Chromosome organization during cell division is achieved through the timely association of proteins with chromatin and is regulated by protein phosphorylation. Kinesin family member 4A (KIF4A) plays an important role in the chromosome organization through the formation of the chromosome scaffold structure. However, the relationship between the function of KIF4A and its phosphorylation remains unclear. Here, we demonstrate that Cdk1-dependent phosphorylation of KIF4A at S1186 is required for chromosome binding and chromosome scaffold formation. The KIF4A mutant, which is not phosphorylated at S1186, was found to localize to the nucleus during interphase but did not accumulate in the chromosome scaffold after nuclear envelope breakdown. In addition, defects in KIF4A phosphorylation were found to disrupt the interaction of KIF4A with the condensin I complex. As a result, the morphology of the chromosomes was observed to be laterally decondensed, without condensin I in the chromosome scaffold. Additionally, a defect in chromosome segregation, chromosome bridge formation, was often observed. Although both KIF4A and condensin I disappeared from the chromosomes, the chromosomal localization of condensin II was not affected. Collectively, our novel results revealed that Cdk1-dependent KIF4A phosphorylation at S1186 is a trigger for chromosomal organization during early mitosis. In eukaryotes, DNA is highly compacted into chromosomes during cell division. Indeed, chromosome condensation is essential for the faithful segregation of chromosomes from one generation to the next. This process is achieved through the concerted action of various condensation factors, including chromosome-binding proteins –3, post-KIF4A, known as a chromokinesin, is a type of motor protein kinesin that binds to both microtubules and chromosomes . SeveralPrevious studies have indicated that phosphorylation of scaffold proteins regulates the chromosome organization. In the initial stage of chromosome condensation, Cdk1 phosphorylates the condensin II subunit CAP-D3 at T1415, which triggers mitotic chromosome assembly by recruiting another kinase, Plk1, to the chromosome scaffold . Msp1 reIn this study, we demonstrated that the phosphorylation of KIF4A at S1186 is required for its chromatin-binding ability during mitosis. In addition, this phosphorylation depends on Cdk1 activity. Lack of KIF4A phosphorylation at S1186 showed a phenotype similar to that of chromosomes observed in KIF4A-knockdown cells. In this phenotype, condensin I is diffused from the chromosome and the shape of the chromosome changes to be both wider and shorter. Collectively, our novel results suggest that the phosphorylation of KIF4A at S1186 by Cdk1 triggers lateral chromatin compaction by loading condensin I onto the chromosome scaffold.2 at 37°C. For the kinase inhibition experiments, HeLa cells were cultured in the medium containing 0.1 μg/mL nocodazole for 14 h to arrest the cell cycle at mitosis, and further treated with 10 μM MG132 for 1 h. The mitotic cells were then treated with 10 μM RO-3306 (RO), 4 μM ZM447439 (ZM), or 100 nM BI2536 (BI) for 2 h. Cells expressing EGFP-KIF4A WT or EGFP-KIF4A S1186A were cultured in the medium containing 1 μg/mL doxycycline and 2 μg/mL puromycin.HeLa Tet-On 3G, the human cervical cancer cell line, was purchased from Clontech, and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 800 μg/mL G418 in a humidified atmosphere of 5% COThe following primary antibodies were used for immunostaining (IF) and western blotting (WB): rabbit polyclonal anti-KIF4A , rabbit polyclonal anti-phosphorylated KIF4A S1186 (WB-1:500), rabbit polyclonal anti-CAP-H1 , rabbit polyclonal anti-GFP , rabbit polyclonal anti-CAP-H2 (WB-1:500), rat polyclonal anti-CAP-H2 , rabbit polyclonal anti-CAP-D2 (WB-1:500), rabbit polyclonal anti-CAP-D3 (WB-1:500), rabbit polyclonal anti-histone H3S10 phosphorylation , chicken polyclonal anti-GFP , rabbit polyclonal anti-histone H3 , and mouse monoclonal anti-α-tubulin . Anti-CAP-H1, anti-CAP-H2, anti-CAP-D2, anti-CAP-D3 and anti-phosphorylated KIF4A S1186 antibodies were produced in rabbits by immunization against CAP-H1 [GTEDLSDVLVRQGD], CAP-H2 [KRFQTYAAPSMAQP], CAP-D2 [TTPILRASARRHRS], CAP-D3 [SRRSLRKTPLKTA], and KIF4A [KKTPPAP(pS)PFDLPE] peptides, respectively (Eurofins).BglII and SalI restriction enzyme sites into both ends of the KIF4A sequences, followed by purification and digestion with BglII (NEB) and SalI (NEB). The pEGFP-C1 plasmid was also digested with the same restriction enzymes. The digested KIF4A cDNA fragment was then cloned into the digested pEGFP-C1 plasmid by using a DNA ligation kit (Takara). The pEGFP-C1-KIF4A plasmid was then digested with NheI and SmaI, and inserted into the pTRE 3G plasmid (Clontech) digested with EcoRV using a DNA Blunting Kit (Takara).The EGFP-KIF4A expression vector was constructed by cloning the amplified KIF4A cDNA fragment into the pEGFP-C1 plasmid. KIF4A cDNA that was originally cloned inside the pF1K vector was purchased from the Kazusa DNA Research Institute. The KIF4A cDNA fragment was first amplified using primers that introduced To obtain KIF4A phosphorylation mutants, serine (S) or threonine (T) residues at four KIF4A phosphorylation sites  were substituted with alanine (A) or aspartic acid (D), which contains GCT or GAC DNA sequences, using a PrimeSTAR mutagenesis basal kit (Takara). All EGFP-KIF4A WT and mutant plasmid DNA sequences were confirmed.HeLa Tet-On 3G cells were transfected with pTRE3G-EGFP-KIF4A WT or mutant plasmids using Lipofectamine 3000 (Invitrogen). Linear puromycin marker DNA (Takara) was also transfected to select EGFP-KIF4A-expressing cells.5′-CCAUAUGCUCAGUGCUACATT-3′) using Lipofectamine RNAiMAX (Invitrogen). After incubation for 48 h, the cells were collected and used for analysis.To knockdown endogenous KIF4A and CAP-D2, HeLa cells were transfected with 150 nM KIF4A siRNA (5′-CAGGTCCAGACTACTACTC-3′) and 150 nM CAP-D2 siRNA  by cytocentrifugation  at 1,300 rpm for 10 min. The chromosome spreads or cells cultured on the poly-L-lysine-coated coverslip were fixed with 2% para-formaldehyde (PFA) in PBS for 15 min and permeabilized with 0.2% Triton X-100 in PBS for 5 min at room temperature (RT). Samples were blocked with 1% bovine serum albumin in PBS for 30 min at RT. Samples were then incubated with primary antibodies at RT for 1 h, followed by secondary antibodies labeled by Alexa Fluor 488 or 594 at RT for 1 h. DNA was counterstained with Hoechst 33342. Samples were mounted onto glass slides using Vectashield mounting medium (Vector Laboratories) or p-phenylenediamine.Images were taken with a DeltaVision microscope (AppliedPrecision) equipped with a 1.42 NA PlanApo N 60× oil immersion objective (Olympus) and a cooled CCD camera . The z-stack distance was 0.2 μm; raw 3D images were deconvoluted using constrained iterative deconvolution and converted to 2D images with maximum projection using softWoRx (AppliedPrecision). Confocal images were obtained with an A1R+ system (Nikon) with GaAsP detectors under the small pinhole setting (0.4AU) and applied deconvolution  to enhance resolution. Laser lines at 405 nm for Hoechst 33342, 488 nm for GFP, and 561 nm for Alexa 594 were used for excitation. An objective lens with NA of 1.49  was used. The brightness and contrast of the images were adjusted using ImageJ software.2 chamber set at 37°C. A 1.42 NA PlanApo N 60× oil immersion objective (Olympus) was used to observe cells at 3-min time intervals.Cells were cultured in poly-L-lysine-coated 35-mm glass bottom dishes (Matsunami). Before observation, the DNA was counterstained with Hoechst 33342 for 15 min, followed by replacing the medium with phenol red-free DMEM containing 10% FBS, 2 mM L-glutamine, and 20 mM HEPES. Time-lapse observation was performed using a Delta Vision microscope (Applied Precision) equipped with a CO2, 100 mM NaCl, 1 mM DTT, 0.25 mM PMSF, and 0.3% Triton X-100) supplemented with Complete protease inhibitor (Roche) and PhosSTOP phosphatase inhibitor mixture (Roche), followed by incubation for 30 min on ice. Insoluble chromatin pellets were collected by centrifuging at 16,100 g for 10 min at 4°C. The supernatant was mixed with the same volume of 2× Laemmli sample buffer and collected as chromatin-unbinding proteins (CUB). Insoluble chromatin pellets were washed again