Subunits Α1 Research Articles

Molecular dynamics exploration of cacophony protein interactions with brood volatiles in honey bee colonies

The cacophony protein is a key component of honey bee biology that has been investigated concerning hygienic behavior. The voltage-gated calcium channel subunit α1 appears to play a significant role in modulating neuronal activity associated with the detection and response to compromised brood within honey bee colonies. Despite its essential role, the molecular mechanisms underpinning hygienic behavior remain incompletely understood. Recent research suggests that the expression of the cacophony protein is linked to the efficiency of hygienic behavior, where colonies with enhanced hygienic behavior exhibit distinct patterns of cacophony expression. Understanding the role of cacophony in honey bee behavior provides insights into the molecular mechanisms underlying social interactions within bee colonies, with implications for enhancing pollination services and mitigating threats to honey bee populations. Therefore, the present study focused on unraveling and characterizing its structure and function, a bioinformatics approach involving sequence and phylogenetic analysis. Homology modeling developed three-dimensional models of cacophony protein for Apis cerana and A. mellifera. Molecular docking studies were performed for various brood pheromones and volatiles, triggering hygienic behavior in honey bees, against cacophony providing computational insights into their molecular interactions. Binding free energy calculations using MM/PBSA and MM/GBSA consistently demonstrate a higher affinity of γ-octalactone for AcerCAC protein (−21.77 ± 3.01 kcal/mol and −24.81 ± 4.07 kcal/mol respectively) compared to AmelCAC protein (−20.03 ± 3.01 kcal/mol and −21.47 ± 3.19 kcal/mol respectively). This study lays a theoretical foundation for further studies regarding the mechanism of interaction ofcacophony with hygienic behavior.

Trans-Ferulic acid reduces the sedative activity of diazepam in thiopental sodium-induced sleeping mice: A potential GABAergic transmission.

trans-Ferulic acid (TFA), a bioactive compound found in many plants, has been recognized for its diverse pharmacological activities, including potential neurological benefits. Previous studies suggest that TFA exerts anxiolytic effects via GABAergic pathways. This study aimed to investigate the sedative effects of TFA and its possible molecular mechanisms through in vivo and in silico approaches. Adult Swiss mice were randomly divided into six groups (n=7): control (vehicle), standard (DZP: Diazepam at 3mg/kg, p.o.), three test groups (TFA at 25, 50, and 75mg/kg, p.o.), and a combination group (TFA: 50mg/kg with DZP: 3mg/kg, p.o.). Thirty minutes post-treatment, thiopental sodium (TS) at 40mg/kg was administered to induce sedation, and latency as well as duration of sleep, were observed for up to 4h. In silico studies were conducted with GABAA receptor subunits (α1 and β2) to elucidate the possible molecular interactions. The results demonstrated that TFA significantly reduced latency and extended sleep duration in a dose-dependent manner compared to the control. Additionally, TFA combined with DZP further significantly (p<0.001) enhanced these effects. In silico analysis revealed that TFA and DZP exhibited strong binding affinities with the GABAA receptor subunits (α1 and β2) in the identical binding sites, with binding energies of -6.8 and-8.7kcal/mol, respectively. Collectively, TFA exerted a mild sedative effect in TS-induced sleeping mice and modulated the activity of DZP, likely through interactions with GABAA receptors. TFA showed promising activity as a potential candidate for managing sleep disorders such as insomnia.

Somatostatin-expressing inhibitory neurons with mTORC1 activation in cortical layers 4/5 are involved in the epileptogenesis of mice

Aim: Patients with tuberous sclerosis complex (TSC) which is caused by hyperactivation of mechanistic target of rapamycin complex 1 (mTORC1) often show giant cells in the brain. These giant cells are thought to be involved in epileptogenesis, but the underlying mechanisms are unknown. In this study, we focused on mTORC1 activation and γ-amino butyric acid (GABA)ergic signaling in somatostatin-expressing inhibitory neurons (SST-INs) using TSC-related epilepsy model mice. Methods: We analyzed the 8-week-old Tsc2 conditional knockout (Tsc2 cKO) mice, which have epileptic seizures that are cured by sirolimus, an mTORC1 inhibitor. After the occurrence of epileptic seizures was confirmed, Tsc2 cKO mice were treated with vehicle or sirolimus. Then, their brains were investigated by hematoxylin and eosin staining, immunohistochemical staining and immunoblotting assay. Results: As in TSC patients, giant cells with hyperactivation of mTORC1 were found in the cerebral cortex of Tsc2 cKO mice. These giant cells were mainly SST-INs in the cortical layers 4/5. Giant cells showed decreased expression of GABA type A receptor subunit α1 (GABAAR-α1) compared with normal size cells in control mice and Tsc2 cKO mice. In addition, decreased GABAAR-α1 expression was also confirmed by immunoblotting assay of the whole cerebral cortex. In the cerebral cortex of sirolimus-treated Tsc2 cKO mice, whose epileptic seizures were cured, decreased GABAAR-α1 expression was recovered to the same level as in control mice. Conclusions: These results suggest that the epileptic seizures in Tsc2 cKO mice are caused by the deregulation of GABAergic signaling through mTORC1 activation of SST-INs localized in cortical layers 4/5.

