These observations could be related to oxidative stress, which is the major mechanism of CIT toxicity. Residues of CIT were detected in all collected tissues, except for muscle and egg white from layers in the lowest dose group, and egg white from layers in the highest dose group. CIT concentrations in plasma ranged between 0.1 (laying hens in lower dose group) and 20.8 ng/mL (pigs). In tissues, CIT concentrations ranged from 0.6 (muscle) to 20.3 µg/kg (liver) in pigs, while concentrations in chickens ranged from 0.1 (muscle) to 70.2 µg/kg (liver). Carry-over ratios from feed to edible tissues were between 0.1 and 2% in pigs, and between 0.1 and 6.9% in chickens, suggesting a low contribution of pig and poultry tissue-derived products towards the total dietary CIT intake for humans.Citrinin (CIT) is a polyketide mycotoxin occurring in a variety of food and feedstuff, among which cereal grains are the most important contaminated source. Pigs and poultry are important livestock animals frequently exposed to mycotoxins, including CIT. Concerns are rising related to the toxic, and especially the potential nephrotoxic, properties of CIT. The purpose of this study was to clarify the histopathological effects on kidneys, liver, jejunum and duodenum of pigs, broiler chickens and laying hens receiving CIT contaminated feed. During 3 weeks, pigs (n = 16) were exposed to feed containing 1 mg CIT/kg feed or to control feed (n = 4), while 2 groups of broiler chickens and laying hens (n = 8 per group) received 0.1 mg CIT/kg feed (lower dose group) and 3 or 3.5 mg CIT/kg feed (higher dose group), respectively, or control feed (n = 4). CIT concentrations were quantified in plasma, kidneys, liver, muscle and eggs using a validated ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method. Kidneys, liver, duodenum and jejunum were evaluated histologically using light microscopy, while the kidneys were further examined using transmission electron microscopy (TEM). Histopathology did not reveal major abnormalities at the given contamination levels. However, a significant increase of swollen and degenerated mitochondria in renal cortical cells from all test groups were observed ( Aspergillus, Monascus and Penicillium species [50) ranges from 19 mg/kg to 134 mg/kg body weight (bw), assessed after intravenous and oral administration in rabbits, respectively [Mycotoxins are structurally diverse toxic fungal secondary metabolites. Citrinin (CIT) is a polyketide mycotoxin, mainly produced by  species . CIT has species ,3,4,5,6. species . In Russ species . Several species . In gene species ,14,15,16 species ,17,18,19ectively ,21.Monascus fermented products  [50 values were reported in the literature, except for turkey poults and ducklings (56 and 57 mg/kg bw per day respectively) [CIT toxicity is of particular interest in pigs and poultry, as they are economically important livestock. Moreover, their diet consists mainly of cereals and other grains, which are major sources of CIT. For pigs, few studies on CIT toxicity exist. Sandor et al.  did not per day) . Hence, per day) . For pouctively) . Feed coctively) . Howeverctively) . In thismax of males and females = 3 h) compared to broilers . Furthermore, elimination half-lives differed significantly between species . Differences in toxicokinetics could be reflected in species-dependent sensitivity to toxicity. A lower oral bioavailability could imply a higher concentration in the intestines, resulting in impaired intestinal health. Furthermore, long persistence in the body and the relatively high distribution volume observed in broilers (3 L/kg) could possibly lead to carry-over in edible tissues such as kidneys, liver and eggs. In laying hens, carry-over to eggs and muscles was indeed demonstrated by Abdelhamid and Dorra [Meerpoel et al.  demonstrnd Dorra , reportiIt is clear that carry-over of CIT is possible, and moderate histopathological effects could be observed at low concentrations, but the number of studies regarding carry-over of CIT is limited. As CIT shows different toxicokinetic characteristics in pigs and chickens, it is important also to perform toxicity studies, since different outcomes could be expected. Since CIT has a longer residence time in pigs compared to broiler chickens, the longer exposure time could lead to higher toxic effects in pigs. On the other hand, the distribution volume of CIT was tenfold higher in broiler chickens, indicating that higher concentrations of CIT could be obtained in organs/tissues. Although acute and short-term toxicity studies of several species have been performed in the past, the European Food Safety Authority (EFSA) stated in its scientific opinion on CIT that there is still missing data, especially concerning the transfer of CIT into organs . Moreove13C13-CIT after injection of neat solvent. The results for apparent recovery and precision fell within the acceptance criteria, with only few small exceptions , while there was no significant difference between the controls and pigs after cessation of CIT exposure (p = 0.33). Likewise, a significant difference was observed in broiler chickens and laying hens from the highest dose groups, compared to controls (p = 0.001 for both). However, while there was also a significant difference between broilers from the low dose group and controls (p = 0.003), no statistically significant difference was demonstrated between laying hens from the low dose group and controls (p = 0.8). After 3 days of cessation of CIT administration, the broiler chickens and laying hens from the highest dose group still had a significantly higher number of degenerated mitochondria compared to the control (p = 0.001 for both) (The number of degenerated mitochondria differed significantly between the control pigs and pigs receiving CIT contaminated feed (or both) .Carry-over of CIT to edible tissues was demonstrated in livestock, indicating that humans can possibly be exposed to CIT after meat consumption, although to a limited extent. In a recent study, chicken breast meat sold on the Belgian market was analysed for CIT, and a maximum concentration of 0.2 µg/kg was found (unpublished data). Furthermore, no CIT was detected in pork derived products. Therefore, carry-over CIT into fresh edible chicken tissue was a plausible reason for CIT contamination, but the risk is rather low. The carry-over ratio in pig tissues ranged from 0 (no CIT detected) to 2% (highest observed concentration in liver), and from 0 to 6.9% (broiler liver) in chickens. Relatively high CIT concentrations were found in liver tissue of chickens. Remarkably, in both broilers and layers, CIT carry-over ratios were higher in the low dose group (0.1 mg/kg feed), namely 0.1–6.9% and 0.2–4.3%, compared to the higher dose groups (3 and 3.5 mg/kg), namely 0.1–2.3% and 0.1–0.8%, respectively. This was especially the case for liver tissue and could possibly indicate a saturation in distribution. Similar observations were made in previous mycotoxin residue studies, in which higher carry-over factors were calculated for low contaminated feed, compared to feed with a higher mycotoxin content. Fraeyman et al.  reported1/2el for males and females of 18 versus 5 h, respectively). After 3 weeks, CIT administration was ceased, and CIT plasma concentrations rapidly declined from day 22 onwards in both pigs and broilers. However, concentrations in layer hens in the high dose group did not decline but remained rather constant. Using the depletion data, a T1/2el of approximately 6 h was roughly calculated for pigs, while the T1/2el for broilers and laying hens was estimated to be 24 h and 36 h, respectively. Interestingly, earlier observations from a toxicokinetic study in broilers, where CIT was administered in a single oral dose of 0.25 mg/kg bw, revealed a plasma elimination half-life of 5 h and no CIT was detected after 72 h post administration [1/2el for pigs is not in accordance with the previously performed toxicokinetic study with an administered P.O. dose of 0.05 mg/kg bw (T1/2el = 18 h). HO-CIT concentrations were also measured in plasma and edible tissue. The metabolite was found in higher concentrations than its parent compound CIT in all species studied. This indicates that CIT was extensively metabolized into HO-CIT. The same trends concerning CIT were observed for HO-CIT comparing broilers and layer hens. A human toxicokinetic study also reported higher HO-CIT concentrations compared to CIT in urine, while no HO-CIT could be detected in plasma [Based on literature data, high concentrations were expected in chicken muscle tissue, since Abdelhamid and Dorra  detectedstration . The rean plasma . In accop = 0.33). This indicates a possible recovery of renal cells when CIT exposure was ceased. However, after 3 days of cessation of CIT administration, the broiler chickens and laying hens from the highest dose group still had a significantly higher number of degenerated mitochondria compared to the control. Therefore, it seems that recovery from mitochondrial damage is a slower process in chickens compared to pigs. Interestingly, depletion of CIT was slower in broiler chickens and laying hens compared to pigs, which could explain the presence of degenerated mitochondria even after cessation of CIT administrationNo ultrastructural damage was observed in kidneys, liver and intestines. However, changes in renal mitochondria were visually observed. Although these observations are difficult to interpret without further confirmation, it is of interest to point out that mitochondrial swelling and misshapen appearance are consistent with the concept that the lesions of this organelle are crucial in the mechanism of CIT toxicity. Studies have reported multiple effects of CIT on mitochondrial function, impairing mitochondrial respiration ,41. At hNo severe toxic effects were expected, since a previous study of broiler chickens fed a diet containing 65 mg/kg feed did not reveal any clinical signs of toxicity. In pigs, no clinical sigs of toxicity were observed at 20 µg CIT/kg bw per day (22), but our study demonstrates slight changes in mitochondria after administration of 50 µg CIT/kg bw per day in pigs, and 10/250 µg/kg bw in broiler chickens and laying hens.This study demonstrates that the presence of CIT in animal feed can lead to its transfer into edible tissues, although with low carry-over rates. Although it is unlikely that high amounts of CIT will be found in animal feed, it is shown that this toxin tends to accumulate in tissues of animal origin, and a contribution to the total dietary CIT intake of humans is plausible. Depletion of CIT in plasma and tissues was rapid in pigs, but slow in chickens, especially in layers. Furthermore, it was demonstrated that exposure to 1 mg CIT/kg feed for pigs, 0.1 and 3 mg/kg for broiler chickens and 0.1-3.5 mg/kg for