with CSK1 buffer. The chromatin pellets were re-suspended in 1× Laemmli sample buffer and collected as chromatin-binding proteins (CB) after sonication. All samples were heated to 95°C for 5 min and subjected to SDS-PAGE using 5%–10% or 5%–20% gradient polyacrylamide gels . Proteins were transferred to PVDF membranes (Millipore) using a PoweredBlot Ace transfer system (ATTO). The membranes were then blocked by Bullet Blocking One . Primary and secondary antibodies were diluted in Signal Enhancer HIKARI for WB and ELISA  and reacted with proteins at RT for 1 h. After each antibody reaction, the membrane was washed with TBST . Immunoreactive bands of proteins were then detected using a 1% NBT/BCIP stock solution (Roche) in alkaline phosphatase buffer . The membrane image after band detection was captured using a WSE-5200 Printgraph 2M (ATTO). The brightness and contrast of the images were adjusted, and the band intensity was quantified using ImageJ software.Whole cell extracts (WCE) were obtained by sonicating cells in 1× Laemmli sample buffer after washing with PBS. For chromatin extraction, cells were washed with PBS and the cell pellets were re-suspended in freshly prepared CSK buffer  containing 0.1 μM okadaic acid, protease inhibitors , and 2 U/μL OmniCleave endonuclease (Ar Brown), and incubated on ice for 20 min. Insoluble chromatin pellets were collected by centrifuging at 15,000 rpm for 10 min, with the supernatant collected as the mitotic extract (input). Mitotic extracts were rotated with anti-GFP antibody  or anti-SMC2 antibody  for 1 h at RT. As a negative control, the antibody was reacted with a mitotic extract prepared from HeLa cells without expressing GFP-KIF4A. The antibody-binding complex was recovered using Dynabeads (Thermo) and dissolved in 1× Laemmli sample buffer. The immunoprecipitates were then analyzed by WB.For immunoprecipitation, endogenous KIF4A was depleted from HeLa cells expressing EGFP-KIF4A WT or S1186A by RNAi treatment. The cells were cultured in the medium containing 0.1 μg/mL nocodazole for 14 h, and mitotic cells were collected by shake-off. The cells were washed with PBS and cell pellets were re-suspended in lysis buffer  before further analysis using ImageJ software. After subtraction of cytoplasmic DNA and target protein signals, signal intensity of the target protein in the stained DNA region was obtained. The protein fluorescence intensity was normalized to the DNA fluorescence intensity as per following calculation: [protein intensity/DNA intensity]. Statistical analysis was carried out by performing Student’s t-test using Microsoft Excel software.In this study, we analyzed the function of KIF4A phosphorylation during mitosis. Four phosphorylation sites of KIF4A, namely, T799, S1001, T1181, and S1186, were targeted. The phosphorylation of KIF4A at T799 during mitosis has been previously reported to be important for regulating the mitotic spindle ; howeverTo examine the effects of KIF4A phosphorylation on chromosome morphology, spread chromosomes were prepared from EGFP-KIF4A WT- or non-phosphorylatable mutant-expressing cells. The chromosome morphology of EGFP-KIF4A T799A-, T1001A-, and T1181A-expressing cells was similar to that of KIF4A WT-expressing cells; however, chromosomes in EGFP-KIF4A S1186A-expressing cells showed expanded chromosome morphology, shorter chromosome length and wider width, which was also observed in both KIF4A and CAP-D2-depleted cells . The phoTo estimate the function of KIF4A phosphorylation at S1186, the localization of EGFP-KIF4A S1186A was compared with that of EGFP-KIF4A WT throughout the cell cycle. In the interphase, both EGFP- KIF4A WT and S1186A were localized to the nucleus . After nTo determine the timing of KIF4A phosphorylation at S1186, a rabbit polyclonal anti-phosphorylated KIF4A S1186 was produced. This antibody detected EGFP-KIF4A WT, T799A, S1001A, T1181A, but not EGFP-KIF4A S1186A . KIF4A kNext, we determined the kinase that phosphorylates KIF4A at S1186. There are three major kinases known to be involved in protein phosphorylation during mitosis: Cdk1, aurora kinase B, and Plk1. Mitotic-arrested cells were treated with the kinase inhibitors RO-3306 (Cdk1 inhibitor), ZM447439 (aurora kinase B inhibitor), or BI2536 (Plk1 inhibitor). Then, the phosphorylation level of KIF4A at S1186 was then detected by WB. Phosphorylation levels were drastically decreased in Cdk1-inhibited cells, whereas phosphorylation levels were largely unchanged in the other kinase-inhibited cells . These fAs shown above , the chrThe amounts of the chromosome scaffold proteins were examined by WB analysis. The chromatin-binding ability of KIF4A was decreased in the S1186A mutant compared with the KIF4A WT , GFP. SiBecause KIF4A interacts with the condensin I complex, and the lack of KIF4A phosphorylation at S1186 resulted in the delocalization of these proteins from chromosomes, their interaction may be regulated by KIF4A phosphorylation. To examine this issue, mitotic cell extracts were prepared from EGFP-KIF4A WT- or S1186A-expressing cells, and immunoprecipitation against GFP or SMC2 was performed. In EGFP-KIF4A WT-expressing cells, EGFP-KIF4A co-precipitated with SMC2 and a condensin I subunit, CAP-D2, but the amount was decreased in S1186A-expressing cells . There wKIF4A and condensin are major chromosome structural proteins identified in our proteomics study of human metaphase chromosomes , 3. BothProtein phosphorylation is known to regulate protein function in many cellular activities, including chromosome condensation , 26. ForThe loss of KIF4A phosphorylation at S1186 caused defects in the accumulation of both KIF4A and condensin I in the chromosome scaffold , and theCdk1 forms a complex with cyclin B and promotes mitosis by protein phosphorylation. KIF4A has at least two phosphorylation sites for Cdk1, namely, T1161  and S118KIF4A localizes not only to the chromosome scaffold, but also to the spindle midzone during late mitosis , and it In addition to Cdk1, KIF4A is phosphorylated by aurora B and Plk1, although these phosphorylation sites have not been identified. The phosphorylation of KIF4 by aurora B is required for its interaction with condensin I , and thiS1 Fig(A) The expression of endogenous KIF4A was suppressed by siRNA transfection against 3’ UTR of KIF4A (lower band). However, the expressions of GFP-KIF4A WT and S1186A were not affected by siRNA transfection (upper band). (B) The mitotic cell extracts were subjected to WB analysis using anti-GFP (upper panel), anti-KIF4A S1186 phosphorylation (middle panel), and anti-α-tubulin antibodies. Using anti-KIF4A S1186A phosphorylation antibody (middle panel), three bands were detected. The middle band (indicated by *) was a non-specific band, because the band was appeared in the cell extracts prepared from KIFA knockdown cells. The upper band shows GFP-KIF4A S1186 phosphorylation levels, but it also includes background signal derived from non-specific proteins detected in untransfected HeLa cells. The signal intensity of GFP-KIF4A S1186A-expressing cells was similar to that of untransfected cells, indicating the KIF4A mutant was not phosphorylated at S1186. The lower band shows phosphorylation level of endogenous KIF4A S1186 and the intensity was decreased by KIF4A knockdown. (C) The specificity of anti-KIF4A S1186 phosphorylation antibody was confirmed using cell extracts prepared from GFP-KIF4A WT-expressing cells and non-phosphorylatable mutants. The band detected by anti-KIF4A S1186 phosphorylation antibody disappeared only in EGFP-KIF4A S1186A-expressing cells.(TIF)Click here for additional data file.S2 Fig(A) HeLa cells expressing EGFP-KIF4A WT or S1186A were observed from prophase to telophase of mitosis. EGFP-KIF4A WT showed localization on chromosomes throughout mitosis, and its accumulation at midbody was observed in late mitosis. In contrast, EGFP-KIF4A S1186A did not localized on chromosomes after nuclear envelop breakdown, and diffused into cytoplasm. The accumulation at midbody in late mitosis was observed similar to that observed for EGFP-KIF4A WT. (B) The GFP/DNA signal intensity ratios at each mitotic stage in living cells were obtained from EGFP-KIF4A WT and S1186A-expressing cells (n = 20).(TIF)Click here for additional data file.S3 FigAll blotting results used in (TIF)Click here for additional data file.


