Presence Of Agonist Research Articles

Sodium Houttuyfonate Ameliorates DSS-induced Colitis Aggravated by Candida albicans through Dectin-1/NF-κB/miR-32-5p/NFKBIZ Axis Based on Intestinal microRNA Profiling.

Our previous research indicated that Sodium houttuyfonate (SH) can effectively ameliorate dextran sulfate sodium (DSS)-induced colitis exacerbated by Candida albicans. However, the underlying protective mechanism of SH remains unclear. Therefore, in this study, a mice colitis model was infected with C. albicans, and the total colonic miRNAs were assessed. Furthermore, the differentially expressed miRNAs were enriched, clustered, and analyzed. Moreover, based on the dual luciferase analysis of NFKBIZ modulation by miR-32-5p, the in vitro and in vivo therapeutic effects of SH on inflammatory response, fungal burden, oxidative stress, and apoptosis were assessed at transcriptional and translational levels in the presence of agonist and antagonist. A total of 1157 miRNAs were identified, 84 of which were differentially expressed. Furthermore, qRT-PCR validated that SH treatment improved 17 differentially expressed miRNAs with > fourfold upregulation or > sixfold downregulation. Similar to most differentially altered miRNA, C. albicans significantly increased Dectin-1, NF-κB, TNF-α, IL-1β, IL-17A, and decreased miR-32-5p which negatively targeted NFKBIZ. In addition, SH treatment reduced inflammatory response and fungal burden in a colitis model with C. albicans infection. Further analyses indicated that in C. albicans infected Caco2 cells, SH inhibited fungal growth, oxidative stress, and apoptosis by increasing Dectin-1, NF-κB, NFKBIZ, TNF-α, IL-1β, IL-17A, and decreasing miR-32-5p. Therefore, SH can ameliorate the severity of colitis aggravated by C. albicans via the Dectin-1/NF-κB/miR-32-5p/NFKBIZ axis.

NanoGraphene Clot: A New Fibrinogen-Mimic Hemostatic Material.

Trauma is the leading cause of death for adults under the age of 44. Internal bleeding remains a significant challenge in medical emergencies, necessitating the development of effective hemostatic materials that could be administered by paramedics before a patient is in the hospital and treated by surgeons. In this study, we introduce a graphene oxide (GO)-based PEGylated synthetic hemostatic nanomaterial with an average size of 211 ± 83 nm designed to target internal bleeding by mimicking the role of fibrinogen. Functionalization of GO-g-PEG with peptides derived from the α-chain of fibrinogen, such as GRGDS, or the γ-chain of fibrinogen, such as HHLGGAKQAGDV:H12, was achieved with peptide loadings of 72 ± 6 and 68 ± 15 μM, respectively. In vitro studies with platelet-rich plasma (PRP) under confinement demonstrated aggregation enhancement of 39 and 24% for GO-g-PEG-GRGDS and GO-g-PEG-H12, respectively, compared to buffer, while adenosine diphosphate (ADP) alone induced a 5% aggregation. Compared to the same materials in the absence of ADP, GO-g-PEG-GRGDS achieved a 47% aggregation enhancement, while GO-g-PEG-H12 a 25% enhancement. This is particularly important for injectable hemostats and highlights the fact that our nanographene-based materials can only act as hemostats in the presence of agonists, reducing the possibility of unwanted clotting during circulation. Further studies on collagen-coated wells under dynamic flow revealed statistically significant augmentation of PRP fluorescence signal using GRGDS- or H12-coated GO-g-PEG compared to controls. Hemolysis studies showed <1% lysis of red blood cells (RBCs) at the highest PEGylated nanographene concentration. Finally, whole human blood coagulation studies reveal faster and more pronounced clotting using our nanohemostats vs PBS control from 3 min and below (blood is clotted with 10% CaCl2 within 4-5 min), with the biggest differences to be shown at 2 and 1 min. At 1 min, the clot weight was found to be ∼45% of that between 4 and 5 min, while no clot was formed in PBS-treated blood. Reduction of CaCl2 to 5 and 3%, or utilization of prostaglandin E1, an anticoagulant, still leads to clots but of smaller weight. The findings highlight the potential of our fibrinogen-mimic PEGylated nanographene as a promising non-hemolytic injectable scaffold for targeting internal bleeding, offering insights into its platelet aggregation capabilities under confinement and under dynamic flow as well as its pronounced coagulation abilities.

