Eriocheir sinensis) and their complete coding sequences (CDS) were obtained. Sequence analysis revealed deduced amino acid sequences of 915, 775 and 683 amino acids, respectively. Gene expression analysis revealed a broad tissue distribution for all three isoforms, with high CPT 1α and CPT 2 mRNA levels in the hepatopancreas of males and females. In males, CPT 1β was highly expressed in gill, heart, brain ganglia and muscle, while in females, CPT 1β-mRNA levels were relatively high in muscle, hepatopancreas and ovary tissue. The effects of dietary fish oil replacement on the expression of the three CPT isoforms in the hepatopancreas during gonadal development were investigated using five experimental diets formulated with replacement of 0, 25, 50, 75 and 100% fish oil by 1:1 rapeseed oil: soybean oil. The results showed that Diets 2# and 5# yielded higher CPT 1α and CPT 2 mRNA expression in males (P < 0.05), while in females, expression of all three CPT isoforms increased then declined in the hepatopancreas with increasing dietary fish oil replacement. The observed changes in CPT gene expression varied in different isoforms and gender, suggesting the three CPT genes might play different roles in fatty acid β-oxidation in E. sinensis.The carnitine palmitoyltransferase (CPT) family includes CPT 1 and CPT 2 that transport long-chain fatty acids into the mitochondrial compartment for β-oxidation. In this study, three isoforms  of the CPT family were cloned from Chinese mitten crab ( Mitochondrial β-oxidation of fatty acids is central to the provision of energy in most organisms, but long-chain fatty acids (LC-FA) cannot cross the mitochondrial inner membrane directly in animals , 2. CarnIn mammals, the CPT family is composed of four different isoforms encoded by distinct genes: a liver isoform (CPT 1α or L-CPT 1), a muscle isoform (CPT1β or M-CPT1), a brain isoform (CPT 10γ) and CPT 2 –11. The Gene expression and enzyme activity of CPT family members varies between different tissues, which suggests that the β-oxidation of fatty acid is a complex pathway in mammals, and activity of CPT enzymes is controlled by various regulatory mechanisms , 14. A wOncorhynchus mykiss) [Sparus aurata) [Lateolabrax japonicas) [Pelteobagrus fulvidraco) [Ctenopharyngodon idella) [Abnormal fat deposition in farmed fish is a significant problem for the development of the aquaculture industry, which may be related to over-feeding , imbalan mykiss) , gilthea aurata) , Japanesponicas) , yellow vidraco)  and gras idella) . In fish idella) , 29.Eriocheir sinensis  is an economically important aquaculture species in China, with annual aquaculture production of ~800,000 tons in 2016 [E. sinensis, which contains 30–80% lipids depending on developmental stage and gender [E. sinensis accumulate lipids in the ovaries, and mature ovaries contain around 30% lipids based on dry weight [E. sinensis is considered a good model for studying lipid metabolism in crustaceans [Chinese mitten crab  in 2016 . The hepd gender . During y weight , 33. Thestaceans –36. Howestaceans , 38.E. sinensis farming, and vegetable oil can be used to replace a proportion of fish oil in feed [E. sinensis has not been reported. Therefore, in the present work, the complete coding sequence (CDS) for each of the three CPT isoforms was obtained from the draft genome of E. sinensis and cloned by PCR [Formulated diets have been widely used in  in feed , 39. Howd by PCR . The isoRefer to Zhao et al. , five isThe contents of moisture, crude protein, and ash in experimental diets were analysed according to AOAC procedures . Total lAlternanthera philoxeroides) was transplanted at 1.0 m × 1.0 m intervals on the bottom of each pond to provide shelter and to help maintain water quality. Each of the five dietary treatments (Diets 1#-5#) were repeated in quadruplicate, with 50 crabs per replicate. Prior to commencement of the experiment, crabs were acclimated in the ponds for 3–5 days until initiation on the 15th September 2014 and lasted for 30 days.Experiments were conducted at Chongming mitten crab research station, Shanghai Ocean University, Shanghai, P.R. China. In late August 2014, pond-reared crabs that had completed puberty moult were obtained from outdoor earth ponds at the research station , 45. TheCrabs were fed one of the five experimental diets once daily at 18:00 with a ration of approximately 1–3% of total biomass depending on the residual feed, water temperature and weather. Every morning at 9:00, excess feed and dead crabs were recorded. During the experimental period, a water depth of 70 cm was maintained throughout, and 30–50% of water in each pond was exchanged every 2 weeks. dissolved oxygen was maintained at ~4 mg/L, ammonia and nitrite were maintained below 0.5 mg/L and 0.15 mg/L, respectively and the pH fluctuated between 7.0 and 9.0.At termination of the feeding trial, crabs were fasted for 24 h and then were treated with cold shock method to minimize suffering before harvest. Hepatopancreas tissue from eight male crabs and eight female crabs from each treatment (two crabs from each replicate) were randomly sampled to investigate the effects of fish oil replacement on CPT gene expression. Meanwhile, 12 tissues from five male crabs  and 10 tissues from five female crabs  fed Diet 1# were collected for testing of CPT tissue expression. Samples were immediately frozen in liquid nitrogen and stored at -80°C until use.E. sinensis. Three putative CPT isoforms  were found and PCR-specific primers were designed to amplify full-length cDNAs  according to the manufacturer’s protocol, and stored at -80°C. RNA concentration was measured using a Q5000 ultraviolet spectrophotometer , and RNA integrity was evaluated by 1% agarose gel electrophoresis. First-strand cDNAs were synthesised using a reverse first-strand cDNA synthesis kit . PCR amplification was conducted at 95°C for 5 min followed by 35 cycles of 95°C for 30 s, 55°C for 30 s, 72°C for 2 min, and a final extension of 72°C for 10 min. Amplified products of the expected size were gel-purified using a TIANgel Midi Purification Kit , cloned into the pMD19-T vector , and the resulting construct containing the target DNA was transformed into Escherichia coli DH5α cells. Positive clones were selected by blue-white spot screening and confirmed by DNA sequencing.Detailed searches using bioinformatics tools and available databases were employed to identify putative CPT candidate genes in the draft genome of th cDNAs . Total Rhttp://blast.ncbi.nlm.nih.gov/). Transmembrane domains of proteins were predicted using TMHMM server v.2.0 (http://www.cbs.dtu.dk/services/TMHMM). Signal peptides were predicted with SignalP v.4.1 . CPT gene family members were predicted using the conserved domains profile (http://smart.embl-heidelberg.de/). Isoelectric point (pI) and molecular weight were calculated using ProtParam within the ExPASy server (http://web.expasy.org/protparam/). Multiple protein sequence alignment was performed using CrustalW program, and phylogenetic trees were constructed by Bayesian inference with MEGA 6.0 [Sequences were verified and analysed using the NCBI BLAST tool  using a SYBR RT-PCR Kit . The total reaction volume was 10 μL containing 5 μL of 2× SYBR Master Mix, 1 μL of diluted cDNA, 0.2 μL (10 mM) of forward and reverse primers and ROX Reference Dye II, and 3.4 μL of Rnase-free dHt method .P < 0.05 was considered statistically significant).Statistical analysis was performed using SPSS 18.0 . Results are presented as mean ± standard error (SE). Statistical significance was determined using one-way analysis of variance (ANOVA) and post-hoc Duncan’s multiple range tests , CPT 1β (GenBank accession no. MH037159) and CPT 2 (GenBank accession no. MH037160). Sequence analysis revealed that CPT 1α, CPT 1β and CPT 2 have a full-length CDS of 2748, 2328 and 2052 bp, encoding polypeptides of 915, 775 and 683 amino acid residues, with a theoretical pI of 9.07, 8.79 and 7.93 and a molecular weight of 104.98, 88.25 and 76.29 kDa, respectively. The instability index (II) of the CPT 1β protein was computed to be 38.37, which classifies the protein as stable. However, the instability index (II) of the CPT 1α and CPT 2 proteins were computed to be 51.03 and 41.58, respectively, which classifies them as unstable.In the present study, the CDS was obtained for of each of the three CPT isoforms of The identities of CPT family members and the other representative CPT amino acid sequences were investigated via multiple sequence alignment using Clustal W . SequencE. sinensis clustered together with CPT 1 from other species (vertebrates and invertebrates), separate from the CPT 2 cluster. In addition, CPT 1α and CPT 1β clustered most closely to Folsomia candida CPT 1α (58% identity) and Drosophilla melanogaster CPT 1 (60% identity). CPT 2 formed one monophyletic sub-branch, and clustered with other CPT2 proteins . By contrast, CPT 1β expression was highest in gill and heart, followed by muscle, brain ganglia and hepatopancreas. For CPT 2, expression levels were ordered hepatopancreas > heart > gill > brain ganglia > thoracic ganglia > seminal vesicle > intestine > haemolymph > germinative area > accessory gland. CPT 1β mRNA expression was generally higher than CPT 1α and CPT 2 expression in the same tissue, except in the hepatopancreas. CPT 1α and CPT 2 displayed similar expression patterns among the tested tissues.All three CPT isoforms were expressed in all tissues tested, including brain ganglia, thoracic ganglia, hepatopancreas, stomach, intestine, heart, gill, haemolymph, muscle, germinative area , seminal vesicle , accessory gland  and ovary females; . ExpressIn female crabs , CPT 1α P < 0.05). Although the expression level of CPT 1β tended to increase with increasing replacement of dietary fish oil, no significant differences were found among the five treatments (P > 0.05)CPT mRNA expression levels of the three isoforms in the hepatopancreas following the five dietary treatments are shown in P < 0.05). Expression of CPT 1β in Diet 2# was significantly higher than in Diets 3#-5# (P < 0.05). CPT 2 was expressed most highly in Diet 2#, but there were no significant differences among the five treatments (P > 0.05).For female crabs , mRNA exSynechogobius hasta [Ctenopharyngodon idellus [Danio rerio, Sparus aurata, Lates japonicas and fish others [Members of the CPT family play an important role in the control and regulation of fatty acid β-oxidation . In mammus hasta , and fiv idellus . Sequench others , 26, 49.h others , and cDNE. sinensis . Interestingly, duplication of CPT 1α was not observed, and CPT 1γ was not present. The deduced polypeptides share high homology with CPT proteins from other species. Moreover, prediction of protein domains suggests that CPT 1β and CPT 2 in E. sinensis include an acyltransferase choActase domain. The N-terminal domain (residues 1–150) of mammalian CPT 1 contains mitochondrial targeting information and has been implicated in the insertion of the protein in the outer mitochondrial membrane [E. sinensis CPT 1β. However, CPT 2 in E. sinensis has no N-terminal domain, which is important for sensitivity to malonyl-CoA [E. sinensis and other fish and mammal species. In previous studies, CPT 1 reported to include two transmembrane domains most fish and mammals [E. sinensis isoforms belong to the CPT family.In the present study, we successfully identified and characterised three CPT isoforms from membrane . Our cur mammals , 52, 53.E. sinensis in malonyl-CoA sensitivity should be investigated in future studies.