Background: Clinical management of ischemic events and prevention of vascular disease is based on antiplatelet drugs. Given the relevance of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) as a candidate target in thrombosis, the main goal of the present study was to identify novel antiplatelet agents within the existing inhibitors blocking PI3K isoforms. Methods: We performed a biological evaluation of the pharmacological activity of PI3K inhibitors in platelets. The effect of the inhibitors was evaluated in intracellular calcium release and platelet functional assays, the latter including aggregation, adhesion, and viability assays. The in vivo drug antithrombotic potential was assessed in mice undergoing chemically induced arterial occlusion, and the associated hemorrhagic risk evaluated by measuring the tail bleeding time. Results: We show that PI3K Class IA inhibitors potently block calcium mobilization in human platelets. The PI3K p110δ inhibitor Idelalisib inhibits platelet aggregation mediated by ITAM receptors GPVI and CLEC-2, preferentially by the former. Moreover, Idelalisib also inhibits platelet adhesion and aggregation under shear and adhesion to collagen. Interestingly, an antithrombotic effect was observed in mice treated with Idelalisib, with mild bleeding effects at high doses of the drug. Conclusion: Idelalisib may have antiplatelet effects with minor bleeding effects, which provides a rationale to evaluate its antithrombotic efficacy in humans. Cardiovascular diseases (CVD), specially ischemic heart disease and stroke, are one of the main causes of death in the world . PlateleCurrent antiplatelet drugs used for the prevention and treatment of thrombosis target four major classes of proteins, including cyclooxygenase 1 (aspirin), P2Y12 , integrin αIIbβ3 , and protease-activated receptor 1 (PAR1) (vorapaxar) [Intracellular platelet signaling pathways, such as those mediated by phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), are target candidates for anti-thrombosis drug development. Indeed, PI3K is required for platelet activation and adhesion as well as thrombus formation . To dateDue to the potential of PI3K inhibition as an antithrombotic therapy, the primary goal of the present study was the identification of novel antiplatelet drugs able to target different isoforms of PI3K. This was done by performing a pharmacological evaluation of PI3K inhibitors. To address this objective, we developed a phenotypic assay to measure the intracellular calcium mobilization in real time upon drug-induced platelet activation. Selected PI3K inhibitors were further evaluated in in vitro human platelet functional assays, including aggregation and adhesion assays as well as viability assays. Finally, the in vivo antithrombotic efficacy of the selected drug was assessed in a murine model of chemically-induced arterial occlusion and the hemorrhagic risk evaluated by measuring the tail bleeding time.Given the relevance of PI3K in GPVI-mediated platelet activation, we evaluated the effect of five PI3K Class IA inhibitors on intracellular calcium mobilization following platelet activation with the GPVI-specific agonist collagen-related peptide (CRP) A. The in50 0.1, 0.3, and 1.6 µM, respectively , α2β1 (collagen), and PAR1/PAR4 (thrombin).As shown in Taken together, these results showed that TGX-221 and Idelalisib inhibit platelet aggregation mediated by GPVI activation with a potential preference for this pathway. On the contrary, PIK-75 inhibitor did not show any selectivity among different platelet signaling pathways since it fully blocked both CRP and collagen-induced platelet aggregation and partially blocked thrombin-induced aggregation.The p110µµ inhibitor TGX-221, which also targets PI3K catalytic subunit δ, was already reported as a potential platelet inhibitor in thrombosis . We examTo further investigate the main activation pathways mediating the inhibition of platelet aggregation by Idelalisib, we studied aggregation in both platelet-rich plasma (PRP) and washed platelets in response to different agonists.Idelalisib (10 µM) blocked CRP-XL-induced platelet aggregation, compared with vehicle control, in both PRP and washed platelets from a cohort of human blood donors A,B. The 50 for CRP-induced aggregation of 1.9 μM  in both CRP- and rhodocytin-induced platelet aggregation responses. Notably, the inhibitory effects of Idelalisib on ITAM-mediated signalling pathways were dose-dependent H,I, withf 1.9 μM J, and 3.f 1.9 μM K. Thus, 2, p = 0.032). Moreover, the drug caused a major reduction in platelet aggregation in this test . These results further support the concept that Idelalisib may have a role in preventing platelet mediated thrombosis.In a separate series of experiments, we examined the effect of p110δ PI3K inhibition on platelet adhesion to type I collagen under static conditions by measuring the absorbance of the enzymatic reaction between p-nitrophenyl phosphate and acid phosphatase A. VehiclTo investigate more in detail the potential of Idelalisib as antithrombotic, in vivo tail bleeding and a ferric chloride-induced arterial thrombosis assays were performed in a murine model. Mice treated with Idelalisib (20 mg/kg) showed a plasma concentration of 5.1 ± 2.0 µM after 1 h .In the tail bleeding assay in mice A, IdelalImportantly, 9 out of 11 (80%) mice treated with Idelalisib showed bleeding times <10 min. By comparison, 66% of mice treated with ASA or clopidogrel, established antiplatelet therapies, bled over 10 min .FeCl3-induced arterial thrombosis was undertaken to further assess the antithrombotic potential of Idelalisib D. IntereThe core findings from the present study are: (i) PI3K Class IA inhibitors potently block calcium mobilization in human platelets; (ii) the drug Idelalisib inhibits platelet aggregation mediated by ITAM receptors GPVI and CLEC-2, without eliciting toxicity; (iii) platelet adhesion to collagen is also inhibited, as well as platelet adhesion and aggregation under shear (Impact-R test with whole blood), in samples treated with Idelalisib; and (iv) a potent antithrombotic and a mild bleeding delay effect was observed in mice treated with Idelalisib.Antiplatelet therapy has improved during the last decade to significantly enhance the antiplatelet efficacy, while reducing the side effects such as bleeding risk or thrombocytopenia . One sucSince PI3K inhibitors represent favorable candidates for the prevention of thrombosis, several pharmacological approaches have been carried out in order to elucidate the role of PI3K isoforms in platelet function . PI3Kβ hThe present study focused on a promising antiplatelet drug, Idelalisib, which targets the δ catalytic subunit of PI3K, which has been poorly investigated in this context. This isoform is almost specific to hematopoietic cells and seems to play a minor role in platelet activation. The first-in-class oral selective inhibitor of p110δ isoform Idelalisib has been approved for the treatment of hematological malignancies including chronic lymphocytic leukemia (CLL), follicular B-cell non-Hodgkin lymphoma (FL), and small lymphocytic lymphoma (SLL) ,16, and Consistent with the previous known role of PI3K in GPVI signalling , we repoThrombosis studies in mice demonstrated that Idelalisib treatment resulted in decreased arterial thrombosis without affecting the bleeding time at the same level than other antiplatelet drugs. Bleeding is a major complication of common antiplatelet therapy in humans and a concern when developing new antiplatelet therapies . The preCollectively, our results suggest that selective PI3Kδ inhibition with Idelalisib prevents platelet activation by ITAM-related signalling pathways, especially GPVI. We also show that this inhibition leads to an abrogation of platelet adhesion to collagen and a thrombus formation reduction in mice associated with minor bleeding. Therefore, our study indicates Idelalisib may have antiplatelet effects and provides a rationale to evaluate its antithrombotic therapeutic efficacy in humans.®, Greiner Bio-One International GmbH, Kremsmünster, Austria), and processed in less than 30 min after the extraction.Fresh blood was obtained from healthy volunteers who were not under chronic medication, or under antiplatelet drugs for the previous 10 days. Blood samples were collected in coagulation 3.2% sodium citrate tubes  were incubated with FLIPR Calcium 4 reagent  in a 384-well transparent bottom plate  and supplemented with Probenecid  0.07% for 1 h at 37 °C. Platelets were co-incubated for 10 min with the PI3K inhibitor (10 μM) and inhibitors of secondary mediators ) under non-aggregating conditions ). Then, the GPVI-specific agonist Collagen-Related Peptide (CRP) was added to the platelet suspension at different doses. Both PI3K inhibitors and CRP were automatically dispensed by FDSS7000EX Functional Drug Screening System . Intracellular Ca2+ mobilization was assessed by measuring fluorescence changes (480 nm emission and 540 nm excitation wavelengths) in real time for 15 min after the addition of the agonists   by carrying out a nonlinear regression fit for log(inhibitor) vs. relative CRP activity using a Hill slope of −1, and top and bottom constraints of 100 and 0, respectively.Washed platelets . After that, PRP or washed platelets were stimulated with the selected agonists for 5 min. The agonists used in the assay were: CRP-XL , Horm® collagen , rhodocytin , thrombin receptor activating peptide (TRAP) , and thrombin .Aggregations were performed in platelet-rich-plasma (PRP) or washed platelets previously warmed at 37 °C for 4 min without stirring and for 1 min with constant stirring at 1200 rpm in a Chrono-log8 platelets/mL) were incubated for 10 min with 10 μM Idelalisib  or 0.1% DMSO  as vehicle. Then, 1 μL of Calcein-AM 0.1 mg/mL  was added to the suspension and incubated for an additional 20 min at room temperature. Calcein-AM binding was measured in triplicate by an Accuri C6 flow cytometer . Data were analyzed comparing the average of the percentage of positive events in the presence of the inhibitor with the average of the percentage of positive events in the absence of the inhibitor (vehicle). To measure the potential toxicity of inhibitors, a viability assay based on Calcein-AM binding measured by flow cytometry was performed. Washed platelets (2.5 × 108 platelet/mL) were added to 96-well assay plates  previously coated overnight with 5 μg/mL Horm® collagen , and incubated for 1 h at 37 °C. Unbound platelets were separated by washing the plates with PBS three times. Then, p-nitrophenyl phosphate-based solution was added to plates for 40 min with agitation. Reactions were stopped with 50 μL of 3 M NaOH and the absorbance (405 nm) was measured in a Tecan Infinite® M1000 plate reader . Each assay was run three times and absorbance values (arbitrary units) were normalized with the negative control (BSA condition).Washed platelets , as previously described. [−1) leading to platelet adhesion and aggregation to the plastic. After washing the well and staining with May-Grünwal solution, the plate surface covered with stained objects (%SC), indicating platelet adhesion, and the average size of the objects AS (μm2), indicating platelet aggregation, were quantified using the inverted light microscope connected to a camera and an image analyzing software. Median values were calculated from 7 images taken per well.Venous blood was obtained from five healthy volunteers, into buffered 3.2% sodium citrate. Blood samples were incubated with either vehicle (DMSO 0.1%) or Idelalisib 10 μM (10 min at RT) an immediately assayed on the Impact-Rscribed.  