Uncovering PPAR-γ agonists: An integrated computational approach driven by machine learning

Peroxisome proliferator-activated receptor gamma (PPAR-γ) serves as a nuclear receptor with a pivotal function in governing diverse facets of metabolic processes. In diabetes, the prime physiological role of PPAR-γ is to enhance insulin sensitivity and regulate glucose metabolism. Although PPAR-γ agonists such as Thiazolidinediones are effective in addressing diabetes complications, it is vital to be mindful that they are associated with substantial side effects that could potentially give rise to health challenges. The recent surge in the discovery of selective modulators of PPAR-γ inspired us to formulate an integrated computational strategy by leveraging the promising capabilities of both machine learning and in silico drug design approaches. In pursuit of our objectives, the initial stage of our work involved constructing an advanced machine learning classification model, which was trained utilizing chemical information and physicochemical descriptors obtained from known PPAR-γ modulators. The subsequent application of machine learning-based virtual screening, using a library of 31,750 compounds, allowed us to identify 68 compounds having suitable characteristics for further investigation. A total of four compounds were identified and the most favorable configurations were complemented with docking scores ranging from −8.0 to −9.1 kcal/mol. Additionally, the compounds engaged in hydrogen bond interactions with essential conserved residues including His323, Leu330, Phe363, His449 and Tyr473 that describe the ligand binding site. The stability indices investigated herein for instance root-mean-square fluctuations in the backbone atoms indicated higher mobility in the region of orthosteric site in the presence of agonist with the deviation peaks in the range of 0.07–0.69 nm, signifying moderate conformational changes. The deviations at global level revealed that the average values lie in the range of 0.25–0.32 nm. In conclusion, our identified hits particularly, CHEMBL-3185642 and CHEMBL-3554847 presented outstanding results and highlighted the stable conformation within the orthosteric site of PPAR-γ to positively modulate the activity.

Impaired T-Cell Proliferation in Chronic Lymphocytic Leukemia Patients Is Linked to Disturbed Lipid Metabolism and Homeostasis

Background Successful T-cell based anti-cancer therapies require proliferation and longevity of effector T cells, features that highly depend on metabolic processes. T cells from chronic lymphocytic leukemia (CLL) patients are characterized by acquired T-cell dysfunction, resulting in impaired activation, proliferation and cytotoxicity upon in-vitro activation (van Bruggen et al, Blood 2019). Whilst the role of glucose metabolism in activated T-cells is extensively studied, the contribution of other energy sources is less clear. Lipid metabolism has only recently come into focus as an important source of energy for immune cells. Aims Molecular and functional analysis of the lipid metabolic pathway in T cells from CLL patients. Methods Peripheral blood mononuclear cells (PBMCs) of untreated CLL patients and, as controls, age-matched healthy donors (HD) were studied. T cells at baseline and after TCR stimulation were analyzed molecularly using transcriptomics and lipidomics, and extensively characterized at protein level by flow cytometry and confocal imaging. Lipid metabolism was manipulated through culturing cells in lipoprotein deficient serum (LPDS), or in the presence of agonists and inhibitors of specific regulatory pathways. Results We found the uptake of lipids, specifically cholesterol, during T-cell activation essential for proliferation, which was evidenced by a nearly absent proliferative capacity of HD T cells in the lack of exogenous lipids which was fully rescued by re-addition of cholesterol-rich low-density lipoproteins (LDL). As CLL T cells portray an altered proliferation pattern even when cultured in normal medium, transcriptomic analysis was performed to investigate the expression of key lipid metabolism genes. CLL T-cells had reduced expression of the master transcription factors SREBP1/2, governing lipid and cholesterol metabolism, when stimulated in the presence of their autologous CLL cells (Figure 1A). Accordingly, downstream protein involved in the synthesis and desaturation of lipids and LDL uptake (specifically FASN, LDL-R and SCD) were significantly reduced in CLL compared to HD T cells. Remarkably, despite reduced capacity for LDL uptake, an accumulation of neutral lipids was found in CLL T cells especially upon stimulation (Figure 1B), a feature earlier described in dysfunctional macrophages. The lipid accumulation observed in CLL T cells was supported by the lack of expression of ATGL, a crucial enzyme for the utilization of neutral lipids for energy production and building of new membranes. In line with this, mitochondrial lipid oxidation was decreased in stimulated CLL T cells compared to HD. To investigate the composition of the accumulated lipids, lipidomics analysis was performed, revealing differential abundance of specific lipid classes in stimulated CLL versus HD T cells. Triglycerides were increased while cholesterol esters and phospholipids were reduced in CLL T cells. Triglycerides likely constitute the neutral lipids accumulation found in these cells. As cholesterol esters and phospholipids are essential for proper membrane formation, reduced levels of these species can contribute to the reduced proliferative capacity of CLL T cells. Summary/conclusion Altogether, the results presented here demonstrate a disturbed lipid composition and utilization in CLL T-cells, which contributes to T-cell dysfunction on at least two levels: decreased bioenergetics and decreased proliferation. Likewise, lipid deprivation due to both decreased uptake and a misbalanced lipidome leads to altered T-cell function. Therefore, strategies to increase lipid uptake, synthesis and utilization in order to rebalance homeostasis could ameliorate CLL T-cell dysfunction and specifically proliferation-defects in CLL.