Ticks secrete hundreds to thousands of proteins into the feeding site, that presumably all play important functions in the modulation of host defense mechanisms. The current review considers the assumption that tick proteins have functional relevance during feeding. The feeding site may be described as a closed system and could be treated as an ideal equilibrium system, thereby allowing modeling of tick–host interactions in an equilibrium state. In this equilibrium state, the concentration of host and tick proteins and their affinities will determine functional relevance at the tick–host interface. Using this approach, many characterized tick proteins may have functional relevant concentrations and affinities at the feeding site. Conversely, the feeding site is not an ideal closed system, but is dynamic and changing, leading to possible overestimation of tick protein concentration at the feeding site and consequently an overestimation of functional relevance. Ticks have evolved different possible strategies to deal with this dynamic environment and overcome the barrier that equilibrium kinetics poses to tick feeding. Even so, cognisance of the limitations that equilibrium binding place on deductions of functional relevance should serve as an important incentive to determine both the concentration and affinity of tick proteins proposed to be functional at the feeding site. Biological function is central to the understanding of life and organismal biology. The context that function confers is exemplified in the frustration felt when a gene or protein of interest can only be annotated as a hypothetical protein with unknown function. Conversely, the ease of annotation by homology has made genome sequencing and assignment of function to orthologs an almost mundane task . Even soin vitro inhibition of blood clotting and that injecting this extract into various animals led to prolongation of blood coagulation in vivo. He also injected what may very well be the first fractionation of tick proteins to show that this purified preparation retained its inhibitory properties. Sabbatani’s deduction has been confirmed by extensive research into tick–host interactions that established a veritable pharmacopeia of bioactive components secreted by ticks during feeding  that range for most biological systems from the fM to mM range  is determined for inhibitors of enzyme active sites and reflect the concentration necessary to reduce enzyme activity by half. It is generally used in the characterization of inhibitors using enzyme kinetics and depends on both enzyme affinity for substrate and inhibitor affinity for enzyme. Even so, it is similar to the KD and should produce similar values when enzyme activity is measured at physiological concentrations. Another measurement analogous to the KD is the IC50 value of an inhibitor. The IC50 value  is the concentration at which 50% inhibition of function is observed. This is generally determined for complex processes such as inhibition of platelet aggregation, blood clotting, or cell migration. This value would ultimately depend on the concentration of receptor or number of cells used in an assay, but would be close to the KD if physiologically relevant receptor or cell concentrations were used. Another measure that may indicate physiological relevance is the stoichiometric inhibition ratio (SI). This ratio indicate physiological relevance when an inhibitor interact with an enzyme or receptor close to equimolar ratios, since this imply high affinity, so that for example an SI = 2 imply saturation of receptor at 2KD. This may be seen for suicide inhibitors that bind irreversible to the enzyme active site. In enzyme kinetics the Km (Michaelis constant) is the substrate concentration that allows an enzyme to attain half Vmax. Vmax is the maximum reaction rate of the enzyme when saturated with substrate. While the Km is dependent on rate constants rather than ligand concentration it is also an indication of affinity and provide an estimate of relevant concentrations of substrates where an enzyme will function. For example, if a given agonist functions at concentrations 10-fold lower than the Km, the enzyme will not be able to effectively remove this agonist from the system and neutralize its effect. The KD, Ki, IC50, SI, and Km are therefore all useful to assess the physiological relevance of any biological activity. The reader is referred to Le Chatelier’s principle states that an equilibrium system will tend to counteract changes to the system to maintain its equilibrium. In biological systems this is overcome by enzymes that create intermediate states to catalyze non-reversible chemical reactions. In the case of receptor-ligand or protein-inhibitor binding, the affinity of the receptor for the ligand and the relative receptor and ligand concentrations determines the bound state at equilibrium . The affmM range . The KD ilibrium . At a lintration . ConcentOrnithodoros kalahariensis  needs to be present at the feeding site at higher concentrations than the tability , contribtability .The equilibrium state is an ideal closed state and the feeding site may be considered as such. However, while the feeding site may have a defined volume approximating a closed state, the feeding site itself may be dynamic with constant changes in concentration of both host and tick proteins. Ticks ingests blood meal and salivary components, since ticks alternately salivate and ingest blood meal , therebyA critical question remains as to what extent the feeding site may be reduced to a biochemical reaction in a test tube, i.e., can we measure the feeding site, calculate the feeding site volume and determine the amount of saliva secreted by the tick to derive estimations of biological concentrations at the feeding site? However, the feeding site is a complex and dynamic environment that changes constantly as determined by both the tick and the host . For exaO. kalahariensis has been characterized in detail. They each make up ∼5% of the total soluble salivary gland protein, comprising ∼20% of the total soluble protein content  comprise ∼23% of the total soluble protein , which may indicate that these proteins may not have been completely purified at the time of their characterization, or may reflect that they derived from whole body extracts and not tick salivary glands, which may suggest that these inhibitors originate from multiple organs  and Rhipicephalus appendiculatus complement inhibitor (RaCI)  for C3b  . OMCI bir (RaCI) . In bothr (RaCI) . Other cariensis . These inM range . A recenaffinity . These ipathways , 2003. Tpathways . These ireported . Of thes for C3b . As suchpM range . It woulIIbβ3) antagonists that inhibit platelet aggregation induced by any agonist  (KD) for fibrinogen is ~1.4 μM, while fibrinogen occur in 10-fold excess  . This cod excess . It is t50 ~ 8 nM (50 ~ 5 nM) of binding of the monoclonal antibody Gi9 to the platelet collagen receptor GPIa-IIa (α2β1) confirmed targeting of this receptor by TAI (Tick adhesion inhibitor (TAI) inhibits adhesion of platelets to collagen with an IC0 ~ 8 nM . Competir by TAI . The colr by TAI . The affr by TAI . It is tKD ~ 72 nM, thereby inhibiting ligand-induced inflammation in tissues (Longistatin binds to the V domain of the receptor for advanced glycation end products (RAGE) with a  tissues . It has  tissues . The con tissues  and longKD ~ 47 nM and inhibits T-cell receptor ligation induced activation resulting in immunosuppression , chemotaxis may only be efficiently inhibited somewhere along the concentration gradient and not across the whole range, especially since competition of evasins for different chemokines might occur.In the case of chemokines where leukocytes respond to concentration gradients for directional chemotaxis, the measured gradients suggest that these may range across low pM to several hundred nM . NaturalIxodes scapularis (Ticks feed from a feeding cavity where blood pools . The feeapularis . For theKm ~ 1 mM for ATP and ADP and high turnover number (106 s-1) and Kcat/Km ratio (109 M-1s-1)  hydrolyse ATP that function in inflammation , as well M-1s-1) . These n M-1s-1) , it woul M-1s-1) . In all 10-helix, with a C-terminal α-helix anchored to the side of the barrel by disulphide bonds. This gives lipocalins the distinct appearance of a cup with an open end, where ligands can access the cup and bind in the cavity inside the barrel. Specificity is conferred by residues inside the barrel, as well as four loops that allow access to the barrel  (4 for its neutrophil receptor BLT1 ranges from 0.1 to 2 nM (4 scavengers and their high concentrations (μM) at the feeding sites would make them relevant competitors at the feeding site.Leukotriene B~950 nM) . The aff to 2 nM . Again, 4 and LTD4 have KD values below 2 nM and also occur at μM concentrations  and their low affinities (low nM) suggest that they would be able to neutralize TXA2 binding to its receptor during feeding . Such an activator would need to be present at similar or higher concentrations than the target enzymes to be functionally relevant. In the case of ixonnexin, plasminogen and tPA occur at plasma concentrations of ~2 μM and 100 pM, respectively  by forming a enzymatically productive ternary complex that forms plasmin to promote fibrinolysis . Ixonnexectively . While nectively . It has ectively .The adrenomedullins are a special case of tick-derived inhibitors that bind to host calcitonin-receptor-like receptor and receptor activity-modifying protein receptor complexes to cause vasodilation . These iR. appendiculatus, where it presumably play a role in regulation of blood vessel tonus and blood flow , a potent vasodilator, at ~40–500 ng/ml saliva . Concent ~0.7 nM . Another ~0.7 nM . The eff ~0.7 nM . As yet,KD values for many proteins. To address this, ticks may employ various alternative strategies that allow adequate expression and optimal use of secreted proteins at the feeding site that allow ticks to circumvent or eliminate the problems posed by an equilibrium system  during the slow pre-feeding phase is required before the rapid engorgement phase can occur , albeit with low affinity and did not compete for the receptor. Several lines of evidence converged on moubatin as scavenger of TXA2: the evidence that moubatin belonged to the lipocalin family  is important in accessing functional relevance. As example, the case of moubatin may be considered. Originally moubatin was identified as a specific inhibitor of collagen-induced platelet aggregation with an ICregation . Subsequcollagen . At high ~ 10 μM . The leetagonist . Subsequhomologs . In retrn family ; the facn family ; the inhc U46619 , and thec U46619 . The gaiavengers . It not in silico. As such, recent advances in technology, both in next-generation transcriptome sequencing and proteomics, has allowed an unprecedented view of salivary gland dynamics from a systems perspective (I. scapularis sampled every 24 h until detachment and indicated differential expression for a large number of lipocalins annotated as histamine-binding proteins (I. scapularis lipocalins with biogenic amine binding motifs, failed to find any histamine-binding lipocalins, but only identified serotonin-binding lipocalins with a single binding site (Ixodes ticks, the results thus far do not support it. Similarly, bioinformatic analysis predicted high affinity binding of histamine and serotonin in lipocalins from Ixodes ricinus, for which none of the critical residues involved in biogenic amine binding was conserved (Ixodes persulcatus without experimental verification of affinities or target enzymes (While the majority of functions found in ticks may be assigned functional significance , enough spective . This haproteins . The dising site . While tonserved . Molecul enzymes . While sIt may be considered whether it really matters whether a measured function is relevant during feeding, given the emerging recognition that all proteins may be moonlighting to some extent. From this perspective, any function present in tick saliva should be relevant at some level and our goal for the next few decades would be to assign functions to salivary proteins, whether relevant or not. A more comprehensive understanding would later emerge once we have gathered enough data to truly assess relevance. This position is appealing since it buys some time for dubious functions. It is, however, also a philosophical “everything goes” viewpoint , that maThe use of tick salivary proteins as therapeutic agents within a clinical or pharmaceutical setting remains an important and promising goal . From thFunctional relevance is determined by the concentration of tick proteins at the feeding site as well as their affinity for their respective host targets. The current review showed that the majority of proteins found in tick saliva or salivary glands thus far characterized will be functional at the tick feeding site. It was also shown how ticks may circumvent the problems presented by an equilibrium system. Even so, inferring functional relevance without estimating concentration or affinity at the feeding site remains a risky endeavor. Future aims in salivary gland biology should focus on quantification of protein concentration secreted during feeding as well as in the actual feeding site. This should provide more accurate estimates of functional relevance.BM conceptualized the study and wrote the manuscript.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


