Membrane-bound Receptors Research Articles

Estradiol decreases the excitability of RA projection neurons in adult male zebra finches.

Zebra finches are essential animal models for studying learned vocal signals. The robust nucleus of the arcopallium (RA) plays an important role in regulating singing behavior. Our previous study showed that castration inhibited the electrophysiological activity of RA projection neurons (PNs) in male zebra finches, demonstrating that testosterone modulates the excitability of RA PNs. Testosterone can be converted into estradiol (E2) in the brain through aromatase; however, the physiological functions of E2 in RA are still unknown. This study aimed to investigate the electrophysiological activities of E2 on the RA PNs of male zebra finches through patch-clamp recording. E2 rapidly decreased the rate of evoked and spontaneous action potentials (APs) of RA PNs, hyperpolarized the resting membrane potential, and decreased the membrane input resistance. Moreover, the G-protein-coupled membrane-bound estrogen receptor (GPER) agonist G1 decreased both the evoked and spontaneous APs of RA PNs. Furthermore, the GPER antagonist G15 had no effect on the evoked and spontaneous APs of RA PNs; E2 and G15 together also had no effect on the evoked and spontaneous APs of RA PNs. These findings suggested that E2 rapidly decreased the excitability of RA PNs and its binding to GPER suppressed the excitability of RA PNs. These pieces of evidence helped us fully understand the principle of E2 signal mediation via its receptors to modulate the excitability of RA PNs in songbirds.

The grapevine LysM receptor-like kinase VvLYK5-1 recognizes chitin oligomers through its association with VvLYK1-1.

The establishment of defense reactions to protect plants against pathogens requires the recognition of invasion patterns (IPs), mainly detected by plasma membrane-bound pattern recognition receptors (PRRs). Some IPs, also termed elicitors, are used in several biocontrol products that are gradually being developed to reduce the use of chemicals in agriculture. Chitin, the major component of fungal cell walls, as well as its deacetylated derivative, chitosan, are two elicitors known to activate plant defense responses. However, recognition of chitooligosaccharides (COS) in Vitis vinifera is still poorly understood, hampering the improvement and generalization of protection tools for this important crop. In contrast, COS perception in the model plant Arabidopsis thaliana is well described and mainly relies on a tripartite complex formed by the cell surface lysin motif receptor-like kinases (LysM-RLKs) AtLYK1/CERK1, AtLYK4 and AtLYK5, the latter having the strongest affinity for COS. In grapevine, COS perception has for the moment only been demonstrated to rely on two PRRs VvLYK1-1 and VvLYK1-2. Here, we investigated additional players by overexpressing in Arabidopsis the two putative AtLYK5 orthologs from grapevine, VvLYK5-1 and VvLYK5-2. Expression of VvLYK5-1 in the atlyk4/5 double mutant background restored COS sensitivity, such as chitin-induced MAPK activation, defense gene expression, callose deposition and conferred non-host resistance to grapevine downy mildew (Erysiphe necator). Protein-protein interaction studies conducted in planta revealed a chitin oligomer-triggered interaction between VvLYK5-1 and VvLYK1-1. Interestingly, our results also indicate that VvLYK5-1 mediates the perception of chitin but not chitosan oligomers showing a part of its specificity.

Barbamide Displays Affinity for Membrane-Bound Receptors and Impacts Store-Operated Calcium Entry in Mouse Sensory Neurons.

Marine cyanobacteria are a rich source of bio-active metabolites that have been utilized as leads for drug discovery and pharmacological tools for basic science research. Here, we describe the re-isolation of a well-known metabolite, barbamide, from Curaçao on three different occasions and the characterization of barbamide's biological interactions with targets of the mammalian nervous system. Barbamide was originally discovered as a molluscicidal agent from a filamentous marine cyanobacterium. In our hands, we found little evidence of toxicity against mammalian cell cultures. However, barbamide showed several affinities when screened for binding affinity for a panel of 45 receptors and transporters known to be involved in nociception and sensory neuron activity. We found high levels of binding affinity for the dopamine transporter, the kappa opioid receptor, and the sigma receptors (sigma-1 and sigma-2 also known as transmembrane protein 97; TMEM97). We tested barbamide in vitro in isolated sensory neurons from female mice to explore its functional impact on calcium flux in these cells. Barbamide by itself had no observable impact on calcium flux. However, barbamide enhanced the effect of the TRPV1 agonist capsaicin and enhanced store-operated calcium entry (SOCE) responses after depletion of intracellular calcium. Overall, these results demonstrate the biological potential of barbamide at sensory neurons with implications for future drug development projects surrounding this molecule.