MALAT1 promotes platelet activity and thrombus formation through PI3k/Akt/GSK-3β signalling pathway

BackgroundIschaemic stroke and other cardiovascular illnesses are characterised by abnormalities in the processes of thrombosis and haemostasis, which rely on platelet activity. In platelets, a wide variety of microRNAs (long...

Profile of CD8+ T-cells in a Stressed beta2- Adrenergic Receptor Knockout Mouse Model during Chlamydia muridarum Genital Infection.

&#x0D; Chlamydia trachomatis is the most common bacterial sexually transmitted disease. A negative correlation between the immune system and stress has been reported. We have demonstrated that cold-induced stress (CIS) increases the intensity of Chlamydia muridarum genital infection in a mouse model, but the status of CD8+ T cells is still unknown. This study profiles CD8+ T cell’s interaction with stress hormone; norepinephrine (NE) and the beta2-adrenergic receptor (β2-AR) in the presence of agonists and antagonists; and examines cytokines produced in the β2-AR knockout (KO) stress mouse model during C. muridarum genital infection. We hypothesize that CD8+ T cells produce high-level IFN-g and low-level TNF-a in stressed β2-AR KO mice than C57BL/6J wildtype (WT). Two groups of mice were cold-water stressed for 21 days, infected with C. muridarum, and then sacrificed for CD8+ T cell isolation. Viability was tested. The proliferation of CD8+ T cells was carried out for 72 h and culture supernatant was collected for cytokine production using ELISA. To confirm genital tract infection, Chlamydia muridarum isolation is underway. Our data show that the viability of CD8+ T cells treated with NE was significantly reduced compared to cells treated with LPS or Con A, indicating the suppressive effect of NE on immune cells. Stressed β2-AR KO mice displayed increased production of protective cytokines compared to stressed WT mice, indicating the absence of β2-AR leads to the functional restoration of protective immune cells.&#x0D;

α-Ketoglutarate Inhibits Thrombosis and Inflammation by Prolyl Hydroxylase-2 Mediated Inactivation of Phospho-Akt.

BackgroundPhospho-Akt1 (pAkt1) undergoes prolyl hydroxylation at Pro125 and Pro313 by the prolyl hydroxylase-2 (PHD2) in a reaction decarboxylating α-ketoglutarate (αKG). We investigated whether the αKG supplementation could inhibit Akt-mediated activation of platelets and monocytes, in vitro as well as in vivo, by augmenting PHD2 activity.MethodsWe treated platelets or monocytes isolated from healthy individuals with αKG in presence of agonists in vitro and assessed the signalling molecules including pAkt1. We supplemented mice with dietary αKG and estimated the functional responses of platelets and monocytes ex vivo. Further, we investigated the impact of dietary αKG on inflammation and thrombosis in lungs of mice either treated with thrombosis-inducing agent carrageenan or infected with SARS-CoV-2.FindingsOctyl αKG supplementation to platelets promoted PHD2 activity through elevated intracellular αKG to succinate ratio, and reduced aggregation in vitro by suppressing pAkt1(Thr308). Augmented PHD2 activity was confirmed by increased hydroxylated-proline and enhanced binding of PHD2 to pAkt in αKG-treated platelets. Contrastingly, inhibitors of PHD2 significantly increased pAkt1 in platelets. Octyl-αKG followed similar mechanism in monocytes to inhibit cytokine secretion in vitro. Our data also describe a suppressed pAkt1 and reduced activation of platelets and leukocytes ex vivo from mice supplemented with dietary αKG, unaccompanied by alteration in their number. Dietary αKG significantly reduced clot formation and leukocyte accumulation in various organs including lungs of mice treated with thrombosis-inducing agent carrageenan. Importantly, in SARS-CoV-2 infected hamsters, we observed a significant rescue effect of dietary αKG on inflamed lungs with significantly reduced leukocyte accumulation, clot formation and viral load alongside down-modulation of pAkt in the lung of the infected animals.InterpretationOur study suggests that dietary αKG supplementation prevents Akt-driven maladies such as thrombosis and inflammation and rescues pathology of COVID19-infected lungs.FundingStudy was funded by the Department of Biotechnology (DBT), Govt. of India (grants: BT/PR22881 and BT/PR22985); and the Science and Engineering Research Board, Govt. of India (CRG/000092).