Changes in D4 and GABAA subunit α3 receptors in the thalamic reticular nucleus caused by unilateral lesion of the globus pallidus.

The thalamic reticular nucleus controls information processing in thalamocortical neurons. GABAergic neurons present in this nucleus express the α3 subunit of post‑synaptic GABAA receptors, which bind GABA from globus pallidus neurons. Pallidal neurons, in turn, have dopaminergic D4 receptors in their axon terminals. The thalamic reticular nucleus connects reciprocally with the thalamus, and it receives afferents from the brain cortex, as well as from other brain structures that have an important role in the modulation of the thalamic network. Based on the above, the purpose of this study was to assess the electrophysiological and molecular effects of unilateral lesion of the globus pallidus on the electric activity of the thalamic reticular nucleus. Two‑month‑old male rats were used. The right globus pallidus was lesioned with quinolinic acid. Seven days after the lesion, ipsilateral turning was registered, confirming the lesion. Afterward, electrophysiological evaluation of the right thalamic reticular nucleus' electrical activity was performed. Subsequently, mRNA expression for D4 receptors and subunit α3, as well as protein content were assessed in the right reticular nucleus. Pallidum lesion caused an increase in firing frequency and decreased firing bursts of reticular neurons. In addition, dopaminergic D4 mRNA, as well as protein increased. In contrast, GABAergic GABAA subunit α3 expression was suppressed, but protein content increased. These results show that the globus pallidus regulates firing in reticular neurons through D4 receptors and subunit α3 of GABAA receptor in the reticular nucleus of the thalamus.

Hypoxia induces reversible gill remodeling in largemouth bass (Micropterus salmoides) through integrins-mediated cell adhesion

Gill remodeling is an important strategy for fish to cope with hypoxia, and many of the teleost possess this ability, but the underlying mechanism is not well understood. To investigate the mechanism of hypoxia-induced gill remodeling, largemouth bass (Micropterus salmoides) exposed to hypoxia (dissolved oxygen level: 2.0 ± 0.2 mg L−1) for 7 days, followed by 7 days of reoxygenation. Hypoxia tests were also performed on primary gill cells from largemouth bass. We found that hypoxia-induced gill remodeling increased the respiratory surface area of the gills. This change in gill morphology was reversible and recovered after reoxygenation. A reduction in the number of mucous cells and rearrangement of mitochondria-rich cells (MRCs) were observed during gill remodeling. After 7 days of reoxygenation, the number of mucous cells and the position of the MRCs were restored. Hypoxia resulted in a 2.92-fold increase in the number of primary gill cells that underwent migration over a 12-h period. The mRNA levels of nine integrin subunits (α1, α2, α5, α7, α8, α10, αL, β1 and β2) were significantly up-regulated after 12 h of hypoxia in vivo, and the changes in the expression of these subunits were consistent with the HIF-1α trend. Immunohistochemistry showed that integrin β1 protein levels were significantly increased and were abundantly expressed in the interlamellar cell mass after exposure to hypoxia. Taken together, the results of the present study demonstrated that changes in mucosal cells and MRCs play an important role in hypoxia-induced gill remodeling in largemouth bass and that these changes are regulated by integrins.

BCAM (basal cell adhesion molecule) protein expression in different tumor populations

