Palytoxin (PLTX) is one of the most poisonous substances known to date and considered as an emergent toxin in Europe. Palytoxin binds to the Na+-K+ ATPase, converting the enzyme in a permeant cation channel. This toxin is known for causing human fatal intoxications associated with the consumption of contaminated fish and crustaceans such as crabs, groupers, mackerel, and parrotfish. Human intoxications by PLTX after consumption of contaminated fishery products are a serious health issue and can be fatal. Different reports have previously explored the acute oral toxicity of PLTX in mice. Although the presence of palytoxin in marine products is currently not regulated in Europe, the European Food Safety Authority expressed its opinion on PLTX and demanded assessment for chronic toxicity studies of this potent marine toxin. In this study, the chronic toxicity of palytoxin was evaluated after oral administration to mice by gavage during a 28-day period. After chronic exposure of mice to the toxin, a lethal dose 50 (LD50) of 0.44 µg/kg of PLTX and a No-Observed-Adverse-Effect Level (NOAEL) of 0.03 µg/kg for repeated daily oral administration of PLTX were determined. These results indicate a much higher chronic toxicity of PLTX and a lower NOAEL than that previously described in shorter treatment periods, pointing out the need to further reevaluate the levels of this compound in marine products. Palythoa [Ostreopsis [Ostreopsis cf. ovata producer of 5.6 pg of PLTX per cell in the Adriatic Sea [Ostreopsis blooms have been reported in the Mediterranean Sea during the last 20 years as summarized in several reports [The marine toxin palytoxin (PLTX) is one of the most toxic marine phycotoxins known to date. Since its first isolation, about 50 years ago, from marine corals of the genus Palythoa  and latetreopsis ,3,4, the reports ,8,9,10,  reports ,12,13.Ostreopsis blooms [The increment in toxic blooms of PLTX in Europe raised high concern and the urgent need to obtain toxicity data useful for the future regulation of this toxin in marine products captured in European coasts. Besides causing human intoxications, after consumption of marine crustaceans and fish ,15,16,17s blooms ,19,20 ans blooms ,22.+/K+-ATPase), an enzyme crucial to maintain the sodium and potassium gradients across all the cell membranes, in a non-selective cation channel [The mechanism of action of palytoxin is well established because the toxin transforms the sodium-potassium pump  has recommended a maximum of 30 µg PLTX and analogues/kg of shellfish meat , and in Based on the initial results for the acute toxicity of PLTX in mice , we furt50) for PLTX of 0.44 µg/kg  from 0.22 to 0.9 µg/kg, R2 = 0.9122, df (degrees of freedom) = 7), although the survival times differed considerably between the individual mice fed with each dose of PLTX as depicted in As shown in All over the treatment period, mice weight was monitored weekly. As shown in Even at PLTX doses as low as 0.1 µg/kg, animals fed with the toxin administered daily by gavage exhibited symptoms typical of the toxin that are summarized in After euthanasia, the animals were subjected to macroscopic observation on necropsy. As shown in On day 27 after the beginning of treatment, mice were housed individually in metabolic cages and the last dose of toxin was administered by gavage, before monitoring food consumption and urine and faeces production over a 24 h period. As shown in t = 2.959; df = 9; p = 0.016) and 3.5 µg/kg .Urine parameters were evaluated in samples collected during the 24 h period before sacrifice, after mouse placement in individual metabolic cages. The animals employed for this analysis were the mice that survived the 28 days treatment period. Urine parameters analyzed included colour, clarity, specific gravity, protein, glucose, ketones, blood/hemoglobin, bilirubin, and urobilinogen. Urine parameters in control and PLTX-treated mice are summarized in −, Na+ and K+), alanine transaminase (ALT), aspartate transaminase (AST), lactate dehydrogenase (LDH), and creatine kinase (CK). As shown in n = 6) and 52 ± 5 U/L (n = 5) in animals dosed with 0.03 µg/kg. ALT blood levels were significantly increased to 163 ± 46 U/L in seven animals dosed with 0.1 µg/kg PLTX . In mice fed orally with PLTX at 0.3 µg/kg ALT blood levels were 103 ± 49 U/L, 116 ± 49 U/L in mice fed with 1 µg/kg PLTX, 128 ± 46 U/L in the four mice fed with 3.5 µg/kg PLTX, and 49 U/L in one mice that survived 10 days after daily PLTX dosage. Besides mean AST blood levels (n = 6) in control mice, 210 ± 23 U/L in mice dosed daily with 0.03 µg/kg (n = 5), 535 ± 206 U/L (n = 7) in mice treated with 0.1 µg/kg PLTX, 869 ± 435 U/L (n = 5) in mice fed with 0.3 µg/kg PLTX, 343 ± 113 U/L (n = 6) in mice fed orally with PLTX at 1 µg/kg, 301 ± 47 U/L (n = 4) in mice fed with 3.5 µg/kg, and 194 U/L in one mice after 10 days administration of PLTX at 10 µg/kg. Statistical analysis of AST data did not yield significant differences between the control group and the PLTX-treated animals with the exception of mice fed with 3.5 µg/kg , but the levels of AST in 0.1, 0.3, 1 and 3.5 groups were above the normal reference range (59–247 U/L for the IDEXX). In contrast, creatine kinase blood levels (n = 6), 708 ± 220 U/L for the group of mice dosed with 0.03 µg/kg (n = 5), 1781 ± 572 U/L (n = 6), 3040 ± 1319 U/L (n = 5), 512 ± 99 U/L (n = 5), 654 ± 208 U/L (n = 4), and 814 U/L (n = 1) for the groups of mice dosed with 0.1, 0.3, 1, 3.5 and 10 µg/kg PLTX, respectively. Statistical comparison of creatine kinase data did not show significant differences between control animals and animals treated daily with PLTX. Nevertheless, in this case, the blood level of CK in the animals dosed daily with 0.1 and 0.3 µg/kg of PLTX was well above the IDEXX reference range, which is between 68 and 1070 U/L, with some animals showing CK levels well above 1070 U/L. As shown in n = 6) in the control group, 1904 ± 338 U/L in three animals dosed with 0.03 µg/kg, 6230 ± 2448 U/L (n = 6) in the 0.1 µg/kg group, 2716 ± 841 U/L (n = 3) for the 0.3 µg/kg group, 1626 ± 325 U/L (n = 4) in the 1 µg/kg group, 1275 ± 296 U/L (n = 4) in the 3.5 µg/kg group, and finally 1914 U/L in one mouse at the highest dose employed. In this case, no statistically significant differences were observed between control animals and those that received a daily PLTX dose. However, two out of the six mice fed with 0.1 µg/kg PLTX had LDH levels well above the IDEXX reference range for blood LDH, which is between 1105 and 3993 U/L.At the end of the 28 days of treatment, mice were euthanized, and biochemical analysis was performed in blood extracted by cardiac puncture immediately after euthanasia or, in some cases, after sudden death of animals. Blood samples were analyzed as in the previous study  and blood levels B, were 1d levels C were 50+/K+ ratio and on blood chloride levels, this dysregulation of electrolytes levels is in agreement with the previously reported alterations produced by a single oral dose of PLTX [Next, the effect of repeated exposure of mice to low doses of PLTX on blood electrolyte levels was evaluated . Statist of PLTX .+ blood values in control animals were 155 ± 1 mmol/L (n = 4) while in PLTX-treated animals, sodium levels were 156 ± 2 mmol/L (n = 5) at 0.03 µg/kg, 152 ± 2 mmol/L in seven animals dosed with 0.1 µg/kg of PLTX. At the dose of 0.3 µg/kg of PLTX, five animals have mean blood Na+ values of 149 ± 1 mmol/L and significant differences were observed versus control animals . Similarly, blood sodium levels were 159 ± 2 mmol/L (n = 6) and 158 ± 2 mmol/L (n = 4) in animals dosed with 1 and 3.5 µg/kg PLTX, respectively. Moreover, at the highest dose employed (10 µg/kg), the blood sodium level was 150 mmol/L, but only one mouse was evaluated. Interestingly, an increase in potassium levels was found in blood samples as indicated in + levels were 6.8 ± 0.2 mmol/L (n = 4), 8.6 ± 0.1 mmol/L (n = 5) in mice fed daily with 0.03 µg/kg PLTX , 9.3 ± 0.3 mmol/L in seven mice fed with 0.1 µg/kg PLTX  and 8.6 ± 0.5 mmol/L (n = 5) in mice treated with 0.3 µg/kg PLTX . Despite the short survival time of the animals treated with the highest PLTX dose, blood K+ levels were well above the normal range in one mouse that survived 10 days of treatment. Due to the decrease in blood sodium concentration and the increase in blood potassium levels, the ratio Na+/K+ decreased (+/K+ was 23 (n = 4) and it decreased to 19 ± 1 , 18 ± 1  and 19 ± 1  in animals treated with 0.03, 0.1 and 0.3 µg/kg PLTX, respectively. Next, the effects of oral PLTX on blood chloride levels were evaluated  in animals fed daily with 0.03 µg/kg PLTX, 120 ± 1 mmol/L  after the 0.1 µg/kg treatment, 117 ± 1 mmol/L (p = 0.0192) after the 0.3 µg/kg treatment, and 119 ± 1 mmol/L (p = 0.0047) after the 1 µg/kg treatment.As shown in ecreased C, especivaluated D. ChloriSince, after necropsy, macroscopic changes in the digestive tract of PLTX-treated animals were observed, and some mice dosed with PLTX at 1 and 3.5 µg/kg exhibited significant weight loss, transmission electron microscopic studies (TEM) of the stomach were performed. Samples from control animals and from animals treated with PLTX were obtained immediately after euthanasia and evaluated. First, and in view of the macroscopic alterations of the stomach with dark-yellow liquid and dilatation with gas accumulation, mucous cells were evaluated. After TEM analysis, some evident changes in the structure of mucous cells, which were identified by their electron-lucent granules, were appreciated as Aside from mucous cells, the gastric epithelium secretes hydrochloric acid and digestive enzymes, and since palytoxin altered blood ionic homeostasis, the acid-producing cells of the stomach  were also analyzed by TEM. As shown in 50 ranging from 510 to 767 µg/kg. Nevertheless, in the latest opinion of the EFSA experts on PLTX-related risks [50 and the chronic NOAEL for oral PLTX. In this context, the data presented in this report constitute the first initial approach for the chronic evaluation of the PLTX toxicity after daily ingestion. Moreover, as far as we know, this is the first work that evaluates the chronic toxicity of this potent marine toxin following the OECD recommendations to evaluate the chronic toxicity of chemicals [50 of 0.44 µg/kg and a NOAEL of 0.03 µg/kg. In view of the high increase of toxicity after repeated exposure to PLTX, it is crucial to consider a representative scenario in which successive exposures to low doses of PLTX might arise, especially for the population living near coastal areas affected by Ostreopsis blooms. Indeed, besides oral toxicity, many reports have described toxic corneal reactions [So far, the available in vivo toxicity studies with palytoxin have shown a high acute oral toxicity with LDed risks , the lachemicals . The dateactions ,46, as weactions ,20,47,4850 of 757 µg/kg have been reported after a single dose of PLTX and an observation period of 24 h [50 of 510 µg/kg was reported by other authors [Until now, different in vivo toxicities for PLTX in mice have been reported, depending mainly on the time of exposure of the animals to the toxin. For instance, a NOAEL of 300 µg/kg and a LD of 24 h . In the  authors , while n authors  but stom authors . However authors ,35,38.