We observed PRMT-specific patterns of expression and activity during myogenesis. PRMT4 and -5 gene expression was unchanged, while PRMT1 mRNA and protein content were significantly induced. Cellular monomethylarginines (MMAs) and symmetric dimethylarginines (SDMAs), indicative of global and type II PRMT activities, respectively, remained steady during development, while type I PRMT activity indicator asymmetric dimethylarginines (ADMAs) increased through myogenesis. Histone 4 arginine 3 (H4R3) and H3R17 contents were elevated coincident with the myonuclear accumulation of PRMT1 and -4. Collectively, this suggests that PRMTs are methyl donors throughout myogenesis and demonstrate specificity for their protein targets. Cells were then treated with TC-E 5003 (TC-E), a selective inhibitor of PRMT1 in order to specifically examine the enzymes role during myogenic differentiation. TC-E treated cells exhibited decrements in muscle differentiation, which were consistent with attenuated mitochondrial biogenesis and respiratory function. In summary, the present study increases our understanding of PRMT1, -4, and -5 biology during the plasticity of skeletal muscle development. Our results provide evidence for a role of PRMT1, via a mitochondrially mediated mechanism, in driving the muscle differentiation program.Despite the emerging importance of protein arginine methyltransferases (PRMTs) in regulating skeletal muscle plasticity, PRMT biology during muscle development is complex and not completely understood. Therefore, our purpose was to investigate PRMT1, -4, and -5 expression and function in skeletal muscle cells during the phenotypic remodeling elicited by myogenesis. C Theseproteins . As a reproteins . It has proteins , demonstproteins –9. Furthproteins ,10. For proteins . Therefoin vivo [Myf5 (myogenic factor 5). Furthermore, reconciling conflicting reports of PRMT5 function during distinct phases of the myogenic program presents additional challenges to achieve clarity with respect to PRMT activity during skeletal muscle development [Though the presence of arginine methylation in skeletal muscle was first reported almost five decades ago , only rein vivo ,13. Furtin vivo –17. Howein vivo , to consin vivo ,18,19, tin vivo . Along tin vivo , recent in vivo  showed telopment ,15,20–22On balance, the conflicting literature on PRMT expression and activity during myogenesis may be due to differences in the PRMTs examined, experimental timecourse utilized, as well as models of muscle differentiation employed in these studies. These inconsistencies make it difficult to elucidate PRMT biology in skeletal muscle. A comprehensive examination of PRMT1, -4, and -5 expression and function throughout a complete timecourse of myogenesis is required in order to clarify and expand our knowledge of the roles of PRMTs in skeletal muscle plasticity. Thus, the purpose of the present study was to examine PRMT1, -4, and -5 expression and function during the conditions of skeletal muscle remodeling evoked during myogenesis. We hypothesized that PRMT expression and activity would be dynamic during muscle development, and that these alterations would exhibit enzyme-specific patterns. A secondary objective, designed to complement a study identifying a critical role for PRMT1 in muscle regeneration that was published during the preparation of this manuscript , was to 2C12 mouse myoblasts (MBs)  were used in the present study. C2C12 cells were grown in the presence of growth medium (GM), which comprised Dulbecco’s modified Eagle’s medium  supplemented with 10% FBS (Invitrogen Life Technologies), and 1% penicillin/streptomycin (Invitrogen Life Technologies). The cultured dishes were maintained in a 5% CO2 atmosphere at 37°C. Myogenic differentiation was induced when ~90% cell confluence was attained, after which GM was replaced with differentiation medium . Cells were grown in DM for 7 days, with DM changed every 48 h. Five experimental timepoints were employed to characterize myogenesis in vitro. The first was the MB stage, which was when cells reached ~90% confluence. The second to fifth timepoints occurred throughout the fusion and growth of myotubes (MT), including day 1 (D1) MT (24 h after the transition from GM to DM), day 3 , day 5 , and day 7 . At the specified timepoints, C2C12 cells were washed three times with Dulbecco’s PBS  and processed for subsequent analyses.Commercially available C2C12 MBs were cultured until ~90% confluence and then induced to differentiate in medium treated with vehicle  or treated with TC-E 5003  2.1 µl; 0.1 µM TC-E; Tocris, Bristol, United Kingdom), a highly selective and specific antagonist of PRMT1 [Cof PRMT1 . Four exg for 15 min. The protein concentrations of the supernatants were determined using the Pierce BCA Protein Assay kit  using BSA as the standard.Cells were scraped in ice-cold RIPA buffer  supplemented with cOmplete Mini Protease Inhibitor Cocktail  and PhosSTOP (Roche). Samples were sonicated (Fisher Scientific) 5× 3 s on ice at 100% power. The samples were spun at 20000×2C12 cells at the MB stage as well as at D1, D3, D5, and D7 of muscle differentiation according to procedures as described previously [g for 15 min, the supernatant and pellet (P0) were separated. The pellet (P0) was resuspended in 1000 µl of STM buffer, vortexed for 15 s, and then centrifuged at 500×g for 15 min. The nuclear pellet (P1) was then suspended in 250–400 µl of STM buffer and spun at 100×g for an additional 15 min while the supernatant was discarded. The sample was again separated and the pellet (P5) was kept; 400 µl of NET buffer was added to the tube before vortexing and then placed on ice for 30 min. The sample was then sonicated at 100% power for 10× 2 s with 30 s in between the bouts. The sample was spun at 9000×g for 30 min before being separated and the supernatant was kept and labeled as the nuclear fraction. Protein concentration of the cellular fractions were determined using the BCA Protein Assay kit.Nuclear fractions were isolated from Ceviously , with mome2a; 39705, Active Motif, Carlsbad, U.S.A.), histone 3 arginine 8 symmetric dimethylation (H3R8me2s) , histone 3 arginine 17 asymmetric dimethylation (H3R17me2a) , H4 , H3 , MMA , ADMA , SDMA , peroxisome proliferator-activated receptor γ co-activator-1α (PGC-1α) , and total oxidative phosphorylation (OXPHOS) cocktail . After incubation, the blots were washed 3× 5 min in TBST, and appropriate horseradish peroxidise-linked secondary antibodies were applied. The bound antibodies were visualized by ECL  and the membrane was imaged with Alpha Innotech imaging equipment . ImageJ (NIH) was employed for densitometry.For whole cell lysates, 20–50 μg of cellular protein was loaded into each lane of 10 or 12.5% polyacrylamide gels and resolved by SDS/PAGE. For nuclear fractions, 40 μg of protein was loaded into each lane. Gels were transferred on to nitrocellulose membranes  after which membranes were stained with either Ponceau S or amido black  in order to normalize loading across samples, as previously described ,26. Memb2 dishes. At each experimental time point, cells were washed three times with PBS and fixed with 4% paraformaldehyde for 30 min. After fixation, cells were washed again in PBS and incubated in 0.25% Triton-X in PBS for 5 min. Following the incubation, blocking was performed with 10% goat serum for 60 min. Subsequently, cells were incubated with embryonic myosin heavy chain (eMHC) primary antibody  overnight at 4°C. The next morning, cells were washed in PBS and incubated in secondary antibody  for 60 min. Following incubation, the cells were washed in PBS and DAPI (1:20000) was applied and incubated for 5 min. Cells were then washed and the coverslips were carefully removed from the plate. Coverslips were mounted using DAKO fluorescence mounting medium . Three images were taken from each coverslip. The images were viewed using a fluorescence microscope  at 20× magnification. MT length and width were determined by calculating the average of the five longest and widest MTs using 4×4 eMHC stained immunofluorescence images. The fusion index was calculated by identifying the percentage of eMHC positive cells that had two or more myonuclei. The area fraction was calculated by determining the percentage of eMHC positive cells relative to the total surface area of the image. Cell metrics were determined using Nikon Eclipse Ti-E software (ver. 4.4.2).Cells were cultured on three 15-mm glass coverslips that were inserted at the bottom of individual 35 × 10 mm2C12 cells using TRIzol reagent (Invitrogen Life Technologies) according to the manufacturer’s protocol. After treating with Turbo DNaseI (Invitrogen Life Technologies) for 30 min, RNA was further purified with RNeasy columns . RNA concentration and integrity (i.e. A260/A230) was measured using a Nanodrop instrument (Thermo Scientific) before being reverse transcribed into cDNA. Superscript III (Invitrogen Life Technologies) was used to synthesize cDNA from total RNA, according to the manufacturer’s instructions. Target cDNA levels were compared by qPCR in reactions containing either SYBR green  or GoTaq qPCR Mastermix , forward (F) and reverse (R) primers, and cDNA. qPCR was conducted over 45 cycles at 95°C for 15 s and at 60°C for 1 min, preceded by an initial 95°C for 10 min. The ΔΔCT method was used to calculate the expression of the genes of interest with the average of 18S rRNA (18S), GAPDH, and 40S ribosomal protein S11 (RPS11) utilized as the internal control [Total RNA was extracted from C control ,29. Prim2C12 cells were washed with PBS and removed from the 100 mm × 20 mm culture plate using trypsin-EDTA. Two culture plates were combined together and the sample was centrifuged at 100×g at room temperature for 3 min. The supernatant was discarded and the pellet was resuspended in mitochondrial respiration medium (MiR05) which contained: 0.5 mM EGTA, 10 mM KH2PO4, 3 mM MgCl2.6H2O, 60 mM potassium lactobionate, 20 mM HEPES, 20 mM taurine, 110 mM sucrose, and 1 g/l BSA. Cells were subsequently counted using Trypan Blue and a hemocytometer (Invitrogen). Samples were treated with 3 μg/106 cells/ml digitonin for 5 min at 37°C on a digital rocker. Following permeabilization, samples were centrifuged at 800×g for 3 min. The permeabilized cells were resuspended in MiR05 buffer and were used for determination of mitochondrial oxygen (O2) consumption at 37°C using the Oroboros Oxygraph-2 K . The