Basal Cell Adhesion Molecule (BCAM), a receptor for laminin subunit α5, plays a crucial role in the pathogenesis of various malignancies. Notably, evidence of hypermethylation at multiple immune checkpoints in patients with low BCAM expression suggests these individuals may respond favorably to immunotherapy using ICIs (immune checkpoint inhibitors). This finding lays the foundation for the hypothesis that BCAM may serve as an important biomarker in cancer patients. To investigate this potential, we evaluated BCAM expression patterns in 3114 patients from both discovery and validation cohorts, spanning seven cancer types, using quantitative immunofluorescence (QIF). We also explored the correlation between BCAM and PD-L1 expressions within these cohorts, aiming to establish its potential predictive value for immunotherapy response. Our findings indicate that BCAM was highly expressed in ovarian (79.2%) and lung (78.5%) tumors, with lower yet significant expression in breast (37.7%), head and neck (31.3%), and bladder-urothelial tumors (27.6%). Notably, high BCAM expression was associated with better OS in NSCLC. More importantly, BCAM expression did not correlate with PD-L1 protein expression in any of these tumors, highlighting its independent predictive potential. The widespread expression of BCAM across multiple tumor types, coupled with its lack of correlation with PD-L1 expression, highlights its potential as a predictive novel biomarker across various cancer types.

Wrinkle-Improving Effect of Novel Peptide That Binds to Nicotinic Acetylcholine Receptor.

Wrinkles, one of the most common signs of aging, are primarily caused by the continuous contraction of muscles. Muscle contraction is induced by the binding of acetylcholine (ACh), released at the neuromuscular junction, to nicotinic acetylcholine receptor (nAChR) present on the muscle cell surface. In this study, we aimed to develop a wrinkle-improving peptide that inhibits the binding of ACh to nAChR using peptide phage display technology. Our peptide showed a remarkably high binding affinity to nAChR subunit α1, with a value below 1 µM, and was found to inhibit the action of ACh through its interaction with these receptors. Furthermore, it increased collagen synthesis in skin cells and upregulated the expression of the aquaporin-3 (AQP3) and hyaluronan synthase-2 (HAS2) genes. These results confirm that the peptide effectively inhibits muscle contraction and enhances skin elasticity and hydration, contributing to its wrinkle-reducing effects. Clinical studies on humans observed significant improvement in wrinkles after three weeks of use, with substantial reduction observed after six weeks. In conclusion, these findings demonstrate the efficacy of the peptide (named Medipep) in reducing wrinkles.

Neuronal nicotinic acetylcholine receptor antibodies in autoimmune central nervous system disorders.

Neuronal nicotinic acetylcholine receptors (nAChRs) are abundant in the central nervous system (CNS), playing critical roles in brain function. Antigenicity of nAChRs has been well demonstrated with antibodies to ganglionic AChR subtypes (i.e., subunit α3 of α3β4-nAChR) and muscle AChR autoantibodies, thus making nAChRs candidate autoantigens in autoimmune CNS disorders. Antibodies to several membrane receptors, like NMDAR, have been identified in autoimmune encephalitis syndromes (AES), but many AES patients have yet to be unidentified for autoantibodies. This study aimed to develop of a cell-based assay (CBA) that selectively detects potentially pathogenic antibodies to subunits of the major nAChR subtypes (α4β2- and α7-nAChRs) and its use for the identification of such antibodies in "orphan" AES cases. The study involved screening of sera derived from 1752 patients from Greece, Turkey and Italy, who requested testing for AES-associated antibodies, and from 1203 "control" patients with other neuropsychiatric diseases, from the same countries or from Germany. A sensitive live-CBA with α4β2-or α7-nAChR-transfected cells was developed to detect antibodies against extracellular domains of nAChR major subunits. Flow cytometry (FACS) was performed to confirm the CBA findings and indirect immunohistochemistry (IHC) to investigate serum autoantibodies' binding to rat brain tissue. Three patients were found to be positive for serum antibodies against nAChR α4 subunit by CBA and the presence of the specific antibodies was quantitatively confirmed by FACS. We detected specific binding of patient-derived serum anti-nAChR α4 subunit antibodies to rat cerebellum and hippocampus tissue. No serum antibodies bound to the α7-nAChR-transfected or control-transfected cells, and no control serum antibodies bound to the transfected cells. All patients positive for serum anti-nAChRs α4 subunit antibodies were negative for other AES-associated antibodies. All three of the anti-nAChR α4 subunit serum antibody-positive patients fall into the AES spectrum, with one having Rasmussen encephalitis, another autoimmune meningoencephalomyelitis and another being diagnosed with possible autoimmune encephalitis. This study lends credence to the hypothesis that the major nAChR subunits are autoimmune targets in some cases of AES and establishes a sensitive live-CBA for the identification of such patients.

Possible association of G6PC2 and MUC6 induced by low‑dose‑rate irradiation in mouse intestine with inflammatory bowel disease.

