point phonons of A1g symmetry and the first-order scattering of E2g phonons of graphene, respectively. The other Raman peaks of interest are second-order bands at 2670 cm−1 and 2920 cm−1 assigned to the 2D band and a combination of the D + G band, respectively [The crystalline structures of pristine rGOequation . The XRDectively . The int0.584 nm ,51. The 4 nm [HT . General4 nm [HT ,54. WhenVxOy see c, box [53G1 and rGOHT with scan rates ranging from 2.5 to 200 mV/s, in 1 M Na2SO4 electrolyte. The CV curves show a quasi-rectangular behavior, confirming the double-layer capacitance of these composite electrodes. The vanadium-rich composite (V3G1) shows broader redox peaks with retention of the quasi-rectangular shape in the potential window of 0.0–+0.8 V. The CV from graphene derivatives are in stark contrast to the hybrids, which are nearly rectangular loops, indicative of almost an ideal supercapacitor. Specifically, all hybrid electrodes displayed well-defined pseudocapacitive behavior at a scan rate of 5 mV/s with two redox peaks at +0.18/+0.3 V (cathodic I/anodic II) and +0.45/+0.5 V (cathodic III/anodic IV) related to presumably faradaic (redox) reactions relating to the conversion between different vanadium oxidation states. Moreover, the VNBs have a layered crystal structure and multivalent oxidation states of vanadium ions. These properties facilitate the insertion and extraction of alkali-metal electrolyte ions  in the vicinity of the electroactive material. The electrochemical sodium-ion insertion process can be expressed as follows [+ into layered VNBs with simultaneous electron transfer, i.e., the partial reduction of V5+ to V4+ (and vice versa during oxidation), and thus, provides pseudocapacitance to VxGy composites. v1/2), and the quasi-linear behavior, especially at higher scan rates, is reminiscence of diffusion-limited (mass transport) phenomena and reflective of heterogeneous diffusion attributed to the composite nature of hybrid electrodes. The magnitude of the current observed is governed by the Randles–Ševćik equation for a reversible transfer process, A is the geometric area of the electrode (cm2), α is the transfer coefficient , F is the Faraday constant (C·mol−1), D is the diffusion coefficient (cm2/s), C is the concentration (mol/cm3), v is the scan rate (V/s), R and T are the usual constants, n (=1) is the total number of electrons transferred in the electrochemical process and n′ (=0.6) is the number of electrons transferred before the rate determining step. The analysis of the current helped to determine the D coefficient that ranged between 2 × 10−9 and 8 × 10−9 m2·s−1 for all of the samples studied. The larger integrated area CV curves for hybrids implies that they have higher specific capacitance (Cs), as discussed below. The specific gravimetric capacitance (Cs) of the hybrid electrodes is calculated according to the following equation: m is the mass of the active electrode material (g) measured using a micro-balance. Cs with the scan rate for all of the electrodes that show a gradual decrease in Cs with the scan rate peaking at a scan rate of 5 mV·s−1, being 450, 350 and 180 F·g−1 for V1G3, V3G1 and rGOHT, respectively, as an upper bound, considering that the average mass of the electrode material is 5 mg. We also measured charging-discharging (V–t) profiles shown in t is the discharge time after IR drop, ΔV is the discharge potential window after IR drop and m is the mass of the single electrode material, respectively. The factor of 2 is used because series capacitance is formed in a two-electrode cell system. The V–t profiles between 0 and 1.0 V at different current densities and cyclability of various electrodes exhibits stable performance for more than 500 cycles, and the fading in Cs is possibly due to internal resistance and polarization of the electrodes. The Cs values are comparable at a specific current density 10 A·g−1 to those determined using CV curves. Moreover, Cs of the hybrid electrodes was significantly larger than that of rGO-only electrodes with values of ~60–70 F·g−1. Generally, the rate capability is strongly influenced by ion diffusion in the electrolyte, the surface adsorption of ions on the electrode materials (electrode/electrolyte accessibility) and the charge transfer in or on the electrode. At higher scan rates, any of these three processes is relatively slower, which limits the rate, lowering the Cs. Nevertheless, the higher specific capacitance is attributed to smaller size vanadium pentoxides nanobelts, resulting in high specific capacitance. Although GNS have a supplementary (supercapacitive) contribution to the hybrid composites, they have excellent electronic conductivity and, thus, shuttle the electrons between vanadium-oxide nanostructures and the current collector. Therefore, the chemical integration between rGOHT-vanadium pentoxide nanobelts (VNB) into a single system enhanced the electrochemical behavior of pseudocapacitive electrodes. The synergistic effects of chemical bridging  and V4+/V5+ ions), the crumpled and flower-like surface morphology promoted tailored properties and interfaces and topologically interconnected network architectures [mGn) composite materials. Following the + into layered VNBs with simultaneous electron transfer, i.e., the partial reduction of V5+ to V4+ (and vice versa during oxidation) and, thus, provide pseudocapacitance to VmGn composites. It is important to note that the composite electrodes possess higher Cs values than those of the constituents. follows ,62: V2O5tectures ,64,65. WZ″ versus Z′) are shown in Rs, equivalent series resistance (ESR), which includes ionic or bulk resistance of the electrolyte solution combined with interface resistance, intrinsic and contact resistance at the electroactive material/current collector [Rct, charge transfer resistance, ZW, Warburg impedance, Cdl, double layer capacitance, as in Randles’ equivalent circuit; and (2) the constant phase element (CPE) equivalent circuit [Ls) to account for self-inductance of the wire leads, a capacitive element (Ce), solution (Rs) and charge transfer resistance (Rct), Warburg impedance (ZW) and CPE , replacing the capacitor used to characterize double-layer capacitance (Cdl) in Randles’ circuit. By definition [ZCPE = Yo = Qo (ω)n en/2-iπ; Qo = 1/|Z| at ω = 1 rad/s, n = 1 is for an ideal capacitor and n = 0, a pure resistor. In principle, the ESR from Circuit Model 1 determined from the high-frequency intercept at real Z′ is smaller for the composite hybrid electrodes (<1.5 Ω) than those of only component electrodes (see Rct) caused by the pseudocapacitive behavior of electroactive material (electrode), double-layer charging on the electrode surface (Cdl) and Warburg impedance (ZW) [Ce) is representative of the dispersion of time-constants in the samples . The dimensionless parameter n in HT and the porous network. HT, V2O5, V1G3 and V1G1. For the macroelectrode and diffusion layer of infinite thickness, ZW = Rct λ/(ω)1/2 and kET is the heterogeneous kinetic rate constant on the electrode and D is the diffusion coefficient of the redox species. The increasing slope trend exhibits the capacitive nature related to the film charging mechanism that is typically characteristic for mesoporous electrodes. The relatively lower Rct values for all of the hybrid electrodes studied reflect an enhancement in the electronic and ionic conductivities of vanadium pentoxides adsorbed on or intercalated within GNS (see D and k). Finally, the slope of the Nyquist plots in the low frequency region tends to increase with the presence of underlying graphene support films, reflecting a decrease in the Warburg resistance (ZW) or faster electrolyte ion diffusion into the hybrids. Clf variation derived from the following relationship: Clf peaks for V1G3 and V3G1 hybrid electrodes are consistent with the results obtained above using CV and galvanostatic measurements. The presence of rGOHT creates more electroactive sites and tailored interfaces, in turn facilitating the access of electrolyte ions and making electron transport between GNS and VNBs easier. This corroborates the presumption that these hybrids can be electrically simulated to an assembly of parallel RC omponents normal to the electrode surface. The energy density In order to further understand the reasons behind the unique performance of hybrid electrodes, electrochemical impedance spectroscopy (EIS) data were analyzed to investigate electrode kinetics and to quantify electronic and ionic contributions besides determining various circuit elements in bulk electrolyte and at the electrode/electrolyte interface. Nyquist plots  . The indnce (ZW) , and thence (ZW)  assume taterials . These r2 and V2O5 on the graphene sheet  are shown in EF, and this behavior changes marginally with different graphene-vanadium oxide (VO2 and V2O5) interfaces, contributing toward enhanced electroactive sites. The partial DOS calculations also show that the electron contributions of V d states prevail and the other contributions of the 2s and 2p oxygen states. 