Haemonchus contortus, are particularly important. The excessive use of anthelmintic compounds to treat infections and disease has led to widespread resistance to these compounds in nematodes, such that there is a need for new anthelmintics with distinctive mechanisms of action. With a focus on discovering new anthelmintic entities, we screened 400 chemically diverse compounds within the ‘Pandemic Response Box’  for activity against H. contortus and its free-living relative, Caenorhabditis elegans—a model organism. Using established phenotypic assays, test compounds were evaluated in vitro for their ability to inhibit the motility and/or development of H. contortus and C. elegans. Dose-response evaluations identified a compound, MMV1581032, that significantly the motility of H. contortus larvae (IC50 = 3.4 ± 1.1 μM) and young adults of C. elegans (IC50 = 7.1 ± 4.6 μM), and the development of H. contortus larvae (IC50 = 2.2 ± 0.7 μM). The favourable characteristics of MMV1581032, such as suitable physicochemical properties and an efficient, cost-effective pathway to analogue synthesis, indicates a promising candidate for further evaluation as a nematocide. Future work will focus on a structure-activity relationship investigation of this chemical scaffold, a toxicity assessment of potent analogues and a mechanism/mode of action investigation.Parasitic nematodes cause diseases in livestock animals and major economic losses to the agricultural industry worldwide. Nematodes of the order Strongylida, including  Haemonchus contortus (family Trichostrongylidae), also known as the ‘barber’s pole worm’, which causes haemonchosis—a disease of ruminant livestock, such as sheep and goats. Haemonchosis is characterised by anaemia, bottle jaw, lethargy, poor wool/milk production and/or sudden death [H. contortus is transmitted orally via a direct life cycle [Parasitic roundworms (nematodes) cause infections and diseases (nematodiases) in humans and animals that have a major adverse socioeconomic impact worldwide ,2. In huen death ,6. H. cofe cycle ; animalsfe cycle .H. contortus [Treatment with anthelmintic compounds is an important component of the control of trichostrongyloid nematodes . Compounontortus ,9. Howevontortus ,11. Resiontortus ,13. Whilontortus ,14.H. contortus within the Pathogen (n = 400 compounds) and Stasis (n = 400 compounds) Box’ collections. Both tolfenpyrad and AG-1295 underwent chemical optimisation, structure-activity relationship (SAR) and/or toxicity studies, although a safe lead compound was not attained [Pandemic Response Box’, which contains 400 structurally diverse and well-curated compounds with activities against key pathogenic bacteria (n = 201 entities), funguses (n = 46) and viruses (n = 153), including highly significant pathogens, such as the bacterium Staphylococcus aureus and the virus Influenza A.Although the rate of discovery and development of novel anthelmintics has been relatively slow, with only monepantel  and derqattained ,32,33. TH. contortus genome, transcriptome, proteome and lipidome [H. contortus. In addition, the closely related free-living nematode, Caenorhabditis elegans, provides a powerful comparator for such mechanistic studies [C. elegans has been comprehensively annotated and made publicly available from WormBase (www.wormbase.org [C. elegans belongs to the same evolutionary clade (V) as H. contortus, with these two species sharing 7361 one-to-one orthologous genes [C. elegans is a powerful surrogate for H. contortus and can be used to infer the functions of orthologous gene or protein targets [In addition to the availability of chemical libraries through collaborations with organisations such as MMV, progress in ‘multi-omics’ research has complemented anthelmintic drug discovery—with significant advances in the understanding of the lipidome ,41,42,43 studies ,45,46. Tbase.org ; accesseus genes ,35,39,48Pandemic Response Box against H. contortus and C. elegans to identify active compounds; (ii) assessed the potency of anthelmintic activity of (hit) compounds; and (iii) evaluated the toxicity of these hit compounds against HepG2 cells, in order to identify candidate compounds for medicinal chemistry optimisation.Here, we (i) undertook a whole-organism, phenotypic primary screen of compounds from the H. contortus xL3s: MMV1581032 reduced xL3 motility by ≥100% at 90 h, inhibited larval development and induced a ‘straight’ (Str) phenotype after seven days; MMV1593539 reduced larval motility by 66%, inhibited larval development and also induced a Str phenotype. Two compounds had marked activity on C. elegans in the transition from the L4 to the adult stage  of 3.4 ± 1.1 μM (maximum motility inhibition: 76%) and compound MMV1593539 had an IC50 of 3.5 ± 0.98 μM (maximum motility inhibition: 71%)—being comparable to that of moxidectin  in the present dose-response assay. Subsequently, the potency of each compound to inhibit larval development of H. contortus was assessed following incubation for seven days . MMV1581032 was also evaluated for motility inhibition on adult females of H. contortus (An assessment of the potency of two hit compounds to inhibit the motility of for 90 h . Compounven days . MMV1581ontortus . MMV1581ontortus  were: 22C. elegans was estimated following incubation for 40 h (50 of 7.1 ± 4.6 μM (maximum motility inhibition: 96%). MMV1593515 achieved an IC50 value of 0.5 ± 0.11 μM (maximum motility inhibition: 75%). Respective IC50 values for monepantel and moxidectin were 0.01 ± 2.0 × 10−3 μM and 0.04 ± 4.0 × 10−4 μM. Thus, MMV1581032 exhibited marked potency against both H. contortus and C. elegans.An assessment of the potency of two hit compounds to inhibit the motility of for 40 h : MMV158150 ≥ 100 μM) nor mitotoxic (MC50 = 81.1 μM) against HepG2 human hepatoma cells.Given the potential of MMV1581032 as a candidate, its toxicity was assessed by measuring cell death using crystal violet staining . This coH. contortus and/or C. elegans. MMV1581032 was shown to be the most potent of these compounds against both H. contortus and C. elegans, with IC50 values of 3.4 μM and 7.1 μM, respectively, and killed adult females of H. contortus at 100 µM in vitro after 24 h. Thus, MMV1581032 presented as a suitable candidate for further evaluation.The screening of 400 diverse, drug-like molecules identified three compounds , designaH. contortus (3.5 μM) but not C. elegans (>100 μM). The reduction in both larval motility and development of H. contortus was comparable with that of moxidectin, but less than that of monepantel. MMV1593539 induced a ‘straight’ (Str) phenotype in H. contortus xL3s at 100 μM—a phenotype induced by other compounds, such as tolfenpyrad—a pyrazole-5-carboxamide [Staphylococcus aureus. Notably, genes encoding pyruvate kinase  against C. elegans. Vorapaxar, an anti-platelet drug (PAR-1 antagonist), was first described in 2008 and is currently commercially available as Zontivity® for the treatment of people with a history of heart attacks [C. elegans, the human muscarinic receptor ortholog, GAR-3, has been implicated in the regulation of muscle contraction [C. elegans, although a mode of action study would be needed to test this hypothesis. Despite the low potency against H. contortus, voraxapar might exert anthelmintic activity against other socioeconomically important parasitic nematodes of the order Strongylida , given the promising potency against C. elegans, which is within the same evolutionary clade (V) as the Strongylida [Compound MMV1593515—a natural himbacine derivative, also known as vorapaxar —displaye attacks ,55. Inte attacks ,57. In Ctraction —thus, itongylida . Howeverongylida ,61,62,63H. contortus and C. elegans) and development (H. contortus), leading to a Str phenotype at 100 μM (like compound MMV1593539). The potent in vitro effect on the most pathogenic and reproductively active stage of H. contortus [Compound MMV1581032—a quinoline derivative, also known as “ABX464” (cf. )—affecteontortus  encouragontortus . A preliontortus  as a guiClinicalTrials.gov, NIH; accessed on 15 September 2021). The anti-inflammatory properties of ABX464 are a consequence of RNA splicing-interference [ABX464  was first reported as a novel anti-HIV molecule [rference . Campos rference ,69. Uponrference . As an arference ,70; miR-rference ,70. Althrference .C. elegans, Kaminsky et al. [acr-23 encoded a target of monepantel and then identified and validated its homologue in H. contortus (designated mptl-1) [C. elegans suggests that such a functional genomic approach might be useful to study this compound’s target and mode of action [H. contortus [Currently, the mode/mechanism of action of ABX464 in nematodes is unknown, but could be explored using genomic, transcriptomic and/or proteomic methods. Genomic and transcriptomic approaches, similar to those employed for the discovery of the target of the anthelmintic monepantel ,72,73, cy et al.  establisf action ,46. It if action , should ontortus ,38,43. Iontortus ,45,46). Pandemic Response Box compound library from MMV, Geneva, Switzerland, contains 400 structurally diverse, drug-like compounds . Individual compounds were supplied at concentrations of 10 mM or 2 mM in 10 μL of (100%) dimethyl sulfoxide (DMSO). Prior to screening, compounds were individually diluted to 40 μM in sterile Luria-Bertani broth ; this supplemented LB was designated LB*.The LB; cf. ,75). LB . LB PandHaemonchus contortus  sodium hypochlorite (NaOCl) for 20 min at 38 °C [g (5 min) and resuspended in LB* at a concentration of 200 exsheathed L3s (xL3s) per 50 µL  was maiain; cf. ,76 and iH. contortus were collected from the abomasa of sheep infected for 10 weeks, washed three times in phosphate-buffered saline  and then three times in RPMI 1640 media supplemented with 4 mM l-glutamine, 100 U/mL of penicillin, 100 µg/mL of streptomycin and 0.25 µg/mL of amphotericin B . Using a dissecting microscope, female and male worms were separated in RPMI* (38 °C), and female worms immediately used (within ~1 h at 38 °C) to test compounds  was maintained in the laboratory under standard conditions at 20 °C on nematode growth media (NGM) agar plates, with Escherichia coli OP50 as a food source [v/v) sodium hypochlorite and 170 mM sodium hydroxide for 4–8 min at 22–24 °C to release eggs [g, 2 min). After washing, the egg pellet was suspended in 8 mL of M9 buffer in a 15 mL tube and gently agitated for 24 h at 22–24 °C (room temperature) to produce first-stage larvae (L1s); 45 h prior to screening, synchronised C. elegans L1s were inoculated on to NGM plates containing 500 µL of E. coli OP50 (~3000 larvae per