Receptor Function Research Articles

Dual Activation-Induced Marker Combinations Efficiently Identify and Discern Antigen-Specific and Bystander-Activated Human CD4+ T Cells.

Identifying activated T lymphocytes and differentiating antigen-specific from bystander T cells is crucial for understanding adaptive immune responses. This study investigates the efficacy of activation-induced markers (AIMs) in distinguishing these cell populations. We measured the expression of commonly used AIMs (CD25, CD38, CD40L, CD69, CD137, HLA-DR, ICOS, and OX40) in an in vitro T-cell activation system and evaluated their sensitivity, specificity, and positive predictive value. We demonstrated that individual AIMs, while specific in detecting activated CD4+ T cells, poorly discriminate between antigen-specific and bystander activation, as assessed by a discriminative capacity (DC) score we developed. Our analysis revealed that dual AIM combinations significantly enhanced the ability to distinguish antigen-specific from bystander-activated T cells, achieving DC scores above 90%. These combinations also improved positive predictive value and specificity with a modest reduction in sensitivity. The CD25hi/ICOShi combination emerged as the most efficient, with an average sensitivity of 84.35%, specificity of 99.7%, and DC score of 90.12%. Validation through T-cell cloning and antigen re-stimulation confirmed the robustness of our predictions. This study provides a practical framework for researchers to optimize strategies for identifying and isolating antigen-specific human CD4+ T lymphocytes and studying their phenotype, function, and T-cell receptor repertoire.

Metabolomic profiling of germinated and non-germinated Lablab purpureus seeds: antioxidant properties and α-amylase inhibitory activities for diabetes management

Diabetes is characterized by prolonged hyperglycemia and disruptions in carbohydrate, lipid, and protein metabolism, stemming from inadequate insulin production, impaired insulin receptor functioning, or a combination of both. Conventional diabetes medications like biguanides and sulphonylureas, are widely used and raise concerns about potential side effects with prolonged usage. In this context, legumes emerge as promising candidates due to their significance in traditional diets globally and associated health benefits. Despite being challenging to digest due to anti-nutritive factors, germination, a simple bioprocessing technique, significantly enhances nutritional aspect of the seeds. This study focuses on Lablab purpureus, an underutilized legume, employing a metabolomic approach to explore compounds in germinated and non-germinated seeds. Metabolomic profiling identified 125 compounds in non-germinated and 80 compounds in germinated seeds, revealing unique compounds in each type with potential health benefits. The study identified therapeutically important metabolites such as alkaloids, flavonoids, terpenoids, and saponin in both the germinated and non-germinated seeds. A notable change in the phytochemical composition (total phenol, flavonoid, and total ascorbic acid content) of germinated seeds was observed compared to the non-germinated seeds flour. An increased fold change (1.15, 1.5 and 1.65) was observed in the total phenol, flavonoid, and total ascorbic acid content in germinated seeds compared to non-germinated seeds, alongside higher antioxidant levels in terms of DPPH, ABTS, and FRAP. The IC50 value for α-amylase inhibitory activity was noted to be 2.05 ± 0.05 mg/ml in germinated samples while 0.79 ± 0.00 mg/ml was observed in the non-germinated Lablab purpureus seeds. Therefore, displaying greater α-amylase inhibitory activity in the non-germinated seeds, possibly due to their unique biochemical composition. Nevertheless, even germinated seeds demonstrated appreciable α-amylase inhibitory activity. Therefore, these findings suggest that germination process significantly influences seed biochemistry and helps to raise the phytochemical composition, while the unique composition of the metabolites in the non-germinated seeds could have impact on the α-amylase inhibitory activity. Thus, study suggests Lablab purpureus as a promising functional food source with diverse health-promoting attributes, particularly in diabetes management.Graphical

Understanding paralogous epilepsy–associated GABAA receptor variants: Clinical implications, mechanisms, and potential pitfalls