β-oxidation of fatty acids is a complex pathway in mammals, and the activity of CPT enzymes is controlled by various regulatory mechanisms including transcriptional regulation, physiological regulation by malonyl-CoA, and pharmaceutical regulation . CPT 1 iE. sinensis were determined to evaluate their physiological roles. In general, mRNA expression of all three CPT isoforms was detected in all tested tissues, albeit at varying levels , the highest CPT 1α mRNA levels occur in heart and white muscle [E. sinensis were higher than in other tissues. The hepatopancreas is a major site for lipolysis, and our results suggest that CPT 1α might be a major player in the degradation of fatty acids in E. sinensis. CPT 1β was predominantly expressed in gill, heart, muscle and hepatopancreas in males, and in muscle, hepatopancreas and ovary tissue in females, with very low expression in other tissues. This distribution is in agreement with previous results for CPT 1β in S. aurata, P. fulvidraco and C. idella [E. sinensis, CPT 2 was abundantly expressed in the hepatopancreas in both males and females, similar to CPT 1α. High expression of all three CPT isoforms in the hepatopancreas of E. sinensis further suggests that it is a major site of lipid metabolism in Crustacea and indicates differences in lipid metabolism between males and females.The tissue distribution profiles of CPT isoforms in g levels . CPT 1α,by CPT 1 . In blune muscle , and in e muscle , 27. Sim. idella , 25, 27.. idella . In E. sE. sinensis.Regulation of CPT gene expression has received considerable interest to improve our better understanding of the regulation of fatty acid β-oxidation in commercial aquaculture species. Fish oil (FO) produced by wild fisheries is a major lipid resource in aquatic feed. However, due to increasing costs and limited supplies of global FO , 63, it E. sinensis, the results of the present study showed that CPT 1α and CPT 2 expression levels in the hepatopancreas first increased then decreased with increasing dietary FO replacement. This may be due to a significant increase in dietary C18:1n9 and C18:2n6 levels in Diet 2#, since some of these LC-FAs may be used for energy provision via CPT 1α and CPT 2. An increase in dietary vegetable oils can lead to high lipid deposition in the hepatopancreas [E. sinensis may be due to the different species. However, expression of CPT 1β tended to increase with increasing FO replacement, but no significant differences were found, which suggests that replacement of dietary FO made no difference to CPT 1β mRNA expression. This result further indicates that CPT 1β might principally regulate fatty acid oxidation in muscle in male E. sinensis.In mammals, long-chain fatty acids  can significantly increase CPT 1 mRNA levels compared with medium-chain fatty acids (8:0 and 10:0), but they have no effect on CPT 2 mRNA expression . In malepancreas . This suE. sinensis, the highest CPT 1α and CPT 2 expression levels were detected in the hepatopancreas of animals fed Diets 4# and 2#, respectively, and expression was decreased following consumption of Diet 5#. A similar result was observed in a previous study on E. sinensis in which CPT 1 mRNA levels were significantly increased in the 1:1 fish oil: linseed oil (FLO) group relative to the FO group [In female FO group . SeveralFO group , 71. In FO group , 72. TheE. sinensis. CPT 1α and CPT 1β have two transmembrane domains, while CPT2 has none. All three CPTs are highly expressed in the hepatopancreas in both males and females, and CPT 1β is also highly expressed in male gill, heart and muscle, and female ovaries and muscle. Dietary fish oil replacement had a significant effect on mRNA levels in the hepatopancreas for all three CPT isoforms. These results indicate that the CPT isoforms may engage in variedly physiological roles related to fatty acid β-oxidation in E. sinensis, but the underlying molecular mechanisms need to be clarified in future studies.In summary, three CPT isoforms  were identified and characterised in


Pseudomonas aeruginosa is an opportunistic bacterium associated with healthcare infections in intensive care units (ICUs), ventilator-associated pneumonia (VAP), surgical site infections, and burns. This bacterium causes 75% of death in burned patients, since it can develop a persistent biofilm associated with infections, express several virulence factors, and antibiotic-resistance mechanisms. Some of these virulence factors are proteases such as elastase and alkaline protease, or toxic metabolites such as pyocyanin and is one of the few microorganisms able to produce cyanide, which inhibits the cytochrome oxidase of host cells. These virulence factors are controlled by quorum sensing (QS). In this work, 30 P. aeruginosa clinical strains isolated from burned patients from a tertiary hospital in Mexico City were studied. Antibiotic susceptibility tests were done, and virulence factors  were determined in presence of an N-acylhomoserine lactonase, AiiM able to hydrolyze a wide range of acyl homoserine lactones. The treatment reduced significantly the activities of elastase and alkaline protease, and the production of pyocyanin and HCN in all producer strains but not the secretion of toxins through the type III secretion system. Our work suggests that AiiM treatment may be an effective therapy to combat P. aeruginosa infection in burn patients. Pseudomonas aeruginosa is an opportunistic bacteria associated with healthcare infections in intensive care units (ICUs), ventilator-associated pneumonia (VAP), central line-associated blood stream infections, surgical site infections . HCN is a poison that inhibits respiration by inactivating cytochrome oxidase C  with Gram negative card identification, some biochemical tests included were oxidase, indole production, growth at 42°C, arginine dihydrolase and glucose oxidation/fermentation. The origin of each clinical isolate is shown in Randomly 200 strains were selected® (CLSI) M07-A10  and were set into a heat block at 95°C for 5 min. Tubes were centrifuged, and the supernatant was added into a new tube.2, 200 μM dNTP′s, 0.2 μM primer forward and reverse, 0.026 U/μL Taq polymerase . The amplification conditions used were: 95°C 10 min, 95°C 30 s, 58°C 45 s and 72°C 50 s during 35 cycles, 72°C 5 min and finally 4°C . Amplification products were loaded into a 1% agarose gel stained with SYBR® green I  and visualized with Gel DOCTM XR + with Image LabTM software . P. aeruginosa PAO1 was used as positive control in each one of the systems and Δ lasR/rhlR PAO1 as negative control of transcriptional regulators.Genes related to Las and Rhl systems were amplified  in a finEscherichia coli M15/pQE30 AiiM was grown overnight (ON) in Luria Bertani broth with 100 μg/mL of carbenicillin (Sigma Aldrich C1389) and 50 μg/mL of kanamycin (Sigma Aldrich K1876), afterward, 10 mL of the ON cultures were taken and inoculated into 1 L of terrific broth with carbenicillin and kanamycin as above described, cultures were incubated at 37°C 220 rpm, optical density (OD) at 600 nm was measured until the culture reached an OD of 0.5 and immediately after, it was induced with 500 μM IPTG. The cultures were incubated at 37°C 220 rpm for 6 h and centrifuged at 10,000 rpm for 40 min. Pellets were resuspended in 40 mL of purification buffer  and 500 μM PMSF (Sigma Aldrich P7626), sonicated  and centrifuged at 10,000 rpm during 40 min. The supernatants were passed through a 0.2 nm filter and loaded onto Protino® Ni-TED resin  for purification of His-tagged proteins previously equilibrated with 3 column volumes (CV) of 20 mL of purification buffer, then filtered protein extracts were passed through the column, after that, 2 additional CV of purification buffer were passed. Protein was eluted with 150 mM imidazole (Sigma Aldrich I5513) in 2 CV of purification buffer. AiiM fractions with higher purity were selected, concentrated with polyethylene glycol 35 KDa (Sigma Aldrich 946-46) into dialysis tubing cellulose membrane (Sigma Aldrich D9777). Afterward, dialysis was done to remove imidazole using dialysis buffer . SDS-PAGE was done to estimate the amount and purity of AiiM. Protein was quantified by its absorbance at 280 nm with NanoDrop 2000 , using an extinction coefficient (Abs 0.1%) of 1.08. Aliquots of protein were made and stored at −20°C until they were used.AiiM construction was provided by Dr. Toshinari Maeda . BrieflyN-butyryl-DL-homoserine lactone , 1 mM N-(3-oxooctanoyl)-L-homoserine lactone , 1 mM N-decanoyl-DL-homoserine lactone , 1 mM N-(3-oxodecanoyl)-L-homoserine lactone , and N-(3-oxododecanoyl) homoserine lactone . 60 mM NaOH was used as positive control since it can hydrolyze HSL molecules and the reaction was stopped with the addition of 2N HCl. Several concentrations, from 250, 100, 50, 25, 10, and 5 μg/mL of purified AiiM were tested. Time exposition was also varied; 24 h, 2 h, 1 h, 30 min, 20 min, 10 min, and 5 min. All experiments were performed in triplicate. Chromatographic conditions were: column temperature 25°C, sample temperature 25°C, injection volume 10 μL, flow rate 1 mL/min, detection 205 nm. Elution mixture was made with 50 mM phosphates buffer pH 2.9:acetonitrile. For C4-HSL the relation was 90:10, 3OC8-HSL 60:40, C10-HSL 60:40, 3OC10-HSL 60:40, and 3OC12-HSL 50:50.To evaluate the HSL lytic activity of the purified AiiM, an analytical assay was developed in an Alliance HPLC system  with a Symmetry  C18 Column . Both short and long acylated chains were included, 1 mM P. aeruginosa PAO1 and Δ lasR/rhlR PAO1 growth kinetics. A 50 mL flask with 5 mL of LB was inoculated with each one of the strains at an initial OD600nm of 0.05 with and without 5 μg/mL of AiiM, samples were taken each hour for 12 h.Grow curves were analyzed to test the effect of AiiM on P. aeruginosa PAO1 ΔlasR/rhlR and ΔlasI/rhlI PAO1 mutants according to methods previously described (600nm of 0.05 in 5 mL of LB with and without AiiM (5 μg/mL), samples were incubated for 18 h at 37°C and 220 rpm, and centrifuged at 14,000 rpm for 2 min. 50 μL of the supernatant were taken and set into 950 μL of elastase buffer  with 2.5 mg of elastin-congo red (Sigma Aldrich reference E0502) as substrate. Tubes were incubated at 37°C, 220 rpm for 2 h, centrifuged at 14,000 rpm for 5 min and the released die in the supernatant was measured at 495 nm with a spectrophotometer SmartSpec Plus . All determinations were performed by triplicate.Elastase was determined for clinical strains and escribed , with so600nm of 0.05 with and without AiiM (5 μg/mL). AiiM protein was added at the beginning of the