layer hens during 3 weeks does not cause toxic effects in kidneys, liver, jejunum and duodenum, after histopathological inspection using light microscopy. However, the present study demonstrated mitochondrial changes, using transmission electron microscopy, which might play a vital role in the mechanism of nephrotoxicity. Changes were already visible at relatively low doses of CIT.13C-labelled CIT (13C13-CIT) as internal standard (IS) in acetonitrile (ACN) was purchased from Biopure-Romer Labs , with a concentration of 10.6 µg/mL. All stock solutions were stored at −20 °C. From the individual stock solutions, a CIT/HO-CIT standard mixture of 10 µg/mL each and a 13C13-CIT solution of 100 ng/mL were prepared in MeOH. A Milli-Q SP Reagent water system  was used to obtain ultrapure water. MeOH , ethyl acetate , hydrochloric acid  were purchased from BioSolve BV , ACN  was acquired from VWR International , whereas sodium chloride , ammonium acetate (98%) and glacial acetic acid  were purchased from Merck. Magnesium sulfate  was procured from Sigma-Aldrich .CIT was purchased from Fermentek  and HO-CIT  was obtag, 10 min, 4 °C). The ACN phase was evaporated under a gentle nitrogen stream at 40 °C and the dry residue was reconstituted in 250 µL of MeOH/water . The limit of detection (LOD) and limit of quantification (LOQ) were 0.05 and 0.1 ng/mL for CIT and 0.01 and 0.1 ng/mL for HO-CIT, respectively.A previously developed and validated method from Meerpoel et al.  was used13C13-CIT was added at 5 µg/kg. After an equilibration period of 15 min, 3 mL of a HCl 1M solution was added and samples were left at room temperature for another 15 min. Next, 5 mL of EtAc was added, followed by shaking for 30 min using an overhead shaker . Subsequently, 1.5 ± 0.05 g of MgSO4 and 0.4 ± 0.04 g of NaCl were added. Samples were first agitated by hand for 30 s to avoid aggregation of the salts, followed by shaking using the overhead shaker for 3 min. Next, samples were centrifuged for 5 min at 3676× g. An aliquot of 1 mL supernatant was transferred and evaporated to dryness under a gentle stream of nitrogen at 40 °C. Extracts were reconstituted in 0.2 mL of injection solvent , vigorously vortexed for 1 min and subjected to ultracentrifugation for 10 min at 9167× g. Finally, extracts were transferred into autosampler vials for analysis. Samples of breast, back muscle, kidney, liver and skin were minced, homogenized using a meat grinder from Moulinex and stored at −20 °C until extraction. In a 50 mL extraction tube, 1.00 ± 0.05 g of homogenized sample was weighed. The samples were spiked with CIT/HO-CIT to construct a matrix-matched calibration curve at the following concentrations: 0.1–0.5–1–5–10–50–100–200 µg/kg. To each sample, 13C13-CIT was added at a concentration of 5 µg/kg After leaving the samples 30 min for equilibration, 10 mL of an acidified saline solution was added, consisting of 10% (m/v) NaCl in acidified water ), followed by 20 mL of extraction mixture consisting of EtAc, ACN and HAc . Samples were extracted for 1 h at room temperature using an overhead shaker . Subsequently, 6.0 ± 0.2 g of MgSO4 and 1.5 ± 0.1 g of NaCl were added. Samples were first agitated by hand for 30 s to avoid aggregation of the salts, followed by shaking using the overhead shaker for 3 min. Next, samples were centrifuged for 5 min at 3676× g. An aliquot of 1 mL supernatant was transferred and evaporated to dryness under a gentle stream of nitrogen at 40 °C. Extracts were reconstituted in 0.2 mL of injection solvent , vigorously vortexed for 1 min and subjected to ultracentrifugation  for 10 min at 9167× g. Finally, extracts were transferred into autosampler vials for analysis.A 4.00 ± 0.02 g homogenized sample was weighed in a 50 mL extraction tube. The samples were spiked to construct a matrix-matched calibration curve at the following CIT/HO-CIT concentrations: 0.1–0.5–1–5–10–50–100–200 µg/kg. To each sample, The obtained extracts were analysed using a previously developed UHPLC-method .The developed method was in-house validated, using spiked blank liver, kidney, skin with adhering fat, muscle, egg yolk and egg white samples obtained from healthy, untreated broiler chickens or pigs. A 3-day validation was performed on pig muscle tissue, while method performance was evaluated on skin with adhering fat, liver, kidney and eggs by a 1-day validation. Linearity, carry-over, apparent recovery, within-run and between-run precision, LOQ and LOD were determined in accordance with the recommendations and guidelines defined by the European Commission .Linearity of a matrix-matched calibration curve was evaluated using the coefficient of determination (R²) and confirmed by calculation of goodness-of-fit (g) (%), which takes into account the difference between nominal value of the calibration curve and the calculated concentration (Equation (1)).r) and on 3 different days . RSD values should be as low as possible, <30% considered as acceptable. The LOQ was established as the lowest point of the calibration curve that could be analysed with good accuracy and precision within the recommended ranges, as confirmed by analyzing six samples spiked at the lowest concentration level. The LOD was defined as the lowest concentration that could be detected with a signal-to-noise (S/N) ratio of ≥3. Absence of carry-over was evaluated by injecting neat solvent directly after the highest calibrator. If a peak was present within the same retention time range of CIT and HO-CIT (± 2.5%), it had to be below the LOD. Apparent recovery (%) was determined in triplicate as the ratio of the measured concentration divided by the actual (spiked) concentration, evaluated at 3 levels . The acceptance criteria for apparent recovery were: 50% to 120%, 70% to 110% and 80% to 110% for concentration levels ≤1 µg/kg, between 1 and 10 µg/kg and > 10 µg/kg, respectively. Within-run and between-run precision were determined by calculating the residual standard deviation (RSD) in % from 6 spiked samples at 3 concentration levels  on 1 day , Aspergillus niger (n = 4), A. alutaceus (n = 4) and Penicillium citrinum (n = 2) from the “Mycothèque de l’Université Catholique de Louvain, MUCL” was constituted. The potential ability of CIT production by these strains was screened at small scale in wheat grains or rice grains (3 replicates of 20 g of grains per fungus) after adjusting the humidity by adding Milli-Q water to obtain a humidity of 24–25%. These were inoculated and cultivated for 3 weeks on sterilized aliquots (wheat or rice grains). Incubation was stopped by autoclaving at 121 °C for 20 min and samples were thereafter dried at 50–60 °C for 48 h, ground and sieved (< 250 µm), homogenized and stored at −20 °C until analysis by LC-MS/MS [Penicillium citrinum MUCL 29781 was selected for CIT production in contaminated feed. Biomass of contaminated wheat grains (kg amounts) was then produced via cultivation of the selected fungus in the same conditions as described above. Control material was identically prepared by replacing the inoculum substrate with sterilized water.Incurred CIT contaminated rice and wheat batches were in-house produced through the following steps. First, a biobank of available strains of CIT reference material (wheat flour) was prepared, checked, and found to be sufficiently homogenized with the final concentration of 404.0 ± 19.7 µg/g. Note that this material was checked to be free of mycotoxins such as aflatoxins B1, B2, G1, G2, T2-toxin, HT2-toxin, fumonisin B1, B2, B3, deoxynivalenol, zearalenone and ochratoxin A. CIT content in control material was 0.0003 µg/g and free of the other tested mycotoxins.Before the reference material was mixed with commercial pig feed, the feed was first analysed for CIT with a validated UHPLC-MS/MS method  to make Two wheat reference materials were prepared and checked to contain 2.0 ± 0.4 µg/g (batch 1) and 53.2 ± 3.3 µg/g (batch 2).To reach a concentration of 0.1 mg/kg and 3 mg/kg feed, 2 kg of batch 1 and 2.25 kg of batch 2 was mixed respectively with 40 kg of broiler feed . The commercial feed was first checked for CIT content, revealing a low, unquantifiable concentration  and 71.6 ± 2.0 µg/g (batch 2). To reach a concentration of 0.1 mg/kg and 3.5 mg/kg feed, 1 kg of batch 1 and 1 kg of batch 2 was mixed respectively with 20 kg of broiler feed . The commercial feed was free of CIT. Feed concentrations correspond to a daily CIT intake of 0.01 and 0.25 mg per kg bw, assuming a daily feed intake of 177 g and an average bw of 1.66 kg. These intakes are the same as the intakes used for broiler chickens.The same commercial feed used to prepare the contaminated diets was used as control feed. The pig and layer feed did not contain CIT, while traces of naturally occurring CIT (<LOQ) were detected in the broiler feed. The animal experiments were approved by the Ethical Committee of the Faculty of Veterinary Medicine and the Faculty of Bioscience Engineering of Ghent University (EC 2017/105). All animal experiments were performed according to Directive 2010/63/EU  and the 2. Four pigs were kept in a separate pen and served as control. For 3 weeks, the pigs received CIT contaminated feed (1 mg/kg) in an amount according to their body weight [® containing ethylenediaminetetraacetic acid (EDTA). The collected blood samples were centrifuged (3676× g for 10 min) within 2 h after collection and plasma was stored at −20 °C. After 3 weeks (day 21), 8 pigs  were euthanized via intravenous administration of an overdose of pentobarbital , while the remaining pigs received again control feed ad libitum. From the euthanized animals, blood, both kidneys, liver, muscle tissue , duodenum, jejunum and skin with adhering fat were collected and immediately prepared for CIT residue analysis and histopathological investigation. For the residue analysis, the collected organs were stored at −20 °C within 1 h. Procedures for histopathology are described in Twenty conventional pigs  were housed according to Directive 2010/63/EU  and the y weight , once da® containing EDTA. The collected blood samples were centrifuged (3676× g for 10 min) and the plasma was stored at −20 °C. After 3 weeks, 2 experimental animals and 1 control animal per group were euthanized (day 21), while the remaining animals again received control feed. From the euthanized animals, blood, kidneys, liver, breast muscle tissue (approximately 100 g), duodenum, jejunum and skin with adhering fat were collected and immediately prepared for CIT residue analyses and histopathological investigation. For the residue analysis, the collected organs were stored at −20 °C within 1 h. Procedures for histopathology are described in Twenty broilers  and 20 layers  were housed according to Directive 2010/63/EU  and the The collected tissues during the animal trials  were extracted and analyzed using the