Briefly,C57BL/6J mice  were kept under controlled environmental conditions  with a 12 h light/dark cycle and free access to chow and water. At the time of the study, mice were 8–12 weeks old. Equivalent numbers of males and females were included. According to the oral drug administration protocol, mice were immobilized and Idelalisib, 20 mg/kg or vehicle , was introduced directly into the stomach with an oral gavage feeding tube. One hour after the treatment, tail bleeding and arterial thrombosis assays were performed. A separate set of animals treated with other established antiplatelet drugs were also used as positive controls. Acetyl salicylic acid  was dissolved in water at 1 mg/mL and given by a single intraperitoneally injection of 100 mg/kg in mice 2–4 h before the experiment, as previously reported . Moreove® Synergy, Winooski, VT, USA). Bleeding times were performed blind to the mice treatment.After anesthetizing mice with isoflurane, tails were amputated 3 mm from the tip and submerged into saline solution at 37 °C. Bleeding time was recorded until the bleeding stopped (no further bleeding within 1 min) or for 10 min at maximum. A quantitative estimation of the amount of blood was determined by measuring the hemoglobin (Hb) content of blood collected in saline. For this purpose, tubes were centrifuged and erythrocyte lysis buffer  was added to samples. Absorbance was assessed at 575 nm in a microplate reader  to the left carotid artery for 2 min. A Doppler ultrasound flow probe  and a flow meter  were used to continuously register the blood flow. Occlusion time was specified as the time elapsed from the withdrawal of FeCl3 to the lack of blood flow (≤0.01 mL/min) for at least 3 consecutive minutes. Experiments were stopped after 30 min if no occlusion occurred. Animals were anesthetized by intraperitoneal injection (xylazine hydrochloride 10 mg/kg + ketamine hydrochloride 10 mg/kg). Thrombus formation was induced by applying a piece of filter paper (5 × 1 mm) soaked with a 7.5% ferric chloride solution  and centrifuged at 2235 g for 60 min. Then, 4 μL of the clean supernatant was injected into the LC–MS/MS system. Waters Acquity tandem quadrupole mass spectrometer Xevo TQD  was used for the analysis. For HPLC separation, Waters Acquity UPLC BEH C18 column was subjected to a flow rate of 0.6 mL/min by using a mobile phase consisting in 0.1% formic acid in water (Solvent A) and acetonitrile (Solvent B) in the following gradient elution: 0–0.1 min, 5% B; 0.1–1 min, 5% B, 1–2 min, 5–100% B; 2–2.1 min, 5% B; and 2.1–3.0 min, 5% B. The software used for instrument control and data analysis was MassLynx™ version 4.1 SCN 940 . The MS/MS detection was conducted operating in positive ion mode by monitoring the fragmentation of m/z 416.11 → 176.1 m/z. The calibration curve of Idelalisib was constructed by plotting the peak area ratio (PAR) in relation to each known concentration of the drug. The slope and correlation coefficient (r2 = 0.999493) were determined from the known concentrations of the drug by linear regression analysis. The unknown drug concentrations were determined by extrapolating PAR values from each sample to the calibration curve.Whole blood from mice was collected after 1 h-Idealisib treatment by retro-orbital bleeding in tubes with citrate. Platelet-poor plasma (PPP) was isolated by centrifugation of whole blood (1500 g for 5 min at RT). Idelalisib (purity 99.98%) was purchased from MedChemExpress . Formic acid, methanol and acetonitrile were purchased from Scharlab . Idelalisib at 10 mM stock solution was prepared in acetonitrile. This stock solution was diluted in 20% acetonitrile at the concentrations of 1000, 500, 250, 125, 62.5, 31.2, 15.6, and 7.8 μM. The calibration curve was constructed by the analysis of mouse plasma samples spiked with the mentioned varying concentrations of Idelalisib. For sample preparation prior to LC–MS/MS, a liquid extraction was carried out. Briefly, individual aliquots of 50 μL of plasma samples were dissolved 1:1 in acetonitrile–water solution (t-test as well as ANOVA as appropriate. p values of <0.05 were considered significant.Normality of data was checked using the Shapiro-Wilk normality test. Non-parametric tests were used when the data did not pass the normality tests. Differences between groups were analyzed by paired or unpaired


Sweat-collecting patches (hereafter referred to as ‘sweat patches’) were applied to the test persons’ skin. One strand of hair and one sweat patch were collected daily from each patient. Analyses were performed using GC–MS/MS (hair) and LC–MS/MS . After 4 days of skin contact, methadone was detectable in the formerly drug-free hair strands in all 15 study participants. EDDP was detectable in 34 of 75 hair strands, with the maximum number of positive results (11 EDDP-positive hair strands) being detected after 5 days. These results show that transfer of methadone and EDDP to drug-free hair is possible through close skin contact with individuals taking part in methadone substitution programmes. A correlation between serum concentration, sweat concentration and substance concentration in hair strands could not be demonstrated, but a tendency towards higher concentrations due to longer contact time is clearly evident.In cases where there is a question as to whether children have come into contact with drugs, examinations of their scalp hair are frequently carried out. Positive test results are often discussed in the forensic community due to the various possible modes via which drugs and their metabolites can be incorporated into the hair. These include drug uptake by the child , but also contamination of hair via contact with the sweat from drug users. In this study, the possibility of methadone and its metabolite EDDP being incorporated into children’s hair by contact with sweat from persons undergoing opiate maintenance therapy (methadone) was examined. The transfer of methadone and EDDP via sweat from methadone patients ( Cases concerning the detection of drugs and metabolites in children´s hair are frequently discussed within the forensic and toxicological science community, as well as receiving attention from the media and political arenas , 2. In 2Excessive sweating, known to be a side effect of methadone therapy, has resulted in both methadone and EDDP  being detected in the sweat of substitution patients . Under sAccording to Kintz et al. sweat contamination could be considered one reason resulting in positive hair tests in children . NeverthThe transfer, via sweat from substitution patients to a child’s hair, of racemic methadone  or levomethadone (l-methadone) and their shared metabolite EDDP, was simulated by means of test subjects being exposed to prolonged dermal contact with drug-free children’s head hair. The children’s head hair, donated by the Bundesverband für Zweithaar-Spezialisten e.V., when the hair length was insufficient for the production of wigs, received ethical committee approval for its use in the study.Hair/skin contact was achieved via mesh-encased belted pouches containing five strands (average weight of 0.7 g per strand) of drug-free children’s head hair (checked in advance by GC–MS/MS for the absence of methadone and EDDP) Fig. . The belAdditional sweat-absorbing patches  were placed on the test subject’s skin (on their back) in order to collect the subject’s sweat in a ‘pure’ form .A blood sample was collected from each participant at the beginning of the study prior to ingesting their daily dose of d,l-methadone/l-methadone. The sample was analysed with regard to methadone and EDDP blood concentrations. Hair samples were not taken from the participants as no consent had been obtained for hair sampling .The final study group consisted of 15 persons ; each of whom had been in a methadone substitution programme for between two to 15 years. Eleven subjects received l-methadone solution  and 4 subjects received d,l-methadone as Methaddict® HEXAL tablets . For an overview of the study group, see Table Hair strand from hair-pouch: one hair strand each day, from day 2 to day 6 of the study period.Sweat patch: one sweat patch each day, from day 2 to day 6 of the study period.Blood sample (serum tubes): one sample on day 1, prior to consuming the daily dose of d,l-methadone or l-methadone, respectively.The blood specimen (serum), drug-free hair and exposed hair from hair pouches were prepared for toxicological analysis and tested for the presence and concentration of methadone and EDDP by validated and court-approved chromatographic analysis procedures . For the determination of methadone and EDDP in sweat (patches), a method was established and validated, based on the method published by Brunet et al. 2008 . An enanCertified reference standards of d,l-methadone and EDDP (each 1 mg/mL in methanol) as well as deuterated standards  were purchased from Lipomed AG . For calibration purposes, standard solution mixtures and internal standard solutions  were prepared in methanol.Methanol , 2-propanol (HPLC grade), dichloromethane , water (Ultra LC–MS grade), acetonitrile (Ultra LC–MS grade) and lactic acid 80% were supplied by Carl Roth . Ethyl acetate (CHROMASOLV Plus HPLC 99.9%) was purchased from Honeywell , and formic acid (LC–MS grade) from Thermo Scientific .2PO4) and sodium dihydrogen phosphate monohydrate (NaH2PO4*H2O) provided by Merck . Urea (99.5%) was purchased from GE Healthcare Bio-Science AB .All other chemicals were of analytical grade: ammonium hydroxide solution (25%), 0.1 M hydrochloric acid (titration solution) and acetic acid (100%) provided by Carl Roth; sodium hydroxide (pure pellets), sodium chloride, ammonium chloride, sodium acetate, potassium dihydrogen phosphate (KH2PO4*H2O in bi-distilled water and the pH was adjusted to 6.0 by adding 1 M NaOH (utilising a pH electrode). 0.5 M sodium acetate buffer (pH 4.0) was made up of 0.089 mol sodium acetate and 0.411 mol acetic acid in bi-distilled water and the pH was adjusted to 4.0 by adding 1 M acetic acid solution . Artificial sweat solution was prepared according to Brunet et al. (2008) [0.1 M and 1 M acetic acid solutions (HAc), as well as 1 M and 2 M NaOH solutions, were prepared in bi-distilled water. 0.1 M phosphate buffer was made up of NaH. (2008) . High-puSweat patches (PharmCheck Sweat Patch Kit) were purchased from PharmChem Inc. .Drug-free children’s scalp hair was provided by an organisation that collects hair donations from children to make wigs for children who have lost their hair as a result of cancer treatment . The hair, provided from four donors, was of insufficient length to manufacture wigs. This hair was analysed for the absence of drugs of abuse prior to it being used in this study. Strands of one pouch each came from a single source donation.Validations were conducted according to guidelines of the Society of Toxicological and Forensic Chemistry (GTFCh) , 15. In Testing for linearity was done by analysing the calibration in 2 (sweat patches), 5 (hair samples) or 6 (serum samples) replicates, followed by evaluating linearity by the Mandel test, outliers by the Grubbs test and variance homogeneity by the Cochran test (each 99% level of significance). Accuracy and imprecision were evaluated over the linear range using two QC samples at higher and lower concentrations of d,l-methadone and EDDP. QC samples were measured on 3 (patches), 5 (hair) or 8 (serum) samples on different days in duplicates. The determination of the concentrations was based on a daily calibration.Additionally, a possible generation of EDDP formation from methadone by hot GC inlet, as described elsewhere for urine testing, was evaluated . At the All statistical evaluations were undertaken using the Valistat software version 2.04 provided by Arvecon , or Microsoft® Excel 2016.Quantification in serum was performed by a validated routine method used to determine several benzodiazepines, z-drugs, d,l-methadone and their metabolites in a target screening by LC–MS/MS combined with a MultiPurposeSampler (MPS). For this study, only d,l-methadone and EDDP in serum were analysed. Briefly, after centrifugation, 0.25 mL serum supernatant were separated, to which 1 mL 0.1 M phosphate buffer (pH 6.0) and 25 µL of the internal standard mixture of d,l-methadon-d9 and EDDP-d3  were added. Further sample preparation was done fully automatically by the MPS device. Conditioning of SPE cartridges  was by MeOH, high-purity water and 0.1 M phosphate buffer. After sample loading, cartridges were washed with phosphate buffer, MeOH/water , 0.1 M hydrochloric acid, an additional time with MeOH/water (30/70) and finally with 0.1 mL MeOH followed by drying with nitrogen. Analytes were eluted with ethyl acetate/ammonium hydroxide 25% . After evaporation, the residue was dissolved with water (Ultra LC–MS grade) and acetonitrile . Five microlitres were injected into the LC–MS/MS