Although there are several types of radiation exposure, it is debated whether low‑dose‑rate (LDR) irradiation (IR) affects the body. Since the small intestine is a radiation‑sensitive organ, the present study aimed to evaluate how it changes when exposed to LDR IR and identify the genes sensitive to these doses. After undergoing LDR (6.0 mGy/h) γ radiation exposure, intestinal RNA from BALB/c mice was extracted 1 and 24 h later. Mouse whole genome microarrays were used to explore radiation‑induced transcriptional alterations. Reverse transcription‑quantitative (RT‑q) PCR was used to examine time‑ and dose‑dependent radiation responses. The histopathological status of the jejunum in the radiated mouse was not changed by 10 mGy of LDR IR; however, 23 genes were upregulated in response to LDR IR of the jejunum in mice after 1 and 24 h of exposure. Upregulated genes were selected to validate the results of the RNA sequencing analysis for RT‑qPCR detection and results showed that only Na+/K+ transporting subunit α4, glucose‑6‑phosphatase catalytic subunit 2 (G6PC2), mucin 6 (MUC6) and transient receptor potential cation channel subfamily V member 6 levels significantly increased after 24 h of LDR IR. Furthermore, G6PC2 and MUC6 were notable genes induced by LDR IR exposure according to protein expression via western blot analysis. The mRNA levels of G6PC2 and MUC6 were significantly elevated within 24 h under three conditions: i) Exposure to LDR IR, ii) repeated exposure to LDR IR and iii) exposure to LDR IR in the presence of inflammatory bowel disease. These results could contribute to an improved understanding of immediate radiation reactions and biomarker development to identify radiation‑susceptible individuals before histopathological changes become noticeable. However, further investigation into the specific mechanisms involving G6PC2 and MUC6 is required to accomplish this.

Neonicotinoids differentially modulate nicotinic acetylcholine receptors in immature and antral follicles in the mouse ovary†.

Neonicotinoids are the most widely used insecticides in the world. They are synthetic nicotine derivatives that act as nicotinic acetylcholine receptor agonists. Although parent neonicotinoids have low affinity for the mammalian nicotinic acetylcholine receptor, they can be activated in the environment and the body to positively charged metabolites with high affinity for the mammalian nicotinic acetylcholine receptor. Imidacloprid, the most popular neonicotinoid, and its bioactive metabolite desnitro-imidacloprid differentially interfere with ovarian antral follicle physiology in vitro, but their effects on ovarian nicotinic acetylcholine receptor subunit expression are unknown. Furthermore, ovarian nicotinic acetylcholine receptor subtypes have yet to be characterized in the ovary. Thus, this work tested the hypothesis that ovarian follicles express nicotinic acetylcholine receptors and their expression is differentially modulated by imidacloprid and desnitro-imidacloprid in vitro. We used polymerase chain reaction, RNA in situ hybridization, and immunohistochemistry to identify and localize nicotinic acetylcholine receptor subunits (α2, 4, 5, 6, 7 and β1, 2, 4) expressed in neonatal ovaries (NO) and antral follicles. Chrnb1 was expressed equally in NO and antral follicles. Chrna2 and Chrnb2 expression was higher in antral follicles compared to NO and Chrna4, Chrna5, Chrna6, Chrna7, and Chrnb4 expression was higher in NO compared to antral follicles. The α subunits were detected throughout the ovary, especially in oocytes and granulosa cells. Imidacloprid and desnitro-imidacloprid dysregulated the expression of multiple nicotinic acetylcholine receptor subunits in NO, but only dysregulated one subunit in antral follicles. These data indicate that mammalian ovaries contain nicotinic acetylcholine receptors, and their susceptibility to imidacloprid and desnitro-imidacloprid exposure varies with the stage of follicle maturity.

Heme oxidation of NO-receptor soluble guanylyl cyclase is an important contributor to cerebrovascular and cognitive dysfunctions