validated UHPLC-MS/MS method described above. Carry-over rate (%) was defined as the ratio of the mean CIT concentration in the tissues determined at day 21 and the concentration in feed, multiplied by 100.Samples of kidneys, liver, duodenum and jejunum were prepared. From the cortex region of each kidney, 4 samples of 0.5 cm³ were taken . The livers were divided into 4 regions of equal size, and from each region, 2 samples of 0.5 cm³ were taken. From the intestines, aliquots of 2 cm from the jejunum (n = 2) and duodenum (n = 2) were rinsed with 0.9% normal saline. All samples were immediately fixed in phosphate buffered saline containing 4% formaldehyde and left for 24 h at room temperature. The samples were dehydrated in graded concentrations of ethanol. The fixed tissues were embedded in paraffin, after which tissue sections of 5 µm were stained with hematoxylin and eosin (H&E) using standard procedures . Microsc4 for 20 min, a 1:1 mixture of absolute alcohol and acetone for 10 min, and acetone for 30 min). Next, samples were impregnated in graded mixtures of acetone and epoxy resin  for 2 h each, and in pure epoxy resin overnight. Afterwards, the embedded samples were left at 60 °C for 3 days to allow polymerization of the resin. Ultrathin sections (± 90 nm) were cut with a Leica Ultramicrotome (EM UC6) using a diamond knife . Each sample was deposited on formvar coated grids. Samples were contrasted with 1% uranyl acetate and 1.33% lead citrate. Images were made by a Quemesa charge-coupled device camera . Toxicity of CIT was assessed by a double blinded histological examination of the kidneys by an independent expert in histopathology. Size and shape of mitochondria were inspected. Furthermore, signs of apoptosis  were evaluated and the number of degenerated mitochondria were counted in 30 randomly selected cells per animal.For histopathological examination of kidney tissue, 3 aliquots (1 mm³) of the cortex region from both kidneys were transferred to individual test tubes containing Karnovsky fixative  and stored at 4°C overnight. In the following sample preparation step, the samples were washed with 0.1 M cacodylate buffer and post-fixed with buffered 1% osmium tetroxide for 1.5 h at room temperature. Samples were rinsed afterwards with double-distilled water followed by dehydration in a graded series of ethanol .For the histopathological samples, statistical differences between groups were determined with a two tailed, unpaired Student’s


NOD-like receptors (NLR) are intracellular sensors of the innate immune system, with the NLRP3 being a pro-inflammatory member that modulates cardiac ischemia-reperfusion injury (IRI) and metabolism. No information is available on a possible role of anti-inflammatory NLRs on IRI and metabolism in the intact heart. Here we hypothesize that the constitutively expressed, anti-inflammatory mitochondrial NLRX1, affects IRI and metabolism of the isolated mouse heart.13C-glucose or 0.4 mM 13C-palmitate under normoxic conditions, followed by LC-MS analysis and integrated, stepwise, mass-isotopomeric flux analysis (MIMOSA).Isolated C57Bl/6J and NLRX1 knock-out (KO) mouse hearts were perfused with a physiological mixture of the essential substrates  and insulin. For the IRI studies, hearts were subjected to either mild (20 min) or severe (35 min) ischemia and IRI was determined at 60 min reperfusion. Inflammatory mediators  and survival pathways  were analyzed at 5 min of reperfusion. For the metabolism studies, hearts were perfused for 35 min with either 5.5 mM NLRX1 KO significantly increased IRI , following severe, but not mild, ischemia. The increased IRI in NLRX1 KO hearts was associated with depressed Akt signaling at early reperfusion; other survival pathways or inflammatory parameters were not affected. Metabolically, NLRX1 KO hearts displayed increased lactate production and glucose oxidation relative to fatty acid oxidation, associated with increased pyruvate dehydrogenase flux and 10% higher cardiac oxygen consumption.Deletion of the mitochondrially-located NOD-like sensor NLRX1 exacerbates severe cardiac IR injury, possibly through impaired Akt signaling, and increases cardiac glucose metabolism. The innate immune system is an important player in cardiac ischemia-reperfusion injury (IRI), serving as the first defense system against infection or injury through engagement of pattern recognition receptors (PRRs) such as the cell membrane-associated toll-like receptors (TLRs) and intracellular NOD-like receptors (NLRs) , 2. In gNLRX1 has been found to attenuate kidney IRI . So far,NLRX1 has also been found to affect metabolism. Recent studies have reported that NLRX1 may limit fatty acid oxidation (FAO) and promote glycolysis in hepatocytes from a non-alcoholic fatty liver disease (NAFLD) model , or reduTherefore, in this study we employed the isolated Langendorff-perfused mouse heart model to address the hypothesis that NLRX1 affects cardiac IRI and cardiac metabolism.-/- (NLRX1 KO) mice were generated as reported previously  118 NaCl, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, 0.5 EDTA, 2.50 CaCl2, with mixed substrates 5.5 D-glucose, 0.5 L-glutamine, 1 lactate, 0.1 pyruvate, 1%(w/v) albumin – 0.4 mM palmitic acid sodium salt, 0.05 L-carnitine and 30 mU/L insulin. Substrate concentrations were chosen to reflect in vivo concentrations of the non-fasted condition  experimentation.Within each protocol . After reperfusion, hearts were blotted dry, weighted, and stored at -20°C for determination of infarct size by TTC staining within 1 week.severe IRI: similar as described for mild IRI, except that ischemic duration was extended to 35 min .molecular characterization: following 35 min no-flow ischemia and 5 min reperfusion, hearts were immediately homogenized and partly centrifuged to obtain mitochondrial, cytosolic and whole-cell fractions for determination of survival kinases  and inflammatory cytokines.carbohydrate metabolism: 5.5 mM non-labeled glucose in the perfusate was replaced by 5.5 mM [U-13C6] glucose : 99%; Cambridge Isotope Laboratories, Andover, USA). After 25 min perfusion, the pulmonary artery was sampled to obtain venous oxygen tension levels [analyzed by blood gas analyzer (Siemens Rapidpoint500)]. At 35 min perfusion, hearts were immediately frozen in liquid nitrogen and stored at -80°C until further examination. Influent samples were then collected to determine arterial oxygen tension by blood gas analyzer .long-chain fatty acid metabolism: similar treatment as described for glucose metabolic characterization, except that 0.4 mM non-labeled palmitate was replaced by 0.4 mM [U-13C16] palmitate : 98%; Cambridge Isotope Laboratories, Andover, USA) .The frozen hearts in IRI protocols were cut into 1mm thick slices and immersed in 1% triphenyltetrazolium chloride (TTC) solution (pH=7.4) for 20 min at 37°C on a shaker at 300 rpm. Subsequently, the slices were incubated in 4% formalin (pH = 7.4) for 2 h at room temperature and scanned afterwards. Infarct size was determined using Image J software, by an investigator not aware of treatment allocation.For the molecular characterization protocol, hearts were quickly minced in cold homogenization buffer (1.5 ml per heart) containing 0.25 M sucrose, 0.02 M HEPES, 1 mM dithiothreitol (DTT) and phosphatases and protease inhibitors on ice, and homogenized in ice-cold glass-Teflon potter at 1200 rpm/min. Then 750 μl homogenate was centrifuged at 10,000g for 10 min at 4°C to gather mitochondria in the pellet. The supernatant was the cytosolic fraction. The pellets were lysed in homogenization buffer supplemented with 0.5% triton X-100 and 1mM glucose-6-phosphate (to detach HKII from mitochondria ) at room-/-mice) were frozen in liquid nitrogen and stored at -80° until analysis. The whole heart was homogenized, lysed and sonicated in a similar fashion as described above for the “remaining 750 μl homogenate”. The whole-heart homogenate of these hearts was used for detection of NLRX1 by Western blot (WB) and determination of whole-heart metabolic enzyme activities.Hearts immediately excised from mice  and the fatty acid oxidation enzyme short-chain-3-hydroxyacyl-CoA dehydrogenase (SCHAD) were determined spectrophotometrically at 25°C as previously reported . In shor-20 (Sigma) and incubated with the complementary secondary fluorescence antibody  for 1 h at room temperature before they were washed again and scanned with an Odyssey scanner(Li-cor). Within one blot, the intensity of all bands was normalized to band with highest intensity.NLRX1, HKII, Akt, AMPK, STAT3, and PDH proteins were determined by Western blotting. HKII was determined in cytoplasmic and mitochondrial fractions; all other proteins in whole-cell fractions. Lowry assay was used to determine protein concentration. Western blotting was conducted as described previously . BrieflyIL-6 and TNFα were determined in the supernatant of the 10,000 g 10 min centrifuged whole heart homogenized tissue, using ELISA kits  following manufacturer’s instructions. Final quantification was normalized by whole protein concentration in the samples, as determined by Lowry assay.15N5-monophosphate (100 µM) was added to each sample. Subsequently, 425 µl water, 500 µl methanol and 1 ml chloroform were added to the same 2 ml tube before thorough mixing and centrifugation for 10 min at 14.000 rpm. The top layer, containing the polar phase, was transferred to a new 1.5 ml tube and dried using a vacuum concentrator at 60°C. Dried samples were reconstituted in 100 µl methanol/water . Metabolites were analyzed using a Waters Acquity ultra-high-performance liquid chromatography system coupled to a Bruker Impact II™ Ultra-High Resolution Qq-Time-Of-Flight mass spectrometer. Samples were kept at 12°C during analysis and 5 µl of each sample was injected. Chromatographic separation was achieved using a Merck Millipore SeQuant ZIC-cHILIC column . Column temperature was held at 30°C. Mobile phase consisted of (A) 1:9 acetonitrile:water and (B) 9:1 acetonitrile:water, both containing 5 mM ammonium acetate. Using a flow rate of 0.25 ml/min, the LC gradient consisted of: 100% B for 0-2 min, ramp to 0% B at 28 min, 0% B for 28–30 min, ramp to 100% B at 31 min, 100% B for 31–35 min. Mass spectrometry (MS) data were acquired using negative and positive ionization in full scan mode over the range of m/z 50–1200. Data were analyzed using Bruker TASQ software version 2.1.22.3. Isotope ratios and correction for contribution of naturally occurring isotopes were calculated using IsoCorrectoR  from simplifications in isotope patterns from the primarily oxidative metabolism of the perfused hearts , as a stepwise flux analysis program to estimate relative glycolytic and mitochondrial rates . To thisHaving ruled out any M+1 acetyl CoA, all heavy citrate isotopomers are a consequence of