system.Analysis of serum extracts was done using an HPLC system (model 1200SL) and a tandem mass spectrometer (model 6460A) from Agilent Technologies , equipped with electrospray ionisation (ESI) source and integrated jet stream technology. MassHunter Workstation Software LC/QQQ Data Acquisition  was used to control the system. SPE was conducted fully automatically using a MPS Autosampler controlled by the Maestro Software .Chromatographic separation of serum extracts was performed with a Nucleoshell RP 18 EC  column . As mobile phase, water (Ultra LC–MS grade) with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B) was used. Initial conditions were 10% of solvent B, increased over 7 min to 50%, and further increased over 4 min to 90%, where it was held for 2 min. The system was returned to initial conditions and held for a further 2 min, resulting in a total runtime of 20 min.ESI was operated in positive mode and parameters were set to 3500 V capillary voltage, 325 °C gas temperature, 12 L/min sheath gas flow, 45 psi nebulizer, 375 °C sheath gas temperature, 10 L/min gas flow and 500 V nozzle voltage. Nitrogen was used as collision gas, applying different collision energies (CE). Quantifier transitions were 310.2 > 265.1 (CE 9 V) for methadone, 319.3 ≥ 268.2 (CE 9 V) methadone-d9 as well as 278.2 ≥ 249.1 (CE 21 V) for EDDP, 282.2 ≥ 235.2 (CE 29 V) EDDP-d3. Qualifier transitions were 310.2 ≥ 105 (CE 25 V) for methadone, as well as 278.2 ≥ 234.1 (CE 29 V) for EDDP.Quantification of d,l-methadone and EDDP in serum samples was implemented with a calibration range from 25 to 500 µg/L. During validation, a LOD of 6.8 and 6.3 µg/L for d,l-methadone and EDDP, respectively, as well as a LOQ of 25 µg/L for the detection of both analytes, was determined.The external QC samples (OSD level 2 and level 4) were purchased from Medichem .Sweat patches were folded and placed in a test tube to which sodium acetate buffer (pH 4.0) was added. Sealed tubes were shaken on a horizontal shaker for 10 min. Following this, the buffer was transferred into a further test tube, the extraction procedure with buffer was repeated and the aliquots were combined. Conditioning of SPE cartridges  was by MeOH and sodium acetate buffer. After sample loading, cartridges were washed with sodium acetate buffer, MeOH/water , 0.1 M hydrochloric acid, an additional time with MeOH/water (30/70), then 0.1 mL MeOH followed by drying in nitrogen. Analytes were eluted using ethyl acetate/25% ammonium hydroxide . Eluates were evaporated to dryness with nitrogen at room temperature. Dried residues were dissolved in water (Ultra LC–MS grade) and acetonitrile . Five microlitres were injected into the LC–MS/MS system.The LC–MS/MS system and software were identical to that used in the analysis of serum . The chromatographic conditions, as well as ionisation settings and transitions, were identical to the quantification of serum . Blank sweat patches were evenly moistened with 750 µL of artificial sweat and allowed to dry for 2 h at room temperature.One hundred microlitres of QC solutions, working solution and internal standard solution were added to sweat patches. QC solutions were separately diluted, daily prepared and added to blank sweat patches to yield a total of 15 ng, 400 ng for d,l-methadone and 7.5 ng, 200 ng for EDDP.Deuterated standards were diluted to a concentration of 20 ng/patch and 10 ng/patch for d,l-methadone-d9 and EDDP-d3 respectively.During validation, calibration curves were freshly prepared by spiking sweat patches or elution solvent (ethyl acetate/25% ammonium hydroxide); sweat patches were premoistened with artificial sweat and with a working solution to obtain 5 to 1,000 ng/patch for d,l-methadone and 2.5 to 500 ng/patch for EDDP. The relative response of matrix calibration and solvent calibration was found to be comparable. Subsequently, solvent calibration curves were used as the number of sweat patches was restricted and the usage of solvent calibration was more practical.One half of each hair strand was used; the other part was retained for any necessary reanalyses. The first aliquot used the whole length of strand, washed two times with 5 mL DCM each and dried room temperature (approx. 20 min). Subsequent grinding was performed up to 5 min in a ball mill  with stainless steel balls. Twenty-five milligrammes of the powdered hair were transferred to a test tube, fortified with 25 µL ISTD solution . For hair extraction, 1 mL of 0.1 M HCl was added and closed tubes were placed in an ultrasonic bath for 3 h. After centrifugation, supernatants were neutralised by adding 1 M NaOH and adjusted to pH 6.0 with phosphate buffer.Clean-up of the prepared hair samples was done by SPE with Strata Screen-C cartridges  following an in-house protocol. In brief, sorbent was conditioned with MeOH and 0.1 M phosphate buffer; after the sample load, it was washed with 0.1 M HAc, high-purity water and MeOH, dried and eluted with alkaline elution mixture ). The extracts were then evaporated to dryness with nitrogen at 30 °C and reconstituted in 25 µL ethyl acetate. Prepared sample extracts were carefully crimped into amber glass vials.Sample SPE clean-up of hair samples was performed in an automated ASPEC GX 274 System with two 406 Dual Syringe Pumps .Measurements of hair sample extracts were conducted on a 7890A / 7000B gas chromatography-tandem mass spectrometry (GC–MS/MS) system equipped with a Split/Splitless inlet . A deactivated 4.0 mm ID Single Taper Liner with CarboFrit  and a GC column Zebron ZB-5MSi 15 m × 0.25 mm × 0.25 µm (Phenomenex) were used. The MassHunter Workstation Software GC/QQQ Data Acquisition  was used to control the GC–MS/MS-system.One microliter of the reconstituted extract was injected splitlessly at 200 °C for transfer onto the GC column. Separation was performed with a constant helium flow of 1.1 mL/min and the following temperature program: 80 °C (1 min), 15 °C/min, 290 °C (3 min), resulting in a total GC run time of 18 min. Nitrogen was used as the collision gas. Quantifier transitions were 72 ≥ 56 (CE 20 V) for methadone, 78 ≥ 59 (CE 20 V) methadone-d9 as well as 277 ≥ 220 (CE 20 V) for EDDP, 280 ≥ 220 (CE 20 V) EDDP-d3. Qualifier transitions were 294 ≥ 223 (CE 10 V) for methadone, 303 ≥ 226 (CE 10 V) methadone-d9 as well as 262 ≥ 170 (CE 20 V) for EDDP, 265 ≥ 171 (CE 20 V) EDDP-d3.Quantification of d,l-methadone and EDDP in hair samples was implemented within a calibration range of 50 to 5000 pg/mg. Pooled blank hair fortified with calibration standards and ISTD was used for calibration. Matrix blank samples that had been tested as negative were taken from volunteers of the Institute of Legal Medicine . Each sample sequence contained a blank hair sample (including ISTD) as well as external and internal QC samples. The external QC sample (DHF 1/18-B) was purchased from ACQ SCIENCE . An internal QC sample, consisting of pooled drug-positive hair , was prepared by the Institute of Legal Medicine . During validation, a LOQ of 50 pg/mg and a LOD of 25 pg/mg for the detection of d,l-methadone and EDDP were determined. Accuracy of quantitation above an upper limit of detection, by means of linear extrapolation, was demonstrated for 10,000 and 15,000 pg/mg (96.5–99.9%).The results of the analyses of hair and sweat patches were recorded and are published here in anonymous or pseudonymised form and have been compared with data obtained from test subjects’ blood samples and information about body weight and daily dose of d,l-methadone/l-methadone.All 15 persons completed the 6-day study phase. Beginning at the second study day (day 1), one strand of hair from the hair pouch and one sweat patch were collected each day Fig. , in ordeR2 = 0.4194). Moreover, persons with the same dose  showed clearly different blood concentrations.The concentrations of methadone were in the range of 15.6 µg/L to 633.5 µg/L (mean = 282.6 µg/L). The dosage of methadone ranges from 0.1 to 2.06 mg/kg of body weight. There was no clear correlation between methadone dosage and the concentrations of methadone and EDDP in the blood. The participant with the lowest dose of 0.1 mg l-methadone/kg had the lowest blood concentrations of methadone and EDDP. However, the person with the highest dose of 2.06 mg methadone/kg did not have the highest blood concentrations . However, no other influence could be found between BMI and the amount of methadone and EDDP per patch.In general, an increase in the amount of methadone per sweat patch from day 2 to 6 could be observed when looking at the data from individual participants. However, in 7 persons, there are one or two outliers each, which do not strictly follow this trend. In 10 subjects, the highest amount of methadone per sweat patch was present on day 6, as expected. In 4 subjects, the highest dosage was already observed on day 4 and in one subject on day 5. Exemplary data of methadone and EDDP concentrations in sweat patches over the study period can be seen in Fig. The amounts of EDDP per sweat patch were clearly lower than the amounts of methadone. They varied less than the methadone amounts and did not follow a clear trend. The highest levels of EDDP per sweat patch were found in only 6 people on the last day of the study. The median ratios of methadone to EDDP per sweat patch for all persons and days ranged from 15 to 402. However, within one person, the ratios of methadone to EDDP per sweat patch varied less Table . One couAfter 1 day of skin contact with the study participants, methadone was already detectable in 10 of 15 hair samples. After 4 days, methadone was detectable in the hair samples of all 15 study participants. Observation of EDDP provides a different picture: After 1 day of skin contact, EDDP could already be detected in 3 samples; the maximum of 11 EDDP-positive hair samples could be measured after 5 days of skin contact. In total, EDDP was detectable above the LOD in 34 of 75 hair samples . For the remaining sample series, there were several (between 1 and 3) concentrations that did not follow the rising trend.The ratio of methadone to EDDP in hair could only be calculated in those samples where EDDP was also detectable. EDDP was not detectable in 41 of the 75 hair samples. Therefore, no methadone to EDDP ratio could be determined for 4 study participants  (Table R2 = 0.4631).In the EDDP-positive hair samples of the other 11 study participants, the ratios of methadone to EDDP were in a markedly narrower range compared to those in the sweat patches. The mean values varied between 13 and 31. A moderate correlation could be seen between hair and patch ratios in the 9 patients in which both were calculated  of methadone to EDDP in the sweat patches. The other two of these 4 study participants had methadone to EDDP ratios between 100 and 200 in the sweat patches.The present collective is too small for statistically sound statements. However, all results show a tendency and are indicative.It has been suggested that drugs may be incorporated into hair: (i) from the blood during hair formation; (ii) from sweat and sebum after formation and (iii) from the external environment after formation and after the hair has emerged from the skin . It mustOur investigations have shown that the transfer of methadone and its metabolite EDDP to previously drug-negative hair is possible through close skin/sweat contact with persons treated with methadone. This finding is of great significance for the interpretation of the results of analyses on children’s hair. If the children have close physical contact with methadone substitution patients or methadone ingesting persons, e.g., with their parents, contamination of the child’s hair by methadone- and EDDP-containing sweat is clearly possible. The assumption that the detection of the metabolite EDDP in a child’s hair indicates that the child may have ingested methadone themselves, as has been previously expressed , is thusA correlation between methadone dosage and the concentrations of methadone and EDDP found in the blood samples of study participants could not be established. This observation was also made in previous studies and can be explained by a high inter-individual variance in pharmacokinetic properties , 20. HowIn the hair, the reported ranges