plate) and allowed to develop to fourth-stage larvae (L4s) at 20 °C. L4s were collected from plates and washed twice with sterile M9 buffer by centrifugation  to remove E. coli OP50, and then resuspended in LB* at a concentration of 125 larvae per 50 µL , monepantel/abamectin , moxidectin  and compound MIPS-0018666  DMSO and LB* + 1% (v/v) DMSO were used as negative controls. Test compounds as well as positive and negative controls were distributed amongst two flat-bottom 384-well microplates . Added to each well were 80 xL3s of H. contortus in 20 μL of LB* to give a final volume of 40 μL. Plates were then placed in a CO2 incubator . At 90 h, worm activity was captured using a WMicroTracker ONE unit . Over a period of 15 min, disturbance of an infrared beam in individual wells was recorded as a worm ‘activity count’. Activity counts were then normalised to the positive and negative controls using the program Prism  to remove plate-to-plate variation. The screening plates were then returned to the incubator  for an additional three days to observe compound effects on larval development. At day seven, worms were fixed with 40 μL of Lugol’s solution . Larval development was identified microscopically and recorded. Additionally, a compound that induced a non-wildtype phenotype  in H. contortus worms was recorded. A compound that reduced xL3 motility by ≥70% and/or inhibited larval development or induced an abnormal phenotype (comparative to the negative control) was recorded as a ‘hit’ compound. The performance of the assay was monitored using the Z’-factor [An established high-throughput phenotypic screening assay  was used666; cf. ), were u’-factor  calculatC. elegans. Compounds were assessed for their effect on the motility of worms at a concentration of 20 μM in LB*. Three compounds, monepantel, moxidectin and M-666 were used as positive controls . Two solutions of LB* + 0.2% (v/v) DMSO and LB* + 1% (v/v) DMSO were used as negative controls. Test compounds and positive and negative controls were distributed amongst two flat-bottom 384-well microplates. Added to each well were 50 C. elegans in 20 μL of LB* to give a final volume of 40 μL. Plates were then placed in an incubator  at 20 °C for 40 h. At 40 h, the motility of C. elegans (in the transition from L4 to the adult stage) was captured using a WMicroTracker ONE unit. Over a period of 15 min, disturbance of an infrared beam in individual wells was recorded as a worm ‘activity count’. Activity counts were then normalised to the positive and negative controls using the program Prism v.9.1.0 to remove plate-to-plate variation. The screening plates were then returned to the incubator at 20 °C. At day 5, worms were fixed with 40 μL of Lugol’s solution. Any compound that induced a non-wildtype phenotype (viewed under microscope) in C. elegans worms was identified. A compound that reduced worm motility by ≥70% and/or induced an abnormal phenotype (comparative to the negative control) was recorded as a ‘hit’ compound. The Z’-factor was calculated in the same manner as in An established assay  was emplH. contortus. Test compounds were assessed individually for an effect on the motility of xL3s . Two compounds, namely monepantel and moxidectin (prepared in the same manner as the test compounds), were used as positive controls. A solution of LB* + 0.25% (v/v) DMSO was used as a negative control. The test compounds and positive control compounds were arrayed in triplicate across individual flat-bottom 96-well microplates , with six wells on each plate containing the negative control. Added to each well were 300 xL3s of H. contortus in 50 μL of LB* to give a final volume of 100 μL. Plates were then placed in a CO2 incubator . The motility of xL3s was measured  at 90 h, and larval development established at 168 h of incubation with compound, as described previously [10-transformed and fitted using a variable slope four-parameter equation, constraining the highest value to 100% using ordinary least squares fit model. The development inhibition and phenotypes of larvae were examined using a microscope [An established dose-response assay  was empleviously . For indcroscope , and resC. elegans. Test compounds were assessed individually for an effect on the motility of C. elegans  in the transition from the L4 to young adult stage. Two compounds, monepantel and moxidectin, were used as positive controls and prepared in the same manner as the test compounds. A solution of LB* + 0.25% (v/v) DMSO was used as a negative control. The test compounds and positive control compounds were arrayed in triplicate across individual flat-bottom 96-well microplates, with six wells on each plate containing the negative control. Added to each well were 100 C. elegans in 50 μL of LB* to give a final volume of 100 μL. Plates were then placed in an incubator at 20 °C for 40 h. At 40 h, worm activity was captured using a WMicroTracker ONE unit. Over a period of 15 min, disturbance of an infrared beam in individual wells was recorded as a worm activity count. Raw ‘activity counts’ for each well were normalised to the negative controls. The compound concentrations were log10-transformed and fitted using a variable slope four-parameter equation, constraining the highest value to 100% using the ordinary least squares fit model employing Prism v.9.1.0.A newly-established dose-response assay  was emplH. contortus using an established assay [v/v) DMSO only were included in triplicates on the same plate. Four adult females were added to each of the triplicate wells containing either the test compound or the controls and placed in a CO2 incubator  for 1 day. A video recording (30 sec) of each well was taken at 1 h, 2 h, 3 h, 5 h and 24 h during the total incubation period to assess the reduction in worm motility, which was scored as 3 (“good”), 2 (“low”), 1 (“very low”) or 0  on adult females of ed assay . The comnt”; cf. ). For eaTM or 4 mM L-glutamine; cat. no. 10566016 or 11966025, respectively; Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 25 mM d-glucose (cytotoxicity) or d-galactose (mitotoxicity), 10% inactivated foetal bovine serum (FBS), 100 IU/mL of penicillin, 100 µg/mL of streptomycin and 0.25 µg/mL of amphotericin B (denoted DMEM*). Monepantel and moxidectin (prepared in the same manner as the test compound) were included as positive control compounds. Two compounds, doxorubicin  and M-666  DMSO was used as a negative control. HepG2 cells were seeded into wells of a 96-well plate in 80 µL of DMEM* (at 5.5 × 104 cells per well) and allowed to adhere for 16 h at 37 °C and 5% (v/v) CO2 at >90% humidity prior to incubation with individual compounds, at a final volume of 100 µL. For the assessment of mitochondrial toxicity, cells were starved of serum (DMEM* without FBS) for 4 h prior to the incubation with individual compounds [50) and half-maximal mitotoxic concentration (MC50) values, compound concentrations were log10-transformed, baseline-corrected using a respective positive control (doxorubicin or M-666) and fitted using a variable slope four-parameter equation employing the ordinary least squares fit model using Prism v.9.1.0.A cell viability assay was employed to evaluate the cytotoxicity and mitotoxicity of one hit compound, MMV1581032, against HepG2 human hepatoma cells . The test compound was serially-diluted  in Dulbecco’s modified eagle medium (DMEM; with GlutaMaxompounds ,85. Follompounds . The abs


A trial was conducted to investigate the effects of supplemental levels of Mn provided by organic and inorganic trace mineral supplements on growth, tissue mineralization, mineral balance, and antioxidant status of growing broiler chicks.4·H2O or MnPro. When Mn was supplied as MnPro, supplements of zinc, copper, iron, and selenium were supplied as organic minerals, whereas in MnSO4·H2O supplemented diets, inorganic salts were used as sources of other trace minerals. Performance data were fitted to a linear-broken line regression model to estimate the optimal supplemental Mn levels.A total of 500 male chicks (8-d-old) were used in 10-day feeding trial, with 10 treatments and 10 replicates of 5 chicks per treatment. A 2×5 factorial design was used where supplemental Mn levels  were provided as MnSO4·H2O, ADG, FCR, Mn balance, and concentration in liver were optimized at 59.8, 74.3, 20.6, and 43.1 mg supplemental Mn/kg diet, respectively. In MnPro fed chicks, ADG, FCR, Mn balance, and concentration in liver and breast were optimized at 20.6, 38.0, 16.6, 33.5, and 62.3 mg supplemental Mn/kg, respectively.Manganese supplementation improved body weight, average daily gain (ADG) and feed conversion ratio (FCR) compared with chicks fed diets not supplemented with Mn. Manganese in liver, breast muscle, and tibia were greatest at 50, 75, and 100 mg supplemental Mn/kg diet, respectively. Higher activities of glutathione peroxidase and superoxide dismutase  were found in both liver and breast muscle of chicks fed diets supplemented with inorganic minerals. In chicks fed MnSOLower levels of organic Mn were required by growing chicks for performance optimization compared to inorganic Mn. Based on the FCR, the ideal supplemental levels of organic and inorganic Mn in chick feeds were 38.0 and 74.3 mg Mn/kg diet, respectively. Manganese (Mn) is a bioactive element required for multiple postabsorptive physiological processes in poultry, such as carbohydrate, lipid, and amino acid metabolism, as well as cartilage and bone development, and antioxidant defense . For fasManganese is also a component of Mn-containing superoxide dismutase (SOD), an essential antioxidant metalloenzyme responsible for free radical scavenging in mitochondria by catalyzing the dismutation of superoxide anions produced from the electron transport chain to molecular oxygen and hydrogen peroxide . MitochoManganese requirements of growing broilers are described by the National Research Council  based onFrom the mid-2000s, organic trace minerals (OTM) received considerable attention due to their potential benefits and advantages compared with inorganic salts. After decades of investments and research efforts, OTM have proved to be more bioavailable than carbonates, oxides, and sulphates traditionally used by industry . One poiSecondly, Mn interacts with other trace minerals in many physiological processes either antagonistically or synergistically. A classic antagonistic interaction of Mn has been reported with iron (Fe). Both minerals share two common transport protein in intestine, the cellular importer divalent metal transporter 1 (DMT1) and the cellular exporter ferroportin 1 ,14. EvidAnimals