Following combination treatment with seviteronel and radiation, increased binding of AR occurred at DNA damage response genes, including genes involved both in homologous recombination and non-homologous end joining. This trend was not observed with combination treatment of enzalutamide and RT, suggesting that seviteronel may have a different mechanism of radiosensitization compared to other AR inhibitors. Enzalutamide and seviteronel treatment also had different effects on AR and AR target genes as measured by immunoblot and qPCR. These results implicate AR as a mediator of radioresistance in AR+ TNBC models and support the use of seviteronel as a radiosensitizing agent in AR+ TNBC.Increased rates of locoregional recurrence (LR) have been observed in triple negative breast cancer (TNBC) despite multimodality therapy, including radiation (RT). Recent data suggest inhibiting the androgen receptor (AR) may be an effective radiosensitizing strategy, and AR is expressed in 15–35% of TNBC tumors. The aim of this study was to determine whether seviteronel (INO-464), a novel CYP17 lyase inhibitor and AR antagonist, is able to radiosensitize AR-positive (AR+) TNBC models. In cell viability assays, seviteronel and enzalutamide exhibited limited effect as a single agent (IC50 > 10 μM). Using clonogenic survival assays, however, AR knockdown and AR inhibition with seviteronel were effective at radiosensitizing cells with radiation enhancement ratios of 1.20–1.89 in models of TNBC with high AR expression. AR-negative (AR−) models, regardless of their estrogen receptor expression, were not radiosensitized with seviteronel treatment at concentrations up to 5 μM. Radiosensitization of AR+ TNBC models was at least partially dependent on impaired dsDNA break repair with significant delays in repair at 6, 16, and 24 h as measured by immunofluorescent staining of γH2AX foci. Similar effects were observed in an  Most patients with TNBC receive multimodal therapy, including surgery, chemotherapy, and radiation therapy (RT), yet TNBC patients still experience the highest rates of locoregional recurrence of any breast cancer subtype. Due to the lack of molecular targeted therapies available for these patients, as well as their intrinsic insensitivity to radiation therapy  or blocking the binding of androgens to their cognate receptor (AR inhibition) –17. ProdBeyond the role of the androgen receptor in driving cancer cell proliferation, previous work in prostate cancer and breast cancer has demonstrated the role of AR in mediating DNA repair and in the DNA damage response following radiation therapy –29. Thesin vitro and in vivo and that radiosensitization is mediated, at least in part, through the delayed repair of dsDNA breaks. The mechanism of radiosensitization, however, appears to be different with seviteronel treatment compared to enzalutamide due to differences in AR binding to DNA damage response genes following treatment with seviteronel and radiation.Previous work by our group has shown that AR is a mediator of radioresistance in TNBC and that enzalutamide-mediated AR inhibition is sufficient to sensitize AR+ TNBC cells to RT . Here we2 incubator at 37°C. All cell lines were also authenticated using DNA fingerprinting by short tandem repeat (STR) profiling and routinely tested for mycoplasma (monthly).Cell lines were obtained from American Type Culture Collection (ATCC) and grown from frozen samples, including MDA-MB-453 and ACC-422 (AR+ TNBC), MCF-7 , and MDA-MB-231 (AR− TNBC). SUM-159 and SUM-185PE (AR+ TNBC) cells were obtained from the University of Michigan stocks generously provided by Dr. Stephen Ethier who isolated the cells while at the University of Michigan. MDA-MB-453 and MDA-MB-231 cells were grown in DMEM (Invitrogen) with 10% fetal bovine serum . ACC-422 cells were grown in MEM (Gibco) supplemented with 15% FBS (Invitrogen) and 1X Insulin-Transferrin-Selenium-Ethanolamine . SUM-185 cells were grown in Ham's F-12 media (Gibco) with 5% FBS (Invitrogen), with 0.01M HEPES (Gibco #H3375), 1 μg/mL Hydrocortisone (Sigma #H4001), and 1X ITS-X (Gibco). SUM-159 cells were grown in Ham's F-12 media (Gibco) with 5% FBS (Invitrogen), 0.01M HEPES, 1 μg/mL Hydrocortisone, 1x antibiotic-antimycotic , and 6 μg/mL insulin (Sigma #I9278). MCF-7 cells were grown in DMEM (Gibco) with 5% FBS (Invitrogen). MDA-MB-453, ACC-422, MCF-7, and MDA-MB-231 cells were supplemented with 1% penicillin/streptomycin (Thermo #15070063). All cell lines were grown in a 5% CO5 cells/well, ACC-422 2.5 × 105 cells/well). The following day, cells were pretreated with charcoal stripped serum (Atlanta Biologics S11650H) for 24 h. Cells were stimulated with 10 nM dihydrotestosterone  and 5 μM enzalutamide (MedChemExpress HY-70002) or seviteronel (Innocrin) and harvested at indicated time points following treatment. Lysis was performed using RIPA buffer (Thermo Fisher 89901) containing protease and phosphatase inhibitors . Proteins were detected using antibodies for phospho-DNAPKcs , total DNAPKcs , androgen receptor , GAPDH , and β-Actin . Secondary antibodies were obtained from Cell Signaling .Cells were plated in 6-well plates in media containing FBS  cells were grown for five days and stained with 0.5% crystal violet. SUM-159 (750 cells/well), MDA-MB-231 , MDA-MB-453 , SUM-185 , and ACC-422  cells were grown for three days, and cell viability was assessed using Alamar Blue (Thermo Fisher DAL1100). Plates were read on a microplate reader (Cytation 3), and growth was calculated relative to the vehicle control (DMSO). IC50 values for were calculated with GraphPad Prism, and results are reported as the mean ± SEM of three independent experiments. To model hormone deplete conditions, media contained charcoal stripped serum (CSS), without phenol-red were used for MDA-MB-453 and MDA-MB-231 (Gibco 21063-029) and ACC-422 (Gibco 51200-038) cells. Phenol-red free media for ACC-422 cells was supplemented with 2 mM L-glutamine (Sigma G7513). Cells were plated in media containing FBS and were pretreated overnight with CSS before treatment with enzalutamide or seviteronel in concentrations from 1 nM to 10 μM.Cells were plated at single cell density in 6 well-plates and allowed to adhere overnight. Cells were then treated with seviteronel at various doses for 1 h before radiation (2–8 Gy) treatment. Cells were grown for one to four weeks before fixing with methanol/acetic acid and staining with crystal violet. Colonies of 50+ cells were counted and analyzed with the linear quadratic model. Plating densities are outlined in 5 cells/well on coverslips in 12 well-plates and allowed to adhere overnight. The following day plates were pretreated with seviteronel (1 and 5 μM) for 1 h before radiation treatment. Cells were fixed at designated time points after radiation using 4% paraformaldehyde. Foci were stained with an anti-phospho-histone H2AX (ser139) antibody (Millipore 05-636), and a fluorescent goat anti-mouse secondary antibody (Invitrogen A11005). Pictures were taken of >100 cells per condition, and γH2AX foci were scored visually by a blinded observer. MDA-MB-453 cells containing ≥ 15 γH2AX foci were scored as positive, and ACC-422 cells with ≥ 10 γH2AX foci were scored as positive. Results were pooled for statistical analysis.Cells were plated at a density of 2.5 × 106 cells were resuspended in 50% Matrigel (BD Biosciences) with PBS. In addition, one 12.5 mg 60 day release DHT pellet (Innovative Research of America) was implanted at the nape of the neck at the time of cell injections. When tumors reached ~80 mm3, mice were treated with one of the following treatment conditions: vehicle control , seviteronel (75 mg/kg), 2 Gy of RT given once a day for 6 days, or combination of seviteronel with RT with 8 mice in each treatment group. Vehicle control and seviteronel (75 mg/kg) were both administered orally, once daily during treatment. Mice treated with both seviteronel and RT were given seviteronel for 24 h before RT. Tumor growth was measured with digital calipers using the equation: V = L*W2*π/6. Body weight was measured weekly to assess weight loss and toxicity of therapy. All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Michigan and comply with regulatory standards.MDA-MB-453 cells were subcutaneously injected bilaterally into the flanks of female CB17-SCID mice. 4.0 × 10DHT was obtained from Sigma (D-073). Enzalutamide was obtained from MedChemExpress (HY-70002). Seviteronel was obtained directly from Innocrin Pharmaceuticals and solubilized in DMSO. Tamoxifen was obtained from MedChemExpress (HY-13757A) and solubilized in DMSO. For cellular assays including clonogenic survival and γH2AX immunofluorescence assays, seviteronel was administered 1 h before radiation treatment.6 cells/dish and allowed to adhere overnight before treatment with enzalutamide (1 μM), seviteronel (1 μM), or DMSO control for 18 h before 4 Gy radiation. Cells were harvested 6 h after radiation and crosslinked using formaldehyde . ChIP was performed following the protocol from the HighCell# ChIP kit (Diagenode C01010062) using the anti-androgen receptor antibody (Millipore 06-680). Following immunoprecipitation, DNA was purified using the iPure kit v2 (Diagenode C03010015) and diluted 1:5 for qPCR. qPCR was performed using Fast SYBR Green Master Mix (Thermo Fisher 4385612). Percent of input is reported as the mean ± SEM of two independent experiments. Primers for qPCR are listed in Cells were plated in 10 cm dishes with 4.0 × 10RNA was harvested from cells using QIAzol and extracted using the miRNeasy mini kit (Qiagen 217004). Reverse transcription was performed using SuperScript III Reverse Transcriptase (ThermoFisher 18080085) with random primers (Thermo Fisher 48190011), and cDNA was diluted 1:5. Comparative qPCR was performed using Fast Sybr Green Master Mix (Thermo Fisher 4385612). Plates were read using a QuantStudio6 Flex Real Time qPCR system and analyzed using a comparative method to no treatment control. Relative expression was calculated as compared to gene expression of an untreated control and reported as the mean ± SEM of three independent experiments. Primers for qPCR are listed in siRNAs targeting AR were Dharmacon ON-TARGETplus siRNA individual oligos . siAR #1: GGAACUCGAUCGUAUCAUU, siAR #2: UCAAGGAACUCGAUCGUAU. Cells were transfected with siRNA using Opti-MEM (Invitrogen 31985-062) and Lipofectamine RNAiMAX (Invitrogen 13778-150). For clonogenic survival assays, cells were transfected with siRNA and irradiated, then plated at cell densities as outlined in Irradiation of cells and mice was performed at the University of Michigan Experimental Irradiation Core using a Philips RT250 . In keeping with previous studies, the dose rate was ~2 Gy/min as previously described , 31.in vitro experiments were performed using a two-sided Student's t-test to compare gene expression between cells treated with DMSO and enzalutamide. For immunoblot comparisons, a one-way ANOVA with Dunnett's multiple comparisons test was used. For in vivo studies, a one-way ANOVA was used to compare tumor volume, and a log-rank test was used to compare survival curves. Synergy between seviteronel with radiation was assessed using the fractional tumor volume (FTV) method for in vivo experiments as previously described . While the selective estrogen receptor modulator tamoxifen is able to inhibit viability of ER+ MCF-7 cells in a dose-dependent manner  of 1.28–1.47. There was also a significant decrease in surviving fraction of cells at 2 Gy . ComparaTo further address how AR is involved in the radiation response, radiosensitization was assessed via clonogenic survival assays with seviteronel-mediated AR inhibition in multiple AR+ models of TNBC. Doses of seviteronel were selected to be 10–100 fold lower than the IC50 of the drug to evaluate radiosensitizing effects independent of cytostatic or cytotoxic effects of seviteronel as a single agent. In AR+ TNBC cell lines, treatment with seviteronel provided a dose-dependent increase in radiosensitivity. In MDA-MB-453 cells, treatment with seviteronel led to significant radiosensitization with radiation enhancement ratios from 1.21 to 1.50 and a significant decrease in the surviving fraction of cells at 2 Gy . Similarin vitro. In breast cancer cell lines that lack AR, however, treatment with seviteronel does not lead to radiosensitization  or a significant decrease in the surviving fraction of cells at 2 Gy . Similartization , suggestAQP3 and SEC14L2  in AR+ TNBC  were treated with seviteronel alone, there was no change in levels of γH2AX positive cells at 2, 6, 16, or 24 h in MDA-MB-453 cells , enzalutamide only, seviteronel only, radiation alone, or the combination of enzalutamide and radiation, or seviteronel and radiation. When cells were treated with seviteronel or enzalutamide alone, AR was recruited to regulatory regions of nd PRKDC . When coin vivo where there was a significant reduction in tumor volume and a delay to tumor doubling and tripling times in mice with AR+ TNBC xenograft tumors treated with seviteronel and radiation. Mechanistically, we report differential binding of AR to target genes in the presence of enzalutamide and seviteronel, suggesting different mechanisms of action between the two drugs.Here we show that although seviteronel and enzalutamide exhibited limited effect as a single agent (IC50 > 10 μM), AR knockdown and AR inhibition with seviteronel were effective at radiosensitizing AR+ TNBC models with radiation enhancement ratios of 1.20–1.89. Radiosensitization of AR+ TNBC models was at least partially dependent on impaired dsDNA break repair. Similar effects were observed These findings should be taken in the broader context of anti-androgens as a therapeutic strategy in breast cancer. Other groups have investigated how AR inhibition may be a therapeutic strategy for aggressive TNBC tumors. Clinical trials with enzalutamide as monotherapy have demonstrated that AR inhibition is safe and efficacious , and patin vitro and in vivo. Indeed, this study demonstrates that although AR inhibition with seviteronel alone is not sufficient to inhibit the viability of AR+ TNBC cells in vitro, seviteronel does inhibit proliferation in vivo and sensitizes cells to radiation treatment both in vitro and in vivo  models are still needed. Future work will also seek to understand the differences in how enzalutamide and seviteronel affect the ability of AR to bind DNA and activate the transcription of downstream target genes. Our results suggest that seviteronel has a unique mechanism of radiosensitization compared to the second generation anti-androgen enzalutamide. Indeed, these results suggest that AR is increasingly recruited to binding sites of DNA damage response genes involved both in HR and NHEJ following treatment with seviteronel and radiation. This may suggest that AR remains bound to these regions but may not be activating transcription of these genes. This may be due to co-repressor recruitment at these sites (instead of co-activator) or stalling of the transcriptional machinery. Thus, although seviteronel is found more frequently bound to promoter regions of NHEJ and HR genes, there does not seem to be a functional improvement of DNA repair efficacy or efficiency, suggesting that the mechanism of radiosensitization with seviteronel is different than that previously reported for enzalutamide. Although the details of these mechanistic differences remain unresolved, additional studies are underway to investigate the mechanism of AR-mediated radiosensitization both with enzalutamide and seviteronel to understand how these AR inhibitors are differentially affecting the radiation response. Another limitation is the disparate findings on the effect of seviteronel  in vivo , 2, 5. TIn summary, TNBC continues to be a clinically challenging disease entity with limited/no effective molecularly targeted therapies. With the identification of AR+ TNBC subtype, interest in targeting AR in these patients continues. The data reported herein provide the preclinical rationale for continued clinical investigation of anti-androgens as a general class of molecularly targeted therapies for the targeted treatment of AR+ TNBC and specifically for the further investigation of seviteronel as a radiosensitizing agent in women with radioresistant AR+ TNBC.The datasets generated for this study are available on request to the corresponding author.The animal study was reviewed and approved by Institutional Animal Care and Use Committee (IACUC) at the University of Michigan.AM, BC, EO, KW-R, LM, ML, AP, AZ, CR, SW, AS, and SN conducted experiments and analyzed the data. All authors contributed to manuscript idea and revision, read and approved the submitted version.JE was previously employed by Innocrin Pharmaceuticals Inc. JE is no longer employed by Innocrin. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The study provides detailed surface reconstructions of two AE1 cell domains and two AE2 cells, showing AE1/AE1, AE1/AE2 and AE2/AE2 contact sites, basolateral microvilli pits at AE2 cells and small AE1 processes beneath AE2 cells. Furthermore, we show reconstructions of a surfactant secretion pore, enlargements of the apical AE1 cell surface and long folds bordering grooves on the basal AE1 cell surface. The functional implications of our findings are discussed. These findings may lay the structural basis for further molecular investigations.Thin type 1 alveolar epithelial (AE1) and surfactant producing type 2 alveolar epithelial (AE2) cells line the alveoli in the lung and are essential for normal lung function. Function is intimately interrelated to structure, so that detailed knowledge of the epithelial ultrastructure can significantly enhance our understanding of its function. The basolateral surface of the cells or the epithelial contact sites are of special interest, because they play an important role in intercellular communication or stabilizing the epithelium. The latter is in particular important for the lung with its variable volume. The aim of the present study was to investigate the three-dimensional (3D) ultrastructure of the  Both are essential for normal lung function: AE1 cells cover the majority of the alveolar surface with thin cytoplasmic extensions that participate in a very thin blood gas barrier and AE2 cells serve as the regeneration source for the alveolar epithelium and secrete the pulmonary surfactant, which lowers the surface tension at the air liquid interface and, thus, prevents alveolar collapse ) after having created snapshots with 3dmod.Figures were prepared in the GNU Image Manipulation Program (gimp 2.10.12; For a general overview of sample preparation techniques and the volume EM workflow, see .