of methadone concentrations were between 0.25 and 80.8 ng/mg; the ranges of EDDP between 0.05 and 7.76 ng/mg. The corresponding ratios in those studies (methadone-to-EDDP) were calculated as between 1.4 and 32.2 , 23–25. For the sweat patches, comparable data for methadone and EDDP are rare. Fucci et al. found 120 to 2160 ng methadone and 25–535 ng EDDP/patch in 10 patients . GambeluAn additional aim of our study was to examine if the concentrations or ratios in hair due to sweat contamination would be different to the concentrations or ratios resulting from the ingestion of methadone. In the event of such a distinction being observed, a better interpretation of children’s hair results would be possible. As could be shown, there is a relevant overlapping of these levels, making discrimination between ingestion and contamination based on ratios impossible. Although this result is consistent, considering that a relevant part of methadone and EDDP in hair may be the result of sweat from the individual .To examine the applicability of our data, results were compared to those from selected real cases of hair analysis in living children found in literature—although few cases were suitable for this kind of comparison—as most of the published results are related to fatal or non-fatal methadone intoxications in children , 7, 18. n = 5 [n = 10 [Although thoroughly conducted, some limitations have to be taken into consideration with respect to our study. Firstly, only 15 patients could be included and a greater cohort would have been desirable. It should be noted that several published study populations were even smaller . Se [n = 10  and even [n = 10 , 27, 28, [n = 10 .The strength of our study is the simultaneous sampling of blood and sweat together with the sweat-contaminated hair samples, giving a comparison of concentrations and ratios of methadone and EDDP. Additionally, this is the first time that the transfer of a drug and its metabolite via sweat into substance-free hair was proven in a structured prospective study. Importantly, we could show that the ratio in sweat and sweat-contaminated hair need not be the same.In summary, the route of transfer of methadone and EDDP by methadone substitution patients to drug-free hair could be proven. The question of the transfer route by sweat and close contact also arises, however, for all substances (of abuse) . Very liOur study has shown that an external contamination by transfer of methadone and EPPD from sweat into hair matrix is possible, although there was no correlation found between blood concentrations, sweat concentrations and substance concentrations in hair samples. Following this result, the transfer of methadone and EDDP to children’s hair by sweat from direct contact with drug users is now a plausible explanation for the presence of these substances in children’s hair. Detection of the metabolite EDDP does not necessarily prove an oral intake of methadone by the child. Moreover, there is a high inter-individual variability and a relevant overlapping of methadone/EDDP ratio in sweat and in hair. Therefore, it is not possible to draw conclusions as to the source of the methadone in children’s hair. This study demonstrates once more that results of hair analysis of children must be interpreted particularly carefully.


Assessment of measurable residual disease (MRD) identifies small numbers of acute myeloid leukemia (AML) cells that may remain after initiating treatment. The achievement of MRD negativity (no detectable AML cells remaining) typically predicts better outcomes for patients with AML. Some patients with AML have disease characteristics that put them at a higher risk of treatment failure or relapse; while outcomes for patients with high-risk AML are historically poor with traditional chemotherapy regimens, newer chemotherapy formulations  and targeted therapies may be more effective in achieving MRD negativity in these patients. Currently, there is no agreement on the best method for determining whether a patient has achieved MRD negativity, and the use of several different methods makes it difficult to compare outcomes across studies. Despite these challenges, regular monitoring of patients for the achievement of MRD negativity will become increasingly important in the routine management of patients with high-risk AML.Mounting evidence suggests measurable residual disease (MRD) assessments are prognostic in acute myeloid leukemia (AML). High-risk AML encompasses a subset of AML with poor response to therapy and prognosis, with features such as therapy-related AML, an antecedent hematologic disorder, extramedullary disease (in adults), and selected mutations and cytogenetic abnormalities. Historically, few patients with high-risk AML achieved deep and durable remission with conventional chemotherapy; however, newer agents might be more effective in achieving MRD-negative remission. CPX-351  demonstrated MRD-negativity rates of 36–64% across retrospective studies in adults with newly diagnosed high-risk AML and 84% in pediatric patients with first-relapse AML. Venetoclax (BCL2 inhibitor) demonstrated MRD-negativity rates of 33–53% in combination with hypomethylating agents for high-risk subgroups in studies of older adults with newly diagnosed AML who were ineligible for intensive therapy and 65% in combination with chemotherapy in pediatric patients with relapsed/refractory AML. However, there is no consensus on optimal MRD methodology in AML, and the use of different techniques, sample sources, sensitivity thresholds, and the timing of assessments limit comparisons across studies. Robust MRD analyses are needed in future clinical studies, and MRD monitoring should become a routine aspect of AML management. Morphologic assessments are often used to determine response to treatment in acute myeloid leukemia (AML) . HoweverIDH1/2 and DNMT3A and pediatric patients having a higher prevalence of cryptic fusions and translocations [High-risk AML encompasses a subset of AML with poor response to therapy and prognosis. High-risk features are defined by cytogenetic and molecular features, as well as response to therapy. Recent advances in genomic testing have identified that pediatric and adult AML populations are biologically distinct, with adults having a higher prevalence of mutations such as ocations . Howeverocations ; AML witocations . In addiocations ,10. Manyocations ,18,19,20In this article, we review the importance and the current landscape of MRD assessment in patients with high-risk AML, focusing on the adult subsets of therapy-related AML, AML with myelodysplasia-related changes (including an antecedent hematologic disorder), and adverse-risk AML per the European LeukemiaNet and National Comprehensive Cancer Network classifications, and on pediatric patients regarding the protocols for high-risk disease. Other high-risk AML subtypes are not discussed due to the lack of MRD data.Various techniques are currently utilized to assess MRD in AML, and these are summarized in NPM1 mutation), whereas next-generation sequencing (NGS) examines a panel of genes of interest at once [DNMT3A, TET2, and ASXL1), CHIP , or in pre-leukemic diseases such as myelodysplastic syndrome may not reflect residual AML [Molecular approaches are also frequently used to assess MRD: polymerase chain reaction (PCR)-based methods  monitor changes in particular genes where real-time quantitative PCR (qPCR) platforms are developed .Several new therapies were approved for the treatment of AML in recent years, many of which may induce deeper responses, sparking an interest in assessing MRD to improve the management of patients with AML. Numerous studies have since demonstrated the predictive nature of MRD assessment in patients with AML both in adult and in pediatric patient populations; these studies are summarized in Several studies also demonstrated that adult and pediatric patients with AML who retain MRD positivity after induction treatment have worse outcomes following HCT compared to patients with MRD negativity . For exaSince MRD assessments are not routinely used in AML, there are inconsistencies in procedure between studies, complicating inter-study comparisons. The sample source often differs between studies; while some studies perform MRD assessment on peripheral blood, others use bone marrow samples . AlthougThere is a large degree of variability regarding the timing of MRD assessments used in clinical studies . At thisThere is also no standardized, predictive threshold for MRD negativity, with different thresholds used for different MRD methods . The EurAlthough intensive chemotherapy followed by HCT is generally recognized as offering the best chance for long-term remission, historically, few patients with high-risk AML have achieved deep and durable remission with conventional chemotherapy regimens. Some novel chemotherapy formulations and regimens may be more effective in achieving MRD-negative remission in this population. The combination of targeted therapies, such as FLT3 inhibitors, with intensive chemotherapy is also a promising strategy to improve the depth of remission and outcomes in patients who have targetable mutations. Refinements in HCT conditioning regimens may also help to deepen and/or prolong remissions prior to and after transplantation. Unfortunately, limited MRD data are published for patients with high-risk subtypes of AML .WT1 qPCR (n = 38) methods, respectively. Although not statistically significant, a trend for MRD negativity and improved 12-month CIR  was observed; however, 12-month OS was 71% versus 84% for MRD-negative and MRD-positive patients, respectively (p = 0.414) [−3), including 8/14 (57%) patients who had available MRD data and proceeded to HCT; so far, OS among transplanted patients was not significantly different between patients with versus without MRD negativity [−3); further, all 23 patients proceeded to HCT and continued to exhibit MRD negativity at the time of HCT [p = 0.01) and among patients who proceeded to HCT (p = 0.02), but not in a multivariable analysis for OS [TP53 (36% vs. 8%) [CPX-351 is a dual-drug liposomal encapsulation of daunorubicin and cytarabine in a 1:5 molar drug ratio. Based on results from a phase 3 study of CPX-351 versus conventional 7 + 3 chemotherapy in older adults (aged 60 to 75 years) with newly diagnosed secondary AML, CPX-351 was approved for the treatment of newly diagnosed therapy-related AML and AML with myelodysplasia-related changes (AML-MRC). The phase 3 study protocol, unfortunately, did not include MRD assessment; however, a few non-randomized studies with relatively small sample sizes have assessed MRD status after CPX-351 treatment. In an Italian compassionate use program of CPX-351 in older adults with newly diagnosed therapy-related AML or AML-MRC , MRD neg= 0.414) . In a Fr= 0.414) , 28 patigativity . In a Ges for OS . Finally vs. 8%) .In the Children’s Oncology Group (COG) AAML1421 study of pediatric patients with AML in first relapse who were treated with CPX-351 followed by FLAG chemotherapy , 16 patients achieved CR or CR with incomplete platelet recovery (CRp) after an initial cycle of CPX-351 treatment, 12 (75%) of whom achieved MRD negativity (based on MFC) . Of the In the phase 3 study of CPX-351 versus conventional 7 + 3 chemotherapy, CPX-351 improved median OS among patients who achieved CR or CRi, as well as the likelihood of proceeding to HCT and median OS landmarked from the date of HCT , suggest−3 based on MFC) occurred in 67/164 (41%) of evaluable patients who achieved CR or CRi, including 18/44 (41%) patients with secondary AML and 16/48 (33%) patients with poor-risk cytogenetics [−3, and these results were confirmed among patients with secondary AML or poor-risk cytogenetics [Venetoclax is an oral BCL2 inhibitor approved in combination with a hypomethylating agent or low-dose cytarabine (the United States only) for the treatment of newly diagnosed AML in adults who are considered ineligible for intensive induction chemotherapy. Although treatment with intensive chemotherapy followed by HCT provides the greatest likelihood of achieving long-term remission, some patients do achieve MRD negativity with less-intensive therapies. The phase 3 VIALE-A trial