Trypanosoma cruzi-infected mice had a marked reduction of cardiac inflammation, fibrosis and galectin-3 expression when compared to controls. Serum concentrations of galectin-3, IFNγ and TNFα, as well as cardiac gene expression of inflammatory mediators were reduced after DMS treatment. The gene expression of M1 marker, iNOS, was decreased, while the M2 marker, arginase1, was increased. DMS-treated mice showed an improvement in exercise capacity. Moreover, DMS caused a reduction in parasite load in vivo. DMS inhibited the activation of lymphocytes, and reduced cytokines and NO production in activated macrophage cultures in vitro, while increasing IL-1β production. Analysis by qRT-PCR array showed that DMS treatment modulated inflammasome activation induced by T. cruzi on macrophages. Altogether, our results demonstrate that DMS, through anti-parasitic and immunomodulatory actions, can be beneficial in the treatment of chronic phase of T. cruzi infection and suggest that S1P-activated processes as possible therapeutic targets for the treatment of Chagas disease cardiomyopathy.Chagas disease cardiomyopathy is a parasite-driven inflammatory disease to which there are no effective treatments. Here we evaluated the therapeutic potential of N,N-dimethylsphingosine(DMS), which blocks the production of sphingosine-1-phosphate(S1P), a mediator of cellular events during inflammatory responses, in a model of chronic Chagas disease cardiomyopathy. DMS-treated,  Trypanosoma cruzi infection, both in the cardiac and in the digestive form, are associated with the occurrence of an inflammatory reaction1. Chronic Chagas disease cardiomyopathy (CCC) involves cardiac myocytes undergoing necrosis and cytolysis via various mechanisms, and areas of myocellular hypertrophy and mononuclear cell infiltration occur4. In response to the myocardial damage, fibrotic areas occur and may contribute to the disruption of the cardiac conduction system and appearance of dysrhythmias, as well as to myocardial thinning and cardiac hypertrophy5. Given the lack of an effective specific therapy, CCC is treated similarly to all other heart failure syndromes using therapies to mitigate symptoms6. Therefore, the development of new alternative treatments for CCC is needed.The pathological manifestations of chronic Chagas disease, caused by 7. Among them, sphingosine-1-phosphate (S1P), produced by phosphorylation of sphingosine (Sph) by sphingosine kinases (SphK1 and SphK2) in response to various stimuli, plays important roles in several cellular processes, including cell growth and cell trafficking9. The balance of Sph and S1P determines the progress of many diseases and there is evidence that sphingolipid metabolism and the expression of S1P receptors (S1PR1-5) are altered in inflammatory processes10. S1P drives the differentiation of different immune cell types, inducing changes in their functional phenotypes and regulating production of pro-inflammatory cytokines and eicosanoids. In particular, S1P has emerged as a central regulator of lymphocyte egress12.Sphingolipid metabolites are emerging as important lipid signaling molecules in both health and diseasein vitro assays.Due to the persistent inflammation found in CCC, which is a hallmark of the disease, and the critical role of S1P-activated pathways on the regulation of inflammation, we hypothesized that N,N-dimethylsphingosine (DMS), a pan SphK inhibitor, has a beneficial effect in chronic Chagas disease. Thus, in the present study we investigated the effects of DMS in a murine model of chronic Chagas disease cardiomyopathy, as well as its mechanisms of action on T. cruzi were treated with DMS or vehicle   Fig. . Inflammne) Fig. . Adminisne) Fig. . Gene exent Fig. . Similarent Fig. .Figure 113. To evaluate the effects of DMS on the expression of this important mediator of inflammation and fibrosis, we performed confocal microscopy analysis in the heart tissue. Vehicle-treated, T. cruzi-infected mice had a high expression of galectin-3, while a reduction of galectin-3 expression was seen after DMS treatment  or anti-CD3 plus anti-CD28. A concentration-dependent inhibition of lymphoproliferation was seen when DMS was added to the cultures  to T. cruzi-infected macrophages significantly increased the activation of caspase 