the ligation of either M+0 or M+2 acetyl carbons with the isotopomers of oxaloacetate. Since, acetyl CoA does not equilibrate across both the mitochondrial and cytosolic compartments, five independent mass isotopomer distribution analyses (MIDA) were performed to identify the enrichment of the mitochondrial acetyl CoA  indicated that seven experiments were adequate for each parameter to reach significance. Statistics were conducted using IBM SPSS statistics version 24 . Figures were made in GraphPad Prism 8.0 . A value of P < 0.05 indicates statistical difference.Data are expressed as mean ± SD, except data in -/-mice . Thus, the presence of NLRX1 protects against severe but not mild IRI.We evaluated whether NLRX1 influenced IR injury in isolated hearts Figure 3-/- 0.83 ± 0.12, P=0.01, -/- group compared with WT group  or mtHK activity (Suppl). In addition, no differences were observed in cytosolic HK II  or mtHKII amount  between NLRX1-/- group and WT group. Finally, analysis of cardiac inflammatory cytokines at early reperfusion demonstrated similar levels of IL-6 and TNFα between the two genotypes .We investigated whether NLRX1 effects on severe IR injury could be associated with changes in cardiac survival signaling pathways at early reperfusion Figure 4-/- and control (WT) through glycolysis up to PEP. Despite identical M+3 PEP enrichments, the pyruvate enrichment was lower in knockouts indicating a dilutive contribution from an unlabeled source . Even with lower pyruvate enrichment compared to control, acetyl CoA and all subsequent mitochondrial metabolites were more enriched relative to controls suggesting a greater contribution of pyruvate oxidation relative to beta-oxidation in knockouts.The sequential enrichments of metabolites in the pathway from glucose through glycolysis through the PDH reaction and into and around the TCA cycle are shown  enrichment of precursor and product are the same, then the precursor is the only pathway contributing to the synthesis of the product. Dilutions in the product enrichment relative to the precursor indicate contributions from other unlabeled pathways. For instance, in the heart pyruvate, beta-oxidation, acetate, and ketones all can contribute to the synthesis of mitochondrial acetyl CoA and so if pyruvate is the only source of label, the dilution of acetyl CoA relative to pyruvate indicates the contribution of carbohydrate oxidation to the TCA cycle. In the controls, the enrichment of mitochondrial acetyl CoA relative to pyruvate  was ~20% indicating that in these perfused hearts, carbohydrates only contributed one fifth of the oxidized carbon through the pyruvate dehydrogenase (PDH) complex. Remarkably, except for the enrichment in acetyl CoA coming from pyruvate, the precursor-product relationships  were identical throughout glycolysis and the TCA cycle  at a rate that is a function of glycolytic lactate exit. The premise we used for analysis of this latter part of glycolysis is that lactate M+3 enrichment is a function of the rate of PEP enrichment flowing through pyruvate kinase into lactate dehydrogenase, and that in steady state an increased lactate enrichment reflects increased LDH flux in the direction of lactate generation. Thus, a higher enrichment of heavy lactate  is qualitatively indicative of a greater rate of fermentative glycolysis . With similar PEP enrichments, lactate enrichments were higher in NRLX12 as compared to WT hearts  worsens severe, but not mild, acute IRI, 2) impairs Akt signaling at early reperfusion without changes in other survival protein or inflammatory mediators, 3) increases glucose oxidation relative to fatty acid oxidation with PDH as key regulatory step affected by NLRX1, 5) elevates lactate generation, and 4) increases oxygen consumption and cardiac mechanical function.in vivo renal ischemia-reperfusion in mice  , the actThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by Animal Ethics Committee of the Academic Medical Center, Amsterdam, Netherlands, and performed in accordance with the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes.HZ and CZ designed the study. HZ, YX, and DB performed the experiments. HZ, YX, RN, SG, SH, MW, RK, and CJZ provided materials, performed measurements, and analyzed the data. HZ and CZ wrote the manuscript. RN, PZ, MH, NW, SH, and MW revised critically the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases R01 DK 108283 to RK and the Chinese Scholarship Council (201706280114 and 201806270257).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.


A theoretical analysis revealed that lysosomal fusion is sufficient to explain the enlargement of lysosomal sequestration capacity. In conclusion, we demonstrated that extracellular TKIs, GF and IM, induced NAADP/Ca2+ mediated lysosomal fusion, leading to enlargement of the lysosomal compartment with significantly increased sequestration capacity for these drugs without apparent lysosomal biogenesis.Lysosomal sequestration of anticancer therapeutics lowers their cytotoxic potential, reduces drug availability at target sites, and contributes to cancer resistance. Only recently has it been shown that lysosomal sequestration of weak base drugs induces lysosomal biogenesis mediated by activation of transcription factor EB (TFEB) which, in turn, enhances their accumulation capacity, thereby increasing resistance to these drugs. Here, we addressed the question of whether lysosomal biogenesis is the only mechanism that increases lysosomal sequestration capacity. We found that lysosomal sequestration of some tyrosine kinase inhibitors (TKIs), gefitinib (GF) and imatinib (IM), induced expansion of the lysosomal compartment. However, an expression analysis of lysosomal genes, including lysosome-associated membrane proteins 1, 2 , vacuolar ATPase subunit B2 (ATP6V1B2), acid phosphatase (ACP), and galactosidase beta (GLB) controlled by TFEB, did not reveal increased expression. Instead, we found that both studied TKIs, GF and IM, induced lysosomal fusion which was dependent on nicotinic acid adenine dinucleotide phosphate (NAADP) mediated Ca Lysosomes were discovered in the 1950s by Christian de Duve . These m+ ATPase maintaining a low pH. Soluble acid hydrolases include proteases , nucleases, glycosidases, lipases, phospholipases, phosphorylases, and sulfatases methyl]-4-methoxyphenyl]-2,3,4,9-tetrahydro-1H-pyrido  indole-3-carboxylic acid, was purchased from Tocris Bioscience .Gefitinib  was purchased from Selleckchem . Imatinib mesylate  was kindly provided by Novartis . Bafilomycin A1 (BafA1), selective inhibitor of the vacuolar H2 atmosphere at 37 °C. Cells were obtained from the European collection of authenticated cell cultures .Human chronic myelogenous leukemia K562 cells and human promyelocytic leukemia HL-60 cells were cultured in the RPMI-1640 medium supplemented with a 10% calf foetal serum and antibiotics in 5% CO5/mL) were incubated in the growth medium with appropriate IM concentration in the absence or presence of BafA1 for 6 h in 5% CO2 atmosphere at 37 °C. Cell pellets were extracted using ice cold 4% (w/v) formic acid (FA) in water on their separation from the growth medium by centrifugation through a layer of silicone oil. Cell extracts were clarified by centrifugation , diluted with extraction solution and analysed by liquid chromatography coupled with a low-energy collision tandem mass spectrometer (LC/MS/MS). Details are given elsewhere [Cells  were incubated in the growth medium with appropriate GF concentration in the absence or presence of BafA1 for 6 h in 5% CO2 atmosphere at 37 °C. Cell pellets were extracted using ice cold 5% (w/v) FA in water upon their separation from the growth medium by centrifugation through a layer of silicone oil [g × 10 min at 4 °C), diluted with extraction solution and analysed or stored at −80 °C.Cells  and B  from 0–4 min (35%→95% of solvent A), from 4–6 min (95% of solvent A), from 6–7 min (95%→35% of solvent A) and from 7–10 min (35% of solvent A); the flow rate at 0.3 mL/min; the sample injection volume at 5 µL. The API 3200 triple quadrupole mass spectrometer  with the TurboIonSpray interface in the positive ion mode was applied for quantification of analytes. Ion spray probe parameters were set for standard: needle voltage 5500 V and temperature 400 °C. The mass spectrometer was operated in the multiple-reaction monitoring (MRM) mode. GF was monitored by MRM transition 447 > 128. The mass-dependent parameters were optimized: the collision energy and the declustering potential for GF standard were 46 V and 35 V. Data were acquired using Analyst® software .Quantitative analysis of GF was done using liquid chromatography coupled with a low-energy collision tandem mass spectrometer (LC/MS/MS) during one run. The HPLC system UltiMate 3000 , a HyperClone BDS C18, 5 µm, 150 × 2.0 mm HPLC column  and a guard C18, 4.0 × 2.0 mm precolumn  were used. The chromatographic parameters were optimized: the binary gradient of mobile phase A  XP antibody (1:1000), rabbit monoclonal anti-LAMP2 (D5C2P) antibody (1:1000), and rabbit monoclonal anti-ATP6V1B2 (D2F9R) antibody ((1:1000); Cell Signaling Technology, Denvers, MA, USA).To confirm equal protein loading, immunodetection was performed with the rabbit polyclonal anti-β-actin antibody ((1:2000); Sigma-Aldrich, St. Louis, MO, USA). The signal was detected using a horseradish peroxidase-conjugated secondary antibody ; Dako, Glostrup, Denmark). Products were visualized using an enhanced chemiluminesence .d-galactosidase (GLP) was determined by measurement of their enzymatic activity in cell lysates [d-galactopyranoside as a substrate. The reaction mixture was incubated at 37 °C for 30 min and then the enzymatic reaction was stopped by adding 50 µL of 1 M Tris buffer, pH 10.7. The relative fluorescence of released 4-methylumbelliferone was monitored at 365/445 nm.Expression of acidic phosphatase (ACP) and β- lysates ,31. Cell lysates . The enzWe used the method described previously . BrieflyLysosomes were visualized by confocal laser scanning microscopy . Due to photobleaching of FITC sequential scanning mode was applied, starting with excitation of FITC by a 488 nm line of argon laser and signal detection by a 505–550 nm emission filter, followed by subsequent DAPI excitation by a diode laser at 405 nm and emission at 430–470 nm. Optical sections through a central plane of each cell were performed with the following set-up: oil immersion objective 100×, zoom 2.8×, C.A. 200, image size 1024 × 1024 px, scan 20 µs/px. At the beginning of experiment, the laser intensity was set based on control samples omitting primary antibody. To increase the visibility of signal in all images, the FITC channel was exported with brightness levels limited from 0–4095 to the range of 40–2140.The ImageJ software  was used for image analysis. Lysosomal morphology