RI expression and cellular distribution in rats mirror that of humans.Designing biologically informative models for assessing the safety of novel agents, especially for cancer immunotherapy, carries substantial challenges. The choice of an in vivo system for studies on IgE antibodies represents a major impediment to their clinical translation, especially with respect to class‐specific immunological functions and safety. Fcε receptor expression and structure are different in humans and mice, so that the murine system is not informative when studying human IgE biology. By contrast, FcεMOv18 IgE, a human chimeric antibody recognizing the tumour‐associated antigen folate receptor alpha. We created an immunologically congruent surrogate rat model likely to recapitulate human IgE‐FcεR interactions and engineered a surrogate rat IgE equivalent to MOv18. Employing this model, we examined in vivo safety and efficacy of antitumour IgE antibodies.We are developing TNFα, a mediator previously linked with IgE‐mediated antitumour and antiparasitic functions, alongside evidence of substantially elevated tumoural immune cell infiltration and immunological pathway activation in tumour‐bearing lungs.In immunocompetent rats, rodent IgE restricted growth of syngeneic tumours in the absence of clinical, histopathological or metabolic signs associated with obvious toxicity. No physiological or immunological evidence of a “cytokine storm” or allergic response was seen, even at 50 mg/kg weekly doses. IgE treatment was associated with elevated serum concentrations of MOv18 IgE, in conjunction with efficacy and immune activation, supporting the translation of this therapeutic approach to the clinical arena.Our findings indicate safety of  The antitumour activity of hMOv18 IgE was superior to the equivalent hMOv18 IgG in a syngeneic rat model, and in two human ovarian carcinoma xenograft mouse models reconstituted with human immune cells.Although human exposure to exogenous IgE has been reported,Here, we report the design and implementation of a syngeneic tumour model in immunocompetent rats to examine IgE safety and compare antitumour efficacy with that of the equivalent IgG2b. Surrogate rat MOv18 IgE and IgG2b antibodies (rMOv18) were generated. Treated rats were subjected to clinical, histopathological and metabolic evaluation. Immune cell recruitment and immune mediator release were interrogated to provide preclinical insights into this most appropriate immunocompetent host.2See 2.1Chimeric mouse/human antibodies, hMOv18 IgE and IgG1 were prepared as before.2.2rMOv18 IgE and IgG2b antibodies were purified using MEP (4‐mercapto‐ethyl‐pyridine) HyperCel™ Mixed‐Mode Chromatography Sorbent  and affinity chromatography using a HiTrap™ Protein G HP column , respectively.2.32.3.12 and ii) % tumour occupancy  surface area [mm2]).Female Wistar Albino Glaxo (WAG/RijCrl) rats (Charles River) were maintained and handled in accordance with the Institutional Committees on Animal Welfare of the UK Home Office in compliance with The Home Office Animals Scientific Procedures Act, 1986. Rats were injected intravenously with CC531tFR tumour cells and subsequently treated with rMOv18 antibodies on days 1 and 14 or on days 1, 7, 14 and 21. Animals were humanely euthanized, and lung tumour burden was determined by i) mean number of surface‐visible metastases/cm2.3.2Animals were examined twice daily for clinical signs of toxicity or changes in behaviour and appearance. On dosing days, animals were examined after dose completion and approximately 1 and 4 hours thereafter. Animals were weighed at the start of treatment and weekly thereafter. General toxicities were evaluated according to the Federation of European Laboratory Animal Science Association (FELASA) working group report on pain and distress in laboratory rodents2.3.3Blood samples were drawn into EDTA and lithium heparin collection tubes and analysed for haemoglobin concentration, total white blood cell count, neutrophil count, and creatinine, alanine aminotransferase and aspartate aminotransferase concentrations  clinical chemistry analyser). Full necropsies were performed at day 30 following tumour challenge. Tissues were preserved in neutral buffered formaldehyde before processing for histological evaluation.2.4Rat tissues , spleen, lymph nodes, liver, kidney, lungs, colon and brain) were submerged in neutral buffered formaldehyde, embedded in paraffin wax, cut at a nominal thickness of 4‐5 μm, stained with haematoxylin and eosin and analysed on a Zeiss Axiophot microscope  and NIS Elements imaging software (Nikon).2.5t test. Nonparametric data were compared using the Mann‐Whitney U test. All statistical analyses were performed using GraphPad™ Prism software . P values were represented as follows: *P < .05, **P < .01, ***P < .001, ****P < .0001. Error bars represent SD and SEM in in vitro figures, and SEM in in vivo figures and histological analyses. All results represent at least 2 independent experiments.Parametrically distributed data were compared with an unpaired two‐tailed Student's 33.1 rMOv18 IgE and IgG2b, surrogates of the original mouse/human chimeric hMOv18 IgE and IgG1, respectively. Rat IgG2b isotype was chosen as equivalent to mouse IgG2a/b and human IgG1, based on complement activation and antibody‐dependent cell‐mediated cytotoxicity (ADCC) functions ‐expressing rat RBL‐2H3 mast cells and to primary rat peripheral blood monocytes CD172 + ) expressing FcεRI(αγ2). The rMOv18 IgG2b bound primary rat monocytes (expressing FcγRI) and RBL‐2H3 cells (expressing low rFcγRII/FcγRIII levels). Both rat IgE and IgG2b bound to human FRα‐expressing, but not to FRα‐negative, tumour cells   and confirmed significantly reduced lung metastases growth with 10 mg/kg rMOv18 IgE compared with rMOv18 IgG2b or PBS  Figure C. IgE adWe concluded that administration of rMOv18 IgE to tumour‐bearing rats induced no clinical, histopathological or metabolic signs of a type I hypersensitivity reaction, even at weekly 50 mg/kg doses.3.4IgE antitumour efficacy is likely to function by recruitment of host immunity. We therefore investigated whether inhibition of lung metastases in IgE‐treated rats was associated with immune cell infiltration and activation. We measured growth inhibition of lung metastases with 4 (weekly) doses of rMOv18 IgE between 1, 10 and 50 mg/kg  to identify gene sets correlated with treatment. This yielded evidence of significant enrichment of gene expression associated with immune activation in IgE‐treated tumours, compared with enrichment of cell cycle‐associated pathways with vehicle treatment Figure E. The IgP = .0044)  Figure A‐B.11 Pr) Figure C. On the) Figure D. These Together these findings point to immune cell recruitment, and activation of immunological pathways and mediators associated with IgE treatment, in the absence of any evidence of overt toxicities, or of any cytokine profiles that would indicate signs of cytokine storm or allergic response activation.4Identification of an appropriate animal model for studies involving IgE functions, including cancer immunotherapy, especially with regard to class‐specific immunological efficacy and safety, represents a major hurdle in clinical translation of this antibody class. Previous studies have reported substantial evidence of comparable or superior antitumour efficacy of hMOv18 IgE compared to its IgG counterpart.Here, we report findings in support of the in vivo safety of a tumour‐targeting IgE in an immunologically relevant immunocompetent syngeneic rat tumour model, selected because of similarities in the structure and distribution of FcεRI between humans and rats. We describe upregulation of immune‐associated pathways in lung metastases of IgE‐treated rats, associated with antitumour efficacy, and evidence of elevated serum concentrations of the pro‐inflammatory mediator TNFα. By contrast, neither the classical “cytokine storm”‐related cytokine IL‐6 nor the key allergic inflammatory cytokine, IL‐4, was upregulated. Despite the activation of immunological pathways associated with IgE treatment, no evidence of systemic or organ‐specific adverse events was detected.We completed the rat model by generating surrogate rat IgE and IgG. We present a simple process for cloning heavy and light chains of any antibody clone into a single mammalian expression vector and report serum‐free production by mammalian cells by scale‐up in bioreactors. These processes are easily adaptable for generating antibodies of any isotype or specificity. Furthermore, the only currently reported purification method for monoclonal rat IgE is for a dinitrophenyl‐specific rat IgE (DNP IgE), purified by an affinity column based on antigen specificity.Administration of rMOv18 IgE and IgG2b to tumour‐bearing rats demonstrated only three toxicities, observed with both isotypes. Accordingly, none of the anaphylaxis‐specific toxicities detailed in previously published criteriaWe previously reported that human IgE cross‐linked on the surface of human monocytes triggered monocyte activation and TNFα upregulation; furthermore, human IgE antitumour ADCC was associated with enhanced TNFα. In parallel, we detected TNFα‐expressing macrophage subsets and elevated concentrations of TNFα in lung metastases of IgE‐treated rats.The immunocompetent syngeneic tumour model may also pose certain limitations, including lack of human FRα expression by normal rat tissues, and unlikely cross‐reactivity of rat FRα homologue with rat MOv18 IgE. Although observed in the circulation of a proportion of patients with FRα tumours such as ovarian carcinomas and mesotheliomas,In conclusion, we designed and implemented an immunologically relevant and physiologically representative tumour model which supports the preclinical safety of an antitumour IgE antibody, MOv18 IgE. Simultaneously, using this model, we report efficacy of the therapy in inducing tumour immune cell infiltration and immune pathway activation in the absence of physiological or immunological evidence of cytokine storm or allergic reaction. Our findings support the translation of IgE into the clinic and contribute to the rapidly growing field of AllergoOncology. Design of this and other immunologically congruent models may also help delineate IgE class‐specific functions in allergic inflammation, allergen immunotherapy and antiparasitic responses.SNK and JFS are founders and shareholders of IGEM Therapeutics Ltd. FON is an employee of Sanofi US. All other authors declare no conflict of interests.D.H.J. and M.N. designed experiments. D.H.J., M.N., H.J.B., T.D., G.M., L.S., P.K., K.M.I., S.C., P.G., N.W., C.L., S.K., C.S., H.L., S.C., M.F. and N.D. performed the research. D.H.J., M.N., H.J.B., T.D., G.M., L.S., P.K., K.M.I., S.C., P.G., N.W., C.L., S.K., C.S., H.L., S.C., M.F., N.D., S.N.K. and J.F.S analysed the data. D.H.J., M.N., H.J.B., T.D., G.M., F.O.N., P.S.J., H.J.G., P.J.B., S.T., S.N.K. and J.F.S interpreted the data. D.H.J., H.J.B., T.D., G.M., F.O.N., P.S.J., H.J.G., P.J.B., S.T., S.N.K. and J.F.S wrote the manuscript. L.S., P.K., K.M.I., S.C., P.G., N.W., C.L., S.K., C.S., H.L., S.C., M.F. and N.D. all generated or provided key reagents. F.O.N., P.S.J., H.J.G., P.J.B. and S.T. discussed the data. S.N.K. and J.F.S conceived this study. S.N.K. designed the study, supervised and coordinated the project. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