cells (1 × 106 cells/chamber) were placed into separate sealed chambers and the following substrates were added : glutamate , followed by malate , ADP , cytochrome c , pyruvate , and succinate . The rate of O2 consumption was recorded and expressed as picomoles/second/million cells using DatLab software (Oroboros Instruments Corp.).At each timepoint of interest, Cttests were used to identify differences between means during the myogenesis timecourse experiments. A two-way ANOVA with Bonferroni post-hoc tests was used for analysis of the PRMT1 inhibition experiments. Statistical analyses were performed on the raw datasets prior to the conversion to fold differences. Sample sizes for all experiments were n=3–6, with each n being the mean of two or three biological replicates. Statistical significance was accepted at P<0.05. Data in graphical summaries are presented as mean ± S.E.M.A one-way ANOVA with Bonferroni post-hoc test and Student’s 2C12 cells progressed morphologically from mononucleated MBs to robust MTs as expected throughout the differentiation timecourse (P<0.05). Myogenin protein content was significantly greater at all differentiating timepoints in comparison with the MB stage.To begin our characterization of PRMT expression and function during myogenesis, we first sought to confirm the progression of muscle development throughout the experimental timecourse. To this end, we examined cell morphology, eMHC expression, as well as myogenin transcript and protein levels, which all represent established histological and molecular markers of myogenesis ,31. Qualmecourse B,C, whicPRMT4 and PRMT5 mRNA levels remained unchanged throughout myogenesis  at D3, as compared with the MB stage, and remained elevated  at D5 and D7 compared with MB  in D3–D7 cells in response to PRMT1 inhibition  in oxygen consumption in D3–D7 TC-E-treated cells following the addition of the CI substrate pyruvate, as compared with VEH-treated cells , reveals a role for the enzyme specifically during muscle differentiation.Relative to other PRMTs, PRMT4 and -5 expression and function during myogenesis have been extensively studied ,22,38,45c et al.  who showin vitro and in vivo conditions. For example, MBs that lack mtDNA fail to differentiate into MTs [Mitochondrial biogenesis is necessary for the myogenic program to proceed under both into MTs . Moreoveinto MTs . We suspinto MTs ,50 and ainto MTs  in mitocinto MTs  showed tinto MTs .PGC-1α levels were also blunted as a function of PRMT1 inhibition in skeletal muscle. The co-activator stimulates the transcription of mitochondrial genes located in nuclear and mitochondrial genomes by, for example interacting with nuclear respiratory factor 1 and mitochondrial transcription factor A (Tfam), respectively ,54. Notain vivo work [In summary, our results demonstrate that the expression and activities of PRMT1, -4, and -5 display differential responses during skeletal muscle development. Indeed, PRMT1 biology was particularly responsive to myogenic cues. We also show that inhibition of PRMT1 in skeletal muscle cells results in morphological deficiencies, as well as decrements in mitochondrial biogenesis and respiratory function. Thus, complementing recent ivo work , this in


Reported efficacy of platelet-rich plasma (PRP) in regenerative medicine is contradictory. We validated the effects of PRP on proliferation of canine bone marrow-derived multipotent mesenchymal stromal cells (K9BMMSCs) in vitro. PRP was extracted from blood of six dogs with osteoarthritis. K9BMMSCs were established from bone marrow and characterized for CD90 and CD19 expression by immunocytochemistry. Effects of PRP concentrations on viability of matching autologous K9BMMSCs were validated using MTS assay.Positive CD90 and negative CD19 expression confirmed MSC origin. PRP at 40% volume/volume concentration increased, while PRP at 80 and 100% v/v concentrations suppressed viability of tested K9BMMSCs.PRP concentration plays an important role in K9BMMSCs viability, which could affect tissue repairs in vivo.The online version of this article (10.1186/s12917-019-2010-x) contains supplementary material, which is available to authorized users. Platelet-rich plasma (PRP) is an enriched plasma containing variety of growth factors, including the platelet derived growth factor (PDGF), vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β), fibroblast growth factor (FGF), and insulin-like growth factors I and II  , 2. ThesOA is a painful and debilitating orthopedic condition, affecting both humans  and compIn this study, we have evaluated the effects of PRP concentration on the cell viability of the autologous canine bone-marrow derived multipotent mesenchymal stromal cells (K9BMMSCs) harvested from client-owned dogs with a history of OA in vitro. These findings will help streamline the methodology for using PRP as one of the standards of care treatment for injuries.We successfully isolated K9BMMSC cells from six dogs diagnosed with OA Table . K9BMMSCPRP was separated from blood collected from the same dogs as the K9BMMSC cells using the Dr.PRP PRP kit as shown in Table **p < 0.01) in K9BMMSC#1, 82% (*p < 0.05) in K9BMMSC#2, 30% (*p < 0.05) in K9BMMSC#3 cells, 43% (**p < 0.01) in K9BMMSC#4 cells, 33% (*p < 0.05) in K9BMMSC#5 cells, and 29% (**p < 0.01) in K9BMMSC#6 cells when compared to cells cultured in DMEM/F12 media only. In addition, PRP at 20 and 50% (v/v) concentrations also significantly increased cell viability by 40 and 64% , respectively in K9BMMSC#5 cells and 24 and 42% , respectively in K9BMMSC#6 cells. In K9BMMSC#1 cells, a significant 42% (***p < 0.001) reduction in cell viability was observed after treatment with 20% (v/v) PRP concentration. In remaining K9BMMSC cell lines, no significant differences between 20% or 50% (v/v) PRP concentration treated cells and untreated cells were observed. Likewise, 60% (v/v) PRP concentration did not have effect on cell viability of K9BMMSCs , respectively in K9BMMSC#1 cells, by 43 and 39% , respectively in K9BMMSC#2 cells, and by 16 and 22% , respectively of K9BMMSC#3 cells , respectively, was observed in K9BMMSC#6 cells.The K9BMMSC cells were treated with PRP at concentrations of 20, 40, 50, 60, 80, and 100% (v/v) in complete DMEM/F12 media for 72 h and cell viabilities were assessed by MTS assay. PRP at 40% (v/v) concentration significantly increased cell viability by 20%  concentration had elongated, spindle shaped morphology as shown in Fig. PRP is an attractive treatment option for patients with joint injuries, including tendon and ligament injuries, especially in the realm of sports medicine and orthopedic surgery . Numerou6 /mm3) promoted intestinal anastomotic healing in rats in vivo, while high PRP concentrations (5 × 106 /mm3) impaired healing [6 /mm3); whereas low concentrations (0.16–0.37 × 106 /mm3) was not effective and high PRP concentrations (1.85–3.2 × 106 /mm3) inhibited bone regeneration [Recent studies have focused on optimizing the concentrations of PRP used for treatments due to variable effects on viability of co-cultured cells in vitro –30 and i healing . Similarneration . Comparaneration .While our studies and numerous others demonstrate that concentrated PRP are not beneficial for cell culture, the described optimal concentrations vary between studies. These differences in the volume ratios, the quality and yield of PRP between studies can be attributed to preparation of PRP using variety of PRP preparation kits and procedures . FurtherOur results demonstrated that the concentration and volume of PRP affect the viability of K9BMMSC in vitro, which might have an effect on the treatment outcomes of OA in dogs in vivo. We confirmed that PRP at 40–50% concentrations (v/v) increased cell viability of K9BMMSCs, while high concentrations at 80–100% (v/v) inhibited cell viabilities in four out of the six tested K9BMMSC cell lines.Six client-owned dogs with naturally-occurring OA were enrolled in this study according to the protocol approved by the Institutional Animal Care and Use Committee at the University of Tennessee (UT-IACUC). The owners signed the informed consent forms to agree to enroll their dog. Inclusion criteria at admission included clinical evidence of OA of the elbow or hip with a unilateral limb lameness greater than 5% between limbs on screening by force plate (FP) evaluation. Exclusion criteria included: presence of other major illness, orthopedic problems unrelated to our study, orthopedic surgery within the last 6 months, or skin infection of injection site for more than one week. Details regarding the dog’s breed, passage number of cells used in MTS assay and number of isolated platelets are presented in the Table Approximately 4 ml of bone marrow was harvested from the proximal humerus of the lame limb of dog using a bone marrow aspirate needle Fig. a while t2 incubator at 37 °C. Cells were passaged when they reached 70–90% of confluence  to remove blood clots. The mixture of bone marrow with PBS was slowly pipetted over 15 ml of Lymphocyte Separation Medium (LSM)  and centrifuged at 1,000 rpm speed. The middle layer containing K9BMMSCs was gently isolated and washed with PBS  following manufacturer’s instructions. Briefly, 18 ml of blood was drawn from the jugular vein with an anticoagulant syringe and placed in a blue PRP container. After centrifugation, separated PRP layer at the middle of the container  concentrations in complete DMEM/F12 media and incubated for an additional 72 h. After treatment, cell viability of K9BMMSCs were measured using the MTS assay  following manufacturer’s instructions. The obtained absorbance at 490 nm  from PRP-treated cells was normalized to untreated cells and reported as mean ± SEM.Images of K9BMMSCs in culture were captured by a MicroPublisher 3.3 camera  attached to a Vista Vision microscope  using the Q-Capture Pro7 software (QImaging).t-test to establish difference between the PRP treated and control groups. Results were considered statistically significant at *p < 0.05, **p < 0.01, and ***p < 0.001.Statistical analyses were conducted using the Student’s paired two-tailed Additional file 1:Figure S1. High PRP (v/v) concentrations inhibit K9BMMSCs in vitro. The K9BMMSCs treated with different concentrations of PRP (v/v) in DMEM/F12 complete media for 72 h. Images of the cell morphology changes in K9BMMSC#3, K9BMMSC#4, and K9BMMSC#6 cells cultured in 0, 20, 50, and 80% PRP (v/v). (PPTX 2630 kb).