was evaluated in at least 250 cells for each treatment.p < 0.05) and ** or ## for the very significant result (p < 0.01).Data are reported as the means ± S.D. Statistical analyses were performed using SigmaPlot 11.0 software package . Statistical significance was determined by a Student’s t-test (when the means of only two groups was to be compared), and one-way ANOVA (when means of more than two groups were to be compared). We used * or # for the significant result  leukemia and acute myeloid leukemia (AML), respectively. At the present time, patients with CML are successfully treated with TKIs  and clinical trials are underway to test TKIs for the treatment of AML ,36. GiveNot surprisingly, we found that GF and IM significantly accumulated in lysosomes of cancer cells . The absLAMP1, LAMP2, and ATPGB2 (vacuolar ATPase subunit B2), indicated no change in their expression at protein level  and transcription factor E3 (TFE3) ,26,39,402+ storage organelles which may on release, induce hetero or homotypic fusion. Ca2+ driven lysosomal membrane fusion with other lysosomes, lysosome related organelles, endosomes, or plasma membrane, regulate important cellular functions such as autophagy, endocytosis, exocytosis, and phagocytosis [2+ release, as inside the lysosomal membrane, there are several calcium channels that serve as NAADP receptors, including the two pore channels 1 and 2  with high affinity for NAADP, and transient receptor potential melastatin 2 (TRPM2) with low affinity for NAADP [2+ release has yet to be elucidated. We believe that our experimental model system is also well suited to identifying other components required for lysosomal fusion, i.e., membrane proteins and lipids that regulate their docking and subsequent fusion.An increase in the lysosomal accumulation capacity may thus be an aspect of lysosomal biogenesis which combines both biosynthetic and membrane trafficking pathways. Apart from other functions, lysosomes serve as Caocytosis ,42. Althor NAADP . Which o2+ mediated lysosomal fusion, leading to enlargement of the lysosomal compartment with a significantly increased sequestration capacity for these drugs without apparent lysosomal biogenesis.We demonstrated that extracellular TKIs, GF and IM, induced NAADP/Ca


Staphylococcus aureus strains are fast emerging, leading to life-threatening situations  associated with delayed healing and chronic inflammation. Electrospinning is one of the most widely used techniques for the fabrication of nanofibers (NFs), induced by a high voltage applied to a drug-loaded polymer solution. Particular attention is given to electrospun NFs for pharmaceutical applications  and tissue regeneration . However, there is a paucity of reports related to their application in diabetic wound infections. Therefore, we prepared eco-friendly, biodegradable, low-immunogenic, and biocompatible gelatin (GEL)/polyvinyl alcohol (PVA) electrospun NFs (BNFs), in which we loaded the broad-spectrum antibiotic cephradine (Ceph). The resulting drug-loaded NFs (LNFs) were characterized physically using ultraviolet-visible (UV-Vis) spectrophotometry (for drug loading capacity (LC), drug encapsulation efficiency (EE), and drug release kinetics determination), thermogravimetric analysis (TGA) , scanning electron microscopy (SEM) , and Fourier-transform infrared spectroscopy (FTIR) . LNFs were further characterized biologically by in-vitro assessment of their potency against S. aureus clinical strains (N = 16) using the Kirby–Bauer test and 3--2,5-diphenyltetrazolium bromide (MTT) assay, by ex-vivo assessment to evaluate their cytotoxicity against primary human epidermal keratinocytes using MTT assay, and by in-vivo assessment to estimate their diabetic chronic wound-healing efficiency using NcZ10 diabetic/obese mice (N = 18). Thin and uniform NFs with a smooth surface and standard size (<400 nm) were observed by SEM at the optimized 5:5 (GEL:PVA) volumetric ratio. FTIR analyses confirmed the drug loading into BNFs. Compared to free Ceph, LNFs were significantly more thermostable and exhibited sustained/controlled Ceph release. LNFs also exerted a significantly stronger antibacterial activity both in-vitro and in-vivo. LNFs were significantly safer and more efficient for bacterial clearance-induced faster chronic wound healing. LNF-based therapy could be employed as a valuable dressing material to heal S. aureus-induced chronic wounds in diabetic subjects.Diabetic wound infections caused by conventional antibiotic-resistant  Diabetes is a noncommunicable chronic metabolic disease characterized by elevated levels of blood glucose (hyperglycemia), which over time leads to serious organ damage . The most common is type 2 diabetes (T2D), usually in adults, which occurs when the body becomes resistant to insulin or does not make enough insulin. Type 1 diabetes, once known as juvenile diabetes or insulin-dependent diabetes, is a chronic condition in which the pancreas produces little or no insulin by itself. About 422 million people worldwide have diabetes, the majority living in low-and middle-income countries, and 1.6 million deaths are directly attributed to diabetes each year. Both the number of cases and the prevalence of diabetes have been steadily increasing over the past few decades. There is a globally agreed target to halt the rise in diabetes and obesity by 2025 [Importantly, more than 20% of the diabetic population develops wounds, most commonly in the legs and feet ,4. ExterRecent studies aimed to develop therapeutics that will promote proper tissue repair and improve impaired wound healing. In this regard, new therapeutic formulations using synthetic and/or natural polymeric materials have been reported, but there is still a paucity of such reports in relation to diabetic skin wounds ,10,11.S. aureus, remains the main challenging issue [Cephradine (Ceph) is a first-generation cephalosporin antibiotic commonly prescribed intravenously in surgical prophylaxis, pre-operative, and post-operative procedures to prevent the infection and spreading of wounds ,13. Despng issue ,15,16. Hng issue ,13,17.Herein, we decided to investigate the outcomes  of newly developed electrospun Ceph-loaded GEL/PVA NFs, abbreviated as LNFs, for diabetic skin wound healing.Electrospinning is considered the gold standard method to produce NFs ,22,23,24GEL is a biocompatible, biodegradable, low-immunogenic, and cost-effective biopolymer that has countless applications in the pharmaceutical industry  ,19,20. GPVA is a non-toxic, water-soluble synthetic polymer that acts as a glue during electrospinning, subsequently enhancing the mechanical properties of electrospun NFs  ,31,32,33Considering the above-mentioned features of GEL, PVA, Ceph, and the electrospinning method, it was worth developing and evaluating LNFs both as a potential antimicrobial delivery system and as a material dressing for accelerated diabetic wound healing. With the aim to reach this important goal, we prepared and characterized electrospun LNFs both in-vitro and in-vivo in an environmentally friendly manner.To the best of our knowledge, this is the first study reporting the development and testing of LNFs for both diabetic wound infection and healing.S. aureus strains (N = 16 from 24 diabetic patients) and primary cultures of human epidermal keratinocytes were kindly gifted from hospital wards.PVA , gelatin , Dimethyl sulfoxide (DMSO), MTT Assay Kit, Muller-Hinton agar (MHA), Muller-Hinton broth (MHB), nutrient agar (NA), and Dulbecco’s Modified Eagle Medium (DMEM) were all purchased from Sigma–Aldrich, Seoul, Korea. Ceph was provided by Bio-Labs Pharmaceutical, Islamabad, Pakistan. A thermostatic incubator (model: DHP-9052), high-speed refrigerated centrifuge (model: TGL20MC), and multifrequency ultrasonic cleaning machine (model: NB-600 DTY) were purchased from Zhengzhou Nanbei Instrument Equipment Co. Ltd., Zhengzhou, Henan, China. Sample stirring and heating were carried out with a heating magnetic stirrer (model: IKA-RCT-B) purchased from IKA group, Guangzhou, China. A microplate reader (model: Infinite 200 Pro) was purchased from Tecan Trading AG., Männedorf, Switzerland. A thermogravimeter (model: Thermo-plus TG 8120) was purchased from Rigaku Corp., Tokyo, Japan. An SEM (model: Quanta 450 FEG) was purchased from Thermo–Fisher Scientific Inc., San Diego, CA, USA. A UV–Vis spectrophotometer (model: Lambda 950) was purchased from PerkinElmer, Beaconsfield, Buckinghamshire, UK. An FTIR instrument (model: FTIR-4100) was purchased JASCO Inc., Easton, PA, USA.LNFs were fabricated by electrospinning, without using a cross-linking agent, following a similar approach previously reported .Briefly, homogenous GEL solution (50 mg/mL) was prepared in distilled water (D/W) under constant magnetic stirring (340 rpm) at 60 °C for 1 h. In parallel, PVA (50 mg/mL) was dissolved in D/W under continuous magnetic stirring (340 rpm) at 80 °C for 5 h. The PVA solution was then added to the GEL solution at different volumetric ratios of GEL:PVA , and the mixture was stirred (340 rpm) at room temperature (RT) for 30 min to achieve a homogenous solution. The blank GEL/PVA electrospun NFs (BNFs) were fabricated by transferring each GEL/PVA blend preparation into a 5-mL syringe with a blunt-end 22 G needle. Each GEL/PVA blend solution was then ejected at a feeding/flow rate of 1 mL/hr using the infusion pump NE-300 . The distance between the needle tips to the grounded aluminium collector was kept constant at 14 cm. A high voltage of 30 kV was applied to the needle at a relative humidity of 50–60%.w/v) to the PVA solution under continuous magnetic stirring (340 rpm) at room temperature (25 °C) for 30 min, followed by the addition of the GEL solution. The blend was subjected to the same electrospinning process as described for BNFs. The resulting electrospun NFs (BNFs and LNFs) were washed several times with D/W at room temperature to carefully remove any remaining unentrapped drug. They were eventually dried overnight and stored in a desiccator prior to analyses [Under an optimized volumetric ratio (5:5 GEL:PVA), LNFs were fabricated by adding Ceph  was dissolved in 1 mL of slightly acidic (0.5% acetic acid) aqueous solution to extract Ceph from the nanofiber mats and form a solution. After 24 h, a 1:10 dilution of the solution was made in D/W and analyzed for the total Ceph content by spectrophotometry at a wavelength of 256 nm (UV) using a standard curve.The percent rate of the Ceph-loading capacity (LC) and encapsulation efficiency (EE) were determined as follows:For microstructure analysis and surface fiber morphology, SEM of LNFs was performed following a protocol previously published . BNFs weThe micrographs were analysed by using the ImageJ open-source image processing program .3. Then, they were pelletized under vacuum. BNFs and free Ceph