2 on graphene causes charge transfers from the adsorbates to the substrate of 0.1 e and 0.19 e, respectively. The exact opposite is observed for V2O5 on graphene. For VO2 and V2O5 on graphene, some charge from the vanadium 4s orbital is transferred to the empty 4p orbital. Additionally, for V2O5 (the vanadium average charge is 21.6 e), we have four oxygens with a charge of about 8.5 e, whereas the fifth oxygen has a charge of about 9 e. On the contrary, for VO2 (vanadium average charge is 21.8 e), the charge for oxygens is about 8.5 e. This implies that the fifth oxygen in V2O5 is pulling electrons from the substrate. This is consistent with the statement that the graphene conductivity is never smaller than the minimum value of the quantum conductivity limit. Thus, it is possible to suppose that graphene can retain its unique properties in graphene/VxOy systems similar to other transition metal oxide-graphene interfaces [xOy at the nanoscale. It is conceivable that nanostructures and the thin layer being accessible to the electrolyte result in the maximum utilization of the metal oxides .Density functional theory (DFT) ab initio calculations help to elucidate the fundamental properties at graphene-metal oxide and graphene oxide-metal oxide interfaces, particularly in terms of lattice stability, electronic structure (electron density of states (DOS))  and inteterfaces ,14. MoreHT having a large surface area, implying supercapacitive behavior, which can be practically used to deliver high power and energy density along the Ragone plot metrics. The three-dimensional multiplexed and highly conductive pathways provided by the rGOHT scaffold architectural support also ensure rapid charge transfer and conduction due to the larger accessible geometric surface area. We simulated ac impedance spectroscopy data with conventional and CPE circuit models, which was useful in determining various equivalent circuit parameters. We attribute the overall good performance due to the synergistic effects from supercapacitive graphene as an elastic and electrically-conductive matrix and pseudocapacitive VNBs, allowing faster ion transport across the electrolyte.In summary, a series of graphene nanosheets/vanadium pentoxide nanobelt hybrids with different weight ratios was prepared by a facile hydrothermal decomposition method. We systematically performed microscopic structural and electrochemical properties/performance studies. The dimensionality of graphene nanosheets combined with vanadium pentoxides creating tailored interfaces with tunable properties plays a significant role and is a key factor in determining the response of these materials towards electrochemical (re)activity. The electrochemical properties showed higher specific capacitance for graphene-rich composites with optimized VO loading as compared to VO-rich ones and the constituents, and this decreased with increasing scan rate, as anticipated. We attribute this to the fact that the nanostructured VO is involved in the faradaic reaction (pseudocapacitance) coupled with highly conducting rGO


We have seen a notable increase in the application of PD-1/PD-L1 inhibitors for the treatment of several solid and hematogenous malignancies including metastatic melanoma, non-small-cell lung cancer and lymphoma to name a few. The need for biomarkers for identification of a suitable patient population for this type of therapy is now pressing. While specific biomarker assays have been developed for these checkpoint inhibitors based on their respective epitopes, the available studies suggested the clinical utility of these biomarker assays is for response stratification and not patient selection. Further improvement in assay development is needed to utilize this type of assay in identification of ideal patient population for this therapy. Immune modulation therapies have seen an impressive growth over the last decade . RecentlHowever, not all patients respond to PD-1/PD-L1 inhibitors. Thus, predicting the likelihood of response to treatment would aid in appropriate patient selection for these drugs. Immunohistochemistry (IHC) biomarker assays for respective PD-1/PD-L1 inhibitors were designed to screen for the presence of specific PD-1/PD-L1 epitopes as well as to estimate the percentage of T cells or tumor cells expressing this receptor or ligand. At this time, 4 FDA-approved IHC biomarker assays have been designed . Their aAmong the first generation of these drugs, Pembrolizumab (an anti PD-1 antibody) was approved for treatment of NSCLC –16 and mSeveral studies examining the usefulness of PD-L1 IHC assays have demonstrated a direct correlation of response rate to PD-L1 expression level. The distinction between a companion assay and a complementary assay should be underlined here. A companion assay is one that is considered to be essential to the use of its corresponding drug. Pembrolizumab is FDA approved only when used in conjunction with the Dako 22C3. Conversely, the other bioassays are considered complementary in that their use is recommended in order to optimize appropriate patient selection but is not considered mandatory for the use of its associated drug . The cutPembrolizumab is currently approved for the treatment of NSCLC, advanced HNSCC and advanced melanoma. Its companion IHC biomarker assay, Dako 22C3, is used to detect PD-L1 in all three types of malignancies. It is the only assay that has FDA companion status . This exNivolumab is currently approved for the treatment of squamous and non-squamous NSCLC, advanced RCC and advanced melanoma. Its companion PD-L1 IHC biomarker assay, Dako 28–8, is only used in tumor tissue from NSCLC and melanoma . In the Durvalumab is currently approved for the treatment of NSCLC and bladder cancer. Its companion IHC biomarker assay is Roche Ventana SP263 . The SP2. The associated SENS and SPEC are 76 and 61%, respectively. The cutoff for urothelial cancer is 1%. The ORR+ and ORR- are 27% and 15%, respectively [Atezolizumab is currently approved for treatment of metastatic NSCLC and urothelial cancer . Its comectively . HoweverIn general, the average specificity for these assays is 58%. Thus, approximately 42% of patients who are not likely to respond to treatment are considered PD-L1+. Furthermore, the average overall sensitivity of these assays with their respective cutoff levels is 72%; suggesting an average of 28% of patients who are considered PD-L1 negative may benefit from this type of treatment. The estimates are based on the figures seen in table.The mechanism of PD-L1 expression is complex. Several factors appear to influence both PD-L1 expression and response to treatment –52. BRAFModification of specific checkpoints in anti-tumor immune response has resulted in significant improvement for the treatment of various malignancies. The relationship between tumor expression of PD-L1 and patient outcome has been established. However, the currently available IHC biomarker assays could not provide clinically meaningful identification of responders and non-responders , 67, 68.Several tumor and patient characteristics appear to influence response to PD-1/PD-L1 inhibitor and should be considered when selecting patients for this treatment. Providing several tissue samples and obtaining tissue samples at different time intervals may allow for more accurate determination of appropriate patient for treatment.A direct comparison of the clinical utility of these diagnostic assays for lung cancer was recently completed. In the Blueprint PD-L1 IHC Assay Comparison Project, the Pathology Committee of the International Association for the Study of Lung Cancer joined efforts with 6 of the commercial stakeholders  to compare these tests. 3 of the 4 assays showed similar results but the SP142 demonstrated significantly less expression . The int