Introduction: Decreased cerebral blood flow (CBF) is one of the major hemodynamic alterations leading to neurodegeneration and age-related cognitive decline. Nitric Oxide (NO)-dependent vasomotor reactivity is central in regulating cerebrovascular hemodynamics and adequate brain blood perfusion. NO receptor Soluble Guanylyl Cyclase (sGC) is the primary mediator of NO-dependent vasodilation. Oxidative stress, often persisting in diseased vasculature, renders sGC insensitive to NO and impairs the NO signaling. The objective of our study was to investigate the role of sGC oxidation in regulation CBF and brain function. Hypothesis: Oxidative stress disrupts NO/cGMP signaling in brain by decreasing sGC activity via oxidation of sGC heme. Approach: To assess causal effect of sGC oxidation on NO signaling function we administered ODQ, a sGC-specific heme oxidizing agent, to simulate long-term effect of sGC oxidation in cerebral vasculature. Two groups of young wild-type male and female mice (4 months old C57BL/6) were treated intraperitoneally (IP) twice a week for one month with ODQ (20 mg/kg) or a solvent as control. Changes in cerebrovascular reactivity and memory function were assessed and compared in treated and control groups. Using a non-invasive pulsed Doppler ultrasound-based system we assessed the changes in blood flow velocity (PVF, % of baseline) in the mouse middle cerebral artery (MCA) of male and female groups. MCA vasodilation was induced by bolus IP injection of NO-donor sodium nitroprusside (SNP) or BAY 58-2667, an allosteric NO-independent sGC activator. Memory function was assessed by the Novel Object Recognition Task (NORT, RI, recognition index) in all experimental groups. Western blotting and densitometry assessed the expression of major sGC subunits (α1, α2 and β1) in cleared brain lysates from ODQ-treated and control groups. Results: We observed a significantly blunted vasodilative response to SNP (1 mg/kg) in ODQ-treated mice vs controls of both genders (PVF, max response at 2 min post-injection, males 73.9 ± 10.4% (n=7/7) vs 58.5 ± 8.6 %, p=0.01; females (n=6/6) 77.4 ± 10.6 % vs 61.9 ± 5.4 %, p=0.01). Changes in response to NO-donor were more pronounced in males than females. The difference in BAY 58-2667 response between ODQ-treated and control groups was not statistically significant. ODQ significantly reduced the long-term memory in males but not female, according to NORT (RI, males (n=7/7) 0.58 ± 0.13 vs 0.69 ± 0.05; p=0.01; females (n=6/6) ODQ 0.59 ± 0.17 vs 0.73 ± 0.1, p=0.13). No statistically significant difference in expression of sGC subunits between control and ODQ-treated (n= 4/4) of male and female groups was determined. Conclusions: ODQ treatment significantly impairs vasodilation response to NO-donor but not to NO-independent sGC activator in mice MCA. ODQ treatment negatively affects long-term memory function in male, but not female mice. ODQ treatment does not significantly affect the expression of sGC subunits. Together our data suggest that the oxidation of sGC heme moiety impairs the regulation of cerebral blood flow of both genders and impairs long-term memory function gender-specifically in young mice. This research was supported by National Institute of Health grants RAG071999A (Iraida Sharina), HL139838, and American Heart Association 23TPA1070711 (Emil Martin). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Lactuca sativa L. Extract Enhances Sleep Duration Through Upregulation of Adenosine A1 Receptor and GABAA Receptors Subunits in Pentobarbital-Injected Mice.

We investigated the effects of Lactuca sativa L. extracts (Lactuc) on pentobarbital-induced sleep in mice to elucidate the mechanisms underlying its impact on sleep quality. Mice were randomly assigned to five groups: control, positive control (diazepam 2 mg/kg b.w.), and three groups orally administered with Lactuc (50, 100, and 200 mg/kg b.w.). After 2 weeks of oral administration and intraperitoneal injections, the mice were killed. We found that the Lactuc-administered groups had significantly reduced sleep latency and increased sleep duration compared with the control group. Furthermore, the oral administration of Lactuc induced a significant increase in mRNA expression and protein expression of adenosine A1 receptor in the brains compared with the expressions in the control group. In addition, the Lactuc-administered groups exhibited significantly higher levels of mRNA expressions of GABAA receptors subunits α2, β2, γ1, and, γ2 in the brain tissue. Therefore, we suggest that Lactuc could be used to develop natural products that effectively improve sleep quality and duration.

Abstract 4281: BMP signaling impaired telocytes-Foxl1+disrupt the extracellular matrix network enabling the development of colitis-associated cancer