care and use procedures described in this section were in accordance with Brazilian Legislation on Animal Experimentation and Welfare, and the experimental protocol was approved by the Ethics Committee on the Use of Farm Animals (CEUAP-UFV) of the Universidade Federal de Viçosa, (protocol 111/2014) before the initiation of the trial.4·H2O), which corresponded to 50% of the dose recommended by NRC [ad libitum access to feed and demineralized water throughout the 10-d experimental period via plastic feeders and cup drinkers. Photoperiod was set at 12 h natural light/12 h artificial light. Both temperature and humidity were set according to genetic guideline recommendations.A total of five hundred 1-d-old male Cobb 500 chickens obtained from a local commercial hatchery were used in this study. During the first seven days of age, the birds were fed a pre-starter diet based on corn and soybean meal formulated to meet or exceed nutritional recommendations of Rostagno et al , except d by NRC . The pre4·H2O, 30.3% Mn). Chicks were randomly assigned to ten treatments with 10 replicates of five birds per treatment. A semi purified basal diet was formulated to meet or exceed Rostagno et al [4·H2O) and the source of other trace minerals (organic or inorganic) as follows: i) basal diet supplemented with OTM supplement without supplemental Mn; ii) OTM+100 mg Mn/kg feed supplemented as MnPro; iii) basal diet supplemented with ITM supplement without supplemental Mn; and iv) ITM + 100 mg Mn/kg of feed supplemented as MnSO4·H2O. Diets supplemented with OTM containing 0 and 100 mg Mn/kg as MnPro were mixed in appropriate ratios to produce five diets with supplemental organic Mn concentrations of 0, 25, 50, 75, and 100 mg Mn/kg of feed. The same procedure was adopted with diets supplemented with ITM, to obtain the five levels of manganese. The OTMs were supplied as Bioplex Fe (15% Fe), Bioplex Zn (15.0% Zn), Bioplex Cu (11.4% Cu), and selenium yeast as Selplex (0.236% Se), whereas ITMs sources included iron sulphate (21.9% Fe), zinc sulphate (22.9% Zn), copper sulphate (25.1% Cu), and sodium selenite (51.6% Se) in ITM, and iodine was added as calcium iodide (86% I) in both trace mineral supplements. Except for Mn, all trace minerals were supplied to meet or exceed NRC [Prior to the experimental period, all chicks were weighed and assigned to treatment groups so that initial body weight BW, 174.6±1.0 g) was similar among experimental treatments. A 2×5 fractional factorial arrangement design was used in this study, where two Mn sources were added to basal Mn deficient diets at the supplemental Mn levels of 0, 25, 50, 75, and 100 mg Mn/kg diet. The organic source of Mn under study was manganese proteinate , whereas inorganic Mn was supplied as manganese sulphate , and feed conversion ratio (FCR). From d 13 to 17 post-hatch, trays covered with plastic were placed underneath pens for total excreta collection. Total excreta were collected daily, weighed, and frozen (−20°C). At the end of the collection period, excreta from each pen were homogenized. On d 18 post-hatch, one bird per cage (10 birds/treatment) was randomly selected and sacrificed by cervical dislocation. Subsequently, the breast, liver, and left and right tibia were collected and stored at −20°C. The bird had the breast muscle, liver, and left and right tibia collected, stored at −20°C, and subsequently, were ether extracted for 4 h in a Soxhlet extractor. Posteriorly, these samples, as well the samples of excreta, were freeze-dried for 72 h at −80°C under 800 mbar of pressure , ground in a stainless ball mill , and finally analyzed in the atomic absorption spectrophotometer  at Animal Nutrition Laboratory  to obtain the Mn, Cu, Zn, Fe, Ca, and P concentrations as described by AOAC [Data were analyzed as a completely randomized design under an incomplete 2-way (source×levels) factorial assay with inorganic and OTMs supplement without Mn supplementation (zero level) and Mn supplement levels as organic Mn in OTM supplement and inorganic Mn in ITM supplement. In this context, the traditional factorial analysis was updated to an incomplete factorial design . This apwhere Y is the dependent variable, X is the dietary Mn concentration, and β0 is the intercept, β1 and β2 are the linear and quadratic coefficients, respectively. Additionally, performance data were fitted to linear broken-line model (LBL) using NLMIXED procedure of SAS  software to estimate the supplemental Mn level which optimized performance responses assessed. The LBL model was expressed as:where Y is the dependent variable, X is the dietary Mn concentration, β0 is the value at the plateau, β1 is the slope and β2 is the Mn supplemental at the break point . Statistical significance was considered as 0.05 for all executed analysis. The term “tendency” was used for situations in which p-value is between 0.05 and 0.10.For both Mn sources under study, the analyzed values of Mn in experimental diets were close to those expected, as well as the concentrations of Fe, Cu, and Zn .4·H2O (4·H2O supplementation increased and decreased (p<0.05) linearly and were quadratically (p< 0.05) affected by increasing dietary MnPro supplementation  nor main effects of sources (p>0.05) were noticed on chick performance responses assessed . Chicks 4·H2O  increaseentation .4·H2O fed chicks. Chick intake, excretion, and retention of Mn increased (p<0.05) gradually in each supplemental Mn level, achieving the highest values at 100 mg Mn/kg diet. Manganese intake, excretion, and retention were linearly increased (p<0.05) as supplementation Mn levels increased. Chicks fed diets without supplemental Mn exhibited the lowest Mn balance (p<0.05) compared with the other Mn supplemental levels. Manganese balance was linearly and quadratically influenced (p<0.05) by Mn supplementation. Chicks fed dietary MnPro or MnSO4·H2O supplementation levels exhibit a linear increase (p<0.05) in Mn intake, Mn excretion, and Mn retention (p<0.05) .Manganese supplemental levels and sources influenced the intake, excretion, retention, and balance of Mn in broiler chicks . No inte(p<0.05) . Chicks 4·H2O supplementation elicited a linear increase (p<0.05) in Mn concentration in liver and tibia and quadratic increase (p<0.05) in liver (No interactive effects between Mn source and Mn supplementation levels (p>0.05) were noticed on Mn tissue concentration . The higin liver .4·H2O supplemented diets exhibited a higher activity of GSH-Px in the liver and breast muscle (p< 0.05) compared with MnPro fed chicks. Breast muscle total-SOD activity tended to be higher (p = 0.081) in MnSO4·H2O fed chicks. The activity of GSH-Px in breast muscle tended (p = 0.074) to increase linearly as dietary MnPro supplementation increased  was absorbed by broiler chicks. Yet, the authors reported that such rate was 2.8% when no Mn source was added to basal diet. Such findings may be clearly supported by our results. As detailed in 4·H2O fed chicks (20.6 mg Mn/kg).Previous reports have suggested that Mn supplementation did not affect ADG and FCR of growing chicks, and that cereal-based diets containing 19 to 26 mg Mn/kg from cereal grains in the diet without a supplemental Mn source, could support performance objectives ,4. Our f4·H2O to a low Mn basal diet, Conly et al [4·H2O supplemented diets, respectively. Liver is the primary organ responsible for regulating Mn body status through biliary excretion [4·H2O, or amino acid chelated Mn), Mn concentrations in broiler tibia were gradually increased up to 800 mg supplemental Mn/kg diet. Similarly, when investigating the Mn supplementation on broiler diets, Conly et al [4·H2O supplemented diets , achieving the greatest value at the highest level studied. Although Mn content in breast muscle and tibia were unaffected by Mn sources, the analysis of levels in each source suggests that Mn concentration in breast increased until 75 mg Mn/kg only when Mn was supplemented as MnPro. According to polynomial regression model estimates, Mn concentration in breast reached its maximum values at supplemental Mn level of 67.5 mg/kg, and for chicks fed MnPro, maximum deposition was achieved at 62.3 mg/kg. We noticed that Mn concentrations in tibia and liver approximately doubled in chicks fed the highest supplemental Mn level compared with chicks fed diets without supplemental Mn. Such outcomes differ from those reported by Lu et al [4·H2O affected breast muscle Mn content of growing chicks fed diets supplemented at 100 and 200 mg Mn/kg, and from those reported by Yan and Waldroup [4·H2O. Our results suggest that Mn concentration in breast muscle are mainly influenced by proteinate Mn source, i.e. MnPro.Manganese levels influenced liver Mn concentrations; whose greatest value was at 50 mg supplemental Mn/kg diet. Despite being statistically different, the means of liver Mn content from chicks fed 50 and 100 mg Mn/kg diet were quite similar numerically (11.5 vs 11.3 mg Mn/kg). Likewise, after adding tribasic manganese chloride or MnSOly et al  reportedxcretion . When prxcretion . Chicks xcretion  who repoly et al  noticed Waldroup  who obse2O2) and hydroxyl, which disrupt the redox balance in cells, and cause oxidative damage to tissues [2O2, GSH-Px ends the process by detoxifying H2O2.Although Mn levels did not affect antioxidant enzyme activity, we observed that, curiously, chicks fed inorganic minerals supplemented diets exhibited higher activity of GSH-Px in breast muscle and liver, and higher total-SOD activity in liver compared with chicks fed organic minerals. Organic minerals are potentially more bioavailable than the inorganic forms, so it was expected that they would support higher total-SOD activity. It is worth highlighting, however, that although trace minerals modulate the activity of antioxidant enzymes like SOD, GSH-Px, and catalase, they may also act as pro-oxidant agents ,29. Free tissues ,31. BecaTo the best of our knowledge, this is the first research where the ideal level of organic Mn for broiler chicks was estimated using a supplement in which the other trace minerals  were provided as organically complexed metals. As hypothesized, when compared with inorganic Mn, lower levels of organic Mn are required by growing chicks for performance optimization. Overall, estimates of supplemental Mn levels required to optimize Mn balance and Mn tissue concentration in chicks were lower than those levels to optimize feed conversion ratio in chicks. Based on the feed conversion response, the ideal supplemental levels of organic and inorganic Mn for broiler chicks were 38.0 and 74.3 mg Mn/kg diet, respectively.