P = 0.01), but not with overall survival within tumor types. PD-L1 status and dMMR are overlapping putative response biomarkers in immunoncology. Clinical trials with biomarker enrichment restricted to PD-L1+ or dMMR may be inadequate to capture the subset of patients who may benefit from immune mediated therapies. More robust immunotherapy biomarkers and careful clinical trial design are warranted.The identification of biomarkers associated with response to therapeutic agents is central to optimizing patient outcomes. Expression of the immune checkpoint proteins PD-1/L1, and DNA mismatch repair deficiency (dMMR) status may be predictive response biomarkers for immunotherapies, but their overlap requires further study. We prospectively conducted PD-L1 and MMR immunohistochemistry (IHC) on 430 consecutive patients with advanced gastrointestinal (GI) cancers, genitourinary (GU) cancers or rare cancers between June 2012 and March 2016. Overall 393/430 (91.4%) patients were evaluable for PD-L1 expression by IHC. The frequency of tumor PD-L1 positivity (PD-L1+) was 16.5% (65/393). Among anatomic tumor sites PD-L1+ was 28.6% in melanoma, 22.2% in GC, 20.9% in CRC, 12.5% in BTC, 7.1% in GU cancer, 6.7% in HCC, 0% in pancreatic cancer and 0% in sarcoma. Among the 394 evaluable for MLH1/MSH2 expression cases, 18 patients (4.5%) had dMMR tumors. The dMMR was most common in GC (7.1%) followed by 6.7% in HCC, 4.4% in CRC, and 2.7% in sarcoma. Of the 365 patients evaluable for both PD-L1 and MLH1/MSH2 expression, there was a significant association between the PD-L1 expression and MLH1/MSH2 loss ( Immune evasion is a hallmark of cancer and the discovery and therapeutic targeting of immune checkpoints has redefined the treatment of multiple tumors types . BroadlyThe mismatch repair (MMR) system is of pivotal importance for the rectification of DNA sequence mismatches during DNA replication, and loss of function of one of the MMR proteins  leads to high rates of mutations that accumulate in repetitive nucleotide regions (microsatellites). Microsatellite instability (MSI), also termed MMR deficiency (dMMR), may have an oncogenic potential when it occurs in coding regions of genes involved critical cellular function . The majTo date, trials of checkpoint inhibitors have been characterized by overall response rates in the 15-25% range, but durable benefit in responding patients. Beyond PD-1/L1 IHC and MMR status, candidate predictive response biomarkers include tumor-infiltrating lymphocytes, T cell receptor (TCR) clonality, and immune gene signatures among others –24. Biomn = 203, 47.2%) followed by gastric cancer (GC) , GU cancer , sarcoma , biliary tract cancer (BTC) (n = 16. 3.7%), hepatocellular carcinoma (HCC) , melanoma , pancreatic cancer , with 13 (3.0%) other various tumor types. Over three quarters  of patients had stage IV disease at enrollment and 173 of 338 stage IV patients (51.2%) had 2 or more metastatic sites.Clinicopathologic characteristics of 430 patients are listed in Table Nearly all patients  were evaluable for PD-L1 expression by IHC. Among all evaluable sample 16.5% (65/393) were PD-L1+ using the ≥1% threshold defined for our study  were not sufficient to analyze MLH1/MSH2 expression by IHC. Among the 394 patients available to evaluate the MLH1/MSH2 expression, 18 patients (4.4%) had MMR-deficient (dMMR) tumors with the loss of either MLH1 or MSH2 expression. Table P = 0.01). In the overall cohort (n = 430), PD-L1 expression was observed in 38.9% (7/18) of dMMR tumors (MLH1/MSH2 loss), 15.2% (57/376) of MMR proficient (pMMR) tumors, and 2.8% (1/36) tumors with unknown MMR status.Among the 365 patients evaluable for both PD-L1 and MLH1/MSH2 expression, we analyzed the correlation between PD-L1 and MMR status    between patients with and without PD-L1 expression and/or MLH1/MSH2 loss  Figure . In 81 m) Figure P = 0.01). Further, in cohorts of advanced GC, CRC, and sarcoma there was no association between PD-L1 status, MMR status, and survival.The identification of prognostic and predictive biomarkers in oncology is of central importance. Prospectively validated predictive response biomarkers can identify patients most likely to benefit from a given therapy while sparing potential physical and socioeconomic toxicity in those unlikely to benefit. In the rapidly evolving field of immunoncology, checkpoint overexpression (PD-1/L1) by IHC and more recently MMR status (IHC and/or PCR) are emerging biomarkers, though little is known about their relationship to each other –8, 20. IP < 0.0001) [PD-L1 expression is a dynamic process and overexpression is reliably detected by multiple IHC-validated antibodies in tumor and or immune cells . To faci 0.0001) . However 0.0001) , 31, 32.MLH1, MSH2, MSH6, PMS2) accumulate genomic alterations and dMMR is a proxy for elevated tumor mutational burden and presumably neoantigen load [Tumors with germline or acquired somatic alterations in the main mismatch repair (MMR) genes  dMMR tumors MLH1/MSH2 loss), 15.2% (57/376) of MMR proficient (pMMR) tumors, and 2.8% (1/36) tumors with unknown MMR status  were tested for PD-L1 and MLH1/MSH2 expression from samples taken at the time of diagnosis. All study participants provided written informed consent before study entry and were monitored in accordance with the Declaration of Helsinki. The following clinicopathologic characteristics were collected for all patients: age, gender, primary tumor site, number of metastatic sites, site of metastasis, treatment and survival.Patients with pathologic confirmation of advanced gastrointestinal (GI) cancer, genitourinary (GU) cancer or rare cancers at Samsung Medical Center between June 2012 and March 2016  was performed on an automated immunostainer . Antigen retrieval was performed for 92 min with CC1 and the antibody was incubated for 120 min in 37°C in Ventana BenchMark XT. Signal visualization was achieved with the Optiview DAB IHC detection kit  and Optiview Amplification kit . PD-L1 expression was evaluated on tumor cells. The proportion of PD-L1-positive (PD-L1+) cells was estimated as a percentage of total tumor cells. Consistent with several reported clinical trials, specimens were categorized as IHC negative or positive if < 1% or ≥ 1% of cells were stained by PD-L1 mAb, respectively  and MSH2 . IHC staining procedures were conducted using a Bond-max autoimmunostainer  using Bond™ Polymer refine detection, DS9800 . MLH1 and MSH2 proteins were determined to be preserved in a case when nuclear staining in the tumor cells of the case was observed. Loss of expression of MMR proteins (MLH1- or MSH2-) was determined when nuclear staining in the tumor cells was not observed. In the present study, MMR-deficient or MSI tumors was defined as the loss of MLH1 and/or MSH2 expression.Descriptive statistics were reported as proportions and medians. Data were also presented as number (%) for categorical variables. Correlation between the status of PD-L1 and MLH1/MSH2 expression was analyzed using the t-test or the Fisher's exact test as appropriate, or one-way analysis of variance (ANOVA). Overall survival (OS) was defined as the time from the first treatment to the date of death. Kaplan-Meier estimates were used in the analysis of all time to event variables, and the 95% confidence interval (CI) for the median time to event was computed.The institutional review board of the Samsung Medical Center (SMC) approved the study. The methods in this study were carried out in accordance with the approved guidelines by SMC and all protocols were approved by the ethics committees of SMC.