Use of complementary biophysical methods to characterize ligand binding to IL-17A.

Interleukin-17A (IL-17A) is a pro-inflammatory cytokine produced by subset 17 of T helper cells and is emerging as one the key mediators for chronic inflammation and autoimmune disease. IL-17A signaling occurs through its membrane-bound receptors, IL-17RA and IL-17RC. Antibodies that neutralize IL-17A or its receptor (IL-17RA) deliver efficacy but, no small molecule IL-17A antagonists have yet progressed. Small molecule inhibition of protein-protein interactions is difficult due to lack of well-defined pockets to target and inherent conformational flexibility of IL-17A makes designing inhibitors even more challenging. To characterize binding of small molecule ligands to IL-17A, we used a variety of biophysical methods including SPR, NMR and a relatively new method called Second Harmonic Generation (SHG). Conformational flexibility of IL-17A was a great hurdle in obtaining high quality X-ray structures. In absence of X-ray crystal structures, the team was dependent on biophysical data to make design decisions. Along with studying direct binding of compounds to IL-17A, we devised competition experiments to determine binding site specificity for ligands that bind to IL-17A. We eventually were successful in obtaining X-ray crystal structures to study the binding interactions in detail. Data obtained from these complementary methods matched very well boosting confidence in the chemical matter. The affinity of ligands to IL-17A correlated with inhibition of IL-17A:receptor interaction in the cell-based functional assays.

Molecular characterization and expression of CD96 in red seabream (Pagrus major)

CD96 is a membrane-bound receptor discovered in humans in 1992 that is mainly present in natural killer cells and T cells derived from haematopoietic cells and performs immune functions. Based on the sequence of CD96 obtained from red seabream (Pagrus major), phylogenetic analysis with other species, infections of normal fish, Streptococcus iniae and red sea bream iridovirus (RSIV), and expression analysis was conducted using real-time polymerase chain reaction. Phylogenetic analysis showed the highest homology with Sparus aurata, and multiple sequence analysis confirmed the conservation of major domains between different fish species. Normal fish high expression results were confirmed in the head kidney, and spleen, which are the haematopoietic organs of the fish. High expression levels were confirmed in the gills, liver, spleen, and kidney on day three after RSIV infection. After S. iniae infection, high expression was confirmed in the gills and liver on day one, and high expression was confirmed in the spleen from 12 hours. These results show that PmCD96 functions as an immune gene in P. major and is considered a basic research case for CD96 in fish’s hematopoietic organ immune system.

Maternal and Fetal Outcome in Pregnant Women With Critical COVID-19 Treated With Tocilizumab in a Tertiary Care Hospital in Dubai.

Pregnancy, complicated by coronavirus disease 2019 (COVID-19), results in higher hospitalization and mortality rate. Pathogenesis of COVID-19 is similar to any other systemic inflammatory condition but results in a cytokine storm ofhigher magnitude causing severe acute respiratory distress syndrome and multiorgan failure. Tocilizumab, a humanized monoclonal antibody, targets soluble and membrane-bound IL-6 receptors and is used in the treatment ofjuvenile idiopathic arthritis, rheumatoid arthritis, and cytokine release syndrome. However, studies exploring itsrole in pregnancy are minimal. Hence, this study was done to study the effect of tocilizumab on maternal and fetal outcomes incritical COVID-19 pregnant women. A retrospective study was conducted on 28 pregnant women with critical COVID-19 who received tocilizumab. Clinical status, chest x-ray, biochemical parameters, and fetal well-being were monitored and documented. The discharged patients were followed up through telemedicine. On treatment with tocilizumab, improvement was seen in thenumber of zones and patterns of chest x-ray, along with 80% reduction in the c-reactive protein (CRP) levels. Based on the WHO clinical progression scale, 20 patients improved by theend of first week, and by the end of first month, 26 patients became asymptomatic. Two patients died during thecourse of the disease.No fetal adverse effects were noted. Based on the encouragingresponse and as tocilizumab did not impartany adverse effects on the pregnancy, tocilizumab may be administered as an adjuvant to criticalCOVID-19 pregnant women in their second and third trimesters.