The widespread use of pesticides to control agricultural pests is a hot topic on the public scene of environmental health. Selective pest control for minimum environmental impact is a major goal of the environmental toxicology field, notably to avoid unintended poisoning in different organisms. Anticoagulant rodenticides cause abnormal blood coagulation process; they have been widely used to control rodents, allowing inadvertent primary and secondary exposure in domestic animals and non-target predatory wildlife species through direct ingestion of rodenticide-containing bait or by consumption of poisoned prey. To report toxic effect, the most common approach is the measurement of liver or plasma residues of anticoagulant rodenticides in dead or intoxicated animals showing clinical symptoms. However, one major challenge is that literature currently lacks a hepatic or plasma concentration threshold value for the differentiation of exposure from toxicity. Regarding the variation in pharmacology properties of anticoagulant rodenticides inter- and intra-species, the dose-response relationship must be defined for each species to prejudge the relative risk of poisoning. Beyond that, biomarkers are a key solution widely used for ecological risk assessment of contaminants. Since anticoagulant rodenticides (AR) have toxic effects at the biochemical level, biomarkers can serve as indicators of toxic exposure. In this sense, toxicological knowledge of anticoagulant rodenticides within organisms is an important tool for defining sensitive, specific, and suitable biomarkers. In this review, we provide an overview of the toxicodynamic and toxicokinetic parameters of anticoagulant rodenticides in different animal species. We examine different types of biomarkers used to characterize and differentiate the exposure and toxic effects of anticoagulant rodenticide, showing the strengths and weaknesses of the assays. Finally, we describe possible new biomarkers and highlight their capabilities. Pesticides are today the main means of pest control, making it possible to mitigate the economic, environmental and health consequences when pest population outbreak occurs . NeverthMore and more primary or secondary non-target intoxications with anticoagulant rodenticides are reported in the literature including mostly birds, especially raptors, but also a minor part of domestic animals such as cats, dogs and horses, and other wildlife species such as dears, polecats, martens, foxes, and very relevant literature reviews are already available on this topic –13. Few Rodents control using anticoagulant rodenticides was introduced officially in 1948 , answeriThe risk of a pesticide used to control target species by causing their death is to select living target species that are less or not sensitive to this pesticide, the latter specimens are said “resistant.” In 60's; the first resistant rodent population has been described . To dealAnticoagulant rodenticides are included in baits of different types and forms  depending on the application site and the targeted rodents . The preThere are two types of exposures to consider. Primary exposure occurs when a non-target animal directly eats the bait, while secondary exposure occurs when a predator ingests preys previously exposed to AR. Prevention is different for each case. Considering the primary exposure, the goal is to avoid the access to the bait by ensuring their correct storage and using them in secured bait station as previously discussed. Considering the prevention of the secondary exposure, the matter is more complex. Indeed, AR is a long-acting poison and targeted rodents may die within 2–6 days after ingestion. Moreover, rodenticides may influence gradually rodents' behavior. Actually, poisoned rodents become weakened with reduced appetite and motionless, lose their nocturnal disposition and positive thigmotactic behavior; they are therefore more prone to predation –35. ThusIn domestic animals, the majority of exposures are primary ones. They are often the consequences of a misuse or an improper storage of baits allowing domestic animals to access to AR. According to a French veterinary poison control center (CNITV), AR exposures represent 10% of the total call to the center and dogs accounted for 82.8% of AR exposure incidents, followed by cats with <10% . The petSince the majority of AR exposures are accidental, owners notice the event and treatment is administered before symptoms occur. According the CNITV data, SGAR are reported in more than 60% of exposure cases  . This inLivestock can also be exposed and intoxicated to natural anti-vitamin K like dicoumarol after the ingestion of moldy clover fodder , 41 or ltaxa and decreases with the distance to the bait boxes  in suburban area . Howeverit boxes , 46. ThePredators can be secondary exposed to AR from target and non-target species, according to their diet . The expHQ) is a cofactor of Gamma-Glutamyl CarboXylase (GGCX) enzyme that performs a gamma-carboxylation of glutamate residue of some proteins called vitamin K dependent proteins (VKDP). VKDP have to go through this post-translational gammacarboxylation to be able to chelate calcium and activate their physiological properties (HQ is oxidized and oxygenated into vitamin K epoxide (VitKOX). Since dietary intake is often insufficient to meet the vitamin K need (OX has to be recycled in VitKHQ to maintain the gamma-carboxylase activity. The recycling is performed by the vitamin K epoxide reductase enzyme (VKORC1) and it takes place in two stages  enzyme leading to the formation of Vitamin K Quinone (VitKQ) and (2) a bielectronic reduction of VitKQ in VitKHQ mostly catalyzed by the VKORC1 enzyme and as an alternative minor pathway by the consensual NAD(P)H:Quinone Oxidoreductase 1 (NQO1) enzyme that is not the sole alternative reduction path  a dion path –71.Anticoagulant rodenticides are inhibitors of VKORC1 . When thSGAR molecules are little more potent than FGAR ones to inhibit VKORC1 of susceptible rat . NeverthWhile vitamin K dependent clotting factors are the main VKDP affected by an AR exposure, long-term exposure may affect other VKDP like osteocalcin (OC) or matrix Gla protein (MGP). These proteins are involved in bone formation and energetic metabolism for osteocalcin , 76 and AR molecules are rapidly and efficiently absorbed after ingestion . MoreoveAs presented above, all stereoisomers of SGAR are not found in exposed non-target animals . This obAR are metabolized through cytochrome P450. Cytochromes involved in the AR degradation are different according to the nature of the molecule and its stereochemistry . These sNot all individuals respond equally to exposure to AR. According to their species, sex or genetic, animals will be more or less susceptible to AR. A large part of the differences of susceptibility to AR might be explained by a difference of VKORC1 sequence among species. Another possible source that may explain differences in sensitivity is the vitamin K dependent clotting factors pool and its rate of decrease after the inhibition of VKORC1 by AR. This mechanism is proved in the difference of susceptibility between female and male rats . Indeed,Finally, food could be a pathway of resistance. Indeed, a vitamin K rich diet might reduce the action and the efficiency of AR exposure by providing the antidotes to the animals. However, this kind of resistance has not been clearly demonstrated or identified in wildlife populations , 110.Gallus domesticus showed greater metabolic activity of warfarin compared to rat whereas very little warfarin metabolism was demonstrated in owls which can explain the recurrence of reported poisoning in these species  (Rattus norvegicus) (Otospermophilus beecheyi)   in Japanvegicus)  and of aeecheyi) . The oveeecheyi) , 117. Deeecheyi)  liver diConsidering the extent of AR exposure in domestic and wildlife fauna, it is essential to monitor it. The first clues of an animal exposure are clinical signs suggesting of an acute exposure. However, the consequences of chronic exposure, even if less known, as well as animals that are asymptomatic but at a high risk of exposure must also be considered in AR exposure monitoring.Clinical signs of rodenticide intoxication are quite similar for higher vertebrate species: hemorrhagic syndrome, with main localizations being respiratory  and digestive  tracts, and less specific symptoms including lethargy, anorexia, pallor or ataxia, that are usually the first signs to be noticed –122. CliNumerous case reports in dogs provide extensive descriptions of the clinical signs observed. Apart from general and non-specific signs, and respiratory and digestive bleeding disorders, tachycardia and dyspnea with polypnea are reported, as well petechiae and ecchymoses on the skin and mucosa, and pallor , 119. SoRuminants are apparently less often exposed than other domestic species , and display clinical signs less frequently  . Case reAR toxicosis in wild animals, especially birds of prey, is also quite well-described in literature. Similar signs are reported, observed ante- or post-mortem, with a great focus on overt hemorrhagic lesions  , 131. InIn laboratory rats, transient early hemoglobinuria (associated with oxidative stress) with consecutive late hematuria (associated with anticoagulation) were observed after poisoning with brodifacoum, that could be used as novel clinical biomarkers of AR poisoning, if generalizable to other species . MoreoveThus, hemorrhage are important markers of acute AR toxicity in higher vertebrate species. Moreover, they can be used in both alive and dead animals. Nevertheless, hemorrhages are not specific of AR intoxication, as they might be the consequence of trauma (consecutive to AR intoxication or not) or diseases like leptospirosis  or otherWhen hemorrhages occur in living animals, the therapeutic response to vitamin K injection may allow to reinforce or reject an AR intoxication hypothesis. However, false positive occurs in the case of vitamin K deficiency  or of genetic VKORC1 activity deficiency . HoweverClinical signs of a chronic exposure are currently not sufficiently characterized to be used to suspect an AR exposure at the individual scale. More and more studies suggest that the effects of AR are not confined to coagulation alone. Indeed, the activity of VKDP unrelated to coagulation may also been impacted by AR. More precisely the bone metabolism may be influenced by the decrease of osteocalcin carboxylation and vascular calcification prevention by that of MGP. Decrease in bone density and increased risk of fractures , have been investigated in AR-exposed predatory birds, without any conclusive result . HoweverLynx rufus) to notoedric mange, with some fatal outcomes, by interfering with the immune system of the exposed animals , unusual infectious susceptibility for no obvious reason (immunodeficiency virus…) and vascular calcification at necropsy. Moreover, chronic exposure with only slight clinical signs may not be identified, the criteria for suspicion of asymptomatic exposure are also relevant.Due to the difference of susceptibilities of animal species to AR, to the level of exposure or to the delay after exposure (few hours to few days), some animals may not present any clinical sign whereas they have been exposed to AR. If it is not a problem for the animal itself; it can be, depending on the situation, for other individuals, for animals in the food chain or even for humans. This animal can be a source for secondary AR exposure. Indeed, this animal or its products can be consumed.In the case of domestic animals, as exposures are mainly primary and occurs in a controlled environment, asymptomatic exposure to AR is suspected when animals were able to get access to the bait. There are few studies on the risk of the consumption of products from exposed animal. However, as AR can be excreted in milk and eggs , 85, it Considering wildlife, it is more complicated as it is not possible to suspect if the animals have been exposed (no controlled environment). It is an issue for the wildlife, as exposed animal can be a source of secondary exposure for predator. Thus, it is a major concern for hunted species like wild boar which can be a source of human exposure. According to the exposure studies on small mammals, predators and wild boar, wild animals should be suspected of being exposed when they are found close to areas with significant human activity  , 49, 51.In the presence or absence of clinical signs, a suspicion of exposure to AR should be confirmed and its biological consequences assessed, in order to measure the impact of these molecules on non-target species. This monitoring will make it possible to measure the effectiveness and relevance of new practices and methods for controlling rodent populations with a view to reducing the exposure of non-target populations.Biomarkers are measurable parameters at biochemical, histological, immunological, physiological, or organismic levels, that are used as indicators of certain biological states or conditions . They arIn the field of ecotoxicology, there is a constant need to detect and assess the impact of pollution, particularly in cases of low concentrations of increasingly complex mixtures of contaminants. This has led