Conversely, BPAF induced RACK1 up-regulation via androgen receptor (AR) activation, as confirmed by treatment with AR antagonist flutamide. Indeed, a biased agonism profile for BPA and BPAF for GPER was suggested based on their different binding modes revealed by our molecular docking. Altogether, our data suggest that RACK1 could represent an important target of EDCs and serves as a screening tool for their immunotoxic potential. Furthermore, RACK1 can be exploited to unmask multiple molecular interactions of hormone-active substances to better dissect out their mechanisms of action.Receptor for activated C kinase 1 (RACK1) has an important role in immune activation, and is regulated through a balance between glucocorticoid and androgen levels. We have previously demonstrated that RACK1 expression can serve as a marker for evaluation of immunotoxic profiles of hormone-active substances, such as endocrine-disrupting chemicals (EDCs). In this study, we investigated the effects of three bisphenols  on RACK1 expression and on the innate immune responses in the THP-1 human promyelocytic cell line, a validated model for this investigation. BPA and BPAF reduced RACK1 promoter transcriptional activity, mRNA expression, and protein levels. However, BPS had the opposite effect. As expected, these results on RACK1 were paralleled by lipopolysaccharide (LPS)-induced interleukin-8 (IL-8) and tumor necrosis factor-α (TNFα) production. Since BPA and BPAF induced RACK1 expression in the presence of glucocorticoid receptor (GR) antagonist mifepristone, a role of G-protein-coupled estrogen receptor (GPER) has been considered due to their known estrogenic profile. Therefore, additional molecular effects of BPA and BPAF were unmasked after treatment with different inhibitors of well-known pivotal players of GPER-mediated signaling. BPA exerted its effects on RACK1  According to the World Health Organization (WHO), an endocrine disrupting chemical (EDC) is “an exogenous substance or mixture that alters function(s) of the endocrine system and consequently causes adverse effects in an intact organism, or its progeny, or (sub)populations” . Due to in vitro and in vivo studies have shown the immunotoxic potential of BPA  through which the bisphenols can affect the immune system, and thence to search for molecular biomarkers to develop a method to rapidly predict immunotoxicity of novel bisphenols before they come into widespread use.Bisphenol A (BPA) is the most widely used and studied of the bisphenols, and its effects on the immune system at the cellular level were recently reviewed by l of BPA . In contl of BPA . Howeverl of BPA . Indeed,p,p’-dichlorodiphenyltrichloroethane  and p,p’-dichlorodiphenyldichloroethylene   , and the,p’-DDE) . In the in silico tools: Endocrine Disruptome  uses automated flexible molecular docking with Yeti/AutoDock to consider all compound orientations and conformations in the receptor binding site. This is combined with the multidimensional Quantitative Structure-Activity Relationships (mQSAR) software (Quasar) to define compound orientation, position, different solvation, protonation and conformation, for induced-fit models and the tautomeric state. This tool does not consider agonist or antagonist modes of action on AR and GR. The results are given as the predicted toxic potential  and the concentration at which the compound is expected to bind to the nuclear receptor (up to 100 µM considered) .® (EHU068001) for GRα were purchased from Sigma Aldrich . Lipopolysaccharide (LPS) from Escherichia coli serotype 0127:B8 and the cell culture media and supplements were from Sigma Aldrich . The mouse anti-human RACK1 monoclonal antibody (sc-17754) was from Santa Cruz Biotechnology . The mouse monoclonal anti-β-tubulin antibody (T0198) was from Sigma Aldrich . Electrophoresis reagents were from Bio-Rad . Chemicals were diluted in dimethyl sulfoxide (DMSO) for 50 mM stock solutions, and sequentially properly diluted.BPA (PubChem CID: 6623), BPAF (PubChem CID: 73864), BPS (PubChem CID: 6626), Mifepristone (RU486) (PubChem CID: 55245), Flutamide (PubChem CID: 3397) and esiRNA MISSION6 cells/mL in RPMI 1640 without phenol red containing 2 mM L-glutamine, 0.1 mg/ml streptomycin, 100 IU/ml penicillin, gentamycin 10 μg/ml, 50 µM 2-mercaptoethanol, supplemented with 5% dextran-coated charcoal-treated fetal bovine serum (DCC-FBS) and cultured at 37°C in 5% CO2 incubator. Preliminary experiments were conducted to identify non-cytotoxic concentrations [cell viability >80% (CV80)]. Cytotoxicity was assessed by propidium iodine staining and the CV80 determined for all compounds. For the different experiments, cells were then treated with increasing concentrations of BPA, BPAF and BPS (0.001–10 μM) or DMSO as vehicle control  as detailed in the legends.The THP-1 cell line was originally derived from an acute monocytic leukemia patient, and was from American Type Culture Collection . For experiments, THP-1 cells, were diluted to 10RACK1 gene promoter region between nucleotides −1744 and +361, and includes the glucocorticoid responsive element (GRE) sequence. The pGL4.32 vector  luciferase-reporter construct plasmid DNA was also used . DNA was quantified and assayed for purity using Quantus™ fluorometer . Transient transfections were performed in 24-well plates; for each well, 5 × 105 cells were seeded in RPMI 1640 medium without phenol red, and with 5% DCC-FBS, 1% antibiotics and supplemented with 1% L-glutamine. Transfections were carried out using Lipofectamine® 2000 , following the manufacturer instructions. Each luciferase reporter construct plasmid DNA was co-transfected with the pRL-TK renilla luciferase expressing vector to measure the transfection efficiency . During transfection, THP-1 cells were incubated at 37°C in 5% CO2, and then treated with the selected compounds for the times and at concentrations specified in figure legends. Cells were then lysed , following the manufacturer specifications. Luminescence was measured with a 20/20n Luminometer , with 10 s integration time.Plasmids preparation, transient transfections and luciferase assays were performed as previously described . The Δ1 lso used . PlasmidRACK1 gene expression was determined by qPCR using the Taq-Man™ PCR technology. Primers were from Applied Biosystems. PCR reactions were performed in duplicate, according to the standard protocols suggested by the manufacturer. For each PCR reaction, 10 ng total RNA was used. 18S ribosomal RNA was used as an endogenous reference. Quantification of the transcripts was performed according to the 2−ΔΔCT method.Quantitative PCR (qPCR) was performed as previously described . Total RRACK1 expression was determined at the protein level in cell lysates by immunoblot analysis. After the treatments, the cells were harvested, washed with phosphate-buffered saline (PBS) 1X, centrifuged, lysed in 100 µl homogenization buffer  and sonicated for 10 s. Protein content was measured using Bradford assay. Samples for Western blotting were prepared by mixing cell lysates with sample buffer , 20% glycerol, 6% β-mercaptoethanol, 0.1% bromophenol) and denatured at 95°C for 5 min. Equivalent amounts of extracted protein (10 μg) were electrophoresed into 10% SDS-PAGE under reducing conditions. The proteins were then transferred to polyvinylidene fluoride (PVDF) membranes , blocked in 5% (w/v) bovine serum albumin (BSA), 1X TBS, 0.1% Tween-20 for 1 h with gentle shaking. Proteins were visualized using primary antibodies diluted in 5% (w/v) BSA, 1X TBS, 0.1% Tween-20 for RACK1 (1:1000) and β-tubulin . In all of the experiments, the immunoreactivity was detected using host-specific secondary IgG peroxidase-conjugated antibodies , and developed using enhanced chemiluminescence . Immunoblot images were acquired with Molecular Imager Gel Doc XR (BioRad), and the optical density of the bands was determined using the ImageJ software . The relative densities of the bands are expressed as arbitrary units, and are normalized to the control samples run under the same conditions.Immunoblot analysis was performed as previously described . RACK1 eCytokine production was assessed using TNF-α  and IL-8 specific sandwich ELISAs . To induce cytokine secretion, 10 ng/ml LPS was added with the 24 h bisphenol exposure, which was prolonged for 3 h for TNF-α and for 24 h for IL-8 assays. Cell-free supernatants obtained by centrifugation at 300 × g for 5 min were stored at − 80°C until measurement. Limits of detection were IL-8 2.6 pg/ml for IL 8, and 22 pg/ml for TNF-α. Results are expressed in pg/mL .The three-dimensional (3D) structure of GPER, generated by homology modeling, was recovered from the web server GPCR-ITASSER . The modt-tests or analysis of variance (ANOVA), followed, when significant, by an appropriate post hoc test, as indicated in the Figure legends. In all of the reported statistical analysis effects were designated as significant if the p value was <0.05.Data are expressed as means ± standard error of mean (SEM) of at least three independent experiments. Statistical analysis was performed using the InStat software, version 7.0 . Significant differences were determined using Student’s in silico programs: Endocrine Disruptome  or DMSO as vehicle control, according to literature data . Upon 10 µM BPA treatment, we observed this correlation for both TNF-α  or DMSO as vehicle control. As shown in CK1 mRNA  but wereCK1 mRNA . The sliCK1 mRNA .Since a strong correlation between RACK1 expression and TNF-α release has been demonstrated, in accordance with the slight but significant RACK1 down-regulation observed after BPAF treatment, a low decrease in LPS-induced TNF-α secretion is reported . AccordiRACK1 promoter activity  identified in the RACK1 promoter region  was downloaded from the web server GPCR-ITASSER . GPCR-ITConversely, as depicted in RACK1 expression. As for the other bisphenols, THP-1 cells were treated for 6, 16, 18 and 24 h with increasing concentrations of BPS (0.001–10 μM) or DMSO as vehicle control. As shown in RACK1 promoter activity at any of the concentrations tested, while 16 h treatment induced significant RACK1 up-regulation only at 10 μM BPS .Finally, we investigated the effects of another BPA analogue, BPS, on 0 μM BPS . In agre0 μM BPS , which w0 μM BPS . Similar0 μM BPS . ConsistRACK1 expression, we investigated whether this increase was AR-mediated. THP-1 cells were, therefore, pre-treated with flutamide for 1 h. As shown in RACK1 promoter activity  . Indeed,In term of hazard identification, our results indicate that high concentrations of exposure to BPS predispose cells to an enhanced immune response, which, depending on the context, could be detrimental, i.e. it can favor the onset of autoimmune diseases, allergic reactions to unrelated antigens, misregulated inflammation to mention some possible consequences. Conversely, BPA and BPAF are associated in a decreased immunostimulation, with a greater effect of BPA compared to BPAF, that should be considered indicative of immunotoxicity. In this regard, different BPA occupational exposure studies performed on Chinese workers at BPA manufacturers and epoxy resin reported in urine samples ≤10 μM BPA concentrations, suggesting that in these workers immunological implications can occur due to BPA exposure . Howeverin vitro strategy where as a screening, molecular modelling and docking simulation to assess the affinity for steroid hormones receptors together with the RACK1 promoter activity should be the initial step, followed by RACK1 mRNA and/or protein expression to confirm that changes in the promoter activity have an impact on cellular RACK1 level, and finally, the physiological consequences of its modulation, can be investigated by evaluating immune functions. The other advantage in the use of RACK1 is that its expression can capture the complex interplay of transcriptional and non-transcriptional events associated with exposure to hormonally active compounds targeting more than one hormonal system and the resulting biological consequence, as we previously demonstrated (To conclude, we propose an nstrated . Results