+-K+-ATPase pump is indispensable for different cellular functions, and its inhibition results in an excess of extracellular K+ and accumulation of intracellular Na+ because, in the presence of toxin the Na+-K+- ATPase can no longer pump K+ into the cell or pump Na+ out of the cell [+-K+ inhibition [+-K+-ATPase was transformed in an open cation channel in the presence of PLTX [+ concentration would affect all the ion transport systems coupled to the sodium gradient like the Na+/H+ exchanger, the Na+/Ca2+ exchanger, the H+-K+-ATPase pump and hence, the osmotic balance [+-K+-ATPase with omeprazol [As the molecular target of PLTX, the Nathe cell , the reshibition  and coulhibition ,29,35,38hibition . Regardi of PLTX ,25 which balance ,53. In tmeprazol ,55. In ameprazol , as obseOstreopsis blooms in Europe, raise the need to perform additional studies on the oral in vivo toxicity of this potent marine toxin in order to prevent human illness and accurately regulate the food levels of this emerging marine toxin.Altogether, the new findings described in the present study, together with the increase in 50 of 0.44 µg/kg and a NOAEL of 0.03 µg/kg.Daily feeding of female Swiss mice by gavage with different PLTX doses for 28 days resulted in an oral LDIn vivo studies were performed with Swiss female mice weighing an average of 21 to 25 g in each group (4 weeks old). All in vivo studies employed in the manuscript followed the European directives (EU directive 2010/63/EU) and the Spanish legislation , and were performed accordingly with the principles approved by the Institutional Animal Care Committee of the Universidade de Santiago de Compostela under procedure number 011/14, authorized on 14 August 2014 (MR110250) and under procedure number 06/19/LU-002, authorized on 1 March 2019 (A12X00509).Palythoa tuberculosa, (purity 88.9% and CAS Number: 77734-91-9) and dissolved in water. Before administration, the stock PLTX solution was diluted in 0.9% saline solution to achieve each dose. For the treatment, mice received a daily oral dose of PLTX by gavage  or the same volume of control saline. Animals were observed closely for the first 2 h and periodically thereafter, until sacrifice of surviving animals at the end of the treatments. During the course of the experiments, moribund animals or animals obviously in pain or showing signs of agony, as well as animals with weight loss higher than 10% and enduring distress, were euthanized and considered in the interpretation of the test results in the same way as animals that were euthanized at the end of the 28 days treatment period. During treatment, mice were housed in rooms with constant temperature, humidity, and a controlled photoperiod at the animal facilities of the School of Veterinary Medicine of the University of Santiago de Compostela (Code: AE-LU-002). During experiments, control and PLTX-treated mice were weighted weekly at day 0 (day of the fist treatment), day 7, day 14, day 21 and day 28, and food consumption was also registered at the same intervals. Euthanasia was performed on day 28. After the last PLTX-dosage (day 27), control and PLTX-treated mice were placed in metabolic cages for 24 h to monitor urine and faeces production and obtain samples. Finally, animals were euthanized in a CO2 chamber, and immediately after death confirmation by cervical dislocation, blood and tissue samples were collected. All animals in the study were subjected to a full necropsy, including detailed visualization of heart, liver, lungs, kidneys, spleen, stomach, duodenum, rectum, and cerebrum.The PLTX used in the experiments was purchased from Fujifilm Wako Pure Chemical Corporation  extracted from g . The computer controlled biochemical analyzer IDEXX Catalyst Dx  was used for the analysis of plasma parameters. In most of the samples, 300 µL of plasma were used to evaluate electrolyte levels , alanine transaminase, aspartate transaminase, lactate dehydrogenase, and creatine kinase. Initially, all the markers were evaluated in undiluted samples, but if the marker level in plasma was above the reference range of the analyzer, an automatic dilution with physiological saline was performed. Samples with altered visual properties that could lead to the misinterpretation of the biochemical parameters  were not further processed.Blood was obtained directly from the ventricle of euthanized mice as previously described ,57 and cUrine colour, clarity, protein, glucose, ketones, blood hemoglobin, bilirubin, and urobilinogen analysis were performed using a reflectance photometer (IDEXX VetLab UA), which reads and evaluates IDEXX UA strips. Urine specific gravity was measured with a refractometer. Because for some mice a small amount of blood or urine were obtained, it was not possible to measure all the parameters for each animal.3) were fixed by immersion  containing in mM: 137 NaCl, 8.2 Na2HPO4, 1.5 KH2PO4 and 3.2 KCl) for 2 h at 4 °C and rinsed three times with PBS. Postfixation by immersion in 1% OsO4 in 0.1 M cacodylate trihydrate buffer was performed for 60 min. After, tissues were rinsed and dehydrated in graded ethanol solutions, including one bath with 70% ethanol and 0.5% uranyl acetate, rinsed in propylene oxide, and embedded in Epon 812 . A Leica Ultracut UCT ultramicrotome  was used to obtain ultrathin sections of the tissue samples. Samples were counterstained with uranyl acetate and lead citrate, and ultrastructural analysis of 1 mm2 thick samples was performed with a JEOL JEM-1011 Transmission Electron Microscope .Stomach samples preparation for transmission electron microscopy was performed according to previously described procedures ,57,58. Cp-value of < 0.05 was considered statistically significant. ANOVA repeated measures followed by the Dunnett’s comparison test was used for statistical comparisons.Statistical analyses were carried out using GraphPad Prism 5 . All values are expressed as mean ± SEM. A


CXP (3 mg/kg) was intraperitoneally injected for 5 days. RV (0.5 or 50 mg/kg) was intraperitoneally injected for 10 days from the first day of CXP administration. Auditory brainstem response (ABR) thresholds were measured before and within 3 days at the end of the drug administration. Cochlear tissues were harvested, and the outer hair cells were examined using cochlear whole mounts. The mRNA expression of NFκB, IL6, IL1β, and CYP1A1, and protein levels of aryl hydrocarbon receptor (AhR) and cytosolic and nuclear receptor for advanced glycation endproducts (RAGE) were evaluated. The ABR threshold increased in the 50 mg/kg RV and CXP groups at 4, 8, 16, and 32 kHz. The 0.5 mg/kg RV + CXP group demonstrated decreased hearing thresholds at 4 and 32 kHz compared to the CXP group. Cochlear whole-mount analysis revealed loss of outer hair cells in the 50 mg/kg RV and CXP groups and partial prevention of these cells in the 0.5 mg/kg RV + CXP group. The mRNA expressions of NFκB, IL6, and IL1β were increased in the 50 mg/kg RV and CXP groups compared to the control group. In contrast, these levels were decreased in the 0.5 mg/kg RV + CXP group compared to the CXP group. The mRNA expression of CYP1A1 was increased in the CXP group, while it was decreased in the 0.5 mg/kg RV + CXP group compared to the control group. The protein levels of AhR and cytosolic RAGE decreased in the 0.5 mg/kg RV group. Low-dose RV had partial otoprotective effects on CXP ototoxicity. The otoprotective effects of RV may be mediated through anti-oxidative (CYP1A1 and RAGE) and anti-inflammatory  responses. High-dose RV exerted an inflammatory response and did not ameliorate CXP-induced ototoxicity.Previous preclinical studies have demonstrated the otoprotective effects of resveratrol (RV) at low doses. This study aimed to investigate the dose-dependent effects of RV in rats with cisplatin (CXP)-induced hearing loss. Sprague-Dawley rats (8-weeks old) were divided into six treatment groups ( RV is tape skin . A moder−5–10−4 M) has been tested for cancer chemoprevention effects, which is much higher than in the in vivo doses [The effects of RV vary depending on its bioavailability and dose. The bioavailability of RV is dependent on the administration route and is affected by the food matrix. When administered orally, the sulfated form is the main metabolite in human plasma and limits the bioavailability of RV . The absvo doses . In addivo doses .A number of preclinical studies have reported the protective effects of RV in animals with hearing loss induced by aging ,10,11, nWe hypothesized that there exists a dose range for the otoprotective effect of RV. To test this hypothesis, rats were administered low (0.5 mg/kg/day) or high (50 mg/kg/day) doses of RV based on a previous study that demonstrated no protective