Rate Of Desensitization Research Articles

Assessment of neuromodulatory effects of Origanum punonense danin essential oil on AMPA receptor function using whole-cell patch-clamp technique

Introduction: The quest for natural compounds with neuromodulatory properties has gained significant momentum in neuropharmacology. Among these, Origanum punonense (O. punonense) essential oil (EO) stands out due to its rich bioactive profile, particularly characterized by carvacrol. This study aimed to elucidate the effects of O. punonense EO on α-amino-3‑hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR), crucial synaptic plasticity, and cognitive function mediators. Methods: Leaves of O. punonense were collected along the Dead Sea coast in Jericho. After washing, it was dried in the shade for 15 days and ground. Essential oil was extracted using a microwave-ultrasonic method, with ultrasonic waves to enhance extraction. Using whole-cell patch-clamp techniques, we studied the effects of O. punonense EO on AMPAR kinetics in HEK293t cells transfected with homomeric and heteromeric subunits. Results: O. punonense EO significantly reduced AMPAR's whole-cell currents, indicating a targeted modulation of synaptic responses. Oil administration inhibited the currents for glutamate receptor subunits GluA1 (reduced from 885±58 pA to 342±20 pA), GluA1/2 (from 627±77 pA to 209±36 pA), GluA2 (from 1125±112 pA to 256±37 pA), and GluA2/3 (from 474±79 pA to 125±31 pA). The EO decreased the desensitization rate significantly for GluA2 and GluA2/3 (p < 0.01) and for GluA1/2 (p < 0.05), except for GluA1. In addition, the EO increased the deactivation rate significantly for GluA2 and GluA2/3 (p < 0.01) and for GluA1/2 (p < 0.05), with no effect observed on GluA1. Conclusions: This study highlights the possible properties of O. punonense EO and suggests future research to understand its medicinal benefits in neurodegenerative diseases.

Gating mechanism of the human α1β GlyR by glycine

Glycine receptors (GlyRs) are members of the Cys-loop receptors that constitute a major portion of mammalian neurotransmitter receptors. Recent resolution of heteromeric GlyR structures in multiple functional states raised fundamental questions regarding the gating mechanism of GlyR, and generally the Cys-loop family receptors. Here, we characterized in detail equilibrium properties as well as the transition kinetics between functional states. We show that, while all allosteric sites bind cooperatively to glycine, occupation of 2 sites at the α-α interfaces is sufficient for activation and necessary for high-efficacy gating. Differential glycine concentration dependence of desensitization rate, extent, and its recovery suggests separate but concerted roles of ligand-binding and ionophore reorganization. Based on these observations and available structural information, we developed a quantitative gating model that accurately predicts both equilibrium and kinetical properties throughout the glycine gating cycle. This model likely applies generally to the Cys-loop receptors and informs on pharmaceutical endeavors.

Human P2X4 receptor gating is modulated by a stable cytoplasmic cap and a unique allosteric pocket.

P2X receptors (P2XRs) are a family of ATP-gated ion channels comprising homomeric and heteromeric trimers of seven subunits (P2X 1 - P2X 7 ) that confer different rates of desensitization. The helical recoil model of P2XR desensitization proposes the stability of the cytoplasmic cap sets the rate of desensitization, but timing of its formation is unclear for slow-desensitizing P2XRs. We report cryo-EM structures of full-length, wild-type human P2X 4 receptor in apo, antagonist-bound, and desensitized states. Because the apo and antagonist-bound structures of this slow-desensitizing P2XR include an intact cytoplasmic cap while the desensitized state structure does not, the cytoplasmic cap forms before agonist binding. Furthermore, structural and functional data suggests the cytoplasmic cap is stabilized by lipids to slow desensitization and that P2X 4 is further modified by glycosylation and palmitoylation. Finally, our antagonist-bound inhibited state structure reveals features specific to the allosteric ligand-binding pocket in human receptors that empower the development of small-molecule modulators.