evaluated venetoclax combined with azacitidine (n = 286) versus azacitidine alone n = 145) in adult patients with newly diagnosed AML who were considered ineligible for intensive chemotherapy; 25% of patients had secondary AML, 33% had AML-MRC, and 37% had poor-risk cytogenetics . In thisgenetics . Those wgenetics . In a st in adultIn a phase 1 study in pediatric patients with relapsed/refractory AML who were treated with venetoclax in combination with cytarabine with or without idarubicin, 20 patients achieved CR or CRi, 13 of whom achieved MRD negativity  .Although MRD data are available for venetoclax combination regimens, including data from a randomized study, these data are for a mixed population that includes a large proportion of patients who were not identified as having high-risk subtypes of AML. Further, these patients were also identified as not being suitable for intensive chemotherapy, where the relevance of MRD as a surrogate endpoint is still to be determined. Similar to the case with CPX-351, robust MRD data are needed for venetoclax regimens to help guide treatment decisions for this population.In a study of 244 patients with high-risk AML or myelodysplastic syndrome undergoing their first HCT, sequential transplantation regimen FLAMSA-Bu (fludarabine/amsacrine/cytarabine-busulfan) was compared with a fludarabine-based reduced-intensity conditioning regimen . MRD posThe objective of maintenance therapy is to maintain MRD negativity, reduce the risk of relapse, and prolong survival. The phase 3 QUAZAR AML-001 trial found that CC-486  maintenance therapy significantly prolonged OS and relapse-free survival versus placebo (n = 234) irrespective of MRD status in patients with AML who were in the first remission after intensive chemotherapy . While mAlthough recent evidence suggests MRD assessments can be predictive of clinical outcomes in patients with favorable- or intermediate-risk AML, the results for patients with high-risk disease remain controversial and warrant further investigation. Additional studies are also needed to optimize and standardize the methodology and timing of MRD assessment in patients with AML, particularly those with high-risk disease. Further, the relevance of MRD as a prognostic indicator may be different in patients with high-risk AML who receive less-intensive therapy instead of intensive chemotherapy as induction due to fitness status. While MRD data were inconsistently collected in past clinical trials in AML, future clinical trials should include robust MRD analyses, as it is likely that CR with MRD negativity will become a key efficacy endpoint in the new drug era. MRD monitoring should also become a more routine aspect of AML management outside of clinical trials, as it can provide important information on patient outcomes and help to inform treatment decisions, such as the selection of HCT conditioning regimen and the need for post-HCT strategies in patients with MRD-positive disease. Achieving MRD negativity should be the main goal of novel therapies, and combination regimens may be more effective at inducing MRD negativity.


Arabidopsis wild-type, cpk5 mutant and CPK5-overexpressing plants (exhibiting constitutive priming) by introgressing the calcium indicator R-GECO1-mTurquoise that allows internal normalization through mTurquoise fluorescence. Aequorin-based analyses were included for comparison. Based on the R-GECO1-mTurquoise data, CPK5-OE appears to reinforce an “oscillatory-like” Ca2+ signature in flg22-treated local tissues. However, no change was observed in the flg22-induced calcium response in the systemic tissues of plants that had been pre-challenged by a priming stimulus – neither in wild-type nor in cpk5 or CPK5-OE-lines. These data indicate that the mechanistic manifestation of a plant immune memory in distal plant parts required for enhanced pathogen resistance does not include changes in rapid calcium signaling upstream of CPK5 but rather relies on downstream defense responses.Upon pathogen recognition, a transient rise in cytoplasmic calcium levels is one of the earliest events in plants and a prerequisite for defense initiation and signal propagation from a local site to systemic plant tissues. However, it is unclear if calcium signaling differs in the context of priming: Do plants exposed to a first pathogen stimulus and have consequently established systemic acquired resistance (SAR) display altered calcium responses to a second pathogen stimulus? Several calcium indicator systems including aequorin, YC3.6 or R-GECO1 have been used to document local calcium responses to the bacterial flg22 peptide but systemic calcium imaging within a single plant remains a technical challenge. Here, we report on an experimental approach to monitor flg22-induced calcium responses in systemic leaves of primed plants. The calcium-dependent protein kinase CPK5 is a key calcium sensor and regulator of the NADPH oxidase RBOHD and plays a role in the systemic calcium-ROS signal propagation. We therefore compared flg22-induced cytoplasmic calcium changes in  Plants that experience a primary attack by microbial pathogens not only induce rapid local immune responses but are also able to build a more long-term systemic immune memory, the so-called systemic acquired resistance (SAR). Primed plants are sensitized toward a secondary infection and often induce faster and stronger immune reactions that ultimately impede pathogen growth. Defense priming requires an initial recognition of a pathogen , the initiation of local immune reactions, a defense signal propagation from local to distal sites of the plant (systemic tissues), and the onset and maintenance of SAR involving phytohormone-dependent changes in gene expression and defense metabolite synthesis .2+]cyt) is one of the earliest events in plant cells upon pathogen recognition, and many studies applying different calcium indicators reported on distinct [Ca2+]cyt patterns in response to PAMPs, e.g., bacterial flg22 -based transcriptional reprogramming, or on epigenetic modifications. But it is unclear whether and how calcium signaling contributes to the mechanism necessary to acquire an immune memory. Do plants that have experienced a first priming pathogen stimulus and have consequently established SAR display a different [Ca2+]cyt change pattern in response to perception and recognition of a second triggering pathogen stimulus?A transient change of the cytoplasmic calcium concentration  systems such as aequorin, cameleon YC3.6 or more recently GCaMP/R-GECO1 and their derived variants , in which a calcium sensor and protein kinase effector domain are united within a single molecule (Kd ∼100 nM) for enzyme activation. This is near the resting [Ca2+]cyt of an un-challenged plant cell, thus rendering the enzyme highly responsive to small [Ca2+]cyt changes , and of the phytohormone SA  stimulus.In plant cells, changes in the intracellular [Caand CPKs . Among tmolecule . Severalmolecule . Among t changes . Besides changes . Furtherrmone SA . When chount SAR . CPK5 is2+]cyt calculation bias if GECI levels differ between lines or during time course of experiments  upon flg22 stimulation did not significantly differ between plants of different priming status – either through pre-exposure to flg22 priming stimulus, “super-priming” (through overexpressing CPK5) or reduced priming (cpk5 background). These data indicate that the systemic immune signaling causal to enhanced pathogen resistance in SAR does not employ alterations in the early [Ca2+]cyt change but relies on downstream defense processes.The GECI R-GECO1 has been successfully used in live cell imaging to resolve PAMP-triggered calcium transients with a high sensitivity but, due to its intensiometric fluorescence readout, suffered from a  sucrose, 0.8% [w/v] phytoagar (Duchefa), pH 5.7 adjusted with KOH, or grown in soil. Seeds were stratified for 2 days at 4°C, and after transfer to individual pots or jiffy-7 soil , the plants were maintained under short-day conditions . Seedlings carrying constructs with fluorophores were screened for fluorescence of the respective fluorescence protein using a fluorescence stereo zoom microscope  before the transfer to individual pots. Plants were grown for a total of 6 weeks under short day condition. For aequorin-based calcium assays, seeds were first surface sterilized and stratified for 2 days at 4°C and grown in liquid MS medium for 8–10 days under long day conditions . Flg22 and an inactive flagellin variant from Agrobacterium tumefaciens were synthesized on an in-house peptide synthesizer and used as previously described  Colombia-0 wildtype plants were transformed by floral dip method. Plasmid pGGZ-RW253 (pUBQ10-N-decoy-R-GECO1-GSL-mTurquoise-tHSP18.2M-HygR) was generated via GreenGate cloning using previously published modules (RGmT lines were harvested and crossed with the mutants cpk5 (SAIL_657C06), rbohD (SALK_070610), and fls2 (SALK_062054) and the CPK5-overexpressing line CPK5#7 that have been described before (cpk5 (SAIL_657C06) and the CPK5-overexpressing line CPK5#7.For generating transgenic  modules . Seeds od before . The gend before . Seeds o2) and the samples were incubated for recovery in a phyto chamber or growth cabinet overnight. The samples were incubated under light at 22–24°C for at least 1 h before imaging. Confocal laser scanning microscopy was performed in bottom imaging mode on a Zeiss LSM 780 or 880 system (Zeiss) using a 40 × water immersion objective  at a zoom factor of 0.8. 16-bit images were acquired every 4 s with a frame size of 512 × 512 pixels and a pinhole of 200. mTurquoise and R-GECO1 were excited with 458 or 561 nm, respectively and an emission-range between 470 and 540 nm for mTurquoise or 590 and 640 nm for R-GECO1 was used for detection. Gain was set to 850 and laser intensity settings were adjusted individually to have comparable baseline intensity values for each experiment. For flg22 treatments, 50-fold and for ATP treatments 10-fold concentrations of the respective agent were prepared in water and added in a 1:50 or 1:10 volume ratio to the imaging chamber to avoid sample movement. The image processing was performed using Fiji  or 5 min (ATP) base line ratio for each ROI before flg22 or ATP treatment (R/R0). For each ROI the following parameters were analyzed: maximal signal change after treatment; time until maximal signal change; number of local maxima, and time between first two local maxima. ROI_1 and the selected ROIS with the highest number of local maxima were chosen for comparison of different genotypes. The graphs of single ROIs were generated in R, using R Studio  incl. the packages tidyverse and reshape2  were coated with a thin layer of medical adhesive . After 10 min of evaporation of volatile medical adhesive components excess medical adhesive was removed by washing three times with water. The leaf abaxial epidermis was then glued to the medical adhesive layer, and magic tape  was taped to the adaxial epidermis and pulled away. Mesophyll cell layers were then removed carefully by affixing and pulling away with magic tape, leaving a single-layer of lower epidermis behind that was attached to the IBIDI slide. Immediately the coverslip was immersed with 1 ml plant imaging buffer  using a 1 × objective and a zoom factor of 10. Optical filters for mTurquoise  with an exposure time of 1.5 s and for R-GECO1  exposure time of 1.8 s were used for fluorescence detection. For image processing the following steps were conducted for R-GECO1 and mT channels using Fiji: Gaussian blur filter set to 1, conversion to 32 bit, threshold adjustment (stack histogram) and the mean gray values of the whole image area were used for calculating fluorescence intensities. Analyses revealed higher signal intensities for R-GECO1 and mTurquoise channels in the CPK5#7 crossed line, probably indicating a higher protein abundance    were mix2) or 200 nM flg22 as priming-stimulus using a needleless syringe. After 48 h, a 4 mm diameter leaf disk was harvested from a systemic leaf and fixed adaxial on a 8 well chambered coverslip (IBIDI) using medical adhesive (Ulrich Swiss) and immersed with distilled water. Selection of local and systemic leaves as described in 0 = F0) via dividing each of