Structural Arrangement Produced by Concanavalin A Binding to Homomeric GluK2 Receptors.

Kainate receptors are members of the ionotropic glutamate receptor family. They form cation-specific transmembrane channels upon binding glutamate that desensitize in the continued presence of agonists. Concanavalin A (Con-A), a lectin, stabilizes the active open-channel state of the kainate receptor and reduces the extent of desensitization. In this study, we used single-molecule fluorescence resonance energy transfer (smFRET) to investigate the conformational changes underlying kainate receptor modulation by Con-A. These studies showed that Con-A binding to GluK2 homomeric kainate receptors resulted in closer proximity of the subunits at the dimer–dimer interface at the amino-terminal domain as well as between the subunits at the dimer interface at the agonist-binding domain. Additionally, the modulation of receptor functions by monovalent ions, which bind to the dimer interface at the agonist-binding domain, was not observed in the presence of Con-A. Based on these results, we conclude that Con-A modulation of kainate receptor function is mediated by a shift in the conformation of the kainate receptor toward a tightly packed extracellular domain.

Computational Modeling to Explain Why 5,5-Diarylpentadienamides are TRPV1 Antagonists.

Several years ago, the crystallographic structures of the transient receptor potential vanilloid 1 (TRPV1) in the presence of agonists and antagonists were reported, providing structural information about its chemical activation and inactivation. TRPV1’s activation increases the transport of calcium and sodium ions, leading to the excitation of sensory neurons and the perception of pain. On the other hand, its antagonistic inactivation has been explored to design analgesic drugs. The interactions between the antagonists 5,5-diarylpentadienamides (DPDAs) and TRPV1 were studied here to explain why they inactivate TRPV1. The present work identified the structural features of TRPV1–DPDA complexes, starting with a consideration of the orientations of the ligands inside the TRPV1 binding site by using molecular docking. After this, a chemometrics analysis was performed (i) to compare the orientations of the antagonists (by using LigRMSD), (ii) to describe the recurrent interactions between the protein residues and ligand groups in the complexes (by using interaction fingerprints), and (iii) to describe the relationship between topological features of the ligands and their differential antagonistic activities (by using a quantitative structure–activity relationship (QSAR) with 2D autocorrelation descriptors). The interactions between the DPDA groups and the residues Y511, S512, T550, R557, and E570 (with a recognized role in the binding of classic ligands), and the occupancy of isoquinoline or 3-hydroxy-3,4-dihydroquinolin-2(1H)-one groups of the DPDAs in the vanilloid pocket of TRPV1 were clearly described. Based on the results, the structural features that explain why DPDAs inactivate TRPV1 were clearly exposed. These features can be considered for the design of novel TRPV1 antagonists.