#2458 Factors associated with cytomegalovirus infection in kidney transplant patients: analysis from a nationwide registry data

Abstract Background and Aims Cytomegalovirus (CMV) has had a significant impact on solid organ transplantation. It can affect allograft function and increase patient morbidity and mortality. We have made several efforts to prevent CMV infection after kidney transplantation (KT), but we have not yet been able to prevent it satisfactorily. Drugs were used as preventive therapy or preemptive therapy, but they did not completely prevent the diseases. Therefore, it will be possible to reduce CMV infection by taking actions against factors that can easily cause CMV infection. We investigated factors that make CMV infection more likely and explored the effects of CMV infection using The Korean Organ Transplantation Registry (KOTRY) datasets. Method We used the data from the KOTRY datasets. A total of 7545 KT recipients were analysed. Results The mean age of the subjects was 49.7 ± 11.5 years (range, 19-80). Among the 7545 subjects, 58.6% were male. Three hundred and twenty-four (4.2%) subjects had CMV infection (CMV group). There were 4827 (64%) living donors, 2717 (36%) cadaveric donors, and 1338 (17.3%) ABO incompatible donors. Of the total, 95% were transplanted from positive to positive CMV IgG, and 1.9% were from positive to negative. Although preventive treatment is performed in the second cases, in most cases preemptive therapy is performed. The age was 49.6 ± 11.5 in the no CMV group and 51.4 ± 12.6 in the CMV group (P = 0.013). There was no difference in sex, weight and BMI in both cases. The causes of end stage renal disease were glomerulonephritis, DM, and hypertension in that order, and there was no difference in the rates of diabetes and hypertension between the two groups. There was no difference in the desensitization rate at 24% in the no CMV group and 23% in the CMV group. At the time of discharge after surgery, the tacrolimus dose was 6.4 mg/d±3.6 in the no CMV group and 6.3 ± 3.8 mg/d in the CMV group (P = 0.89). Tacrolimus concentration (C0) was 8.0 ± 2.6 ng/ml in the no CMV group and 8.9 ± 2.4 ng/ml in the CMV group. It was higher in the CMV group (P = 0.002). When divided into 3 tertiles based on C0, the break points were 6.7 and 8.9 ng/ml. There was a higher incidence of CMV in the third tertile group compared to the first and second tertiles, but there was no difference between the first and second groups. When the metabolic rate (tacrolimus concentration divided by dose) was divided into three groups, CMV occurred more frequently in the group with the lower metabolic rate, at 3.9% in the group with the lowest tertile and 2.3% in the highest tertile (P = 0.046). At 6 months, tacrolimus dose was 4.3 ± 2.4 mg/d in the no CMV group and 3.9 ± 2.3 mg/d in the CMV group, which was lower in the CMV group (P = 0.005). Tacrolimus concentration was 6.8 ± 2.4 ng/ml in the no CMV group and 6.4 ± 2.3 ng/ml in the CMV group, which was lower in the CMV group (P = 0.004). There was no difference according to metabolic rate. At discharge (62.5 ± 25.7 vs. 54.5 ± 26.5), at 6 month (56.0 ± 22.3 vs. 47.9 ± 19.0), and at 1 year (57.2 ± 20.0 vs. 48.8 ± 19.6 mL/min/1.73 m2), eGFR in the No CMV group was higher than that in the CMV group (P &amp;lt; 0.005). One thousand two hundred and sixty-four (16.8%) patients showed acute rejection within 1 year. Acute rejection occurred within 1 year in 36.4% of the CMV group and 15.9% of the no CMV group (P &amp;lt; 0.001). Delayed graft function occurred in 4.6% of the CMV group and 5.5% of the no CMV group (P &amp;lt; 0.001). Conclusion CMV was more common in older people, those with higher tacrolimus concentrations, and those with lower tacrolimus metabolic rates. The risk increases when the concentration exceeds 8.9 ng/ml, and after reducing the dose, the dose and concentration were lower in the CMV group at 6 month and 1 year. Although the dose was later reduced in the CMV group, more acute rejections occurred within 1 year, which suggests that CMV infection can trigger rejections. Delayed graft function occurred less frequently in the CMV group, which may be related to the higher concentration of tacrolimus.

Evaluating the Neuroprotective Potential of Novel Benzodioxole Derivatives in Parkinson's Disease via AMPA Receptor Modulation.