effect of RV at 50 mg/kg in the vascular endothelial function in rats .p < 0.001 for 4 kHz, 26.25 ± 2.63 vs. 49.29 ± 1.95 dB SPL, p < 0.001 for 8 kHz, 27.50 ± 2.50 vs. 46.43 ± 3.57 dB SPL, p = 0.001 for 16 kHz, and 41.25 ± 1.25 vs. 61.43 ± 2.94 dB SPL, p = 0.02 for 32 kHz). Cisplatin administration induced elevation of the hearing threshold. The CXP group demonstrated increased hearing threshold after CXP injection at 4, 8, 16, and 32 kHz . The 50 mg/kg RV + CXP group demonstrated increased hearing threshold after CXP injection at 4, 8, 16, and 32 kHz, and the hearing thresholds after drug administration (post) were not significantly different from those of the CXP group . The 0.5 mg/kg RV + CXP group also demonstrated increased hearing threshold after CXP injection at 4, 8, 16, and 32 kHz, but the hearing thresholds after drug administration (post) were lower than those of the CXP group at 4 and 32 kHz  for 4 kHz, 35.91 ± 1.82 vs. 46.25 ± 4.91 dB SPL, p = 0.034 for 8 kHz, 33.64 ± 1.55 vs. 41.88 ± 4.76 dB SPL, p = 0.071 for 16 kHz, and 41.82 ± 1.26 vs. 40.00 ± 2.04 dB SPL, p = 0.576  for 32 kHz).Among the six groups, four groups (other than control and 0.5 mg/kg RV groups) demonstrated ABR threshold shifts following drug administration . AlthougCochlear whole-mount examination revealed a loss of outer hair cells in the 50 mg/kg RV group . CXP admNFκB, IL1β, and IL6 (1.63 ± 0.14 (p = 0.001), 2.22 ± 0.39 (p = 0.03), and 2.25 ± 0.24 (p = 0.02) fold change, respectively) compared to the control group  compared to the control group. The 0.5 mg/kg RV group and 0.5 mg/kg RV + CXP group did not show a significant change in mRNA levels of NFκB, IL1β, and IL6, compared to the control group.The CXP group showed significantly higher mRNA levels of ol group . The 50 NFκB, IL1β, and IL6 . The 50 mg/kg RV + CXP group did not show any significant difference in mRNA levels of NFκB, IL1β, and IL6 compared to the CXP group.Compared to the CXP group, the 0.5 mg/kg RV + CXP group showed decreased mRNA levels of CYP1A1 was increased in the 0.5 mg/kg RV group but decreased in the CXP group compared to the control group . CYP1A1 mRNA level, which was decreased in the CXP group, was increased in the 0.5 mg/kg RV + CXP group but not in the 50 mg/kg RV + CXP group .The mRNA expression level of p = 0.006). CXP administration increased the AhR protein level in the CXP group and the 50 mg/kg RV + CXP groups . AhR protein levels showed a decreased tendency in the 0.5 mg/kg RV group and an increased tendency in the 0.5 mg/kg RV + CXP group compared to the control group, although there was no statistical significance .AhR protein levels decreased following administration of a high dose of RV . The 50 p = 0.001). The CXP group did not show any significant difference in cytosolic RAGE protein level compared to the control group . Both 0.5 mg/kg RV + CXP and 50 mg/kg RV + CXP groups showed lower cytosolic RAGE protein levels compared to the control group . The nuclear RAGE protein levels were not different among the groups.Cytosolic RAGE protein level decreased following administration of a high dose of RV . The 50 CYP1A1 transcript and the decreased CYP1A1 mRNA expression in CXP rats was reversed following co-administration of low-dose RV with CXP. In contrast, high-dose RV increased the auditory thresholds. High-dose RV did not attenuate the mRNA expression levels of pro-inflammatory cytokines. High-dose RV alone elevated the mRNA expression level of NFκB. Both low- and high-dose RV alone decreased AhR and cytosolic RAGE protein levels. Although they did not reduce AhR, cytosolic RAGE expression was decreased in the groups co-administered high or low doses of RV along with CXP. By investigating the dose-dependent otoprotective effects of RV on relevant molecules, this study contributes substantially to the existing body of knowledge in the field.In this study, we demonstrated that low-dose RV partially reduced the auditory threshold shifts in CXP-induced hearing loss rats. The increased expression of the pro-inflammatory cytokines IL1β, IL6, and cytosolic RAGE in CXP rats were attenuated in the low-dose RV + CXP rats. Low-dose RV elevated NFκB, IL6, and IL1β.In this study, we found that low-dose RV reduced hearing loss and inflammatory responses. A few prior studies have reported the otoprotective effects of low to moderate doses of RV ,15,17,18NFκB, IL6, and IL1β, and anti-oxidative effects of CYP1A1 and cytosolic RAGE. CXP increases oxidative stress and the pro-inflammatory cytokines NFκB and IL6 [κB/IL6 and IL1β [The hearing preservation effects of low-dose RV observed in this study may be mediated by the anti-inflammatory effects of  and IL6 . RV has  and IL6 . In diab and IL6 . In mousand IL1β . In the and IL1β . The incand IL1β .NFkB and IL6 and decrease of anti-oxidative molecule of CYP1A1 may mediate the ototoxic response of a high dose of RV in this study. Previous studies reported the pro-oxidant effects of a high dose of RV in the cardiovascular system [CYP1A1 was reported to be mediated by AhR, the suppression of AhR expression with a high dose of RV could be linked with the insufficient activation of CYP1A1 [The high dose of RV did not show otoprotective effects in the present study. A few previous studies have suggested the adverse effects of RV overdose ,29. The r system ,31. Becaf CYP1A1 . In addif CYP1A1 . Mitochof CYP1A1 . In addif CYP1A1 . A previf CYP1A1 . Howeverf CYP1A1 . The cumf CYP1A1 ,17,18,20n = 12/group; The animal experiments performed in this study were approved by the Institutional Animal Care and Use Committee of the CHA University Medical School . All experimental procedures complied with the guidelines of the Institutional Animal Care and Use Committee of the CHA University Medical School. Seventy-two female Sprague-Dawley rats (8-weeks old) were divided into six groups  were used for cochlear whole mounts. The cochleae were fixed in 4% paraformaldehyde solution. After decalcification, the cochlear outer hair cells were dissected. Then, tissues were soaked in 0.3% Triton blocking solution. The 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) solution was applied to the tissues for 1 h, and the tissues were then mounted on slides and examined under a light microscope. The number and arrangement of cochlear outer hair cells were examined using confocal microscopy with a stack image under 400-fold magnification .2 deep freezer. Total RNA was extracted within 24 h of tissue collection using TRIzol™ Reagent . The purified RNA was checked for purity and quantity by measuring the 260/280 nm absorbance ratio using a Micro UV-Vis spectrophotometer . Only samples with a 260/280 ratio > 1.8 and a 260/230 ratio > 1.5 were used for qRT-PCR. Maxime™ RT Pre Mix (Oligo (dT)15 Primer)  was used for reverse transcription. Nuclear factor-κB (NFκB), interleukin-1β (IL1β), interleukin-6 (IL6), and cytochrome P450 1A1 (CYP1A1) were reverse transcribed and PCR-amplified using the primers listed in ® DNA polymerase at 95 °C for 15 min, followed by 50 cycles of 95 °C for 10 s, 60 °C for 15 s, and 72 °C for 15 s. The amplification efficiency (E) of each amplicon was determined using a 10-fold serial dilution of positive control complementary DNA (cDNA) and calculated from the slopes of the log input amounts (20 ng–2 pg of cDNA) that were plotted according to the crossing point values using the formula E = 10 − 1/slope. All primer efficiencies were confirmed to be high (>90%) and comparable. The calculated mRNA levels were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) according to the formula 2−ΔΔCt, and expressed as a percentage of the reference gene.Five rats per group  were used for quantitative reverse transcription-polymerase chain reaction (qRT-PCR). qRT-PCR was performed as described previously . The memFive rats per group  were used for Western blotting. Two rats per group  were used to measure AhR protein level, and the remaining three rats in each group  were used to measure protein levels of cytosolic/nuclear RAGE.g for 5 min. The pellet was resuspended in 400 μL of cytoplasmic extraction reagent I by homogenizing. The suspension was then incubated on ice for 10 min followed by the addition of 22 μL of cytoplasmic extraction reagent II. The mixture was vortexed for 5 s, incubated on ice for 1 min, and centrifuged for 5 min at 16,000× g. The supernatant (cytoplasmic extract) was transferred to a microcentrifuge tube. The insoluble pellet was resuspended in 200 μL of nuclear extraction reagent by vortexing for 15 s, incubated on ice for 10 min, and centrifuged for 10 min at 16,000× g. The resulting supernatant constituted the nuclear extract.Nuclear and cytosolic fractions from cochlear tissue were extracted using the NE-PER Nuclear Cytoplasmic Extraction Reagent kit . Briefly, the tissue was washed twice with cold PBS and centrifuged at 500× Radioimmunoprecipitation assay buffer  was used for tissue lysis. The protein concentration was evaluated using a Bio-Rad Protein Assay Kit. The proteins were separated using 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gels were transferred to polyvinylidene difluoride membranes  and soaked in blocking buffer ) for 1 h. The membrane was incubated in primary antibodies against AhR , nuclear RAGE , cytosolic RAGE , β-actin , and HDAC1 . The membranes were then incubated with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies ; Abcam, #ab97023). The protein bands were then visualized using an enhanced chemiluminescence kit . Protein expression levels were calculated using ImageJ gel analysis software  and compared with the expression levels of β-actin (for cytosolic proteins) or HDAC (for nuclear RAGE).t-test within each group and unpaired t-test between groups. The differences in mRNA and protein levels were analyzed using unpaired t-tests. The values are presented as mean ± standard deviation. Statistical significance was defined as p < 0.05. All analyses were performed using SPSS 21.0 .Changes in ABR thresholds were analyzed using paired CYP1A1 expression might be linked to the otoprotective effects of low-dose RV. The increased expression of pro-inflammatory cytokines IL6 and IL1β in CXP rats was attenuated in the low-dose RV + CXP rats. However, high-dose of RV did not exert otoprotective effects in CXP-induced hearing loss rats.Low-dose RV partially preserved hearing in cisplatin-induced hearing loss rats. The increased anti-oxidative effects involving