cultures, samples were incubated for 18 h at 37°C and 220 rpm, and centrifuged at 14,000 rpm for 2 min. 50 μL of supernatant were taken and added into 950 μL of protease buffer with 2.5 mg of Hide-Remazol brilliant blue R (Sigma Aldrich reference H6268) as substrate. Tubes were incubated at 37°C, 220 rpm for 20 min centrifuged at 14,000 rpm 5 min and the supernatant was measured at 595 nm with a spectrophotometer SmartSpec Plus .Alkaline protease was determined according to methods previously described , with so600nm 0.05 with and without AiiM (5 μg/mL). AiiM protein was added at the beginning, samples were incubated 18 h at 37°C and 220 rpm. One milliliter of supernatant was taken, centrifuged at 14,000 rpm for 5 min, and then 800 μL of supernatant were set into new 1.5 mL conic tube and 400 μL of chloroform was added, each tube was mixed in vortex for 2 min. Tubes were centrifuged for 5 min at 14,000 rpm, 300 μL of the organic phase were taken and deposited into a new tube, 800 μL of 0.2 N HCl were added and mixed for 2 min in vortex then samples were read at 520 nm . In addition another 3 cultures per strain with AiiM 0.5 μg/mL were grown to an OD600nm of ∼ 1.0, supplemented with a final concentration of 30 μM of 3OC12-HSL, and incubated until 18 h of incubation were completed, supernatants were collected and pyocyanin concentration and elastase activity determined.For these control experiments, the PAO1 reference strain and the clinical isolate P809 were used. Three independent cultures per strain were inoculated in LB medium at an initial OD600nm 0.05 in 5 mL of LB and grown at 37°C, with 220 rpm shaking during 18 h, with AiiM 5 μg/mL and without AiiM enzyme. LB cultures then were centrifuged 14,000 rpm for 5 min. Supernatants were separated in new tubes. For long chain HSL detection Agrobacterium tumefaciens NT1 pZLR4 , 1 mM N-(3-oxodecanoyl)-L-homoserine lactone  and N-(3-oxododecanoyl) homoserine lactone  were used as positive controls and the molecules treated with AiiM 5 μg/mL as negative controls. Plates were incubated at 28°C and results were observed, a positive reaction associated to the production of long chain HSL was observed as a green halo and inactivation of the signals when the halo was absent.To identify autoinducer production and its inactivation by AiiM, each one of the 30 clinical strains were grew up onto MacConkey agar plates, then one colony was taken and inoculated into 5 mL of LB for ON growth. After that, new cultures were inoculated at an initial ODT1 pZLR4  was usedP. aeruginosa strains  were grown overnight in LB medium. Bacteria were diluted 1:200 into 4 mL of a modified LB medium supplemented with 10 mM MgCl2, 0.5 mM CaCl2 and 5 mM EGTA (pH 7.4) in the presence or absence of 5 μg/mL of AiiM, and grown at 37°C to an OD600nm of 0.8 to 1.0. 1 mL of each culture was collected into a microcentrifuge tube and bacteria were pelleted by centrifugation. The resulting pellet was resuspended in 200 μl of 1× Laemmli SDS sample buffer normalized for OD600nm. The supernatant was centrifuged once again and the resulting supernatant was transferred into a clean tube. Supernatant proteins were precipitated overnight at 4°C by adding trichloroacetic acid to a final concentration of 10%, pelleted by centrifugation and resuspended in 20 μL of 1x Laemmli SDS sample buffer containing 10% saturated Tris base normalized for OD600nm. Samples were separated by 15% SDS-PAGE, transferred onto a nitrocellulose membrane and probed for the presence of the effectors ExoS and ExoU by immunoblotting. Detection was performed using the Immobilon Western chemiluminescent HRP substrate (Millipore), and bands were visualized on X-ray films (Carestream MXB Film).For type III secretion assays Escherichia coli ATCC® 25922 was used as a pansusceptible strain. Disks were incubated for 1 and 10 min, 2 and 24 h. A suspension of 0.5 McFarland was made with E. coli ATCC® 25922 and was plated onto Müller Hinton agar , tubes were incubated at 37°C until their use. All experiments were made by triplicate; the inhibition diameter was measured using a Vernier device.As ß-lactams have a lactone ring, we performed the inactivation disk method to determine whether AiiM would be able to inactivate this kind of antibiotics. Briefly, 2 mL of 0.8% isotonic saline solution (ISS) was added into sterile 12 mm × 75 mm tube, disks of 30 μg ceftazidime , 30 μg cefepime , 10 μg imipenem , and 10 μg meropenem . One set of disks containing ISS was used as negative control, another set of all antibiotics above mentioned with 5 μg/mL of AiiM, and finally, since NaOH can break the lactone ring another set of all antibiotics was used as positive control with 60 mM NaOH. P. aeruginosa. The most common burn etiology was fire (66.7%) followed by scalds (23.3%) and electrical burns (10%). Medians of hospital length of stay were 53 days (8-303), the mean total body surface area was 40% (10-85%) with a mortality rate of 26.6% (n = 8). P. aeruginosa strains were isolated from urine (n = 8), quantitative biopsies (n = 8), blood (n = 6), endotracheal aspirates (n = 3), bronchoalveolar lavage fluid (n = 2), catheter tips (n = 2), and qualitative biopsy (n = 1).All clinical isolates were obtained from burned patients infected with P. aeruginosa and other non-fermentative Gram negative rods; therefore colistin represents the last treatment option for these types of infections  and Rhl system (rhlI and rhlR) were found in all the clinical strains. P. aeruginosa PAO1 was used as positive control and ΔlasR/rhlR PAO1 was used as negative control (data not shown).In order to determine if Las/Rhl systems were present in AiiM protein was obtained at a purity of >90% as judged by SDS-PAGE. After elution with 150 mM imidazole, fractions were collected, and those in which AiiM was present were concentrated into dialysis tubing cellulose membrane with polyethylene glycol, then protein concentration was determined and kept at −20°C until used. The purified protein consists of a single band of ≈ 27 kDa, compared with a theoretical molecular mass of 27.2 kDa .In order to prove AiiM activity against acylated chains of diverse HSL, cleavage was determined by HPLC. Five HSL autoinducers with both short and long chains were tested . First HSL alone was run to identify retention times , later tP. aeruginosa with and without AiiM were done to determine if its addition had any effect in the growth rates. As expected, there was no difference in the growing dynamics between these cultures .Once it was confirmed that AiiM did not affect activity , activitactivity  there weactivity , and sevactivity . At the Agrobacterium tumefaciens NT1 pZLR4 , carbapenems , aminoglycosides (amikacin and gentamicin), fluoroquinolones (ciprofloxacin and levofloxacin), and piperacillin/tazobactam. Moreover, one strain was resistant to colistin, which is the last antibiotic resource.Burn injuries are one of the most common and devastating forms of trauma and patients with serious thermal injury require immediate specialized care in order to minimize morbidity and mortality . P. aeruaumannii . In a reaumannii , P. aeruP. aeruginosa growth kinetic.AiiM showed a wide activity and was able to cut all HSL molecules tested, consistent with a previous report by P. aeruginosa clinical strains isolated from diabetic foot using the lactonase SsoPox with a substitution in the amino acid 263 changing a tryptophan to isoleucine, in order to increase the enzymatic activity, using 0.5 mg/mL of protein, they found some strains with certain tolerance to the SsoPox addition. In our study, AiiM reduced elastase and alkaline protease activities, pyocyanin and HCN concentrations, and we did not find any strain with tolerance against AiiM treatment, in spite that AiiM was used at a 100 times lower concentration than SsoPox. Moreover, AiiM effectivity is much higher than the effectivity of small molecule QS inhibitors such as brominated furanones and 5-fluorouracil, that cannot inhibit QS-virulence factor production of several of the clinical strains tested, and that are very toxic to some of them (In contrast, AiiM significantly reduced the four QS-dependent virulence factors tested in our study following a dose response pattern; moreover, others have demonstrated that AiiM had very good activity in a mouse model of acute pneumonia  and redu of them .lasR/rhlR mutant of P. aeruginosa PAO1, T3SS effector toxins are secreted at the same levels than in the wild-type strain, demonstrating that this virulence factor is not positively regulated by QS (Nevertheless, for type III secretion, no inhibition by AiiM was found, which is consistent with recent findings showing that in a Δed by QS , insteaded by QS . These hed by QS . Moreoveed by QS  must be P. aeruginosa infections is the fact that lasR defective mutants are often found in infections and although in principle these mutants will produce low levels of QS-dependent virulence factors, this is not always the case due to a rewiring of the virulence factor regulation (One possible limitation of the utilization of AiiM and other QQ enzymes for treating gulation . And theSince AiiM is a member of the alpha/beta hydrolases superfamily and several antibiotics are inactivated by metallo ß-lactamases, reducing clinical options to treat infections , we testin vivo tests in burn infection models are lacking, our work suggests that AiiM treatment may be an effective addition for the treatment of P. aeruginosa infections, and since research by other groups had shown also the utility of the lactonase SsoPox against clinical isolates from diabetic foot patients in vitro (in vivo using an amoeba model (in vivo studies using acylases are scarce, recently it was shown the PvdQ in addition to their inhibitory properties in vitro (Caenorhabditis elegans model was also able to increase survival, reduce damage and decrease bacterial loads in a pulmonary infection mice model (Although in vitro  and in vba model , and in ba model  lactonasin vitro  and in Cce model . Hence QP. aeruginosa isolated from burned patients and no AiiM tolerant strain was found. However, it had no inhibitory effect against the T3SS.AiiM showed a strong activity against C4-HSL, 3OC8-HSL, C10-HSL, 3OC10-HSL, and 3OC12-HSL. It reduced elastase and alkaline protease activities as well as pyocyanin and HCN concentrations in all tested clinical strains of All datasets generated for this study are included in the article/Pseudomonas aeruginosa clinical strains used in this study were isolated as part of routine clinical hospital procedures to diagnose infection and hence ethical approval was not required, according to the National Institute of Rehabilitation ethical committee. All bacterial isolates were stored as part of laboratory and epidemiology necessities.LL-J, GG-R, MH-D, DR-M, PN, MD-G, DL, JS-R, and DD-R performed the experiments. RF-C, TM, BG-P, GG-R, LL-J, and RG-C designed the study, supervised the project and discussed the results. LL-J wrote the manuscript with input from all authors.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.