Abstract The relationship between colon stem cells (SCs), its surrounding niche microenvironment like the extracellular matrix (ECM) and the mesenchymal niche cells is complex and dynamic. ECM proteins are not only a scaffolding system for SCs, but their biophysical properties also contribute to localizing signals and creating gradients leading to the SCs homeostasis. Foxl1+-Telocytes (TCFoxL1+) are mesenchymal niche cells implicated in cell-cell communication and the production of BMP ligands. Upon chronic inflammation, mice lacking BMP signaling in TCFoxL1+ (BmpR1aΔFoxL1+), developed colonic tumors with microenvironment exhibiting abnormal TCFoxL1+ structure, increased BMP expression and myofibroblastic-like cancer-associated fibroblasts (myCAF) subpopulation. Yet, how the TCFoxL1+ influences SCs niche ECM proteins during tumorigenesis is less understood. We aimed to investigate how the loss of BMP signaling in TCFoxL1+ affects ECM network impacting the niche microenvironment promoting the development of CAC. DSS-based chronic inflammatory challenge in mutant and control mice, quantitative-MS-based strategy was performed to determine the proteome of the transformed regions solely in epithelial-mesenchymal tissue following colon deconstruction. Expression of the proteins of interest were validated by Western Blots or immunostainings. Quantitative analysis of collagen network was assessed using phyton pipeline U-net in Sirius red-stained colon analyzed under polarized light. Differential significant expression of 798 proteins, with 342 upregulated and 456 downregulated, were found in the tumors of mutant mice compared to controls. Mfap5 involved in CAF activation was the highest upregulated protein. We found upregulated, laminin subunits α4 and β2, decorin, lumican as well as transgelin (myCAF marker), which are proteins known for contributing to the stiffening of the ECM during colorectal cancer. Adamdec1 which prevents aberrant ECM accumulation and thrombospodin-1, an ECM glycoprotein whose low levels have been related to tumor growth, were both found to be downregulated. Data extracted from Python's U-Net pipeline demonstrated that tumors, in mutant mice, exhibited higher frequencies of straighter collagen fibers compared to wavier fibers in controls. Collagen fiber distribution was shown to present an even angle distribution in controls whereas BmpR1aΔFoxL1+ mice presented a preferential orientation. These results suggest that defective TCFoxL1+ commits the surrounding niche microenvironment, especially its ECM, toward the development and progression of CAC. Citation Format: Vilcy Reyes Nicolas, Alain B. Alfonso, Jennifer Raisch, François M. Boisvert, Marc-Antoine Lauzon, Nathalie Perreault. BMP signaling impaired telocytes-Foxl1+disrupt the extracellular matrix network enabling the development of colitis-associated cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4281.

Mutation V65I in the β1 Subunit of the Nicotinic Acetylcholine Receptor Confers Neonicotinoid and Sulfoxaflor Resistance in Insects.

Neonicotinoids have been widely used to control pests with remarkable effectiveness. Excessive insecticides have led to serious insect resistance. Mutations of the nicotinic acetylcholine receptor (nAChR) are one of the reasons for neonicotinoid resistance conferred in various agricultural pests. Two mutations, V65I and V104I, were found in the nAChR β1 subunit of two neonicotinoid-resistant aphid populations. However, the specific functions of the two mutations remain unclear. In this study, we cloned and identified four nAChR subunits (α1, α2, α8, and β1) of thrips and found them to be highly homologous to the nAChR subunits of other insects. Subsequently, we successfully expressed two subtypes nAChR (α1/α2/α8/β1 and α1/α8/β1) by coinjecting three cofactors for the first time in thrips, and α1/α8/β1 showed abundant current rapidly. Acetylcholine, neonicotinoids, and sulfoxaflor exhibited different activation capacities for the two subtypes of nAChRs. Finally, V65I was found to significantly reduce the binding ability of nAChR to neonicotinoids and sulfoxaflor through electrophysiology and computer simulations. V104I caused a decrease in agonist affinity (pEC50) but an increase in the efficacy (Imax) of nAChR against neonicotinoids and reduced the binding ability of nAChR to sulfoxaflor. This study provides theoretical and technical support for studying the molecular mechanisms of neonicotinoid resistance in pests.

Altered extracellular matrix dynamics is associated with insulin resistance in adolescent children with obesity.

The objective of this study was to examine the hypothesis that abdominal and gluteal adipocyte turnover, lipid dynamics, and fibrogenesis are dysregulated among insulin-resistant (IR) compared with insulin-sensitive (IS) adolescents with obesity. Seven IS and seven IR adolescents with obesity participated in a 3-h oral glucose tolerance test and a multi-section magnetic resonance imaging scan of the abdominal region to examine body fat distribution patterns and liver fat content. An 8-week 70% deuterated water (2 H2 O) labeling protocol examined adipocyte turnover, lipid dynamics, and fibrogenesis in vivo from biopsied abdominal and gluteal fat. Abdominal and gluteal subcutaneous adipose tissue (SAT) turnover rates of lipid components were similar among IS and IR adolescents with obesity. However, the insoluble collagen (type I, subunit α2) isoform measured from abdominal, but not gluteal, SAT was elevated in IR compared with IS individuals. In addition, abdominal insoluble collagen Iα2 was associated with ratios of visceral-to-total (visceral adipose tissue + SAT) abdominal fat and whole-body and adipose tissue insulin signaling, and it trended toward a positive association with liver fat content. Altered extracellular matrix dynamics, but not expandability, potentially decreases abdominal SAT lipid storage capacity, contributing to the pathophysiological pathways linking adipose tissue and whole-body IR with altered ectopic storage of lipids within the liver among IR adolescents with obesity.