We revealed the X-ray structure of PPARγ co-crystallized with SR1664 bound to the alternate binding site of PPARγ and confirmed that this blocks the phosphorylation of Ser273. 11 containing a bioorthogonal functional group.Peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-regulated transcription factor that plays crucial roles in adipogenesis, lipid metabolism, and glucose homeostasis. Several PPARγ ligands possess anti-diabetic activity and they commonly inhibit the phosphorylation of PPARγ at serine 273 (Ser273). The recently reported PPARγ ligand SR1664, which selectively blocks the phosphorylation of PPARγ without classical agonism, has potent anti-diabetic activity, indicating that the inhibition of Ser273 phosphorylation is sufficient to provoke anti-diabetic effects. In this study, we revealed the X-ray structure of PPARγ co-crystallized with SR1664 bound to the alternate binding site of PPARγ and confirmed that the alternate site binding of SR1664 blocks the phosphorylation of Ser273. Furthermore, using covalent inhibitors as chemical tools, we demonstrated that the inhibition of phosphorylation is attributed to the occupation of a specific site which is a hydrophobic region between helix 3 and β3–β4 at the binding pocket of PPARγ. In high-fat diet-induced obese mice, we confirmed the anti-diabetic activity of our covalent inhibitor SB1453 that was designed to bind at the specific site in PPARγ for blocking the phosphorylation of Ser273. Lastly, the target selectivity of SB1453 was demonstrated by fluorescence-based visualization of target proteins complexed with the covalent probe  Unlike glitazones, SR1664, which inhibits the phosphorylation of Ser273 without altering the transcriptional activity of PPARγ, exerts potent in vivo anti-diabetic effects without causing fluid retention and weight gain.Although glitazones have remarkable effects in the treatment of type II diabetes, their use has declined because of their serious adverse effects including weight gain, fluid retention, and congestive heart failure.via strong hydrogen bonding with helix 12  complexed with SR1664 and the SRC1 coactivator peptide to a resolution of 2.20 Å  and S1a†helix 12 , blue.16helix 12 , green.1To determine the phosphorylation-inhibiting effect of SR1664 bound at the alternate site, we investigated using GW9662, a synthetic irreversible PPARγ inhibitor that covalently binds to cysteine 313  on helix 3 (H3).1), by replacing the phenyl group with a methyl group , we confirmed that SB1404 covalently bound to Cys313 on H3 and completely blocked the binding of rosiglitazone at the canonical binding pocket of PPARγ  and S3†.2) and SB1406 (3) containing 2-(benzyloxy)phenyl and 3-(benzyloxy)phenyl groups, respectively, instead of the phenyl group in GW9662  of 0.36 Å, and there was no significant difference in the positioning of the residues around the specific binding site Fig. S6, which i5N-(2-substituted phenyl)-2-chloro-5-nitrobenzamide as a suitable molecular framework and synthesized a series of covalent inhibitors of PPARγ phosphorylation containing different R groups to effectively occupy this specific binding site -2-chloro-5-nitrobenz-amides possessing hydrophobic R substituents efficiently blocked Cdk5-mediated PPARγ phosphorylation at Ser273, and the occupation of R substituents at the hydrophobic region between H3 and β3–β4 of PPARγ appears to be sufficient for reducing the flexibility of this region, which inhibits the in vitro phosphorylation of PPARγ by Cdk5.Based on the mechanistic and structural understanding of PPARγ phosphorylation, we selected ing site . As show by Cdk5 . Taken tN-(2-substituted phenyl)-2-chloro-5-nitrobenzamides, we selected SB1405 (2) for a further in vivo study because of its excellent inhibition potency in the in vitro Cdk5 assay  and the subsequent hydrophilic space (blue) (9) and SB1453 (10) containing hydrophilic piperazine moieties attached to the additional benzene rings to improve solubility (in vitro Cdk5 assay (in vitro (Among the k5 assay  and the k5 assay . Howevere (blue) . On the lubility . After sk5 assay . This ink5 assay . Howeverin vitro .via Oil Red O staining.To examine the agonistic activity in PPARγ-mediated transcription, we conducted two cellular assays. First, using a luciferase reporter gene assay in HEK-293T cells expressing full-length PPARγ with tandem PPAR response elements (PPRE), we confirmed that SB1451 and SB1453 had almost no classical transcriptional agonism compared with that of rosiglitazone . Secondlin vivo reduction of PPARγ phosphorylation and the resulting anti-diabetic activity than SB1453. Previous studies clearly demonstrated that SR1664, a selective inhibitor of PPARγ phosphorylation without classical agonism, exerts in vivo anti-diabetic effects and causes changes in the expression of diabetic genes that were dysregulated as a result of PPARγ phosphorylation in obese animals.We evaluated the anti-diabetic activity of SB1451 and SB1453 in animal models using DIO mice that are insulin-resistant with an increased level of phosphorylated PPARγ at Ser273.in vivo treatment of glitazones.We also investigated several adverse effects, including fluid retention and cardiac hypertrophy, which have been observed following treatment with glitazones.11 containing a terminal acetylene group on the piperazine moiety to enable the fluorescence labeling of target proteins via a bioorthogonal click reaction  to gain more information about its binding mode . As expereaction . Despiteof PPARγ .11 was incubated with the proteome of HEK-293T cells expressing either murine PPARα, PPARδ, or PPARγ, followed by a copper-catalyzed click reaction with an azide-containing Cy5 to visualize the proteins complexed with probe 11. The resulting proteome was separated by gel electrophoresis, and target proteins were visualized via fluorescence scanning. Predominantly, the fluorescence-labeled protein was only detected in the PPARγ-transfected cells, indicating that probe 11 efficiently binds to PPARγ, and not to PPARα or PPARδ (Fig. S924First, we confirmed the PPAR subtype selectivity of our covalent inhibitor. Probe δ Fig. S9. Then, wδ Fig. S9. These lδ Fig. S9. In factin vitro and in adipose cells. We also demonstrated that SB1453 exerts potent anti-diabetic effects in DIO mice by blocking PPARγ phosphorylation at Ser273 in the white adipose tissue without several of the adverse effects associated with glitazones. Lastly, through fluorescence-based visualization of the target proteins complexed with the covalent probe 11 containing a bioorthogonal functional handle, we confirmed that our potent anti-diabetic agent SB1453 selectively binds to PPARγ. This study provides a useful guideline for the structure-based rational design of SPPARγMs that effectively inhibit the phosphorylation of Ser273 for the development of anti-diabetic PPARγ ligands. It can be expected that new classes of PPARγ-targeting anti-diabetic drugs will be developed in accordance with this guideline.In this study, we identified that an alternate site binding of SR1664 effectively blocks the phosphorylation of PPARγ at Ser273. By comparing this binding mode with that of the conventional PPARγ ligand rosiglitazone, we found that ligand binding at a specific binding site, the hydrophobic region between H3 and β3–β4, is closely related to the inhibition of PPARγ phosphorylation at Ser273. To determine the functional effect of PPARγ ligand binding at the specific site, we rationally designed and synthesized irreversible covalent inhibitors as chemical tools. By analysing the data from biochemical assays, X-ray crystallography, and HDX mass spectrometry with these covalent inhibitors, we found that occupation of the specific binding site in PPARγ by small-molecule ligands causes the change in the dynamic nature of β-sheet and directly correlates with the inhibitory effects on the phosphorylation of PPARγ at Ser273. This structural insight led us to rationally design the improved covalent inhibitors SB1451 and SB1453, which effectively inhibit the phosphorylation of PPARγ at Ser273 Supplementary informationClick here for additional data file.Supplementary informationClick here for additional data file.


Taenia crassiceps metacestode (cysticerci) was carried out. The results obtained from both initial velocity and product inhibition experiments suggest the enzyme follows a two-site ping-pong bi bi kinetic mechanism, in which both substrates and products are bound in rapid equilibrium fashion. The substrate GSSG exerts inhibition at moderate or high concentrations, which is concomitant with the observation of hysteretic-like progress curves. The effect of NADPH on the apparent hysteretic behavior of TGR was also studied. At low concentrations of NADPH in the presence of moderate concentrations of GSSG, atypical time progress curves were observed, consisting of an initial burst-like stage, followed by a lag whose amplitude and duration depended on the concentration of both NADPH and GSSG. Based on all the kinetic and structural evidence available on TGR, a mechanism-based model was developed. The model assumes a noncompetitive mode of inhibition by GSSG in which the disulfide behaves as an affinity label-like reagent through its binding and reduction at an alternative site, leading the enzyme into an inactive state. The critical points of the model are the persistence of residual GSSG reductase activity in the inhibited GSSG-enzyme complexes and the regeneration of the active form of the enzyme by GSH. Hence, the hysteretic-like progress curves of GSSG reduction by TGR are the result of a continuous competition between GSH and GSSG for driving the enzyme into active or inactive states, respectively. By using an arbitrary but consistent set of rate constants, the experimental full progress curves were successfully reproduced in silico.A kinetic study of thioredoxin-glutathione reductase (TGR) from As a meic group . As in mic group , a selenic group . Unlike ic group , thus maic group –7. Inter mansoni , suggest mansoni , 10, inv Taenia crassiceps were reported [ Echinococcus granulosus [ Taenia solium cysticerci [ Fasciola hepatica [ Fasciola gigantica [ Schistosoma mansoni [μM or higher have been used [ T. crassiceps was investigated. Furthermore, the effect of NADPH on the atypical kinetic behavior of TGR was also studied. Based on both the kinetic and the crystallographic evidence on TGR, a comprehensive model consistent with all the experimental observations is put forward and tested through in silico simulations. All the experimental observations regarding the kinetic behavior of the enzyme were successfully reproduced by the model.The presence of the enzyme has been demonstrated in animals , 4, humareported . Particureported . Such atreported . The samanulosus  and Taensticerci  and in thepatica , Fascioligantica , and Sch mansoni . Thus, t mansoni . However mansoni . Such mo mansoni . On the een used , 14–17. 