We demonstrate a water-based synthetic route to fabricate composite inverse opals for simultaneous detection of ascorbic acid (AA), dopamine (DA), and uric acid (UA). Our process involves the conformal deposition of poly (PEDOT) and PEDOT/Au on the skeletons of Ni inverse opals via cyclic voltammetric scans (CV) to initiate the electropolymerization of 3,4-ethylenedioxythiophene (EDOT) monomers. The resulting samples, Ni@PEDOT, and Ni@PEDOT/Au inverse opals, exhibit a three-dimensional ordered macroporous platform with a large surface area and interconnected pore channels, desirable attributes for facile mass transfer and strong reaction for analytes. Structural characterization and material/chemical analysis including scanning electron microscope, X-ray photoelectron spectroscopy, and Raman spectroscopy are carried out. The sensing performances of Ni@PEDOT and Ni@PEDOT/Au inverse opals are explored by conducting CV scans with various concentrations of AA, DA, and UA. By leveraging the structural advantages of inverse opals and the selection of PEDOT/Au composite, the Ni@PEDOT/Au inverse opals reveal improved sensing performances over those of conventional PEDOT-based nanostructured sensors.  Ascorbic acid (AA), dopamine (DA), and uric acid (UA) are three important chemicals present in our central nervous system and serum. The AA, also known as vitamin C, plays critical functions in our body including hormonal activation, anti-oxidation, immunity enhancement, and tissue regeneration ,2. The tThe sensing of AA, DA, and UA is feasible electrochemically because these chemicals exhibit a two-electron oxidation reaction at sufficient oxidizing potentials. However, the challenge is to distinguish them simultaneously with tailored electrode designs because a typical glassy carbon electrode (GCE) reveals similar oxidizing potentials for these three compounds and hence, their resulting voltammetric responses are overlapped, leading to low resolutions and poor sensitivities . To overRecently, Au nanoparticles have been shown as an effective electrochemical sensor because of their impressive chemical stability and biocompatibility, as well as superior electric conductivity for electrocatalytic action . TherefoTo date, the synthesis of PEDOT is often conducted in organic solvents for desirable electric conductivity and porosity ,32. In c2 at 65 °C for 12 h in a reactor equipped with a bladed pitched paddle impeller. Next, 160 g styrene , 0.005 g sodium 4-styrenesulfonate , and 0.2 g potassium bicarbonate  were mixed to form a homogeneous solution. Afterward, 0.4 g potassium persulfate  was added as the initiator and the solution was stirred at 370 rpm for 16 h at 60 °C. Lastly, the mixture was dried at 65 °C to evaporate residual solvent, and the remaining PS microspheres were re-suspended in anhydrous ethanol at 25 °C and sonicated for 24 h to form a homogeneous PS colloidal suspension of 0.5 wt% loading.Polystyrene (PS) microspheres with an average diameter of 820 nm were synthesized via an emulsifier-free emulsion polymerization process. First, 1000 mL deionized water was deoxygenated in N2 ITO was used as the working electrode, and a 6 × 6 cm2 stainless steel plate was employed as the counter electrode. The distance between these two electrodes was 1 cm. At 10 °C and an electric field of 6 V cm−1, the PS microspheres were driven toward the working electrode to form highly-ordered close-packed colloidal crystals. Afterward, the PS colloidal crystals were placed in an oven at 90 °C in air for 48 h. The PS colloidal crystals were constructed by a vertical electrophoresis step in which a 2 × 2 cm2 PS colloidal crystals as the working electrode and a 4 × 4 cm2 Ni plate as the counter electrode. The aqueous electrolyte contained 0.5 M NiSO4·6H2O , 0.125 M NiCl2·6H2O , and 0.3 M H3BO3 . The Ni plating was carried out by imposing a galvanostatic current of 2.5 mA cm−2 for 1 h at 25 °C. Afterward, the sample was immersed in toluene  at 60°C for 48 h, followed by a heat treatment at 350 °C for 2.5 h in 95% Ar + 5% H2 to remove the PS colloidal crystals completely to obtain Ni inverse opals. We adopted the Watt’s electroplating solution to backfill the PS colloidal crystals with Ni. The electroplating entailed 2 × 2 cm−1 at 25 °C for 3 cycles. The electropolymerization of 3,4-ethylenedioxythiophene (EDOT) to PEDOT was conducted using 2 × 2 cm2 Ni inverse opals as the working electrode, a 2 × 2 cm2 Pt plate as the counter electrode, and an Ag/AgCl as the reference electrode. The electrolyte was aqueous solution of 0.05 M EDOT monomer , 0.1 M lithium perchlorate , and 0.01 M sodium dodecyl sulfate . The resulting sample was denoted as Ni@PEDOT inverse opals. A conformal PEDOT coating was deposited on the Ni inverse opals by carrying out CV scans in a potential range between −0.6 and 1.1 V with a scan rate of 20 mV s−1 at 25 °C for 3 cycles. The deposition was performed using 2 × 2 cm2 Ni inverse opals as the working electrode, a 2 × 2 cm2 Pt plate as the counter electrode, and an Ag/AgCl as the reference electrode. The aqueous solution contained 0.05 M EDOT monomer, 0.1 M lithium perchlorate, 0.01 M sodium dodecyl sulfate, and 0.5 mM hydrogen tetrachloroaurate(III) trihydrate . The deposition process involved the oxidation of EDOT to PEDOT and the reduction of Au ions simultaneously to form Ni@PDOT/Au inverse opals. For comparison purposes, we also prepared planar Ni@PEDOT films on ITO substrates using identical processes for sequential plating of Ni and PEDOT.A conformal PEDOT/Au coating was deposited on the Ni inverse opals by conducting CV scans in a potential range between −0.6 and 1.1 V with a scan rate of 20 mV s+, Waltham, MA, USA) was used to analyze the compositions and oxidation states of constituents in our samples. A surface cleaning step was adopted by ion beam etching before XPS analysis. The complex impedance measurements were carried out via a potentiostat  in 0.1 M phosphate-buffered saline (PBS) at the open circuit potential. The amplitude for the electric stimulation was 10 mV, and the frequency range was from 1 to 100 kHz. A 2 × 2 cm2 Pt foil was used as the counter electrode. The distance between the working and counter electrode was 1.5 cm. A scanning electron microscope  was utilized to observe the morphology of PS colloidal crystals, Ni inverse opals, Ni@PEDOT inverse opals, Ni@PEDOT/Au inverse opals, and planar Ni@PEDOT films. Their atomic ratios were obtained by an energy dispersive X-ray spectroscope  equipped on the SEM. In each analysis, five positions were randomly selected for average. Raman spectroscopy analysis was performed using a Jobin Yvon Labram HR 800 microscope  and the excitation source was a 632.8 nm HeNe laser. X-ray photoelectron electron spectroscope  with various concentrations of AA, DA, and UA. The sensing behaviors of Ni@PEDOT inverse opals, Ni@PEDOT/Au inverse opals, and planar Ni@PEDOT films for AA, DA, and UA were explored by CV scans using a potentiostat . A 2 × 2 cm2, and explored their potential applications in chemical sensing and photonic devices  (μM)–4.34 (R2 = 0.984), j (μA cm−2) = 0.403 × [DA] (μM)–4.95 (R2 = 0.99), and j (μA cm−2) = 0.709 × [UA] (μM)–4.07 (R2 = 0.976), respectively. The corresponding detection limit was 29.22 [AA], 20.3 [DA], and 14.67 μM [UA], respectively. For Ni@PEDOT inverse opals shown in −2) = 0.145 × [AA] (μM)–1.07 (R2 = 0.996), j (μA cm−2) = 0.583 × [DA] (μM) + 10.98 (R2 = 0.991), and j (μA cm−2) = 1.23 × [UA] (μM) + 21 (R2 = 0.991), respectively. The corresponding detection limit was 10.88 [AA], 10.88 [DA], and 4.93 [UA] μM, respectively. Despite the planar Ni@PEDOT film revealed a wrinkled morphology for better conductivity, the Ni@PEDOT inverse opals, with a three-dimensional ordered macroporous structure with interconnected pore channels, still outperformed the planar Ni@PEDOT film in both sensitivity and detection limit.−2) = 0.266 × [AA] (μM)–3.65 (R2 = 0.978), j (μA cm−2) = 1.041 × [DA] (μM) + 4.31 (R2 = 0.995), and j (μA cm−2) = 1.126 × [UA] (μM)–3.03 (R2 = 0.979), respectively. The corresponding detection limits were 5.98 [AA], 5.98 [DA], and 2.98 [UA] μM, respectively. In comparison with those of Ni@PEDOT inverse opals, the Ni@PEDOT/Au inverse opals clearly demonstrated greater sensitivities toward AA and DA, as well as lower detection limits for AA, DA, and UA. This superior performance of Ni@PEDOT/Au inverse opals suggested that the Au nanoparticles in the PEDOT structure facilitated the electron transfer and promoted the oxidation of AA, DA, and UA. As for Ni@PEDOT/Au inverse opals shown in We demonstrated the conformal deposition of PEDOT and PEDOT/Au on the skeletons of Ni inverse opals using CV cycling in an aqueous solution. The inverse opaline structure allowed for a large surface area with interconnected pores, desirable features for simultaneous detection of AA, DA, and UA. The impregnation of Au nanoparticles in the PEDOT further improved electron transfer, resulting in even better sensing performance as compared to those of conventional nanostructured PEDOT-based counterparts. Our water-based synthesis is environmentally friendly and our results indicated that by combining the structural features of inverse opals and the composite of PEDOT/Au, we were able to overcome the intrinsic limitation of relatively poor PEDOT films from aqueous solution to show better performances over PEDOT-based sensors from more popular organic processing route. In addition, the unique structure of inverse opals made possible the detection of AA, DA, and UA via a straightforward CV technique.