Enzyme-Instructed Peptide Assembly Favored by Preorganization for Cancer Cell Membrane Engineering.

Innovative methods for engineering cancer cell membranes promise to manipulate cell-cell interactions and boost cell-based cancer therapeutics. Here, we illustrate an in situ approach to selectively modify cancer cell membranes by employing an enzyme-instructed peptide self-assembly (EISA) strategy. Using three phosphopeptides (pY1, pY2, and pY3) targeting the membrane-bound epidermal growth factor receptor (EGFR) and differing in just one phosphorylated tyrosine, we reveal that site-specific phosphorylation patterns in pY1, pY2, and pY3 can distinctly command their preorganization levels, self-assembling kinetics, and spatial distributions of the resultant peptide assemblies in cellulo. Overall, pY1 is the most capable of producing preorganized assemblies and shows the fastest dephosphorylation reaction in the presence of alkaline phosphatase (ALP), as well as the highest binding affinity for EGFR after dephosphorylation. Consequently, pY1 exhibits the greatest capacity to construct stable peptide assemblies on cancer cell membranes with the assistance of both ALP and EGFR. We further use peptide-protein and peptide-peptide co-assembly strategies to apply two types of antigens, namely ovalbumin (OVA) protein and dinitrophenyl (DNP) hapten respectively, on cancer cell membranes. This study demonstrates a very useful technique for the in situ construction of membrane-bound peptide assemblies around cancer cells and implies a versatile strategy to artificially enrich cancer cell membrane components for potential cancer immunotherapy.

GPR4 in the pH-dependent migration of melanoma cells in the tumor microenvironment.

Due to its high metastatic potential, malignant melanoma is one of the deadliest skin cancers. In melanoma as well as in other cancers, acidification of the tumor microenvironment (=TME, inverse pH-gradient) is a well-known driver of tumor progression and metastasis. Membrane-bound receptors, such as the proton-sensitive GPCR (pH-GPCR) GPR4, are considered as potential initiators of the signalling cascades relevant to malignant transformation. In this study, we investigated the pH-dependent migration of GPR4 wildtype/overexpressing SK-Mel-28 cells using an impedance-based electrical wounding and migration assay and classical Boyden chamber experiments. Migration of GPR4 overexpressing SK-Mel-28 cells was enhanced in a range of pH6.5-7.5 as compared to controls in the impedance-based electrical wounding and migration assay. In Boyden chamber experiments, GPR4 overexpression only increased migration at pH7.5 in a Matrigel-free setup, but not at pH6.5. Results indicate that GPR4 is involved in the migration of melanoma cells, especially in the tumor periphery, and that this process is affected by pH in the TME.

Isomeric Activity Cliffs-A Case Study for Fluorine Substitution of Aminergic G Protein-Coupled Receptor Ligands.

Currently, G protein-coupled receptors (GPCRs) constitute a significant group of membrane-bound receptors representing more than 30% of therapeutic targets. Fluorine is commonly used in designing highly active biological compounds, as evidenced by the steadily increasing number of drugs by the Food and Drug Administration (FDA). Herein, we identified and analyzed 898 target-based F-containing isomeric analog sets for SAR analysis in the ChEMBL database-FiSAR sets active against 33 different aminergic GPCRs comprising a total of 2163 fluorinated (1201 unique) compounds. We found 30 FiSAR sets contain activity cliffs (ACs), defined as pairs of structurally similar compounds showing significant differences in affinity (≥50-fold change), where the change of fluorine position may lead up to a 1300-fold change in potency. The analysis of matched molecular pair (MMP) networks indicated that the fluorination of aromatic rings showed no clear trend toward a positive or negative effect on affinity. Additionally, we propose an in silico workflow (including induced-fit docking, molecular dynamics, quantum polarized ligand docking, and binding free energy calculations based on the Generalized-Born Surface-Area (GBSA) model) to score the fluorine positions in the molecule.