1, whereas infection by T. cruzi alone induced a slight increase of caspase 1 activity  during 1 and 24 h and total RNA was extracted for gene expression analysis, compared to uninfected macrophage cultures , whereas 2 genes were found altered only at 24 h time point (48 h of infection)  Fig. . DMS treon) Fig. . When T.k13 Fig. , Il6, Ilp1a Fig. . Additiolls Fig. . Analysiays Fig. .Figure 76. Therefore, the development of therapeutic strategies aiming at modulation of inflammation without affecting parasite control is of great interest. Here we show, using a mouse model of chronic Chagas cardiomyopathy which reproduces the pathological findings observed in human hearts, a potent effect of DMS, causing reduction of heart inflammation and fibrosis, modulation of pro-inflammatory mediators and improvement of exercise capacity. Importantly, the residual parasite load found in mice chronically infected with T. cruzi was reduced by DMS treatment.Persistent inflammation is one of the hallmarks of chronic Chagas disease cardiomyopathy, and leads to a progressive destruction of the myocardium and heart dysfunctionin vitro reinforces that, in addition to a reduction of cell migration, a direct effect of the drug on immune cells may cause the potent immunomodulatory effect of DMS observed in vivo in our model. In line with this idea, it was recently shown that FTY720, a recently approved drug that inhibits the S1P pathway, also modulates the activation of human T lymphocytes and leads to a reduction of IFNγ production16.In our study, a marked reduction of inflammation was seen after DMS treatment, as shown by two different analyses, morphometry and qRT-PCR for quantification of CD45 expression. Moreover, the production of IFNγ and TNFα, two cytokines known to promote the chronic Chagas myocarditis, were significantly reduced in the heart, as well as in the serum, showing a local and a systemic effect of DMS. The fact that DMS modulates the activation of lymphocytes and macrophages 19. The expression of SphK1 is an important factor regulating the proliferation of cardiac fibroblasts20. SphK1-transgenic mice which overproduces endogenous S1P showed 100% occurrence of cardiac fibrosis, involved with activation of the S1P3-Rho family small G protein signaling pathway and increased ROS production21. Additionally, DMS-treated mice had a reduced expression of TGFβ and galectin-3, two pro-fibrogenic factors that stimulate the proliferation and production of extracellular matrix proteins by cardiac fibroblasts23. Here we observed a reduction on fibrotic area in the heart of DMS-treated mice, corroborating the importance of S1P signaling for heart fibrosis.All of the three major S1P receptor (1–3) subtypes are also expressed in cardiac fibroblasts and participate in cardiac remodeling by the activation of signaling pathways through S1P. Moreover, both FTY720 and DMS have been shown to reduce fibrosisin vivo, despite causing a reduction on the production of pro-inflammatory factors, such as IFNγ and TNFα, known to play important roles in resistance to T. cruzi infection25. We showed here that DMS has not only a direct effect on the parasite, causing several cellular alterations and death of trypomastigote forms, but also an indirect effect, by inducing the increase of NO and ROS production in infected macrophages in vitro. These results are in accordance with a previous report showing S1P down regulating iNOS expression in macrophages through the inhibition of NF-κB, AP-1 and/or STAT-1 activation26. This suggests that the inhibition of S1P production by DMS treatment may lead to an increased NO production, also contributing to the in vivo antiparasitic mechanisms of DMS.An important finding was the antiparasitic effect of DMS, leading to a reduction of parasite load 27. The reduction of parasitism by benznidazole, a drug used to treat Chagas disease, reduces cardiac alterations during the chronic phase of infection28. Additionally, the reinforcement of immunological tolerance to myocardial antigens also caused reduction of inflammation in a mouse model of T. cruzi infection29. Thus, the fact that DMS affects both inflammatory cells as well as the parasite suggests that these effects may contribute to the modulation of inflammation seen after DMS