MTDH) is an important oncogene, which is overexpressed in most cancers and responsible for apoptosis, metastasis, and poor patient survival. Here we show that the expression levels of Mtdh and phosphorylated p38 mitogen-activated protein kinase (MAPK) are significantly increased, whereas those of the microRNA-30 family members (miR-30s) are considerably reduced in the glomeruli of DN rat model and in high glucose (HG)-induced conditionally immortalized mouse podocytes (MPC5). These levels are positively correlated with podocyte apoptosis rate. The inhibition of Mtdh expression, using small interfering RNA, but not Mtdh overexpression, was shown to inhibit HG-induced MPC5 apoptosis and p38 MAPK pathway, and Bax and cleaved caspase 3 expression. This was shown to be similar to the effects of p38 MAPK inhibitor (SB203580). Furthermore, luciferase assay results demonstrated that Mtdh represents the target of miR-30s. Transient transfection experiments, using miR-30 microRNA (miRNA) inhibitors, led to the increase in Mtdh expression and induced the apoptosis of MPC5, whereas the treatment with miR-30 miRNA mimics led to the reduction in Mtdh expression and apoptosis of HG-induced MPC5 cells in comparison with their respective controls. Our results demonstrate that Mtdh is a potent modulator of podocyte apoptosis, and that it represents the target of miR-30 miRNAs, facilitating podocyte apoptosis through the activation of HG-induced p38 MAPK-dependent pathway.Apoptosis, one of the major causes of podocyte loss, has been reported to have a vital role in diabetic nephropathy (DN) pathogenesis, and understanding the mechanisms underlying the regulation of podocyte apoptosis is crucial. Metadherin ( However, cellular and molecular pathomechanisms responsible for podocyte apoptosis in DN remain largely unknown.Podocyte loss is emerging as a central pathological mechanism underlying diabetic nephropathy (DN). Podocyte apoptosis is one of the major causes of podocyte loss6 or lysine-rich CEACAM1 co-isolated (LYRIC)7) in this process. A comprehensive and convincing body of data highlights MTDH as an important oncogene that is overexpressed in all previously investigated cancers.8 It is involved in cancer cell proliferation, survival, autophagy, migration, invasion, apoptosis, angiogenesis, metastasis, and treatment resistance.9 In addition to its role in tumorigenesis, MTDH is a pleiotropic protein involved in various physiological and pathological processes, including development,10 neurodegeneration,11 inflammation,14 and epithelial–mesenchymal transition (EMT).15 MTDH has a role in these processes through the regulation of various signaling pathways, including transcription factor nuclear factor-κB (NF-κB), GTPase HRAS, phosphoinositide 3-kinase (PI3K)/AKT, p38 MAPK, and WNT pathways.16 Recently, Eisenrech et al.17 demonstrated that podoplanin, a factor involved in cancer biology but poorly characterized and functionally analyzed in podocytes, is antiapopotic in AngII-induced human podocytes, and regulated by miR-29b. However, the expression and role of MTDH in podocytes have not been investigated before.Here we evaluated the involvement of metadherin 18 Previous studies have implicated p38 MAPK in a variety of cellular activities, including cell survival, death, proliferation, differentiation, and transformation.19 It was shown to regulate apoptosis by modulating the activities of the Bcl-2 family proteins.20 Furthermore, P38 MAPK was implicated in podocyte apoptosis induced by doxorubicin (Adriamycin)21 and high serum lipopolysaccharide levels.22p38 MAPK is one of the important member of MAPKs, activated by proinflammatory cytokines or cellular stress, such as genotoxic, osmotic, hypoxic, or oxidative stress.23 Extensive investigations showed that MTDH represents a target gene of several miRNAs.16 miR-30 family consists of five members, miR-30a–miR-30e, and it was shown that they are involved in hepatocyte EMT,24 cancer cell apoptosis and autophagy,25 and cardiomyocyte apoptosis.26 Moreover, Li et al.27 reported that the overexpression of miR-30a-5p inhibits cell proliferation, colony formation, and induces apoptosis in liver cancer cells by targeting MTDH. Members of miR-30 family were shown to be abundantly expressed in podocytes, and the downregulation of their expression facilitates podocyte apoptosis in glomerulosclerosis.28Data obtained over the past decade have demonstrated that microRNAs (miRNAs) have vital roles in the cell growth, proliferation, differentiation, apoptosis, and stress response.In this study, we investigated the potential involvement of Mtdh in podocyte apoptosis, together with the interactions between the members of miR-30 family and the changes in Mtdh expression. We demonstrate here that MAPK signaling is required for Mtdh-mediated induction of podocyte apoptosis.The results obtained using electron microscopy showed the evidence of glomerular basement membrane thickening, effacement, and focal fusion of the podocyte foot process in db/db mice .d-glucose  for 48 h. Consistent with the TUNEL staining, flow cytometry analysis showed that the rate of apoptosis was significantly increased in HG-treated group compared that in control groups  staining combined with WT-1 (podocyte nucleus marker) immunofluorescent staining. WT-1 immunostaining showed a significant reduction in the number of podocyte in the glomeruli of db/db mice . In contl groups . We thenl groups . Result l groups , as welll groups , in compTo determine whether Mtdh is expressed in podocytes, we performed double fluorescence staining of nephrin (podocyte marker) and Mtdh. Our results demonstrated that Mtdh is expressed not only in the tubules but also in the glomeruli of the investigated mice. Furthermore, the colocalization of Mtdh and nephrin indicated that Mtdh is predominantly expressed in glomerular podocytes. The intensity of Mtdh staining was shown to be much stronger in db/db mice than in db/m mice . WesternMtdh knockdown or overexpression. The results obtained using flow cytometry showed that the rate of apoptosis of HG-induced MPC5 cells transfected with small interfering RNAs (siRNAs) targeting Mtdh (si-Mtdh) considerably decreased compared with the negative control (NC) group (We further investigated the role of Mtdh in the induction of apoptosis by C) group . SuccessC) group . In addiC) group .Following the overexpression of Mtdh in MPC5 cells, we showed that the apoptosis rate was significantly elevated in the Mtdh overexpression group compared with control vector-treated cells . SimilarTo investigate whether Mtdh-induced apoptosis of HG-induced MPC5 cells is mediated by the p38 MAPK pathway, we measured the expression of phosphorylated (p)-p38 MAPK in the glomeruli from DN rats and HG-induced MPC5 cells. The expression levels of p-p38 MAPK were shown to be significantly increased in the DN glomeruli  and HG-iThe identification of putative Mtdh-regulating miRNAs, using TargetScan v5.1 , showed that 3′-untranslated region (UTR) of Mtdh contains seed sequences of miR-30s and that these sequences are highly conserved in diverse species . TherefoNext, luciferase reporters, containing normal or mutated binding sites for miR-30s, were constructed. The obtained results demonstrated that miR-30a, -30b, -30c, -30d, and -30e mimics can significantly inhibit the luciferase activity of the wild-type Mtdh 3′-UTR reporter, but not that of the NC, and that this inhibition was reduced when the mutant reporter, with mutated miR-30-binding site, was used .To assess the effects of miR-30s on the expression of Mtdh, we transiently transfected MPC5 cells with miR-30 inhibitors, synthetic miRNA mimics, or their NCs. As shown in MPC5 were transfected with miR-30 inhibitors, mimics, or the respective NCs. Flow cytometry analysis demonstrated that the transfection of cells with miR-30 inhibitors significantly increased the percentage of apoptotic cells compared with the NC group . Convers1 Podocytes are exposed to various stimuli, such as hyperglycemia, fatty acids, growth factors, cytokines, and hormones, in diabetes mellitus. It has been demonstrated that, both in vivo and in vitro, HG levels lead to the activation of a series of event cascades in the kidneys, which may result in podocyte apoptosis. These may include increased oxidative stress, one of the most studied stimuli,30 the activation of RAS, the induction of TGF-β, and the formation of advanced glycation end products.31 Langer et al.32 and Huang et al.33 demonstrated that HG (30 mM) induces podocyte apoptosis in vitro. Consistent with this, we showed that the apoptosis rate of podocytes is significantly increased in db/db mice, a well-recognized mouse model of type 2 diabetes, and that the increase in glucose levels is sufficient to induce apoptosis in MPC5. However, we suggest a different mechanism of HG-induced podocyte apoptosis, supported by the results presented in this study that showed that the