Acute promyelocytic leukemia (APL) is a special type of acute myeloid leukemia Thrombosis is at increased risk complication in patients with this disease. However, the risk factors of thrombosis related to Chinese APL patients are not fully understood.In this study, clinical and laboratory data of 44 consecutively Chinese APL patients were collected and analyzed.P = 0.01), PAI-1 4G4G (P = 0.0009), bcr3 (P = 0.027), CD15 (P = 0.005), and FLT3-ITD mutation (P = 0.0008) were statistically significant. Using multivariate analysis, the risk factors of venous thrombosis in APL were CD15 (P = 0.043), PAI-1 4G4G (P = 0.009), WT-1 (P = 0.043) and FLT3/ITD (P = 0.013), respectively.One arterial and 6 venous thrombosis occurred in 44 patients, including 22 males and 22 females, with a median age of 44 years (range from 18 to 74 years). The ratio of male and female gender, age, white blood cell count, hemoglobin, platelets, disease risk stratification, CD2, Khorana score, differentiation syndrome (DS) and gene mutation related to prognosis of APL, including DNMT3A, TET2, IDH1, IDH2, NRAS and ASXL1 in the two groups with and without thrombosis were not statistically significant. The detection rate of PAI-1 genotype 4G4G was 71.4% (5/7) in 7 patients with thrombosis, while the detection rate of PAI-1 genotype 4G4G in 37 patients without thrombosis was 8.1% (3/37). The differences between the two groups in WT-1 (Our results suggested the PAI-1 gene 4G4G type, CD15, WT-1 and FLT3-ITD mutations excluding DNMT3A, TET2, IDH1/2, NRAS and ASXL1 are risk factors of thrombotic events in Chinese APL patients. Acute promyelocytic leukemia (APL) is a special type of acute myeloid leukemia with cytogenetic characteristics of t 15; 17)  formation the PML/RARa fusion gene [5; 17  , reverse transcription polymerase chain reaction (RT-PCR) for detection of PML-RARA fusion transcripts. The FLT3/ITD and WT1 gene mutations and epigenetic modifier genes (EMG) mutations related to prognosis of APL were detected in 44 patients by PCR and next generation sequencing (NGS). Four color flow cytometry  was used to detect the surface antigens of leukemia cells. Monoclonal antibodies were HLA-DR, CD2, CD3, CD4, CD5, CD7, CD8, CD9, CD10, CD11b, CD13, CD14, CD15, CD16, CD19, CD20, CD22, CD33, CD34, CD38, CD56, CD64, CD71, CD117, CD123, MPO and CD45.9/L by infusion of platelets, and the fibrinogen level was controlled above 1.5 g/L by fresh frozen plasma infusion. The patients did not use antifibrinolytic drugs, such as tranexamic acid, and did not have a central venous catheter during the induction therapy.All patients were treated with all-trans retinoic acid plus anthracyclines and/or arsenic trioxide. During the induction treatment, the platelet count of patients was controlled above 30 × 10The activated prothrombin time (APTT), prothrombin time (PT) and fibrinogen were detected by Automatic Coagulation Analyzer . D-dimer was measured by immunoturbidimetry. The normal range of them is shown as follows: APTT 25–35 s, PT 6–14 s, fibrinogen 2–4 g /L and D-dimer 0–0.5 μg/ml.Plasminogen activator inhibitor-1 (PAI-1) gene 4G/5G polymorphism was performed in 7 APL patients with TE and 37 APL patients without TE as previously described .From the day of admission to our department to the last day of follow-up, the thrombotic events and bleeding of APL patients were evaluated and recorded. Bleeding severity classification criteria was assessed using the International Society on Thrombosis and Haemostasis (ISTH) bleeding assessment tool . Symptom9/L and fibrinogen > 1.5 g/l, patients received the intravenous injection of heparin for 2 weeks and then received warfarin sequential therapy for another 3 months. During the short-term treatment of thrombosis, the warfarin dosage was adjusted based on the international normalized ratio (INR) value. Antiplatelet drugs were used in the treatment of arterial thrombosis, and the platelet count was maintained > 100 × 109/L during treatment. Three months of anticoagulation and antiplatelet therapy, the recanalization of blood vessels of patients would be detected by Color Doppler Ultrasound and CT or MRI image [In the acute phase of venous thrombosis, under the condition of ensuring platelets value > 30 × 10RI image , 21.P < 0.05 is considered statistically significant. The differences between groups were analyzed by log rank test. Univariate Cox proportional regression model and Multivariate Cox Proportional Regression model were used to identify risk factors. The odds ratio (OR) and 95% confidence interval (CI) were calculated by social science statistical software package.We used chi square and Fisher’s exact method to analyze the relationship between variables, by which The study population included 44 APL patients, 22 (50%) males and 22 (50%) females with a median age of 42 years (range 18–74 years). These population were all newly diagnosed APL patients admitted to our department from January 2013 to December 2019 and received treatment according to the PETHEMA APL99 protocol. Among them, 7 APL patients had thrombotic events, none of them had previous history of thrombosis and family history of thrombosis. One patient had arterial thrombosis (2.3%) and 6 patients had venous thrombosis (13.6%). No APL patients showed the symptoms of bleeding and thrombosis simultaneously. The clinical characteristics of APL patients with thrombotic events were shown in Table The rate of arterial thrombosis was 1/44 (2.3%), this patient was a female, aged 53 years, when the disease was first diagnosed, she felt weakness of the limbs. There was no history of thrombosis, hypertension, diabetes and hyperlipidemia of the female patient. The cerebral infarction of the patient was confirmed by CT and MRI, and then she was treated with antiplatelet drug. The rate of VTE was 6/44 (13.6%), the ratio of male to female was 4/2, and the median age was 37 years (22–67 years). Venous thrombosis occurred in 6 cases of lower extremity venous thrombosis, among which 4 cases occurred on the left and 2 cases on the right. All of those cases occurred during the induction of all-trans retinoic acid after diagnosed. The median time to diagnosis of thrombotic events was 14 days (9–26 days). Six patients with venous thrombosis were treated with unfractionated heparin (UFH) anticoagulation under the premise of platelet and fibrinogen supplementation and treated with sequential administration of warfarin later. After anticoagulant therapy for 2 weeks, the symptoms and signs related to thrombosis were improved. Moreover, serious bleeding and recurrent TE have not been observed during the 3 months of anticoagulant therapy.P = 0.68), age (P = 0.823), white blood cell count (P = 0.077), hemoglobin (P = 0.409), platelet (P = 0.334), disease risk stratification (P = 0.475), CD2 (P = 0.737) and differentiation syndrome (P = 0.562). There were significant differences between the two groups in PAI-1 4G4G (P = 0.0009), bcr3 (P = 0.027), CD15 (P = 0.005). To demonstrate that whether additional gene mutations involved in thrombotic events in APL patients, we analyzed the results of gene mutation related to prognosis of APL (See Table P = 0 .01) and FLT3-ITD muations (P = 0.0008), excluding DNMT3A (P = 0.44), TET2 (P = 0.43), IDH1 (P = 0.6), IDH2 (P = 0.66), NRAS (P = 0.66), ASXL1 (P = 0.9). In Multivariate Cox Proportional Regression, as shown in Table P = 0.043, OR 17.35, 95% CI 1.093–275.406), WT-1  and FLT3-ITD mutations , respectively. As shown in Table The clinical characteristics of the two groups of APL patients with thrombotic events were shown in Table  P = 0.82, white bin P = 0.09, plate P = 0.47, CD2 (P P = 0.0009). Moreover, in Multivariate Cox Proportional Regression, the risk factor of venous thrombosis was PAI-1 4G4G , suggesting a high-risk factor related to thrombotic event.Additionally, risk factors of hereditary thrombophilia, including antithrombin III, protein C, protein S, antiphospholipid antibodies, lupus anticoagulant and homocysteine, were evaluated in these 7 APL patients with thrombotic events. However, these risk factors of thrombophilia were not identified. The detection rate of PAI-1 genotype 4G4G was 71.4% (5/7) in 7 APL patients with thrombotic events, while the detection rate of PAI-1 genotype 4G4G in APL patients without thrombotic events was 8.1% (3/37). There was significantly statistical difference between the two groups regarding whether thrombotic events occurred or not . Venous thrombosis occurred in the disease induction treatment phase. The median time of thrombotic events occurred 14 days (range 9–26 days). Six patients with venous thrombosis were given heparin anticoagulant therapy under the premise of platelet and fibrinogen supplementation, leading to the complete recanalization of the vein, while no serious bleeding events being observed. The rate of venous thrombosis is consistent with the data reported in the previous literature . Other sCurrently, the risk factor of thrombosis in APL patients has not been fully discovered. Previous studies reported that these factors increased the risk of APL thrombosis, including male, high score performance status (PS), high white blood cell count and platelet count, low fibrinogen levels, hypoalbuminemia, PML/RARa fusion gene variant, CD2/CD15 and FLT3-ITD positive.P = 0.043, OR 17.35, 95% CI 1.093-275.406), suggesting high-risk factors associated with thrombotic events.Our results showed that there was no significant difference between thrombotic events and gender ratio, age, white blood cell count, hemoglobin, platelets, disease risk stratification and CD2. In addition, PML/RARa (bcr3) and CD15 were statistically significant. Moreover, in Multivariate Cox Proportional Regression, the risk factor of venous thrombosis in APL was CD15 . Similarly, results of this study showed that PAI-1 4G/4G was detected in 71.4% (5/7) of 7 patients with thrombosis, and PAI-1 gene polymorphism was 4G/5G  in 2 patients. The detection rate of PAI-1 4G/4G in the patients without thrombotic events was 8.1% (3/37), and the difference was statistically significant (P = 0.0009). In Multivariate Cox Proportional Regression, the risk factor of venous thrombosis was PAI-1 4G4G , suggesting a high-risk factor related to thrombotic event.PAI-1 is a key regulator of endogenous fibrinolytic activity . It is rThrombotic events are significant complications in malignant patients. Thrombosis risk is well defined in patients with solid tumors, and Khorana score is well validated for these patients. However, the value of Khorana scoring system in predicting the thrombotic events risk of hematological malignant diseases remains to be evaluated , 30. HenP = 0.043, OR 9.14, 95% CI 1.072-77.825) and FLT3/ITD , respectively (Table Lee YG et al.  suggesteIn conclusion, this study showed that the rate of thrombotic events in Chinese APL patients is consistent with the data reported previously. The WT-1 and FLT3/ITD mutations, PAI-1 gene 4G4G, CD15 positive expression are the risk factors of thrombotic events in Chinese APL patients. A better knowledge of risk factors of thrombosis is crucial to improve the management and outcome of thrombotic events in APL. However, the relatively small sample size and retrospective design are the limitations of this study. In addition, due to retrospective features, we have not performed a cumulative frequency analysis of thrombotic events in Chinese APL patients in our center, and it remains to be further explored in well designed clinical trials.