2′5′ ADP–Sepharose 4B was obtained from Amersham Pharmacia Biotech . All others chemicals were obtained from Sigma Chemical Company  and used without further purification. Water purified by reverse osmosis was used in the preparation of solutions. T. crassiceps was used as a source of TGR. The cysticerci growth conditions, as well as its extraction, rupture, and preparation of a crude homogenate have been described elsewhere [The HYG strain oflsewhere . T. crassiceps was purified to homogeneity as previously described [TGR from the cytosolic fraction of larvalescribed . Enzyme The disulfide reductase activity assays of TGR were carried out in an Agilent 8453 uv/visible spectrophotometer (Hewlett Packard) fitted with a thermostated cell holder. All the kinetic experiments were performed at 25°C in 0.1 M Tris/HCl buffer (pH 7.8) containing 1 mM EDTA (buffer A) in a final volume of 1 mL.−1 cm−1 for NADPH was used in the calculations of initial velocities. In those experiments dealing with the effect of GSSG on the reduction of oxidized thioredoxin (Trx), human Trx was used as substrate. In this case, the reaction mixture contained 100 μM NADPH, human Trx at the corresponding concentration, and 2 mM GSSG at a final volume of 120 μL in buffer A. The reaction was started by adding a small TGR aliquot.The GSSG reductase activity of TGR was determined by following the decrease in absorbance at 340 nm as a consequence of NADPH oxidation . Both NA+ was determined spectrophotometrically by reading the absorbance of an aliquot of the corresponding stock solution at either 340 nm (ε = 6220 M−1 cm−1) or 259 nm (ε = 18000 M−1 cm−1) for NADPH or NADP+, respectively. As regards GSSG, its concentration in the stock solution was calculated through enzyme assays by mixing an aliquot of the disulfide with TGR in the presence of an excess of NADPH. After exhaustion of GSSG, its concentration was determined from the total change in absorbance at 340 nm.The concentration of both NADPH and NADP−1 cm−1 for protein bound FAD [The monomer concentration in the stock solutions of TGR was determined from its absorbance at 462 nm using a molar extinction coefficient of 11.3 mMound FAD .μM of the disulfide were used in order to avoid the strong substrate inhibition observed at moderate or high concentrations of GSSG. This decision is warranted because the initial portion of the saturation curve is very sensitive to discriminate between sequential and ping-pong kinetic mechanisms [KmA and KmB are the corresponding Michaelis-Menten constants, while Kia represents the dissociation constant for A. The inhibitory ability of NADP+ on the GSSG reductase activity of TGR was determined by analyzing its effect on the initial velocities under steady-state conditions. NADP+ was incubated in buffer A with NADPH and GSSG under nonhysteretic conditions and the reaction was started by adding a small enzyme aliquot. Data obtained in the presence of the product NADP+ as the inhibitor were fitted to either a competitive . An initial estimate for the Ki value was obtained by plotting initial velocity data obtained over a broad range of GSSG concentrations in a semilog fashion [m represent the concentration of substrate at the maximum point of the curve, while S1 and S2 correspond to the substrate concentration at the two points where velocity is half that at the maximum.In order to elucidate the kinetic mechanism of TGR, initial velocity data were obtained by varying the concentration of both NADPH and GSSG . For thechanisms . A globaive (see ) or an uive (see ) model on method  for a pi fashion  and the In those kinetic experiments carried out at high concentrations of GSSG, the magnitude of the apparent lag time observed in the full time progress curves was estimated as described elsewhere .Fitting of data to the different velocity equations was made by nonlinear regression analysis using Sigma Plot software. No weighting of data was applied. Kinetic parameters are given as mean ± standard deviation.When the initial velocity patterns did not allow a clear distinction between alternative kinetic mechanisms, a model discrimination analysis was needed. The following rules were used in the data analysis.(I) The initial selection of a particular kinetic model was based on visual inspection of the corresponding double-reciprocal plot. When this preliminary analysis did not lead to a clear model discrimination, data were fitted to both alternative velocity equations.χ2 values, then the model consistent with additional kinetic evidence  was chosen as the most plausible.(II) If the two alternative kinetic models had equal or very similar In order to compare the experimental full progress curves obtained under different concentrations of NADPH, GSSG, and enzyme, with that predicted by the mechanistic model (see discussion), a curve fitting procedure was performed. In this case the fitting procedures were carried out with the Dynafit software  version (i) In all cases, the best set of rate constants pertaining to the ping-pong reaction cycle were fixed , representing ten microscopic rate constants (reactions 1 to 6 of the model).(ii) In the fitting procedure of the full progress curves obtained at moderate or high concentrations of GSSG, two rate constants were allowed to be fitted in order to find the best fit values. One of such constants pertains to a reaction associated with the reversible binding of GSSG to the inhibitory site (reactions 7 and 9 of the model), while the other microscopic rate constant was chosen from any of the two reactions responsible for the atypical full progress curves  For long full time courses, the best fitting value for either GSSG or enzyme concentrations, or both, was also searched, allowing a variation of ±10% of the experimental value.Simulation of the full time courses of TGR was performed with the Dynafit software  version  S. mansoni TGR (PDB code 2X8C) was obtained from the RCSB Protein Data Bank (http://www.rcsb.org). The structure of GSSG was recovered from the Homo sapiens glutathione S-transferase μ2 (PDB code 1YKC) using the Hic-up server (http://xray.bmc.uu.se/hicup/). The structures of TGR and GSSG were processed with AutoDockTools (ADT) version 1.5.4 (http://mgltools.scripps.edu) [http://vina.scripps.edu) [For molecular docking, the protein structure ofpps.edu) . Essentipps.edu) . For TGR Kia value (4.98 × 10−17μM) with a very high variation coefficient (7.4 × 1017%). To confirm the ping-pong kinetic model suggested by the initial velocity patterns, a product inhibition study with NADP+ was performed. The results are shown in χ2 values . In order to elucidate this point, the effect of NADP+ on the initial velocity patterns, with GSSG as the variable substrate, was also analyzed. The resultant double-reciprocal plots showed a parallel lines pattern, strongly suggesting an uncompetitive inhibition , confirming the lack of a reversible connection during the NADP+ dissociation step. Hence, it can be concluded that the inhibition of the product NADP+ on the GSSG reductase activity of TGR is the result of the formation of a dead-end complex with the unmodified enzyme, explaining the competitive inhibition pattern of NADP+ with NADPH as the variable substrate. In Km, kcat as well as the specificity constant kcat/Km for both NADPH and GSSG of T. crassiceps TGR are summarized. These values are consistent with those previously reported [hibition . Clearlyectively , 32. To ectively . Hence, reported .Reduced glutathione was a very poor inhibitor product. At 8 mM GSH, barely 10% inhibition was detected. Between 1 and 4 mM GSH, a moderate but consistent activating effect (up to 25%) was observed. Due to the micromolar concentrations of GSSG present in the GSH stock solutions, its effect as an inhibitor product was not further explored. T. crassiceps TGR is strongly inhibited. Such inhibition is concomitant with the appearance of the hysteretic-like progress curves [Km ( in silico simulations using the mechanism-based model (see discussion) either in the presence or in the absence of the reactions involved in the generation of the hysteretic-like full time courses. The results of the simulations revealed no significant difference when the initial velocity data obtained under the two above-mentioned conditions were compared , no evidence for substrate inhibition or hysteretic kinetics was observed [When the alternative biological disulfide Trx was assayed at relatively high concentrations , giving a surface electrostatic potential which is more similar to the corresponding site on GR than that of the homologous TR [ S. mansoni [ F. gigantica [ T. crassiceps and the associated binding enthalpy was estimated as – 7.1 kcal mol−1. This figure is significantly lower than the value of – 24.6 kcal mol−1 obtained by isothermic titration calorimetry for the binding of GSSG to the active site of human GR [μM for Ki obtained in the present work from the substrate inhibition data is consistent with such result. μM). In the range from 50 mM to 600 mM NaCl, an inhibition up to 70% of the initial velocity was observed.From the X-ray crystallographic analysis ofrevealed . It is l mansoni  and F. gigantica  revealedhuman GR , suggest E. granulosus [ F. hepatica [ S. mansoni [ E. granulosus TGR) was replaced with serine, the lag time observed at a high concentration of GSSG was fully abolished. However, by decreasing the protein concentration of the mutant enzyme in the reaction mixture, the hysteretic-like progress curve was regained [TGR represents an atypical case in the disulfide reductase family of enzymes. Although the enzyme retains the homodimeric nature which is common to this set of oxidoreductases, it is outstanding in tertiary structure, total number of redox centers, and wide substrate specificity. Thus, in TGR a Grx-like domain has been appended to the N-terminal end of the animal TR module, conferring to the enzyme additional catalytic abilities, notably the reduction of GSSG at significant rates, as well as catalysis of thiol-disulfide exchange reactions, including deglutathionylation of mixed disulfides protein-glutathione , 4, 6. Fanulosus  was prophepatica  and S. m mansoni  also sho T. crassiceps cysticerci follows a ping-pong bi bi kinetic mechanism with the NADPH/GSSG couple as the substrates, in which dissociation of NADP+ from the enzyme during the catalytic cycle is an irreversible event. Interestingly, the inhibition patterns obtained with NADP+ are consistent with a variant of the ping-pong bi bi kinetic mechanism typical for two-site enzymes [ S. mansoni TGR [+) can occur either before or after binding of the second substrate , explaining the atypical inhibition patterns observed with T. crassiceps TGR. Thus, TGR can be considered as an additional example of enzymes where the active site is split into halves.The results described in the present work show that TGR from enzymes –36. Accosoni TGR , 9 is co in silico simulations performed either in the presence or in the absence of the reactions responsible for the unusual kinetic behavior of TGR (see below) revealed such analysis is warranted. As shown in Fig. μM, reaching a maximum (about 8%) at the highest concentrations of GSSG, both at a low and at a high NADPH concentration. Such differences are in the range of the experimental uncertainty obtained in the determination of initial velocities.On the other hand, the initial velocity data obtained in a broad range of GSSG concentrations revealed a strong substrate inhibition . The oveAs regards the hysteretic-like full progress curves of TGR observed at moderate or high concentrations of GSSG, the results obtained in the present work revealed the presence of an additional complexity. Thus, the enzyme assays performed at low concentrations of the electron donor NADPH showed an initial burst-like stage, followed by the lag stage. The initial stage of fast NADPH consumption is barely noticeable in the enzyme assays carried out in the presence of high concentrations of both GSSG and NADPH , 16, 17.In order to build a comprehensive model to explain the atypical kinetic behavior of TGR, the following observations regarding the normal performance of the enzyme must be stated. These are based on all the kinetic and structural evidence available.  