Parkinson's disease (PD) is a significant health issue because it gradually damages the nervous system. α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors play a significant role in the development of PD. The current investigation employed hybrid benzodioxole-propanamide (BDZ-P) compounds to get information on AMPA receptors, analyze their biochemical and biophysical properties, and assess their neuroprotective effects. Examining the biophysical characteristics of all the subunits of the AMPA receptor offers insights into the impact of BDZ-P on the desensitization and deactivation rate. It demonstrates a partial improvement in the locomotor capacities in a mouse model of Parkinson's disease. In addition, the in vivo experiment assessed the locomotor activity by utilizing the open-field test. Our findings demonstrated that BDZ-P7 stands out with its remarkable potency, inhibiting the GluA2 subunit nearly 8-fold with an IC50 of 3.03 μM, GluA1/2 by 7.5-fold with an IC50 of 3.14 μM, GluA2/3 by nearly 7-fold with an IC50 of 3.19 μM, and GluA1 by 6.5-fold with an IC50 of 3.2 μM, significantly impacting the desensitization and deactivation rate of the AMPA receptor. BDZ-P7 showed an in vivo impact of partially reinstating locomotor abilities in a mouse model of PD. The results above suggest that the BDZ-P7 compounds show great promise as top contenders for the development of novel neuroprotective therapies.

Neto proteins differentially modulate the gating properties of Drosophila NMJ glutamate receptors.

The formation of functional synapses requires co-assembly of ion channels with their accessory proteins which controls where, when, and how neurotransmitter receptors function. The auxiliary protein Neto modulates the function of kainate-type glutamate receptors in vertebrates as well as at the Drosophila neuromuscular junction (NMJ), a glutamatergic synapse widely used for genetic studies on synapse development. We previously reported that Neto is essential for the synaptic recruitment and function of glutamate receptors. Here, using outside-out patch-clamp recordings and fast ligand application, we examine for the first time the biophysical properties of recombinant Drosophila NMJ receptors expressed in HEK293T cells and compare them with native receptor complexes of genetically controlled composition. The two Neto isoforms, Neto-α and Neto-β, differentially modulate the gating properties of NMJ receptors. Surprisingly, we found that deactivation is extremely fast and that the decay of synaptic currents resembles the rate of iGluR desensitization. The functional analyses of recombinant iGluRs that we report here should greatly facilitate the interpretation of compound in vivo phenotypes of mutant animals.

Engineering a human P2X2 receptor with altered ligand selectivity in yeast

P2X receptors are a family of ligand gated ion channels found in a range of eukaryotic species including humans but are not naturally present in the yeast Saccharomyces cerevisiae. We demonstrate the first recombinant expression and functional gating of the P2X2 receptor in baker’s yeast. We leverage the yeast host for facile genetic screens of mutant P2X2 by performing site saturation mutagenesis at residues of interest, including SNPs implicated in deafness and at residues involved in native binding. Deep mutational analysis and rounds of genetic engineering yield mutant P2X2 F303Y A304W, which has altered ligand selectivity towards the ATP analog AMP-PNP. The F303Y A304 variant shows over 100-fold increased intracellular calcium amplitudes with AMP-PNP compared to the WT receptor and has a much lower desensitization rate. Since AMP-PNP does not naturally activate P2X receptors, the F303Y A304 P2X2 may be a starting point for downstream applications in chemogenetic cellular control. Interestingly, the A304W mutation selectively destabilizes the desensitized state, which may provide a mechanistic basis for receptor opening with suboptimal agonists. The yeast system represents an inexpensive, scalable platform for ion channel characterization and engineering by circumventing the more expensive and time-consuming methodologies involving mammalian hosts.

Opioid Analgesic as a Positive Allosteric Modulator of Acid-Sensing Ion Channels.