to an increasing number of studies on the development of indicators of the biological effects of contaminants on organisms.A common dichotomy for biomarkers in ecotoxicology is between biomarkers of exposure, and biomarkers of effect. While biomarkers of exposure are relevant for hazard identification , biomarkers of effect are used for hazard assessment, as they assess the response of the exposed organism to the particular xenobiotic or complex mixture . A thirdBiomarkers are sought at different levels of the organisms , 149. ThIt is worth noting that misapplication or misinterpretation of biomarkers in ecotoxicology may lead to erroneous conclusions. The first potential biases arise from handling conditions of samples, including their collection and storage that might affect the final quantitative and qualitative assessment of the biomarker. Sample relevance may decrease due to biological (decomposition process including other living organisms) and physicochemical  factors that cause potential instability of the biomarkers. In general, ante-mortem samples are better processed because they are taken fresh from a live animal. This does not apply to those taken from animals found in the wild without knowing the time of their death therefore ignoring the post-mortem interval before their collection. Carcasses should ideally be collected upon animal death and specimens should be fixed in liquid nitrogen transported and stored at −80°C to ensure biomarker preservation. For an accurate interpretation of circumstantial changes, a thorough understanding of the post-mortem process of the biomarker in different organs regarding to its constancy over time and environmental factors  is necessary.Interspecies extrapolation is not always possible, as well as translation from the laboratory to the field situation, and the impact of non-pollution-related variables  should be considered as confounding factors . The relFor non-target fauna poisonings by anticoagulant rodenticides (AR), while clinical biomarkers are relevant for the identification of individual poisonings in domestic and wild animals, ecotoxicological biomarkers are needed to assess the extent of exposure to AR and the sublethal effects of these contaminants on wildlife populations. Based on the criteria that have been defined to identify relevant biomarkers in the environmental risk assessment (ERA) for chemicals , we prop- Development stage: is the biomarker only an idea, or used for research purposes or sufficiently standardized to be used on the field?- Dead or alive: does the biomarker is usable for a dead animal, a living animal or both?- The availability of usual values or thresholds for the considered species.- Compatibility with treatment with vitamin K: can biomarker be used to assess AR exposure or potential effects during treatment?- Sensitivity: does biomarker can detect limited exposure to AR or confirm a symptomatic exposure?- Specificity: are there confounding factors for the biomarker?- The toxicological significance of the biomarker (relationship between response and impact to the organism) must be demonstrated.Non-invasive detection and quantification of AR can be performed in plasma , 152–155AR screenings can be relevant to confirm and quantify exposure to these chemicals. For domestic animals, it can be a valuable tool for confirming the diagnosis of AR intoxication in cases where ingestion has not been observed,  but it iAs feces are the main route of AR elimination, their use to monitor exposure to AR has been widely investigated, both for domestic species  , 155 andTyto alba). More recent research has been conducted by combining both published data and results from surveillance programs in Canada and using logistic regression to estimate the probability of toxicosis associated with different liver AR residues. It described a significant likelihood of toxicosis in 5% of individuals with liver concentrations of 20 ng/g wet weight and in 20% of individuals with concentrations of 80 ng/g wet weight  measured on citrated plasma samples is considered a sensitive test to confirm AR contamination, as it is the first coagulation parameter to be altered after AR exposure, due to the short half-life of factor VII (6.2 h) . It shouPT as a diagnostic tool for AR contamination is widely described in dogs , 168. BaIn ruminants, same mechanisms are expected, and there are also some reports of increased PT after AR exposure in cattle , sheep , 125, anWhile PT seems a good biomarker of AR intoxication in domestic animals, no linear correlation was found between AR concentrations and extent of PT prolongation neither in dogs  nor in sin vitro clot formation, and thus may be more sensitive in detecting prolongation of clotting time by AR. Still, as the PT variation after AR exposure is usually marked, and with the availability and standardization of PT reagents, the PIVKA test was quite abandoned as a diagnostic tool, especially as results of this test can also be increased by deficiencies in factors II, VII, and X because of heredity and acquired coagulopathies other than anticoagulant poisonings, and is sensitive to the presence of heparin (It is worth noting that some research has previously been performed on the use of Proteins Induced by Vitamin K Antagonism or Absence (PIVKA) for the sensitive diagnosis of AR intoxication in dogs . The PIV heparin , 173.in vitro coagulation. Great variations  can occur when using human or mammalian  thromboplastin for avian clotting assays  dosing might be an important source of biomarkers as the inhibition of the recycling mechanism of vitamin K has direct consequences on those proteins. Nevertheless, the challenge is not only to dose VKDP but also to dose separately carboxylated and under-carboxylated VKDP (ucVKDP). Indeed, AR molecules act on the post-translational gamma-carboxylation mechanism but not on the genetic expression of VKDP.The dosing of vitamin K dependent clotting factors might be a good indicator of AR intoxication. These dosages are based either (i) on the evaluation of the activity of the clotting factor under consideration through a reaction cascade (close to the coagulation cascade) leading to the production of a chromogenic factor and where the dosed factor is the limiting factor, or (ii) on the supplementation of plasma depleted in the factor considered. Only carboxylated clotting factors should be assessed. As carboxylated clotting factors decrease faster than increase in clotting times arises, their dosing allows early detection of intoxication. For example, in rats and after an ingestion of a lethal dose of AR; the factor VII and factor X activity are halved in about 4 and 5 h, respectively, while prothrombin time doubles in 10 h . HoweverIf vitamin K dependent clotting factors are linked to the acute toxicosis of AR, other VKDP might be linked to their chronic effects. The two major non-clotting VKDP are osteocalcin (OC) and Matrix gla protein (MGP).Osteocalcin limits bone formation without impairing bone resorption or mineralization . Under-cConversely, under-carboxylated MGP (ucMGP) is well-known to be in favor of vascular calcification , 190, whMGP and OC are small proteins currently measured by immunoassays. Their sequences are variable between species, which raises the question of assay methods. Their assay will require the development of specific antibodies in the absence of methods to evaluate their activity. Moreover, as they are proteins, their stability at room temperature in animal cadavers will be a challenge. These biomarkers will eventually be used as research tools, but their use in surveillance studies seems difficult in the future. On the other hand, they could be dosed on live animals as well as on dead animals, from blood (OC and MGP), bone (OC), vascular, pulmonary and renal tissues (MGP).In mammals, two different enzymes possess Vitamin K epOxide Reductase activity (VKOR) and are coded by paralogous genes. The enzyme called VKORC1 is mainly located in the liver and is the main contributor to the recycling of Vitamin K needed for activation of clotting factors, while the enzyme called VKORC1L1 does not sustain coagulation but maintain the Vitamin K cycle for carboxylation of other VKDP , 193.In human, sensibility to warfarin before an anticoagulant treatment can be assessed by testing alleles coding for VKORC1 to detect the presence of potential variants susceptible to adapt the proper dose of warfarin , 195. Thin vitro against VKORC1 of each species. Two methods are currently described to measure VKOR activity, the DTT-driven VKOR assay , from bacterial origin. All those vitamers share a common 2-methyl-1,4-naphtoquinone cycle, corresponding to vitamin K3, derived from synthesis or catabolism. Vitamin K1 has a phytyl side chain at the 3-position, while MKs have several repeating prenyl units, the number of units being given as a suffix (menaquinone-n or MK-n) . MenaquiVKORC1 is partly responsible for the recycling of vitamin K: when the VKOR activity is impaired by AR, it can be assumed that it has an impact on the concentrations of those vitamers, with a possible shift between epoxide forms and quinone forms. A dose-response relationship was suggested between ratio of epoxide and quinone forms of vitamin K1 in plasma and concentrations of two AR, phenprocoumon and warfarin . It was The measurement of vitamin K forms is usually performed by chromatography , that can be coupled with fluorescence  or mass Cytochromes P450 (CYPs) are a superfamily of enzymes containing heme as a cofactor that function as monooxygenase. CYPs are the major enzymes involved in drug metabolism, accounting for about 75% of the total metabolism of xenobiotics. Most xenobiotics are inactivated by CYPs, although some are bioactivated. The detection and quantification of specific metabolite(s) resulting from this metabolization can be a biomarker of exposure. In the case of AR and vitamin K, the pathways of metabolization are not really well-defined. Vitamin K1 metabolism would involve different CYPs. In humans, CYP4F11 and F2 have been identified. The metabolism of warfarin in humans involves CYP2C9, but also CYP1A2, 2C19, and 3A4. In rats CYP1A1, CYP2B1, CYP2C6, CYP2C11, and CYP3A2  are invoMany drugs may increase or decrease the activity of various CYP isozymes either by inducing the biosynthesis of an isozyme (enzyme induction) or by directly inhibiting the activity of the CYP (enzyme inhibition). Many studies deal with Cytochromes P450 as biomarkers of xenobiotic exposure in the liver but also in the blood  thanks to plasma exosomes e.g., –214]. Th. Th214)]Metabolic profiling, also called metabolomics, consists in a screening of a maximum of metabolic parameters, in a fresh biological sample, that should as far as possible be relevant to the precise state or condition of the organism from which the sample come from at the time of the sampling. The usual but non-exhaustive parameters studied are: the evolution of protein expression level, of metabolites, of biological parameters , of cell number and relative types, of redox and acido-basic parameters, etc. The parameters are as well qualitative for example by studying the structure of a molecule or the morphology of a cell, as quantitative, either relatively to a given parameter, either through absolute quantification .One classical use of metabolic profiling is the research of very specific biomarkers for disease or exposure to xenobiotics –219. It The limitation of this method is its current technical difficulty and the requirement of very advanced data analysis , includiLynx rufus) used RNA sequencing on whole blood to investigate genes and cellular processes affected by sublethal exposure to AR. Differential expressions of genes involved in xenobiotic metabolism, endoplasmic reticulum stress response, epithelial integrity and immune function were identified  to assess the sublethal effects of AR.On the other side, genomics is the study of global gene expression patterns. It addresses all genes and their inter relationships in order to identify their combined influence on the organism , and relentified . In partIf rodenticide anticoagulants are currently essentials to manage pest rodent populations, wildlife and domestic animal exposure to AR is a major concern. The exposure level of wildlife is important close to area with human activities. However, it is difficult to assess the consequences of exposure on the non-target animal population, since there are great differences of susceptibilities between species. The biomarkers can be useful tools in this task by highlighting AR effect on non-target species and thus to open the way to an evaluation of pest control practice in order to decrease AR impact. While some biomarkers seem promising, they are currently underdeveloped for AR monitoring purpose. Some of the biomarkers proposed in this study could be developed for systematic use in AR monitoring exposure in the field, while others, which are more complex to implement, could certainly only be developed for research purposes.AR, M-AM, XS, SL, and VL: conceptualization, writing—original draft preparation, and review. All authors contributed to the article and approved the submitted version.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.