Leishmania infantum mimotopes (B10 and C01) identified by phage display showed to be antigenic and immunogenic for visceral (VL) and tegumentary (TL) leishmaniasis; however, their biological targets in the parasites have not been identified. The aim of the present study was to investigate the native antigens expressing both mimotopes, and to use them in distinct immunological assays. For this, a subtractive phage display technology was used, where a combinatorial library of single-chain variable fragments (scFv) was employed and the most reactive monoclonal antibodies for each target were captured, being the target antigens identified by mass spectrometry. Results in immunoblotting and immunoprecipitation assays showed that both monoclonal scFvs antibodies identified the β-tubulin protein as the target antigen in L. infantum. To validate these findings, the recombinant protein was cloned, purified and tested for the serodiagnosis of human leishmaniasis, and its immunogenicity was evaluated in PBMC derived from healthy subjects and treated or untreated VL patients. Results showed high diagnostic efficacy, as well as the development of a specific Th1 immune response in the cell cultures, since higher IFN-γ and lower IL-10 production was found.Two  Leishmania infantum (=L. chagasi) and L. donovani species, while tegumentary leishmaniasis (TL) is caused mainly by L. major and L. tropica species in the Middle East and central Asia, and by L. braziliensis, L. amazonensis and L. mexicana in the Americas [Leishmaniasis is an infectious disease caused by protozoan parasites of the genus Leishmania, through the bite of infected phlebotomine sand flies . Among cAmericas .L. infantum species being the main parasite species responsible for the disease in dogs and humans [Brazil is responsible by about 90% of VL cases, with the d humans . Prophyld humans . Therefod humans .Leishmania infantum antigens, being expressed in the B10 and C01 phage clones [L. infantum infection in BALB/c mice, and a specific Th1 immunity primed by high levels of IFN-γ, IL-12 and GM-CSF was observed before and after challenge [L. amazonensis [Leishmania expressing B10 and C01 mimotopes  were still not identified.We have previously identified two mimotopes through the phage display technology, which mimic epitopes of e clones . These mhallenge . In addizonensis . The B10zonensis , since tThe diagnosis of VL has been based on clinical evaluation and the application of distinct laboratory strategies, such as parasitological and immunological methods ,10. ParaL. infantum parasites capable of expressing the B10 and C01 epitopes, by means of a reverse engineering process selecting monoclonal antibodies specific to those two mimotopes, through subtractive phage display technology. For this, a combinatorial library of single-chain variable fragments (scFv) was used, and the most reactive antibodies for each target were captured, being the native antigens identified by mass spectrometry. Results showed the recognition of the unique target antigen in parasites, namely β-tubulin, as expressing both epitopes. Then, the recombinant protein was cloned, purified and evaluated in immunological assays in the human VL.The present study was developed to identify antigen targets in L. infantum SLA. The scFv-E8 anti-B10 phage demonstrated higher reactivity to B10 phage and L. infantum SLA . In addition, the immunogenicity of B10 and C01 clones was evaluated in human cells, when cell supernatants were collected and the cytokine production was evaluated by capture ELISA. Results showed that clones, as well as their synthetic peptides induced higher IFN-γ and lower IL-10 levels in PBMCs collected from untreated and treated VL patients and healthy subjects. The β-tubulin protein was also used, and results showed higher IFN-γ/IL-10 ratio in all groups, thus demonstrating the potential immunogenic of this protein in human cells , which were capable of capturing the β-tubulin protein in immunoprecipitation reactions, as evidenced by mass spectrometry, immunoblotting and ELISA assays. This fact can be explained due to the recognition of different epitopes and their ligation sites in the same protein structure. Interestingly, predicted residues defined a conformational epitope of β-tubulin, which is in direct contact with residues of the hypervariable loops of the CDR3 light chain (CDR-L3), corroborating with the notion that kappa and lambda light chains of the CDR-L3 may present different roles in the adaptive immune response [We have previously shown that two phage clones, B10 and C01, which were recognized by antibodies in VL dogs sera, were protective against s for VL ,7,8. In  disease ,7. In thresponse .Tubulins are members of a family of globular proteins responsible for the cell cytoskeleton . In LeisThe infection in humans with Leishmania parasites can results in distinct clinical manifestations, depending on their ability to evade the immune response of the host by means of a well-structured orchestra of host-parasite interactions . ModulatL. braziliensis promastigotes and amastigotes employing serum samples of TL patients allowed to the identification of antigenic proteins in the parasites. In this context, one could speculate about the diagnostic potential of these molecules. Between the identified targets, β-tubulin was showed to be recognized by antibodies in sera from both cutaneous and mucosal leishmaniasis patients [The limitations found by the use of conventional parasitological and immunological techniques to diagnose VL have led to an increasing exploration of new targets to develop an accurate diagnosis, aiming to detect the disease in infected subjects; but not in these non-infected individuals but living in endemic areas of VL . In addipatients . In thisLeishmania. This strategy can be considered innovative, and could well help in other studies where phage display technology is employed, when mimotopes are selected as biological targets against distinct diseases, allowing the identification of the native antigens expressing these epitopes in microorganisms.The diversity created by gene recombination and somatic hypermutation makes protein sequencing of monoclonal antibodies a challenge . Mass spL. infantum β-tubulin as a potent theranostic antigen on leishmaniasis. These immunogenic domains of β-tubulin may be used in vaccine formulation for protection against Leishmania spp. infection, as well as differential components for the serodiagnosis of disease in L. infantum-infected dogs and humans.In conclusion, we have applied a reverse engineering technology through phage display selection using a combinatorial scFv antibody library against phage clones expressing two target mimotopes, which led us to the identification of Experiments were performed in compliance with the national guidelines, as set forth by the Institutional Animal Care , and the Committee on the Ethical Handling of Research Animals from the Federal University of Minas Gerais (UFMG), who approved this study under protocol number 225/2017 (25 September 2017).L. infantum (MOM/BR/1970/BH46) strain. Parasites were grown at 24 °C in Schneider’s medium , supplemented with 20% heat-inactivated fetal bovine serum , 20 mM l-glutamine, 200 U/mL penicillin, and 100 µg/mL streptomycin, pH 7.4. The Leishmania antigenic extract (SLA) was prepared from stationary promastigotes, after few passages in liquid culture, as described elsewhere [8 promastigotes per mL, in a volume of 5 mL, were washed 3 times in 5 mL of cold sterile phosphate-buffered saline (PBS 1×). After five cycles of freezing and thawing, the suspension was centrifuged at 8000× g for 20 min at 4 °C, and the supernatant containing SLA was collected in 500 μL aliquots and stored at −70 °C, until use. The protein concentration was estimated by Bradford method [Experiments were carried out using the lsewhere . Brieflyd method .6 clones. The variable regions were derived from nine different V gene families, including three VH gene families  and six VL subgroups ; demonstrating that scFv fragments were distributed across the full repertoire of antibody germ-line genes. Then, the bio-selection cycles of scFv antibody clones specific to the mimotopes expressed in B10 and C01 phages were performed as described [®M-270 Epoxy, Invitrogen Life Sciences, Waltham, MA, USA) were linked to each phage clone , according the manufacturer’s instructions. To screen the scFv library and select clones, beads coupled with the wild-type and irrelevant phages (20 µL) were placed sequentially into a microtube and incubated with the scFv library (50 µL) for 1 h each, respectively, when scFv clones that interacted with wild-type and irrelevant phages were removed. Next, the supernatant was collected by using a magnetic apparatus and transferred to two new tubes containing beads coupled with the target B10 and C01 mimotopes, and the incubation for 1 h at 37 °C was performed. The supernatant was discarded, and bound phages were washed 10 times with PBS/0.05% Tween 20; being then eluted with 100 μL of 100 mM glycine-HCl, pH 2.2, for 10 min at room temperature (RT), and the solution pH was neutralized by adding Tris base . Then, selected clones were transferred to a XL1-Blue culture for amplification and titration. Clones were extracted from E. coli XL1-Blue bacterial cells infected in the bio-selection cycles for further transformation in E. coli TOP10 [g for 15 min at 4 °C, and the supernatant containing the soluble scFv clones was transferred to another 96-well plate and stored at 4 °C.For the selection of the specific monoclonal antibodies, a scFv phage library was developed according described . Brieflyescribed ,43,44. I2O2, 2 mg of ortho-phenylenediamine, and 0.1 M citrate-phosphate pH 5.0; when the absorbance was read in an ELISA reader  at 492 nm.For the analysis of scFv clones’ expression, an ELISA experiment was performed. For this, supernatants containing soluble scFv antibodies were incubated in high affinity microplates, and the incubation was processed for16 h at 4 °C. Then, plates were washed with washing solution (PBS 1× and Tween 20 0.05%) and blocked with PBS 1× and BSA 3%, for 1 h at 37 °C, followed by two washing using only PBS 1× and three washes with washing solution. After, plates were incubated with anti-HA horseradish peroxidase-conjugated antibody , which was 1:2500 diluted in PBS 1× and BSA 0.3%, for 1 h at 37 °C. Then, plates were washed again as described earlier, and reactions were developed by adding 30% H11 plaque-forming unit (PFU) or L. infantum SLA (2.0 μg/well) for 16 h at 4 °C. A wild-type clone (1 × 1011 PFU per well) was used as control. Plates were washed 3 times with washing solution, and blocked with 100 μL from a solution composed by PBS 1× and BSA 5%, for 1 h at 37 °C. Then, 