Tafalgin (Taf) is a tetrapeptide opioid used in clinical practice in Russia as an analgesic drug for subcutaneous administration as a solution (4 mg/mL; concentration of 9 mM). We found that the acid-sensing ion channels (ASICs) are another molecular target for this molecule. ASICs are proton-gated sodium channels that mediate nociception in the peripheral nervous system and contribute to fear and learning in the central nervous system. Using electrophysiological methods, we demonstrated that Taf could increase the integral current through heterologically expressed ASIC with half-maximal effective concentration values of 0.09 mM and 0.3 mM for rat and human ASIC3, respectively, and 1 mM for ASIC1a. The molecular mechanism of Taf action was shown to be binding to the channel in the resting state and slowing down the rate of desensitization. Taf did not compete for binding sites with both protons and ASIC3 antagonists, such as APETx2 and amiloride (Ami). Moreover, Taf and Ami together caused an unusual synergistic effect, which was manifested itself as the development of a pronounced second desensitizing component. Thus, the ability of Taf to act as a positive allosteric modulator of these channels could potentially cause promiscuous effects in clinical practice. This fact must be considered in patients' treatment.

Probiotics and other adjuvants in allergen-specific immunotherapy for food allergy: a comprehensive review.

This review delves into the potential of manipulating the microbiome to enhance oral tolerance in food allergy, focusing on food allergen-specific immunotherapy (FA-AIT) and the use of adjuvants, with a significant emphasis on probiotics. FA-AIT, including oral (OIT), sublingual (SLIT), and epicutaneous (EPIT) immunotherapy, has shown efficacy in desensitizing patients and achieving sustained unresponsiveness (SU). However, the long-term effectiveness and safety of FA-AIT are still under investigation. Probiotics, particularly strains of Lactobacillus, play a crucial role in enhancing immune tolerance by promoting regulatory T cells (Tregs) and modulating cytokine profiles. These probiotics can induce semi-mature dendritic cells, enhance CD40 expression, inhibit IL-4 and IL-5, and promote IL-10 and TGF-β, thus contributing to mucosal defense and immunological tolerance. Clinical trials combining probiotics with FA-AIT have demonstrated improved desensitization rates and immune tolerance in food-allergic patients. For example, the combination of Lactobacillus rhamnosus with peanut OIT resulted in a significantly higher rate of SU compared to the placebo group, along with notable immune changes such as reduced peanut-specific IgE and increased IgG4 levels. The review also explores other adjuvants in FA-AIT, such as biologic drugs, which target specific immune pathways to improve treatment outcomes. Additionally, nanoparticles and herbal therapies like food allergy herbal formula 2 (FAHF-2) are discussed for their potential to enhance allergen delivery and immunogenicity, reduce adverse events, and improve desensitization. In conclusion, integrating probiotics and other adjuvants into FA-AIT protocols could significantly enhance the safety and efficacy of FA-AIT, leading to better patient outcomes and quality of life.

Desensitization in patients with hypersensitivity to platinum and taxane in gynecological cancers.

Exposure to paclitaxel and carboplatin has the risk of developing hypersensitivity reactions (HSRs), which could necessitate using less effective treatments to avoid anaphylaxis. Desensitization to platinum and taxane HSRs can be used to complete chemotherapy according to the standard regimen; therefore, this study investigated rates and benefits of successful desensitization in patients with gynecologic cancers (GC). We collected data from 241 patients with GC who had at least one cycle of platinum or taxane chemotherapy. The rate of HSRs and successful desensitization were evaluated, and an outcome analysis was conducted. The rate of HSRs to platinum and taxane was 6.39% and 13.07%, respectively. We observed a 100% success rate of desensitization in our cohort. Patients with HSR were significantly younger (57.1 vs. 64.9 years, p = 0.030) in the taxane cohort. Importantly, the overall survival (OS) of patients with platinum and taxane HSRs who underwent desensitization was comparable to that of patients with no HSRs (platinum vs. controls; median OS 60.36 vs. 60.39 months, p = 0.31; taxane vs. controls; OS 80.29 vs. 60.00 months, p = 0.59). Thus, we show that desensitization for platinum and taxane HSRs is safe and effective, resulting in an outcome that is well comparable to patients without HSR. Based on these observations, desensitization procedures might be considered as standard of care before switching to less effective treatment for patients with GC.

Deciphering the Biophysical Properties of Ion Channel Gating Pores by Coumarin-Benzodiazepine Hybrid Derivatives: Selective AMPA Receptor Antagonists.