Toxoplasma gondii infection. In this study, we inserted a B cell epitope (SAG182−102 or SAG1301−320), a CD8+ cell epitope (HF10 or ROP7), and a CD4+ cell epitope (AS15) of T. gondii into a truncated HBcΔ(amino acids1–149) particle to construct four chimeric VLP vaccine formulations, i.e., HBcΔH82, HBcΔH301, HBcΔ R82, and HBcΔ R301. When these chimeric HBc particles were expressed in Escherichia coli, they showed icosahedral morphology similar to that of the original VLPs and were evaluated as vaccine formulations against acute and chronic toxoplasmosis in a mouse model (BALB/c mice (H-2d). All these chimeric HBc VLPs induced strong humoral and cellular immune responses with high IgG antibody titers and interferon(IFN)-γ production. Only the mice immunized with HBcΔH82 showed prolonged survival time (15.6 ± 3.8 vs. 5.6 ± 0.8 days) against acute infection with RH tachyzoites and decrease in brain parasite load  against chronic infection with Prugniuad cysts, as compared to the findings for the control group. These findings suggest that HBc VLPs would act as an effective carrier for delivering effective multiple antigenic epitopes and would be beneficial for developing a safe and long-acting vaccine against toxoplasmosis.There is no effective protective vaccine against human toxoplasmosis, which is a potential threat to nearly a third of the world population. Vaccines based on virus-like particles (VLPs) have been highly successful in humans for many years, but have rarely been applied against  Toxoplasma gondii, an obligate intracellular protozoan, is the causative agent of toxoplasmosis. The disease is a severe health threat for fetuses, newborns, and immunocompromised individuals  are diverse nanoparticles  that are formed by structural viral proteins, such as capsids, and can self-assemble. They resemble viruses but are noninfectious because they lack viral genetic materials , 5. VLPsin vitro (Hepatitis B virus (HBV) core antigen (HBc) has been extensively used as a VLP platform for many years . It is hin vitro . It can in vitro . Variousin vitro , 10.+ T cells that can secrete interferon (IFN)-γ and develop cytotoxic activity against infected cells (+ cytotoxic T lymphocyte (CTL)-mediated resistance to T. gondii cysts in the brain is absolutely correlated with the major histocompatibility complex (MHC) class I Ld allele in mice  and rhoptry proteins (ROPs), are antigens that are recognized by murine T lymphocytes and are vaccine candidates against toxoplasmosis , a CD8+ cell epitope (HF10 or ROP7), and a CD4+ cell epitope (AS15). The efficacy of these vaccines in eliciting humoral and cellular immune responses in BALB/c mice was examined. Immunization of these mice activated CD8+ T cells to produce IFN-γ, activated B cells to produce antibodies, and protected against subsequent challenge with type I and type II strains of T. gondii. Our results indicated that a VLP-based immunization with complex and heterogeneous antigenic epitopes is a promising vaccine approach to protect against toxoplasmosis.In this study, we designed four HBc VLP vaccine formulations, i.e., HBcd) were purchased from Shandong University Laboratory Animal Center . All the mice were bred and maintained in small groups inside separate cages under a 12-h light/dark cycle (lights switched on at 7:00 am). Water and food were available ad libitum to all the mice throughout. The protocol was approved by the Institutional Animal Care and Use Committee of Shandong University (Contract LL201602044). All efforts were made to minimize animal suffering and the number of mice used.Female 6–8 week old BALB/c mice (H-2T. gondii (type I), was kindly provided by Professor Jilong Shen . Cultures of RH tachyzoites were propagated in human foreskin fibroblast (HFF) cells in Dulbecco's modified Eagle's medium  supplemented with 10% (vol/vol) fetal bovine serum , as previously described (T. gondii (type II), was a kind gift from Professor Xingquan Zhu . The cysts of the Pru strain were maintained in Kunming mice by oral passage of infectious cysts in the mice, as previously described  and a B cell epitope (SAG182−102 or SAG1301−320) of T. gondii were inserted between amino acids 78 and 79 of HBcΔ particles, with a Gln (Q) and Asp (D) linker at both ends (+ cell epitope (AS15) was fused into the C terminal of HBcΔ particles. The final constructs were named HBcΔ, HBcΔH82, HBcΔH301, HBcΔR82 and HBcΔR301. The amino acid sequences of the inserted B, CD8+, and CD4+ cell epitopes are provided in Δ, HBcH82Δ, HBcH301Δ, HBcR82Δ, and HBcΔR301, were verified by DNA sequencing. The recombinant plasmid pET-30a(+)/HBcΔ was used as a negative control.When used as a carrier protein to present antigens, HBc particles have historically been truncated at amino acid 149 to avoid incorporation of host RNA by the C-terminal tail [amino acids (aa) 150−183], which contains the RNA/DNA-binding site of the viral capsid . The truoth ends . A CD4+ Escherichia coli BL21(DE3) (Novagen) cells transformed with the recombinant plasmids, i.e., pET-30a (+)/HBcΔ, HBcΔH82, HBcΔH301, HBcΔR82, and HBcΔR301, were grown on Luria-Bertani (LB) agar plates containing kanamycin (50 μg/ml). Individual colonies were cultured at 37°C with shaking at 220 rpm in 3 ml LB medium supplemented with kanamycin (50 μg/ml). On the next day, the night cultures were transferred to 500 ml fresh LB medium with kanamycin for amplification. When the optical density (OD600) of the cultures reached 0.6–0.8, expression of the chimeric proteins was induced by adding 0.5 mM isopropyl-β-D-thiogalactopyranoside. The cultures were grown for an additional 16–18 h at 11°C and harvested by centrifugation at 4,000 × g for 20 min at 4°C. Resuspended cell pellets were disrupted by sonication, subjected to 12% sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE), and stained with Coomassie Blue to analyse chimeric VLPs expression.The procedure for expression of the chimeric proteins was performed as previously described , with miE. coli transformants were collected and resuspended in lysis buffer . These cells were broken up and the chimeric proteins were released by sonication at 400 W for 4 s, performed at 8-s intervals for a total of 20 min. Most of the target proteins existed in the form of inclusion bodies in the E. coli expression system. After the inclusion bodies were collected by centrifugation at 10,000 × g for 20 min at 4°C, they were washed three times with wash buffer  and dissolved in lysis buffer  in a certain ratio overnight. Then, the supernatant was added gradually to the buffer , stirred slowly, and gradient-diluted. Then the protein solution was dialyzed against the same buffer overnight in the dialysis bag . After the supernatant was recovered, His-tagged chimeric HBc VLPs were purified using Ni2+ iminodiacetic acid (IDA) affinity chromatography gel (Novagen), following the procedure recommended by the manufacturer.The recombinant proteins were purified as described in a previous study . The celΔ protein was used as a negative control in all three experiments. Endotoxin contamination in the chimeric HBc VLPs was analyzed using Limulus amebocyte lysate(LAL)  , followed by detection with horseradish peroxidase (HRP) Affinipure goat anti-mouse IgG (H+L) . HBc, China) .+ cell epitope (HF10 or ROP7), a B cell epitope (SAG182−102 or SAG1301−320), and a CD4+ cell epitope (AS15) were synthesized (>95% purity) by Synpeptide Co., Ltd.  and stored at −80°C until use.Four multiepitope peptides, i.e., H82, H301, R82, and R301, containing a CD8Δ, HBcΔH82, HBcΔH301, HBcΔR82, and HBcΔR301, were adsorbed onto carbon-formvar-coated copper grids (200 mesh) and negatively stained with 1% phosphotungstic acid as previously described  or 50 μl PBS (blank control) on Days 0, 14, and 28 as previously described .The mice were segregated into groups of 23 each and immunized subcutaneously with 50 μg of a recombinant protein, i.e., HBcT. gondii infection, 10 mice per group were infected intraperitoneally with a lethal dose (1 × 103 tachyzoites) of the RH strain and were monitored over 20 days as performed previously . All the mice were euthanized at 45 days after the challenge and their brains were removed and homogenized in 1 ml PBS. The T. gondii cyst burden in the mouse brains was confirmed by analyzing 10 μl samples of each cerebral homogenate with a light microscope as previously described  as previously described  was adsorbed overnight onto 96-well plates  at 4°C in 50 mM carbonate buffer (pH 9.6). After blocking with 1% low fat milk in phosphate-buffered saline (PBS) containing 0.05% Tween 20 (PBST) for 1 h at 37°C, the mouse serum was diluted in PBS (1:25) and incubated at 37°C for 1 h. After washing three times with PBST, the anti-mouse-IgG, IgG1 or IgG2a HRP-conjugated antibodies (Sigma-Aldrich) were added. Peroxidase activity was detected using 10 mg/ml 3,3′,5,5′-tetramethylbenzidine  and stopped by adding 50 μl 2M H2SO4. The results were recorded as the absorbance at 450 nm and detected with a Thermo Scientific Multiskan  FC Microplate Photometer. All assays were performed in triplicate.The escribed . Briefly+ and CD8+ T lymphocytes subsets of immunized mice were analyzed by flow cytometry as previously described , anti- mouse CD4-FITC (eBiosciencesA), and anti-mouse CD8-PE (eBiosciences) for 30 min at 4°C in the dark. All these cells were analyzed with a Cytoflex S Flow Cytometer , and the data were analyzed with the CytExpert software.The percentages of the CD4escribed . Briefly6 cells/ml) were stimulated with 10 μg/ml of the recombinant proteins, i.e., HBcΔ, H82, HBcΔH82, H301, HBcΔH301, R82, HBcΔR82, R301, or HBcΔR301, or 5μg/ml Con A (Sigma-Aldrich) (positive control), or 50 μl PBS (blank control), as previously described . The survival percentage was analyzed with the Kaplan-Meier test, and survival curves were compared using the log-rank test. Data have been expressed in terms of mean ± standard deviation (SD) values. Values of Δ, HBcΔH82, HBcΔH301, HBcΔR82, and HBcΔR301, is shown in Δ, HBcΔH82, HBcΔH301, HBcΔR82, and HBcΔR301. After digestion with restriction endonucleases, these plasmids were