Postural control is the ability to maintain equilibrium and orientation in a gravitational environment. Patients with chronic subjective dizziness have some troubles with their postural stability. The present study aimed to assess the benefit of home-based vestibular rehabilitation in patients with chronic subjective dizziness using computerized dynamic posturography. Therefore, 100 subjects, between 19 to 86 years, diagnosed with dizziness were included in the study. Computerized dynamic posturography was performed to assess postural stability. Vestibular rehabilitation programs included exercises tailored to the particular needs of each patient. After vestibular rehabilitation, patients were re-examined using the same tests. Posturographic data were analyzed and compared for before and after vestibular rehabilitation findings. The mean composite scores before the intervention  was significantly (p<0.01) lower than the mean composite scores after vestibular rehabilitation . This result is found to be statistically significant. In conclusion it could be suggested that the effectiveness of vestibular rehabilitation could be verified by means of computerized dynamic posturography as a concrete method. Dizziness, is an umbrella term which is used to define a subjective sensation of disorientation or involuntary motion that could be related to several factors ,2. ChronVestibular rehabilitation (VR) has become widely used in the management of patients with dizziness, disequilibrium, and gait instability . VR is aVestibular rehabilitation attempts to improve the competence and well being of the individual who has a vestibular pathology in the performance of daily life activities. It also aims to restore spatial orientation as much as possible. It stimulates visual stabilization, reduces discomfort during head movements, and leads to more excellent stability in body posture while both moving and relaxing .Computerized Dynamic Posturography (CDP) is a test used to evaluate the general assessment of balance, combining labyrinthine, visual and somatosensory data. The most commonly used test is the Sensory Organization Test , consistThe study aims to determine whether home-based vestibular rehabilitation improves postural control of patients with chronic subjective dizziness with computerized dynamic posturography.The study was approved by Hacettepe University Faculty of Medicine Non-Interventional Clinical Research Ethics Committee . According to the national legislations and institutional rules and procedures, all patients sign an informed-consent approving analysis of their clinical records, and publication of the anonymous data.Subjects were identified through a retrospective medical record review of patients seen for vestibular and balance rehabilitation between January 2010 and November 2018 at the Balance Rehabilitation Program of the Hacettepe University Audiology Clinic. A hundred subjects diagnosed with chronic subjective dizziness were included in the present study. Inclusion and exclusion criteria were defined as:Inclusion Criteria: Individuals of both genders over 18 years old who presented with chronic vestibular dysfunction, dizziness or balance impairment and completed the vestibular rehabilitation program.Exclusion Criteria: Individuals with severe visual and/or hearing impairment, orthopedic disorders that limited the performance of proposed activities, nervous system injuries that resulted in additional motor and/or sensitive damages, and/or peripheral vestibular diseases of the benign paroxysmal positional vertigo or Meniere disease.On the initial visit, the physical therapist / audiologist performed a complete medical, symptom, and functional history, and a physical evaluation. The physical evaluation included a neuromuscular assessment, oculomotor and VOR testing such as saccadic and pursuit eye movements, optokinetic, gaze and spontaneous nystagmus, head impulse test, positional and movement testing, balance testing, and gait evaluation. Next, the individuals with dizziness were subjected to CDP with Equitest SystemTM—Version 4.0 equipment, produced by NeuroCom InternationalTM- USA. SOT, one to CDP's subtest was performed. A harness was provided for safety. The step width during standing was standardized according to the subjects' height.The conventional SOT is a test that consists of 6 different conditions used to identify relative contribution of the three main sensory systems involved in balance: namely somatosensory, visual and vestibular systems. The 6 conditions include are as follows: (1) eyes open with fixed surface and visual surround, (2) eyes closed with a fixed surface, (3) eyes open with fixed surface and sway referenced visual surround, (5) eyes closed with a sway referenced surface, and (6) eyes open with sway referenced surface and visual surround. By controlling the usefulness of the sensory  information through sway referencing and/or eyes-open/closed conditions, the SOT protocol systematically eliminates useful visual and/or support surface information and creates sensory conflict situations –10.Three trials were taken for each condition. A fall was defined as a patient taking a step, grabbing the wall or side the platform, opening their eyes during an eyes closed condition, or when the patient exceeded their limits of stability. If the operator stopped the trial, the trial was scored a 0.Based on the data under each condition, Equitest is capable of calculating the Average for each of them, an index for somatosensory, visual and vestibular functions and the relationship between visual and vestibular information, known as "visual preference," in addition to a balance index, all of which are described below:Somatosensory function (SOM): Average of condition 2 / Average of condition 1Visual function : AverageVestibular function (VEST): Average of condition 5 / Average of condition 1Visual preference: Average of condition 3+6 /Average of condition 2+5 .The "Equilibrium Score" is based on the assumption that a normal person can sway 12.5 degrees without losing balance within the limits of stability . When caOnce the first posturography session had been performed for each of the individuals to be studied, the home-based vestibular rehabilitation (VR) program was begun. A customized exercise program was developed for each subject according to the results of the assessment and included the following interventions as indicated: gaze stabilization, balance and gait training, and habituation exercises. The vestibular rehabilitation program was tailored as follow: (a) if just the VEST parameter was under the normal limits vestibulo-ocular reflex (VOR) exercises, (b) if the VEST and SOM parameters were under the normal limits together VOR exercises on foam-pad and (c) if the VIS and VEST parameters were under the normal limits VOR and foam pad exercises have been suggested . AlthougAt the end of the 4–6 weeks of vestibular rehabilitation, patients were reassessed using computerized dynamic posturography.Data were analyzed using SPSS 21.0  program. A p-value below 0.05 was considered statistically significant. Descriptive analysis (mean and standard deviation) included demographic factors such as age and sex. A paired t-test was used to compare SOT scores before and after vestibular rehabilitation.In total, 100 individuals  who diagnosed with dizziness were evaluated. The mean age of the subjects was 47.24 ± 15.57 years.The comparison of average results under each of the various computerized dynamic posturography conditions studied, before and after vestibular rehabilitation, can be found in The number of patients who have normal and abnormal results before and after vestibular rehabilitation in SOM, VIS, VEST and PREF parameters of SOT was given in The primary purpose of the present study was to investigate whether the computerized dynamic posturography is beneficial for assessing the effectiveness of vestibular rehabilitation in subjects with chronic subjective dizziness. Our results revealed that the performing computerized dynamic posturography before and after vestibular rehabilitation is useful to determine the effectiveness of vestibular rehabilitation.The main problem encountered when dealing with subjective dizziness and/or disequilibrium is the difficulty to measure symptoms objectively. Distinguishing normal and abnormal balance function is an important component of the diagnostic investigation of all patients with dizziness and balance complaints. Posturography has yielded results on sway on the sensory organization test that have been associated with a variety of balance disorders ,15. PostA customized vestibular rehabilitation therapy program is an important treatment modality for patients who have dizziness, disequilibrium and gait disturbances . After dMost clinical studies on the effectiveness of vestibular rehabilitation have relied on subjective measures such as questionnaires and scales focused on assessing changes in symptom severity ,13,17,18In the present study, CDP used to determine the effectiveness of vestibular rehabilitation. For this purpose, subjects were administered SOT before vestibular rehabilitation to specify which system or systems used to maintain balance was disoriented and then the vestibular rehabilitation program was tailored based on this data. After the vestibular rehabilitation program, reassessment was performed again with SOT to check if there is an improvement or not. The strong correlation was found in equilibrium scores for all conditions and composite scores before and after vestibular rehabilitation.Medeiros et al. (2003), has found a significant improvement condition 1 and condition 5 of the equilibrium scores and of the composite balance score in ten children with vestibular symptoms . Cass etSince none of the individuals with a diagnosis of CSD presented with an identifiable ontological contribution, we speculated that observed decreades in level of dizziness could be explained by vestibular rehabilitation. However, such improvement could be explained by the reduced anxiety experienced by the patients . This faIn this study statistical analysis implemented on SOT showed that the significant improvement in the composite score is primarily due to improvements under condition 5 and 6. The improvement in sensory analysis of final vestibular function was also significant, further indicating objective and effective improvement under condition 5, which performed with an unstable platform and with closed eyes, revealing the subject’s use of the vestibular information in maintaining an upright position. This finding is compatible with other studies in the literature ,25,27.It is crucial to determine which system is disoriented in maintaining balance to provide beneficial vestibular rehabilitation. Moreover, it is also essential to assess the effectiveness of vestibular rehabilitation. It could be speculated that Computerized Dynamic Posturography could be a beneficial method to determine the disoriented system and the effectiveness of vestibular rehabilitation. It could provide objective data to compare the results of a vestibular rehabilitation.S1 Data(XLSX)Click here for additional data file.