Interference of neuronal TrkB signaling by the cannabis-derived flavonoids cannflavins A and B

Cannflavins A and B are flavonoids that accumulate in the Cannabis sativa plant. These specialized metabolites are uniquely prenylated and highly lipophilic, which may permit their interaction with membrane-bound enzymes and receptors. Although previous studies found that cannflavins can produce anti-inflammatory responses by inhibiting the biosynthesis of pro-inflammatory mediators, the full extent of their cellular influence remains to be understood. Here, we studied these flavonoids in relation to the Tropomyosin receptor kinase B (TrkB), a receptor tyrosine kinase that is activated by the growth factor brain-derived neurotrophic factor (BDNF). Using mouse primary cortical neurons, we first collected evidence that cannflavins prevent the accumulation of Activity-regulated cytoskeleton-associated (Arc) protein upon TrkB stimulation by exogenous BDNF in these cells. Consistent with this effect, we also observed a reduced activation of TrkB and downstream signaling effectors that mediate Arc mRNA transcription when BDNF was co-applied with the cannflavins. Of note, we also performed a high-throughput screen that demonstrated a lack of agonist action of cannflavins towards 320 different G protein-coupled receptors, a result that specifically limit the possibility of a TrkB transinactivation scenario via G protein signaling to explain our results with dissociated neurons. Finally, we used Neuro2a cells overexpressing TrkB to show that cannflavins can block the growth of neurites and increased survival rate produced by the higher abundance of the receptor in this model. Taken together, our study offers a new path to understand the reported effects of cannflavins and other closely related compounds in different cellular contexts.

Role of stress-related glucocorticoid changes in astrocyte-oligodendrocyte interactions that regulate myelin production and maintenance.

Repeated activation of stress responses and elevated corticosteroids result in alterations of neuronal physiology and metabolism, and lead to disturbances of normal connectivity between neurons in various brain regions. In addition, stress responses are also associated with anomalies in the function of glial cells, particularly astrocytes and oligodendrocytes, which in turn may further contribute to the mechanisms of neuronal dysfunction. The actions of corticosteroids on astrocytes are very likely mediated by the presence of intracellular and cell membrane-bound CORT receptors. Although apparently less abundant than in astrocytes, activation of CORT receptors in oligodendrocytes also leads to structural changes that are reflected in myelin maintenance and plasticity. The close interactions between astrocytes and oligodendrocytes through extracellular matrix molecules, soluble factors and astrocyte-oligodendrocyte gap junctions very likely mediate part of the disturbances in myelin structure, leading to plastic myelin adaptations or pathological myelin disruptions that may significantly influence brain connectivity. Likewise, the intimate association of the tips of some astrocytes processes with a majority of nodes of Ranvier in the white matter suggest that stress and overexposure to corticosteroids may lead to remodeling of node of Ranvier and their specific extracellular milieu.

Dissecting Interleukin-6 Classic and Trans-signaling in Inflammation and Cancer.

Interleukin-6 (IL-6) is a cytokine synthesized by many cells in the human body. IL-6 binds to a membrane-bound receptor (IL-6R), which is only present on hepatocytes, some epithelial cells, and some leukocytes. The complex of IL-6 and IL-6R binds to the ubiquitously expressed receptor subunit gp130, which forms a homodimer and thereby initiates intracellular signaling, e.g., the JAK/STAT and MAPK pathways. Proteases can cleave the membrane-bound IL-6R from the cell surface and generate a soluble IL-6R (sIL-6R), which retains its ability to bind IL-6. The IL-6/sIL-6R complex associates with gp130 and induces signaling even on cells which do not express the IL-6R. This paradigm has been called IL-6 trans-signaling, whereas signaling via the membrane-bound IL-6R is referred to as classic signaling. We have generated several molecular tools to differentiate between both pathways and to analyze the consequences of cellular IL-6 signaling in vivo. One of these tools is soluble gp130Fc, which selectively inhibits IL-6 trans-signaling. This protein under the WHO name Olamkicept has successfully undergone phase II clinical trials in patients with autoimmune diseases. Here, in this chapter, we describe several molecular tools to differentiate between IL-6 classic and trans-signaling and to analyze the consequences of cellular IL-6 signaling in vivo.