confirmed by agarose gel electrophoresis. The plasmids of chimeric HBc VLPs  were digested into large fragments (4193 bp) and small fragments  by the 5'-ApaI and 3'-XhoI endonucleases, respectively, and the non-chimeric HBc particle (HBcΔ) was 4,193 bp and 1,503 bp correspondingly, which indicated that the recombinant plasmids contained inserts , HBcΔH82 (23.2 KD), HBcΔH301 (23.0 KDa), HBcΔR82 (23.0 KDa) and HBcΔR301 (22.8 KDa) was observed in the supernatants of cellular lysates obtained by sonication  demonstrated good capability for self-assembly and had icosahedral morphology similar to the original corresponding non-chimeric VLPs (HBcΔ) (A truncated version of HBc (aa 1–149), lacking a protamine-like domain (aa150–183), is known to be sufficient for self-assembly into capsid particles . TEM anas (HBcΔ) . These pT. gondii antibodies, sera from the immunized mice were tested with ELISA. Total IgG antibodies were detectable as early as 4 weeks after the first vaccination in the mice immunized with HBcΔH82 and HBcΔR82 proteins compared with the mice immunized with PBS (p < 0.001). The IgG antibody levels in the sera of mice immunized with the recombinant proteins containing the SAG1301−320 epitope  also increased. In contrast, the mice injected with HBcΔ, H82 and R82 did not have significant antibody titers as compared to those of the control group (PBS).To detect the levels of anti-with PBS . These tΔH301, HBcΔR82, R301, and HBcΔR301 proteins, especially with HBcΔH82  did in the immunized mice. However, for the conformational B cell epitope SAG182−102, the strong IgG antibody response was induced only after it was loaded onto the HBc VLP platform (HBcΔH82 and HBcΔR82) in mice, which can maintain its natural conformation. This specific immune response was of the Th1 type.To determine whether a Th1 and/or a Th2 humoral response was induced by vaccination with the recombinant proteins, the levels of specific IgG1 and IgG2a subclasses were measured. Increased IgG2a production was observed in the sera of mice immunized with H301, HBcΔ, H82, HBcΔH82, H301, HBcΔH301, R82, HBcΔR82, R301, or HBcΔR301, or PBS alone (blank control), the percentages of CD4+ and CD8+ T lymphocytes subsets in the spleens of immunized mice were evaluated by flow cytometry 2 weeks after the third immunization. The percentages of CD4+ T cells in all the mice immunized with the recombinant proteins containing the CD4+ T cell epitope (AS15) , especially the chimeric HBc particles ,were higher than those for the PBS group , HBcΔR82 and HBcΔR301 (p < 0.01), and with H82, H301, R82, and R301 (p < 0.05) proteins were significantly higher than those of the mice immunized with PBS (ΔH82 (p < 0.001) and HBcΔH301(p < 0.01) vaccinated mice had higher IL-2 levels than the control group (PBS) did (ΔH82 protein compared with those of PBS (+ T cell epitopes (HF10 and GRA720−28) could both induce robust Th1-type cellular immune responses in mice. When these two peptides were loaded onto the carrier of HBc VLPs, the immune effect was more pronounced, especially for the HF10 peptide (HBcΔH82 and HBcΔH301). In addition, splenocytes from all groups of mice proliferated to comparable levels in response to ConA  did . Howevere of PBS . All the to ConA .T. gondii infection, immunized mice were challenged with 1 × 103 RH tachyzoites 2 weeks after the final immunization. The survival rates of the challenged mice are shown in Δ (5.7 ± 0.9 days), H82 (6.1 ± 1.2 days), H301 (6.0 ± 1.4 days), HBcΔH301 (8.1 ± 1.5 days), R82 (5.9 ± 1.2 days), HBcΔR82 (8.8 ± 1.3 days), R301 (6.3 ± 1.1 days), and HBcΔR301 (6.6 ± 1.2 days). The mice vaccinated with the chimeric HBc VLPs HBcΔH82 (15.6 ± 3.8 days), HBcΔH301(8.1 ± 1.5 days), HBcΔR82(8.8 ± 1.3 days), and HBcΔR301 (6.6 ± 1.2 days) were partially protected compared with the control group (PBS). The longest survival time was observed in the mice immunized with HBcΔH82 protein, and one mouse even survived after 20 days of challenge.To evaluate whether the recombinant proteins could induce effective protection against acute T. gondii chronic infection was also evaluated by oral challenge the immunized mice with 30 Pru strain cysts. At 45 days after the challenge, the mice vaccinated with HBcΔH82  and HBcΔH301  proteins had fewer cysts than the PBS immunized mice did , CD8+ T cell epitope (HF10), and CD4+ T cell epitope (AS15), induced substantial resistance to acute and chronic infection with T. gondii.The resistance provided by this vaccine formulation to 82−102 or SAG1301−320), a CD8+ cell epitope (HF10 or ROP7), and a CD4+ cell epitope (AS15) into truncated HBcΔ (aa1–149) to construct four chimeric HBc VLPs, i.e., HBcΔH82, HBcΔH301, HBcΔR82, and HBcΔR301. When the chimeric HBc particles were expressed in E. coli, they showed icosahedral morphology similar to that of the original HBc protein. All these chimeric HBc VLPs induced strong humoral and cellular immune responses, along with high IgG antibody titer and IFN-γproduction. Prolonged survival time and decreased cyst burden in the brain were observed only in mice vaccinated with the HBcΔH82 protein.In this study, we inserted a B cell epitope (SAG1E. coli), which is a dangerous component for vaccines used in humans and animals  and the other with T = 4 symmetry (120 dimers), and have a diameter of approximately 30 nm or 34 nm, respectively. The truncated HBc (aa1–149) forms ~ 95% of T = 4 capsids and ~ 5% of T = 3 capsids  successfully assembled into icosahedral particles and were similar to the original non-chimeric HBc VLPs (HBcΔ). Most of these particles were uniform in size.Hepatitis B virus (HBV) causes both acute and chronic infections of the liver . It is a animals . Therefo capsids , 38. In 82−102 or SAG1301−320) and a CD8+ cell epitope (HF10 or ROP7) in the site between Asp78 and Pro79 and inserted a CD4+ cell epitope (AS15) into the C-terminus of the truncated HBcΔ(aa1–149) to construct the chimeric HBc VLPs. The malaria  vaccine MalariVax (ICC-1132), produced by Apovia, has a similar foreign insertion site of MIR and the C terminal of HBc VLP and has already undergone Phase I clinical trials  (T. gondii protein extract in porous nanoparticles (DGNP) have been reported; all of these enhanced the immunogenicity of the antigens derived from the parasite , on the HBc molecule . It is al trials . In addiheir own . HBc-VLPparasite .T. gondii (82−102) peptide (H82 and R82) did not induce higher IgG titers than PBS, but (SAG182−102) did after insertion into the MIR of HBc (HBcΔH82 and HBcΔR82). These findings may be attributable to the fact that most B cell epitopes required to induce the desired humoral immune responses have to maintain their native conformation found in the protein  (HF10) and ROP7 (IPAAAGRFF) epitopes can elicit robust T cell responses during the chronic phase . In addition, the peptide AS15, which was presented by the Ab MHC class II molecule to the CD4+ T lymphocytes  HF0 and ROPic phase . GRA6 isic phase . ROP7 isic phase . It is s T cells . Consistphocytes , successT. gondii strains can be broadly classified into three clonal lineages, which differ in their virulence. Type I strains  are highly virulent in mice and are used in survival-based acute infection models. Low-virulence strains  have high cyst-forming ability in mice and are used in chronic infection models (ΔH82 showed prolonged survival time and lesser brain cysts than mice immunized with PBS. These results are consistent with the above findings that only the mice immunized with HBcΔH82 successfully triggered both strong humoral and cellular immune responses in all the vaccinated mice, and these two immune responses were interrelated and acted synergistically to increase protection against acute and chronic infection with T. gondii (+ T cells induced by AS15. Although most of them differentiate into Th1 cells triggered by IFN-γ secreted from the activated CD8+ T cells, a small number of CD4+ T cells still differentiate into TH2 cells, resulting in a slight but significant increase in IL-10 and IL-4 secretion. Similar activation of a T helper immune response with high levels of Th1 and low levels of Th2 related cytokines has been elicited by many other promising T. gondii vaccines also (ΔH301 protein, which contains the same peptide HF10 as HBcΔH82. This may indicate that the useful features of HF10, as an immunodominant Ld gene restricted epitope whose region was found to control cyst formation, can be utilized well on this VLP carrier platform. Immunization of Ld mice with an HF10-containing vaccine comprising palmitic acid moieties or a monophosphoryl lipid A derivative was found to induce potent IFN-γ production from Ld-restricted CD8+ T cells and protect mice from Pru strain challenge (Most n models , 53. Then models , 54. In . gondii . Increas. gondii , 55. A pnes also , 56, 57.hallenge .T. gondii on their surface and elicited potent humoral and cellular responses. The mice vaccinated with the HBcΔH82 formulation survived after lethal and sublethal doses of parasite challenge. To our knowledge, no VLP vaccine against T. gondii that simultaneously contains a conformational B epitope, an Ld-restricted CD8+ T cell epitope, and an Ab-restricted CD4+ T cell epitope has been reported yet. Our study provides a novel and efficient strategy to develop vaccines against infection with this parasite. However, the significant differences between mice and humans, including those in MHC molecules, should not be ignored. Therefore, finding proteins or epitopes that are immunogenic and protective to the human body is an important topic for future research on vaccines against T. gondii; this also requires in-depth understanding of the molecular mechanisms governing the activation of the immune system in humans.In conclusion, the HBc VLPs displayed a high density of effective antigenic epitopes of JG, SH, and HC designed experiments. JG, XS, and WS performed experiments and analyzed data. JG wrote the manuscript. AZ, HC, and CZ revised the manuscript. KA, GP, and HZ provided advice for experiment. All authors read and approved the final 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.