Amauroderma rugosum (AR) is a dietary mushroom in the Ganodermataceae family whose pharmacological activity and medicinal value have rarely been reported. In this study, the antioxidant capacity and neuroprotective effects of AR were investigated. The aqueous extract of AR was confirmed to contain phenolic compounds, polysaccharides, and triterpenes. The results of 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) and total antioxidant capacity assays revealed that AR extract scavenged reactive oxygen species. Moreover, AR extract decreased the cytotoxicity, oxidative stress, mitochondrial dysfunction, and apoptosis of PC12 cells induced by 6-hydroxydopamine (6-OHDA). In addition, 6-OHDA upregulated the expressions of proapoptotic proteins and downregulated the Akt (protein kinase B)/mTOR-  and MEK (mitogen-activated protein kinase kinase)/ERK-  dependent signaling pathways. These effects of 6-OHDA were abolished or partially reversed by AR extract. Furthermore, the neuroprotective effects of AR in 6-OHDA-treated PC12 cells were significantly abolished by Akt and MEK inhibitor. Thus, AR extract possesses neuroprotective effects, probably through its antioxidant and antiapoptotic effects. These findings suggest the potential application of AR in the prevention or treatment of oxidative stress-related neurodegenerative diseases such as Parkinson's disease. To date, Parkinson's disease remains the second most common neurodegenerative disease worldwide, and its incidence is increasing in people over 60 years of age . It is cAlthough the pathological mechanisms of Parkinson's disease remain elusive, accumulating scientific evidence suggests that oxidative stress-induced cell injury plays an indispensable role in the degeneration of dopaminergic neurons . Interruin vitro and in vivo Parkinson disease models. It is taken up by dopaminergic neurons via dopamine and norepinephrine transporters and is subsequently oxidized intracellularly, thereby releasing ROS including hydrogen peroxide, superoxide, and hydroxyl radicals [Various experimental models have been established for investigating the role of oxidative stress in dopaminergic neuronal degeneration. These models involve the use of neurotoxins such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), rotenone, 1,1′-dimethyl-4,4′-bipyridinium dichloride (paraquat) and 6-hydroxydopamine (6-OHDA) . The comradicals –12.Amauroderma rugosum (AR) is a basidiomycete in the Ganodermataceae family. This mushroom has a black stipe and a white surface covered with numerous pores. A notable characteristic of the mushroom is that its surface becomes red when it is scratched. Hence, it is also known as “blood Linzhi” in Chinese. Although AR is commonly consumed by people in China and South Asia, very few scientific studies have explored its beneficial effects on health or its medicinal value. Nevertheless, a previous study reported that the extract of AR mycelia has antioxidant and anti-inflammatory effects in lipopolysaccharide-stimulated RAW 264.7 cells. Thus, AR might be a potential therapeutic agent or health supplement useful in the management of oxidative stress-related diseases [diseases . The aimw/v) trypsin containing 1 mM ethylenediaminetetraacetic acid were purchased from Invitrogen , and 6-OHDA, 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH), vitamin C, dimethyl sulfoxide (DMSO), Akt inhibitor IV, MEK inhibitor (PD 98059), and 3--2,5-diphenyltetrazoliumbromide (MTT) were purchased from Sigma-Aldrich . Nerve growth factor (NGF) was obtained from R&D Systems . A total antioxidant capacity assay kit was purchased from Abcam . A lactate dehydrogenase (LDH) cytotoxicity assay kit was purchased from Cayman Chemical . A Caspase 3/7 activity detection kit was obtained from Promega . Antibodies for western blotting were purchased from Cell Signaling Technology . All chemicals were dissolved in appropriate solvents and stored at −20°C before use to maintain their chemical stability.Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), 4′,6-diamidino-2-phenylindole (DAPI), penicillin–streptomycin, and 0.25% , an organic farm that had been granted an organic crop production certificate by the Hong Kong Organic Resource Centre. The samples were dried in an oven and ground into powder. A reflux system for the extraction process was used to prepare the crude extract. Two grams of the powdered sample was extracted with 50 mL of distilled water at 95 ± 2°C for 60 min. The crude extract was centrifuged at 4000 rpm for 20 min. Afterward, the supernatant was collected, and the sample residue was reextracted twice via the steps described above. Subsequently, all extracts were pooled, filtered, and concentrated to 80 mL with a rotary evaporator. The extract was stored at -20°C until further use.μL of 10% Folin-Ciocalteu phenol reagent was added to 50 μL AR extract and incubated in dark at room temperature for 3 min. Afterwards, 100 μL of 10% Na2CO3 was added to the mixture for 1 h. The absorbance at 750 nm was measured with a microplate absorbance reader. Gallic acid was used as a standard phenolic compound. All determinations were expressed as mg gallic acid equivalent per g (mg GAE/g).To measure the total phenolic content of AR extract, 50 μL water. Total polysaccharide content was measured by adding 2.5 μL phenol (80%) and then 125 μL concentrated sulfuric acid. After incubation for 10 min, the mixture was shaken and then incubated at 30°C for 20 min. The absorbance at 490 nm was measured with a microplate absorbance reader using glucose as standard. The results were expressed as mg glucose equivalent per g (mg GE/g).Before the measurement of the total polysaccharides, 0.1 mL AR extract was precipitated with 1 mL of 95% ethanol overnight at 4°C. The precipitate was collected by centrifugation at 10,000 rpm for 10 min at 4°C. Then, the precipitate was dissolved in 50 μL AR extract was transferred to 15 mL tube and evaporated to dryness using nitrogen flow. Then, 0.4 mL 5% vanillin–acetic acid solution and 1 mL perchloric acid were added into the tube, mixed and incubated at 60°C for 15 min. Afterwards, 5 mL acetic acid was added and incubated at room temperature for 15 min. The absorbance at 549 nm was measured with a microplate absorbance reader. A solution of oleanolic acid was used as the standard. The results were expressed as mg oleanolic acid equivalent per g (mg OA/g).To measure the total triterpenes, 100 μL of AR extract was mixed with 195 μL of DPPH solution (24 mg/L) in a 96-well plate. The reaction proceeded in the dark for 60 min. Afterward, the absorbance of the reaction mixture at 515 nm was measured with a microplate absorbance reader. Vitamin C dissolved in distilled water served as the positive control. The SC50 was estimated as the concentration of extract that scavenged 50% of the free radicals.The free radical scavenging capacity (SC) of AR extract was measured with DPPH assays. Briefly, 5 2+ to Cu+. The resulting Cu+ formed a colored complex with a specific dye reagent in the assay kit. In brief, 5 μL of AR extract was diluted into 100 μL with deionized water and then mixed with 100 μL of Trolox standard in a 96-well plate. The reaction was performed by the addition of 100 μL of Cu2+ working solution, and the plate was shaken for 90 min in dark at room temperature. The absorbance at 570 nm was measured with a microplate absorbance reader.The total antioxidant capacity (TAC) of AR extract was evaluated with a TAC Assay Kit, which measured the ability of antioxidants to reduce Cu2. For the experiments with 6-OHDA, PC12 cells in DMEM with low serum (0.5% FBS) were seeded in 12- or 96-well plates. The cells were incubated with different concentrations of AR extract (0-2 mg/mL) for 2 h and then treated with 500 μM 6-OHDA for 24 h. The differentiation of PC12 cells was described previously [PC12 rat pheochromocytoma cells were obtained from the American Type Culture Collection . The cells were cultured in DMEM supplemented with 10% heat-inactivated FBS and 1% penicillin-streptomycin and then incubated at 37°C in a humidified atmosphere with 5% COeviously . NeuronaThe cell viability was measured with MTT assays according to the manufacturer's protocol. In brief, the cultured medium was discarded, and the cells were incubated with MTT solution  for 4 h at 37°C. Dimethyl sulfoxide was then added to lyse the cells and dissolve the violet formazan crystals that had formed inside the cells. The absorbance at 570 nm was measured with a microplate absorbance reader.Cellular injury was determined by measurement of the LDH released into the culture medium. LDH activity was measured with a detection kit according to the manufacturer's instructions. The absorbance at 490 nm was measured with a microplate absorbance reader.μg/mL) for 20 min. The images were then captured by fluorescence microscopy . The PC12 cells were also resuspended in binding buffer and then stained with annexin V-FITC and PI (1.0 mg/mL) for 20 min. The stained cells were analyzed immediately with a flow cytometer . Ten thousand events were counted for each sample. The data were analyzed in the FlowJo software .After drug treatment, the PC12 cells were washed twice with cold PB and stained with DAPI (2.0 μg/mL) for 20 min. A portion of the cells was photographed by a fluorescence microscopy, and the remaining cells were washed twice with warm PBS and examined by flow cytometry. The intensity of red fluorescence  and green fluorescence  was determined in ImageJ . The ratio of red/green fluorescence was calculated for semiquantitative assessment of mitochondrial polarization states.The PC12 cells were incubated with JC-1 dye (3 3 cells/well) were seeded into a Seahorse XF 24 well culture microplates and incubated overnight at 37°C in a humidified atmosphere with 5% CO2. After drug treatment, the cultural medium was replaced with Seahorse base medium and incubated in a non-CO2 incubator for 1 h. PC12 cells were sequentially treated with 1 μM oligomycin (Oligo), 1 μM carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP), and 1 μM rotenone plus 1 μM antimycin A (R + A). OCR was calculated using the Seahorse software. After finishing the assay, the cells were lysed with RIPA buffer (200 μL/well), and the protein concentration was measured by bicinchoninic acid assay. OCR was normalized to the protein content and presented as pmol/min/μg protein.Mitochondrial oxygen consumption rate (OCR) was measured using Seahorse XFe24 Analyzer . PC12 cells  was determined in the ImageJ software.ROS was detected with CM-HCaspase 3/7 activity in PC12 cells was evaluated with a caspase 3/7 activity detection kit according to the manufacturer's protocol. Luminescence signals were recorded with a microplate absorbance reader.Protein was extracted from PC12 cells with lysis buffer containing 1% phenylmethylsulfonyl fluoride and 1% protease inhibitor. Lysates were centrifuged at 12,500 × g for 20 min at 4°C, and the supernatant was collected. The total protein concentration was determined with bicinchoninic acid assays. Equal amounts of protein were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then electrically transferred onto a polyvinylidene difluoride membrane, which was subsequently blocked with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween-20 for 1 h. The membrane was subsequently incubated with primary antibodies against mTOR, phospho-mTOR (Ser2448), Akt, phospho-Akt (Ser473), ERK1/2, phospho-ERK1/2 (Thr202/Tyr204), MEK, phospho-MEK (Ser217/221), cleaved-PARP (Asp214), cleaved-caspase 3 (Asp175), cleaved-caspase 9 (Asp315), or GAPDH overnight at 4°C. After being washed with PBS, the membrane was incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. After repeated washes with PBS, proteins were visualized by enhanced chemiluminescence. Images of protein bands were captured, and densitometric measurements of band intensity were performed with a ChemiDoc XRS Molecular Imager .p < 0.05 was considered statistically significant.Data are expressed as the mean ± standard deviation (SD) of at least three independent experiments. Statistical analyses were performed with one-way ANOVA followed by Tukey's multiple comparison test (two or more groups) in the GraphPad Prism 6.0 software . Major contents of AR extract including total phenolic compounds, polysaccharides, and triterpenes were measured by chemical assays. The content of total phenolic compounds of AR extract was 5.53 ± 0.11 mg GAE/g of dry weight. The content of total polysaccharides in AR extract was 1.12 ± 0.23 mg GE/g of dry weight. The content of total triterpenes was 3.20 ± 0.14 mg OA/g of dry weight.50 of AR extract and vitamin C in DPPH assays was 0.58 mg/mL and 1.34 μg/mL, respectively. Similarly, AR extract and vitamin C reduced Cu2+ radicals in TAC assays in a concentration-dependent manner  to scavenge DPPH• free radicals increased with the tested concentration Figures . The SC5 Figures .