the following created 600 images over the R-GECO1 signal at time point t0 (F/F0) using the image calculator tool in Fiji. The resulting mean gray value was used for further calculations. The RGmT ratio in the beginning and at the end of the assay was calculated via dividing R-GECO1 mean gray values by mTurquoise mean gray values using the acquired raw data.Three fully developed “local” leaves of 6-week-old plants were infiltrated with either mock-treatment  (max) are shown. An aequorin-expressing line in the rbohD background  . To facickground  served aLeaf material was homogenized in liquid nitrogen in a laboratory mill and incubated with a 2:1 weight ratio of 2x SDS sample buffer  glycerol; 2% (w/v) SDS; 0.004% (w/v) bromophenol blue) for 5 min at 95°C for subsequent SDS-Page western blotting and immunodetection. For RGmT detection the α-RFP  primary antibody was used. For visualization a HRP-conjugated secondary α-rabbit-HRP antibody  was applied. Blots were developed using enhanced chemiluminescence  detection.RNA, isolated with Trizol reagent, was treated with DNAseI (to remove any remaining DNA contamination) and cDNA was synthesized using the RevertAid kit (Thermo Scientific). For realtime quantitative PCR (qPCR), amplicons from cDNA samples were analyzed on an Mx3005P qPCR system  after amplification with Maxima SYBR Green qPCR Master Mixes (Thermo Scientific). Relative gene expression values of the genes-of-interest were calculated with the comparative CT method  using th2+]cyt changes in response flg22 perception, we generated Ca2+ reporter lines expressing R-GECO1-mTurquoise (RGmT) in cpk5 and CPK5-OE (CPK5#7) backgrounds by crossing. Since all GECI lines have been reported to exhibit some level of reduced growth  of 6 ± 2 cells for improved resolution of the Ca2+ dynamics  over 40 min, and a more pronounced spiky response for ROIS (2+ response is specific since no flg22-induced rise in [Ca2+]cyt was detected in the fls2 receptor mutant (2+ response traces between experiments (2+ peak is 9.2 ± 5.8 min (n = 36). Here, the large variation in onset of Ca2+ signaling may be attributable to technical restrictions related to poor flg22 diffusion/accessibility in the tight space between the leaf surface and the coverslip in the bottom imaging mode. Interestingly, however, traces of the overexpressing CPK5#7xRGmT line exhibited a distinct Ca2+ signature characterized by defined peaks and longer intervals between the peaks  the number of peaks within the 40 min recording time, (2) the maximum signal change (amplitude), and (3) the time between the first and second peak. A distribution plot showed a shift toward Ca2+ traces with an increased “number of peaks” in the CPK5#7xRGmT line, which is consistent for both ROI_1 and ROIS  is generally not affected by cpk5 mutation or CPK5 overexpression. However, a significant reduction in the Ca2+ amplitude in the CPK5#7 line and an increase in the cpk5 mutant was observed .We next compared the flg22-induced Ca0–25 min . The ove0–25 min . When thohD line  was usedobserved . Taken t2+ response when exposed to a secondary flg22 trigger. As illustrated in the scheme in 2 as a mock control). After 2 days incubation in a growth chamber, a disk from a systemic leaf was sampled and mounted on a microscope chamber slide, incubated again over-night in a growth cabinet for recovery, and incubated for 1 h in light before the second flg22 treatment and RGmT-based calcium imaging. This modification of the experimental setup was necessary because the preparation of epidermal peels  within our priming experiments and found that the resting [Ca2+]cyt was with statistical significance lower in the CPK5-overexpressing line CPK5#7xRGmT compared to the wild-type or cpk5 carrying RGmT  influence on a stimulus-induced CaSARD1) , phytohormone levels  and expression of key transcription factors (SARD1) . Further2+ response in leaf disks of adult plants upon a triggering flg22 stimulus from plants that had or had not experienced a previous flg22 priming treatment. The leaf disk approach bears the opportunity to average signals over a multitude (>n 103) of cells. While the aequorin-based system  cells  waves of Ca2+ signals induced by flg22 in the CPK5-OE line compared to RGmT  cells . Therefoiscussed . Our dat to RGmT , 2. It i mutants . Likewists  driven by electrochemical gradients of H+ and autoinhibited P-type II Ca2+-ATPases (ACA)  . ACAs haes (ACA) . InteresCa2+]cyt . P-type n domain . Similarrylation . Whethersibility , 2018. Sechanism .2+ peaks” observed in the CPK5-OE line decoded into enhanced basal defense responses within a cell?” Based on what is known from plant immune signaling in pattern-triggered, effector-triggered or systemic immunity in SAR, this will involve the myriad of different Ca2+-sensor, -relay and -effector proteins, such as CaM/CML, kinases or CAMTA transcription factors (2+ transients into downstream defense responses may depend on the “counting” of encoded cytosolic (and/or nuclear) Ca2+ changes (up and downs in [Ca2+]) rather than a monophasic Ca2+ change.A natural follow-up question is: “How are the “more pronounced and defined number of Ca factors . In the 2+ signature upon flg22 treatment in local tissues. This is consistent with CPK5 function as a predominant Ca2+ sensor and effector in pattern-triggered immunity mediating Ca2+ signal synchronization and defense response activation. However, the flg22-induced [Ca2+]cyt increase in systemic cells are not different in the various priming context. Therefore, at least according to our experimental system, rapid Ca2+ changes in systemic tissues do not reflect the plant memory of “having been primed.”In summary, our data provide evidence for an altered CPK5-dependent CaThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.BE, SL, JL, AL, and TR conceived and designed the strategy and experiments. BE, SL, and AL generated the RGmT lines. BE conducted all priming experiments and analyzed the systemic calcium imaging. SL and AL analyzed the local calcium imaging. FT conducted the aequorin-based calcium assays. XJ conducted the molecular characterization of RGmT lines. TG generated the aequorin lines. RW designed and generated the RGmT construct. BE, SL, XJ, AL, and JL prepared the figures. JL, AL, and TR wrote the manuscript. All authors discussed the results and commented on the manuscript.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Nurturing of physician-scientists is an important mission of academic medical institutes. Although the importance of mentorship in developing future physician-scientists internationally is well established, not much information is available about how they are mentored and how national cultures influence the mentoring relationship. This study explores the cultural characteristics of mentoring relationships between senior mentors and junior mentees of Japanese physician-scientists.A qualitative approach has been employed to explore mentoring relationships of Japanese physician-scientists from cultural viewpoints, through semi-structured interviews with 17 mentees who had the experience of working overseas as post-doctoral research fellow. The reflection of their experiences and the perception of mentoring relationships before going abroad were thematically analyzed by applying Hofstede’s model of six cultural dimensions as a theoretical framework.Twelve characteristic themes for mentoring were observed, including trustworthy dependency on mentor, embracing paternalistic mentoring, mentee’s initiative within expectations of mentor based on power distance, a sense of loyalty to mentor/organization/colleagues, family-like relationship with mentor based on collectivism, sense of security on being led by mentor through uncertainty avoidance, motivation by role modeling for the competitive academic world, and adaption of female mentee/mentor to a male-dominated academic structure based on masculinity, a long-term relationship between mentor and mentee, receiving advice for organizational continuity based on long-term orientation, putting work before leisure, and friendly relationship between mentor and mentee outside of work based on indulgence.This study identified the characteristic mentoring relationships of postgraduate mentees of Japanese physician-scientists. Considering the importance of mentoring for physician-scientists in a globalized society, understanding the characteristics of national cultures would help in ensuring culture-sensitive mentoring and would contribute to the development of academic medicine. Nurturing physician-scientists who greatly contribute to the development of medicine is indispensable for academic medical institutions . HoweverMentoring to nurture physician-scientists has garnered attention as an educational strategy to address this critical situation . MentoriMost previous research on mentoring in academic medicine has been conducted in Western countries, and focused on intrapersonal and interpersonal factors affecting mentoring relationships . In addiWhile it is established that cultural diversity would have an impact on mentoring, our understanding is that no specific frameworks of culture have been applied to analyze the cultural differences in mentoring in academic medicine so far. Previous scholarly works have offered many definitions of culture . HofstedThis study explores how cultural characteristics influence the relationship between senior mentors and junior mentees, in the context of career development of Japanese physician-scientists belonging to academic institutions., including PhD trainees for several years after graduating from medical school and the residency training, and are often known as Ikyoku . Th. Th23]. wn needs . Hofstedeginning .Japan has an uncertainty avoidance score of 92, which is one of the highest values among countries; because of frequent disasters in Japan, people are chary of uncertain situations . MenteesJapan has a score of 95 for masculinity, which is one of the highest values among countries . ThroughIkyoku and physicians’ perspectives for their career development might change along with this training reform. There is also an increase in the number of female physicians in the younger generation in Japan and women’s value-based mentoring has become more common. This may have an impact on the culture of mentoring.This indicates that the national culture influences mentoring, and other factors such as organizational culture, generation, and gender should also be considered. Hofstede argues that national culture cannot be altered easily  but someIn a globalized society, the interaction between physician-scientists across the world will increase, and mentoring among different countries, cultures, organizations, and professions will also increase . As cultThis study has several limitations. First, the number of participants was relatively small and mentors’ views were not addressed. Second, there is a possibility of recall bias among the participants who were reflecting on their mentoring relationships that occurred prior to their studies abroad. Third, we analyzed the data from only one cultural definition or perspective, namely Hofstede’s cultural dimensions, although there are many different definitions of culture. Fourth, we only explored one type of mentoring relationship, namely between senior physician-scientists and junior physician-scientists, and we did not include the various types of mentoring relationships, such as peer mentoring, near-peer mentoring, and group mentoring. Fifth, all the participants (mentees) in this study had graduated from medical school before the new clinical training system was introduced, and this may have influenced their perception. Sixth, the participants (mentees) were mostly males, and this could have influenced the themes based on the cultural dimensions; however, this situation represents the current state of mentoring relationship in Japanese academic medicine.This qualitative study revealed the cultural characteristics in mentoring of Japanese physician-scientists and the usefulness of applying Hofstede’s cultural dimensions for understanding mentoring relationships. The findings of this study may assist in ensuring culturally sensitive mentorship henceforth.