Advances in the molecular level understanding of G-protein coupled receptor.

G-protein coupled receptors (GPCRs) represent largest family of plasma membrane-bound receptor proteins that are involved in numerous cellular and physiological functions. Many extracellular stimuli such as hormones, lipids and chemokines activate these receptors. Aberrant expression and genetic alteration in GPCR are associated with many human diseases including cancer and cardiovascular disease. GPCRs have emerged as potential therapeutic target and numerous drugs are either approved by FDA or under clinical trial. This chapter provides an update on GPCR research and its significance as a promising therapeutic target.

Short-term effects of estradiol and bisphenol A on gene expression associated with early head mineralization in the seabass Dicentrarchus labrax

IntroductionNatural and synthetic estrogens are pollutants found in aquatic ecosystems at low concentrations reaching ng.L-1 to μg.L-1. At these concentrations, they are able to interfere with the fish endocrine system. When waterborne exposure occurs at early life stages, when blood estrogens concentrations are low, this may have significant consequences for estrogen-sensitive functions such as skeletal development.MethodsTo better understand how (xeno)estrogens may affect early head mineralization, 12 days post-hatch larvae of the European seabass Dicentrarchus labrax were experimentally exposed for 4 days to the natural estrogen estradiol E2 and to the xenoestrogen bisphenol A (BPA), both used at either regulatory concentration of water quality or a 100 times higher concentration. Head mineralization level was assessed using Alizarin red staining, together with the relative quantification of mRNA expression levels of several genes playing key roles in skeletogenesis and estrogen signaling pathways.ResultsWe showed that (xeno)estrogen exposure at early larval stage increases the expression of skeleton-associated genes: matrix proteins encoding genes (col1a2, col2a1a, col2a1b, bgp1a, bgp1b, sparc), proteolytic enzyme encoding genes (ctsk) and transcription and signaling factors (sox9a, sox9b, ihha, runx2, rankl). Although transcriptional overexpression of these genes was significant in larvae exposed to 40 ng.L-1 E2 and to 1.6 and 160 μg.L-1 BPA, increased mineralization was detected only in E2-exposed larvae, suggesting a difference in head skeleton development and remodeling in BPA-treated larvae.DiscussionOur results suggest that these phenotypic differences could be due to the implication of other estrogenic signaling pathways involving both nuclear and membrane-bound estrogen receptors (ERs and GPERs), but also estrogen-related receptors (ERRs). This study brings new insights into the regulatory mechanisms of skeletogenesis by E2 and BPA and into the effects of waterborne exposure to (xeno)estrogens on the early skeletal development of teleost fishes.

Overexpression of hepatocyte EphA2 enhances liver-stage infection by Plasmodium vivax

The liver is the first destination of malaria parasites in humans. After reaching the liver by the blood stream, Plasmodium sporozoites cross the liver sinusoid epithelium, enter and exit several hepatocytes, and eventually invade a final hepatocyte host cell. At present, the mechanism of hepatocyte invasion is only partially understood, presenting a key research gap with opportunities for the development of new therapeutics. Recently, human EphA2, a membrane-bound receptor tyrosine kinase, was implicated in hepatocyte infection by the human malaria parasite Plasmodium falciparum and the rodent parasite Plasmodium yoelii, but its role is not known for Plasmodium vivax, a major human parasite whose liver infection poses a specific challenge for malaria treatment and elimination. In this study, the role of EphA2 in P. vivax infection was investigated. It was found that surface expression of several recombinant fragments of EphA2 enhanced the parasite infection rate, thus establishing its role in P. vivax infection. Furthermore, a new permanent cell line (EphA2Extra-HC04) expressing the whole extracellular domain of EphA2 was generated. This cell line supports a higher rate of P. vivax infection and is a valuable tool for P. vivax liver-stage research.