μM) treatment, the viability of PC12 cells decreased by 45%, and LDH release increased by 110%  for 24 h. The MTT assays showed that AR extract did not affect the viability of PC12 cells in the concentration range of 0.06 to 2 mg/mL . In addi Figures . AR extr Figures .in vitro antioxidant activity of AR extract was studied in PC12 cells treated with 6-OHDA. The intracellular ROS generation was reflected by the green florescent signal produced by the probe CM-H2DCFDA. AR extract itself had no effect on ROS generation in PC12 cells  and antimycin  into the cells could shut down the electron transfer, allowing the calculation of nonmitochondrial respiration. In comparison with control group, treatment with AR caused a mild increase in basal respiration, maximal respiration, spare respiratory capacity, and a mild decrease in ATP-linked respiration . In contμM of 6-OHDA increased the number of apoptotic cells by 2.6-fold but by only 1.7-fold when the cells were preincubated with AR extract  slightly decreased the cell viability. Notably, both Akt inhibitor  were applied to observe the neuroprotective effect of AR. As shown in nhibitor  and MEK nhibitor  abolisheOxidative stress is widely believed to be involved in the pathogenesis of many age-related diseases, such as neurodegenerative diseases, cardiovascular diseases, and cancer. Some antioxidants have been demonstrated to be effective in the prevention or treatment of oxidative stress-related diseases . For insGanoderma lucidum, also known as “Lingzhi” in Chinese, is a well-known and popular edible and medicinal mushroom in Asia. In traditional Chinese medicine, it is used to promote health and longevity [Ganoderma lucidum exerts significant beneficial effects in neurodegeneration, diabetes mellitus, cardiovascular diseases, and tumor development [Ganoderma lucidum are at least partly attributed to its remarkable antioxidant and free radical scavenging activity. For instance, Ganoderma lucidum extract ameliorates MPTP-induced parkinsonism and protects dopaminergic neurons from oxidative stress via regulating mitochondrial function, autophagy, and apoptosis [Ganoderma lucidum was found to prevent hippocampus neurons from mitochondrial dysfunction and apoptosis by alleviating oxidative stress [Ganodermataceae family may also exhibit potential effects in the prevention or treatment of oxidative stress-related diseases. AR is a species in the Ganodermataceae family whose pharmacological effects have rarely been explored. There were only two studies related to the chemical constituents of AR. These studies reported that ethanolic extract of AR contained phenolic compounds [Ganoderma lucidum. Moreover, the major chemical contents of aqueous extract of AR have never been explored. This missing information is important because water decoction is the traditional method of extraction.ongevity . Numerouelopment . These ppoptosis . In a rae stress . These fompounds , 25, butGanoderma lucidum [Ganoderma lucidum reported in other studies [Ganoderma lucidum [in vitro model of Parkinson's disease, with PC12 cells. These cells are derived from the pheochromocytoma of the rat adrenal medulla, and they exhibit similar characteristics to those of neurons, owing to their common embryonic origin from the neural crest [in vitro model to study neuronal development and neurological disease.In the present study, the results of chemical assays demonstrated that the aqueous extract of AR contained phenolic compounds, polysaccharides, and triterpenes. The contents of total polysaccharides and triterpenes of AR extract were lower than that of aqueous extract of  lucidum , 27. Int studies . Indeed, studies . Therefo lucidum . The antal crest . PC12 cein vitro model for studying the antioxidant and neuroprotective effects of AR extract.In the present study, 6-OHDA was used to induce neurotoxicity in PC12 cells. In line with findings from other studies , 30, ourOxidative stress is considered as one of the major mechanisms contributing to dopaminergic neuron death in Parkinson's disease. Apart from being an important source of ROS production, mitochondria are also susceptible to oxidative stress. Increased oxidative stress can lead to mitochondrial dysfunction, which in turn triggers the further generation of ROS. Therefore, a vicious circle may form, in which oxidative stress and mitochondrial dysfunction feed each other forward . The incin vitro and in vivo models [In addition, emerging evidence has indicated that the accumulation of ROS causes mitochondrial damage and induces apoptosis via the upregulation of proapoptotic protein expression , resulting in the loss of dopaminergic neurons in Parkinson's disease. The expression of cleaved-caspase 3/8/9 in dopaminergic neurons is significantly higher in people with Parkinson's disease than in healthy individuals , 36. Simo models , 38. Theo models . In thisin vivo animal studies and in vitro neuronal and neuroepithelial cell studies have demonstrated that MEK and ERK promote neuron cell survival and proliferation by antagonizing cell death and apoptosis [Beyond apoptotic proteins, previous studies have reported that Akt/mTOR- and MEK/ERK-dependent signaling pathways regulate cell survival and proliferation in different types of cells, including endothelial cells, cancer cells, and neuronal cells –44. Disrpoptosis , 47. HenIn conclusion, this study has demonstrated that AR can protect PC12 cells from 6-OHDA-induced oxidative stress, mitochondrial dysfunction, and apoptosis. In addition to the direct ROS scavenging effect, AR extract may downregulate proapoptotic proteins and upregulate the Akt/mTOR- and MEK/ERK-dependent signaling pathways. This is the first study reporting the neuroprotective effect of AR. Our findings provide useful information for future investigation of the potential application of AR or its active ingredients in the prevention and treatment of oxidative stress-related neurodegenerative diseases such as Parkinson's disease.


World Health Organization initiated community-based rehabilitation (CBR) in 1978, and by now, it has been an essential process of medical services worldwide. China had strengthened primary health care on building more than 35,000 community health centers (CHCs) in cities, and more than 34,000 township health centers (THCs) in the rural area. Nevertheless, it remains unclear that if these primary health centers could provide optional rehabilitation services for disabilities. And this study aims at evaluating the supply capacity of rehabilitation service in primary health centers of Chengdu, a regional center city of southwest China.We conducted a general investigation of primary health centers in Chengdu, a city located in southwest China with more than 15 million population. Our investigation covered all of Chengdu’s 390 primary health centers from October to November 2016. We researched these primary health centers on basic rehabilitation services, diseases, and rehabilitation equipment quantity and quality, and traditional Chinese medicine (TCM) physiotherapy.P < 0.001, 95% confidence interval (CI) ),number of rehabilitation bed .Rehabilitation therapy is available in 88.9% (337 of 379) of all primary health centers. Meanwhile, CHCs slightly surpass THCs with an available rate of 92.2% (106 of 115) and 87.5% (231 of 264), respectively. Traditional Chinese Medicine (TCM) physiotherapy is available in 97.1% (368 of 379) of all primary health centers, 97.3% (112 of 115) of CHCs and 97.0% (256 of 264) of THCs. Quantitative analysis showed that substantial factors which could make an impact on the number of patients per year contain: categories of rehabilitation disease (CBR and TCM physiotherapy has become accessible for disabilities in most basic health centers of Chengdu City, whereas, available rate of CBR in THCs is lesser than in CHCs, which suggests an imbalance in primary health service development between rural and urban area. Categories of rehabilitation diseases, and the number of rehabilitation beds constitute co-factors that make an impact on the CBR capacity of basic health centers. Global Disability Action Plan ).number of rehabilitation bed  To authors’ knowledge, by Sep 2019, this survey is the ever first general survey of rehabilitation capacity of primary health care in China from a literature study, as well as the first analysis of factors that influence rehabilitation service capacity in the primary health system. In the general survey, we found more than 88% of CHCs and THCs involved in this survey have been well equipped to provide rehabilitation services. But, the imbalance between urban and rural community health centers remains prominent, even though the government has made efforts to minimize the gap. For nearly the same average number of disabilities , CHCs’ number of patients is 1.56 times as THCs’ (7456.9 vs 4762.6). Compared to THCs, CHCs obviously equipped with higher-quality equipment, more rehabilitation doctors (1.9 vs 1.2) and more rehabilitation counseling (189.1 vs 37.6). Reasons that make this kind of gap between urban and rural may be various, such as economic level, physician attraction, transportation facilities, and citizens’ health awareness. Consequently, this phenomenon of the urban-rural imbalance inspires the government and community to shift more financial support and health education to the rural area to improve the rehabilitation capacity of primary health centers there.P = 0.05, 95%CI ). For instance, β value of categories of rehabilitation diseases is − 1.136, which means primary health centers house more than 9 categories of rehabilitation diseases would attract a larger number of patients, comparing to those with less than 9. It indicates that increase the types of rehabilitation diseases would improve CBR service capacity, which should be considered during the planning of the primary health care system. Following the same logic, increasing the number of rehabilitation beds and physicians should also be included in the plan when possible.As to factors that were analyzed by multivariable ordinary logistic regression, categories of rehabilitation diseases and the number of rehabilitation bed are considered statistically significant. However, we should not ignore the number of rehabilitation physicians as a potential factor even though it is not statistically significant (Since 2009, the Chinese government has been putting a large number of resources to improve its primary health care system. For example, in 2014, the expenditure on primary care reached ¥110 billion .Along with the giant leap in the primary health care system under a powerful and effective push from China government on medical services, the rehabilitation capacity of these primary health centers also advanced in both hardware and software. However, in the southwest of China, there are dozens of regions troubling in short of appropriate medical service, and inevitable lack of affordable and convenient rehabilitation services, because of poverty, inadequate transportation and lack of health awareness. Chengdu, as the central city of southwest China, has a powerful and comprehensive influence on regional development in every aspect, including economy, education, culture, technology, and medicine. The outcome of our study, therefor, may be representative to entire southwest China rather than a city.Due to poor education and training systems on general practitioners, rehabilitation services could not be well delivered to disabilities, even though CHCs have been equipped with the newest equipment and technology. Hence, creating an optimal general practitioner training system would be another driver that could further improve rehabilitation service capacity in the primary health system.In this survey covering all 390 basic primary health centers of Chengdu city, we eventually received 379 intact replies that could be used in analysis. Other 11 primary centers were ruled out from analysis due to incomplete data, which may lead to unknown bias in the results. Another limitation is that we selected the number of patients as a proxy of CBR service, which we think could reflect the scale and service quality of a primary health center. Patients did not know exactly how a primary health center is, they would select which is better according to their experience and impression during accepting CBR service. Hence, patients’ subjective judgement decides whether to accept CBR service in CHCs and then has an influence on our results.Rehabilitation service capacity of primary health facilities in Chengdu took a giant leap in the past 20 years and reached a relatively high level in both quantity and quality. However, there are still many flaws and shorts during the rapid development of the primary health system and rehabilitation service. For instance, the imbalance between CHCs and THCs is one of the challenges that need to be addressed. Our study shows that the government could take measures directly correlated with rehabilitation service capacity to improve CBR service capacity, such as house more categories of rehabilitation diseases in the center, train more rehabilitation physicians and augment the number of rehabilitation beds.Additional File 1. Self-evaluation form of basic medical service capacity of primary medical institutions in Chengdu


A key open problem with multiparty session types (MPST) concerns their expressiveness: current MPST have inflexible choice, no existential quantification over participants, and limited parallel composition. This precludes many real protocols to be represented by MPST. To overcome these bottlenecks of MPST, we explore a new technique using weak bisimilarity between global types and endpoint types, which guarantees deadlock-freedom and absence of protocol violations. Based on a process algebraic framework, we present well-formed conditions for global types that guarantee weak bisimilarity between a global type and its endpoint types and prove their check is decidable. Our main practical result, obtained through benchmarks, is that our well-formedness conditions can be checked orders of magnitude faster than directly checking weak bisimilarity using a state-of-the-art model checker.