50 μL of scFv supernatants were added, and the incubation occurred for 2 h at 37 °C. The anti-HA peroxidase-conjugated antibody (1:2500 diluted) was added, and the incubation occurred for 1 h at 37 °C. After washing, reactions were developed according described above, and optical densities were read in an ELISA reader at 492 nm.For the analysis of specificity of the scFv antibody clones, polystyrene microplates  were coated with 1 × 10g for 10 min at 25 °C, in order to form two phases and the upper phase containing the scFv antibody was collected. The technical procedure was repeated three times, when the limulus amebocyte lysate commercial kit  was used to evaluate the endotoxin level in the purified samples. The purified scFv clones showed near 96% purity and reached a yield of approximately 30.0 mg/L). To identify the corresponding native antigens in Leishmania for each obtained scFv, selected clones were coupled to beads, and 100 μL of mouse anti-His mAb Mag Beads  were added with 100 μL of purified scFv (E8 and C10) clones, according to the manufacturer´s instructions. After washing, L. infantum SLA (1 mg/mL) was added and, after incubation and additional washings, 20 μL of the sample buffer were added, and the incubation for 5 min at 100 °C was performed. The immune-precipitated molecules with the monoclonal antibodies were eluted from beads by using a magnetic apparatus, and they were submitted to mass spectrometry (CID-MS/MS).For the purification of the scFv antibody-display phage clones, His-tagged scFv antibody fragments were immobilized in a metal (Nickel) affinity chromatography column  using a HPLC system (AKTATM purifier), according to the manufacturer’s instructions. The purified scFv clones were concentrated using a 10 kDa Amicon apparatus , and the protein concentration was estimated as described elsewhere . Extracthttp://www.ncbi.nlm.nih.gov) and VBASE2 (http://www.vbase2.org) programs [http://raptorx.uchicago.edu/), and scFv antibody interaction with the target protein : P21148) was evaluated by the Pathdock program (http://bioinfo3d.cs.tau.ac.il/PatchDock/). Structural representations were rendered by the PyMOL Molecular Graphics System program (http://www.pymol.org).The sequencing was performed in heavy- and light-chain variable region genes of selected antibodies, by using the MegaBACE 1000 sequencer . Reactions were developed employing the DyEnamic ET Dye Terminator Cycle Sequencing Kit , following the manufacturer’s instructions. The nucleotide sequence was determined using MMB4 (5′-GCTTCCGGCTCGTATGTTGTGT-3′) and MMB5 (5′CGTTTGCCATCTTTTCATAATC-3′) primers, and antibody variable domain sequences and V gene families were analyzed by the Ig-BLAST (programs . The thrL. infantum genomic DNA by using the (GCTAGCATGCGTGAGATCGTTTCCTG) and (5′GAGCTCCTAGTAGGCCTCCTCCTC) primers. The PCR amplified products were analyzed on agarose gels, excised from the gels, purified, digested with restriction enzymes and ligated to the pET28a-TEV vector. The recombinant plasmid was introduced to Escherichia coli BL21 Arctic Express (DE3) cells  by electroporation using a MicroPulser Electroporation Apparatus . The recombinant protein was expressed by adding 1.0 mM IPTG  for 3 h at 37 °C, with shaking at 200× g per min. Cells were lysed by sonication by applying a continuous pulses of 30 s, with 15 s of interval between them, at 38 MHz, and centrifuged at 13,000× g for 20 min at 4 °C. The purified protein was passed on a 5 mL HIS-Trap column  attached to an FPLC  system, and thus through a polymyxin-agarose column (Sigma), in order to remove residual endotoxin content. Aiming to evaluate the purity of the recombinant protein, bacterial total extracts before and after IPTG induction, as well as the purified recombinant protein (10 µg each) were submitted to a 10% SDS-PAGE.The region codifying the β-tubulin (XP_001468164.1) gene was cloned from the 2O2 30 vol., and stopped by adding 10 mL distilled water.To verify the reactivity of the recombinant protein with the specific monoclonal antibodies, β-tubulin (10 µg) was submitted to a 10% SDS-PAGE and blotted onto a nitrocellulose membrane . Then, membranes were blocked with a solution composed by PBS 1× plus Tween 20 0.05% (PBS-T) added with 5% albumin solution and incubated for 1 h at 37 °C. After, they were washed in PBS-T and incubated with the anti-scFv monoclonal antibodies (1:100 diluted in PBS-T) for 1 h at 37 °C. Membranes were washed with PBS-T and the anti-HA peroxidase conjugated antibody (1:5000 diluted in PBS-T) was added, at which time a new incubation was developed for 1 h at 37 °C. As control, an anti-Histidine monoclonal antibody  and the anti-IgG rabbit peroxidase conjugated antibody  were used. Reactions were developed by adding 12.5 mg chloronaphtol, 25.0 mg diaminobenzidine, and 20 µL Hn = 40; including 24 males and 16 females, with ages ranging from 23 to 59 years), of non-infected individuals living in an endemic area of disease , sera from patients with TL , Chagas disease  and leprosy  were used. The VL diagnosis was confirmed by clinical evaluation and PCR to evaluate the presence of L. infantum kDNA. Also, none of the patients had been previously treated with antileishmanial drugs before samples collection. Healthy subjects did not present any clinical signal or suspect of leishmaniasis. Patients with Chagas Disease were confirmed by Chagatest® v.3.0 kit recombinant ELISA kit and Chagatest® hemagglutination inhibition kit (HAI). Leprosy patients were confirmed by clinical evaluation, immunological assays, and DNA detection through RT-PCR. For the ELISA experiments, 96-well plates  were sensitized with the recombinant β-tubulin protein or L. infantum SLA , which was diluted in carbonate buffer , for 16 h at 4 °C. After sensitization, plates were blocked with PBS 1× and BSA 5%  for 1 h at 37 °C. After, sera samples (1:200 diluted in PBS 1× and BSA 0.5%) were added to the wells, and the incubation occurred for 1 h at 37 °C. Then, plates were washed 5 times with washing solution (PBS 1× and Tween 20 0.05%) and the anti-human IgG peroxidase antibody , 1:5000 diluted in 1× PBS/0.5% BSA, was added. The incubation was processed for 1 h at 37 °C. After, plates were washed 5 times with the washing solution, and reactions were developed by using 0.1 M citric acid, 0.2 M Na2PO4, 0.05% OPD and 0.1% H2O2; and stopped by addition of H2SO4 2 M. The optical densities were determined in an ELISA reader  at 492 nm.Sera samples from VL patients (n = 10) and healthy subjects (n = 10). Patients  were submitted to the same therapeutic regimen by using a dose of 20 mg Sb+5 per kg during 30 days. None of them suffered from any other infections or had any pre-existing disease. PBMCs were purified by density centrifugation through Ficoll-Hypaque , according described [7) were cultured in complete RPMI 1640 medium, which was composed by the medium plus 20% inactivated fetal bovine serum , 2 mM l-glutamine, 200 U/mL penicillin, 100 µg/mL streptomycin, 50 μM 2-mercaptoethanol, 1 mM sodium pyruvate, and 1× non-essential amino acid, and they were incubated in medium (background control) or stimulated with B10, C01 or irrelevant (NRP) phage clones , with the B10 or C01 peptides (10 µg/mL each), or with β-tubulin and L. infantum SLA , for 5 days at 37°C in 5% CO2. The supernatants were collected, and IFN-γ and IL-10 levels were evaluated by capture ELISA by using commercial kits , according to manufacturer’s instructions. Results were interpolated from a standard curve using recombinant cytokines (in pg/mL).To evaluate the specific cytokine production, blood samples were collected from treated untreated or VL patients (escribed . Cells (TM (version 5.0 for Windows). Statistical analyses were performed by two-way analysis of variance (ANOVA), followed by Bonferroni’s post-test for comparisons among the groups. Differences were considered significant when p < 0.05.The results were analyzed by GraphPad Prism


The use of personal cooling systems to mitigate heat strain on first-responders achieves two potential performance benefits relative to the absence of such cooling: (1) the completion of a workload with less effort; and/or (2) the completion of a greater workload for the same effort. Currently, claims made by manufacturers regarding the capability of their products for use in conjunction with chemical/biological protective clothing remain largely unsubstantiated. The purpose of this investigation was to evaluate the means by which heat strain can be alleviated during uncompensable heat stress in chemical/biological clothing, using the ASTM F2300-10 methodology.−1; 35°C; 49% RH) for up to 120 min while wearing one of four cooling systems and/or a National Fire and Protection Association 1994 Class-3 chemical/biological ensemble. The four cooling methods  and no cooling (CON).Eight healthy males completed five trials of continuous walking , PCM (20 ± 10 min) and SLIV (22 ± 10 min), but no differences for WS (4 ± 7 min). Heart rate, rectal, mean skin, and body temperatures were significantly lower in all cooling conditions relative to control at various matched time points in the first 60 min of exercise. Thermal sensation, comfort and perceived exertion all had significant main effects for condition, and time, there were no differences in their respective interactions.The IV, PCM, and SLIV produced lower heart rate, mean skin, rectal and mean body temperatures in addition to improved work times compared to control. The WS did not improve work times possibly as a result of the cooling capacity of the suit abating, and magnifying thermal insulation. Considering the added time and resources required to implement combination cooling in the form of ice slurry and ice vest (SLIV), there was no significant additive effect for perception, cardiovascular strain, rectal temperature and total trial time relative to the phase change vest or ice vest alone. This may be a product of a “ceiling” effect for work limit set to 120 min as part of ASTM F2300-10. Moderate to high-intensity work in the presence of environmental heat stress forces simultaneous demands upon the cardiovascular system by increasing the need for blood flow for thermoregulation and at the active musculature . The phyUncompensable heat stress in these occupations has been demonstrated to significantly limit the operational times before physiological safety limits are reached . CommercThe scientific literature reflects a strong interest in reducing thermal and cardiovascular strain in the first responder and military occupations, suggesting many types of cooling systems are beneficial for workers when used in conjunction with protective clothing . Though,In aggregate, a number of reviews of occupational cooling tend to favor active cooling systems over their passive