In the 1980s, the identification of specific pharmacological antagonists played a crucial role in enhancing our comprehension of the physiological mechanisms associated with α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors (AMPARs). The primary objective of this investigation was to identify specific AMPA receptor antagonists, namely 2,3-benzodiazepines, that function as negative allosteric modulators (NAMs) at distinct locations apart from the glutamate recognition site. These compounds have exhibited a diverse array of anticonvulsant properties. In order to conduct a more comprehensive investigation, the study utilized whole-cell patch-clamp electrophysiology to analyze the inhibitory effect and selectivity of benzodiazepine derivatives that incorporate coumarin rings in relation to AMPA receptors. The study's main objective was to acquire knowledge about the relationship between the structure and activity of the compound and comprehend the potential effects of altering the side chains on negative allosteric modulation. The investigation provided crucial insights into the interaction between eight CD compounds and AMPA receptor subunits. Although all compounds demonstrated effective blockade, CD8 demonstrated the greatest potency and selectivity towards AMPA receptor subunits. The deactivation and desensitization rates were significantly influenced by CD8, CD6, and CD5, distinguishing them from the remaining five chemicals. The differences in binding and inhibition of AMPA receptor subunits can be attributed to structural discrepancies among the compounds. The carboxyl group of CD8, situated at the para position of the phenyl ring, substantially influenced the augmentation of AMPA receptor affinity. The findings of this study highlight the potential of pharmaceutical compounds that specifically target AMPA receptors to facilitate negative allosteric modulation.

Thiazole Derivatives as Modulators of GluA2 AMPA Receptors: Potent Allosteric Effects and Neuroprotective Potential.

Thiazole carboxamide derivatives were synthesized in this investigation, with a subsequent examination of their impact on GluA2 AMPA receptors. The synthesized compounds, namely MMH-1-5, were subjected to characterization using high-resolution mass spectrometry (HRMS), proton nuclear magnetic resonance (1H-NMR), and carbon-13 nuclear magnetic resonance (13C-NMR). The present work thoroughly investigates the impact of five thiazole derivatives on GluA2 AMPA receptors. This investigation examined their effects on both whole-cell currents and receptor kinetics. In addition, the cytotoxicity of the samples was assessed using the MTS test. The compound MMH-5 had the highest effect level, resulting in a notable drop in current amplitude by a factor of six. Similarly, MMH-4 and MMH-3 also caused major reductions in the current amplitude. The compounds mentioned above also influenced the rates of deactivation and desensitization. MMH-5 and MMH-4 exhibited an increase in deactivation, while MMH-5 showed reduced desensitization. Our research findings highlight the efficacy of MMH-5 as a negative allosteric modulator of GluA2 AMPA receptors, exerting substantial effects on both the magnitude and time course of receptor activity. Significantly, the compound MMH-2 demonstrated noteworthy cytotoxic effects, as evidenced by cell viability rates dropping below 6.79% for all cancer cell lines and 17.52% for the normal cell line (LX-2). Of particular interest is the pronounced cytotoxicity observed in MMH-5, suggesting its potential as a safe neuroprotective agent targeting the AMPA receptor, as indicated by cell viability percentages exceeding 85.44% across all cancer and normal cell lines. Docking simulations were performed to determine possible modes of interaction between MMH5 and the GluA2-AMPA receptor (PDB:7RZ5). The abovementioned facts and the well-documented effects of further thiazole derivatives provide a strong foundation for future research endeavors to enhance tailored treatments for neurological disorders that rely heavily on GluA2 signaling. The present study elucidates the intricate association between thiazole derivatives and GluA2 receptors, providing valuable perspectives on the prospects of enhanced and specific therapeutic interventions for diverse neurological conditions.