Discovery of Molecular Interactions of the Human Melanocortin-4 Receptor (hMC4R) Asp189 (D189) Amino Acid with the Endogenous G-Protein-Coupled Receptor (GPCR) Antagonist Agouti-Related Protein (AGRP) Provides Insights to AGRP’s Inverse Agonist Pharmacology at the hMC4R

The melanocortin receptors (MCRs) are important for numerous biological pathways, including feeding behavior and energy homeostasis. In addition to endogenous peptide agonists, this receptor family has two naturally occurring endogenous antagonists, agouti and agouti-related protein (AGRP). At the melanocortin-4 receptor (MC4R), the AGRP ligand functions as an endogenous inverse agonist in the absence of agonist and as a competitive antagonist in the presence of agonist. At the melanocortin-3 receptor (MC3R), AGRP functions solely as a competitive antagonist in the presence of agonist. The molecular interactions that differentiate AGRP's inverse agonist activity at the MC4R have remained elusive until the findings reported herein. Upon the basis of homology molecular modeling approaches, we previously postulated a unique interaction between the D189 position of the hMC4R and Asn114 of AGRP. To further test this hypothesis, six D189 mutant hMC4Rs (D189A, D189E, D189N, D189Q, D189S, and D189K) were generated and pharmacologically characterized resulting in the discovery of differences in inverse agonist activity of AGRP and an 11 macrocyclic compound library. These data support the hypothesized interaction between the hMC4R D189 position and Asn114 residue of AGRP and define critical ligand-receptor molecular interactions responsible for the inverse agonist activity of AGRP at the hMC4R.

Biochemical Characterization of GPCR-G Protein Complex Formation.

The complex of G protein-coupled receptors (GPCR) and G proteins is the core assembly in GPCR signaling in eukaryotes. With the recent development of cryo-electron microscopy, there has been a rapid growth in structures of GPCR-G protein complexes solved to near-atomic resolution, giving important insights into this signaling complex. Here we describe the biochemical protocol to study the interaction between GPCRs and G proteins before preparation of GPCR-G protein complexes for structural studies. We use gel filtration to analyze the binding properties between GPCR and G protein with the presence of agonist or antagonist, as well as the complex dissociation in the presence of GTP analogue. Methods used in the protocol are affinity purification and gel filtration, which are also commonly used in protein sample preparation for structural work. Therefore, the protocol can be easily adapted for large-scale sample preparation.

Regulatory effect of PGE2 on microbicidal activity and inflammatory cytokines in canine leishmaniasis.

Canine leishmaniasis (CanL) is caused by the intracellular parasite Leishmania infantum. Prostaglandin E2 (PGE2 ) exerts potent regulatory effects on the immune system in experimental model Leishmania infection, but this influence has not yet been studied in CanL. In this study, PGE2 and PGE2 receptor levels and the regulatory effect of PGE2 on arginase activity, NO2 , IL-10, IL-17, IFN-γ, TNF-α and parasite load were evaluated in cultures of splenic leucocytes obtained from dogs with CanL in the presence of agonists and inhibitors. Our results showed that splenic leucocytes from dogs with CanL had lower EP2 receptor levels than those of splenic leucocytes from healthy animals. We observed that NO2 levels decreased when the cells were treated with a PGE2 receptor agonist (EP1/EP2/EP3) or COX-2 inhibitor (NS-398) and that TNF-α, IL-17 and IFN-γ cytokine levels decreased when the cells were treated with a PGE2 receptor agonist (EP2) or PGE2 itself. The parasite load in splenic leucocyte cell cultures from dogs with CanL decreased after stimulation of the cells with PGE2 . We conclude that Leishmania infection of dogs modulates PGE2 receptors and speculate that the binding of PGE2 to its receptors may activate the microbicidal capacity of cells.

Generating Phosphorylation Site Antibodies to Study α2A‐Adrenergic Receptor Phosphorylation by G Protein‐coupled Receptor Kinase 2 in Intact Cells

G protein‐coupled receptors (GPCRs) are transmembrane proteins which are activated by the binding of agonist to create a cell signal response. GPCRs are regulated through phosphorylation by G protein‐coupled receptor kinases. We focus on G protein‐coupled receptor kinase 2 (GRK2) and have previously characterized phosphorylation of the β2‐adrenergic receptor (β2‐AR) by this kinase. Currently, we are trying to determine if the same mutations shown to effect β2‐AR phosphorylation are also important for phosphorylation of the α2A‐adrenergic receptor (α2A‐AR). The α2A‐AR plays a role in regulating insulin, neurotransmitter secretion, and is a pharmaceutical target for pain relief. It differs from β2‐AR in that α2A‐AR has a larger third intracellular loop, containing four consecutive serine residues. These residues can be phosphorylated by GRK2 in the presence of agonist, causing a desensitization response. One way to detect receptor phosphorylation is to use antibodies that specifically recognize phosphorylated receptor. Since there is no commercially available antibody that can detect phosphorylation of α2A‐AR, we developed an antibody, RB617, which detects the phosphorylation of two serine residues. Using this antibody, we have developed methodologies to study GRK2 phosphorylation of α2A‐AR in intact COS‐7 cells. We tested the dose‐response of α2A‐AR to epinephrine and determined the EC50 for GRK2‐dependent phosphorylation to be 35 nM. In the future, we plan to assess the effect of GRK2 mutations previously shown to affect β2‐AR phosphorylation to better understand the role of GRK2 in α2A‐AR desensitization.Support or Funding InformationThis work was funded by NSF MCB0744739 and Siena College Center for Undergraduate Research and Creative Activity (CURCA).