were used as controls.The presence of specific functional chemical groups and their structural interactions in fabricated samples were examined by FTIR spectroscopy . The LNF−1, with 120 scans averaging 4 cm−1 resolution, at a scanning speed of 2 mm/s.Eventually, the pellets were analyzed in the infrared spectral region of 600–4000 cm2) atmosphere at a purge rate of 30 mL/min and a constant rate of 10 °C/min.The thermal stability of the LNFs was evaluated by TGA, following a method previously described . BNFs anThe loss in weight (%) observed in the different decomposition steps of the LNF thermogram was compared to that of BNFs and the free drug.The curve fitting was performed by using Origin Pro 8.0.The Ceph release behavior from GEL/PVA NFs was studied at pH 7.4 and pH 4.8 using UV–Vis spectrometry at a wavelength of 256 nm. These two pH values (7.4 and 4.8) were chosen to understand the drug release kinetics in the case where LNFs would be intravenously injected or topically applied, respectively. Indeed, pH 7.4 represents the strictly normal blood pH for mammals . pH 4.8, which is mildly acidic, represents the normal average skin pH for humans and young mice.Concisely, 10 mg of LNFs was placed inside a dialysis bag which was pre-filled with 5 mL of 1× PBS . This temperature was chosen based on the normal human body temperature which is also suitable for experiments in mice (since the mouse body temperature ranges from 36.5 to 38 °C). The dialysis bag was then hung inside a beaker containing 20 mL of the same buffer, which was shaken (150 rpm) at 37 °C for 24 h. Free Ceph (2 mg/mL) was used as a control; 3 mL of the samples was collected at specific time points from the beaker, which was thereafter replenished with fresh buffer.t is the cumulative amount of drug released at time t; M∞ is the mass of the released drug as time reaches infinity; K (=0.708) is a constant characteristic of the drug-polymer system; n is the diffusion exponent suggesting the nature of the release mechanism .Non-linear regression fitting curve fitting was eventually used to obtain the parameters and their uncertainties. The Ceph release kinetics from LNFs were described using the Korsmeyer–Peppas method, following this equation :Mt/M∞ × echanism . In the S. aureus (N = 16) using both the agar well diffusion method (aka Kirby–Bauer test) and MTT assay, as previously described [The antibacterial activity of LNFs was assessed in-vitro against resistant clinical strains of escribed .S. aureus strains to LNFs was first analyzed by broth microdilution to determine the minimal inhibition concentration (MIC) of free Ceph, which served as the MIC reference. Serial dilutions (up to 10 two-fold dilutions) of the Ceph solution (2 mg/mL) were prepared in MHB medium. The final concentrations of Ceph in the prepared tubes were 0, 2, 4, 8, 16, 32, 64, 128, 256, 512, and 1024 μg/mL, to which 10 μL (1 × 106 colony forming units (CFUs)) of each bacterial inoculum was added. The inoculated tubes were eventually incubated at 37 °C for 24 h. The lowest concentration (8 μg/mL) showing no cell growth represented the MIC, which was used for subsequent agar well diffusion and MTT assays. It is worth mentioning that 6 μg/mL, defined as the breakpoint concentration recommended by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (https://www.eucast.org/ accessed on 1 March 2021), was not a sufficient concentration. Indeed, at 6 μg/mL, only 2 Ceph-sensitive S. aureus strains (or 14 Ceph-resistant S. aureus strains) were detected out of the 16 tested (data not shown).Briefly, the sensitivity of 2·2H2O) with 9.95 mL of 1% sulfuric acid (H2SO4)), was spread on Petri dishes using sterile cotton swabs. Subsequently, 18 mm wells were made in the dishes and sealed. Suspensions (100 µL) of LNFs , BNFs , free Ceph , or sterilized D/W , were added to each well, separately. Then, the plates were incubated at 37 °C for 24 h before examination and measurement of the respective ZIs.In a step further, the zone of inhibition (ZI) of LNFs was measured by agar well diffusion assay. MHA was prepared according to the manufacturer’s instructions, sterilized, and poured into Petri plates. Then, the bacterial suspension, matching the 0.5 McFarland standard (prepared by mixing 0.05 mL of 1.175% barium chloride dihydrate (BaCl50) determination. In this case, IC50 measured the potency of LNFs in inhibiting resistant S. aureus cells, compared to that of BNFs and free Ceph, used as controls. Each S. aureus strain was cultured in a 100 mm petri dish containing MHA (30 mL). After incubation overnight at 37 °C, 10 μL (5 × 105 CFUs) of each bacterial strain suspension was prepared in sterilized D/W by comparison with the 0.5 McFarland (turbidity) standard against black and white backgrounds. Each bacterial suspension was added to each well of plates containing MHA (100 μL) and incubated for 24 h. After incubation, MHA was replaced with fresh 100 μL MHA containing LNFs and Ceph (50 µL) in each well, separately. Sterilized D/W was used as a control. A range of concentrations (0–128 μg/mL) of LNFs, BNFs, and Ceph was used for this assay. The microplates were incubated at 37 °C for 12 h. Subsequently, 20 μL of MTT reagent (5 mg/mL) was added to each well, and the microplate was further incubated for 4 h. S. aureus cells reduced the MTT to the purple color of formazan. The formazan ring was dissolved by adding 120 μL isopropanol, and the production of formazan in each sample was quantified by measuring its absorption at 490 nm using a microplate reader. The absorption was directly related to the number of viable bacterial cells.Furthermore, the effect of LNFs on bacterial cell viability was examined by MTT assay through half-maximal inhibitory concentration  for 24 h. Then, LNFs, BNFs, free Ceph, and D/W were added separately, and the plates were incubated at 37 °C for 24 h. Subsequently, the cells were washed with 1× PBS, treated with MTT reagent, 20 µL (5 mg/mL), and further incubated at 37 °C for 4 h after adding 100 µL of fresh cell culture medium (DMEM/FBS). Eventually, DMSO (150 µL) was added to dissolve the formazan crystals, and the absorption was measured at a wavelength of 490 nm.Briefly, various concentrations (range: 0–128 mg/mL) of LNFs were tested on primary cultures of proliferating human epidermal keratinocytes. BNFs, free Ceph, and D/W were used as controls; 1 × 10The percentage viability of keratinocytes was calculated as follows:https://www.jax.org/strain/004456 accessed on 1 March 2021), was used as a model for diabetic wounds. Twenty NcZ10 male mice 8–10 weeks old, purchased from the Department of Experimental Animals, Soochow University, China, were maintained under standard controlled housing conditions. NcZ10 mice were maintained on high-fat diet. All animal experiments were carried out in accordance with guidelines evaluated and approved by the ethics committee of Soochow University, China (No. 2019LW003).Mouse strain NONcNZO10/LtJ, hereafter abbreviated as NcZ10, considered an emerging model of polygenic, moderate type 2 diabetes (T2D) (n = 18) aged 12–14 weeks with an average body weight of 60 g were used for the development of chronic wounds.NcZ10 diabetic and obese mice (n = 18) because mice can only withstand the burden of one wound. Then, 9 × 108 CFUs of a Ceph-resistant S. aureus suspension (100 μL) were injected into the local wound area of each mouse. After producing infected wounds in the diabetic mice, the mice were divided into three groups (n = 6/group): Group 1: mice were treated with LNFs, Group 2: mice were treated with free Ceph, Group 3: mice were treated with BNFs. LNFs, BNFs, or free Ceph was applied topically (to the wound area) on a daily basis for 11 days. The wound was then covered firmly with a sterilized transparent dressing film (5 × 5 mm2). The wound area of each mouse was measured by a digital vernier caliper on days 1, 7, and 11 after bacterial inoculation [All mice were shaved before a depilatory lotion was applied (to obtain a smooth skin). Buprenex (0.05 mg/kg mouse in 1× PBS) was injected intraperitoneally for pain relief 30 min before surgery and 6 h after surgery. The wound regdox agent Inject 3-amino-1,2,4-triazole (ATZ) (1 g/kg mouse in 1x PBS), was also injected intraperitoneally 20 min before surgery. A wound was created in 30–45 s using a 7 mm skin biopsy punch, tweezers, and surgical scissors. The biopsy punch was lightly pressed onto the anticipated wound location before it warped. The outlined skin was expunged by dragging up the punch centre and cutting it along the outline with surgical scissors. Only one wound developed in each mouse (n = 18) was serially diluted (4-fold dilution) for bacterial counts. Then, 100 µL of the homogenized tissue was added to each NA plate and incubated at 37 °C for 24 h for potential bacterial growth. After incubation, the number of bacterial colonies was estimated by using a colony counter, and the CFU was calculated by the following formula:Furthermore, the eventual bacterial clearance from the diabetic wound was validated by the colony counting method . BrieflySubsequently, all the experimental mice were sacrificed, by cervical dislocation, at the end of the study. Cervical dislocation is a common and appropriate method of euthanasia for mice and small rats (<200 g) since it is largely assumed that it relieves pain and suffering.All experiments were performed at least in triplicate. All data are expressed as the mean ± standard deviation.The statistical analysis of differences was performed using a t-test in OriginPro 2018.p < 0.05 was considered to indicate a statistically significant difference.S. aureus resistance to Ceph constitutes a challenging issue, including for diabetic wound healing [Ceph, a broad-spectrum first-generation cephalosphorin antibiotic, was proved to be effective against soft tissue infections and wounds, but the emerging  healing ,12,13.In the present study, we developed an innovative biocompatible delivery system to heal chronic diabetic wounds by sustained and effective Ceph delivery.Unlike other synthesis protocols, electrospinning is a popular method for generating continuous elongated NFs (at a high aspect ratio and a high degree of fiber orientation) due to its simplicity and versatility ,41. In vw/v) and GEL:PVA . Interestingly, % LC and% EE were found to be 11.4% and 63%, respectively. These data are encouraging, based on our previous study [S. aureus strains-induced wound infection. These data also support the fabrication of electrospun GEL/PVA NFs without the use of a  cross-linking agent [GEL/PVA blends were prepared in D/W at different volumetric ratios , with or without Ceph. LNFs were ecofriendly synthesized by electrospinning . Optimized LNFs were fabricated with Ceph  compared to that of BNFs, with 285 ± 32 nm (v/v) resulted in a significant