Recent discoveries have revealed that genetic variants in γ-aminobutyric acid type A (GABAA) receptor subunits can lead to both gain-of-function (GOF) and loss-of-function (LOF) receptors. GABAA receptors, however, have a pseudosymmetrical pentameric assembly, and curiously diverse functional outcomes have been reported for certain homologous variants in paralogous genes (paralogous variants). To investigate this, we assembled a cohort of 11 individuals harboring paralogous M1 proline missense variants in GABRA1, GABRB2, GABRB3, and GABRG2. Seven mutations (α1P260L, α1P260S, β2P252L, β3P253L, β3P253S, γ2P282A, and γ2P282S) in α1β2/3γ2 receptors were analyzed using electrophysiological examinations and molecular dynamics simulations. All individuals in the cohort were diagnosed with developmental and epileptic encephalopathy, with a median seizure onset age of 3.5 mo, and all exhibited global developmental delay. The clinical data for this cohort aligned with established GABAA receptor GOF but not LOF cohorts. Electrophysiological assessments revealed that all variants caused GOF by increasing GABA sensitivity by 3- to 23-fold. In some cases, this was accompanied by LOF traits such as reduced maximal current amplitude and enhanced receptor desensitization. The specific subunit mutated and whether the mutation occurred in one or two subunits within the pentamer influenced the overall effects. Molecular dynamics simulations confirmed similar structural changes from all mutations, but with position-dependent asymmetry. These findings establish that paralogous variants affecting the 100% conserved proline residue in the M1 transmembrane helix of GABAAR subunits all lead to overall GOF traits. The unexpected asymmetric and mixed effects on receptor function have broader implications for interpreting functional analyses for multimeric ion-channel proteins.

Behavioral decline in Shank3Δex4–22 mice during early adulthood parallels cerebellar granule cell glutamatergic synaptic changes

BackgroundSHANK3, a gene encoding a synaptic scaffolding protein, is implicated in autism spectrum disorder (ASD) and is disrupted in Phelan-McDermid syndrome (PMS). Despite evidence of regression or worsening of ASD-like symptoms in individuals with PMS, the underlying mechanisms remain unclear. Although Shank3 is highly expressed in the cerebellar cortical granule cells, its role in cerebellar function and contribution to behavioral deficits in ASD models are unknown. This study investigates behavioral changes and cerebellar synaptic alterations in Shank3Δex4–22 mice at two developmental stages.MethodsShank3Δex4–22 wildtype, heterozygous, and homozygous knockout mice lacking exons 4–22 (all functional isoforms) were subjected to a behavioral battery in both juvenile (5–7 weeks old) and adult (3–5 months old) mouse cohorts of both sexes. Immunostaining was used to show the expression of Shank3 in the cerebellar cortex. Spontaneous excitatory postsynaptic currents (sEPSCs) from cerebellar granule cells (CGCs) were recorded by whole-cell patch-clamp electrophysiology.ResultsDeletion of Shank3 caused deficits in motor function, heightened anxiety, and repetitive behaviors. These genotype-dependent behavioral alterations were more prominent in adult mice than in juveniles. Reduced social preference was only identified in adult Shank3Δex4–22 knockout male mice, while self-grooming was uniquely elevated in males across both age groups. Heterozygous mice showed little to no changes in behavioral phenotypes in most behavioral tests. Immunofluorescence staining indicated the presence of Shank3 predominantly in the dendrite-containing rosette-like structures in CGCs, colocalizing with presynaptic markers of glutamatergic mossy fiber. Electrophysiological findings identified a parallel relationship between the age-related exacerbation of behavioral impairments and the enhancement of sEPSC amplitude in CGCs.LimitationsOther behavioral tests of muscle strength (grip strength test), memory (Barnes/water maze), and communication (ultrasonic vocalization), were not performed. Further study is necessary to elucidate how Shank3 modulates synaptic function at the mossy fiber-granule cell synapse in the cerebellum and whether these changes shape the behavioral phenotype.ConclusionsOur findings reveal an age-related exacerbation of behavioral impairments in Shank3Δex4–22 mutant mice. These results suggest that Shank3 may alter the function of glutamatergic receptors at the mossy fiber-cerebellar granule cell synapse as a potential mechanism causing cerebellar disruption in ASD.