counterparts . However−1), whilst ice vest influenced mean skin temperature the most (Δ−0.142 ± 0.03°C⋅min−1)  . TherefoFollowing ethical approval from the Queensland University of Technology’s Human Research Ethics Committee, eight healthy males volunteered to participate in this study. The physical characteristics of the recruited sample are displayed in −1; conditions were monitored via wet bulb globe thermometer  approximately positioned at the height of the participants hip. The trials consisted of continuous walking at 4.5 km⋅h−1, 1% grade on a calibrated treadmill for up to 120 min while wearing a personal cooling system and/or a National Fire and Protection Association 1994 Class-3 chemical/biological ensemble. During each trial, standard termination criteria were applied as per F2300-10 (R) ≥ 39.0°C; (2) 120 min of walking duration; (3) heart rate equal to 90% of pre-determined maximum; or (4) impending heat illness . Participants completed a single trial per visit, separated by at least 48 h. Cooling intervention allocation and trial order were randomized (v4 Research Randomizer Form) to ensure a within-participant controlled crossover design.Participants reported to the laboratory on six occasions. The initial visit consisted of familiarization with the various research equipment , environmental conditions, cooling systems, protective clothing, aerobic capacity testing, and anthropometric measurements. The subsequent five sessions consisted of data collection whereby one of four cooling methods and a no cooling control (CON) were implemented in accordance with F2300-10 . Inside F2300-10 : (1) a r−1; 1% grade) fell within the recommendations of the standard (250–400 W) . Phantom calibration was performed before each scan of the participant lying in the supine position. A single scan lasted approximately 6 min and collected segmental and total body composition of fat mass, lean mass, and bone mineral content. Oxygen consumption via indirect calorimetry  was then collected during 8 min of steady-state treadmill walking to ensure the work rate prescribed for all participants (4.5 km⋅h0–400 W) .−1 was implemented before 60 s increments of 1 km⋅h−1 were applied until the fastest speed the participant could maintain without shifting back on the treadmill. Once this pace was achieved, 1% increases in gradient were applied each 60 s until volitional termination. Breath-by-breath gas analysis was undertaken via indirect calorimetry . The gas analysers and pneumotach were calibrated prior to each aerobic capacity test. Participants were fitted with a heart rate monitor and chest strap to allow for calculation of 90% heart rate max, .Aerobic capacity and maximal heart rate were derived from a treadmill-based athletic protocol . An init−1 of water the day before the trial . A USG value < 1.020 classified participants as euhydrated . Those wt-shirt, shorts, sports socks, underwear, and athletic shoes. Participants donned all garments within 6 min, then entered the chamber and stood still for 1 min before commencing walking.The National Fire and Protection Association 1994 Class 3 Extended Response Suit  had a total mass of 2.05 kg; consisting of a one-piece fully encapsulating hooded jumpsuit, including outer gloves and booties (1.35 kg) and a face respirator . For all trials, per F2300-10 , particiThe systems chosen consisted of three passive systems and a single active system. All systems were selected based upon their superior performance, compatibility with the required protective clothing for testing and ensuring at least one active and passive system be comparable. One experimental condition paired an extrinsic (ice vest) and intrinsic (ice slurry ingestion) passive cooling system.Two different cooling vests were tested: (1) an ice-based cooling vest (IV), stored in a -20°C freezer ; and (2) a non-ice-based cooling vest with a melting temperature of 14°C (PCM), stored in a 4°C fridge .−1 from an integrated portable pump  connected to a 2 L reservoir initially containing 90% ice and 10% water. This resulted in ∼10°C water entering the WS when first turned on .Participants donned a three-piece portable battery-operated water-perfused suit  that covered the body, excluding the face, hands, and feet. The WS consisted of tubing sewn into a stretchable pullover, trousers, and hood . Water w−1 of ice slurry  at a rate of 1.25 g⋅kg−1 every 5 min to standardize the ingestion rate , and placed at four sites in accordance with 2, Polar, Finland).After verification of adequate hydration, participants self-inserted a single-use disposable rectal thermistor  12 cm past the anal sphincter to measure TR . During A baseline recording of thermal comfort and sensation was collected inside the chamber in the minute immediately prior to walking. Discrete scale measures of thermal sensation, thermal comfort, and rating of perceived exertion (RPE) were subsequently recorded at 15 min intervals and immediately before trial termination. Thermal sensation was assessed using a modified 13 point scale  with theR and Heart rate, TMean skin and rectal temperatures were used to calculate mean body temperature lated as :R, P-values were obtained by likelihood ratio tests of the full model with the effect in question against the model without the effect in question. Cohen’s d effect sizes between 0 and 60 min were calculated between CON and personal cooling system [Cohen’s d = (Mean Difference/(SDgroup1 +SDgroup2)/2)], and interpreted as small (0.2), medium (0.5), or large (0.8) (d effect sizes in MS Excel (Office 2013), with the linear mixed effect modeling undertaken in the “lme4” package  and inferential statistics (Shapiro-Wilk test). The Greenhouse-Geisser correction was applied when Mauchly’s test of sphericity was violated. A one-way repeated measures analysis of variance (ANOVA) was used to confirm participants arrived in a similar physiological state for each testing day, and for termination times and sweat rates between conditions. We then performed a linear mixed effects analysis of the relationship between the conditions  and the physiological  . ANOVAs  package  in the sp = 0.998), p = 0.208), TR (p = 0.466), p = 0.432), urine color (p = 0.576), urine specific gravity (p = 0.878), urine osmolality (p = 0.775), serum osmolality (p = 0.642), or body mass (p = 0.260).Participants commenced all five trials from a resting physiological baseline, with no significant differences between trials in heart rate (p < 0.001). Post hoc analysis revealed significant differences (p < 0.05) between termination times for IV, PCM, and SLIV and those of CON and WS (p = 0.012), SLIV , PCM , and WS .The eight participants successfully completed all 40 trials without volitional fatigue. Termination criteria and times are outlined in N and WS . The meaR (χ2(4) = 93, p < 0.0001) on average by 0.23 ± 0.03°C, 2(4) = 330, p < 0.0001) by 2.21 ± 0.15°C, 2(1) = 349, p < 0.0001) by 0.62 ± 0.04°C, and heart rate (χ2(4) = 63, p < 0.0001) by 10 ± 1 b⋅min−1. These models were all improved by the addition of an interaction term between time and condition: TR (χ2(4) = 52, p < 0.0001), 2(4) = 11; p = 0.0220), 2(4) = 22; p = 0.0002), and heart rate (χ2(4) = 23; p = 0.0001). Consequently, post-hoc analysis of matched time points between 0 and 60 min, with a Bonferroni adjustment for multiple comparisons, was conducted between CON and each condition for heart rate, TR, ,d = 0.2–1), SLIV (d = 0.3–1), PCM (d = 0.3–0.9), and WS (d = -0.1–0.9); large effects for d = 2.7–3.8), SLIV (d = 3.2–10.1), PCM (d = 1.3–4.6), and WS (d = 1.0–3.9); small to large effects for TR in conditions IV (d = 0.1–0.9), SLIV (d = 0.7–1.8), PCM (d = 0.4–1.4), and WS (d = -0.2–1.2); and large effects for d = 1.5–2.2), SLIV (d = 2.3–4.8), PCM (d = 1.5–2.3), and WS (d = 1.2–1.6).Physiological variables during the trials are displayed in p = 0.002) than that of control; CON 9.3 (± 2.8), IV 7.6 (± 4.3), SLIV 6.1 (± 3.0), PCM 6.9 (± 3.2), and WS 8.1 (± 4.9) mL⋅min−1. Linear mixed models revealed significant main effects for condition (p < 0.0001) and time (p < 0.0001) for thermal sensation, comfort and perceived exertion. The addition of condition to the linear mixed models resulted in lower thermal comfort  by on average 0.61 ± 0.12 au, thermal sensation  by 1.5 ± 0.25 au, and RPE  by 1.75 ± 0.33 au. The addition of an interaction effect of condition by time did not improve the model fit for thermal sensation , thermal comfort , nor RPE .Only mean differences (± SD) in sweat rate for SLIV were significantly lower  and control. Secondly, considering the added time and resources required to implement combination cooling in the form of ice slurry and ice vest (SLIV), there was no significant additive effect for perception, cardiovascular strain, TR, and Often the work intensity, duration and environmental conditions of first responders are dynamic and governed extrinsically, limiting the control of heat illness and injury risk. Personal cooling systems aid in controlling for heat injury risk by significantly reducing the workers thermal and cardiovascular strain. Consider that one (of many) prerequisites to higher work capacities is an ability to extend the time until high deep body temperatures are reached . That isAll four cooling systems implemented in this study had significantly lower cardiovascular and thermoregulatory measures during the first 60 min of walking compared to no cooling. Although, total trial times were significantly less for the perfused water suit relative to the other cooling methods . NotionaAs the particular water-perfused suit tested in our investigation was considered effective for the first hour of our trials , it may still be of interest in operations lasting less than 60 min where chemical/biological protection is used. As there is no one size fits all approach, it is important for key stakeholders to synthesize the relevant resources and literature in order to implement the most pragmatic means of cooling for their workers. With the final decision based upon gauging the occupations specific needs including, the threshold for risk of heat stress , employeWhen interpreting results of heat stress mitigation studies (including our own), readers should remain cognisant that these investigations often lend themselves toward a relatively small sample of unacclimatised, homogeneous, healthy and young male cohort . As a coThe ASTM F2300-10 standard protocol recommends the use of 4, 8 or 14 site mean skin temperature measurements via ISO 9886 , with thR, and In conclusion, three of the four cooling interventions from this study improved work times during prolonged, uncompensable heat stress in chemical/biological clothing. The ice vest, phase change vest and the combination of ice slurry and ice vest saw lower heart rate, This study was carried out in accordance with the recommendations of Human Research Ethics Application by QUT’s University Human Research Ethics Committee with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the QUT’s University Human Research Ethics Committee.AB, MM, GM, and IS conceived and designed the experiments, analyzed the data and wrote the manuscript. AB, MM, SZ, and KS performed the experiments and contributed reagents, materials, and analysis tools.