CLASSIC IL-6 SIGNALING IS ASSOCIATED WITH POOR OUTCOMES IN BURN PATIENTS.

Background: Interleukin (IL)-6 is a multifunctional cytokine with both a proinflammatory and anti-inflammatory role. In many studies, IL-6 increases rapidly after burn injury and is associated with poor outcomes. However, there are two aspects to IL-6; it can signal via its soluble IL-6 receptor (sIL-6R), which is referred to as trans-signaling and is regarded as the proinflammatory pathway. The role of sIL-6R postburn injury has yet to be explored in its entirety. We hypothesized that patients with a lower ratio of IL-6 to sIL-6R would have worse outcomes. Methods: Patients admitted to our burn center within 7 days of injury were included in this study. Patients were divided into two groups based on IL-6 and sIL-6R levels measured within the first 7 days postburn injury. Patients were in the high ratio group if their IL-6/sIL-6R ratio was ≥0.185. Clinical outcomes included organ biomarkers, morbidities, and hospital length of stay. Groups were compared using Student's t test, Mann-Whitney U , and Fisher's exact test as appropriate; a P value of <0.05 was considered statistically significant. Results: We studied 86 patients with a median age of 50 years (36-66 years) and a median total body surface area burn of 18% (10-31). There were 40 patients categorized with a low IL-6/sIL-6R ratio and 46 patients with a high IL-6/sIL-6R ratio. Patients in the high IL-6/sIL-6R ratio group had a significantly greater total body surface area burn ( P < 0.001) and a significantly greater proportion of patients with inhalation injury ( P = 0.001). Levels of IL-6 were significantly higher in patients with a high IL-6/sIL-6R ratio ( P < 0.0001). However, levels of sIL-6R were not significantly different among the low and high groups ( P = 0.965). Mortality was significantly greater in the high IL-6/sIL-6R ratio group (3% vs. 26%; P = 0.002). Conclusions: Interestingly, patients with a higher ratio of IL-6/sIL-6R had significantly greater mortality. Using sIL-6R as a marker for the proinflammatory immune response, we expected patients with a lower IL-6/sIL-6R ratio to have poor outcomes, typically associated with a hyperinflammatory or exaggerated immune response. However, the absolute value of sIL-6R did not differ. This suggests that classical signaling of IL-6 via its membrane-bound receptor, with an anti-inflammatory function, is important.

Fluorescence microscopy imaging of a neurotransmitter receptor and its cell membrane lipid milieu.

Hampered by the diffraction phenomenon, as expressed in 1873 by Abbe, applications of optical microscopy to image biological structures were for a long time limited to resolutions above the ∼200nm barrier and restricted to the observation of stained specimens. The introduction of fluorescence was a game changer, and since its inception it became the gold standard technique in biological microscopy. The plasma membrane is a tenuous envelope of 4nm-10nm in thickness surrounding the cell. Because of its highly versatile spectroscopic properties and availability of suitable instrumentation, fluorescence techniques epitomize the current approach to study this delicate structure and its molecular constituents. The wide spectral range covered by fluorescence, intimately linked to the availability of appropriate intrinsic and extrinsic probes, provides the ability to dissect membrane constituents at the molecular scale in the spatial domain. In addition, the time resolution capabilities of fluorescence methods provide complementary high precision for studying the behavior of membrane molecules in the time domain. This review illustrates the value of various fluorescence techniques to extract information on the topography and motion of plasma membrane receptors. To this end I resort to a paradigmatic membrane-bound neurotransmitter receptor, the nicotinic acetylcholine receptor (nAChR). The structural and dynamic picture emerging from studies of this prototypic pentameric ligand-gated ion channel can be extrapolated not only to other members of this superfamily of ion channels but to other membrane-bound proteins. I also briefly discuss the various emerging techniques in the field of biomembrane labeling with new organic chemistry strategies oriented to applications in fluorescence nanoscopy, the form of fluorescence microscopy that is expanding the depth and scope of interrogation of membrane-associated phenomena.

Understanding the female athlete: molecular mechanisms underpinning menstrual phase differences in exercise metabolism.