increase (p < 0.05) in the average diameter of the electrospun LNFs (v/v) resulted in a significant decrease (p < 0.05) of the average diameter of electrospun LNFs  observed for the nanomaterials are related to CH2 symmetrical and asymmetrical stretching [Briefly, the absorption band at 3400 cmectively ,32. In aThe FTIR profile displayed by LNFs showed a combination of both BNFs and free Ceph spectra (dash lines), confirming Ceph loading into BNFs.2) [The thermal stability of LNFs was estimated by TGA using BNFs and free Ceph as controls . TGA sca2) .10%, which was reached at 150 ± 5 °C, 120 ± 3.5 °C, and 100 ± 4.2 °C for LNFs, BNFs, and free Ceph, respectively. This phenomenon is explained by moisture vaporization being significantly higher (p < 0.05) in NFs than in the free drug [max (325 ± 10 °C). The significant difference (p < 0.05) between WL Tmax for LNFs and BNFs was then contributed by Ceph drug loading. Around 600 °C, the residual weight (RW) was insignificantly different (p > 0.05) between LNFs (21 ± 0.8%), BNFs (20 ± 0.6%), and free Ceph (19 ± 0.7%). At the end of the analysis carried out at the highest temperature (1000 °C), all the samples lost most of their water (≥90%), with no significant differences (p > 0.05) between LNFs (11 ± 1.3%), BNFs (10 ± 1.1%), and free Ceph (9 ± 0.9%).Initially, minimal (10%) onset weight loss (WL) was noticed at Tree drug . After lTaken together, TGA data showed that LNFs are thermostable to withstand elevated temperatures. Importantly, these data also revealed that free Ceph is more susceptible to thermal instability/degradation compared to LNFs. Similar conclusions were reported by our group  and otheIn-vitro Ceph release kinetics from LNFs were evaluated for 24 h by UV–Vis spectrometry in 1× PBS thermostated at normal human/mouse body temperature (37 °C) and adjusted either to normal mammalian blood pH (7.4) or human/young mouse skin surface pH (4.8) A.p < 0.05). This effect was independent of the pH (p > 0.05).After 1 h, the release of free Ceph was about 90 ± 1.25%, whereas the release of Ceph from LNFs was about 60 ± 1.12% (p < 0.05). This effect was independent of the pH (p > 0.05).After 4 h, the release of free Ceph was already recorded as maximal (98 ± 1.21%), whereas the release of Ceph from LNFs was about 80 ± 1.95% (p > 0.05).After 24 h, the release of Ceph from LNFs was 92 ± 2.23% and 95 ± 1.68% at pH 7.4 and pH 4.8, respectively. This effect was pH-independent  and/or drug delivery/intravenous route (pH 7.4), both in humans and young mice ,30.n”, found to be 0.513 at pH 4.8 and 0.512 at pH 7.4 (p > 0.05), the release of Ceph from LNFs ensued mainly by diffusion following the non-Fickian  transport mechanism , only two (12.5%) strains showed susceptibly to Ceph, while the remaining strains  elicited resistant against the breakpoint value of Ceph (6 μg/mL) defined by EUCAST (https://www.eucast.org/ accessed on 1 March 2021). This finding is in agreement with previous studies. For instance, Zaman et al. reported that S. aureus strains are highly resistant to Ceph [S. aureus strains, which was eventually found to be 8 μg/mL.Among the isolated  to Ceph . TherefoDilution methods  are the most appropriate for the determination of MIC values because they offer the possibility to quantitatively estimate the in-vitro antimicrobial  activity of the tested antimicrobial agent in medium . There aS. aureus strains compared to that of free Ceph (p < 0.05). Indeed, ZI averaged 9.56 ± 1.67 mm and 3.23 ± 1.5 mm for LNFs and free Ceph, respectively  than that of free Ceph for which the IC50 value averaged 16.746 ± 1.2 µg/mL . In general, antibiotic-loaded NFs show enhanced antibacterial and antifungal activities by inhibiting cell wall synthesis , protein synthesis, DNA/RNA synthesis, mycolic-acid synthesis, and/or folic acid synthesis. A study carried out with ciprofloxacin (Cipro)-loaded electrospun CS NFs, intended to be used as potential antibacterial wound dressing material, reported improved antibacterial activity compared to that of free Cipro when tested against albicans . For thefradoxil .4 cells/well in 96-well plates) using MTT assay  on primary cultures of proliferating human epidermal keratinocytes (1 × 10TT assay . Free Cep > 0.05).Up to 16 μg/mL, no cytotoxic effect was noticed statistically when the cells were treated either with LNFs or Ceph compared to BNFs (p < 0.05). This cytotoxic effect appeared to be concentration-dependent with the most significant difference noticed at 128 μg/mL (p < 0.01).Nevertheless, from 32 μg/mL, both LNFs and Ceph exhibited significant cytotoxicity compared to BNFs (p > 0.05).Furthermore, no statistical differences in cytotoxicity were observed up to 32 μg/mL when the viability rate (%) of LNF-treated cells was compared to that of free Ceph-treated cells (p < 0.05).However, from 64 μg/mL, a significantly higher rate (%) of viable LNF-treated cells was observed compared to that of free Ceph-treated cells (p > 0.05).We postulate that the LNF-induced lower cytotoxicity (compared to that of Ceph) is more likely due to the indirect contact of Ceph with the keratinocytes. Indeed, in LNFs, Ceph was encapsulated into the biocompatible polymer GEL, and no concentration-dependent cytotoxicity was detected with BNFs compared to untreated cells  of LNFs (Test Group/Group 1) (2). The percent wound closure was reported for each treated group of mice (six mice per group) on days 1, 7, and 11 (after creating the infected wound), in ratio-based calculations relative to the wound on day 1 (100%)  wound healing. For this purpose, we applied the diabetic/obese NcZ10 mouse model in which a single Group 1) A. Ceph (1 (100%) B.p < 0.05) in Group 2, with 5 ± 3.5% (or 95 ± 3.5% unclosed wound area). In addition, the wound closure observed in Group 2 was insignificantly different compared to that of Group 3 (p > 0.05). The wound healing in Group 3 was non-existent, 0 ± 1.25% (100 ± 1.25% unclosed wound area) and comparable to that observed on day 1 (p > 0.05).On day 7, the wound healing efficacy of Group 1 was 15 ± 4.2% (or 85 ± 4.2% unclosed wound area), whereas it was significantly lower (p < 0.01) in Group 2, with 31% ± 4.11% (or 69 ± 4.11% unclosed wound area). This wound healing observed in Group 2 was significantly higher than the effect seen in Group 3 (p < 0.05). The wound healing in Group 3 was non-existent, 0 ± 3.04% (99.8 ± 3.04% unclosed wound area) and comparable to that observed on day 1 or 7 (p > 0.05).On day 11, the wound-healing efficacy in Group 1 was about 65 ± 5.2% (or 35± 5.2% unclosed wound area), whereas it was significantly lower (p < 0.001), which was significantly better than that elicited by BNFs (p < 0.05).Although the progressive wound closure was not completed with a microscopic sample for histopathological study, the stages of the wound-healing process were extensively reviewed by Tolotti et al. (2020) . In addiS. aureus strains. Conversely, although BNFs were found to be safe nanomaterials ex-vivo, they did not exert any benefits alone on wound healing in diabetic mice. Thus, our study demonstrated the high benefit/risk ratio in combining Ceph with BNFs, which would likely act as an adjuvant to improve the outcome obtained with free Ceph to fight resistant S. aureus. Our data revealed that LNFs might be efficiently and safely used in clinical practice as a biodegradable, biocompatible, and low immunogenic dressing material for bacterial clearance, especially to overcome resistance associated with S. aureus-induced diabetic skin wounds.The wound-healing process in diabetic mice was a time-dependent phenomenon. Indeed, topical application of LNFs exerted a greater wound healing compared to that of free Ceph applied under the same experimental conditions, and the greatest wound healing was noticed on day 11. The efficiency of LNF-mediated wound healing appeared to be linked to its capacity to clear bacteria, including the intradermal inoculated resistant S. aureus-induced infection and inflammation. Few recent studies showed possible benefits of using stem cells [To the best of our knowledge, this is the first report that demonstrates the benefits of a topical route for Ceph administration through LNFs, as a dressing material/tissue scaffold, and a sustained drug system delivery. This original nanocarrier could overcome the antibiotic resistance commonly associated with em cells ,8, topicem cells , and bioem cells , for theS. aureus is present in about one-quarter of diabetic infections and is associated with a higher rate of treatment failure, morbidity, and hospitalization cost.Currently, there are inadequate clinical data for the bacterial clearance from human diabetic wounds and chronic diabetic wound healing. Most of the monotherapies failed to treat the multifactorial diabetic chronic skin wounds. Furthermore, a lot of work is still needed for the firm understanding of all stages of diabetic wound healing. Diabetes is synonymous with compromised life quality with the risk of deleterious skin ulcerations. Thus, with the ambition of discovering original and accessible therapeutics, we strived to design an eco-friendly and cost-effective therapeutic strategy for effective healing of diabetic skin wounds. Resistant S. aureus clinical strains both in-vitro and in-vivo. The electrospun LNFs allowed faster wound healing in diabetic/obese NcZ10 mice compared to that of free Ceph, and this appeared to be linked to their bacterial clearance capacity.Ceph-loaded BNFs (LNFs) were rapidly produced in an eco-friendly manner by electrospinning at the optimized GEL:PVA volumetric ratio of 5:5. Ceph, a broad-spectrum cephalosporin antibiotic, is commonly prescribed intravenously in surgical procedures to prevent the infection and spreading of wounds. LNFs were successfully characterized both physically and biologically. Thin and uniform NFs with a smooth surface and standard size (<1000 nm/1 μm) were observed by SEM. FTIR analyses confirmed the drug loading into the (BNFs) nanocarrier. LNFs were significantly more thermostable than free Ceph. Ceph release from LNFs was controlled/sustained through diffusion following a non-Fickian  transport mechanism, which was not the case for free Ceph. Further, LNFs were significantly safer than Ceph in primary keratinocytes, which favoured their application on the skin surface. Importantly, LNFs were significantly more effective than Ceph in eradicating resistant Electrospun LNFs could represent a promising therapeutic option, in terms of potency and safety, to prevent both resistant Staph infections and life-threatening complications in diabetic subjects. LNFs exerted a dual function, as a Ceph delivery nanosystem and as a wound-dressing nanomaterial. The WHO recommends a surveillance program for monitoring antibiotic resistance. The present study is a step in this direction.