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The aim of this study was to investigate the effects of dihydromyricetin (DHM) on rats with comorbid DNP and MDD. After the comorbid model was established, rat behavior changes were monitored by measuring the mechanical withdrawal threshold, thermal withdrawal latency, sugar water preference, immobility time in the forced-swim test, and open-field test parameters. The expressions of P2X7 receptor in the dorsal root ganglia (DRGs), spinal cord, and hippocampus were assessed by quantitative real-time PCR, Western blotting, and double immunofluorescence. We found that hyperalgesia, allodynia, and depressive behaviors of rats with comorbid DNP and MDD were relieved by treatment with DHM or application of a short-hairpin RNA for P2X7 receptor. The expression levels of P2X7, phosphorylated extracellular signal–regulated kinase 1/2, tumor necrosis factor α, and interleukin 1ß were increased in the DRGs, spinal cord, and hippocampus of rats in the model group but restored after DHM or P2X7 short-hairpin RNA treatment. In conclusion, P2X7 receptor in the DRGs, spinal cord, and hippocampus participates in the transduction of DNP and MDD signals. DHM seems to relieve comorbid DNP and MDD by reducing the expression of P2X7 receptor in the DRGs, spinal cord, and hippocampus and may be an effective new drug for the treatment of patients with both DNP and MDD.Diabetic neuropathic pain (DNP) and major depressive disorder (MDD) are common complications of diabetes mellitus and mutually affect each other. As a member of the ATP-gated ion channel family, P2X Given the high prevalence of diabetes mellitus worldwide, diabetic neuropathic pain (DNP) has become a relatively common condition , 2. DNP Depression, also known as major depressive disorder (MDD), is a serious medical condition affecting public health. Mild and severe depression is closely associated with increased mortality rates in patients with diabetes mellitus . Painful7 receptor is a ligand-gated nonselective cation channel receptor  and spinal cord have not been reported.After nerve injury and inflammation, nerve endings release large amounts of adenosine triphosphate, an important neurotransmitter that activates several purinergic receptors and is necessary for many biological and pathological functions , 10. P2Xreceptor –13 that receptor , 15. Mangression –18. Our rol rats . We specAmpelopsis grossedentata, and provides myriad health benefits, including anti-inflammatory, antitumor, and rapid antidepressant-like effects  is the most abundant organic chemical in vine tea, which is made from  effects , 21. Hom effects , suggestreceptor Figure 1Male Sprague–Dawley rats (180–220 g) were provided by the Centre of Laboratory Animal Science of Nanchang University. The procedures of this study were approved by the Animal Care and Use Committee of Nanchang University Medical School and were performed according to IASP’s ethical guidelines for pain research in animals. Rats were housed under controlled conditions at 25°C temperature and 60% humidity, with freely available food and water. Five rats were housed in each cage. The timeline of this study is shown in During the week before the start of the experiment, rats were fed a normal diet. After that, they were fed high-glucose, high-fat diet for 4 weeks. After the end of the 4 weeks, rats were starved for more than 12 h and were then given an intraperitoneal (i.p.) injection of streptozotocin . Blood glucose was measured after food consumption. Rats whose blood glucose levels were higher than 16.7 mmol/l were chosen as having type 2 diabetes mellitus. For the next 5 weeks after injecting STZ, chronic unpredictable stress (CUS) stimuli were given randomly. Meanwhile, we measured responses in several behavioral tests once a week to verify that rats had both DNP and MDD, as follows: thermal withdrawal and mechanical withdrawal tests, sucrose preference (SP) test, forced-swimming test (FST), and open-field test (OFT). The CUS stimuli included food deprivation (24 h), cold swimming , water deprivation (24 h), heat stress , reverse light/dark cycle, no stressor, and clip of the tail (1 min) . Rats we7 receptor short-hairpin RNA (shRNA) treatment group (model + P2X7 shRNA), and (6) scramble shRNA treatment group (model + scramble shRNA). Rats in the control + DHM and model + DHM groups were treated with DHM via i.p. injection once a day, at a dose of 30 mg/kg, for 14 consecutive days.Seventy-two male rats were randomly divided into six groups: (1) control, (2) control + DHM, (3) comorbid DNP and MDD model (model), (4) DHM treatment group (model + DHM), (5) P2X7 or scramble shRNA) and transfection reagent at a ratio of 1:2 (μg/μl) was prepared using the Entranster™ in vivo transfection reagent (Engreen Biosystem Company of Beijing), according to the manufacturer’s instructions. The complex was intrathecally injected into rats of the model + P2X7 and model + scramble shRNA groups. The behavior of rats in the two groups was assessed once a day after the intrathecal injection. The sequences of P2X7 shRNA were as follows: ′-CACCGTGCAGTGAATGAGTACTACGAATAGTACTCATTCACTGCAC-3′ and 3′-CACGTCACTTACTCATGATGCTTATCATGAGTAAGTGACGTGAAAA-5′.The transfection complex consisting of shRNA  was evaluated by observing the withdrawal responses to mechanical stimulation, induced by using the BME-404 electronic mechanical stimulator . The end-face diameter of the test needle, the pressure measurement range, and pressure measurement resolution of the stimulator were 0.6 mm, 0.1 to 50 g, and 0.05 g, respectively. Before evaluation, each rat was placed in a clean glass box positioned on the sieve of a metal frame for an adaptive period of at least 30 min. The test needle touched the place between the third and fourth metatarsus of the left hind paws, until the rat attempted to withdraw its paw. The computer recorded the pressure values automatically. The stimulus alternated between the left and right hind paws at 5-min intervals. The MWT was calculated as the mean of three consecutive stable values, expressed in grams, and was determined by one observer.Thermal withdrawal latency (TWL) was evaluated by measuring the latency to hind paw withdrawal from a thermal stimulus, administered using the BME-410C Thermal Paw Stimulation System . Before evaluation, each rat was placed on a glass plate in a transparent, square, bottomless acrylic box, for an adaptive period of at least 30 min. A beam of radiant heat was oriented at the plantar surface of the rat’s paws. The activation of the beam simultaneously activated a timer. The cutoff time for heat stimulation was 30 s. The light beam was switched off when the animal lifted its paw, and the timing was over. The time on the screen of the apparatus was designated as the TWL and was expressed in seconds. The hind paw withdrawal was tested in triplicate, and hind paws were alternated at 5-min intervals.Before the test, rats were fasted for 24 h and then placed individually in separate cages. Two identical water bottles, one containing 100 ml of 1% sucrose in water and another containing 100 ml of pure water, were placed in every cage at the same time. SP was evaluated by measuring the levels of sugar–water and pure-water consumption in 1 hour. The SP rate was calculated as the ratio of sugar water/total liquid consumption × 100%. This test reflects a lack of pleasure.Rats were placed in an 80-cm-high glass cylinder with a 40-cm inner diameter. Water temperature was approximately 20, and water depth was 30 cm. The immobility time (IT) of rats in the water, i.e., when rats stopped struggling and floated in a fixed shape, and the swimming time of each rat were recorded for 5 min and expressed in seconds.Before the test, rats were placed in the dark for 30 min to adapt to the environment. Then, they were placed in a black box that measured 40 × 60 × 50 cm. Each rat was placed gently in the middle of the box, and the distance navigated by the animal was recorded using a Canon Powershot A610 camera  during a 5-min session. The recorded videos were analyzed and processed using MATLAB  to determine the total distance traveled, expressed in centimeters. The apparatus was cleaned with a 10% ethanol solution before the next animal was introduced into the box.Rats were anesthetized by i.p. injection of 10% chloral hydrate . DRGs, the spinal cord at the level of L4–L5 vertebrae, and hippocampus were isolated immediately after sacrifice from rats in different groups, flushed with ice-cold phosphate-buffered saline (PBS), and stored in RNA Store solution at −20°C until further use. All instruments were treated with DEPC before use.7 forward 5′-GATGGATGGACCCACAAAGT-3′ and reverse 3′-GCTTCTTTCCCTTCCTCAGC-5′.Total RNA was separately isolated from DRGs, spinal cord, and hippocampus using the TRIzol Total RNA Reagent (Beijing TransGen Biotech Co.). Complementary DNA synthesis was performed with 2 µg total RNA using the RevertAid™ HMinus First Strand cDNA Synthesis Kit. The primers were designed using Primer Express 3.0 Software (Applied Biosystems), and sequences were as follows: β-actin forward 5′-TAAAGACCTCTATGCCAACA -3′ and reverse 3′-CACGATGGAGGGGCCGGACTCATC-5′; P2X−ΔΔCT.Quantitative real-time PCR was performed using the SYBR® Green Master Mix in the ABI PRISM® 7500 Sequence Detection System . The expression of each gene was quantified using the ΔΔCT method, with CT as the threshold cycle. The relative levels of target genes normalized to the sample with the lowest CT are presented as 27 , β-actin , extracellular signal–regulated kinases 1/2 , and phosphorylated (p)-ERK1/2  at 4°C overnight. Membranes were washed three times with 1× TBST, 10 min each, incubated for 2 h at room temperature with horseradish peroxidase–conjugated secondary goat anti-rabbit immunoglobulin G and goat anti-mouse immunoglobulin G antibodies  in blocking buffer, and washed again three times with 1× TBST, 10 min each. Labeled proteins were then visualized with enhanced chemiluminescence on a Bio-Rad system. Band intensities were quantified using Image-Pro Plus software, and the intensities of target proteins were normalized against the respective β-actin internal control.After rats were anesthetized, DRGs, spinal cord at L4–L5, and hippocampus were separated and flushed with ice-cold PBS. Tissues were