The high voltage-gated Ca2+ channels (HVCCs) occupy a central role in pancreatic hormone release both as a source of Ca2+ required for excitation-secretion coupling as well as a scaffold for the release machinery. HVCCs are multi-protein complexes composed of the main pore-forming transmembrane α1 and the auxiliary intracellular β, extracellular α2δ, and transmembrane γ subunits. Here, we review the current understanding regarding the role of all HVCC subunits expressed in pancreatic β-cell on electrical activity, excitation-secretion coupling, and β-cell mass. The evidence we review was obtained from many seminal studies employing pharmacological approaches as well as genetically modified mouse models. The significance for diabetes in humans is discussed in the context of genetic variations in the genes encoding for the HVCC subunits.The pancreatic islets of Langerhans secrete several hormones critical for glucose homeostasis. The β-cells, the major cellular component of the pancreatic islets, secrete insulin, the only hormone capable of lowering the plasma glucose concentration. The counter-regulatory hormone glucagon is secreted by the α-cells while δ-cells secrete somatostatin that via paracrine mechanisms regulates the α- and β-cell activity. These three peptide hormones are packed into secretory granules that are released through exocytosis following a local increase in intracellular Ca Therefonnel, γ1 , γ4, γ7 7 and γ8  seem to  subunit . Further subunit . This is systems ,227,229,almitate .2+ influx on excitability, vesicle exocytosis, and gene transcription and regulation has been extensively demonstrated in many excitable cells such as neurons, cardiac myocytes, skeletal muscle, neuro endocrine cells, etc. Critical information regarding the biophysical and pharmacological properties of all HVCC subunit isoforms has been obtained in simplified heterologous co-expression systems such as Xenopus laevis oocytes or HEK293 cells. In this review article, we summarized the knowledge regarding the structure and function of the HVCCs, how this impacts on pancreatic β-cell insulin release and glucose metabolism and, whenever we saw fit, compared these findings with data obtained from research performed in other cell types. The physiological role of Ca2+ channels subunits in pancreatic hormone release and glucose metabolism has been obtained mostly from research performed on transgenic mouse models. Critical experiments have also been performed in isolated human pancreatic islets and cells from healthy or diabetes donors. This provides a very important translational aspect to the research performed in animal models.The crucial role of HVCC Ca2+ influx and therefore altered excitability [2+ concentration and oscillations [V1.2 genetic deletion or pharmacological block reduced the β-cell Ca2+ influx by ~60% (V1.2-α1C subunit is coupled to vesicle release (V1.3 deletion did not affect the β-cell peak Ca2+ influx but resulted in a shift of the voltage dependence of activation towards more positive potentials. CaV1.3 deletion reduced insulin release at lower glucose concentration and, due to smaller β-cell mass, also reduced the total insulin release, thereby leading to glucose intolerance in mice. CaV2.1 pharmacological block resulted in an ~20% reduction in β-cell Ca2+ currents. Although its role on GIIS has been demonstrated only in static experiments, based on its potential contribution to the peak of APs, it is expected that CaV2.1 loss-of-function would slightly reduce both phases of GIIS. If in human β-cells CaV2.1 is coupled to secretory machinery, then its genetic deletion or pharmacological block will reduce both phases of GIIS to a similar extent as CaV1.2 does in mouse β-cells. Similar to CaV2.1, CaV2.3 channels also contribute with ~20% to total β-cell Ca2+ influx. CaV2.3 deletion or SNX-482 application did not alter the first phase but strongly reduced second-phase GIIS due to its proposed role in promoting vesicle trafficking to the release sites (2δ-1, the main α2δ isoforms expressed in pancreatic β-cells, forms a complex with all HVCC isoforms. Consequently, its genetic deletion resulted in ~70% decreased β-cell Ca2+ influx and a strong reduction of both phases GIIS (4 subunit seems to promote only L-type channel membrane incorporation; therefore, its reduced expression is expected to have a similar effect on GIIS as L-type channel block (3 subunit has HVCC-independent functions and its genetic deletion increases IP3R Ca2+ release from intracellular stores, thereby most probably increasing insulin secretion only in the second phase since the first phase is limited by the size of the readily releasable pool of vesicles (Genetic ablation of most HVCC subunits in pancreatic β-cells led to reduced β-cell Catability ,57, redullations ,56, redullations , and alsllations ,56,76. H release A it almose sites , cyan. αses GIIS , red. γ4el block , yellow.vesicles , green.2+ transients. However, such a figure would be mostly speculative since the experiments have either never been performed or the data have been obtained under very different experimental settings. We are confident that these gaps in our knowledge will soon be filled and complemented by additional RNAseq and proteomics data. This will shed light on the known and hitherto unknown roles of the HVCC subunits in pancreatic hormone release and glucose homeostasis in health and diabetes.During the preparation of


Interest in honey utilization in wound approaches is rising in recent years. In order to evaluate the positive effects of buckwheat honey on endothelial responses, we utilized an immortalized endothelial cell line to evaluate cellular responses upon honey exposure, with particular interest in Ca2+ signaling involvement. The results highlight the positive effects of buckwheat honey on endothelial cells’ responses and the central role played by Ca2+ signaling as an encouraging target for more efficacious clinical treatments. Intracellular Ca The integrity of the endothelial monolayer may be compromised by either disturbed blood flow or pathological conditions. The prolonged and/or repeated exposure of the endothelial monolayer to well-known cardiovascular risk factors can induce a dramatic impact on vascular endothelium. The injured endothelium is regarded as the early event that results in the onset of severe vascular disorders, including atherosclerosis, acute myocardial infarction, brain stroke, and aortic aneurysm.It is, therefore, not surprising that novel approaches are crucial for clinicians to achieve the early restoration of a fully competent endothelium in order to gain more effective long-term results after vascular regenerative surgery ,2.2+ signals play a central role in controlling endothelial survival, proliferation, motility, and differentiation, maintaining substantially conserved mechanisms even through the vascular tree variability [2+]i), usually taken back to the resting conditions after agonist removal or even in its presence, thanks to the active extrusion from the cytoplasm [2+]i increase arises from the entry of extracellular Ca2+ through plasma membrane Ca2+-permeable ion channels and by the release of Ca2+ from the endoplasmic reticulum (ER), the main intracellular Ca2+ store. Calcium release from ER is normally due to the rise in cytosolic levels of inositol-1,4,5-trisphosphate (InsP3), a diffusible and ubiquitous intracellular second messenger [Intracellular Caiability . Therefoiability . These kytoplasm . The [Ca2O2) that confers the capability to the honey to begin the wound healing process and trigger the [Ca2+]i mobilization i variation was observed. Such a variation is described by a peak phase subsequent to the addition of honey, followed by a plateau phase leading again to homeostatic [Ca2+]i . The absence of extracellular Ca2+ resulted in a reduction of about 80% of the peak previously observed after the treatment with honey  channel which mediates Caechanism ,14.2+ entry across the TRPM2 channel [2+]i increase caused by honey treatment . Therefore, the observations were repeated in the presence of some inhibitors of the PLC-IP3 pathway: bEND5 was pre-treated with 50 µM 2-APB . In  release   could weaken the structural integrity of the vascular endothelium, causing vascular injury . MoreoveTherefore, there is a need for innovative approaches to hit a prompt renovation of the endothelium to improve any long-term effects after vascular regenerative surgery .A possible novel strategy for the treatment of endothelial injury could be the use of honey, a natural product that in recent decades, as in ancient times, sparks interest for its several virtues and pharmacological properties ,31,32,33In the present study, we investigated the potential effect of honey, in particular buckwheat honey (BH), on an endothelial cell line, bEND5, in terms of regenerative capability.w/v), exhibited a positive effect determining a 2.5–3-fold increase in the wound closure rate. In particular, the concentration of 0.5% was the most effective. The first study, as already demonstrated on other cell lines, i.e., keratinocytes and fibroblasts ,30, highOne of the principal factors in the wound healing process is the intracellular calcium , therefoWe observed that this increase was dose-dependent, displaying the onset of a peak immediately after the addition of BH that came back to homeostatic values in a maximum of 200 s. 2O2 in the extracellular space and this amount of peroxide could enter inside the cells moving through a specific aquaglyceroporin, i.e., aquaporin 3 (AQP3). The entry of H2O2 in the cytoplasm triggers the intracellular signaling that operates through the wound healing process i variation, it was determined the origin of calcium. Under [0Ca2+]i conditions, even with the presence of 2%BH, a peak 2.5–3-fold less than the one observed previously was recorded. This highlighted the extracellular calcium as the main contributor of the [Ca2+]i variation. It is known from the literature that endothelial cells are characterized by a high expression and activity of the TRPM2 channel involved in a conspicuous entry of calcium into the cell , therefore it could be possible to identify a TRPM2 channel as the main access route of extracellular calcium after BH treatment.The traces obtained after pretreatment with econazole are consistent with those observed in [0Ca3 pathway [3R inhibition by using 2-APB [2+]i increase. Since the inhibition of extracellular calcium entry did not result in a complete reduction in the peak, it was decided to consider a possible role of the intracellular stores, focusing the attention on the endoplasmic reticulum (ER). As previously demonstrated on the same cells, calcium from ER plays a fundamental role in the response to stimuli that induces tissue regeneration, in particular the PLC-IP pathway . The PLC pathway  and InsPng 2-APB  or caffeng 2-APB , determi2O2 as a physiological mediator of cellular behavior in response to BH treatment. This critical role is due to the ability of extracellular H2O2 to activate a variety of nonreceptor- and receptor-type tyrosine kinase in heterogeneous cell systems, leading to a tyrosine kinase-dependent IP3 production and consequent calcium release, suggesting an activation of PLC via tyrosine phosphorylation by H2O2-activated tyrosine kinases i:o The activation of a TRPM2 channel determining calcium entry from the extracellular space,o 3 pathway leading to a calcium release from the ER,The activation of a PLC-IPH2+]i increase contributes to the activation of PLC,The i, that, in turn, results in ERK1/2 activation.Accordingly, the addition of honey, through H