In addition to effects in the cell body, α-synuclein accumulation occurs at presynapses where the protein is normally localized. While it is generally agreed that excess α-synuclein impairs synaptic vesicle trafficking, the underlying mechanisms are unknown. in vitro with Hsc70, the chaperone protein that uncoats CCVs at synapses. After introducing excess α-synuclein, Hsc70 availability was reduced at stimulated synapses, suggesting Hsc70 sequestration as a possible mechanism underlying the synaptic vesicle trafficking defects. In support of this hypothesis, increasing the levels of exogenous Hsc70 along with α-synuclein ameliorated the CCV uncoating and vesicle recycling defects. These experiments identify a reduction in Hsc70 availability at synapses, and consequently its function, as the mechanism by which α-synuclein induces synaptic vesicle recycling defects. To our knowledge, this is the first report of a viable chaperone-based strategy for reversing the synaptic vesicle trafficking defects associated with excess α-synuclein, which may be of value for improving synaptic function in PD and other synuclein-linked diseases.α-Synuclein overexpression and aggregation are linked to Parkinson’s disease (PD), dementia with Lewy bodies (DLB), and several other neurodegenerative disorders. In addition to effects in the cell body, α-synuclein accumulation occurs at presynapses where the protein is normally localized. While it is generally agreed that excess α-synuclein impairs synaptic vesicle trafficking, the underlying mechanisms are unknown. We show here that acute introduction of excess human α-synuclein at a classic vertebrate synapse, the lamprey reticulospinal (RS) synapse, selectively impaired the uncoating of clathrin-coated vesicles (CCVs) during synaptic vesicle recycling, leading to an increase in endocytic intermediates and a severe depletion of synaptic vesicles. Furthermore, human α-synuclein and lamprey γ-synuclein both interact  Synaptic defects caused by α-synuclein overexpression are linked to cognitive deficits in Parkinson’s disease (PD) and other diseases. However, the mechanisms by which excess α-synuclein impairs synaptic vesicle trafficking are unknown. Data presented here demonstrate that acute introduction of excess α-synuclein at a classical vertebrate synapse inhibits clathrin-coated vesicle (CCV) uncoating, leading to impaired vesicle recycling. Furthermore, increasing α-synuclein also reduced synaptic levels of Hsc70, the clathrin uncoating chaperone protein, indicating sequestration of Hsc70 as a possible underlying mechanism. Subsequently increasing exogenous Hsc70 restored CCV uncoating and vesicle recycling. This study identifies a novel molecular mechanism underlying the α-synuclein-induced synaptic defects and presents one viable strategy for reversing them.SNCA), as well as several point mutations , which result in atypical aggregation of α-synuclein protein throughout neurons (α-Synuclein is a presynaptic protein whose aberrant aggregation causes neurodegeneration in Parkinson’s disease (PD), dementia with Lewy bodies (DLB), and several variants of Alzheimer’s disease . Inherit neurons . α-Synuc neurons . Though Under physiologic conditions, α-synuclein participates in synaptic vesicle trafficking via its established roles in vesicle clustering/reclustering , SNARE cLamprey giant reticulospinal (RS) synapses provide an excellent model for assessing how excess α-synuclein affects vertebrate synapses. RS synapses are ideal for these studies because they are amenable to acute perturbations of presynaptic processes , thus pein vitro with Hsc70, the chaperone protein that uncoats CCVs at synapses, thus identifying an interaction that may affect synapses in vivo. Indeed, excess α-synuclein reduced Hsc70 availability at stimulated synapses, suggesting Hsc70 sequestration as a possible mechanism underlying the synaptic defects. Consequently, co-injection of exogenous Hsc70 and α-synuclein ameliorated the synaptic vesicle trafficking defects. Thus, Hsc70 is an in vivo target of excess α-synuclein at synapses, and increasing Hsc70 function reverses the deleterious impacts.We report here a mechanism by which excess α-synuclein induces synaptic vesicle recycling defects and present a novel strategy for ameliorating these defects. When introduced acutely, α-synuclein selectively impaired CCV uncoating during synaptic vesicle recycling, leading to a depletion of the synaptic vesicle cluster . We furtCloning of recombinant GST-tagged human α-synuclein and His-tagged bovine Hsc70 used for biochemistry experiments was as described previously . RecombiPetromyzon marinus; 11–13 cm; five to seven years old of either sex) were anesthetized in 0.1 g/l MS-222 . Spinal cord pieces (2–3 cm) were dissected and pinned ventral side up in a Sylgard-lined dish. Axonal microinjections were performed as described in +; pH 7.4) to a pipet concentration of 130–160 μM. In some experiments, recombinant bovine Hsc70 (27 μM) was included in the injection pipet either alone or together with α-synuclein. Proteins were then loaded into glass microelectrodes (20–25 MΩ) and microinjected into giant RS axons using small pulses of N2  delivered through a picospritzer. Fluorescein (10 kDa) or tetramethylrhodamine (70 kDa) dextrans , approximating the molecular weights of α-synuclein and Hsc70, respectively, were included in the pipets and co-injected to visualize the proteins’ diffusion rates in the axons. Axonal injections resulted in a 10–20× dilution of the proteins. Thus, the final axonal concentration of exogenous human α-synuclein was estimated to be ∼7–13 μM, and the final concentration of exogenous bovine Hsc70 was ∼1–3 μM. Axons were subsequently stimulated with action potentials  using current injections  to induce synaptic vesicle exocytosis/endocytosis.All animal procedures were approved by the Institutional Animal Care and Use Committee at the MBL in accordance with standards set by the National Institutes of Health. Lampreys  where the protein concentration was measurable based on the diffusion of the co-injected fluorescent dye , as well as distances farther from the injection site (150–700 μm) where no protein had diffused . Thus, each EM experiment was internally controlled, as shown in https://github.com/audreytmedeiros/Morgan-Lab), which measured the distance from the center of each SV to the nearest point on the active zone. After obtaining measurements for each organelle, a total membrane analysis was performed on each synapse to determine how synaptic membranes were redistributed with each perturbation. Here, SV and CCP/V membrane areas were calculated by multiplying the surface area of a sphere (4πr2) by the number of each type of vesicle at each synapse. Plasma membrane and cisternae areas were obtained by multiplying the length of membrane evaginations and summed cisternae perimeters, respectively, by the section thickness (70 nm). Graphing and statistical analyses, including Student’s t tests and ANOVA, were performed in Origin 7.0 (OriginLab Corp). Data were reported as the mean value per section per synapse.Spinal cords were fixed immediately after stimulation , processed for EM, sectioned at 70 nm, and counterstained with uranyl acetate and lead citrate, as described previously . Images +, 150 mM KCl, 1 mM EDTA, and 1% Triton X-100; pH 7.4) containing protease inhibitors. GST pull downs were performed using 50–100 μg of GST-tagged proteins and either 1–5 mg of rat brain or lamprey CNS extracts or 100 μg of purified recombinant proteins. After performing the pull downs, the bound proteins were run on 10% SDS-PAGE gels and transferred to nitrocellulose membranes. For Western blotting, antibodies were diluted in TBST  with 1% dry milk or 5% BSA. Primary antibodies were used at 1:1000 and included: mouse monoclonal anti-β1/β2-adaptins , mouse monoclonal anti-clathrin heavy chain , mouse monoclonal anti-dynamin , mouse monoclonal anti-synaptojanin-1 , mouse monoclonal anti-auxilin   were prepared in HKET buffer 2SO4, 25 mM KCl, and 2 mM MgAc2 were reacted with an equal volume of 4 μM Hsc70 in the same buffer. Background scattering determined from reactions without cages or CCVs was subtracted from measured scattering values, which were normalized by dividing by the starting scattering value so that the initial scattering in all reactions was 1.0. In some reactions 20 μM recombinant human α-synuclein (rPeptide) was added to the Hsc70 syringe. Various components were also omitted when indicated as controls. Data were plotted using Origin 7.0 software.Clathrin cages were assembled with 1 μM recombinant bovine brain clathrin and 0.1 μM auxilin, as described in + (50 mM) Ringer for 10 min . The remaining synaptic vesicles were of normal size . However, the SVs were slightly more dispersed , which is consistent with the phenotype reported in hippocampal synapses from transgenic mice overexpressing human α-synuclein . Supporting effects on CME, the total numbers of clathrin coats (CCPs+CCVs) were increased 2- to 3-fold . Further dissection of the stages of CME revealed that α-synuclein selectively increased the number of free CCVs (stage 4) without significantly altering the earlier stages of CCP formation . A total membrane analysis shows that the loss of synaptic vesicle membrane area was compensated by an expansion of the plasma membrane, cisternae, and CCP/Vs . Taken together, these data indicate that excess α-synuclein impairs clathrin-mediated synaptic vesicle recycling with selective effects on CCV uncoating . We repeated the pull downs using GST-tagged lamprey γ-synuclein, which is the most highly expressed synuclein isoform in the lamprey giant RS neurons .To determine how α-synuclein impairs clathrin uncoating, we next tested for possible interactions between α-synuclein and several key players that mediate CME at synapses, including those involved in the clathrin uncoating process . Human αno acids . Demonst neurons . Full-le neurons . Furthern = 3; ANOVA p = 5 × 10−5; Tukey’s post hoc). Similarly, lamprey γ-synuclein and its NTD also pulled down bovine Hsc70 in these direct binding assays . To further map the interaction, we additionally tested the binding of α-synuclein to several Hsc70 truncation mutants. One truncation consisted of only the NBD (a.a. 1–383), and the other contained the NBD and SBD but lacked the C-terminal 10 kDa region , and a substrate-binding domain (SBD) with a more variable 10 kDa C-terminal region E. While n = 5; ANOVA p < 0.0001; Tukey’s post hoc; GST: 11.5 ± 2.3 AU; GST-Syn: 97.8 ± 5.5 AU; GST-E46K: 75.2 ± 6.3 AU; n = 3; ANOVA p < 0.0001; Tukey’s post hoc; GST: 6.1 ± 4.4 AU; GST-Syn: 68.0 ± 6.8 AU; GST-A53T: 67.1 ± 3.2 AU; n = 5; ANOVA p < 0.0001; Tukey’s post hoc).We repeated the pull downs using the α-synuclein point mutants that are linked to PD: A30P, E46K, and A53T A. All thin vivo clathrin uncoating defect and in vitro interaction between Hsc70 and α-synuclein suggest that when in excess, α-synuclein may interact with Hsc70 and alter its CCV uncoating function at synapses. Among the possible mechanisms, α-synuclein could interfere with Hsc70 uncoating activity and/or alter Hsc70 availability at synapses. We began testing these possibilities by examining whether α-synuclein affects the ability of Hsc70 to promote clathrin cage disassembly in vitro using a light scattering assay as previously described , which reside along the periphery of the giant RS axons . Next, as with the EM experiments, we injected human α-synuclein to a final axonal concentration of 7–13 μM . Measurements of endogenous α-synuclein range from 3–6 μM . We also quantified the average number of Hsc70 puncta per synapse under the same conditions and obtained similar results. At control synapses, the average number of Hsc70 puncta per synapse increased on stimulation, but this did not occur after α-synuclein treatment . In an independent set of experiments, using action potential stimulation  and a second Hsc70 antibody , we observed similar results . Thus, excess α-synuclein sequesters Hsc70 in vivo and reduces