downregulation of miR-30s leads to Mtdh upregulation, inducing MPC5 apoptosis mediated by the activation of p38 MAPK pathway  and their normal littermates  of both sexes, aged 6 weeks, were purchased from the Model Animal Research Center at Nanjing University, China. During the experiments, all animals were housed in standard animal facilities, at 22–26 °C and 40–70% humidity, and a 12/12 h light/dark cycle. All animal were killed at the age of 29 weeks. The study design and all animal procedures conformed to National Institutes of Health guidelines and were approved by the ethics committee for the experimental use of animals at Southern Medical University, Guangzhou, China (L2015046).Diabetic db/db mice C57BL/KsJ  at 33 °C (permissive temperature) and 5% CO2 in RPMI 1640  containing 10% fetal bovine serum , penicillin (100 U/ml), streptomycin , and 50 IU/ml of recombinant murine IFN-γ . To induce differentiation, the cells were transferred to 37 °C (nonpermissive temperature) for 10–14 days and the medium was replaced with RPMI 1640 containing 5% FBS without IFN-γ. For the experiments, the medium was replaced by Dulbecco's modified Eagle medium (DMEM), containing 5.3 mM glucose  supplemented with 1% FBS. The differentiated podocytes were treated with 5.3 mM glucose (NG group), 5.3 mM glucose+25 mM mannitol , 30 mM d-glucose  for 12, 24, and 48 h, or HG plus SB203580  for 48 h, depending on the experiment.Conditionally immortalized mouse podocytes (MPC5), between passages 12 and 20 , were cultured as previously described.in vivo, we performed double WT-1 and TUNEL7 staining. In brief, renal frozen sections were incubated with anti-WT-1 polyclonal antibody  overnight at 4 °C. After the washing, a fluorescent secondary antibody, Alexa Fluor 546  was applied for 6 h at 4 °C, and the samples were rinsed with phosphate-buffered saline . Afterward, TUNEL staining was performed using the In Situ Cell Death Detection kit , according to the manufacturer's protocol. The specimens were counterstained with 4′,6-diamidino-2-phenylindole  for 10 min, and following this, the images were obtained with a light microscope . Podocyte apoptosis was defined as the double WT-1 and TUNEL staining of a cell, and double labeled/WT-1 staining ratio was calculated.To identify podocyte apoptosis μl of binding buffer, according to the manufacturer's instructions. The solution containing 3 μl of PE Annexin and 3 μl of 7-AAD was added, and the samples were incubated in the dark for 15 min at room temperature. These samples were analyzed using a cytometer (BD Biosciences).MPC5 apoptosis was analyzed by flow cytometry using the PE Annexin V Apoptosis Detection kit (BD Biosciences). MPC5 cells were incubated in six-well plates. Following the treatment with NG, M, or HG for 48 h, or RNA transfection, the treated cells were collected, washed, and resuspended in 300 Mtdh and nephrin (podocyte marker) antibodies were used to investigate the location and expression of Mtdh in frozen renal tissue samples obtained from db/db and db/m mice. The slides were permeabilized with 0.05% Triton X-100  in PBS for 10 min and blocked with 5% goat serum mixed with 2.5% bovine serum albumin for 2 h. Afterward, these sections were incubated with anti-Mtdh antibody  together with anti-nephrin antibody  at 4 °C overnight. Anti-Mtdh and anti-nephrin primary antibodies were detected using Alexa Fluor 546 donkey anti-rabbit and Alexa Fluor 488 goat anti-rabbit  secondary antibodies, respectively, and the samples were incubated with them for 6 h at 4 °C. Cell nuclei were stained with DAPI for 10 min before the observation of the samples under a light microscope (Nikon Corporation).5 cells per well in six-well plates at 70% confluence after the differentiation. These cells were transfected with Mtdh siRNA  or the overexpression  the mixture of the mimics or inhibitors  of all five miR-30 family members at the final concentrations of 50 nM, using Lipofectamine 2000 (Invitrogen) for 6 h in OPTI-MEM (Gibco BRL), according to the manufacturers' instructions. Afterward, OPTI-MEM was replaced with the complete medium containing 1% FBS, and treated with HG for 48 h after the transfection with siRNAs or mimics, whereas the cells treated with miR-30 inhibitors were not treated with HG. The sequences of Mtdh siRNAs were as follows: Mtdh 1: sense: 5′-GGAUGAAGUUGUUAGAAAUTT-3′, antisense: 5′-AUUUCUAACAACUUCAUCCTTUTT-3′ Mtdh 2: sense: 5′-GUCUACCACUUCUGAUUAUTTUTT-3′, antisense: 5′-AUAAUCAGAAGUGGUAGACTT-3′ Mtdh 3: sense: 5′-CGAGCCAUCUAUUACCUUATT-3′, antisense: 5′-UAAGGAAUAGAUGGCUCGTT-3′ and control: sense: 5′-UUCUCCGAACGUGUCACGUTT-3′, antisense: 5′-ACGUGACACGUUCGGAGAATT-3′.MPC5 were seeded at 2 × 10β-actin mRNA or U6 miRNA levels as internal controls. The primers used in this study are listed in miRNAs and total RNA were extracted using TRIzol reagent , and qPCR was performed using a commercial kit  according to the manufacturer's protocol. All experiments were performed in triplicate and the comparative assessment of target gene expression was performed using 43In brief, after mincing and grinding of the tissue, the renal cortex paste was forced through a series of mesh filters with 180-, 100-, 75-, and 70-μm mesh openings. The tissue material remaining on the last sieve was rinsed and collected.Glomeruli were isolated from streptozotocin-induced diabetic rats  and kindly provided by Professor Hong-xin Niu form ZhuJiang Hospital, Southern Medical University, China) using the standard mechanical sieving technique, as previously described.μg) were separated on the 12% sodium dodecyl sulfate-polyacrylamide gels, and transferred to polyvinylidene fluoride membranes . Following the blocking with 5% skimmed milk/TBST for 1 h, the membranes were incubated overnight at 4 °C with primary antibodies against Mtdh , p38 MAPK, phosphorylated (p)-p38 MAPK, pro-caspase 3, and cleaved caspase 3 , β-actin , and Bax . The membranes were probed with the appropriate HRP-conjugated secondary antibodies  for 1 h at room temperature. Protein band intensities were quantified as described previously.44Protein samples were prepared using radioimmunoprecipitation assay buffer, and protein concentrations were determined using the BCA kit . Equal amounts of proteins . In addition, a mutated Mtdh 3′-UTR was constructed  to ACAAATG; GTTTAC (563–569) to CAAATG). MPC5 cells in logarithmic growth phase were seeded into 96-well plates and incubated for 48 h before the transfection. miRNA mimics or NCs and luciferase reporter vector containing the 3′-UTR of Mtdh or the mutant sequence vector were co-transfected into the cells using Lipofectamine 2000 transfection reagent (Invitrogen) in OPTI-MEM (Gibco BRL). Cells were collected 48 h after the transfection and dual-luciferase reporter assay was performed . Firefly luciferase activity was normalized to the corresponding levels of renilla luciferase activity.t-test was used for analyzing the differences between two groups, whereas ANOVA was used for the comparisons between multiple groups, followed by Student's t-test for the determination of differences between groups. P<0.05 was considered statistically significant in all cases.Statistical analyses were performed with SPSS for Windows version 19.0 . All data were from at least three independent experiments. Data are presented as mean±S.E.M. Student's


Protein quality control or proteostasis is an essential determinant of basic cell health and aging. Eukaryotic cells have evolved a number of proteostatic mechanisms to ensure that proteins retain functional conformation, or are rapidly degraded when proteins misfold or self-aggregate. Disruption of proteostasis is now widely recognized as a key feature of aging related illness, specifically neurodegenerative disease. For example, Alzheimer’s disease, Huntington’s disease, Parkinson’s disease and Amyotrophic Lateral Sclerosis (ALS) each target and afflict distinct neuronal cell subtypes, yet this diverse array of human pathologies share the defining feature of aberrant protein aggregation within the affected cell population. Here, we review the use of budding yeast as a robust proxy to study the intersection between proteostasis and neurodegenerative disease. The humanized yeast model has proven to be an amenable platform to identify both, conserved proteostatic mechanisms across eukaryotic phyla and novel disease specific molecular dysfunction. Moreover, we discuss the intriguing concept that yeast specific proteins may be utilized as bona fide therapeutic agents, to correct proteostasis errors across various forms of neurodegeneration. The regulation of cellular protein levels is an indispensable feature that ensures fidelity of fundamental processes within all cell types. Although nascent peptide strands contain the requisite information to fold proteins into functional structures and confer biological activity, the dynamic nature of protein folding as well as the crowded cell environment pose significant barriers in maintaining protein homeostasis or proteostasis Loss of proteostatic control has emerged as a common cellular pathology in seemingly disparate forms of neurodegenerative disease A consensus has arisen supporting the concept that protein misfolding/aggregation and the progression of neurodegenerative disease are inherently linked. This model also suggests that ongoing aberrant protein misfolding and decline in proteostatic machinery function may accelerate the pathogenic cascade Saccharomyces cerevisiae, to elucidate the molecular basis of various pathologies. 