Research should equitably reflect responses in men and women. Including women in research, however, necessitates an understanding of the ovarian hormones and menstrual phase variations in both cellular and systems physiology. This review outlines recent advances in the multiplicity of ovarian hormone molecular signaling that elucidates the mechanisms for menstrual phase variability in exercise metabolism. The prominent endogenous estrogen, 17-β-estradiol (E2), molecular structure is bioactive in stabilizing plasma membranes and quenching free radicals and both E2 and progesterone (P4) promote the expression of antioxidant enzymes attenuating exercise-induced muscle damage in the late follicular (LF) and mid-luteal (ML) phases. E2 and P4 bind nuclear hormone receptors and membrane-bound receptors to regulate gene expression directly or indirectly, which importantly includes cross-regulated expression of their own receptors. Activation of membrane-bound receptors also regulates kinases causing rapid cellular responses. Careful analysis of these signaling pathways explains menstrual phase-specific differences. Namely, E2-promoted plasma glucose uptake during exercise, via GLUT4 expression and kinases, is nullified by E2-dominant suppression of gluconeogenic gene expression in LF and ML phases, ameliorated by carbohydrate ingestion. E2 signaling maximizes fat oxidation capacity in LF and ML phases, pending low-moderate exercise intensities, restricted nutrient availability, and high E2:P4 ratios. P4 increases protein catabolism during the luteal phase by indeterminate mechanisms. Satellite cell function supported by E2-targeted gene expression is countered by P4, explaining greater muscle strengthening from follicular phase-based training. In totality, this integrative review provides causative effects, supported by meta-analyses for quantitative actuality, highlighting research opportunities and evidence-based relevance for female athletes.

A Novel Dual-Antigen Targeting Approach Enables Off-the-Shelf CAR NK Cells to Effectively Recognize and Eliminate the Heterogenous Population Associated with AML

While acute myeloid leukemia (AML) treatment has improved, relapse is still common, even after reduced intensity conditioning allogeneic transplantation. Natural killer (NK) cells are a promising allogeneic cell immunotherapy without the risk of graft-versus-host disease or cytokine release syndrome. We and others have reported that haploidentical NK cells can safely result in 30-50% complete remissions in advanced AML patients when given with high-dose lymphodepleting chemotherapy and exogenous cytokine support. However, while NK cell natural cytotoxicity has a clinical signal in AML, increased specificity and multi-dosing are likely to enhance the efficacy and durability of this treatment strategy. A number of stress ligands, including the MHC-I polypeptide-related sequence A and B (MICA/B), bind to the NK cell activating receptor NKG2D and initiate NK cell killing of transformed and infected cells, and several groups have shown promising results in clinical trials by developing NK cells that carry a NKG2D chimeric antigen receptor (CAR). However, the distal α1 and α2 domains of MICA/B that are recognized by NKG2D can be proteolytically cleaved from the surface of the tumor by metalloproteases, allowing for escape from NK cell-mediated detection and elimination. Wucherpfennig and colleagues have described that the membrane proximal α3 domain remains on the cell surface and may be a potential target for immunotherapy across many cancers, including AML. However, since AML is heterogenous, we hypothesized that clinical success would require dual-targeting and selected CD33 as the ideal complementary targeting approach since CD33 is a validated marker expressed on greater than 80% of AML blasts. To this end, our lab has developed a Tri-specific Killer Engager (TriKE) capable of agonistically ligating the CD16 Fc receptor to CD33 found on AML along with IL-15 co-stimulation to further activate the NK cell response. Here, we demonstrate the efficacy of a multiplexed-gene edited induced pluripotent stem-cell (iPSC) derived NK cell (iNK) product that expresses a high-affinity, non-cleavable version of CD16 (hnCD16), a membrane-bound IL-15 fusion receptor, CD38 knockout to enhance metabolic fitness, and a CAR against the α3 domain of MICA/B (α3 MICA/B) to drive a potent response against AML alone and when combined with anti-CD33 TriKE. In the initial study, the iNK cell backbone (iNK cells without the α3 MICA/B CAR) induced potent activity against the AML cell line HL60, and displayed further enhancement of activity with the addition of anti-CD33 TriKE (GTB-3650), representing combined effects of natural cytotoxicity and antibody-dependent cellular cytotoxicity. To show specificity for α3 MICA/B targeting, the AML cell line THP-1 was stained for the presence of MICA/B by flow cytometry using the 6D4 clone that recognizes the α1/2 domains of MICA/B or the 7C6 clone that uniquely recognizes the α3 domain +/- proteolytic cleavage with trypsin (Fig. 1A-B). Significant expression of the α3 domain was observed on THP-1 cells using the 7C6 antibody, which was highly expressed even after protease treatment. On the other hand, the α1/2 domains of MICA/B were detectable at lower levels using the 6D4 antibody and were undetectable after protease treatment (Fig. 1A-B). To assess a dual targeting approach, THP-1 cells were used as targets in live imaging functional assays under standard conditions (Effector:Target [E:T] 2:1). The iNK cell backbone had modest natural cytotoxicity at 3 hours that was significantly enhanced by the addition of α3 MICA/B CAR. The effect was further improved with more rapid killing kinetics when combined with the anti-CD33 TriKE (Fig. 1C). To evaluate the efficacy of dual-targeting in a more stringent physiologic manner, we mimicked "stress” conditions by minimizing the E:T ratio to 0.25:1. While all effector conditions induced immediate killing in 4 hours at the low E:T ratio, sustained tumor control was only observed with dual-antigen targeting (Fig. 1D). Studies with primary AML targets, +/- preincubation of decitabine and all-trans retinoic acid, known to upregulate NKG2D ligands in AML, are in progress and will be discussed. In summary, dual-targeting strategies using off-the-shelf CAR NK cells targeting α3 MICA/B in combination with antigen-specific TriKE targeting CD33 represent an ideal clinical strategy to enhance efficacy and durability of treatment in advanced AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