Expression of GABAA Receptor Subunits α1 and β2 in Healthy Human Dental Pulp

BACKGROUND: Gamma-aminobutyric acid (GABA), the main inhibitory neurotransmitter in the mammalian central nervous system (CNS), has a well-established role in pain modulation. While numerous studies have delved into the expression of GABA and its receptors in dental pulp, the exact influence of these receptors on dental pain signaling has not been fully elucidated. This study aimed to investigate the gene and protein expression of the two most abundantly expressed GABA type A (GABAA) receptor subunits, GABAA receptor subunit α1 (GABRA1) and β2 (GABRB2), in healthy human dental pulp.METHODS: Six tooth samples were collected from healthy individuals referred for orthodontic treatment. Total RNA was isolated from the pulp tissues of three samples and reverse transcription-polymerase chain reaction (RT-PCR) was performed to assess gene expression of GABRA1 and GABRB2. The other three samples were examined using immunohistochemistry (IHC) for visualization of GABRA1 and GABRB2 proteins within the dental pulp.RESULTS: RT-PCR analysis reported the presence of both GABRA1 and GABRB2 in the dental pulp, and independent t-test analysis revealed that the expression of GABRA1 was significantly higher than GABRB2. The immunohistochemical staining provided compelling visual evidence of the expression of GABRA1 and GABRB2 proteins within the odontoblast layer of dental pulp, clearly indicating their presence in the cell bodies and odontoblastic processes extending into the dentin.CONCLUSION: The presence of α1 and β2 subunits of the GABAA receptor in healthy human dental pulp offers valuable insights for further research into the potential roles of GABAA receptors in dental pain signaling.KEYWORDS: γ-aminobutyric acid, GABAA receptors, GABRA1, GABRB2, dental pain, pain signaling

Normal Proteasome Function Is Needed to Prevent Kidney Graft Injury during Cold Storage Followed by Transplantation.

Kidney transplantation is the preferred treatment for end-stage kidney disease (ESKD). However, there is a shortage of transplantable kidneys, and donor organs can be damaged by necessary cold storage (CS). Although CS improves the viability of kidneys from deceased donors, prolonged CS negatively affects transplantation outcomes. Previously, we reported that renal proteasome function decreased after rat kidneys underwent CS followed by transplantation (CS + Tx). Here, we investigated the mechanism underlying proteasome dysfunction and the role of the proteasome in kidney graft outcome using a rat model of CS + Tx. We found that the key proteasome subunits β5, α3, and Rpt6 are modified, and proteasome assembly is impaired. Specifically, we detected the modification and aggregation of Rpt6 after CS + Tx, and Rpt6 modification was reversed when renal extracts were treated with protein phosphatases. CS + Tx kidneys also displayed increased levels of nitrotyrosine, an indicator of peroxynitrite (a reactive oxygen species, ROS), compared to sham. Because the Rpt6 subunit appeared to aggregate, we investigated the effect of CS + Tx-mediated ROS (peroxynitrite) generation on renal proteasome assembly and function. We treated NRK cells with exogenous peroxynitrite and evaluated PAC1 (proteasome assembly chaperone), Rpt6, and β5. Peroxynitrite induced a dose-dependent decrease in PAC1 and β5, but Rpt6 was not affected (protein level or modification). Finally, serum creatinine increased when we inhibited the proteasome in transplanted donor rat kidneys (without CS), recapitulating the effects of CS + Tx. These findings underscore the effects of CS + Tx on renal proteasome subunit dysregulation and also highlight the significance of proteasome activity in maintaining graft function following CS + Tx.

De novo GABRA1 variants in childhood epilepsies and the molecular subregional effects.