Desensitization and remission after peanut sublingual immunotherapy in 1- to 4-year-old peanut-allergic children: A randomized, placebo-controlled trial

Prior studies of peanut sublingual immunotherapy (SLIT) have suggested a potential advantage with younger age at treatment initiation. We studied the safety and efficacy of SLIT for peanut allergy in 1- to 4-year-old children. Peanut-allergic 1- to 4-year-old children were randomized to receive 4 mg peanut SLIT versus placebo. Desensitization was assessed by double-blind, placebo-controlled food challenge (DBPCFC) after 36monthsoftreatment. Participants desensitized to at least443mg peanut protein discontinued therapy for 3 months and then underwent DBPCFC to assess for remission. Biomarkers were measured at baseline and longitudinally during treatment. Fifty participants (25 peanut SLIT, 25 placebo) with a median age of 2.4 years were enrolled across 2 sites. The primary end point of desensitization was met with actively treated versus placebo participants having a significantly greater median cumulative tolerated dose (4443 mg vs 143 mg), higher likelihood of passing the month 36 DBPCFC (60% vs 0), and higher likelihood of demonstrating remission (48% vs 0). The highest rate of desensitization and remission was seen in1- to 2-year-olds, followed by 2- to 3-year-olds and 3- to 4-year-olds. Longitudinal changes in peanut skin prick testing, peanut-specific IgG4, and peanut-specific IgG4/IgE ratio were seen in peanut SLIT but not placebo participants. Oropharyngeal itching was more commonly reported by peanut SLIT than placebo participants. Skin, gastrointestinal, upper respiratory, lower respiratory, and multisystem adverse events were similar between treatment groups. Peanut SLIT safely induces desensitization and remission in 1- to 4-year-old children, with improved outcomes seen with younger age at initiation.

Electrophysiological Assessment of Newly Synthesized 2,3-Benzodiazepine Derivatives for Inhibiting the AMPA Receptor Channel.

Three major subtypes of ionotropic receptors regulate glutamatergic synaptic transmission, one of which is α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors (AMPARs). They are tetrameric, cation-permeable ionotropic glutamate receptors found across the brain. Abnormalities in AMPA receptor trafficking and synaptic assembly are linked to cognitive decline and neurological diseases such as Alzheimer's, Parkinson's, and Huntington's. The present study will investigate the effects of four novel 2,3-benzodiazepine derivatives on AMPA receptor subunits by comparing their effects on synaptic responses, desensitization, and deactivation rate in human embryonic kidney cells (HEK293T) recombinant AMPAR subunits using whole-cell patch-clamp electrophysiology. All four 2,3-BDZ compounds showed inhibitory activity against all the homomeric and heteromeric subunits tested. While the desensitization and deactivation rates in 2,3-BDZ-1 and 2,3-BDZ-2 decreased and increased, respectively, in the other two compounds (i.e., 2,3-BDZ-3 and 2,3-BDZ-4), there was no change in the desensitization or deactivation rates. These results contribute to a better understanding of AMPARs by identifying potential 2,3-BDZ drugs that demonstrate inhibitory effects on the AMPAR subunits.

Sustained unresponsiveness development in wheat oral immunotherapy: predictive factors and flexible regimen in the maintenance phase.

Background and aim. Immunotherapy may induce sustained unresponsiveness (SU) in which the patient can tolerate the allergen without any severe symptoms after discontinuing immunotherapy. The present study evaluated serum and cutaneous markers for predicting SU in patients with wheat anaphylaxis who underwent oral immunotherapy. Moreover, we investigated the effectiveness of a flexible regimen of 5 to 10 g wheat protein (WP) in the maintenance phase of oral immunotherapy (OIT). Methods. This study was conducted on 19 patients with wheat anaphylaxis who underwent OIT. The result of the skin prick test (SPT), besides specific serum IgE (sIgE) and IgG4 (sIgG4) to WP, were evaluated before the desensitization. The maintenance dose started from the preferred dose of 5 to 10 g WP after the build-up phase, if the patient could tolerate it. All patients were recruited 7 to 9 months after undergoing this flexible regimen, and the results of SPT and sIgE, and sIgG4 levels were obtained once more. The patients underwent oral food challenge (OFC) after a 3-4-week avoidance to evaluate SU. Results. There was an association between mean IgE reduction and SU (p less than 0.0006), while no association was observed between the mean increase in specific IgG4 (p = 0.1) and the mean wheal diameter decrease (p = 0.29). In the present study, a 50% reduction in sIgE was associated with SU. Thirteen patients were considered to have a SU. Moreover, there was no association between the flexible regimen and the desensitization rate. Conclusions. The results revealed that the reduction of 50% sIgE is a predictive factor for SU in patients with IgE-mediated wheat allergy.