OR23-3 Hunger Hormone Asprosin Activates Orexigenic Neurons via SK Currents

Abstract Neurons that co-express the orexigenic agouti-related protein (AgRP) and neuropeptide Y (NPY) are indispensable for normal feeding behavior. Firing activities of AgRP/NPY neurons dynamically fluctuate with energy status and coordinate appropriate feeding behavior to meet nutritional demands. We previously demonstrated that asprosin, a recently discovered fasting-induced glucogenic and orexigenic hormone, crosses the blood-brain barrier and directly activates the orexigenic AgRP/NPY neurons via a cyclic cAMP-dependent pathway. However, intrinsic mechanisms on how asprosin regulates AgRP/NPY neural activities during the fed-to-fasted transition are not fully understood. In the satiated mice with low circulating levels of asprosin, we found that AgRP/NPY neurons expressed high levels of the small conductance calcium-activated potassium channel 3 (SK3) and were inhibited by SK3-mediated potassium currents. Conversely, in 24 hour-fasting mice with high levels of asprosin, AgRP/NPY neurons expressed low levels of SK3 and were activated via inhibition of SK3-mediated potassium currents. Notably, the stimulatory effects of asprosin on AgRP/NPY neurons were blunted in mice with SK3 selectively deleted in the AgRP/NPY neurons, further supporting a mediating role of SK3. Moreover, we also identified protein tyrosine phosphatase receptor δ (Ptprd), a membrane-bound phosphatase receptor, as the orexigenic asprosin-receptor. Deletion of Ptprd in AgRP/NPY neurons abolished asprosin's stimulatory effect on SK3-mediated currents and protected mice from diet-induced obesity. Lastly, increased circulating levels of asprosin promoted hyperphagia, body weight gain, and glucose intolerance. On the other hand, anti-asprosin antibody treatment decreased food intake and body weight by inhibiting AgRP/NPY and other orexigenic hypothalamic neurons via SK currents stimulation. In summary, our data support a model that asprosin acts on Ptprd expressed by AgRP/NPY neurons to inhibit SK3 current, further activating AgRP/NPY neurons to increase food intake and body weight. Our findings also provided preclinical evidence that asprosin-neutralizing antibodies could be used to treat obesity. Presentation: Monday, June 13, 2022 11:45 a.m. - 12:00 p.m.