There is still a high unmet need for the treatment of most patients with cystic fibrosis (CF). The identification and development of new Cystic Fibrosis Transmembrane conductance Regulator (CFTR) modulators is necessary to achieve higher clinical benefit in patients. In this report we describe the characterization of novel potentiators. From a small screening campaign on F508del CFTR, hits were developed leading to the identification of pre-clinical candidates GLPG1837 and GLPG2451, each derived from a distinct chemical series. Both drug candidates enhance WT CFTR activity as well as low temperature or corrector rescued F508del CFTR, and are able to improve channel activity on a series of Class III, IV CFTR mutants. The observed activities in YFP halide assays translated well to primary cells derived from CF lungs when measured using Trans-epithelial clamp circuit (TECC). Both potentiators improve F508del CFTR channel opening in a similar manner, increasing the open time and reducing the closed time of the channel. When evaluating the potentiators in a chronic setting on corrected F508del CFTR, no reduction of channel activity in presence of potentiator was observed. The current work identifies and characterizes novel CFTR potentiators GLPG1837 and GLPG2451, which may offer new therapeutic options for CF patients. However, these treatments result in only minor benefit to these patients (Cystic fibrosis (CF) is the most common genetic  disease in Caucasians, with an estimated 80,000 CF diagnosed cases worldwide . Currentpatients  and thusPotentiators can only function if CFTR is already present at the cell membrane, and work by increasing the opening probability (Po) of CFTR . VX770 iHere we describe the characterization of two chemically distinct clinical candidate potentiators. This characterization was performed by evaluating their activity using higher throughput methodologies such as yellow fluorescent protein (YFP)-halide assays, low throughput patch clamp assays and more clinically relevant assays using primary cells derived from CF patients.CFBe41o- cells were cultured in Eagle’s minimal essential medium (MEM) (Life Technologies) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 1% L-glutamine and 500 μg/ml hygromycin B. The cells were grown on culture flasks coated with 0.01% bovine serum albumin (BSA) (Sigma), 30 μg/ml Purecol (Nutacon), and 0.001% human fibronectin (Sigma). A CFBe41o- cell line stably expressing F508del CFTR harboring an HRP-tag in the 4th extracellular loop was obtained from Professor Gergely Lukacs  . Cells wBronchial epithelial cells isolated from transplanted lungs from wild-type (WT), CF patients homozygous for the F508del mutation or heterozygous G551D/F508del or R334W/F508del patients, were obtained from McGill University  and University of North Carolina . Cells were isolated from lungs obtained from donors undergoing a planned transplantation. These primary cells were cultured directly on type IV collagen-coated polycarbonate Transwell supports with a diameter of 6.5 mm and pore size of 0.4 μm  for 18–25 days in air liquid  interface essentially as previously described .2+ and Mg2+ (Life Technologies) using a Microplate Washer (BioTek). Then, cells were treated with 10 μM forskolin and the desired concentration of potentiator in a volume of 30 μl and the plates were incubated at room temperature for 10 min, a timepoint optimized in previous experiments showing good window (positive control/negative control > 2) and signal to background ratio. The YFP fluorescence was recorded for 2 min, starting immediately before addition of 30 μl NaI buffer  to the wells, using a FDSS/μCell (Hamamatsu) fluorescence reader with a 480 nm excitation filter and a 540 nm emission filter. The capacity of potentiators to increase CFTR channel function was expressed as 1-[fluorescence 36 s after NaI addition (F)/fluorescence before NaI addition (F0)], a timepoint optimized previously, resulting in an optimal signal window. Data was normalized using the formula: normalized response = 100∗ (absolute response – negative control response)/(VX770 response – negative response) as such, VX770 response corresponds to 100. For Dose-response experiments, data was fitted using a 4 parameter hill function of the form Response = Bottom + (Top-Bottom)/(1 + 10ˆ((LogEC50-concentration)∗HillSlope)) to determine EC50 values.CFBe41o- cells were plated in black 384-well microplates at a density of 1,500 cells per well. After 24 h, cells were transduced with adenoviruses containing F508del CFTR and YFP (H148Q/I152L/) F47L using a multiplicity of infection of 30 infectious units per cell for each adenovirus. The next day, for the low temperature corrected assay, cells were incubated at 27°C for 24 h, for the chemically corrected assay cells were treated with corrector for 24 h at 37°C. On the day of analysis, cells were washed five times with Dulbecco’s Phosphate-Buffered Saline (DPBS) containing Ca2+ and Mg2+. Subsequently, cells were treated with 10 μM forskolin and the desired concentration of potentiator in a volume of 40 μl and incubated at room temperature for 10 min, a timepoint optimized in previous experiments resulting in a good window (positive control/negative control > 2) and signal to background ratio. YFP fluorescence was measured using an EnVision plate reader (PerkinElmer). The signal was recorded for 7 s, starting just before injection of 110 μl NaI buffer  into the well with a speed of 150 μl/s, resulting in a final volume of 150 μl. The excitation wavelength was 485 nm and the emission wavelength 530 nm. The capacity of potentiators to increase CFTR channel function was expressed as 1-[fluorescence 7 s after NaI addition (F)/fluorescence before NaI addition (F0)]. Data was normalized using the formula: normalized response = 100∗ (absolute response – negative control response)/(VX770 response – negative response) as such, VX770 response corresponds to 100. For Dose-response experiments, data was fitted using a 4 parameter hill function of the form Response = Bottom + (Top-Bottom)/(1 + 10ˆ((LogEC50-concentration)∗HillSlope)) to determine EC50 values.HEK293 cells were transfected with 10–80 ng of plasmid encoding G551D, G178R, G1349D, S549N, R117H, R334W, or wild type CFTR and 20 ng of plasmid encoding YFP (H148Q/I152L/F47L) using jetPEI (Polyplus transfection). Directly after transfection, cells were seeded in black 96-well plates coated with Poly-D-lysine at a density of 70,000 cells per well. The next day, cells were incubated at 27°C for 24 h, for low temperature correction. When evaluating in a chronic setting, the potentiator concentration range was also added to the cells for 24 h. After this incubation period, cells were washed twice with DPBS with Ca- TetON cells expressing HRP tagged F508del-CFTR  for 15 min. Chemiluminescence was measured using an Envision plate reader (Perkin Elmer).CFBE41odel-CFTR  were see2, 1.2 mM MgCl2, 0.8 mM KH2PO4, 0.8 mM K2HPO4, 5 mM glucose, pH 7.4) on both the basolateral (640 μl) and the apical side (160 μl) and kept at 37°C. Apical amiloride was used to inhibit the endogenous ENaC currents (100 μM) while forskolin (10 μM) was applied on both the apical and basolateral sides to stimulate CFTR. All triggers and compounds used during the experiment were first dissolved in DMSO to a 1000 X concentrated solution, just prior to treatment a 10 X stock was prepared in the NaCl-Ringer solution which was used for addition of the correct concentration of trigger and or compound during the experiment. When using the F508del CFTR mutant, VX809 (3 μM) or GLPG2222 (1 μM) corrector was added on the basolateral side for 24 h to partially rescue F508del CFTR before adding the potentiator. For chronic experiments, the potentiator range was also added to the cells for 24 h and reapplied in the NaCl-ringer buffer prior to the electrophysiological recording. Measurements were done during a 20 min timeframe with recordings every 2 min. The transepithelial potential (PD) and transepithelial resistance (Rt) were measured in an open circuit and transformed to Ieq using Ohm’s law. The increase in Ieq (Δ Ieq) was used as a measure for the increased CFTR activity. EC50 values were generated by measuring impact of different concentrations of compound on Ieq in primary cells. For this purpose each transwell was treated with a different compound concentration. CFTRInh-172 was used at 10 μM to assess the specificity of the response. Each compound concentration was tested in duplicate or triplicate and standard error of mean (SEM) was calculated except for R334W/F508del which was single replicate but had a large concentration range showing a clear concentration-responsive effect. Data was fitted using a 3 parameter hill function of the form Response = Bottom + (Top-Bottom)/(1 + 10ˆ((LogEC50-concentration))) to determine EC50 values.Trans-epithelial clamp circuit (TECC) recordings were performed using the TECC instrument developed and sold by EP Design . During the recording, the epithelial cells were bathed in a NaCl-Ringer solution -Cl (Fisher Biotec), 2 MgCl2 , 5 CaCl2 (Fisher Biotec), and 10 HEPES (Fisher Biotec), pH 7.4, with NMDG , 5 HEPES, and 20 sucrose (Fisher Biotec), pH 7.4, with NaOH (Fisher Biotec). After establishing an inside-out configuration, the patch was perfused with a standard perfusion solution  containing (in mM) 150 NMDG-Cl, 2 MgCl2, 10 EGTA (Fisher Biotec), and 8 Tris (Fisher Biotec), pH 7.4, with NMDG . Recording microelectrodes were made from borosilicate capillary glass with a two-stage pipette puller . The pipette tip was polished with a home-made microforge before use. The pipette solution contained (in mM) 140 NMDG ; P < 0.05 is considered statistically significant.Microscopic kinetic analysis was performed with a program provided by Dr. László Csanády  . ExperimSmall compound libraries, selected on the basis of molecular shape and electrostatic similarity to known potentiators and correctors, were screened for their ability to improve channel activity of low temperature rescued F508del CFTR using a YFP halide based assay. Several hits were identified including several series of structurally related compounds. Two of these series were selected for further medicinal chemistry efforts and optimization, resulting in the molecules GLPG1837 (described by 50 of 11.1 ± 3.6 nM (n = 10) and 3.5 ± 0.2 nM (n = 31) for GLPG2451 and GLPG1837  than on low temperature rescued F508del CFTR in a YFP halide assay [46.6 ± 14.2 nM (n = 6) vs. 3.5 ± 0.2 nM (n = 31) for GLPG1837 and 68.9 ± 25.7 nM (n = 4) compared to 11.1 ± 3.6 nM (n = 10) for GLPG2451  (TECC) compared to 126.2 ± 10.9 nM (n = 56) , data not shown). To further understand this difference, a YFP halide assay was developed using a GLPG2222-like compound (1 μM) to chemically correct F508del CFTR which allows to determine the potency of the novel potentiators in a setting more similar to the HBE setting. In this case, the potency of both GLPG1837 and GLPG2451 corresponded closely to the potency obtained on chemically corrected F508del HBE cells  corrected F508del/F508del CFTR was determined using an electrophysiological readout (TECC) and the data were compared to the data generated in the YFP halide assay. This showed the potency on VX809 corrected F508del/F508del HBEs to be lower  again with a higher channel opening when compared to VX770 (162% for GLPG1837 and 161% for GLPG2451) , in the presence of 3 μM GLPG1837 the Po increased to 0.78 ± 0.04 (n = 5) with open time (τo) and closed time (τc) constants of 1.48 ± 0.41 s and 0.29 ± 0.03 s, respectively, a result comparable to those seen with VX770 (n = 5) with τo = 3.29 ± 0.82 s and τc = 2.18 ± 0.52 s with GLPG1837 (Pre-potentiator Po was 0.04 ± 0.01 with τo = 1.16 ± 0.15 s and τc = 17.86 ± 1.90 s). Addition of GLPG2451 to F508del CFTR resulted in an increase in Po to 0.57 ± 0.05 (n = 4) with τo = 5.05 ± 1.82 s and τc = 4.44 ± 2.23 s (Pre-potentiator Po was 0.05 ± 0.01 with τo = 2.14 ± 0.86 s and τc = 47.88 ± 20.41 s)  after addition of the compounds  activity when compared to the single components (data not shown). The absence of any