Structural effects of divalent calcium cations on the α7 nicotinic acetylcholine receptor: A molecular dynamics simulation study.

The α7 subtype of neuronal nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel protein that is vital to various neurological functions, including modulation of neurotransmitter release. A relatively high concentration of extracellular Ca2+ in the neuronal environment is likely to exert substantial structural and functional influence on nAChRs, which may affect their interactions with agonists and antagonists. In this work, we employed atomistic molecular dynamics (MD) simulations to examine the effects of elevated Ca2+ on the structure and dynamics of α7 nAChR embedded in a model phospholipid bilayer. Our results suggest that the presence of Ca2+ in the α7 nAChR environment results in closure of loop C-in the extracellular ligand-binding domain, a motion normally associated with agonist binding and receptor activation. Elevated Ca2+ also alters the conformation of key regions of the receptor, including the inter-helical loops, pore-lining helices and the "gate" residues, and causes partial channel opening in the absence of an agonist, leading to an attendant reduction in the free energy of Ca2+ permeation through the pore as elucidated by umbrella sampling simulations. Overall, the structural and permeability changes in α7 nAChR suggest that elevated Ca2+ induces a partially activated receptor state that is distinct from both the resting and the agonist-activated states. These results are consistent with the notion that divalent ions can serve as a potentiator of nAChRs, resulting in a higher rate of receptor activation (and subsequent desensitization) in the presence of agonists, with possible implications for diseases involving calcium dysregulation.

Adenosine: Synthetic Methods of Its Derivatives and Antitumor Activity.

Since 1929, several researchers have conducted studies in relation to the nucleoside of adenosine (1) mainly distribution identifying, characterizing their biological importance and synthetic chemistry to which this type of molecule has been subjected to obtain multiple of its derivatives. The receptors that interact with adenosine and its derivatives, called purinergic receptors, are classified as A1, A2A, A2B and A3. In the presence of agonists and antagonists, these receptors are involved in various physiological processes and diseases.This review describes and compares some of the synthetic methods that have been developed over the last 30 years for obtaining some adenosine derivatives, classified according to substitution processes, complexation, mating and conjugation. Finally, we mention that although the concentrations of these nucleosides are low in normal tissues, they can increase rapidly in pathophysiological conditions such as hypoxia, ischemia, inflammation, trauma and cancer. In particular, the evaluation of adenosine derivatives as adjunctive therapy promises to have a significant impact on the treatment of certain cancers, although the transfer of these results to clinical practice requires a deeper understanding of how adenosine regulates the process of tumorigenesis.

Abstract 4885: Diverse microenvironmental agonists induce de novo resistance to Bcl-2 and BTK targeted combination therapy and cancer cell proliferation in B cell malignancies