Neuronal loss in Parkinson’s disease and related brain diseases has been firmly linked to the abundant neuronal protein α-synuclein (αS). However, we have gained surprisingly little insight into how exactly αS exerts toxicity in these diseases. Hypotheses of proteotoxicity, disturbed vesicle trafficking, mitochondrial dysfunction and other toxicity mechanisms have been proposed, and it seems possible that a combination of different mechanisms may drive pathology. A toxicity mechanism that has caught increased attention in the recent years is αS-related lipotoxicity. Lipotoxicity typically occurs in a cell when fatty acids exceed the metabolic needs, triggering a flux into harmful pathways of non-oxidative metabolism. Genetic and experimental approaches have revealed a significant overlap between lipid storage disorders, most notably Gaucher’s disease, and synucleinopathies. There is accumulating evidence for lipid aberrations causing synuclein misfolding as well as for αS excess and misfolding causing lipid aberration. Does that mean the key problem in synucleinopathies is lipotoxicity, the accumulation of harmful lipid species or alteration in lipid equilibrium? Here, we review the existing literature in an attempt to get closer to an answer. SNCA genetic locus duplication/triplication [SNCA-promoter region REP1 [SNCA 3′ region [SNCA somatic copy number gains [SNCA have also been identified in PD patients [EIFG1, PARK2, LRRK2, GBA, SNCA, PINK1, PARK7, VPS35, and ATP13A2. No extra copies of SNCA were detected either, indicating a yet unknown cause for the increased αS levels. Although fPD cases account for less than 10% of all PD in the general population, abnormal accumulation of αS is present in virtually all patients with PD and related synucleinopathies. In the patient brain, αS-rich lesions can be found in neuronal somata  and in neurites . The exact nature of the αS accumulation/aggregation has been under debate, not least since a recent study described Lewy pathology as largely rich in lipids and membranous organelles, although ~20% of these LBs and LNs also contained large (5 nm) αS fibrils [α-Synuclein (αS) is an abundant protein of 140 residues present in all human neuron types. αS biology has attracted great interest given its association with a group of devastating neurodegenerative disorders known as synucleinopathies, the most prevalent of which is Parkinson’s disease (PD), followed by dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). PD is a debilitating and progressive movement disorder that affects nearly 10 million people worldwide, and yet measures that prevent or slow its course are currently lacking. PD pathogenesis has been linked to increased αS expression levels by virtue of lication , variabiion REP1 ,4, the Ser gains . Single-patients ,12,13 an fibrils ,16. This fibrils . While nAs mentioned above, a recent characterization of LBs by correlative light and electron microscopy (CLEM) came to surprising conclusions: fibrillar αS aggregates were found in about 20% of LBs and LNs examined, while abundant crowded organelles such as mitochondria, vesicle clusters and lipid membranes were detected in the core, which were coated with high concentration of non-fibrillar αS . AnotherSaccharomyces cerevisiae  leads to massive accumulation of lipid droplets (LDs) [Drosophila melanogaster, another animal model that lacks endogenous αS, shows age-dependent locomotor dysfunction and neurodegeneration upon transgenic expression of human αS [Drosophila melanogaster model of PD, in which human αS is specifically expressed in photoreceptor neurons, the accumulation of LDs induced by various LD proteins  was synergistically amplified by the co-expression of αS [Drosophila photoreceptor neurons and in human neuroblastoma cells [SNCA [The clearest evidence of αS being upstream of  lipid alterations can be found in one of the simplest model organism: αS expression in ts (LDs) . This fits (LDs) . A lipidts (LDs) . Drosophhuman αS ,38, therhuman αS . In a tron of αS . αS locama cells . As far ma cells . A humanma cells . Brain lma cells . These ima cells . More evma cells ,45,46,47GBA1 gene, the GCase enzyme catalyzes the conversion of glucosylceramide into glucose and ceramide [GBA1 are the most common genetic risk factor for PD [The impact of αS on lipid metabolic pathways has also been studied in a number of human cell culture models. Altered FAs, in particular long-chain PUFAs such as 22:4 and 22:6, have been reported in mesencephalic neuronal cells that stably overexpress human αS as well as in PD/DLB brain lysates . The autceramide . Notablyr for PD ,56,57. Ar for PD . Lipidomr for PD ,60,61. Wr for PD . The cohr for PD ,63, therCole et al. suggested that αS binds LDs and influences lipid metabolism by reducing the turnover of stored TGs within . This meEstablished biomarkers for PD or other synucleinopathies do not exist, and lipid-related biomarkers are no exception. Interesting in the context of this review is a recent study that highlighted lipid alterations in patient vs. control sebum, a non-invasively available biofluid : using Lupstream of lipid alterations, which then become drivers of toxicity (“lipotoxicity”). Admittedly, it was not an easy task to ignore more complicated scenarios that, conversely, involve effects of lipids on αS. There are many indications that lipids also affect αS biology, not least since the discovery of lipid-related PD-risk genes such as GBA [DGKQ [ELOVL7 [In this review we focused as much as possible on summarizing available evidence that αS dyshomeostasis can be unequivocally h as GBA ,56,57, DBA [DGKQ ,72 and E [ELOVL7 . Thus, a [ELOVL7 ,50,60. L [ELOVL7 ,77. In c [ELOVL7 . GBA1 depletion/mutation in dopaminergic neurons differentiated from human PD-iPSCs with a heterozygous GBA1 mutation (N370S/WT) led to accumulation of αS monomers [Beyond neutral lipids, Mazzulli et al. have proposed a “bidirectional pathogenic loop” in which glucocerebroside catabolism affects αS levels, which in turn affect glucocerebroside catabolism. Related to this, inhibition of GCase activity in differentiated SH-SY5Y cells  or GBA1 monomers . Treatmemonomers . A role monomers .Together these data suggest bidirectional interplays between both αS-neutral lipid and αS-glucocerebroside/sphingolipid metabolism. To complicate things even further, neutral lipids and glucocerebroside/sphingolipid metabolism also influence each other. In this context, it was recently demonstrated that enhancing GCAse activity ameliorates both lipid disturbance and αS dyshomeostasis in PD mouse models . It shouOverall, we are convinced that PD, LBD and MSA need to be treated as ‘lipidopathies’ as much as, if not more than, they have traditionally been considered to be ‘proteinopathies’ ,50. Futu


Leishmania infantum is the causative agent of visceral leishmaniasis transmitted by the bite of female sand flies. According to the WHO, the estimated annual incidence of leishmaniasis is one million new cases, resulting in 30,000 deaths per year. The recommended drugs for treating leishmaniasis include Amphotericin B. But over the course of the years, several cases of relapses have been documented. These relapses cast doubt on the efficiency of actual treatments and raise the question of potential persistence sites. Indeed, Leishmania has the ability to persist in humans for long periods of time and even after successful treatment. Several potential persistence sites have already been identified and named as safe targets. As adipose tissue has been proposed as a sanctuary of persistence for several pathogens, we investigated whether Leishmania infantum could be found in this tissue. We demonstrated both in cell cultures and in vivo that Leishmania infantum was able to infect adipocytes. Altogether our results suggest adipocytes as a ‘safe target’ for Leishmania infantum parasites. Leishmania infantum (L. infantum) is the causative agent of visceral leishmaniasis and is transmitted by the bite of female sand flies. According to the WHO, the estimated annual incidence is one million new cases, resulting in 30,000 deaths per year1. The outcome of infection can be variable, ranging from an asymptomatic form in immunocompetent individuals to obvious disease2. These clinical features depend on the species and the immune response of the host3. Symptomatic leishmaniasis is characterized by 3 main forms: cutaneous (CL), mucocutaneous (MCL) and visceral leishmaniasis (VL). CL is a chronic infection with ulcerative skin lesion occurring at the site of inoculation. MCL is generally the result of parasite dissemination from the skin to the naso-oropharyngeal mucosa. VL is the most serious form of leishmaniasis and typically leads to death in a few months in the absence of treatment. It is characterized by irregular fever, weight loss, hepatosplenomegaly, lymphadenopathies and pancytopenia. Currently, there is no human vaccine and treatments are expensive, with WHO guidelines recommending the use of just a few drugs, such as Amphotericin B4. Over time, several cases of relapses have been documented6, which thus call into question the efficiency of current treatments7 and raise the unsolved question of host sites allowing parasite persistence. Indeed, Leishmania has the ability to persist in humans for long periods of time, even after successful treatment8. Several potential persistence sites have already been identified and named as safe targets. These include immature myeloid precursor cells, monocytes, sialoadhesin-positive stromal macrophages of the bone marrow, hepatocytes and fibroblasts8. Previously, L. infantum persistence and development has been demonstrated in intra-abdominal adipose tissue of intraperitoneally infected mice9. Moreover, adipose tissue has been proposed as a sanctuary of persistence for bacteria such as Mycobacterium (M.) tuberculosis10 or M. canettii11, Coxiella burnetii12, viruses such as Human or Simian Immunodeficiency Virus14, and parasites such as Trypanosoma (T.) cruzi or T. brucei and Plasmodium spp.16. Here, we address the issues of whether L. infantum infects adipose tissues and whether adipocytes represent host cells for these parasites.L. infantum was present in brown (BAT) and white (WAT) adipose tissue of infected mice. For this purpose, BALB/c mice were intravenously inoculated with LUC-L. infantum as previously described9. Although bioluminescent parasites were used in this study, parasite burden in adipose tissue was insufficient to obtain an exploitable signal and allow in vivo monitoring of infection. Mice were thus sacrificed 6, 10 and 31 weeks post infection (p.i.). Tissue samples were analyzed for the presence of L. infantum by qPCR, which confirmed chronic infection in the spleen and liver . WAT and BAT from 4 mice infected intravenously were isolated and floating fractions enriched in adipocytes were obtained. The samples were pooled in order to increase the number of parasites and improve detection by qPCR. Separation between adipocytes and stromal cells was checked by amplification of adipocyte marker mRNA  Mitochondrial protein responsible for thermogenic respiration, a specialized capacity of brown adipose tissue, as well as adiponectin, a glycoprotein adipocyte-specific factor18, were specifically detected in the adipocyte fraction. As no parasite DNA was detected in the SVF of each kind of adipose tissue  white adipose tissue and differentiated into white and brite adipocytes. Pre-adipocytes from BAT were differentiated into brown adipocytes. 