treatment.Chagas disease cardiomyopathy may result from multiple pathological mechanisms, including immune responses against the parasite, as well as self-reactive responses against cardiac antigensTrypanosoma brucei SphK causes attenuation of cell division, microtubule elongation at the posterior tip, and altered organelle positioning. SphK inhibitors, such as DMS and safingol, are toxic to T. brucei, both of them having a 10-fold therapeutic index versus human cells, suggesting their potential use against T. brucei infection30. Here we demonstrated that DMS also induces morphological alterations and death in T. cruzi, suggesting that TcSphK may be a candidate target for drug development against T. cruzi. The reduced parasite load on infected macrophages may be also an important mechanism to reduce the dissemination of parasites, since it is likely that T. cruzi-infected macrophages circulate in vivo31.SphK is a highly conserved enzyme in eukaryotes, and while there are two isoforms in mammals, only one is found in trypanosomatids. Depletion of 32. Both Sph analogues are capable of inducing IL-1β production, and our data reinforces the ability of DMS to activate the inflammasome pathway. T. cruzi infection has also been shown to induce inflammasome activation in mice, being a resistance factor to infection by regulating the production of nitric oxide and ROS35. Here we found that DMS induced inflammasome activation in T. cruzi-infected macrophages, as shown by increased caspase-1 activation and IL-1β production. Moreover, DMS increased the production of ROS and NO, which may be contributing to parasite clearance by infected macrophages. Interestingly, the increase on production of nitric oxide was not accompanied by an upregulation of iNOS gene transcription, suggesting that post-transcriptional and post-translational mechanisms are responsible for DMS-induced NO production36. The transcription of genes activated in response to ROS production, however, were increased in DMS-treated macrophages, such as NFEl2l, SOD-1 and catalase 1.A previous study has shown that Sph and its analogues DMS and FTY720 act as a danger-associated molecular patterns (DAMPs), inducing mature IL-1β secretion and promoting inflammasome activation38. Although we did not see interference of PKC and MAPK inhibitors on the effects of DMS in macrophage cultures, further studies are required to demonstrate whether the beneficial effects of DMS in vivo are solely dependent on Sphk inhibition. The fact that DMS may have off-target effects, however, may not be detrimental, since FTY720 and other approved drugs are known to have off-target effects39.In addition to its effects on Sphk, DMS was shown to inhibit the activity of protein kinase C and mitogen-activated protein kinase (MAPK)41. The analysis by PCR array performed in our study indicates that DMS induces the expression of inflammasome genes known to repress NF-κB activation, such as Nlrc5 and Nlrx143, which suggests that inhibition of T. cruzi-induced NF-kB may be linked to the activation of these inflammasome mediators. Both Sph and its analogue FTY720 have already been described as regulators of the NLRP3-inflammasome and IL-1β release from macrophages32. Although NLRP3-inflammasome activation plays a significant role in the activation of IL-1β/ROS and NF-kB signaling of cytokine gene expression for T. cruzi control in human and mouse macrophages, it was observed that NLRP3-mediated IL-1β/NF-kB activation is dispensable since it is compensated by ROS-mediated control of T. cruzi replication and survival in macrophages35. Therefore, our results suggest that, in addition to the NLRP3 inflammasome previously described32, DMS activates other inflammasome pathways.Despite the increased production of IL-1β by DMS-treated macrophages, a reduction of TNFα and IL-6, two proinflammatory cytokines, was found. This may be due to a modulation of NF-κB activation by DMS, as observed in our study and in previous reportsin vitro and in vivo by its antiparasitic and immunomodulatory effects and suggest that inflammasome activation is a promising strategy for the development of anti-Chagas disease treatment. Further studies are required to demonstrate the usefulness of inhibitors of S1P pathway, which are being already used