Despite the beneficial role of antibiotics in reducing bacterial infection in rabbits, there is an indirect harmful influence on human health. Thus, replacing antibiotics with friendly alternatives is a suitable strategy to protect the performance and welfare of rabbits. This study aimed at comparing the effects of including dietary egg lysozyme and zinc bacitracin antibiotic (ZnB) on the productivity and health conditions of rabbits. The results show a marked enhancement of the growth performance, antibacterial capacity, blood health, and antioxidative status in rabbits treated with egg lysozyme compared with those treated with ZnB. Thus, using egg lysozyme is recommended to replace the usage of ZnB in rabbit production.p < 0.05). Nevertheless, the feed conversion ratio (FCR) was markedly decreased by dietary egg lysozyme and ZnB (p < 0.05). Interestingly, dietary egg lysozyme resulted in higher final weight and BWG and lower FCR than rabbits treated with ZnB (p < 0.05). Rabbits treated with egg lysozyme and ZnB additives had markedly lower populations of Clostridium spp. and Escherichia coli (p < 0.05) compared with the control. However, the counts of Lactobacillus and total bacteria were meaningfully increased in the the intestines of rabbits treated with egg lysozyme and ZnB (p < 0.05). The blood total protein and globulin of rabbits fed dietary egg lysozyme and ZnB additives were meaningfully increased (p < 0.05). Blood creatinine was significantly lowered by dietary egg lysozyme compared with the control and ZnB-treated rabbits (p < 0.05). The levels of blood urea, ALT, and AST were markedly lowered (p < 0.05) by dietary egg lysozyme and ZnB. The gene expressions of superoxide dismutase 1 (SOD1) and glutathione peroxidase (GPX) in the liver of rabbits fed dietary egg lysozyme and ZnB additives were markedly upregulated (p < 0.05) compared with the control. Dietary egg lysozyme resulted in higher expression of SOD1 and GPX genes than rabbits treated with ZnB (p < 0.05). In conclusion, the inclusion of egg lysozyme could replace the inclusion of ZnB in the diets of rabbits. Dietary egg lysozyme has beneficial roles in the growth performance and health conditions of animals. The study was performed using 90 multicolored rabbits in three groups (each replicate with thirty rabbits). In the control group, rabbits were fed a diet without zinc bacitracin (ZnB) or egg lysozyme, while the second and third groups were treated with ZnB and lysozyme additive at 100 mg/kg, respectively. After eight weeks, the final weight and body weight gain (BWG) of rabbits fed dietary egg lysozyme and ZnB additives were meaningfully increased ( Rabbit meat is widely consumed as a tasty and nutritious protein source with high commercial value . HoweverAlternatively, lysozyme extracted from eggs was approved as an effective natural growth-promoting and antibacterial agent in rabbit and broiler production ,12. LysoBoth ZnB and lysozyme have antibacterial capacity against Gram-positive bacteria and can actively enhance the growth performance and well-being ,25. HoweTM . The ingredients were pelleted and then mixed with egg lysozyme and ZnB in the presence of molasses to avoid the loss of additives’ functionality and concentration. The feed intake and live body weight were recorded on two fixed days weekly, and then body weight gain and feed conversion ratio were calculated.The study was performed by using APRI-Line weaning male rabbits aged five weeks and weighed 611.32 ± standard deviation (0.36) g/animal. In the farms of Sakha Poultry Research Station and Laboratories, Kafrelsheikh, Animal Production Research Institute, Egypt, 90 multicolored rabbits were distributed into three groups (each group with thirty rabbits). Rabbits were individually housed in galvanized batteries (60 cm × 40 cm × 35 cm) provided with automatic nipples and feeders. Rabbits were kept under controlled hygienic conditions (14 h of light/day) and fed the prepared, tested rations for eight weeks. Rabbits were fed ad libitum, and freshwater was available in each cage through automatic nipples. The temperature and relative humidity were 18–20 °C and 55–65% during the trial, respectively. Rabbits in the control group were fed a diet without zinc bacitracin (ZnB) or egg lysozyme, while the second and third groups were treated with ZnB and lysozyme additive at 100 mg/kg, respectively. The doses of egg lysozyme and ZnB were proposed by following El-Deep et al. , and theEscherichia coli, Clostridium, and Lactobacillus count were performed according to Collins and Lyne [Samples from the cecum were chosen individually from six rabbits from each group, and cecum contents were obtained after slaughtering and filtrated to estimate cecum microflora. Total bacterial count, and Lyne .The blood was collected from the lateral ear vein of slaughtered rabbits (9 rabbits per group). The blood was left to clot at room temperature and then centrifuged at 3000 rpm for 15 min. Sera were then separated and stored at −20 °C in aliquots for individual biochemical estimations. The serum was used to detect alanine aminotransferase (ALT), aspartate aminotransferase (AST), total proteins, albumin, creatinine, urea, triglycerides, and cholesterol were detected according to the manufacturer’s protocol using commercial kits . Globulins concentration in serum was computed by subtracting albumin concentration from total proteins, while the albumin to globulin ratio (A/G) was calculated according to Kaneko et al. . −ΔΔct technique as outlined in Pfaffl [Part of the livers from 6 rabbits per group were collected and immediately frozen in liquid nitrogen. Total RNA was extracted by easy-RED Total RNA Extraction Kits  using the manufacturer procedure. The integrity of RNA was checked by agarose gel electrophoresis, while a NanoDrop spectrophotometer detected the purities and quantities of the samples. The first-strand cDNA was obtained using a kit for HiSenScript cDNA . Specific primers were used to amplify chosen genes with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a housekeeping gene that was stable among the sample groups . The mRNn Pfaffl .p < 0.05. The obtained data were subjected to one-way analysis of variance using SPSS . Differences within means of treatments were tested by Tukey’s test at p < 0.05) compared with the control (p < 0.05). Interestingly, dietary egg lysozyme resulted in higher final weight and BWG and lower FCR and FI than rabbits treated with ZnB (p < 0.05)  of rabbits fed dietary egg lysozyme and ZnB additives were meaningfully increased ( control . Neverth < 0.05) .Clostridium spp. and Escherichia coli (p < 0.05) compared with the control . The inclusion of egg lysozyme resulted in higher counts of Lactobacillus and total bacterial count than dietary ZnB (p < 0.05) (Rabbits treated with egg lysozyme and ZnB additives had markedly low populations of  control . However < 0.05) . p < 0.05) compared with the control (p < 0.05). Blood creatinine was significantly lowered by dietary egg lysozyme compared with the control and ZnB-treated rabbits (p < 0.05) (p < 0.05) by dietary egg lysozyme and ZnB (p < 0.05). No meaningful impacts of egg lysozyme or ZnB were seen on the blood cholesterol and triglycerides (p > 0.05)  . The lev and ZnB . Rabbits > 0.05) .SOD1 and GPX in the liver of rabbits fed dietary egg lysozyme and ZnB additives were markedly upregulated (p < 0.05) compared with the control (SOD1 and GPX genes than rabbits treated with ZnB (p < 0.05).The expressions of  control . DietaryIt becomes necessary to replace antibiotics with natural functional substances in the animal production industry to avoid the negative impacts on animals and human health ,33,34. RThe results show increased growth performance in rabbits treated with dietary lysozyme or ZnB, but rabbits fed lysozyme had superior enhanced growth performance. The results agree with El-Deep et al. , who staLactobacillus count along with decreased Clostridium spp. and Escherichia coli populations in rabbits treated with dietary egg lysozyme or ZnB. Similarly, rabbits treated with lysozyme showed high total bacterial and Lactobacillus counts but lowered Clostridium spp. and E. coli counts [E. coli count. In the same line, rabbits treated with ZnB showed high antibacterial activity against Clostridium perfringens [Lactobacillus) and decreased Clostridium spp. and E. coli counts in this study explain the enhanced feed utilization and thereby growth performance in rabbits treated with lysozyme and ZnB. Herein, the enhancement of feed utilization can be related to the lack of pathogenic bacteria, resulting in an increased number of beneficial bacteria known for their ability to produce digestive enzymes to facilitate the digestion of nutrients in rabbits’ intestines [The enhancement of feed utilization and growth rate is also associated with improved cecal microbial diversity, especially in beneficial populations ,44. The i counts . Brundigi counts  and Gongi counts  also indfringens . Egg lysfringens . Ibrahimfringens  discussetestines . In additestines . HoweverThe evaluation of blood biochemical traits helps in testing the influence of dietary additives on the health status of animals ,48. DietSOD1 is the most abundant isoenzyme in the cellular cytoplasm, which reduces the superoxide radical anions, while GPX is another antioxidant enzyme involved in modifying harmful peroxides to non-harmful hydroxyl compounds to protect cellular structure and function [SOD1 and GPX, which help in protecting the entire body and immune cells from oxidation [SOD1 and GPX) in the liver of rabbits treated with egg lysozyme and ZnB. The upregulation of SOD1 and GPX genes is probably attributed to the role of egg lysozyme and ZnB additives in improving intestinal immunity [Stressful farming conditions induce oxidative stress in rabbits due to the induction of reactive oxygen metabolites (ROS), leading to high lipid peroxidation ,50. In tfunction . The antxidation ,57. The immunity . It has immunity . The results show marked enhancement of the growth performance, antibacterial capacity, blood health, and antioxidative status in rabbits treated with egg lysozyme comparing with those treated with ZnB. Thus, using egg lysozyme is recommended to replace the usage of ZnB in rabbit production.