S. mansoni TGR [4) reduced species as an intermediary in its normal functioning.(i) In the GSSG reductase activity of TGR, the Grx-like domain of the enzyme plays an essential role , 4, 7. Hsoni TGR  supports E. granulosus and S. mansoni, in which either the N-terminal or the C-terminal cysteine residue of the Grx redox motif was replaced [(ii) Natural variants of the enzyme  in which the C-terminal cysteine residue of the redox motif at the Grx-like domain is absent are fully functional in GSSG reduction . This fareplaced , 7, suppreplaced , 38. S. mansoni TGR  As with the related TR, both subunits of TGR are required during the catalytic cycle of the enzyme. Thus, in order to regenerate the reduced state of the nucleophilic cysteine of subunit A, electrons must be shuttled from the selenol-thiol redox center, located at the C-terminal end of subunit B . Howeversoni TGR , the twotransfer . S. mansoni [ E. granulosus [(iv) The X-ray crystallographic structures of both mansoni  and E. ganulosus  TGR haveanulosus . Hence, anulosus , 16.Regarding the complex kinetic behavior of TGR, the following experimental facts must be considered.(i) The profiles of the full progress curve of NADPH consumption and the magnitude of the apparent lag time are dependent on the concentration of the substrates NADPH and GSSG. Thus, the amplitude of both the initial burst-like stage and the inhibited segment of the progress curves can be modified by changing the initial concentration of the substrates in the reaction mixture.(ii) Both the magnitude of the apparent lag time and the profile of the full progress curves of TGR are not those expected for a typical hysteretic enzyme. For the latter, the reported lag times are in the range of seconds or a few minutes and the shape of the curve in the transition zone is due to a single exponential transition , 38–41.μM GSH saturation curves with GSSG as the variable substrate was fully hyperbolic. Up to a concentration of 300 μM of the disulfide no inhibition was observed (data not shown). Such evidence strongly suggests that in the atypical kinetic behavior of TGR thiol/disulfide exchange reactions are involved.(iii) The presence of disulfide reducing reagents  at micromolar concentrations in the reaction mixture lead either to a decrease in the magnitude of the apparent lag time or its full abolition , 14, 15.(iv) The concentration of the enzyme in the reaction mixture is also critical for the presence or absence of the atypical progress curves as well as for the amplitude of the initial fast stage of the reaction. The higher the enzyme concentration, the lesser the magnitude of the apparent lag time , 14.Based on all the above kinetic and the structural evidence on TGR, a mechanism-based model was built to explain its atypical kinetic properties . The maj S. mansoni [ E. granulosus [(i) According to the results obtained in the present work, the normal catalytic cycle of the enzyme  Dissociation of the products NADP  si face of FAD (Ki = 331 ± 79 μM) compared with the affinity of the grx-like domain (Km = 14.4 ± 2.3 μM). GSSG bound at the inhibitory site will block the electron flow from FAD to the C-terminal redox center, thus inhibiting its reduction.(iii) GSSG acts as a noncompetitive inhibitor by binding to the alternative site of either the reduced form of the enzyme (F) or the F-GSSG binary complex . Hence, in the absence of either the redox active motif of the Grx-like domain or the selenocysteine residue, TGR will be still able to reduce GSSG in a GR-like fashion. The low activity of GSSG reductase at the alternative site can be explained as a result of a nonoptimal distance of the disulfide bond of GSSG after binding from the nucleophilic cysteine of the redox active motif ((iv) It is proposed that both the GSSG-F binary and the GSSG-F-GSSG ternary complexes of the enzyme have the ability to reduce GSSG at the low-affinity alternative site  either in the presence or absence of NADPH [ in silico simulations revealed conventional profiles of NADPH consumption in which GSSG acts as an reversible inhibitor but without any atypical behavior ((v) As a result of the residual disulfide reductase activity of both the GSSG-F binary complex and the GSSG-F-GSSG ternary complexes, the enzyme population will be directed into the inactive states I and I-GSSG (reactions 10 and 12 in of NADPH . The forbehavior .(vi) The residual GSSG reductase activity of inhibited TGR will result in a slow but continuous increase in the concentration of the product GSH. The latter will be able to reactivate both I and I-GSSG complexes by reducing its redox active centers (reactions 11 and 13 in  in silico simulations (By deleting from the model the reactivation of the enzyme by disulfide reducing compounds (reactions 11 and 13 of ulations . Instead k7 and k8 ((vii) The effect of NADPH on the hysteretic-like full progress curves of TGR is explained as a result of providing the enzyme species  to which GSSG can bind, both as substrate and as inhibitor. Hence, GSSG will behave as an uncompetitive inhibitor regarding NADPH. The unusual profile of the full progress curves observed at low concentrations of NADPH  but at h7 and k8  suggest (viii) The effect of a high concentration of GSSG on the reduction of the alternative disulfide substrate Trx by TGR further supports the proposal that the binding of GSSG occurs at the putative site located in the neighborhood of the FAD prosthetic group . The abi EgTGR C34S mutant. As reported by Bonilla et al. [The present model also can furnish an explanation of the results obtained with thea et al. , the ful in silico of both initial velocity patterns and full progress curves were carried out. The conditions used to obtain a selected set of the specific rate constants are described in Supplementary Materials. As shown in the supplementary figures, all the experimental observations on the atypical kinetic behavior of TGR are reproduced by the model. These include the GSSG-dependent substrate inhibition  and a product (GSH) for driving the enzyme into inactive or active states, resulting in atypical hysteretic-like progress curves. We have named this kind of kinetic behavior as pseudohysteresis. This kind of atypical progress curve of enzyme activity adds to other bizarre kinetic phenomena such as the true hysteretic behavior , 28 dampA comment concerning a recently published paper dealing with recombinant human TGR . In suchμM GSH is high enough to avoid the GSSG-dependent inhibition of the enzyme. From the total concentration of glutathione of 1.2 mM and a GSH/GSSG ratio of 131 determined in T. crassiceps cysticerci under basal conditions [ E. granulosus TGR is also inhibited by high concentrations of GSSG [Finally, although the substrate inhibition of TGR by moderate or high concentrations of GSSG may, in principle, appear contradictory with the function of the enzyme, it must be taken into account that the degree of inhibition is dependent on the concentration of both GSH and GSSG. As described above, 50 nditions  it appea of GSSG . It is a T. crassiceps follows a two-site ping-pong bi bi kinetic mechanism with GSSG as the substrate. The latter exerts a strong but temporary substrate inhibition, resulting in hysteretic-like progress curves.Thioredoxin-glutathione reductase fromNADPH plays a critical role in the atypical kinetic behavior of the enzyme, by supplying the reduced form of the enzyme to which GSSG binds.A mechanism-based model explaining all the kinetic observations of TGR was developed. A key point of the model is the presence of a low-affinity second binding site for GSSG, with the ability to reduce GSSG at a low rate. in silico simulations of the model, it becomes clear that the hysteretic-like progress curves of the enzyme are the result of a continuous competition between GSH and GSSG for driving the enzyme into active or inactive pathways. Hence, the atypical full progress curves of GSSG reductase activity of TGR must not be considered as a kind of hysteretic behavior.From theAs a corollary of the model, it must be stressed that the maximal slope observed in the atypical full progress curves does not represent the steady-state of enzyme activity.


Intermediate monocytes have been associated with poor outcome, while non-classical CX3CR1-positive monocytes could have reparative function after STEMI. We characterized monocyte subsets and NET markers at the culprit lesion site of STEMI patients (n = 91). NET surrogate markers were increased and correlated with larger infarct size and with fewer non-classical monocytes. Intermediate and especially non-classical monocytes were increased at the culprit site compared to the femoral site. Low CX3CR1 expression of monocytes correlated with high NET markers and increased infarct size. In this translational system, causality cannot be proven. However, our data suggest that NETs interfere with monocytic differentiation and receptor expression, presumably promoting a subset shift at the culprit lesion site. Reduced monocyte CX3CR1 expression may compromise myocardial salvage.Neutrophils release their chromatin into the extracellular space upon activation. These web-like structures are called neutrophil extracellular traps (NETs) and have potent prothrombotic and proinflammatory properties. In ST-elevation myocardial infarction (STEMI), NETs correlate with increased infarct size. The interplay of neutrophils and monocytes impacts cardiac remodeling. Monocyte subsets are classified as classical, intermediate and non-classical monocytes. In the present study,  The pathomechanism of coronary thrombosis, ischemia and myocardial scar formation is incompletely understood. We have previously shown that neutrophils release neutrophil extracellular traps (NETs) at the culprit lesion site (culprit site), which might serve as scaffolds for platelets and erythrocytes and could initiate coronary thrombosis2, especially in the setting of superficial plaque erosion3. Coronary NET burden significantly correlated with reduced ST-segment resolution and increased infarct size in ST-elevation MI (STEMI) patients. Degradation of NETs by deoxyribonucleases in circulation is crucial for homeostasis4. High endogenous deoxyribonuclease activity was correlated with reduced infarct size2. Hence, increased NET burden due to impaired degradation appears to be detrimental downstream in the ischemic myocardium, causing microvascular obstruction, inflammation and cardiomyocyte cell death5. NET-releasing neutrophils and monocytes cooperate in inflammation6, thrombus formation7 and cardiac remodeling8. Monocytes can differentiate into macrophages9, which release major proinflammatory mediators like Interleukin-1ß in response to NETs10. Macrophages in turn complement the clearance of NETs11 and are shifted towards an anti-inflammatory phenotype after phagocytosis of