Abstract The Bruton's Tyrosine Kinase (BTK) inhibitor ibrutinib (IBR) is FDA approved for Chronic Lymphocytic Leukemia (CLL) and Mantle Cell Lymphoma (MCL), but de novo resistance and rapid recurrence are common. We showed that venetoclax (VEN), an inhibitor of Bcl-2, induced synergistic cytotoxicity with IBR in cell lines and patient samples ex vivo (Axelrod et al. Leukemia, 2014; Jayappa et al. Blood Adv., 2017) and have initiated a clinical trial to test this combination. However, we noted variable sensitivity to IBR+VEN among CLL/MCL patient samples ex vivo, indicating a high frequency of de novo resistance. Resistance was evident in CLL cells showing an “activation” phenotype (CD5+/CD19+/CD69+) that occurs from interactions with the microenvironment in vivo, suggesting these interactions could induce resistance. This hypothesis was supported when we found that the combination of CpG-ODN, soluble CD40L (sCD40L), and IL10 (“agonist mix”) generated resistance to IBR+VEN in most CLL/MCL samples. In this study, we explored the role of innate immune receptors and other microenvironmental agonists in drug resistance and cancer cell proliferation, singly and in combination. We analyzed induction of apoptosis by IBR+VEN in CLL/MCL patient PBMCs cultured with agonists of various TLRs, NOD1/2, CD40, and IL10R by FACS. Significant resistance to apoptosis by IBR+VEN was noted in most patient samples treated with TLR9 agonist CpG-ODN (10/12) or sCD40L (9/12). IL10 induced modest resistance in a few samples (4/12). TLR1/2, TLR7, and NOD1/2 agonists generated moderate resistance only in MCL samples (N=4). Our preliminary FACS data suggest that CLL cells expressing ZAP-70, a marker of poor disease prognosis, responded with enhanced proliferation to CpG-ODN, sCD40L, and agonist mix as compared to ZAP-70- cells (3/3). In addition, prior exposure to CpG-ODN enhanced the proliferation and drug resistance response to sCD40L and vice versa (4/4), predicting mutually reinforcing microenvironmental interactions in vivo. CpG-ODN or agonist mix induced NF-kB dependent over expression of Mcl-1 and Bcl-xL in CLL/MCL cells, and increased ratio of these proteins to the cognate pro-apoptotic proteins (Puma, BIM, and BAD), indicating enhanced dependence of leukemic B cells for Mcl-1 and Bcl-xL. This presumably caused resistance by lowering cancer cell dependence for VEN target Bcl-2 in presence of agonists. In conclusion, several microenvironmental agonists, particularly the agonist of TLR9 and CD40, induce resistance to IBR+VEN and cell proliferation in CLL/MCL. Response to these agonists is enhanced in ZAP-70+ cells and when agonist exposure occurs combinatorially. Drug resistance by these diverse agonists was generated by a convergent mechanism of NF-kB dependent over-expression of Mcl-1 and Bcl-xL, which provide additional targets to overcome drug resistance. Citation Format: Kallesh D. Jayappa, Craig A. Portell, Vicki L. Gordon, Timothy P. Bender, Michael E. Williams, Michael J. Weber. Diverse microenvironmental agonists induce de novo resistance to Bcl-2 and BTK targeted combination therapy and cancer cell proliferation in B cell malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4885.

Platelet activation and aggregation after aneurysmal subarachnoid hemorrhage

BackgroundEndovascular techniques have proven beneficial in the treatment of aneurysmal subarachnoid hemorrhage (aSAH), but with high risk of arterial clotting, emboli and dissection. Platelet activation and alterations in hemostasis may contribute to these complications. We investigated platelet activation and aggregation pathways in aSAH patients who underwent endovascular treatment.MethodsTwo blood samples were taken, in the early days after bleeding and during the period at risk of vasospasm. We studied platelet activation through the expression of GpIIbIIIa and P-selectin as well as aggregation rate in the presence of agonists. Platelets from aSAH patients were compared with those from orthopedic postoperative patients (POSTOP).ResultsPlatelets in aSAH were initially spontaneously activated and remained so over time. aSAH platelets were further activated with rapid aggregation in the presence of agonists, particularly ADP, with behavior comparable to POSTOP platelets.ConclusionsaSAH platelets showed prolonged increases in activation and aggregation. Therapies targeting the ADP pathway might reduce the risk of clotting and ischemic events in this context among patients requiring multiple endovascular procedures.Trial registrationNot applicable.

Human a1A‐adrenergic receptor phosphorylation, internalization and desensitization.