Charged residues in the GABA-a receptor intracellular domain impact channel activation and desensitization.

Opening and closing of GABA receptor (GABAR) channels regulate inhibitory signaling in the brain. Mechanisms underlying channel open, closed, desensitized transitions remain unclear. Although GABAR cryo-EM structures provide details about its extracellular and transmembrane domains, little is understood about its intracellular domain (ICD) and its role in regulating channel gating. We constructed a GABAR homology model based on the 5HT-type-3 receptor, which revealed a helix (MX) at the C-terminal end of M3 that is lysine rich in the α1 (6 lysines) and γ2 (8 lysines) subunits. Charged residues in the α and γ subunits were mutated to alanine. Effects on GABA EC50 were measured using two-electrode voltage clamping of oocytes expressing α1β2γ2L WT and mutant GABARs. αK327A significantly increased GABA EC50 6-fold in comparison to WT and significantly reduced GABAR macroscopic desensitization. Extent of current desensitization after 10 seconds in saturating GABA (10 mM) was 6.86 ± 2.28% (n=7) versus WT (23.7 ± 7.29%, n=10). Neutralizing charge at αK328, K330 and K335 had no functional effects. γK328A, γ2K330A and γ2K332A significantly increased GABA EC50 by 2 to 3 fold. γ2K330A reduced GABAR desensitization (extent after 10 seconds 7.36 ± 2.37%, n=2). Our data indicate structural perturbations in the α and γ MX regions of the GABAR ICD alter GABAR channel activation and desensitization. Interestingly, in our GABAR structural model, α1R312 (in MX) interacts with α1R249 (M1-M2 linker). Single charge reversals at these positions increased GABA EC50 and the rate of GABA desensitization. A charge swap (R312E - E249R) rescued receptor function to WT suggesting a functional electrostatic interaction and that these residues form a salt bridge, providing support for our homology model.

Algorithmic Identification of Patients With Aspirin-Exacerbated Respiratory Disease Using an Electronic Health Record.

To determine whether an electronic health record (EHR) system can be used to identify cases of aspirin-exacerbated respiratory disease (AERD) in an area outside of a regional referral center with low rates of aspirin desensitization therapy. Retrospective chart review single academic tertiary care hospital. Single-site academic tertiary care hospital. Using Epic's SlicerDicer function, an algorithm was created and applied to all patient charts from 2013 to 2021. The algorithm was as follows: "Allergy/Contraindication to NSAIDs OR aspirin" AND "Diagnosis of Nasal polyp AND "Diagnosis of Asthma." Clinical data including demographics, NSAID reaction, and specialist involvement was collected. A total of 54 potential cases of AERD were identified. Thirty-twowere determined to have AERD after chart review, yet 12 of these patients (37.5%) had no mention of AERD within the chart. The 54 patients were stratified into 2 cohorts based on reaction to NSAIDs: respiratory (n = 29) or unspecified (n = 25). Of the patients in the respiratory reaction group, 26 were found to have clinical AERD, demonstrating a positive predictive values (PPV) of 89.7%. The overall PPV was 59.3%. Those with a respiratory reaction to NSAIDS listed in the EHR were more likely to have clinical AERD (odds ratio27.44; confidence interval6.08-123.85; p < 0.0001). Only 2 patients (6.3%) underwent aspirin desensitization. AERD remains under-diagnosed in the study population. The informatics algorithm presented here has a high positive predictive value for identifying clinical AERD patients in a geographical area with low rates of aspirin desensitization and may aid in identifying candidates for expanded treatment options.

Dopamine-induced arrestin recruitment and desensitization of the dopamine D4 receptor is regulated by G protein-coupled receptor kinase-2.