positioned in the spherical part of a 2-ml homogenizer and homogenized in RIPA lysis buffer  containing protease inhibitors. Tissues were ground for 30 min on ice and centrifuged at 4°C at 12,000 rpm for 10 min. The supernatants were collected, diluted with 6× loading buffer, and heated to 95 for 10 min. The protein concentration was calculated with the BCA Protein Assay Kit, and samples were kept at −20°C until use. Proteins in samples from each group (20 μg) were separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, using Bio-Rad electrophoresis device, and transferred onto polyvinylidene fluoride membranes. Polyvinylidene fluoride membranes were blocked with 5% nonfat dry milk in 1× TBST  for 2 h at room temperature, followed by incubation with antibodies against P2XRats were anesthetized with 10% chloral hydrate and were transcardially perfused with 4% paraformaldehyde (PFA). The DRGs, spinal cord at L4–L5, and hippocampus were removed and fixed in 4% PFA at 4°C for 2 h at room temperature. The tissues were immersed in 30% sucrose solution (in 4% PFA) for 24 h, at 4°C, for dehydration; solutions were changed every 8 h. Tissues were cut into 12- or 8-μm-thick slices in a cryostat (Leica). The sections were placed at 37°C for 2 h and then stored at −20°C until use.7, 1:200, Alomone Labs; and mouse anti–glial fibrillary acidic protein [GFAP], 1:200, Millipore) overnight at 4°C. The next day, sections were placed at room temperature for 30 min and washed with PBS (three × 5 min); secondary antibodies  were added on sections at 37°C for 1 h. Sections were washed for 5 min × three times with PBS, sealed with antifade solution, and imaged using a fluorescence microscope . Image-Pro Plus6.0 software was used to analyze the immunofluorescence intensity ratio of P2X7 and GFAP coexpression normalized to control values.Before staining, sections were balanced at room temperature, rinsed with 0.01 M PBS for 5 min × three times, incubated with 0.3% Triton X-100, and washed again with PBS for 5 min × three times. Then, slices were incubated in 10% goat serum for 1 h at 37°C, followed by incubation with the diluted antibodies (rabbit anti-P2XFor assessing the levels of tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β), we performed enzyme-linked immunosorbent assay (ELISA) using Rat PicoKine™ ELISA Kit , according to the manufacturer’s instructions. Experimental samples (diluted to 100 µl) or control were loaded in triplicates, and 100 µl of antibody solution (1× biotinylated anti-rat TNF-α or IL-1β antibody) was added to each well and incubated for 60 min at 37°C. After washing three times with 1× wash buffer, 100 µl of 1× avidin–biotin–peroxidase complex was added to each well and incubated for 30 min at 37°C. Wells were washed five times with 1× wash buffer, and 90 µl of color developing reagent was added to each well and incubated in the dark for 25 min at 37°C. The reaction was stopped by adding 100 µl of stop solution to each well. The absorbance at 450 nm was read using a microplate reader.p < 0.05.Statistical analyses were performed using SPSS 21.0 software. Data were analyzed by one-way analysis of variance. The experimental results are expressed as mean ± standard error of the mean and were considered significant at p< 0.05), thus confirming the efficiency of our model. Two weeks after injection of DHM or 1 week after injection of P2X7 shRNA, the MWT and TWL were significantly lower in the model + P2X7 shRNA and model + DHM groups than in the model group (p < 0.05), indicating that both P2X7 shRNA and DHM treatment relieve neuropathic pain–related behavior in rats with DNP and MDD . Two weeks after injection with DHM or 1 week after injection with P2X7 receptor shRNA, the values for SP and OFT distance were significantly lower in the model + P2X7 shRNA and model + DHM groups than in the model group (p < 0.05). In addition, the IT in the FST was shorter in the model + P2X7 shRNA and model + DHM groups than in the model group (p < 0.05). The above results indicate that treatment with DHM or P2X7 shRNA relieves depression-like behaviors in model rats. Simultaneously, anhedonia improved, as evidenced by the higher values in the SPT and OFT and by the reduced IT in the FST in rats with DNP and MDD, indicating that DHM or P2X7 shRNA may moderate depressive symptoms .The expression levels of P2Xblotting Figure 47 in the DRGs, spinal cord, and hippocampus was detected using double-immunofluorescence labeling for GFAP and P2X7. GFAP marks satellite glial cells (SGCs). We found that P2X7 and GFAP coexpression in these regions were higher in model than in control rats (p < 0.05). However, the treatment with DHM or P2X7 shRNA reversed these changes , while treatment with DHM or P2X7 receptor shRNA significantly reduced them . However, treatment with DHM or P2X7 receptor shRNA reversed this increase    in the Dytokines . A signiactivity , 39. P2Xdiseases , 41. Mordiseases , 43, anddiseases . Our teadiseases , 46. In 7 receptor shRNA reduced the levels of p-ERK1/2 in the model group indicates that P2X7 receptor is associated with ERK pathway activation, involved in DNP and MDD. We found that DHM and P2X7 receptor shRNA treatments had similar effects. Therefore, DHM may inhibit the activation of ERK1/2 pathway by decreasing the expression of P2X7 receptor, reducing the secretion of inflammatory cytokines in peripheral glial cells, restraining pain and depression transduction, and thereby alleviating the symptoms of pain and depression.Mitogen-activated protein kinase is involved in MDD and peripheral nerve injury-induced neuropathic pain, which mainly includes three pathways: ERK, p38 kinase, and c-Jun N-terminal kinase , 48. Man7 receptor, including research with human subjects, is therefore required.One limitation of our study is that we used a rat model. Due to the complexity of DNP and MDD, rat models cannot completely mimic the human symptoms. Moreover, multiple pathways and receptors are involved in the progression of these disorders, and there are drugs other than DHM that may also effectively treat these disorders, such as palmatine . Further7 receptors in the DRGs, spinal cord, and hippocampus participate in the transduction of DNP- and MDD-related signals. DHM decreases P2X7 receptor expression in rats with comordid DNP and MDD, down-regulates ERK1/2 pathway activation, and reduces the release of the inflammatory factors TNF-α and IL-1β. These effects ultimately alleviate DNP and depressive behaviors. We propose that DHM may be an effective new drug for treating patients with comorbid DNP and MDD.In conclusion, we showed that P2XSG conducted the experiments with assistance from YS, HG, LL, CY, XW, LH, and WX. SG, WX, and YG contributed to the experimental design, data analysis and interpretation, and writing.This study was supported by grants from the National Natural Science Foundation of China , Major Disciplines of Academic and Technical Leaders Project of Jiangxi Province (no. 20142BCB22001).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cisplatin increased ROS yield (1.2-fold), and curcuminoid and combination treatments reduced ROS (0.75–0.85-fold). Combination treatments reduced A549 migration (0.51–0.53-fold). Both curcuminoids reduced auditory threshold shifts induced by cisplatin. In summary, cisplatin and the curcuminoids might cause cell death through AIF and caspase-12. The curcuminoids may potentiate cisplatin’s effect against A549 migration, but may counteract cisplatin’s effect to increase ROS production. The curcuminoids might also prevent cisplatin ototoxicity.In this study, we investigated whether the curcuminoids, CLEFMA and EF24, improved cisplatin efficacy and reduced cisplatin ototoxicity. We used the lung cancer cell line, A549, to determine the effects of the curcuminoids and cisplatin on cell viability and several apoptotic signaling mechanisms. Cellular viability was measured using the MTT assay. A scratch assay was used to measure cell migration and fluorescent spectrophotometry to measure reactive oxygen species (ROS) production. Western blots and luminescence assays were used to measure the expression and activity of apoptosis-inducing factor (AIF), caspases-3/7, -8, -9, and -12, c-Jun N-terminal kinases (JNK), mitogen-activated protein kinase (MAPK), and proto-oncogene tyrosine-protein kinase (Src). A zebrafish model was used to evaluate auditory effects. Cisplatin, the curcuminoids, and their combinations had similar effects on cell viability (IC However, curcumin exhibits limited bioavailability, which restricts its efficacy ,15. Syntactivity ,19. CLEFactivity ,21,22. Eactivity ,24,25. A50) values of cisplatin, CLEFMA, and EF24 alone and combination treatments in which either one of the curcuminoids was combined with cisplatin.We utilized a series of biochemical assays along with a zebrafish model of hearing to evaluate whether CLEFMA and EF24 could promote the anti-cancer action of cisplatin and prevent the platinum compound’s auditory side effects. First, we used the MTT assay to ascertain the inhibitory concentration at 50%  and significantly increased for the positive control (1.32-fold of control), but caspase-9 activity decreased for all other treatments .We then investigated whether cisplatin, the curcuminoids, or cisplatin–curcuminoid combinations could affect cancer cells migration using a wound recovery assay . We founWestern blot analysis was used to determine the effect of cisplatin, the curcuminoids, and their combination on the expression of several proteins involved in cisplatin-mediated signaling. We found that all treatment categories exhibited expression of apoptosis-inducing factor (AIF) protein . Similarp < 0.0001; As a final step, we performed the auditory evoked potential technique in a zebrafish model of hearing to determine if treatment with cisplatin, the curcuminoids, and the cisplatin–curcuminoid combinations altered hearing. We found that fish injected with cisplatin exhibited threshold shifts at several frequencies compared to sodium chloride (NaCl) vehicle- or DMSO vehicle-injected fish . When a 50 value of EF24 , Sox2, Wnt/β catenin signaling and the microRNA, miR-146a, through regulation of cyclin J ,53,54. HAs CLEFMA and EF24 are structural analogs of curcumin ,24, and Cisplatin treatment damages DNA in auditory hair cells causing ROS generation, hair cell death, and reduced hearing ,63,64,65We found that cisplatin caused significantly increased hearing threshold shifts (up to 10 dB) at three frequencies compared to both the platinum compound’s NaCl vehicle and the curcuminoid vehicle, DMSO, while both CLEFMA- and EF24-treated fish exhibited less than 5-dB threshold shifts at one frequency (CLEFMA) and two frequencies EF24; . When we2 with passaging every 4–7 days.The non-small cell