24, 48 and 72h post-infection, observation of the cells suggested the presence of intracellular parasites. This infection was observed in all types of adipocytes, and indicated the ability of L. infantum to infect and survive in murine adipocytes in vitro  were taken as a positive control for infection . Mice were maintained and handled according to the regulations of the European Union, the French Ministry of Agriculture and to FELASA  recommendations. Experiments were approved by the ethics committee of the Nice School of Medicine, France (Protocol number: 2017-56).GraphPad Prism 9 was used for statistical analysis. Significance was determined by analysis of variance (ANOVA) using a Bonferroni correction for multiple comparisons. A p-value < 0.05 was considered significant.L. infantum MON-1 (MHOM/FR/94/LPN101), was isolated from a patient with Mediterranean visceral leishmaniasis contracted in the Nice area (South of France). We used this isolate to generate a recombinant L. infantum—expressing the Green Fluorescent Protein reporter (GFP-L. infantum) and L. infantum—expressing the Luciferase reporter (LUC-L. infantum)9. L. infantum promastigotes were routinely grown at 26 °C in Schneider’s Insect Medium (Sigma®) supplemented with NaHCO3 0.4 g/L (Janssen chimica®), CaCl2 0.6 g/L (Fluka Chemika®), Fetal Bovine Serum 10% (Gibco®), 10 mL urine pool for 500 mL of medium, Phenol Red 0,1%, Hepes 10 mM pH 7.3, penicillin/streptomycin 1% (Gibco®), and l-Glutamine 1% (Gibco®).8 parasites were injected by intravenous route in 200 µL of PBS. Control mice were injected with 200 µL of PBS.Briefly the promastigote forms were washed three times in PBS, and 2 × 10BAT from infected BALB/c mice was sampled and freshly minced using a potter. The minced infected BAT was injected by intraperitoneal route in naive BALB/c mice.Briefly BAT and WAT were minced and then digested for 45 min at 37 °C in collagenase type 2. The tissue digest was passed through 250 μm nylon sheets. Floating adipocytes were separated from the SVF after decantation. The floating fraction corresponding to the adipocyte-enriched fraction was carefully removed.®), and then homogenized by Precellys®  in lysis buffer of the Qiagen Kit QIAmp DNA Mini Kit®. DNA extraction was conducted according to the recommendations of Qiagen®. The extracts were kept at − 20 °C for conservation. Quantitative PCR was implemented for detection and quantification of L. infantum targeting minicircle kinetoplast DNA (kDNA). Primers and probes previously described by Mary et al.5 containing 20 pmol of each forward (5′-CTTTTCTGGTCCTCCGGGTAGG-3′) and reverse (5′-CCACCCGGCCCTATTTTACACCAA-3′) primer and 3.33 pmol TaqMan probe (FAM-TTTTCGCAGAACGCCCCTACCCGC-TAMRA) were used for Leishmania screening and quantification5. The assays were performed with a final volume of 10 µL, including the 2.5 µL DNA sample. A standard curve was obtained from the primary DNA extraction source of 2.5 × 107 parasites and diluted serially at a 1/10 rate, which corresponded to 50,000 to 0.05 parasites in 2.5 µL. The PCR program was implemented with two temperature steps of 95 °C and 60 °C for 40 cycles. The standard curve and a pair of negative controls were used for each assay.Each sample of Adipose tissue, liver and spleen (25 mg) was put in a sterile tube of Lysing Kits (Precellys®). The tissue processor STP120 uses alcohol to remove water from tissues and replace it with a medium that allows sectioning of tissue. Thin sections (2.5 µm) were cut with a Microtome Microme HM340E (Leica BIOSYSTEMS®). Sections were deparaffinized by immersing 3× in xylene, rehydrated by successive immersion in ethanol solutions of different percentages  and in water. Unmasking was conducted boiling in 10 mM Sodium Citrate buffer (pH 6.0). Endogenous peroxidase activity was blocked by a solution of 3.0% hydrogen peroxide. The immuno-histochemistry labeling consisted of a primary human antibody directed against Leishmania. This antibody was recognized by an anti-human goat antibody, which was recognized by biotinylated anti-goat, the signal was amplified and revealed by Streptavidin-HRP. The brightfield microscope was an Eclipse Ci upright stand , using objectives 20× dry NA 0.40. Acquisitions were done with a DS—Ri 1 camera .Organs were fixed in 4% PFA (ParaFormAldehyde). Samples were embedded in paraffin automatically with the spin tissue processor STP120  containing 2 mg/mL collagenase A  and 20 mg/mL BSA . The digestion was successively filtrated through 250, 100 and 27 μm nylon sheets, and finally centrifuged at 500×g for 5 min. The pellet containing the SVF was cleared from red blood cells using specific buffer (Sigma) before being plated and maintained in DMEM containing 10% (v/v) fetal calf serum (FCS) until confluence. Differentiation was induced by supplementation with 1 μM dexamethasone , 0.5 mM isobutylmethylxanthine  and 860 nM insulin  for 2 days. Cells were then maintained for 7–10 days in presence of 100 nM insulin for white adipogenesis or a mixture containing 100 nM insulin, 1 μM rosiglitazone  and 0.2 nM triiodothyronine  for brown or brite adipogenesis.The method for generating white, brite and brown adipocytes from stromal vascular fraction (SVF) cells was adapted from a previous publication3T3-L1 fibroblasts were grown at 7% CO2 and 37 °C on coverslips in 35 mm dishes in DMEM, 25 mm glucose, and 10% calf serum, and 1% Penicillin–Streptomycin, and induced to differentiate in adipocytes. Briefly, 2 days after confluence, medium was changed for DMEM, 25 mM glucose, 1% Penicillin–Streptomycin, and 10% fetal calf serum (FCS) supplemented with isobutylmethylxanthine (0.25 mM), dexamethasone (0.25 µM), insulin (5 µg/mL), and pioglitazone (10 µM). The medium was removed after 2 days and replaced with DMEM, 25 mM glucose, 1% Penicillin–Streptomycin, and 10% FCS supplemented with insulin (5 µg/mL) and pioglitazone (10 µM) for 2 days. Then the 3T3-L1 adipocytes were fed every 2 days with DMEM, 25 mM glucose, 1% Penicillin–Streptomycin, and 10% FCS.BMDM (Bone Marrow Derived Macrophage), mouse femurs were removed and purified from the surrounding muscles and connective tissue. Under sterile conditions, the bone marrow was flushed by pressure after needle penetration in epiphyses with BMDM medium containing RPMI and decomplemented FBS 10% and gentamycin 0.001%. The cells were centrifuged  and resuspended in BMDM medium supplemented with M-CSF 10 ng/mL. Cells were seeded at 5 × 105 cells/well.For Blood monocytes were isolated from human healthy blood samples  using EasySep™ Human Monocyte Enrichment Kit (STEMCELL Technologies) according to manufacturer’s instructions. Macrophage differentiation was induced by human M-CSF .19 and differentiated as follow. Cells were cultivated in DMEM containing 10% FCS until confluence. When the cells reached confluence, they were induced to differentiate for 3 days in DMEM/Ham’s F12 (1:1) media supplemented with 10 μg/mL transferrin, 10 nM insulin, 0.2 nM triiodothyronine, 1 μM dexamethasone and 500 μM isobutyl-methylxanthine. The cells were next differentiated into white adipocytes using a media supplemented with 10 μg/mL transferrin, 10 nM insulin, 0.2 nM triiodothyronine or into brite adipocytes in the same media supplemented with 100 nM rosiglitazone.Human adipose tissue primary progenitor cells were from a previous studyTotal RNA was extracted using a TRI-Reagent kit (Euromedex) according to the manufacturer’s instructions. Reverse transcription-polymerase chain reaction (RT-PCR) was performed using M-MLV-RT (Promega). SYBR qPCR premix Ex Taq II from Takara (Ozyme) was used for quantitative PCR (qPCR), and assays were run on a StepOne Plus ABI real-time PCR instrument . The expression of selected genes was normalized to that of the 36B4  and TBP (TATA-box protein) housekeeping genes and then quantified using the comparative-ΔCt method. Primer sequences are available upon request.L. infantum per cell. Human adipocytes were infected after 14 days of differentiation with a ratio of 10:1 GFP-L. infantum per cell. 3T3 cells were infected 8 days after their differentiation with a ratio of 10:1 GFP-L. infantum per cell.The BMDM and adipocytes of pre-adipocyte murine origin were infected after 7 days of differentiation with a ratio of 10:1 GFP-Leishmania-GFP parasite are green. The fluorescent signals were analyzed with a Nikon confocal microscope using a × 60 magnification lens.For epifluorescence acquisition, we used the EVOS FL microscope , using the 10× dry Ph and 20× dry FL objectives. Acquisitions were done with a Sony ICK285AL monochrome CCD, 2/3′′ 1360 × 1024, 1.4 Megapixel camera . For confocal microscopy, F-actin was labeled with phalloidin-TRITC (red), and the nuclei were labeled with DAPI (blue). For ultrastructural analysis, cells were fixed in 1.6% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) at 4 °C, rinsed in 0.1 mol/L cacodylate buffer, and fixed for 1 h in 1% osmium tetroxide and 1% potassium ferrocyanide in 0.1 mol/L cacodylate buffer to enhance the staining of membranes. Cells were rinsed in cold distilled water, quickly dehydrated in cold ethanol, and lastly embedded in epoxy resin. Contrasted ultrathin sections (70 nm) were analyzed under a JEOL 1400 transmission electron microscope (EM) mounted with a Morada Olympus charge-coupled device camera.Supplementary Legends.Supplementary Figure S1.Supplementary Figure S2.Supplementary Figure S3.Supplementary Figure S4.Supplementary Figure S5.Supplementary Figure S6.