additional effect on CFTR activity could be due to both potentiators having the same binding site, as molecules are then competing to bind CFTR or due to the dynamic window of the YFP halide assay which might not allow us to see further increase in CFTR activity. Since the maximal effect on WT or F508del CFTR with different potentiators was similar, but different in the case of G551D, it was decided to evaluate potentiator effects on G551D CFTR using the YFP halide assay  is a potentiator which improves the channel function for CFTR mutants with a gating or a conductance defect . In a clThis paper described the characterization of two chemically distinct potentiators GLPG1837 and GLPG2451 which have recently been identified . Whilst Both GLPG1837 and GLPG2451 are able to potentiate F508del, and some of the Class III and IV mutant CFTR. The pharmacological effects measured with YFP halide assays were found to correlate well to the results from more physiologically relevant assays using patient derived primary cells. In the case of F508del CFTR, the potency observed in the YFP halide assay on low temperature rescued CFTR was higher compared to chemical rescued F508del CFTR in both YFP halide and primary HBE cells. This difference was not observed for VX770 used in the same assay settings, suggesting that the novel potentiators may act on CFTR by a different binding mechanism. However, patch clamp data on F508del CFTR showed both GLPG1837 and GLPG2451 enhance the open probability by prolonging the open time and reducing the closed time, similar to that measured in a similar setting for VX770 . The extWe investigated whether both potentiators could act additively to each other or to other potentiators. On F508del CFTR, no further increase in CFTR was observed after addition of any second potentiator to GLPG1837 or GLPG2451. These data suggest that the potentiators cannot bind at the same time to F508del CFTR, or if they do, no further activation of the channel is possible. On G551D CFTR, however, several of the potentiators yielded a different maximal quenching rate and therefore we investigated possible interactions between potentiators. GLPG1837 and GLPG2451 didn’t show any additive effect to each other nor to VX770, suggesting that the potentiators compete for the same binding site or binding of one hampers binding of the other.All these data demonstrate that the interactions between potentiators and the channel are complex and additional experiments including structural as well as mechanistic studies would be needed to better understand the binding site and mode of action of these potentiators.Recently, two groups published data on the potential inhibitory effect of chronically dosed VX770 on VX809 corrected F508del CFTR leading to reduced levels of VX809 corrected F508del CFTR at the plasma membrane . The cliin vitro characteristics of GLPG1837 on Class III CFTR mutants. GLPG1837 has been dosed in patients harboring a G551D or S1251N CFTR mutation. A phase 1b study evaluating the triple combination of GLPG2451, GLPG2222, and GLPG2737 in F508del CF patients is currently ongoing.Based on the more desirable properties of GLPG1837 and GLPG2451 as potentiators, further testing was performed to determine suitability for use in the clinic. GLPG1837 was evaluated in humans and was well tolerated in healthy volunteers, with single doses up to 2,000 mg and 14 days dosing up to 800 mg . SimilarIn summary, having additional therapeutic options available is of high interest to the CF community. Here, we have characterized two CFTR potentiators with distinct chemical structures GLPG1837 and GLPG2451, the hallmark of which is their higher efficacy for promoting Class III mutant channel gating in comparison to VX770. Having these molecules available to the CF field will enable a better understanding of the molecular mechanism of defects associated with different CFTR variants. Moreover, further testing and optimization of these may pave the way for the development of additional treatment regimens for patients with CF.MG, SM, KC and JVdS conceptualized the data. MG, SM, SVdP, A-SW, KV, AV, KS, OM, T-CH, PS, AS, MJ, LN, and MA investigated the data. KC wrote the original draft. MG, SM, and MA wrote, reviewed, and edited the manuscript. MG, SM, and KC visualized the data.MG, SM, SVdP, A-SW, AV, KV, KS, PS, MJ, JVdS, LN, MA, and KC are employees of Galapagos and may own stock options in that company. AS was an employee of AbbVie. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Controlled potential reduction was applied for the reduction of graphene oxide (GO). The characterization was performed by scanning electron microscopy, X-ray diffraction and cyclic voltammetry. Compared with the poor electrochemical response at bare GCE, a well-defined oxidation peak of sunset yellow (SY) was observed at the MnO2 NRs-ERGO/GCE, which was attributed to the high accumulation efficiency as well as considerable electrocatalytic activity of ERGO and MnO2 NRs on the electrode surface. The experimental parameters for SY detection were optimized in detail. Under the optimized experiment conditions, the MnO2 NRs-ERGO/GCE showed good linear response to SY in concentration range of 0.01–2.0 μM, 2.0–10.0 μM and 10.0–100.0 μM with a detection limit of 2.0 nM. This developed method was applied for SY detection in soft drinks with satisfied detected results.The nanohybrid of electrochemically-reduced graphene oxide (ERGO) nanosheets decorated with MnO Sunset Yellow (SY) is a water-soluble synthetic colorant, extensively used in the food industry because of its excellent color uniformity, low production cost, and high stability. However, the content of SY in foods must be strictly controlled and SY is not allowed to be added to fresh meat because it can cause allergies, diarrhea and other symptoms in sensitive people . When thAt present, some analytical methods for SY detection have been reported, such as spectrophotometry , high pe2O3  solution containing 1.0 M KCl, where the diffusion coefficient of K3[Fe(CN)6] is 7.6 × 10−6 cm2 s−1 [2 and 0.293 cm2 for GCE and MnO2 NRs-ERGO/GCE, respectively. These results showed that the effective surface area of the modified electrode increased obviously, which would improve the current response and decrease the detection limit.In the formula, A is the surface area of the working electrode,  cm2 s−1 . Accordi2 NRs-ERGO/GCE was also studied by chronocoulometry. The corresponding chronocoulometric curves are displayed in D and the adsorption charge Qads can be determined by Equation (8). As shown in the insert of 1/2 was shown as a straight line after background subtraction. The slope was 1.652 × 10−5 C·s−1/2 and the intercept (Qads) was 4.813 × 10−5 C. As n = 1, A = 0.293 cm2, and c = 0.1 mM, D was calculated to be 2.68 × 10−5 cm2·s−1. According to the equation Qads = nFAΓs, the adsorption capacity Γs was 1.70 × 10−9 mol·cm−2. These results confirmed the remarkable enhancement effect of MnO2 NRs-ERGO for SY oxidation.The electrooxidation of SY at the MnO2 NRs-ERGO/GCE by SDLSV. Repetitive determinations were carried out on a single electrode. The used MnO2 NRs-ERGO/GCE could be regenerated easily by voltammetric sweeps between 0.0 V to 1.2 V in a blank solution. The relative standard deviation (RSD) for the peak currents of SY based on seven replicates was obtained as 2.56%. The reproducibility was studied by fabricating seven modified electrodes which were applied for SY detection, the result of RSD with 5.32% revealed the excellent reproducible of MnO2 NRs-ERGO/GCE. The stability of the MnO2 NRs-ERGO/GCE was studied over a two-week period by periodically measuring the peak currents of SY. The electrode remained 94.8% of its initial response value after two weeks, indicating that the MnO2 NRs-ERGO/GCE had acceptable storage stability.A solution containing 10 μM SY was used for the investigation of the repeatability, reproducibility and stability of MnO2+, Cu2+, Fe3+, Ca2+, Mg2+, Cl−, NO3−, SO42−, CO32−, glucose, oxalate, sucrose, glycine, alanine, L-cysteine, L-glutamine, L-serine, caffeine, benzoic acid; 0.5 mM vitamin C; 20 μM amaranth, allura red, brilliant blue, and 10 μM tartrazine, quinoline yellow (peak current change <10%). The results demonstrated that the MnO2 NRs-ERGO/GCE has a good selectivity for SY analysis in real samples.To evaluate the selectivity, the voltammetric response of 10 μM SY in the presence of different alien species were measured. The experimental data showed that no influences on the detection of 10 μM SY are found after addition of 1.0 mM Zn2 NRs-ERGO/GCE. A remarkable enhancement of peak current was observed with the increase of SY concentration. A good linearity was exhibited between the peak current of SY and its concentration in the range 0.01 μM~100 μM with three linear functions:Under the optimized experimental conditions, the quantitative analysis of SY was carried out by SDLSV. 2 NRs-ERGO/GCE is comparable to or superior to that of the previously reported modified electrodes [The limit of detection (LOD) was estimated to be 2.0 nM (S/N = 3). As shown in ectrodes ,15,16,172 NRs-ERGO/GCE for SY determination in real samples was testified in soft drinks with different China’s famous brands . Before analysis by SDLSV, the samples were filtered to remove any suspended solids. The concentration of SY was obtained by the standard addition method. The results are listed in 2 NRs-ERGO/GCE were consistent, which indicates that the new method is accurate and feasible.The practical application of MnO4), graphite powder, manganese sulfate (MnSO4) were provided by Sinopharm Chemical Reagent Co., Ltd. . Sunset yellow (SY) was supplied by Aladdin . All analytical grade reagents were used as received without further purification. 0.04524 g of SY was dissolved in 100.00 mL deionized water to prepare a 1.0 mM standard stock solution. A series of low concentration working solutions were prepared by further dilution of the stock solution with water. 0.3 M citric acid-sodium citrate buffer with a pH of 4.5 was used as supporting electrolyte.Potassium permanganate  at an accelerating voltage of 30 kV and a powder X-ray diffractometer  with Cu Kα radiation (0.1542 nm). Cyclic Voltammetry (CV) was finished on a CHI 660E electrochemical workstation . Second derivative linear sweep voltammetry (SDLSV) was carried out on a JP-303E polarographic analyzer . A traditional three-electrode system for all electrochemical experiments was composed of a bare or modified glassy carbon electrode as working electrode, a platinum wire as auxiliary electrode and a saturated calomel electrode (SCE) as reference electrode. A pH-3c exact digital pH meter  was used for solution pH measurements.2 NRs was synthesized by a hydrothermal method according to Gan et al. [2 NRs dispersions (1.0 mg/mL) were obtained by addition of MnO2 NRs (10 mg) to deionized water (10 mL) and ultrasonication for 1 h. Graphite oxide was prepared using a modified Hummer’s method according to our previous report [2 NRs dispersion  was very slowly dropped into GO aqueous solution (5.0 mL) and ultrasonically dispersed for 2 h. A homogeneous black dispersion was obtained MnOn et al. . MnO2 NRs report . GO was 2O3 slurry. After that, it was washed thoroughly with deionized water and cleared in anhydrous ethanol and deionized water in an ultrasonic bath. 5.0 μL of the obtained MnO2 NRs-GO dispersion was coated on the GCE surface and dried under an infrared lamp, followed by electrochemically reduction at a constant potential of −1.2 V for 120 s in a phosphate buffer solution (pH 6.5). The obtained modified electrode was denoted as MnO2 NRs-ERGO/GCE. For comparison, GO/GCE and ERGO/GCE were also prepared by the similar way.Before modification, the GCE with a diameter of 3 mm was polished on silk with 0.05 μM of α-Al2 NRs-ERGO), and the MnO2 NRs-ERGO-modified GCE exhibited superior electrocatalytic ability towards the oxidation of SY, which can be attributed to the strong catalytic activity of MnO2 NRs, high adsorption capacity and excellent conductivity of ERGO. The developed modified electrode exhibited excellent analytical performance such as fast response, low cost, high sensitivity and selectivity, as well as wide linear range and low detection limit for SY detection.This study provides a simple and practical method for preparing the nanohybrid of electrochemical reduced graphene oxide decorated with manganese dioxide nanorods (MnO