Urinary 1-hydroxynaphthalene + 1-hydroxyacenapthene and 2-hydroxyfluorene presented the highest increments , followed by 1-hydroxyphenanthrene (ca. 2.3 times) and 1-hydroxypyrene (ca. 1.8 times). Additionally, 1-hydroxypyrene levels were higher than the benchmark, 0.5 µmol/mol creatinine, in 5% of exposed workers. Moreover, 3-hydroxybenzo(a)pyrene, biomarker of exposure to carcinogenic PAHs, was detected in 13% of exposed workers. Individual excretion profiles showed a cumulative increase in ∑OHPAHs during consecutive working days. A principal component analysis model partially discriminated workers’ exposure during working and nonworking periods showing the impact of grilling activities. Urinary OHPAHs were increased in grill workers during working days.Grilling activities release large amounts of hazardous pollutants, but information on restaurant grill workers’ exposure to polycyclic aromatic hydrocarbons (PAHs) is almost inexistent. This study assessed the impact of grilling emissions on total workers’ exposure to PAHs by evaluating the concentrations of six urinary biomarkers of exposure (OHPAHs): naphthalene, acenaphthene, fluorene, phenanthrene, pyrene, and benzo(a)pyrene. Individual levels and excretion profiles of urinary OHPAHs were determined during working and nonworking periods. Urinary OHPAHs were quantified by high-performance liquid-chromatography with fluorescence detection. Levels of total OHPAHs (∑OHPAHs) were significantly increased (about nine times;  Kitchen and grillroom workers are daily exposed to several airborne gaseous and particulate pollutants formed during the preparation of foods using different cooking methods . Cooking fumes are thermal oxidative decomposition products containing several hazardous pollutants such as respirable particulate matter (PM), heavy metals, black carbon, heterocyclic amines, and volatile organic compounds including polycyclic aromatic hydrocarbons (PAHs), aldehydes, and carbonyls ,5,6,7,8.PAHs are a group of more than a hundred different aromatic compounds that are produced during the (incomplete) combustion processes of carbonaceous materials. Some PAHs present toxic, mutagenic, and carcinogenic properties, being also known as endocrine disruptors ,19,20,21Several authors already proved the occupational exposure to PAHs in cooks, grill workers, and other kitchen staff as well as in street food vendors ,35,36,37The concentrations of urinary biomarkers of exposure to six different PAHs, namely, naphthalene, acenaphthene, fluorene, phenanthrene, pyrene, and benzo(a)pyrene, was for the first time assessed in European grill workers. Individual levels and excretion profiles of the characterized urinary OHPAHs were determined during a regular working period and in the following resting days to evaluate the contribution of grilling emissions on grill workers’ total exposure to PAHs.All grill workers  from sixAll participants filled a questionnaire adapted from a World Health Organization questionnaire  and prevEach participant collected a spot urine sample in a sterilized container at the end of the working day over a complete working week, including the resting days. After collection, urine samples were coded and frozen at −20 °C.A researcher was daily present at the grillroom area to keep a record on other potential sources of exposure to PAHs.®Light Plus C18  that were preconditioned with 5.0 mL of methanol and 10.0 mL of water. After elution of samples, cartridges were cleaned with 10.0 mL of water and 10.0 mL of methanol/water . Cartridges were then completely dried, eluted with 20.0 mL of methanol/ethyl acetate , and evaporated till dryness (Büchi R200 rotavapor and a Büchi Vac V-500 pump). Extracts were redissolved in 300 µL of methanol. Urine extracts were analyzed in a C18 column  by liquid chromatography  with a fluorescence detection system. Detailed information related with the chromatographic characteristics can be found in Oliveira et al. [Urinary 1-hydroxynaphthalene and 1-hydroxyacenaphthene (1OHNaph + 1OHAce), 2-hydroxyfluorene (2OHFlu), 1-hydroxyphenanthrene (1OHPhen), 1OHPy, and 3OHBaP were extracted according to Oliveira et al. (2016). Briefly, 10 mL of urine was buffered with acetate buffer (pH 5.0) and hydrolyzed with 80 µL of ß-glucuronidase/arylsulfatase from Helix pomatia  for 120 min at 37 °C . After hydrolysis, samples were loaded into Sep-Paka et al.  and Figu2 ≥ 0.999)  . The achUrinary levels of OHPAHs were corrected with the levels of urinary creatinine (µmol OHPAH/mol creatinine), which was determined according to the Jaffe colorimetric method . The detData treatment was performed using SPSS (IBM SPSS Statistics 20) Statistica software . Levels of urinary OHPAHs were expressed as median, percentiles 25–75, and range. Since normality was not observed, urinary levels of OHPAHs were compared with nonparametric tests; a significant level of 5% was considered. When a PAH metabolite was not detected in the urine sample, the value of its respective LOD/√2 was considered . Spearma2 (23.7–35.6 kg/m2)  and presented a mean body mass index (BMI) of 28.3 kg/m6 kg/m2) . A totalg/m2) . Concentrations of creatinine ranged from 0.432 to 2.90 g/L in the urine of grill workers, being these values within the acceptable range of values (0.3–3.0 g/L) defined by WHO [p ≤ 0.001) (−5 − 15.5) versus 0.018 (1.24 × 10−4 − 0.133) µmol/mol creatinine; 1OHPy: 0.086 (0.011–1.09) versus 0.049 (0.013–0.188) µmol/mol creatinine; 1OHPhen: 0.073 (2.51 × 10−4 − 0.719) versus 0.031 (0.016–0.088) µmol/mol creatinine, respectively; p ≤ 0.001) (Urinary concentrations of ∑OHPAHs were significantly higher during a regular working period comparatively with the following resting days (ca. 9 times higher: 2.77 (0.213–42.3) versus 0.298 (0.097–1.66) µmol/mol creatinine, respectively; ≤ 0.001) a. Moreov≤ 0.001) b–e.p ≤ 0.001). Previously, Singh and coworkers [3, the permissible exposure level defined by the American Conference of Governmental Industrial Hygienists [Regarding the individual biomarkers of exposure, the metabolites of naphthalene and acenaphthene (1OHNaph + 1OHAce) and fluorene (2OHFlu) presented the highest increments , followed by 1OHPhen (135%) and 1OHPy (76%)  were significantly lower than the concentrations reported in the literature for other kitchen workers, namely, chefs, bakers, and assistant cooks  and the molecular weight of the individual metabolite with evidences suggesting that lighter compounds are mostly eliminated through the urine, while the heaviest through the feces ,49,50. SUrinary concentrations of OHPAHs presented different profiles of distribution according to subjects’ working and resting periods: 1OHNaph + 1OHAce (74% of ∑OHPAHs) > 2OHFlu (11%) > 1OHPy (9%) > 1OHPHen (6%) in the days performing grilling activities and 1OHNaph + 1OHAce (52%) > 1OHPy (23%) > 1OHPHen (15%) > 2OHFlu (10%) during nonworking days .The different distribution profiles of OHPAHs highlight the impact of abstention and/or participation in regular grilling activities. p ≤ 0.01) and nonworking  periods (p ≤ 0.01), except with 1OHNaph + 1OHAce (p ≤ 0.01) during the working period of subjects. At the grillroom of the studied restaurants, there were two predominant sources of PAHs, the fumes from food processing and those released by the incomplete burning/pyrolysis of charcoal; no other cooking activities  were performed during the sampling periods. The prevalence of these two major sources of PAHs can explain the moderate and even the low correlations found among the urinary levels of OHPAHs in occupationally exposed grill workers. A more complete identification and characterization of the predominant sources and routes of exposure to PAHs would be possible with the simultaneous assessment of PAHs in the breathing air zone and dermal exposed areas of grill workers and the urinary concentrations of their respective metabolites.Spearman correlations were calculated to assess the inter-relation among individual PAH biomarkers of exposure and ∑OHPAHs in the selected group of workers during working and nonworking periods . Levels  periods . Previou+ 1OHAce . In addip ≤ 0.05) (p ≤ 0.01) and 1OHPhen .Regarding the resting period of grill workers, levels of urinary ∑OHPAHs were moderately correlated with the concentrations of 1OHNaph + 1OHAce, 2OHFlu, and 1OHPy  . ConcentPrincipal component analysis (PCA) based on levels of OHPAHs was performed to evaluate if urinary levels of OHPAHs can be used as occupational exposure descriptors for grillroom workers . Two priSince information on the topic is almost inexistent, this study assessed the levels of six PAHs biomarkers of exposure in the urine of grill workers’ during a regular working period. Concentrations of total OHPAHs were significantly increased during subjects’ working period comparatively with the following resting days , being 1OHNaph + 1OHAce (median 2.23 versus 0.098 µmol/mol creatinine) and 2OHFlu (median 0.112 versus 0.018 µmol/mol creatinine) the compounds that increased the most . The lowest increments  were found for 1OHPy, however, about 5% of the collected samples exhibited urinary 1OHPy levels that were higher than the recommended benchmark value . Thus, t