in the clinical setting, as potential candidates for the treatment of Chagas disease cardiomyopathy.In conclusion, our results demonstrate a potent effect of DMS ad libitum. All experiments were carried out in accordance with the recommendations of Ethical Issues Guidelines, and were approved by the local ethics committee for animal use under number 001/15 .Four weeks-old male C57BL/6 mice were used in all experiments. They were raised, maintained in the animal facility of the Center for Biotechnology and Cell Therapy, Hospital São Rafael , and provided with rodent diet and water T. cruzi strain were obtained from culture supernatants of infected LLC-MK2 cells. Infection was performed by intraperitoneal (i.p.) injection of 1000 T. cruzi trypomastigotes in saline and parasitemia was monitored during infection. Groups of chronically infected mice were treated i.p. with N,N-dimethylsphingosine  based on a previous report by Lai and col. (2008)44, 3×/week during two months  was used to exercise the animals. The speed of the treadmill and the intensity of the shock (mA) were controlled by a potentiometer . Total running distance and time of exercise were recorded.45.Electrocardiography was performed using the Bio Amp PowerLab System , recording the bipolar lead I. All data were acquired for computer analysis using Chart 5 for Windows (PowerLab). The EKG analysis included heart rate, PR interval, P wave duration, QT interval, QTc, and arrhythmias. The QTc was calculated as the ratio of QT interval by square roots of RR interval (Bazett’s formula)46.The hearts of all mice were removed and half of each heart was fixed in buffered 10% formalin. Sections of paraffin-embedded tissue were stained by the standard hematoxylin-eosin and Sirius red staining methods for evaluation of inflammation and fibrosis, respectively, by optical microscopy. Images were digitized using a color digital video camera  adapted to a BX41 microscope . Morphometric analyses were performed using the software Image Pro Plus v.7.0 , as described before46. Sections were incubated overnight with anti-galectin-3, diluted 1:50  followed by incubation, for 1 h, with Alexa fluor 633 (1:200)  Nuclei were stained with 4,6-diamidino-2-phenylindole . The presence of fluorescent cells was determined by observation on a FluoView 1000 confocal microscope (Olympus).Sections of formalin-fixed paraffin embedded hearts were used for detection of galectin-3 expression by immunofluorescence as described before47, and the quantification of parasite load was performed as described previously46. To calculate the number of parasites per milligram of tissue, each plate contained an 8-log standard curve of DNA extracted from trypomastigotes of the Colombian T. cruzi strain in duplicate. Data were analyzed using 7500 software 2.0.1 (Applied Biosystems).For DNA extraction, spleen fragments were submitted to DNA extraction using the NucleoSpin Tissue Kit , as recommended by the manufacturer. Primers were designed based on the literature5 cells/mL in a 24 well-plate with rounded coverslips on the bottom in RPMI-1640 medium  supplemented with 10% fetal bovine serum  and 50 µg/mL of gentamycin  and incubated for 24 h. Cells were then infected with trypomastigotes (1:10) for 2 h. Free trypomastigotes were removed by successive washes using saline solution. Cultures were incubated for 24 h to allow full internalization and differentiation of trypomastigotes into amastigotes. Cultures were then incubated in complete medium alone or with test inhibitors for 72 h. Cells were fixed, then stained with hematoxylin and eosin, and submitted to manual counting using an optical microscope (Olympus).Peritoneal exudate macrophages obtained from C57BL/6 mice, four days after thioglicollate injection, were seeded at a cell density of 2 × 10Ptprc, Lgals3, Tnf, Ifnγ, Il10, Tgfβ, Nos2, Chi3l3, Il1β, Arg1, Nfe2l2, Cat and Sod1. All reactions were run in triplicate on an ABI 7500 Real Time PCR System (Applied Biosystems) under standard thermal cycling conditions. A non-template control (NTC) and non-reverse transcription controls (No-RT) were also included. The samples were normalized with 18S and Hprt. The threshold cycle (2-ΔΔCt) method of comparative PCR was used to analyse the results48.RNA was extracted of the