We examined regulatory function of astaxanthin on mRNA expression of nuclear factor κB (NF-κB) p65, interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), and interferon gamma (IFN-γ) in peripheral blood mononuclear cells in pre and postpartum Murrah buffaloes during summer  and winter  seasons.A total of 32 Murrah buffaloes apparently healthy and in their one to four parity were selected from National Dairy Research Institute herd and equally distributed randomly into four groups . All groups were fed according to the nutrient requirement of buffaloes . The treatment group was supplemented with astaxanthin at 0.25 mg/kg body weight/animal/day during the period 30 days before expected date of calving and up to 30 days postpartum.There was downregulation of NF-κB p65 gene in all the groups. NF-κB p65 mRNA expression was lower (p<0.05) in treatment than control group from prepartum to postpartum during summer, while mRNA expression was low only on day 21 after calving during winter season. The mRNA expression of IL-6, TNF-α, and IFN-γ was lower (p<0.05) in treatment than a control group of buffaloes during summer and winter seasons. The mRNA expression of NFkB p65, IL-6, TNF-α, and IFN-γ was higher (p<0.05) in summer than in winter seasons.The xanthophyll carotenoid astaxanthin a reddish-colored C-40 compound is a powerful broad-ranging antioxidant that naturally occurs in a wide variety of living organisms, such as microalgae, fungi, crustaceans, and complex plants. Astaxanthin blocked nuclear translocation of NF-κB p65 subunit and IκBα degradation, which correlated with its inhibitory effect on IκB kinase (IKK) activity. These results suggest that astaxanthin, probably due to its antioxidant activity, inhibits the production of inflammatory mediators by blocking NF-κB activation and as a consequent suppression of IKK activity and IκB-degradation. The maitrations ,6 and antrations ,8 propertrations ,10, redutrations  and is etrations , arteriatrations , and dystrations .Nuclear factor κB (NF-κB) is a well-characterized transcription factor that is known to regulate the expression of a wide range of genes, including those of cytokines, chemokines, and cytokine receptors  that conWe hypothesized that astaxanthin regulated the production of pro-inflammatory cytokines by inhibiting NF-κB activation during different seasons. We studied effect of astaxanthin  as an antioxidant on mRNA expression of NF-κB p65, IL-6, TNF-α, and interferon gamma (IFN-γ) in peripheral blood mononuclear cells (PBMCs) in pre and postpartum Murrah buffaloes during summer and winter seasons.The experiment was approved by the Institutional Animal Ethics Committee  constituted as per the article 13 of the CPCSEA rules, laid down by Government of India. All the ethical guidelines were followed during the course of the experiment.™ 100 bp DNA Ladder (50 µg) were purchased from Thermo Fisher Scientific (LSG) Mumbai, India.Histopaque 1077 from Sigma Chemical Co.  and Dulbecco’s phosphate buffer saline (DPBS) procured from HiMedia Laboratories Pvt. Ltd., India was used for separation of PBMCs. RNeasy (Cat. No-74104) and RNase-Free DNase Set (Cat No. - 79251) were purchased from QIAGEN Pvt. Ltd., New Delhi, India. Nuclease-free water was purchased from Genetix, Biotech Asia Pvt. Ltd (Fermentas life sciences). Revert Aid First strand cDNA synthesis kit , Dream Taq Green PCR Master Mix (2X) (K 1082), DyNAmo Color Flash SYBER Green Qpcr kit (F-416XL), and GeneRulerBubalus bubalis) having an approximate body weight of 600-650 kg, milk yield of (average) 7.6 kg in their one to four parity 30 days before parturition from Livestock Research Center (LRC), National Dairy Research Institute (NDRI), Karnal, were selected for the experiment. The experiments were carried out during two distinct phases coinciding with two seasons of the year, namely winter (December-February) and summer (April-June). From these 32 animals were further randomly divided equally (8 each) into four groups . The experimental animals were given adaptation period of 1 week to get adapted to new surroundings. Control groups of buffaloes during summer and winter seasons remained without provision of astaxanthin supplementation, while treatment groups of buffaloes were managed with astaxanthin supplementation during summer and winter seasons, respectively. The astaxanthin as powder form was fed at 0.25 mg/kg body weight/day [A total of 32 animals, eight each of pregnant buffalo  and was immediately transported to laboratory under refrigeration. Blood samples were collected at 30, 21, 15, and 7 days prepartum, day of calving and 7, 15, and 21 days postpartum from each animal in both seasons.Bovine PBMCs were isolated after blood centrifugation at 3000 rpm at 4°C for 30 min in a refrigerated centrifuge  for separation of lymphocytes. The buffy coat was harvested and re-suspended in 1:1 v/v DPBS. PBMCs were isolated by density gradient centrifugation method using lymphocyte separation medium, Histopaque 1077 (Sigma). The whole content was layered carefully onto the Histopaque to produce a clean interface between the two layers. Further, it was centrifuged at 2000 rpm for 30 min at room temperature. The white opaque mononuclear fraction from the interface was collected between the DPBS and Histopaque. Further, centrifugation was done at 1500 rpm for 7 min for washing the cells with PBS (pH=7.4). Finally, the cell pellet was obtained.Total RNA from PBMC was isolated using RNeasy Mini Kit (Qiagen India Pvt. Ltd.) according to the manufacturer’s protocol. The quality and integrity of isolated RNA was checked by carrying out agarose gel electrophoresis in 1.5% agarose in 1× TAE buffer at 100 Volts for 30 min. The RNA purity was verified by optical density (OD) absorption ratio at λ260/λ280 using Biospec-nano Spectrophotometer . A ratio of ~2.0 was generally accepted as “pure” for RNA. The RNA samples with good purity and integrity were used for cDNA synthesis.For each sample, about 200 ng of total RNA was used for cDNA synthesis using Revert Aid First strand cDNA synthesis kit  by reverse transcription polymerase chain reaction (PCR) according to the manufacturer’s protocol. The RT reaction was carried out at 25°C for 10 min, 42°C for 60 min, and 75°C for 5 min in a thermal cycler .Primers for NF-κB, IL-6, TNF-α, and INF-γ were designed using primer 3.0 software or suggested from published literature. The sequences and expected PCR product length are shown in ΔΔCT method [The RT-PCR reaction was carried out in Applied Biosystems 7500 RT-PCR systems using 0.5 μl of cDNA, 5 μl of Maxima SYBR green qPCR master mix, and 0.5 μl of concerned gene sequence-specific forward and reverse primers (10 pmol), and the final volume of 10 μl was made with nuclease-free water. The RT-PCR program consisted of initial heating at 50°C for 2 min followed by 95°C for 10 min, and samples were amplified for 40 cycles . The final extension at 72°C incubation was continued for a further 10 min GAPDH was used as housekeeping gene. The relative quantification of target gene was done by the 2T method .© (2011) SAS Institute Inc., Cary, NC, USA. Data from different experiments are presented as mean±standard error. The pairwise comparison was drawn using Tukey’s multiple comparison tests. The difference at p<0.05 was considered to be statistically significant. The model performed for each test is as follows:The data were analyzed using SAS Software, version 9.3 of the SAS System, Copyrightij=µ+αi+βj+Hk+eijYij is a dependent variable, μ is the overall mean, αi effect of buffaloes ; βj = effect of treatment j (j=astaxanthin supplemented or non-supplemented); Hk = effect of time period and eij is the random error which is assumed to be independent and normally distributed.Where, YDuring summer season, the NF-κB p65 gene expression of control and treatment groups was downregulated significantly (p<0.05) on day of calving and on day 21 after calving as compared to day 30 before calving, respectively. The NF-κBp65 gene expression decreased significantly (p<0.05) in treatment group as compared to control group on day 7 before calving, on day of calving and during postpartum period. During winter season, the NF-κBp65 gene expression of control and treatment groups was downregulated significantly (p<0.05) on day of calving and on day 21 after calving as compared to day 30 before calving, respectively. The NF-κB p65 gene expression decreased significantly (p<0.05) in treatment groups as compared to control group on day 60 after calving . Gene exDuring the summer season, the IL-6 gene expression in control and treatment groups increased significantly (p<0.05) on the day of calving and day 21 after calving as compared to day 30 before calving, respectively. The IL-6 gene expression decreased significantly (p<0.05) in treatment groups as compared to control group of respective days. During the winter season, the IL-6 gene expression in control and treatment groups increased significantly (p<0.05) on the day of calving and day 21 after calving as compared to day 30 before calving, respectively. The IL-6 gene expression decreased significantly (p<0.05) in treatment groups as compared to control group on day 15, 7 before calving and on day 15, 21 after calving . The IL-th before calving to day 15th after calving, respectively  on the day of calving and day 21 after calving as compared to day 30 before calving, respectively. The TNF-α gene expression was decreased significantly (p<0.05) in the treatment group as compared to control group. During the winter season, the TNF-α gene expression in control and treatment groups increased significantly (p<0.05) on the day of calving and day 21 after calving as compared to day 30 before calving, respectively. The TNF-α gene expression decreased significantly (p<0.05) in the treatment group as compared to control group on day 15ectively . The TNFDuring the summer season, the IFN-γ gene expression in control and treatment groups was upregulated significantly (p<0.05) from day of calving to day 15 then downregulated significantly (p<0.05) on day 21 after calving as compared to day 30 before calving, respectively. The IFN-γ gene expression decreased significantly (p<0.05) in treatment groups as compared to control group from day 7 before calving to day 21 after calving. During the winter season, the IFN-γ gene expression of control and treatment groups was significantly (p<0.05) upregulated on day of calving to day 15 then downregulated significantly (p<0.05) on day 21 after calving as compared to day 30 before calving, respectively . The IFN2O2-stimulated U937 mononuclear cells, and this property was elicited by a restoration of the basal SHP-1 protein expression level and reduced NF-κB (p65) nuclear expression [NF-κB p65 is one of the most important pathways in inflammation and tumors . In our pression . Anotherpression . IL-6 anpression . IFN-γ epression .nd week (2.89-fold) postpartum and subsequently decreased in the 3rd (0.73-fold) and 4th week (0.70-fold) in healthy buffaloes [Gallus gallus) compared to that of control birds [IFN-γ expression increased gradually from prepartum (0.56-fold) up to 2uffaloes . IL-6 anol birds .invivo production of pro-inflammatory cytokines, such as TNF-α, IL-6, and IFN-γ. Furthermore, it suppressed NF-κB activation may be by inhibiting IKK activity. These results support the idea that astaxanthin prevents inflammatory processes by blocking the expression of pro-inflammatory genes as a consequence of suppressing NFkB activation.We showed that NF-κB inhibited LP carried out the research under the supervision of AA. LP collected blood samples, did molecular work and analyzed the data. The manuscript was drafted and revised by LP under the guidance of AA. Both authors read and approved the final manuscript.