Adh genes) to mitigate alcohol toxicity, but these genes have functions beyond alcohol degradation. Previous research has shown that ADH can modulate quorum sensing in Acinetobacter baumannii, a rising opportunistic pathogen. However, the number and nature of Adh genes in A. baumannii have not yet been fully characterized. We identified seven alcohol dehydrogenases (NAD+-ADHs) from A. baumannii ATCC 19606, and examined the roles of three iron-containing ADHs, ADH3, ADH4, and ADH6. Marker-less mutation was used to generate Adh3, Adh4, and Adh6 single, double, and triple mutants. Disrupted Adh4 mutants failed to grow in ethanol-, 1-butanol-, or 1-propanol-containing mediums, and recombinant ADH4 exhibited strongest activity against ethanol. Stress resistance assays with inorganic and organic hydroperoxides showed that Adh3 and Adh6 were key to oxidative stress resistance. Virulence assays performed on the Galleria mellonella model organism revealed that Adh4 mutants had comparable virulence to wild-type, while Adh3 and Adh6 mutants had reduced virulence. The results suggest that ADH4 is primarily involved in alcohol metabolism, while ADH3 and ADH6 are key to stress resistance and virulence. Further investigation into the roles of other ADHs in A. baumannii is warranted.Most bacteria possess alcohol dehydrogenase (ADH) genes ( The NAD+-dependent ADHs (NAD+-ADHs) can be further subdivided into zinc-containing, iron-containing, and short chain ADHs [Alcohol dehydrogenases  are widely present in most bacteria [ain ADHs ,4,5,6. Iain ADHs ,8,9, andain ADHs , biofilmain ADHs , and bacain ADHs .Acinetobacter baumannii, a rising opportunistic pathogen that is increasingly seen in nosocomial and community-acquired infections [A. baumannii upregulated the expression of several proteins involved in stress responses, iron assimilation, phosphate transport, and lipid and carbohydrate anabolism; while at the same time, increased secretion of phospholipase C, acidification of bacterial cultures, and formation of biofilms was also noted [A. baumannii Adh gene (A1S_2098) was strongly induced in the presence of ethanol, but other genes potentially encoding ADHs, including two iron-containing ADHs (FeADHs), were not induced or only slightly upregulated [A. baumannii demonstrated stronger virulence in a Galleria mellonella model of infection [A1S_2098 gene is significantly upregulated during the A. baumannii biofilm formation process, and may play a role in quorum sensing, bacterial growth, and motility [A. baumannii genes potentially encoding ADHs, particularly FeADHs, has not yet been explored in detail.In addition, several studies have shown that the presence of ethanol can induce stress responses in bacteria that lead to increased virulence ,14,15, amotility . HoweverA. baumannii genes identified through in silico methods as potentially encoding FeADHs, and assessed the impact of their expression products on ethanol metabolism, stress responses, and virulence. These results can help to bridge the gaps in our current understanding of ADH function in A. baumannii, and may serve as the basis for future research into other as yet uncharacterized Adh genes. In the long term, the results of such research could potentially advise more effective sanitizing procedures and infection control measures, in order to prevent the survival and spread of A. baumannii in both clinical and community settings.In this study, we sought to elucidate the roles of three Adh genes in A. baumannii , DJ41_136 , and DJ41_1604 . Interestingly, the number of annotated Adh genes is relatively low in terms of the genome size of A. baumannii , as other Gram-negative soil bacteria that are also opportunistic pathogens have significantly more annotated Adh genes, with 44 Adh genes found for Burkholderia psedomallei K96243 (7.42 Mb genome) and 42 Adh genes annotated for Pseudomonas aeruginosa PAO1 (6.26 Mb genome). However, this may be indicative of limited research conducted in A. baumannii, and further studies are needed to better examine this phenomenon.A search of the GenBank database revealed seven genes annotated as possible aumannii . Genomichttps://www.megasoftware.net/, accessed on 31 July 2021) was used to construct a phylogenetic tree. The resulting dendrogram revealed two discrete iron-containing and zinc-containing clades  was lowest for ethanol , three double mutants , and one triple mutant (Δ346) using marker-less mutation. Mutants were confirmed by colony PCR  as a control to calculate relative expression levels  in the ethanol-containing medium . These results suggest that ADH4 plays a significant role in ethanol metabolism, while ADH3, ADH5, and ADH6 may have other key functions that necessitate a high level of expression, regardless of the presence of ethanol.Gene expression patterns of wild-type and mutant strains cultivated in 5 mM citrate medium or 5 mM citrate medium with 0.5% ethanol were compared through transcript analysis, with the n levels . In the  of Adh5 A. Expresg medium A. In mut of Adh5 B–H. In t Adh5  in some FeADHs in other microorganisms [+ as the preferred cofactor [+ [+ binding [Genomic and proteomic analysis of the three FeADHs examined in this study showed that the GGGS cofactor binding site motif (GGGSXXD) was conserved in all FeADHs ,8, and frganisms ,21, resucofactor ,23. It h binding .A. baumannii was able to survive in medium containing only ethanol, 1-propanol, or 1-butanol as the sole energy source , a minimal microbial growth media to which 1% ethanol, 1% 1-propanol, 1% a-butanol, or 5 mM citrate (5 mM citrate medium) was added in accordance with experimental needs. The bacterial strains and plasmids used are presented in Adh gene(s) intended for mutation were cloned into plasmid pk18mobsacB, and transformed into E. coli S17λπ to generate a donor strain for conjugation with A. baumannii. The E. coli donor strains and A. baumannii recipient strains were cultured in LB medium at 37 °C with shaking for 12 to 16 h, after which a 200 μL aliquot of donor bacterial cells was mixed with recipient A. baumannii cells at a 1:20 ratio. The mixed cells were spun down and washed with 60 μL of conjugation buffer  to remove traces of LB medium, and the cell pellet was resuspended in 60 μL of conjugation buffer, then spotted onto a membrane filter  placed on top of 1.5% LB agar. After cultivation at 37 °C for 19 h, filters were washed with conjugation buffer to remove bacterial cells, which were spun down and resuspended in 200 μL of conjugation buffer, then plated to 1.5% LB agar plates containing 50 μg/mL of ampicillin and 50 μg/mL of kanamycin. The first homologous recombination event enables the E. coli donor plasmid, which contains a kanamycin-resistant gene, to be integrated into the bacterial chromosome of A. baumannii recipient cells. Successful recombinants were then cultivated in LB medium containing 20% sucrose but without kanamycin, thus inducing the A. baumannii recombinants to excise the sacB gene in a second crossover event, and thereby enabling the deletion of the target Adh gene(s). Deletion mutants were subsequently confirmed through PCR analysis.Marker-less mutation was performed as previously described , with soA. baumannii Adh genes were cloned into plasmid pQE80L , which was then transformed into E. coli DH5α for recombinant protein production. Ni-affinity chromatography was used for recombinant protein purification. Columns were charged with 1× charge buffer (5 mM NiSO4) for 50 min to enable Ni2+ binding with the column, after which columns were washed with 1× binding buffer  for 50 min. Supernatants containing recombinant proteins were then passed through the column, after which the column was washed with 1× wash buffer , and then bound proteins were eluted using 1X elute buffer . The soluble fraction was collected and passed through Amicon Ultra-15 Centrifugal Filter-10 kDa units  to eliminate proteins smaller than 10 kDA. Retained proteins were concentrated by adding 4× ADH storage buffer  to a concentration of 1× and then centrifuging for 20 min at 4 °C, 4000× g, after which the supernatant was discarded and 1× ADH storage buffer was added, followed by centrifugation for 10 min at 4 °C, 4000× g. Concentrated proteins were divided into aliquots of 250 μL, to which 250 μL of 4×ADH storage buffer and 500 μL of 100% glycerol were added, and then divided into 200 μL aliquots for storage at −80 °C. Purified proteins were used in enzymatic assays as previously described [+, 1 mM semicarbazide, and 100 µL of alcohol  in 1 mL. The OD340 for different protein concentrations was determined every 30 s for three minutes. Specific activity was determined according to the Beer-Lambert Law: Aλ = ελ × C × l, with Aλ representing the OD340 difference between the third and the first minutes; ελ = 6.22 mM−1cm−1, the coefficient of OD340; and C indicating the concentration of produced NADH. The production of 1 µmole of NADH was defined as 1 unit of ADH activity [2HPO4, 80 mM NaH2PO4, pH = 5.8–8), Tris-Cl  and carbonate-bicarbonate buffer .Protein overexpression and purification methods were adapted from our previous study . Brieflyescribed . The enzactivity . Three b600 of 0.1. Bacteria were treated with 5 mM H2O2 or 300 µM tert-butyl hydroperoxide (tert-BHP) for 20 min and 4% NaCl, and viable counts were then determined by dropping 5 µL of cultured medium on LB agar plates 6 times and calculating colony-forming units (CFU) after colonies formed on plates [Single colonies of each strain were inoculated in 3 mL LB medium with corresponding antibiotics, which were cultured for 12–16 h at 37 °C with agitation. The overnight culture was refreshed with fresh medium to an OD600 of 0.6 were collected and centrifuged, and pellets were treated with 0.1 volume of fix solution , then centrifuged at 4 °C at 12,000× g for 10 min. Bacterial pellets were kept at −80 °C before RNA extraction. For RNA extraction, each pellet was suspended in 2 mL of Nucleozol  and processed according to the instruction manual provided by the manufacturer. The concentration of RNA was determined using a NanoDrop 2000c Spectrophotometer . DNA contamination was assessed by conducting PCR analysis on a 2 μL RNA sample, using DNA polymerase in the absence of reverse transcriptase. No PCR products were noted, indicating the absence of DNA in purified RNA samples. Following extraction, 2 µg of RNA from each sample was prepared for reverse transcription with 1× RTase buffer, 0.5 mM dNTP, 5 mM DTT, and 200 U of RTase , and placed at 50 °C for one hour. Quantitative RT-PCR was performed with 100 ng cDNA for each reaction. SYBR Green  was mixed with cDNA and the corresponding primers  [Bacterial cultures at an ODmers see  for PCR zerland) .Adh genes contribute to NADH/NAD+ homeostasis, the Peredox cassette from pRestB-His7tag-peredox-mCherry , a fluorescent biosensor of the cytosolic NADH-NAD+ redox state [EcoRI restriction site into the pWH1266 shuttle vector of A. baumannii and E. coli, to form pWH1266-pereodox-mCherry. This plasmid was then transferred by electroporation at 1.8 kV, 200 W, 25 μF  into wild-type and mutant bacterial strains [+ ratios. Following incubation at 37 °C for one hour, 200 μL of bacterial culture was spotted onto 1.5% LB agar plates containing 6.25 μg/mL of tetracycline, and plates were subsequently cultured at 37 °C for 12 to 16 h to select for successful transformants.To ascertain if ox state ,34, was  strains  to measu600 = 0.3, after which bacterial cells were concentrated by centrifugation. Cell pellets were resuspended in 300 μL of 0.9% NaCl, from which 50 μL of solution was taken and placed on a slide , then mounted with a coverslip  and sealed around the edges using transparent nail polish. Fixatives were not used in this study. The slides were examined under an Eclipse E800 fluorescent microscope , using red  and green  fluorescence filters, and at least three different views were captured for each sample.Successful transformants harboring the pWH1266_peredox-mCherry plasmid were cultured in LB medium containing 6.25 μg/mL of tetracycline at 37 °C for 12 to 16 h until ODA. baumannii ATCC 19606 wild-type and Adh3, Adh4, and Adh6 single, double and triple mutants of each strain. All procedures were performed as previously described, with minor modifications [G. mellonella larvae were selected for the same total weight, and were kept in petri dishes without food prior to infection. Larvae were infected with 5 × 106 CFU of each strain. Overnight cultures of each strain were washed twice with PBS , then diluted in PBS. Bacteria in 10 µL aliquots were injected into the hemocoel of each larva via the last left proleg by a Hamilton syringe. Infected larvae were incubated at 37 °C and scored for survival  every 24 h. Larvae were also scored for melanization over 96 h, according to a previously described scoring method [A virulence comparison was carried out with ications . Ten G. g method .p < 0.05 denoting significance.Data were analyzed using two-way ANOVA tests, with