its availability at stimulated synapses, suggesting a possible mechanism underlying the CCV uncoating defects during synaptic vesicle recycling.Compared to the RS axons B, inset.RS axons . To deteRS axons B. After RS axons . The synRS axons , as wellRS axons C,D. Althm 3–6 μM  to 45 μMm 3–6 μM , suggestm 3–6 μM . After iynuclein D. Next, ynuclein A. In Figynuclein , 2006, aynuclein E. Stimuln = 27 synapses, n = 2 axons; Hsc70: 108.2 ± 9.6 SVs; n = 28 synapses, n = 2 axons; Student’s t test; p = 0.18; SV distribution, <500 nm, control: 79.91 ± 3.12%; Hsc70: 77.36 ± 3.02%; >500 nm, control: 20.09 ± 3.12%; Hsc70: 22.64 ± 3.02%; n = 27–28 synapses, n = 2 axons; ANOVA p < 0.0001; Tukey’s post hoc p = 0.94; plasma membrane, control: 2.61 ± 0.12 μm, n = 27 synapses, n = 2 axons; Hsc70: 2.58 ± 0.18 μm; n = 28 synapses, n = 2 axons; Student’s t test; p = 0.89; #clathrin coats, control: 4.4 ± 0.6 coats, n = 27 synapses, n = 2 axons; Hsc70: 4.8 ± 0.5 coats, n = 28 synapses, n = 2 axons; Student’s t test; p = 0.60; clathrin coat distribution, stage 1, control: 0.14 ± 0.07 CCPs/section; Hsc70: 0.15 ± 0.07 CCPs; stage 2, control: 0.36 ± 0.12 CCPs, Hsc70: 0.15 ± 0.07 CCPs; stage 3, control: 2.39 ± 0.47 CCPs; Hsc70: 2.67 ± 0.30 CCPs; stage 4, control: 1.54 ± 0.26 CCVs, Hsc70: 1.85 ± 0.34 CCVs; n = 27–28 synapses, n = 2 axons; ANOVA p < 0.0001, Tukey’s post hoc). The number of cisternae increased slightly, but their size remained unchanged . Thus, introducing Hsc70 alone produced very little effect on synaptic vesicle trafficking with no measurable effects on CME or synaptic vesicle clusters.The above results suggest that reduced availability of Hsc70 at synapses, and consequently its function, could underlie the α-synuclein-induced defects in CCV uncoating and synaptic vesicle recycling. If so, then increasing Hsc70 levels would be expected to reverse the synaptic defects. To test this, we co-injected recombinant bovine Hsc70  along with human α-synuclein  and examined the effects at lamprey synapses. As a control for these experiments, we first injected Hsc70 alone . As expen = 22–30 synapses, n = 2 axons; Students t test; p = 0.644). Although the SVs still appeared slightly dispersed . The synaptic vesicles were of similar size (diameter) in both conditions . Similarly, the number and size of cisternae were unchanged . Hsc70 additionally restored the total number of clathrin-coated structures (CCPs+CCVs) to control levels . Furthermore, there was a complete rescue of the CCV uncoating defect . Only the plasma membrane evaginations remained larger after Hsc70 and α-synuclein co-injection, indicating some persistent effects on the plasma membrane . The total membrane analysis further corroborated that synapses co-treated with Hsc70 and α-synuclein were similar to controls . Thus, increasing exogenous Hsc70 levels largely reversed the synaptic vesicle trafficking defects caused by α-synuclein, including the deficits in CCV uncoating.We next examined synapses co-treated with both α-synuclein and Hsc70. As before, stimulated control synapses exhibited large vesicle clusters, shallow membrane evaginations, and few CCPs and CCVs A,C. Compispersed F, the peWhile a number of studies have focused on the physiologic roles of α-synuclein at synapses (for review, see By this working model, replenishing Hsc70 at synapses should have the effect of restoring CCV uncoating, thereby freeing clathrin and other limited coat proteins to recycle synaptic vesicles more efficiently B. Indeedin vitro, leading to increased cell viability (Our results corroborate and extend previous reports of an interaction between α-synuclein and Hsc70 . Hsc70 biability . Cross-liability . Similariability . Our resiability . In the iability . The seciability  likely biability . In contiability . In addiiability . The cliiability , and so Drosophila and mouse models of α-synuclein toxicity (Hsc70 was previously identified as a potential target in other α-synuclein-related contexts. In human brains affected by PD, DLB, and related synucleinopathies, Hsc70 and other chaperone proteins are detected in high levels along with α-synuclein . The co-toxicity . Thus, Htoxicity . Our datImportantly, our work also provides new insights into the cellular mechanisms for α-synuclein and Hsc70 involvement in the neurodegenerative process, specifically related to the impacts on CME. Since CME is also involved more broadly in vesicle endocytosis from the plasma membrane and intracellular vesicle trafficking events, these experiments also have wider implications for potential impacts on membrane trafficking throughout the entire neuron. In addition, our work also highlights the need for the type of studies described here, in which the mechanism of phenotypic rescue is characterized in depth, because of the potential complexities of deploying a therapeutic approach that involves increasing the activity of a protein like Hsc70, which is involved in many cellular processes. We find that while Hsc70 fully rescues the CCV uncoating defects induced by α-synuclein and restored the synaptic vesicle cluster, the membrane evaginations persisted, suggesting residual impairment of membrane endocytosis by α-synuclein . Such obDNAJ, the gene that encodes for the Hsc70 co-chaperone, auxilin, in patients with juvenile Parkinsonism or early-onset PD (The findings reported here contribute to a growing body of evidence that PD and other forms of Parkinsonism may be linked to deficits in the clathrin-mediated synaptic vesicle recycling , and an onset PD . In addionset PD . Furtheronset PD . Severalonset PD . Transgeonset PD . Thus, a


Understand the traffic health beliefs and traffic risk behaviors among secondary technical vocational school students, explore the relationship between health beliefs and traffic risk behaviors, and to provide proof and direction for further traffic injury intervention.A cluster sampling method was used to conduct a questionnaire survey of all grade one and grade two students in a secondary technical vocational school in Jiangsu Province in April 2018, to investigate student’ traffic risk behaviors and traffic health beliefs. Behavior number was calculated according to the kinds of risk behaviors, and behavior score was calculated according to behavior frequency.A total of 1079 students were investigated. Traffic risk behaviors were highly prevalent, including walking through a red light (24%), not using the crosswalk (22%), no using a helmet (69%), no using reflective equipment at night (78%). There were significant correlations among the traffic risk behaviors (P<0.001). Of the traffic risk behaviors in the past 30 days, 64% of the students had 3 or over, and 51.5% scored 6 or over. The total health belief score was 3.39±0.74, and for the 7 dimensions, “perceived susceptibility”, 3.68±1.00; “perceived severity”, 3.73±0.98; “perceived benefits”, 3.73±0.96; “perceived barriers”, 3.19±1.04; “cues to action”, 3.10±1.05; “self-motive”, 3.38±1.04; “self-efficacy”, 2.60±0.77. The results of GLM  analysis showed that, “cues to action” and “self-efficacy” significantly affect both traffic risk behavior number and score, with OR of 1.60 (1.16-2.20) and 0.45 (0.33-0.61) for behavior number, and 1.65 (1.07-2.55) and 0.41 (0.27-0.62) for behavior score. “Cues to action” increase the incidence of traffic risk behaviors, which may reflect the fact that peer effect in vocational school students played significant role in traffic safety. “Self-efficacy” decrease traffic risk behaviors and other dimensions of health belief model had no significant effect, which Indicate further intervention direction for traffic safety education. Traffic risk behaviors in secondary technical vocational school students were highly prevalent and clustered. Peer effect in vocational school students should be taken into full consideration to improve traffic safety. Further intervention for traffic safety should increase health education to increase students’ health belief.Secondary technical vocational school, Traffic injury, Risk behavior, Health belief


Imperatorin is a phytochemical isolated from medicinal plants and is widely used in herbal medicine. The aim of this study was to investigate the interactions between imperatorin and diazepam 50 values and the type of inhibition. For in vivo assessment, six rats were pretreated with 50 mg/kg imperatorin for two weeks, six were administered saline, and a single dose of 10 mg/kg diazepam was administered orally to both groups 30 min after the administration of imperatorin.In vitro inhibition of imperatorin was assessed by incubating rat liver microsomes with diazepam to determine ICin vitro metabolism of diazepam via the competitive mechanism of CYP450. The IC50 values of imperatorin to nordazepam and temazepam were 1.54 μM and 1.80 μM, respectively. The inhibitory constant values for temazepam and nordazepam were 1.24 μM and 1.29 μM, respectively. Long-term administration of imperatorin significantly increased the AUC(0-12h), AUC(0-∞), and Cmax of diazepam, while Vz/F and CLz/F were decreased significantly (P < 0.05). In turn, the AUC(0-12h), AUC(0-∞), and Cmax of nordazepam and temazepam decreased significantly, and Vz/F and CLz/F increased significantly (P < 0.05).Imperatorin inhibited the in vitro and in vivo. These results indicated that more attention should be paid when taking diazepam together with food or herbs containing IMP, although further investigation is still needed.This study demonstrates that imperatorin inhibits the metabolism of diazepam both  Cynanchum otophyllum Schneid, Angelica sinensis, and Angelica dahurica , a linear furocoumarin compound, is widely found in the roots of the medicinal plants dahurica . IMP is dahurica . IMP alshimurium . In addihimurium . In a sthimurium . The mulin vivo  is well known to have improved analyte throughput, sensitivity, and chromatographic peak resolution compared with other analytical methods  was purchased from MUST . Diazepam was purchased from Tianjin KingYork Pharmaceutical Co., Ltd. Clonazepam, nordazepam, and temazepam (purity > 98%) were purchased from Sigma-Aldrich , and clonazepam was used as the internal standard (IS). NADPH was purchased from Roche . Ultra-pure water was prepared by a Milli-Q puriﬁcation system . Analytical grade formic acid was provided by Sigma-Aldrich . UPLC grade acetonitrile and methanol were purchased from Merck Company . The BCA Protein Assay Kit was purchased from Beyotime . Blank rat plasma samples were supplied by drug-free rats from the Laboratory Animal Center of Wenzhou Medical University. All other chemicals for this study were reagent grade and used without further puriﬁcation.A UPLC-MS/MS system with ACQUITY I-Class UPLC and a XEVO TQD triple quadrupole mass spectrometer , equipped with an electrospray ionization (ESI) interface, was used to analyze the compounds in positive mode. The UPLC system was comprised of a binary solvent manager and a sample manager with flow-through needle. Masslynx 4.1 software (Waters Corp.) was used for data acquisition and instrument control.