lung cancer cell line, A549, was obtained from American Type Culture Collection . F12K media  and was used with 10% fetal bovine serum  and 1% penicillin/streptomycin (Gibco). Cells were incubated at 37 °C in 5% CO2. Then, the 96-well plates were treated with a dilution series  of either cisplatin, EF24, or CLEFMA at 37 °C in 5% CO2 over the following time intervals: Cisplatin (0–24 or 0–48 h), EF24 (0–24 or 24–48 h), and CLEFMA (0–24 or 24–48 h). A negative control (cells in media with no drug administration), positive control , and media-only blanks were run. After 24 or 48 h, the MTT assay was run for 2 h and absorbance was determined using a spectrophotometer  set at 570 nm and 690 nm. Then, for experiments where cisplatin and a curcuminoid were combined, another set of plates was prepared using the same procedure as above, except the experimental wells were treated (t = 0 h), with the 48-h IC50 value of cisplatin followed by treatment (t = 24 h) with a dilution series  of a curcuminoid for 24 h. Then, the MTT assay was performed to generate a combination treatment IC50 value based on the total concentration of cisplatin with EF24 or CLEFMA. A separate plate with a DMSO (Fisher Scientific)-only treatment was prepared using the same dilution series and the protocol described above to determine whether this solvent used in the curcuminoid preparations affected cellular viability.The colorimetric 3--2,5-diphenyltetrazolium bromide  assay was used to determine the effect of cisplatin, EF24, and CLEFMA (Sigma-Aldrich) against cancer cell viability. A549 cells were seeded at a density of 5000 cells per well in supplemented F12K media in 96-well plates in replicates of three in three separate experiments and incubated for 24 h at 37 °C in 5% CO6 A549 cells in 10 mL of F12K media with 10% fetal bovine serum and 1% penicillin/streptomycin supplementation. Dishes were incubated for 24 h at 37 °C and 5% CO2. Media was then aspirated out of the dishes and replaced with 10 mL of the following treatments : Media only for 48 h (negative control); 100 µM 30% hydrogen peroxide (H2O2) (Sigma-Aldrich) for 48 h (positive control); 10 µM cisplatin for 48 h, 2 µM EF24 for 48 h, 15 µM CLEFMA for 48 h, 10 µM cisplatin for 24 h followed by 10 mL of 2 µM EF24 for another 24 h, and 10 µM cisplatin for 24 h followed by 10 mL of 15 µM CLEFMA for another 24 h. After 48 h, media was aspirated out. Each dish was then washed three times with 2 mL of phosphate-buffered saline (PBS) (Sigma-Aldrich). The cells were then detached by adding 1 mL of Accutase  to each dish. Then, the cells were transferred to individual microcentrifuge tubes and 0.5 mL of PBS was added to each tube. Each tube was spun for 5 min at 1000 rpm. The supernatant was then carefully discarded and the pellet was resuspended with 500 µL of 10 µM ROS indicator dye  in PBS and each tube was incubated for 45 min at 37 °C in 5% CO2. Next, the tubes were centrifuged at 1000 rpm for 5 min. Then, the cells were washed three times with PBS and were resuspended in 1 mL of PBS. Then, 100 µL of cell suspension, containing approximately 100,000 cells, was placed into each of nine wells of a black 96-well plate. Also, 100 µL of PBS was placed into nine wells (blank treatment). Then, the plate was placed into a spectrophotometer and read at 495 nm (excitation) and 527 nm (emission) wavelengths.A spectrophotometric fluorescent assay adapted from ,73 was u2. Wells were treated with one of the following: cisplatin IC50 for 48 h, EF24 IC50 from 24–48 h, CLEFMA IC50 from 24–48 h, cisplatin IC50 for 24 h followed by EF24 IC50 for 24 h, cisplatin IC50 for 24 h followed by CLEFMA IC50 for 24 h, negative control (media with cells), positive control (100 µM 30% hydrogen peroxide (H2O2), and blank (media with no cells). After introduction of the luminogenic substrate, plates were kept in the dark at room temperature. The plates were read at 0.5, 1, 2, and 3 h-timed intervals with a plate reader (BioTek) using the luminescent setting.Luminogenic kits  were used to measure caspase-3/7, -8, and -9 activity. First, 10,000 A549 cells were plated in replicates of three in white 96-well plates (Fisher Scientific) and placed in an incubator for 24 h at 37 °C in 5% CO6 A549 cells and incubated at 37 °C in 5% CO2 for 24 h. Next, the media was aspirated out and each dish was treated with one of the following preparations: 10 mL media only (negative control) for 48 h, 10 mL of 100 µM 30% H2O2 (positive control) for 48 h, 10 mL of IC50 cisplatin for 48 h, 10 mL of IC50 EF24 from 24–48 h, 10 mL of IC50 CLEFMA from 24–48 h, 10 mL of IC50 cisplatin for 24 h followed by IC50 EF24 for 24 h, or 10 mL of IC50 cisplatin for 24 h followed by IC50 CLEFMA for 24 h.Western blot analysis was used to measure AIF or caspase-12 expression. First, seven 10-cm dishes were seeded with 1.0 × 10g, 4 °C). Determination of protein concentration was done using the Bradford assay  and a standard curve of results made using Prism . Samples containing 15 µg of protein (5 µg for EF24 samples) were then loaded into Mini Protean TGX  gel wells. Gels were run in a gel apparatus (Bio-Rad) for approximately 1 h, and samples were then electro-transferred for 60 min to a polyvinylidene fluoride (PVDF) membrane (Bio-Rad) using a Bio-Rad transfer device. Membranes were then incubated with primary antibody obtained from Cell Signaling Technology  against AIF (4642), β-tubulin (2146), caspase-12 (2202), GAPDH (3683), JNK (9252), pJNK (4668), MAPK (4370), pMAPK (4695), Src (2109) or pSrc (2101) diluted 1:1000 overnight. After washing in tris-buffered saline (TBS) and polysorbate 20 , membranes were incubated in 10 mL SignalFire™  and were then fluorescence imaged using an Alpha-Innotech FluorChem HD2 Gel Imaging System .Cells were washed with PBS and then removed using a scraper and transferred into micro-centrifuge tubes. After centrifugation (1500 RPM for 5 min), cells were lysed using sodium dodecyl sulfate buffer  and then protease and phosphatase inhibitor  was added to each tube. Samples were then heated to 95–100 °C followed by sonication and centrifugation . For the cisplatin with curcuminoid treatments, the curcuminoid was added three hours after the cisplatin. This step was to ensure that the cisplatin can enter the cell and the nucleus ,71 beforA Nikon camera  attached to a Nikon TMS microscope was used to take pictures of the wound at 0 h and 24 h after treatment. Then, the area of the wound was measured using Adobe Photoshop’s  lasso function. The percent wound recovery was then calculated by subtracting the cleared area at 24 h from the cleared area at 0 h and then dividing the resultant value by the original cleared area and converting to a percentage.Danio rerio) were obtained from a commercial supplier  and maintained in the Western Kentucky University animal handling facility following Institutional Animal Care and Use Committee protocols . All zebrafish were a mix of male and female adult animals at least six months of age and were maintained according to standard methods [Wild-type zebrafish  technique  was perfFish were prepared for AEP by being anaesthetized with tricaine methanesulfonate (MS-222)  and then placed into a mesh harness suspended 6 cm from the water surface and 22 cm above a University Sound UW-30 underwater speaker  in a 19-L tank containing 27–28 °C water. Electrical interference was minimized by keeping the tank within a Faraday cage inside a sound-attenuation room . Three stainless steel subdermal electrodes  were attached 1–2 mm subdermally into the fish at the following locations: over the brainstem (recording electrode), between the nares (reference electrode), and in the tail musculature (ground electrode). Pure tone pip sound stimuli at eight different frequencies  were presented to the fish and AEP waveforms collected using SigGen and BioSig software running on a TDT physiology system . The sound pressure levels of each frequency were confirmed using a calibrated hydrophone  placed proximate to the fish. Each frequency was tested by decreasing decibel levels in 5-dB steps until an AEP trace was no longer visible. The last sound pressure level at which an AEP trace was visible was noted as the threshold for each frequency, and the frequency thresholds were used to produce audiograms. Between six and eight fish were treated for each treatment category.p value set at 0.05 was used for all statistical analysis unless otherwise noted below. MTT assay IC50 values were calculated in Prism using a relative, nonlinear best fit analysis performed with the sigmoidal, 4PL, x is log(concentration) analysis feature or were calculated using a linear analysis with ED50plus v1.0 online software . MTT assay standard deviation values were calculated using ED50plus. The second-order polynomial (quadratic) function in Prism was used to derive best fit values and linear regression analysis was used to analyze slope difference. AEP, caspase, and ROS assay results were analyzed using a two-way ANOVA and Tukey’s post hoc tests. Migration assay results were analyzed using a one-way ANOVA with a Dunnett’s multiple comparison test. Analysis of cisplatin-curcuminoid drug combinations to determine additive or synergistic effects was performed using the Prism Bliss independence nonlinear analysis feature with the confidence limit set to 95%.GraphPad Prism v6  with the Cisplatin is extensively used in chemotherapy but is limited by the development of resistance and side effects, including permanent hearing loss. The synthetic curcumin analogs, CLEFMA and EF24, may signal in different cell death pathways to potentiate the effect of cisplatin against cancer and prevent its side effects. In this project, we found that cisplatin and the curcuminoids, alone or in combination, had similar effects against A549 non-small lung cancer cell viability. Also, we found that cisplatin treatment acted to increase ROS production, but that the curcuminoids, separately or in combination, reduced ROS yield. Further, the treatments either reduced or left the activity of caspases-3/7, -8, and -9 unchanged. Western blot analysis suggested that the compounds may modulate cell death or survival mechanisms integrating AIF, caspase-12, and MAPK signaling, but not JNK or Src. We also found that the cisplatin–curcuminoid combinations could reduce A549 cancer cell migration. Finally, our AEP analysis suggests that the curcuminoids may be able to reduce cisplatin-induced auditory threshold shifts, but we cannot rule out a role for the curcuminoid vehicle, DMSO, in this effect. Evidently, additional study of cisplatin, CLEFMA, and EF24 could help determine their mechanistic relationships in modulating cancer cell death and ototoxicity and assist in the development of new chemotherapy drugs with reduced side effects.