The a1A‐adrenergic receptor (a1A‐AR) is a G protein coupled‐receptor (GPCR) that signals through the Gq/ phospholipase C/ calcium release pathway. This adrenergic receptor subtype modulates important cellular responses and it is also involved in the pathogenesis of diseases. Phosphorylation of intracellular loops and the carboxyl terminal tail of a1A‐AR is part of their fine regulation, generally associated with desensitization/internalization processes, in which different protein kinases participate, Including second messenger‐activated protein kinases, such as protein kinase C isoforms (PKCs) and G protein‐coupled receptor kinases (GRKs).In silico analysis of the human a1A‐AR amino acid sequenced, suggested a series of possible phosphorylation sites targeted by PKCs, GRKs and also for the glycogen synthase protein kinase‐3 (GSK3). GSK3 is an important element into signal transduction of diverse receptor systems, and the phosphorylation on specific substrates can modulates enzyme activity, subcellular localization, stability of the target protein and protein‐protein interactions. We observed using a1A‐AR immunopurification and mass spectrometry analysis that in cells treated with noradrenaline, oxymetazoline or phorbol esters four serines that are target of GSK3: S229, S258, S352 and S381, are phosphorylated. However, the specific GSK3 inhibitor, SB216763, did not block the a1A‐AR desensitization induced by noradrenaline or oxymetazoline. This suggested that other protein kinases play major roles in such agonist‐induced desensitization. Interestingly, phorbol ester‐induced a1A‐AR desensitization was increased by the GSK3 inhibitor. In cellulo colocalization assays of a1A‐AR‐GSK3, showed increased interaction of these proteins in the presence of agonists and phorbol esters. Interestingly, expression of a GSK3 dominant‐negative mutant interferes with a1A‐AR internalization in response to NA and OXY, but this effect does not occur when cells are pretreated with PMA. All these data suggest the possibility that GSK3 might play a role in the regulation of a1A‐AR function and localization.Partially supported by Grants from Dirección General de Asuntos del Personal Académico (IN200718) and Consejo Nacional de Ciencia y Tecnología (Fronteras 882 and 253156).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Investigating endogenous µ-opioid receptors in human keratinocytes as pharmacological targets using novel fluorescent ligand.

Opioids in skin function during stress response, regeneration, ageing and, particularly in regulating sensation. In chronic pruritus, topical treatment with Naltrexone changes μ-opioid receptor (μ-OR) localization to relieve itch. The molecular mechanisms behind the effects of Naltrexone on μ-OR function in reduction of itching behavior has not been studied. There is an immediate need to understand the endogenous complexity of μ-OR dynamics in normal and pathological skin conditions. Here we evaluate real-time behavior of μ-OR-Endomorphine complexes in the presence of agonist and antagonists. The μ-OR ligand Endomorphine-1 (EM) was conjugated to the fluorescent dye Tetramethylrhodamine (TAMRA) to investigate the effects of agonist and antagonists in N/TERT-1 keratinocytes. The cellular localization of the EM-TAMRA was followed through time resolved confocal microscopy and population analysis was performed by flow cytometry. The in vitro analyses demonstrate fast internalization and trafficking of the endogenous EM-TAMRA-μ-OR interactions in a qualitative manner. Competition with Endomorphine-1, Naltrexone and CTOP show both canonical and non-canonical effects in basal and differentiated keratinocytes. Acute and chronic treatment with Naltrexone and Endomorphine-1 increases EM-TAMRA binding to skin cells. Although Naltrexone is clinically effective in relieving itch, the mechanisms behind re-distribution of μ-ORs during clinical treatments are not known. Our study has given insight into cellular mechanisms of μ-OR ligand-receptor interactions after opioid agonist and antagonist treatments in vitro. These findings potentially offer opportunities in using novel treatment strategies for skin and peripheral sensory disorders.

Single-molecule imaging reveals dimerization/oligomerization of CXCR4 on plasma membrane closely related to its function

Dimerization and oligomerization of G-protein coupled receptors (GPCRs) have emerged as important characters during their trans-membrane signal transduction. However, until now the relationship between GPCR dimerization and their trans-membrane signal transduction function is still uncovered. Here, using pertussis toxin (PTX) to decouple the receptor from G protein complex and with single-molecule imaging, we show that in the presence of agonist, cells treated with PTX showed a decrease in the number of dimers and oligomers on the cell surface compared with untreated ones, which suggests that oligomeric status of CXCR4 could be significantly influenced by the decoupling of G protein complex during its signal transduction process. Moreover, with chlorpromazine (CPZ) to inhibit internalization of CXCR4, it was found that after SDF-1α stimulation, cells treated with CPZ showed more dimers and oligomers on the cell surface than untreated ones, which suggest that dimers and oligomers of CXCR4 tend to internalize more easily than monomers. Taken together, our results demonstrate that dimerization and oligomerization of CXCR4 is closely related with its G protein mediated pathway and β-arrestin mediated internalization process, and would play an important role in regulating its signal transduction functions.