heart samples and macrophage cultures using TRIzol . cDNA was synthetized using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The qPCR was prepared with TaqMan® Universal PCR Master Mix (Applied Biosystems). qRT-PCR assays were performed to detect the expression levels of 6 cells/mL) were incubated in 24 well-plates with supplemented RPMI for 24 h. After washing with saline solution to discard non-adherent cells, infection was performed with trypomastigotes (1:10), for 24 h. Free trypomastigotes were removed by successive washes. Cultures were then incubated with complete medium alone or with DMS (5 µM) in triplicate for 24 h. RNA was extracted using the Rneasy Plus Mini Kit . Quantification of RNA and degree of purity were performed in a spectrophotometer . Sample integrity was observed using a 1% agarose gel. cDNA synthesis was performed using the RT2 First Strand kit (Qiagen). For target gene expression analysis, we used RT2 Profiler PCR Arrays Mouse Inflammasome (Qiagen), the SYBR®Green system and 96-well plates. The 7500 Real Time PCR was used (Applied Biosystems). All experiments were performed in DNase/RNase free conditions. The analysis was performed by Threshold Cycle Method48, obtained by calculating 2-ΔΔCt. The QIAGEN’s qPCR analysis web portal date, available on http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php was used to assist in the analysis, and analysis of differentially expressed genes and pathways prediction were done through free online bioinformatic sites BioVenn (www.cmbi.ru.nl/cdd/biovenn) and Enrichr (amp.pharm.mssm.edu/Enrichr).Peritoneal exudate macrophages  only or with anti-CD3 and anti-CD28 (Themo Fisher Scientific), in the absence or presence of DMS at different concentrations . After 48 h, plates were pulsed with 1 μCi of methyl-3H-thymidine  for 18 h. The plates were harvested and the 3H-thymidine uptake was determined using a β-plate counter . Dexamethasone (Sigma-Aldrich) was used as positive control.Spleen cell suspensions from naive C57BL/6 mice were prepared in DMEM medium  supplemented with 10% FBS and 50 μg/mL of gentamycin. Splenocytes were cultured in 96-well plates at 1 × 10in vivo study were used for galectin-3, TNFα and IFNγ and TGFβ determination. Quantification of cytokines was performed by ELISA, using specific antibody kits , according to manufacturer’s instructions. To estimate the amount of nitric oxide (NO) produced, macrophage culture supernatants were used for nitrite determination by the Griess reaction, as previously described49.Serum samples from the 6) were obtained and infected with T. cruzi. Cultures were then incubated with complete medium alone or with DMS for 30 min. After incubation, macrophages were removed from each well using 0.01% trypsin and labeled with 10 μM of 2′,7′-dichlorofluorescin diacetate (Sigma-Aldrich) for 30 minutes at 37 °C. Cells were then washed and analyzed using a cell analyzer  with FlowJo software .Thioglicollate-elicited peritoneal exudate macrophages (1 × 106) were obtained and infected with T. cruzi as described above. Cultures were then incubated with reagents for 2 h and after that, caspase-1 activity was measured using caspase-Glo® 1 inflammasome assay , according to the manufacturer’s instructions. The luminescence of each sample was measured in a Glomax 20/20 luminometer (Promega).Thioglicollate-elicited peritoneal exudate macrophages (1 × 10T. cruzi trypomastigotes (5x107) were treated with DMS  and incubated for 24 h at 37 °C. After incubation, parasites were fixed for 1 h at room temperature with 2% formaldehyde and 2.5% glutaraldehyde  in sodium cacodylate buffer  for 1 h at room temperature. After fixation, parasites were processed for transmission electron microscopy as previously described50. Images were obtained in a JEOL TEM-1230 transmission electron microscope.P < 0.05.All continuous variables are presented as means ± SEM. Data were analyzed using 1-way ANOVA, followed by Newman-Keuls multiple-comparison test with Prism 5.0 . All differences were considered significant at values of Supplementary materialSupplementary Table S1Supplementary Table S2Supplementary Table S3Supplementary Table S4Supplementary Table S5Supplementary Table S6Supplementary Table S7


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.