9101410In recent years, the field of neurodegeneration has derived significant benefit from the use of baker's yeast, The mislocalized accumulation of the pre-synaptic protein α-synuclein (α-syn) as a result of mutations or gene multiplications is a hallmark feature that defines PD and other disorders collectively termed synucleinopathies 13Drosophila and C. elegans These studies highlight the great complexity in dealing with PD and neurodegenerative disorders in general, yet have confirmed the utility of the yeast system in addressing complex human disease pathology. Yeast models have also been instrumental in identifying other molecular determinants that modify α-syn cytotoxicity. In particular, the functional identification of the GTPase Rab1, as a suppressor of α-syn toxicity, stemmed from initial studies in yeast vps35 gene suggesting that VPS35 is protective against toxicity relating to EIF4G1 upregulation. Likewise, the PD-associated D620N mutation in VPS35 also had a similar effect. This genetic interaction between VPS35 and EIF4G1 was further confirmed in neurons of both C. elegans and transgenic mouse models In addition to Rab GTPases, studies using similar approaches continue to identify novel protein mediators of α-syn related cytotoxicity in PD, including endonuclease G (endoG) and the sorting protein VPS35 Understanding the cellular etiology of ALS, a fatal and intractable neurodegenerative disorder, has also benefitted greatly by use of yeast model systems 99et. al.9The first yeast TDP-43 proteinopathy model by Johnson Pab1) - binding protein, Pbp1 as a specific enhancer of TDP-43 toxicity; overexpression of Pbp1 increased TDP-43 toxicity whereas as Δpbp1 cells showed increased cell survival upon TDP-43 overexpression atxn2), which has been implicated in spinocerebellar ataxia type 2 (SCA2) that is caused by glutamine expansion within the polyQ tract. This interaction between TDP-43 and Pbp1/Atx2 was further validated in Drosophila and humans. Moreover, the physical interaction between TDP-43 and Atx2 was shown to be dependent on RNA binding, as mutations within the RRMs as well as RNase treatment, dissolve the interaction between Atx2 and TDP-43 The success of modifier gene discovery in α-syn yeast models of PD has also propelled the use of similar genome wide screens to elucidate TDP-43 induced toxicity, i.e. yeast overexpression screens have led to the identification of a pool of 40 genes that modify TDP-43 toxicity. This pool of factors contained the Poly (A) binding protein , as having a role in TDP-43 mediated toxicity. Using the fly model, investigators have shown that an Atx2 mutant lacking the PAM1 motif (through which it binds Pab1), cannot interact with TDP-43 and confer toxicity in the retina. Similarly, human PABPC1 was observed to be mislocalized in ALS patients et. al.dbr1 deletion mitigated the toxic effects of wildtype TDP-43 and the mutant TDP-43 Q331K expression. This suppression of TDP-43 toxicity was also validated in the human M17 neuroblastoma cell line and primary neurons using a siRNA-based knockdown approach, suggesting that inhibiting the enzymatic activity of Dbr1 is sufficient to reduce TDP-43 toxicity Act1 gene in the cytoplasm and the suppression of TDP-43 toxicity in Δdbr1 results from interactions of TDP-43 with the accumulated intronic lariats. The postulate derived from these experiments implied that TDP-43 associated toxicity in ALS and FTLD-U arose from a loss of essential TDP-43 RNA interactions 313233TDP-43 toxicity appears to also segregate with modifications in DNA structure. Here, Armakola +1 induced mitochondrial stress, which is a hallmark feature of AD pathology The prior discussion has focused on the role of yeast models as a test bed to explore disease mechanisms in α-syn linked PD and TDP-43 linked ALS, yet yeast has served as an ideal platform to study the cell biology of various aggregation prone proteins that characterize neurodegeneration. Of note, is the recent elucidation that manipulation of the ubiquitin protease system (UPS) in yeast (by enhancing the expression level of Cdc48 and Vms1) can curtail UBBMuch effort has focused on defining genetic susceptibility in neurodegenerative disease, yet this line of investigation has failed to provide new or effective clinical interventions. Indeed, the capacity to actively reverse aggregation and control protein misfolding will be an essential attribute when considering therapeutic solutions for proteinopathies. Provocative evidence now suggests that yeast specific proteins have the capacity to degrade aggregate prone human disease proteins. Although speculative at this point in time, the data does support the conjecture that yeast proteins may be eventually employed to combat human neurodegeneration. In this regard, the yeast specific Hsp104 chaperone disaggregase has shown considerable promise Hsp104 is an AAA+ chaperone of the heat shock protein family which is known for its ability to completely remodel denatured aggregates, conferring “thermotolerance” in yeast. Structurally, Hsp104 is hexameric pore-like structure consisting of an N-terminal domain, a middle domain (MD) flanked by two AAA (AAA-1/2) domains and a C-terminal domain. ATP hydrolysis at the two AAA domains is required for substrate translocation through the pore. The unique M-domain, which is absent in other AAA+ proteins, is suggested to be the molecular switch which mediates the ATPase activity of Hsp104 and co-ordinates interaction with other chaperones of the Hsp70 and Hsp40 families 38in vitro as well as prevent α-syn inclusion formation in vivo37in vitro revealed that the enhanced disaggregase activity was dictated by amino acid identity within the middle domain of Hsp104, and key mutations in this region increased chaperone collaboration leading to disaggregation of a wide range of substrates 3742Metazoans lack the presence of a clear Hsp104 ortholog, yet despite its absence Hsp104 can be stably expressed and can rescue protein aggregation and prevent neurodegeneration The unexpected role of Hsp104 in mitigating human aggregate-sensitive disease proteins raises the prospect that additional unforeseen regulatory mechanisms may exist to manage this critical aspect of cell biology. As such, a novel proteostasis role has been identified for the clan C/D family of proteases, which include metacaspases and caspases. While this protease clan is typically associated with induction of programmed cell death, through broad range protein destruction, a growing body of evidence suggests that discrete activation of these proteases is essential for numerous complex cell behaviors, independent of inducing cell death 4446et al.Most recently, Hill Admittedly, the delivery of yeast proteins as a therapeutic intervention to treat human neurodegeneration is an entirely speculative proposition. However, the emerging data for Hsp104 indicates that measured delivery of this protein in preclinical animal models of PD and Huntington’s disease has no observable off target toxicity 4050The discovery in yeast, that a metacaspase subdues toxic aggregate formation, raises an interesting prospect, i.e. that mammalian/human caspase proteins may have retained similar beneficial proteostatic function(s). Early studies characterizing cellular pathology in neurodegenerative disease have noted that affected and dying neurons display robust elevation in effector caspase activity (caspase 3 and 6), with subcellular distribution to protein inclusions 5255In conclusion, we have demonstrated that deployment of the yeast model has provided exceptional advances in the study of neurodegenerative disorders including PD and ALS. Similar concerted efforts using yeast proteinopathy models have advanced understanding of disease related proteins such as tau, Aβ and prions. Likewise, the concept that yeast specific proteins, such as Hsp104 and/or Yca1, may be engineered to combat toxic human aggregates opens the door to entirely novel avenues of therapeutic intervention. The use of yeast proteins as therapeutic agents needs to be approached with considerable caution, yet the early studies in relevant animal models support the basic premise. Clearly, the ease and flexibility of the yeast model will ensure continued use of this eukaryotic cell as a preferred means to interrogate and define the molecular pathology of neurodegeneration.