◦C. The initial mobile phase consisted of acetonitrile and formic acid water (0.1% formic acid v/v) with gradient elution at a ﬂow rate of 0.4 mL/min and an injection volume of 2 μL. Elution was performed in a linear gradient where the acetonitrile content increased from 10% to 30% within 0.3 min and then continued to increase from 30% to 95% between 0.3 and 2.0 min. The acetonitrile content was maintained at 95% for 0.5 min, then dropped to 10% within 0.1 min. The total run time of the analytes was 3 min. After each injection, the sample manager underwent a needle wash process with methanol–water.Diazepam, nordazepam, and temazepam were separated using a UPLC BEH C18 column  maintained at 40Nitrogen was used as the desolvation gas (1000 L/h) and cone gas (50 L/h). The ion monitoring voltage conditions were as follows: capillary voltage: 2.5 kV; source temperature: 150°C; desolvation temperature: 500°C. Multiple reaction monitoring methods were used for quantitative analysis. The transitions of diazepam, nordazepam, temazepam, and clonazepam are shown in In this study, rats were weighed and anesthetized with 10% chloral hydrate (0.35 mL/100g) followed by hepatic perfusion with ice-cold saline (0.9% NaCl), weighed, and homogenized. Subsequently, the homogenized liquid was centrifuged at 9000 rpm for 30 minutes at 4°C. The supernatant was then removed and centrifuged at 105,000 rpm for 60 minutes under the same conditions. The precipitate was reconstituted with 0.15 mol of a KCl-PBS buffer solution (pH 7.4) and dispensed. After repeating the above procedure, the precipitate was stored in a KCl-PBS buffer solution containing 0.25 mol/L sucrose at -80°C until use. The protein concentrations of RLMs were determined by BCA assay.50  determination, concentrations of 0.1, 0.5, 1, 2.5, 5, 10, 25, 50, and 100 µM IMP were used. For determination of the type of inhibition of IMP on diazepam metabolism, inhibition constant (Ki) values were obtained by incubating IMP  with a series of concentrations of diazepam .The above preparations were mixed and incubated at 37°C for 5 min. NADPH was added to initiate a 30-min reaction process, which was then transferred to ice, and 200 μL acetonitrile was added to stop the reaction. Twenty microliters of clonazepam was added to the incubation system, vortexed for 2 min, and centrifuged at 13000 rpm for 5 min, and a 2-μL aliquot of the supernatant was used for UPLC-MS/MS analysis.Incubation mixtures were prepared in a total volume of 200 μL as follows: 0.44 mg/mL RLMs, 100 mM potassium phosphate buffer (pH 7.4), 50 μM diazepam, 1 mM NADPH, and IMP. For the ICThe assay method was fully validated in terms of precision, accuracy, recovery, matrix effect, and stability, according to the guidelines set by the Male Sprague-Dawley (SD) rats with body weights of 220-250 g were obtained from the Wenzhou Medical University Experimental Animal Center. The animals were kept in a controlled environment at 20–26°C and 55 ± 15% relative humidity under a 12-h light-dark cycle. Diet was prohibited for 12 h before the experiment, but water was freely available. All experimental procedures and protocols were reviewed and approved by the Animal Ethics Committee of Wenzhou Medical University, in line with the Guidelines for the Care and Use of Laboratory Animals.Twelve SD male rats were randomly selected and assigned to either treatment group or control group. Treatment group rats were pretreated with an oral administration of IMP (50 mg/kg) daily for 2 weeks. Control group rats were pretreated with saline for 2 weeks. All rats were given diazepam (10 mg/kg) after the last administration of IMP. 300 μL blood was collected from the tail veins of both groups into centrifuge tubes with heparin according to a time series of 0.083, 0.25, 0.5, 1, 2, 3, 4, 6, 8, and 12h. Each blood sample was immediately centrifuged at 3,000 rpm for 10 min, and the supernatant was separated, transferred to another centrifuge tube, and stored at -20°C until analysis. Before analysis, the plasma sample was thawed to room temperature. An aliquot of 20 μL of the IS working solution (0.5μg/mL clonazepam) was added to 50 μL of plasma sample in a 1.5-mL centrifuge tube, followed by the addition of 200 μL of acetonitrile. The tubes were vortexed for 1 min. After centrifugation at 13000rpm for 5 min, the supernatant (2 μL) was injected into the UPLC-MS/MS system for analysis.50 was calculated with GraphPad v.8 . Statistical analyses were evaluated by student T test . Differences were considered statistically significant when P < 0.05.Non-compartmental analysis was used to calculate the pharmacokinetic parameters using Drug and Statistics software (v.3.2.8). The average plasma concentration–time curve was drawn according to the mean drug concentrations at each time point. The IC50 values of diazepam metabolism to temazepam and nordiazepam  of diazepam significantly increased from 157.25 ± 19.52 to 219.77 ± 41.91 μg/h/L (P<0.05). The Cmax of diazepam significantly increased from 77.05 ± 12.25 to 107.11 ± 30.73 ng/mL (P < 0.05), and the clearance (CLz/F) was obviously decreased from 59.16 ± 7.14 to 42.38 ± 6.57 L/h/kg (P < 0.05). Although the MRT(0-12) of diazepam increased from 3.71 ± 1.7 to 4.36 ± 2.12 h, the difference was not significant (P > 0.05). The t1/2 of diazepam in treatment group rats was shorter compared with control group rats , but the difference was not significant (P > 0.05).(0-12h) and Cmax of nordazepam were significantly decreased from 35.22 ± 8.65 to 23.09 ± 4.11 ng/mL and from 78.55 ± 12.07 to 50.15 ± 9.96 μg/h/L, respectively (P < 0.05). Vz/F and CLz/F in the treatment group increased 2.42- and 1.55-fold, respectively, compared with the treatment group (P < 0.05). Although the MRT(0-12) and Tmax of nordazepam increased from 1.94 ± 0.36 to 2.23 ± 0.31 h and from 0.46 ± 0.1 to 0.63 ± 0.31, respectively, the difference was not significant (P > 0.05). The results obtained from the preliminary analysis of temazepam are compared in (0-12h) and Cmax of temazepam significantly decreased from 18.47 ± 5.45 to 9.96 ± 2.3 ng/mL and from 41.17 ± 6.52 to 21.73 ± 3.73 μg/h/L, respectively (P < 0.05). Vz/F and CLz/F in the treatment group increased 1.73- and 1.86-fold, respectively, compared with the control group (P < 0.05). The MRT(0-12) and Tmax of nordazepam were not significantly different between the treatment group or control group (P > 0.05).The results of the correlational analysis of nordazepam are displayed in Pharmacokinetic interactions between food and drugs may occur during absorption, distribution, metabolism, or excretion. DDIs generally occur through their pharmacokinetic properties, with metabolic drugs having the highest incidence of DDIs . FurocouPrevious studies have used liquid-high-resolution mass spectrometry to detect dozens of metabolites, mainly oxidation products, in the urine of rats orally administered IMP . This inBecause diazepam can rapidly enter the cranial nerve after intravenous injection and cause mild central nervous system inhibition, it is widely used in psychiatric practice. The metabolism of diazepam is intricate. It is first demethylated by CYP3A4 and CYP2C19 in the liver to nordiazepam, and hydroxylated by CYP3A4 to form the active metabolite temazepam. Both nordiazepam and temazepam are further metabolized to oxazepam. Then, the combination of temazepam and oxazepam by glucuronidation and glucuronic acid is largely eliminated. However, they are still biologically active and able to accumulate with continuous application .50 values of temazepam and nordazepam were less than 1.8 μM, consistent with in vivo pharmacokinetic studies. The Ki values for temazepam and nordiazepam were very close, with values of 1.24 μM and 1.29 μM, respectively, indicating that IMP might inhibit diazepam in a competitive manner. IMP and diazepam might bind to the same active center of the enzyme. Conversely, IMP displayed a mixed type of inhibition on human CYP3A4 and CYP2C9 in reported studies , AUC(0-∞) and Cmax of diazepam significantly increased, while CLz/F decreased about 1.4 fold, compared to the control group. For pharmacokinetic parameters of the metabolites of diazepam, nordiazepam, and temazepam, IMP reduced the AUC(0-12h), AUC(0-∞), and Cmax but increased the CL/F. The results of the in vivo experiment revealed that IMP had an influence on the metabolism of diazepam, which causes drug-drug interactions (DDIs). Like the IMP, another furocoumarin, psoralen, is a mechanism-based inactivator of CYP2B6 , Natural Science Foundation of Zhejiang and Zhejiang Pharmaceutical Association Joint Foundation (LYY18H280003), City-level public welfare technology application research project of Lishui (2016GYX32 & 2017GYX15), and CAMS Innovation Fund for Medical Sciences (2018-I2M-1-002).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.


Pseudomonas spp. In addition, FT-IR and MSI spectral data were collected at the same time intervals as for microbiological analyses. Multivariate data analysis was performed using two software platforms  comprising several machine learning algorithms for the estimation of the TVC and Pseudomonas spp. population of the surface of the samples. The performance of the developed models was evaluated by intra batch and independent batch testing. Partial Least Squares- Regression (PLS-R) models from the commercial software predicted TVC with root mean square error (RMSE) values of 1.359 and 1.029 log CFU/cm2 for MSI and FT-IR analysis, respectively. Moreover, RMSE values for Pseudomonas spp. model were 1.574 log CFU/cm2 for MSI data and 1.078 log CFU/cm2 for FT-IR data. From the implementation of the in-house sorfML platform, artificial neural networks (nnet) and least-angle regression (lars) were the most accurate models with the best performance in terms of RMSE values. Nnet models developed on MSI data demonstrated the lowest RMSE values (0.717 log CFU/cm2) for intra-batch testing, while lars outperformed nnet on independent batch testing with RMSE of 1.252 log CFU/cm2. Furthermore, lars models excelled with the FT-IR data with RMSE of 0.904 and 0.851 log CFU/cm2 in intra-batch and independent batch testing, respectively. These findings suggested that FT-IR analysis is more efficient than MSI to predict the microbiological quality on the surface of chicken breast fillets.The objective of this research was the evaluation of Fourier transforms infrared spectroscopy (FT-IR) and multispectral image analysis (MSI) as efficient spectroscopic methods in tandem with multivariate data analysis and machine learning for the assessment of spoilage on the surface of chicken breast fillets. For this purpose, two independent storage experiments of chicken breast fillets (n = 215) were conducted at 0, 5, 10, and 15 °C for up to 480 h. During storage, samples were analyzed microbiologically for the enumeration of Total Viable Counts (TVC) and  Scheme describing data processing and models construction. •Poultry meat’s vulnerability to spoilage demands rapid quality assessment LWT-Food Sci. Technol.methods.•FT-IR and MSI are non-invasive methods applied in a variety of meat products.•SorfML is a web platform providing diverse machine learning algorithms.•FT-IR analysis via lars predicted efficiently microbial loads of TVC. Both spAn important and challenging decision in the development of predictive models with spectral data is the performance of the optimum machine learning algorithm resulting in efficient models that describe more accurately the dynamics of microorganisms during spoilage. Until now, many algorithms have been employed in the rapid assessment of meat quality through several software applications . SorfML The aim of this research was (i) to develop models derived from different analytical instruments (FT-IR and MSI) assessing the microbiological quality of chicken breast fillets during storage at isothermal conditions, (ii) to assess the performance of different machine learning algorithms and analytical platforms, based on a commercial software and a publicly available Web platform, to monitor the population dynamics of spoilage microorganisms during storage, and (iii) to infer on the potential and limitations of each analytical tool.22.1ca. 245–280 g per fillet) were obtained from a Greek poultry industry and transported under refrigeration immediately to the laboratory. The samples were supplied by the industry in plastic packages  and stored aerobically at four isothermal conditions  for up to 480 h depending on storage temperature. At regular time intervals, spectral data (FT-IR and MSI) were collected from the surface of chicken meat samples and correlated with microbiological data. Two independent experiments were undertaken with two different chicken meat batches  and duplicate samples were analyzed from each sampling point and storage temperature. Storage of samples was terminated at 480 h at 0 °C while for the highest storage temperature (15 °C) the duration of the experiments was 168 h. All samples originated from Ross strains broilers with the same feeding, farming and slaughtering conditions. Feeding was