Compositions with prominent content of saturated as well as unsaturated long-chain FFAs showed moderate but significant toxicity both in human EndoC-βH1 beta-cells and rat islets, however, without further measurable metabolic impairments. On the other hand compositions with high content of medium-chain FFAs revealed no toxicity. A composition with 50% of the very long-chain unsaturated FFA erucic acid caused high toxicity with concomitant peroxisomal H2O2 production. The toxicity of FFAs to human EndoC-βH1 beta-cells was dampened in mixtures of FFA compositions with a significant content of medium-chain FFAs, but not with a significant proportion of unsaturated FFAs.An inappropriate diet, particularly excessive consumption of dietary fats and oils, may have a major negative impact on beta-cell function and cause type 2 diabetes mellitus. To investigate this issue, we examined the toxicity of free fatty acid (FFA) compositions mirroring the FFA profiles of various popular edible oils in human EndoC-βH1 beta-cells and in rat islets. For this purpose, we made compositions consisting exclusively of various FFAs in different volumetric percentages mimicking these oils and additionally mixtures of these compositions. Human EndoC-βH1 beta-cells were incubated with different oil compositions and the toxicity, lipid droplet formation, ER-stress, and H A prominent example is olive oil, containing mostly the monounsaturated free fatty acid (FFA) oleic acid, which is thought to have beneficial effects on cardiovascular health and the metabolic state in T2DM3. On the other hand, coconut oil, rich in medium-chain FFAs, has been promoted for positive effects on health1 although clinical trials could not provide solid evidence for this assumption2.Dietary habits can have a major impact on health and may cause obesity and type 2 diabetes mellitus (T2DM). Numerous studies have examined the effects of dietary oil consumptions on human health; however, with conflicting results4. An excessive fat intake may have a major negative impact on beta-cell function. Since individuals consume oils composed of various saturated and unsaturated FFAs, FFA mixtures represent the typical lipid component of the daily diet.In general, saturated FFAs are thought to be harmful due to lipotoxicity, while unsaturated FFAs are considered to be beneficial5.In the present study, we, therefore, compared the effects of different FFA profiles of thirteen popular edible plant oils and butter on human EndoC-βH1 beta-cells and of selected popular oils on isolated rat islets. In addition, we analyzed the toxicity of the former traditional version of old rapeseed oil with its characteristically high content of the very long-chain erucic acid (C22:1). Nowadays it has been replaced due to its toxicity by new rapeseed oil varieties with a very low erucic acid contentWe generated oil compositions mimicking the FFA profiles of these edible oils containing different long-chain saturated, monounsaturated, diunsaturated, polyunsaturated and medium-chain FFAs. Since we did not include other constituents such as antioxidants, we were able to associate differences in toxicity to the different FFA species in these oils.In a last step, we tested the toxic effects of mixtures of the FFA compositions of four different edible oils in order to elucidate whether it is possible thereby to ameliorate the toxic potential of individual edible oil compositions.6 and regularly tested for mycoplasma. Rat pancreatic islets were isolated as described7.Human EndoC-βH1 beta-cells  were cultured11, adapted and listed in The FFA compositions contain FFAs  in different volumetric percentages mimicking various popular edible oils and butter. The percentages of single FFAs for each composition were taken from12 and for EndoC-βH1 beta-cells lipid droplet formation13, H2O2 generation  and isolated rat islets  were incubated with different FFA compositions for 24 or 48 h. Thereafter caspase-3 activitytion see , as wellData are expressed as means ± SEM and tested for significance using analysis of variance (ANOVA) plus Dunnett’s multiple comparison test .To investigate the toxicity of FFA compositions mimicking edible oils we determined the activity of the toxicity marker caspase-3, starting with a group of the most important popular oils in human EndoC-βH1 beta-cells Fig.  as well Exposition of EndoC-βH1 beta-cells to FFA compositions with a significant content of medium-chain FFAs (C8–C14)  resulted in a 2.5-fold increase of caspase-3 activity, which was significantly higher than the toxicity of the composition mimicking present-day rapeseed oil Fig. .Next, we examined four mixtures created from different characteristic FFA compositions Fig. . MixtureThe effects of FFA compositions from the group of the most important popular oils  caused comparable effects in rat pancreatic islets Fig.  as in huHaving shown the toxicity of different FFA compositions, we thought to gain insight into the impact of lipid droplets caused by the FFA compositions. All examined FFA compositions, containing both saturated medium-chain and long-chain FFAs together with unsaturated FFAs induced lipid droplet formation to a comparable extent in EndoC-βH1 beta-cells Fig. .Fig. 2Lin = 3).In addition, we studied the effects of the FFA compositions on ER-stress. None of the FFA compositions had an influence on the ER-stress marker CHOP in EndoC-βH1 beta-cells Fig. . The com2O2 generation in mitochondria and peroxisomes by organelle specific HyPer protein expression. No significant increase of H2O2 generation was observed after treatment with any of the examined FFA compositions in mitochondria  showed a mild but significant toxicity. When comparing the beta-cell toxicity with respect to the proportion of unsaturated FFAs in the compositions the extent of toxicity remained unchanged and was not dependent on the number of double bonds. Thus our results do not provide evidence for a major protective potential of these unsaturated FFAs within the oil compositions. Many popular oils belong to this group, including olive oil. The T2DM risk reduction through oils with a high content of unsaturated FFAs as observed in several studies12. The importance of an exclusion of very long-chain FFAs from nutritional oils is convincingly documented by the great beta-cell toxicity of a composition with a very high content of erucic acid (50%) as found in oils of former old rapeseed varieties.On the other hand, it is obvious that FFAs with high beta-cell toxic potential must be excluded from nutritional oils. In particular since toxicity increases with increasing chain length of the FFAs7. In the present study, we demonstrate that a high content of medium-chain FFAs (C8–C14) in compositions with long-chain FFAs mirroring the FFA profile of coconut or palm kernel oil were able to reduce toxicity of these compositions. Even when mixing FFA compositions mimicking different edible oils, the reduction of toxicity by the medium-chain FFAs could be maintained, when one of the four different edible oils in this mixture contained a high proportion of medium-chain FFAs.Medium-chain FFAs shorter than C16 are not toxic to EndoC-βH1 beta-cellsThe crucial toxicity observations made in human EndoC-βH1 beta-cells were confirmed also in primary rat islets.2O2 production in peroxisomes with concomitant moderate toxicity especially in FFA compositions with very long-chain FFAs, whereas H2O2 production in mitochondria and ER-stress were not involved. Since virtually all oil compositions caused comparable lipid droplet formation in spite of differences in their beta-cell toxicity, lipid droplet formation should not be considered a primary element responsible for the toxicity of edible oils.The analyses showed increased HThe toxicity of FFAs to human EndoC-βH1 beta-cells and rat islets could be dampened by mixtures of FFA compositions mimicking edible oils with a significant content of medium-chain FFAs, but not with a significant proportion of unsaturated long-chain FFAs.Supplemetary Information Tab. S1, Fig. S1, Supplementary Method