TDP-43-mediated proteinopathy is a key factor in the pathology of amyotrophic lateral sclerosis (ALS). A potential underlying mechanism is dysregulation of the cytoskeleton. Here we investigate the effects of expressing TDP-43 wild-type and M337V and Q331K mutant isoforms on cytoskeletal integrity and function, using rat cortical neurons in vitro. We find that TDP-43 protein becomes mislocalised in axons over 24–72 hours in culture, with protein aggregation occurring at later timepoints (144 hours). Quantitation of cell viability showed toxicity of both wild-type and mutant constructs which increased over time, especially of the Q331K mutant isoform. Analysis of the effects of TDP-43 on axonal integrity showed that TDP-43-transfected neurons had shorter axons than control cells, and that growth cone sizes were smaller. Axonal transport dynamics were also impaired by transfection with TDP-43 constructs. Taken together these data show that TDP-43 mislocalisation into axons precedes cell death in cortical neurons, and that cytoskeletal structure and function is impaired by expression of either TDP-43 wild-type or mutant constructs in vitro. These data suggest that dysregulation of cytoskeletal and neuronal integrity is an important mechanism for TDP-43-mediated proteinopathy. It is well established that mutations of the TAR DNA-binding protein 43 (TDP-43), a highly conserved nuclear protein, are causal of Amyotrophic Lateral Sclerosis (ALS), and in the most common variant of Frontotemporal Lobe Dementia, FTLD-U –4. SeverTDP-43 is predominantly a nuclear protein, although it is actively shuttled between the nucleus and the cytoplasm  However,futsch (MAP1B) mRNA in a complex in vivo to regulates its localization and translation in Drosophila motor neurons [TDP-43 is an RNA binding protein which plays an important role in mRNA transport, stability, and translation , 14. TDP neurons . Hence,  neurons . TDP-43  neurons , 18.in vitro and in vivo, TDP-43 mis-localises in axons, coinciding with a reduction in axonal length and an increase in cellular toxicity [Most studies have used transgenic animals and cell lines to investigate TDP-43 pathomechanisms with fewer studies on primary neurons , 19, 20.toxicity . In the Here we compare the effects of transfecting into rat cortical neurons wild-type TDP-43, and two mutant isoforms containing the M337V familial mutation or the Q331K sporadic mutation. Both are missense mutations in the highly conserved glycine-rich C-terminus of TDP-43 which mediates protein-protein interactions . We haveWT-TDP-43, M337V-TDP-43 and Q331K-TDP-43 constructs and GFP control plasmids were as described previously [EB3-RFP construct was a gift from Maddy Parsons’s lab .The GFP-tagged eviously . The EB3Sprague-Dawley rats pregnant with E18 embryos were obtained from Charles River UK, Ltd., located in Manston Road. Margate CT9 4LT. The animals were bred for research purposes. E18 rat cortices were dissected in ice-cold Hanks Buffered Salt Solution , equilibrated for 2 minutes, and incubated with trypsin diluted 1:4 in HBSS for 15 mins at 37°C with intermittent shaking for 15 mins. Trypsin inhibitor solution was then added and the cortices were dissociated by trituration and centrifuged for 5 mins at 1200 rpm. The supernatant was removed, the pellet was re-suspended in 5 ml of pre-warmed HBSS, passed through a 70 µm filter and re-centrifuged as above. The pellet was resuspended in 2ml of pre-warmed HBSS and the number of cells was counted using a haemocytometer. For non-transfected cells, 80,000 cells from the cell suspension was centrifuged at 1800 rpm for 5 mins and the pellet was re-suspended in 400 µl of culture medium per well.6 cells per plasmid construct) to be transfected was centrifuged at 1800 rpm for 5 mins. The neurons were transfected using an Amaxa machine with rat neuron nucleofector solution (Lonza). The cell pellet was resuspended in 100 µl of nucleofector solution with 4 µg of DNA, transferred into the Amaxa cuvette and transfected using the O-003 program. 200 µl of pre-equilibrated culture medium was added and cells were plated at a density of 100,000 cells in 400 µl of culture medium per well. Transfection efficiency was ~70%.For transfection, the required number of cells  in multiwell plates (Nunc). Confocal dishes (Ibidi) were used for live imaging. The culture medium was Neurobasal with 2% B-27, 1% glutamax and 1% penicillin-streptomycin  with 2% horse serum (Sigma-Aldrich). Cultures were incubated at 37°C with 5% COPrimary antibodies used were chick anti-GFP , rabbit anti-TDP43 , rabbit anti-neurofilament-H , rat anti-tyrosinated tubulin  and rabbit anti-Glu-tubulin . Secondary antibodies were anti-rabbit Cy-3-conjugated, anti-chick Alexafluor488, anti-rabbit Alexafluor 568 and anti-rat Alexafluor 568 .Coverslips were fixed by adding 400 µl of 4% paraformaldehyde (PFA)/15% sucrose (Sigma-Aldrich) to each well for 15 mins at room temp, then washed twice with PBS-T  + 0.1% Triton X-100) and rocking. Non-specific binding was blocked using 0.5% bovine serum albumin  in PBS for 1 h at room temp. Coverslips were incubated in primary antibodies diluted as above in 0.1% PBS-T with 0.5% FBS, overnight on a rocker at 4°C, then washed 3 X 10 mins with PBS-T on a rocker plate at room temperature, followed by incubation in secondary antibodies as above in PBS-T for 1–2 h at room temperature with rocking. After further washes, the coverslips were mounted on slides using Fluorsave  with Hoechst reagent (Thermo-Fisher Scientific).Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) was used to detect fragmented DNA associated with cell apoptosis, using the DeadEnd colorimetric TUNEL system (Promega) according to the manufacturer's instructions. Before starting the assay, cells were permeabilised for 5 mins with 100 µl of 20 µg/ml of Proteinase K in PBS-T. Coverslips were washed twice with PBS-T with 2 mg/ml of glycine, then fixed for 5 mins with 4% PFA, and washed twice with PBS-T. After the diaminobenzidine staining step of the assay, coverslips were washed several times with water, fixed with 4% PFA for 10 mins and immunostained with the respective primary and secondary antibodies as above.2 fields from a 13 mm coverslip. A comparison with the Hoechst nuclear staining (Invitrogen) using blue epifluorescence gave the total cell numbers. Three coverslips from three experiments per condition was used to average the number of TUNEL-positive cells.The TUNEL-positive cells appeared as black dots and were manually counted from five different 0.24 mmCortical neuron cultures were plated on white opaque-walled 96 well plates. Cell viability was monitored using a continuous read format with the Promega RealTime-Glo™ MT Cell Viability Assay. The MT Cell Viability Substrate and NanoLuc® Enzyme were added to the culture medium at the dosage specified by the protocol, plates were re-incubated for 4 h and the first luminescence reading was taken 24 h after transfection and every 24 h thereafter using a luminescent plate reader. The average relative light unit (RLU) from three different experiments, as measured by the luminometer, was plotted for the different constructs against the different time-points.2. Transfected neurons that expressed GFP were imaged at 60X with the DIC channel for 30 mins with 2–5 sec intervals using Nikon Elements software.For live imaging, neurons were plated on confocal dishes at a density of 160,000 cells per well in 1 ml of medium. For EB3 imaging, rat cortical neurons were co-transfected with equal quantities of the GFP control plasmid or TDP-43 wild-type and mutant plasmids together with an RFP-tagged EB3 plasmid. Neurons were imaged in a Nikon Live imaging chamber microscope maintained at 37°C with 5% COFor axon length measurements, the length of 100 neurons per transfection per experiment (n = 3) were measured