The dopamine D4 receptor (D4R) is expressed in the retina, prefrontal cortex, and autonomic nervous system and has been implicated in attention deficit hyperactivity disorder (ADHD), substance use disorders, and erectile dysfunction. D4R has also been investigated as a target for antipsychotics due to its high affinity for clozapine. As opposed to the closely related dopamine D2 receptor (D2R), dopamine-induced arrestin recruitment and desensitization at the D4R have not been studied in detail. Indeed, some earlier investigations could not detect arrestin recruitment and desensitization of this receptor upon its activation by agonist. Here, we used a novel nanoluciferase complementation assay to study dopamine-induced recruitment of β-arrestin2 (βarr2; also known as arrestin3) and G protein-coupled receptor kinase-2 (GRK2) to the D4R in HEK293T cells. We also studied desensitization of D4R-evoked G protein-coupled inward rectifier potassium (GIRK; also known as Kir3) current responses in Xenopus oocytes. Furthermore, the effect of coexpression of GRK2 on βarr2 recruitment and GIRK response desensitization was examined. The results suggest that coexpression of GRK2 enhanced the potency of dopamine to induce βarr2 recruitment to the D4R and accelerated the rate of desensitization of D4R-evoked GIRK responses. The present study reveals new details about the regulation of arrestin recruitment to the D4R and thus increases our understanding of the signaling and desensitization of this receptor.

α-Lipoic Acid Derivatives as Allosteric Modulators for Targeting AMPA-Type Glutamate Receptors' Gating Modules.

The ionotropic glutamate receptor subtype α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) is responsible for most excitatory transmission in the brain. AMPA receptor function is altered in numerous neurological illnesses, making AMPA receptors appealing therapeutic targets for clinical intervention. Alpha-Lipoic acid (α-LA) is a naturally occurring compound, which functions as a co-factor in metabolism and energy production. α-LA is an antioxidant with various benefits in treating diabetes, including managing symptomatic diabetic neuropathy. This study will test a novel and innovative strategy to synthesize a new isomer of lipoic acid (R-LA) derivatives (bifunctional NO-donor/antioxidant) in one chemical on homomeric and heteromeric AMPA receptor subunits. We used patch-clamp electrophysiology to examine LA derivatives expressed in human embryonic kidney 293 cells (HEK293) for inhibition and changes in desensitization or deactivation rates. LA derivatives were shown to be potent antagonists of AMPA receptors, with an 8-11-fold reduction in AMPA receptor currents seen following the delivery of the compounds. Furthermore, the LA derivatives influenced the rates of desensitization and deactivation of AMPA receptors. Based on our results, especially given that α-LA is closely connected to the nervous system, we may better understand using AMPA receptors and innovative drugs to treat neurological diseases associated with excessive activation of AMPA receptors.

Efficacy and safety of food allergy oral immunotherapy in adults.

Oral immunotherapy (OIT) is an emerging method for treating food allergy in children. However, data regarding adults undergoing this process are lacking. We retrospectively analyzed the medical records of patients with food allergy aged ≥17 years who completed OIT treatment between April 2010 and December 2020 at Shamir Medical Center. Data were compared with that of children aged 4 to <11 years and adolescents aged ≥11 to 17 treated during the same time period. A total of 96 adults at a median age of 22.3 years who underwent OIT for milk (n=53), peanut (n=18), sesame (n=7), egg (n=5), and tree nuts (n=13) were analyzed and compared with 1299 children and 309 adolescents. Adults experienced more adverse reactions requiring injectable epinephrine, both during in-clinic up-dosing (49% vs. 15.9% and 26.5% for children and adolescents, respectively, p < 0.0001) and during home treatment (22.9% vs. 12.4%, p=0.007 for children, and 17.5%, p=0.23 for adolescents). Most adults (61.5%) were fully desensitized, but the rates of full desensitization were significantly lower than children (73.4%, p=0.013). Significantly more adults (28.3%) undergoing milk OIT failed treatment than children (14.3%, p=0.015) and adolescents (14.1%, p=0.022), while failure rates in adults undergoing OIT for other foods were low (9.3%) and comparable with children and adolescents. OIT is successful in desensitizing most adults with IgE-mediated food allergy. Adults undergoing milk OIT are at increased risk for severe reactions and for OIT failure while failure rates in adults undergoing OIT for other foods are low.