The GABRA1 gene, encoding the GABRAR subunit α1, plays vital roles in inhibitory neurons. Previously, the GABRA1 gene has been identified to be associated with developmental and epileptic encephalopathy (DEE) and idiopathic generalized epilepsy (IGE). This study aims to explore the phenotypic spectrum of GABRA1 and molecular subregional effect analysis. Trios-based whole-exome sequencing was performed in patients with epilepsy. Previously reported GABRA1 mutations were systematically reviewed to analyze the molecular subregional effects. De novo GABRA1 mutations were identified in six unrelated patients with heterogeneous epilepsy, including three missense mutations (p.His83Asn, p.Val207Phe, and p.Arg214Cys) and one frameshift mutation (p.Thr453Hisfs*47). The two missense mutations, p.His83Asn and p.Val207Phe, were predicted to decrease the protein stability but no hydrogen bond alteration, with which the two patients also presented with mild genetic epilepsy with febrile seizures plus and achieved seizure-free status by monotherapy. The missense variant p.Arg214Cys was predicted to decrease protein stability and destroy hydrogen bonds with surrounding residues, which was recurrently identified in three cases with severe DEE. The frameshift variant p.Thr453Hisfs*47 was located in the last fifth residue of the C-terminus and caused an extension of 47 amino acids, with which the patients presented with moderated epilepsy with generalized tonic-clonic seizures alone (GTCA) but achieved seizure-free status by four drugs. The four variants were not presented in gnomAD and were evaluated as "pathogenic/likely pathogenic" according to ACMG criteria. Analysis of all reported cases indicated that patients with mutations in the N-terminal extracellular region presented a significantly higher percentage of FS and DEE, and the patients with variants in the transmembrane region presented earlier seizure onset ages. This study suggested that GABRA1 variants were potentially associated with a spectrum of epilepsies, including EFS+, DEE, and GTCA. Phenotypic severity may be associated with the damaging effect of variants. The molecular subregional effects help in understanding the underlying mechanism of phenotypic variation.

Identifying the prognosis implication, immunotherapy response prediction value, and potential targeted compound inhibitors of integrin subunit α3 (ITGA3) in human cancers

The integrin subunit α3 (ITGA3) is a member of the integrin alpha chain protein family, which could promote progression, metastasis, and invasion in some cancers. Still, its function in the tumor microenvironment (TME), cancer prognosis, and immunotherapy remains unclear. A multifaceted analysis of ITGA3 in pan-cancer utilizing various databases and online web tools revealed ITGA3 was aberrantly expressed in tumor tissues and upregulated in most cancers, which may be related to ITGA3 genomic alterations and methylation modification. In addition, ITGA3 was significantly correlated with the poor or better prognosis of cancer patients, immune-related pathways in hallmark, immune infiltration, and immune checkpoints, revealing a biological function of ITGA3 in the tumor progression, tumor microenvironment, and tumor immunity. We also found that ITGA3 could predict the response to tumor immunotherapy based on cytokine-treated samples and immunotherapy cohorts. ITGA3 may participate in shaping and regulating the tumor microenvironment to affect the tumor immune response, which was a promising immunotherapy response predictive biomarker and potential therapeutic target to work synergistically with cancer immunotherapy to boost the response and efficacy. Finally, potential targeted compound inhibitors and sensitive drugs were screened using databases ConnectivityMap (CMap) and CellMiner, and AutoDock Tools was used for molecular docking.

Oleuropein Stimulates Migration of Human Trophoblast Cells and Expression of Invasion-Associated Markers.

Successful pregnancy establishment requires highly synchronized cross talk between the invasive trophoblast cells and the receptive maternal endometrium. Any disturbances in this tightly regulated process may lead to pregnancy complications. Local factors such as nutrients, hormones, cytokines and reactive oxygen species modulate the invasion of extravillous trophoblasts through critical signaling cascades. Epidemiological studies strongly indicate that a Mediterranean diet can significantly impact molecular pathways during placentation. Therefore, the aim of the current study was to examine whether oleuropein (OLE), one of the main compounds of the Mediterranean diet, may influence trophoblast cell adhesion and migration, as well as the expression of invasion-associated molecular markers and inflammatory pathways fostering these processes. HTR-8/SVneo cells were incubated with OLE at selected concentrations of 10 and 100 µM for 24 h. Results showed that OLE did not affect trophoblast cell viability, proliferation and adhesion after 24 h in in vitro treatment. The mRNA expression of integrin subunits α1, α5 and β1, as well as matrix-degrading enzymes MMP-2 and -9, was significantly increased after treatment with 10 µM OLE. Furthermore, OLE at a concentration of 10 µM significantly increased the protein expression of integrin subunits α1 and β1. Also, OLE inhibited the activation of JNK and reduced the protein expression of COX-2. Finally, a lower concentration of OLE 10 µM significantly stimulated migration of HTR-8/SVneo cells. In conclusion, the obtained results demonstrate the effects of OLE on the function of trophoblast cells by promoting cell migration and stimulating the expression of invasion markers. As suggested from results, these effects may be mediated via inhibition of the JNK signaling pathway.