using neuron J, selecting healthy neurons with an uninterrupted axon that was at least 3 times the length of the cell body.The growth cones of neurons labelled with anti-GFP and anti-tubulin antibodies were imaged using a Zeiss AxioScop at 63X, and images were processed using Volocity or ImageJ software. ImageJ was used to quantitate growth cone area, by thresholding the GFP fluorescence, drawing an outline and measuring the area in pixels. A total of ~70 neurons per construct were analysed from 3 independent experiments. The graphs were drawn based on the average area and Standard error of mean.TM software, which calculates the velocity of individual EB3 traces based on the angle and tangent of the kymograph patterns. A total of 20 movies from 3 independent experiments were analysed. The graphs were drawn based on the average velocity in pixels/frame and standard error of mean. Kymographs were drawn for 10 neurons per construct per experiment (n = 3) using Image J.To quantitate EB3 ‘comet’ velocities along axons, we used kymographs, made using ImageJ and analysed using KymoquantStatistics used a one-way Anova coupled with Dunn's multiple comparison test.The research was approved by the Home Office UK and the ethics committee of Kings College London—College Research Ethics Committee (CREC). The Project was reviewed by the Biomedical Sciences, Medicine, Dentistry and Natural & Mathematical Sciences Research Ethics Sub-Committee (BDM RESC). The rats used in the experiments were humanely killed by cervical dislocation, under the “Schedule 1 to the animals (Scientific Procedure) Act 1986”.in vivo [TDP-43 protein aggregation and mislocalisation is a hallmark of ALS. We have previously shown that TDP-43 shows enhanced cytoplasmic mislocalisation over time in chick spinal motor neurons and in the spinal cord in vivo . Given tIn order to evaluate TDP-43 mis-localisation and aggregation, cortical neurons were transfected with a control (EGFP) construct, with a GFP-tagged TDP-43 wild-type (WT) construct, or with M337V or Q331K mutant constructs (here referred to as M and Q) and fixed at time points from 24 hours to 144 hours. Neurons were immunostained with anti-GFP antibodies to observe the distribution patterns of GFP control or TDP-43 isoforms. At all four time points, the GFP control protein was distributed through the entire neuron including axons and dendrites . At 24 aAs a control for the tagged TDP-43 distribution, neurons were immunostained for tyrosinated tubulin, which is distributed throughout the neuron. Tubulin staining was seen throughout neurons transfected with all constructs at 24–144 hour timepoints, showing an even distribution relative to GFP, and validating the changes in TDP-43 localisation that were observed .Transfected neurons were next double-labelled with antibodies to GFP (green) and TDP-43 (red); the latter detected both the endogenous (rat) protein and the transfected (human) protein. At 24, 72 and 144 hours, TDP-43 protein was detected only in the nuclei of neurons transfected with the GFP control, as expected . At 24 hWe observed that in cultures from 72 hours onwards (data not shown) and particularly at 144 hours, a minority of neurons in the WT and mutant TDP-43 transfected cultures showed evidence of axonal disintegration and cell death. At 144 hours, dying or dead cells appeared as a series of spots, which were GFP-positive  and tubulin-positive –2J'.We have therefore shown that TDP-43 wild-type and mutant proteins were re re-distributed to axons over 0–72 hours, whereas at 144 hours, there is a suggestive appearance of aggregate formation, with evidence of neuronal fragmentation. However, fractionation assays have to be done to ascertain the nature of these aggregates. These data suggest that the mislocalisation of TDP-43 protein in cortical axons precedes neuronal disintegration.in vivo in chick embryo spinal cord [We have previously shown that over-expression of the wild-type and mutant isoforms of TDP-43 causes neurotoxicity nal cord . In ordein vitro, with a slightly increased effect of the Q mutant construct.We also determined cell viability using a bioluminescent assay which is non-lytic and measures the metabolic activity of cells. We found that the GFP-transfected control cells showed greater viability than the TDP-43 transfected neurons at all timepoints, with an increase in cell numbers from 24–72 hours which likely reflects cell division in vitro . There wIn order to characterize the effects of TDP-43 on cortical axon outgrowth, axon lengths were measured at 24–72 hours post-transfection after immunostaining with anti-GFP antibodies. Quantification suggested that control GFP-transfected neurons were longest at all time points whereas at 24 and 48 hours the TDP-43 transfected neurons (wild-type and mutants) show a modest reduction in neuronal length compared to the controls, which were significant at the p < 0.0001 level . At 72 hWe noticed that some transfected cortical neurons had small growth cones, and quantitated this from 24–72 hours post-transfection by immunostaining with anti-GFP and anti-tubulin antibodies, and measuring growth cone areas . GFP-expThe data obtained so far converge on the possibility that TDP-43 associates either directly or indirectly with proteins that affect cytoskeletal integrity, axonal outgrowth and growth cone dynamics. We next tested whether TDP-43 also affects anterograde transport of microtubule plus tip proteins, by studying the dynamics of EB3, a microtubule-associated protein that binds to the plus end of microtubules and regulates and promotes microtubule dynamics and growth . Rat corKymographs show the tracks of EB3 comets, with the gradient of the tracks representing the velocity of movement . The kymThe aim of this study was to shed light on the mechanisms underlying TDP-43-mediated ALS. We have shown that TDP-43 mis-localises in cortical neurons and that the re-distribution is enhanced compared to that previously observed in spinal motor neurons . Over-exTDP-43 wild-type and mutant constructs were neurotoxic and the number of apoptotic cells increased over time, with the TDP-43 mutant Q331K showing the highest number of apoptotic cells across the different time points. The increased toxicity correlates with the progressive re-distribution of TDP-43. This is in accordance with several studies that show a positive correlation between TDP-43 toxicity and TDP-43 mislocalisation . ComparaA number of lines of evidence in this study showed that disruption of cytoskeletal integrity occurs in cortical neurons transfected with TDP-43 isoforms. TDP-43 transfected neurons showed a significant reduction in axon length, consistent with previous findings wherein over-expression of the TDP-43 WT caused a significant reduction in axon outgrowth in primary motor neurons when compared to the TDP-43 mutants . TDP-43 in vivo and reduced neurite outgrowth in chick spinal motor neurons in vitro . The smaller growth cones we observe in this study could explain the shorter axons in vitro and the premature truncation and debundling/de-fasciculation of axons in vivo in chick spinal cord. It is plausible that growth cone defects could in turn lead to perturbations of axonal maintenance, which might contribute to neurodegeneration.Our results complement our previous findings in vivo wherein TDP-43 caused premature truncation and de-fasciculation of motor axons Axonal mRNA transport is considered to be a possible target of RNA-binding protein-mediated neurodegeneration and is a necessity for local translation . Exome-wThe identification of mutations in several cytoskeletal genes in neurodegenerative diseases have highlighted their importance in the disease pathophysiology. Alzheimer’s disease, Parkinsons disease, Tauopathies, ALS, polyglutamine diseases all feature defects in cytoskeleton . Cytoske