neutrophilic proteins12. Monocyte subsets have specific functions in cardiac remodeling after MI13 and are grouped according to their CD14/CD16 expression into classical (CD14++ CD16−), intermediate (CD14++ CD16+) and non-classical (CD14+ CD16++) monocytes14. A recent study has shown that monocyte subsets evolve from classical to intermediate and non-classical monocytes15. Classical monocytes are proinflammatory and produce high levels of cytokines16. Intermediate monocytes are potent antigen-presenting cells, which is reflected by a high HLA-DR expression17. They are proinflammatory18, exhibit a strong phagocytic activity and strongly contribute to oxidative burst. Non-classical monocytes display the highest CX3CR1  expression, which is an important receptor for adhesion and migration. They have a patrolling function and efficiently invade inflamed tissue. Via CX3CR1, they respond to apoptotic signals to remove cell debris and contribute to tissue repair, thus exerting anti-inflammatory properties19. Classical and intermediate monocytes are increased in the periphery in the course of MI and correlate with increased infarct size and poor cardiac function at follow up21, whereas non-classical monocytes were associated with improved recovery of dysfunctional myocardium22.Myocardial infarction (MI) and subsequent ischemic cardiomyopathy are a major disease burden worldwidein vitro and evaluated NET surrogate markers and monocytes subsets in vivo at the culprit site of STEMI patients.In the present study, we investigated the impact of NETs on monocyte subsets STEMI patients n = 91), who received thrombectomy in the course of primary percutaneous coronary intervention (pPCI) at the Department of Cardiology, General Hospital of Vienna, were consecutively included in the study and monocyte subsets were quantified in femoral and culprit site blood samples. An additional characterization of monocyte expression markers was performed in the last 36 consecutive patients. Inclusion criteria were chest pain at the time of pPCI, ST-elevations of ≥2 mm on >1 chest lead or new ST-elevations of ≥1 mm on >1 limb lead within 20 minutes of pPCI. All patients received 250 mg of acetylsalicylic acid and unfractionated heparin to achieve an activated coagulation time of >300 seconds (4000–10000 IE). Criteria for thrombectomy were a vessel diameter of ≥3 mm, a large intraluminal contrast medium filling defect, suggestive of thrombus within 50 mm of the respective ostium without severe calcification, tortuosity, or difficult vascular access. A total volume of 10 to 20 mL culprit site blood were aspirated using a commercial thrombectomy catheter . Femoral whole blood samples were obtained simultaneously from the femoral arterial sheath. All plasma measurements were normalized for femoral whole blood hematocrit as aspiration catheter flushing with 2–4 ml 0.9% sodium chloride leads to dilution of the culprit site blood sample. Exclusion criteria were low culprit site sample volume, intake of immunosuppressants, cardiogenic shock, severe infection, active neoplasia, end stage kidney disease and glycoprotein IIb/IIIa-blocker treatment. Infarct size was estimated by measuring creatine phosphokinase isoform MB area under the curve (CK-MB AUC), which allows an adequate approximation of infarct size . All investigations conformed to the principles outlined in the Declaration of Helsinki.Blood samples were incubated with fluorochrome-labeled antibodies for 30 minutes in the dark. Cells were stained for human CD11a , CD11b , CD14 , CD16 , CD45 , CD56 , CD66b , CD142  CD192 , toll-like receptor (TLR)2 , TLR4 , CX3CR1  and HLA-DR . Erythrocyte lysis by BD FACS lysing solution (BD Biosciences) was followed by 3 washing steps with phosphate-buffered saline (PBS), then cells were resuspended for the measurements. Per panel, 50000 events were counted and the percentage of positive cells or mean fluorescence intensity (MFI) was measured.Monocyte subsets were gated as depicted in Supplementary Fig. Isotype-matched antibodies  were added to negative control samples. Cells were analyzed using a BD FACSCanto II and FACSDiva Software (BD Biosciences).23. Culprit site differential blood counts could be obtained from 69 patients.Total monocyte subset numbers were calculated via monocyte numbers measured in femoral and culprit site differential cell counts similar to a published approach24, with minor modifications. Streptavidin-coated plates were incubated with anti-histone biotin (Roche) for 120 minutes. Plates were washed and incubated with plasma samples or standard  for 90 minutes. After washing, anti-histone H3  was added for 60 minutes. Plates were washed and incubated with goat anti-rabbit HRP conjugate antibody  for 60 minutes, washed, and developed with BM Blue POD Substrate (Roche) for 20 minutes. The reaction was stopped by addition of 2 M H2SO4 and optical density was measured on a Promega GloMax Discover microplate reader .Citrullinated histone H3 (citH3) was measured as describedQuant-iT PicoGreen dsDNA Assay (Invitrogen) according to manufacturer´s instructions. Fluorescence was measured on a GloMax Discover microplate reader (Promega).Double-stranded deoxyribonucleic acid (dsDNA) was quantified using the Human CX3CL1/Fractalkine Quantikine ELISA Kit (R&D Systems) according to manufacturer’s instructions in the same subset of patients, from whom an expression marker analysis was performed by flow cytometry (FC) (n = 34). Optical density was measured on a Promega GloMax Discover microplate reader .CX3CL1 was quantified using the 26. Purity of neutrophils was routinely above 98%. Neutrophils were resuspended in RPMI 1640 (Gibco) + 3% fetal calf serum  and stimulated with 500 nM phorbol-myristate acetate for 4 hours at 37 °C. Supernatant was discarded, and cells were incubated with RPMI containing 3% FCS and 10 U/ml Alu I (Roche) for 30 minutes at 37 °C. Supernatant was collected and centrifuged for 5 minutes, 300 g to remove remaining cellular debris. Concentration of NETs was assessed using PicoGreen as described above. A detailed analysis of NETs produced in vitro can be found in Supplementary Fig. Neutrophils and NETs were isolated from healthy donors according to recently published protocols with minor modifications50 µl of EDTA whole blood drawn from healthy volunteers were stimulated with 500 ng/ml isolated NETs or vehicle control in a polystyrene tube for 1 hour at 37 °C. Blood was washed with 2 ml PBS + 1% FCS. Supernatant was discarded, and the remaining pellet was resuspended in 50 µl PBS. Cells were stained for CD66b (Pacific Blue), CD45 (Pacific Orange), CD142 (Brilliant Violet 711), CD14 (FITC), CX3CR1 (PE), HLA-DR; CD16 (PE-Cy7), CD56 (Alexa Fluor 647), CD11b (Alexa Fluor 700), HLA-DR (APC-Cy7). Cells were lysed using BD FC Lysis Solution for 15 minutes. Samples were washed three times with PBS + 1% FCS and analyzed using an Attune NxT Flow Cytometer (ThermoFisher).s). Normally distributed data are given as mean ± standard deviation (SD) and tested using paired Students t-test. Bonferroni-Holm correction was applied in each set of experiments to correct for multiple testing. Boxplots display the 25th–75th percentile, the whiskers cover the 5th–95th percentile. Extreme outliers (<5th or >95th percentile) are not displayed in the figures. A published trapezoidal formula was used to calculate CK-MB AUC27, with at least 5 consecutive values over a period of 3 days after pPCI. A p-value below 0.05 was considered significant. Statistical testing was performed using IBM SPSS Statistics 22.0 for Windows.Distribution of data was assessed by histograms complemented by Kolmogorov-Smirnov test and Shapiro-Wilk test. Non-parametric data are given as median and interquartile range [IQR] and analyzed using Wilcoxon signed rank test and Spearman rank correlation  were stimulated with isolated NETs tes Fig. . Moreovetes Fig. . Stimulates Fig. .Figure 128, dsDNA was significantly increased at the culprit site compared to femoral plasma . Monocyte percentage of leukocytes was significantly decreased in culprit site whole blood compared to the femoral site , but total monocyte number in differential blood cell counts was not significantly different . Interestingly, classical monocyte percentage was decreased at the culprit site Fig. , whereasIn total cell numbers, intermediate  and non-classical monocytes  were significantly increased, while classical monocytes were not significantly different .We determined surface receptors of monocytes at the culprit site compared to the femoral site by FC in a subset of patients (n = 36). As expected, classical monocytes displayed a constitutively low expression of CX3CR1, while intermediate and especially non-classical monocytes showed distinctly higher levels. The CX3CR1 expression was substantially decreased at the culprit site in all subsets Fig. .Figure 4Soluble CX3CL1 was measured in the same patients and was significantly decreased in culprit site compared to femoral plasma .Classical monocytes displayed increased CD11b expression at the culprit site compared to the femoral site Fig. , CD142 es = −0.517, p < 0.05), intermediate monocytes  and non-classical monocytes .29. Neutrophils and NETs have a detrimental impact on reperfusion and infarct size in STEMI30. It has been proposed that NETs are involved in microvascular obstruction and downstream injury5. In line with previous data, we detected increased levels of NET surrogate markers in culprit site plasma, which correlated with higher enzymatic infarct size  on classical and intermediate monocytes in vitro  were significantly increased compared to the femoral site, and higher dsDNA correlated with lower non-classical monocyte levels Fig. . In spit19. It was shown that repair after arterial injury is compromised in CX3CR1 knockout mice and can be rescued by application of wild type Ly6c-low monocytes39. Similar results were reported from a model of ischemic kidney reperfusion injury40. In contrast, it was shown that CX3CL1 neutralization in a murine myocardial infarction model resulted in improved survival41.High NET surrogate markers correlated with low CX3CR1 expression of non-classical monocytes Fig.  and culp42. We found decreased levels of CX3CL1 at the culprit site compared to femoral plasma. Hypoxia led to reduced CX3CL1 release of endothelial cells in vitro43. Of note, pulmonary endothelial cells responded differently44. Receptor internalization after ligand binding could be an alternative explanation for reduced CX3CR1 expression and CX3CL1 at the culprit site, however, our in vitro data do not support this hypothesis.Soluble CX3CL1 , the major ligand of CX3CR1, was reported to be increased during STEMIin vitro. In vivo, fibrocytes are increased at the culprit site, which appears to also favor adverse tissue remodeling31.Interestingly, NETs also promote differentiation of monocytes into fibrocytes Taken together, these data suggest that extracellular chromatin could promote a differentiated monocyte response and lower CX3CR1 expression on monocytes at the culprit site. This possibly leads to decreased transmigration into the ischemic tissue of non-classical monocytes and impaired myocardial healing. Targeting NETs in acute coronary syndrome may be a promising therapeutic strategy.With the present study, we provide information on monocyte subsets in MI. However, sampling from the culprit and femoral site of STEMI patients only yields a snapshot of ongoing processes. In this human system, causality cannot be established and our findings remain observational. The effect size of presented data from the femoral and culprit site is small, however paired comparisons are from the same circulatory system, therefore also small differences might be very meaningful.Supplementary information