customized by the company and comprised of grain, wheat, maize, soya bean oil and meat and premix for broilers . Chickens were slaughtered after 3 months of age and all stages of production were in compliance to EU regulations .Chicken breast fillets (2.22 (maximum thickness: 2 mm) from the surface of chicken breast fillet was removed aseptically using a sterile stainless steel cork borer (2.5 cm in diameter), scalpel and forceps, added to 100 mL of sterile quarter strength Ringer’s solution  and homogenized in a Stomacher device  for 120 s at room temperature. Serial decimal dilutions were prepared in the same medium and 1.0 or 0.1 mL of the appropriate dilutions were spread or poured on the following media: a) Tryptic glucose yeast agar  for the enumeration of Total Viable Counts (TVC) incubated at 25 °C for 72 h; b) Pseudomonas Agar Base with selective supplement cephalothin-fucidin-cetrimide  for the enumeration of Pseudomonas spp. after incubation at 25 °C for 48 h. After incubation, typical colonies for each microbial group were enumerated and colony counts were logarithmically transformed and expressed as log CFU/cm2. Further on, the primary model of Pseudomonas spp. to determine the kinetic parameters of microbial growth .A slice of 20 cm2.32 and CO2 during storage.Prior to microbiological analysis, the gas composition in the headspace of the packages was analyzed using a Dansensor CheckMate 9900 gas analyzer  to monitor the changes in the concentration (%) of O2.42.4.1MSI spectra were captivated via Videometer-Lab instrument  which frames surface reflectance of samples from 18 different monochromatic wavelengths (405–970 nm), namely: 405, 435, 450, 470, 505, 525, 570, 590, 630, 645, 660, 700, 850, 870, 890, 910, 940 and 970 nm. The organology of this sensor and the image acquisition is thoroughly described in previous publications . The res2.4.2-1. Spectra were obtained at the wavenumber range of 4000 to 400 cm-1 using Spectra Manager Code of Federal Regulations (CFR) software version 2 , by accumulating 100 scans with a resolution of 4 cm-1 and a total integration time of 2 min.FT-IR measurements were performed using a ZnSe 45 HATR  crystal , and a FT-IR-6200 JASCO spectrometer . The measurement crystal shows a refractive index of 2.4 and a depth of penetration of 2.0 μm at 1000 cm2.52.5.1Pseudomonas spp. counts the statistical software Unscrambler © ver.9.7  was used. Prior to analysis, MSI data were pre-treated by Standard Normal Variate (SNV) transformation for the exclusion of collinear and “noisy” data  (independent variables = 829) to minimize baseline shifts and noise , in which nine algorithms were considered for the prediction of TVC counts, namely Partial-least squares (pls)  ; Supportes (pls) ; Supportes (pls) ; K-nearees (pls) ; Principes (pls) ; Least-aes (pls) ; Ridge res (pls) ; Artifices (pls) . Spectraes (pls) . FT-IR sAnother point of attention in the sorfML software analysis was the splitting procedure of the data sets, which consisted of two phases . In the 2.62)  values  , mean ab2)  and accu) values . Supplem33.1Pseudomonas spp. on the surface of chicken breast fillets for each storage condition is presented in 2 in B1 and B2, respectively. Likewise, Pseudomonas spp. was enumerated at the beginning of storage at 2.0 and 2.1 log CFU/cm2 for B1 and B2, respectively. Storage temperature seemed to significantly influence the growth of chicken’s microbiota as inferred by the respective kinetic parameters for TVC and Pseudomonas spp. as derived by the primary growth model of max of Pseudomonas spp. of chicken samples stored at 0 °C were 72.2 h and 0.036 h-1, respectively. On the contrary, samples stored at 15 °C exhibited μmax and lag phase duration of Pseudomonas spp. at 0.241 h-1 and 8.8 h, respectively.The microbial population of TVC and Pseudomonas spp. counts in B1 and B2 presented variations during storage at 0 and 5 °C but always within the range of ± 1 log unit. At the end of storage, TVC and Pseudomonas spp. counts on samples from B1 were 6.2 and 5.7 log CFU/cm2, respectively. Similarly, for B2 samples the level of final TVC and Pseudomonas spp. counts was 6.3 and 5.5 log CFU/cm2, respectively. For B1 at 5 °C the number of TVC and Pseudomonas spp. after a period of 360 h was 6.8 and 6.6 log CFU/cm2, respectively, while for B2 at the same storage conditions, TVC and Pseudomonas spp. counts were 7.6 and 6.2 log CFU/cm2, respectively. This difference in microbial counts was expected as samples of B1 and B2 were collected with an interval of 4 months (winter-spring) to take into account seasonal variation. It is also worth noting that in all storage conditions, the final number of TVC ranged between 6.2-7.6 log CFU/cm2, unlike other studies reporting spoilage level of poultry meat at 7.0–8.0 log CFU/cm2 .TVC and  CFU/cm2 . The low3.22) and spoiled (456 h corresponding to 5.9 log CFU/cm2) chicken breast fillet samples are illustrated in -1 for the prediction of the microbial counts on the surface of samples is highlighted in Typical MSI and FT-IR spectra of fresh  as well as linear parameters  are provided in Pseudomonas spp. on the surface of chicken breast fillets via MSI analysis. More specifically, RMSE and r values ranged between 0.752-1.359 log CFU/cm2 and 0.604–0.876, respectively for the estimation of TVC counts when B1 was used as training set and B2 as testing set. Similar performance was observed for PLS-R model assessing Pseudomonas spp. counts. In this case, the values of r increased from 0.665-0.905, while RMSE exhibited values in the range of 0.724–1.574 log CFU/cm2. Additionally, a graphical approach of these linear models is represented in Pseudomonas spp. counts are illustrated. Beta coefficients of the models are provided in order to comprehend the contribution of specific wavelengths to model development. As demonstrated in Performance metrics  are shown in 2, MAE from 0.993 to 1.710, R2 from -23.368 to 0.246 and accuracy from 27 to 56 %. More specifically, the models developed on B1, predicted TVC population from B2 with around 0.3 higher performance on RMSE values. In contrast to intra-batch case, the highest performance was accomplished by lars with RMSE of 1.252 log CFU/cm2. Model’s optimization with B1 exhibited low values of RMSE (1.251 versus 1.544 log CFU/cm2 for lars model). However, in the case of B2 as a calibration data set, R2 values presented improved values, especially when lars, pls and ridge algorithms were applied.Following the same approach, results for batch-on-batch are provided in 3.4Pseudomonas spp. counts with FT-IR measurements are shown in 2 and 0.679–0.882, respectively. PLS-R model for Pseudomonas spp. via FT-IR exhibited r values of 0.739–0.916 and RMSE values were from 0.683 to 1.077 log CFU/cm2. The influence of each spectral variable is illustrated in -1 contained interesting information and therefore had great impact on model development. Absorption bands of 1230–1403 cm-1 were considered as important for the prediction of TVC and Pseudomonas spp. Beta coefficients of 1432–1498 cm-1 as well as 1549- 1584 cm-1 and 1658- 1704 cm-1 had impact on model construction.The findings of models predicting TVC and 2, MAE from 0.669 to 1.164, R2 from -3.129 to 0.546, and accuracy from 50.0 to 75.9 %. As 2 for RMSE, while lars and svmLinear algorithms performed better with RMSE being at 0.904 and 0.954 log CFU/cm2, respectively for B2.The results for intra batch training for FT-IR data are summarized as a heatmap in 2 while training model on B1 and validating on B2 outperformed the second model around significantly with 55% lower RMSE. More specifically, nnet accomplished the lowest RMSE (0.851 log CFU/cm2) and MAE (0.67 log CFU/cm2) over the other algorithms as well as models trained on B2 and validated on B1.Likewise, batch-on-batch prediction metrics are represented in 4Pseudomonas spp. was 3.1 (±0.29) and 2.1 (±0.15) log CFU/cm2, respectively, which is considered low compared to published data where the respective counts for TVC and Pseudomonas spp. were above 5.0 and 3.5 log CFU/cm2, respectively (ca. 7.0–8.0 log CFU/cm2)  . Unlike CFU/cm2) , Pseudom CFU/cm2 , due to ackaging . The difackaging , alteratackaging  as well ackaging . It is w2. For the prediction of TVC and Pseudomonas spp. counts with PLS-R models, RMSE was 1.359 and 1.574 log CFU/cm2, respectively. It needs to be noted that all developed models presented the tendency of overestimating the predicted counts. The increased RMSE values could be further improved (reduced) by applying alternative algorithms and sample splitting. Indeed, the assessment of TVC counts by sorfML platform showed satisfactory results, especially in the case of intra-batch validation and nnet algorithm. In this model, RMSE presented the lowest value (0.717 log CFU/cm2) while for ridge model RMSE was 0.769 log CFU/cm2. On the contrary, for batch-on-batch validation, three algorithms were considered acceptable for the evaluation of TVC counts, with lars model having RMSE of 1.252 log CFU/cm2 followed by pls and ringle models with1.319 and 1.262 log CFU/cm2, respectively.For MSI spectral data, model performance metrics predicted RMSE from 0.739 to 1.536 log CFU/cmPseudomonas spp. counts were predicted with RMSE being 1.029 and 1.078 log CFU/cm2, respectively. For intra batch testing, nnet algorithm for B1 and lars for B2 were considered effective for the evaluation of TVC counts, with lars having lower RMSE (0.905 log CFU/cm2) than nnet (1.047 log CFU/cm2). In contrast, in batch-on-batch validation, RMSE value for nnet (B1 on B2: 0.912 log CFU/cm2) were higher than lars where RMSE had the lowest value (0.851 log CFU/cm2).FT-IR models showed satisfactory prediction of counts, with performance metrics achieving better values than MSI. For PLS-R models, TVC and 2). Additionally, several models of FT-IR were able to attain respectable prediction on different data sets.The differentiation of model performance for the 2-sensor analysis highlights the important role of splitting process, data set selection and algorithm during model’s optimization. One significant factor for accurate prediction is inter-batch variability. Moreover, MSI results on intra-batch performance and its low RMSE suggested that this analysis could be applicable for internal validation or quality control in the production line. The latter option has been confirmed via experiments performed in the production line of chicken products at industrial level . Furtherk-fold cross-validation which removes only one sample at a time from the training set and considers it as a test set. Subsequently, for this case k is equal to the number of objects. This method may be useful for small database size presenting the problem of the inability to divide the data set into fairly sized subsets for training and test sets. However, this cross-validation approach can lead to overfit when the sample size is not large enough, and thus, results in high prediction error  with Monte Carlo cross validation by repeating the process outlined above for a number of times with different training and test splits and by averaging the performance of all iterations  implemented for PLS-R models is a variant of on error . In conterations . Regardierations .The influence of certain wavelengths to MSI model development was documented via b coefficient values for PLS-R models . Reflect-1 were related to polyglycines, polysaccharides (C–O stretch) and amines (NH2 rock/twist), respectively , amines from free amino acids and amide III .This research has been co-financed by the Evgenia D. Spyrelli: Methodology, Formal analysis, Data curation, Validation, Writing – original draft. Onur Ozcan: Methodology, Software, Formal analysis, Data curation, Visualization. Fady Mohareb: Software, Formal analysis, Data curation, Supervision, Visualization. Efstathios Z. Panagou: Investigation, Supervision, Validation, Writing – review & editing. George- John E. Nychas: Conceptualization, Funding acquisition, Resources, Project administration, Reviewing and Editing.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.