Revisiting the follicle-stimulation hormone receptor expression and function in human myometrium and adipose tissue

BackgroundExtragonadal follicle-stimulating hormone receptor (FSHR) expression at low levels has been shown in several normal and tumor tissues, including myometrium and adipose tissue. FSH-FSHR signaling in the myometrium has been suggested to regulate uterine contractile activity and the timing of labor. In contrast, FSH-FSHR has been linked to the activation of brown/beige fat thermogenesis in adipose tissue. The issue of extragonadal FSHR expression and its functionality remains contentious within the scientific community, as contradictory findings necessitate further independent and critical analyses. Hereby, we re-investigated the FSHR expression and its functionality in normal non-pregnant (M-NP) and pregnant (N-P) human myometrium, as well as in human visceral (VAT) and subcutaneous (SCAT) adipose tissue (AT).MethodsFSHR expression at mRNA (real-time qPCR, RNAscope in situ hybridization) and protein (immunohistochemical staining) levels in adipose tissue, myometrium, and adipocytes were evaluated. Myometrium and adipocytes were treated with recombinant (rh)FSH to study its effects on functional pathways. Myometrium contractile activity was measured using a force transducer with digital output and the DASYLab software unit. Cyclic adenosine monophosphate (cAMP) production by myometrium explants and adipocytes was measured using a cAMP ELISA Kit. The activation of the AKT pathway in myometrium and adipocytes was analyzed by Western blot analysis.ResultsContrary to previous observations, we found no expression of FSHR at either mRNA or protein levels in M-NP, N-P, VAT, and SCAT. Treatment with recombinant human FSH (rhFSH) showed no effect on cAMP production or phosphorylation of AKT in M-NP, N-P, as well as in VAT and SCAT. rhFSH treatment did not influence contractile activity in M-NP, N-P.ConclusionsThese findings suggest that the FSHR signaling pathway does not regulate myometrial contractility during pregnancy. Additionally, the absence of FSHR expression in both VAT and SCAT implied that FSHR does not play a role in the functional signaling pathways in adipose tissues. In conclusion, our findings contradict earlier data on the involvement of FSH-FSHR signaling in regulating myometrial contractility near term, as well as in adipose tissue function.

Genetic variation of HPA axis activity in farm animals and beyond.

Many experimental data in several species clearly demonstrate the important genetic contribution to variations in HPA axis activity. The influence of corticosteroid hormones on adaptive processes and on production traits such as growth rate, feed efficiency, carcass composition and meat quality is a strong impetus to the search for the molecular bases of these differences for efficient genetic selection. Three main sources of genetic variability have been documented so far in farm animal species, the adrenal cortex sensitivity to ACTH regulating corticosteroid hormone production, the bioavailability of corticosteroid hormones and especially corticosteroid-binding globulin capacity, and glucocorticoid receptor function. The effect of single mutations may be dependent on the genetic background and genetic variation of cortisol levels may have different functional consequences depending on the molecular mechanisms responsible for this change. Understanding the genetic basis of HPA axis activity allows the development of genomic tools and breeding technologies aimed at improving adaptive capacity and stress tolerance in farm animals and their use as valuable models for the genetic study of the HPA axis and the correlation with adaptation, metabolism and other functions regulated by adrenal hormones, and associated pathologies (obesity, cardiovascular, etc.). The next step will be to explore HPA axis variability from a system genetics perspective including the multiple sources of variation and their interactions. This multifactorial approach is a prerequisite to the use of the HPA axis phenotypes in the genetic selection for more productive and robust animals, with a high level of production of quality products.

Role of phosphorylated Y1252, Y1336 and Y1472 on NR2B subunits in hypoxia tolerance of neuronal cell in vitro.

The N-methyl-D-aspartate (NMDA) receptors are related to the various functioning of the nervous system. It has been shown that the NR2B subunit plays an important role in neurological hypoxic/ischemic diseases by regulating NMDA receptor function. NR2B tyrosine phosphorylation is also an important regulatory mechanism for NMDA receptor function. However, the mechanism of NR2B tyrosine phosphorylation in hypoxic/ischemic injury is still unclear. Therefore, in the present study, we aimed to further clarify the changes in NR2B tyrosine phosphorylation in hypoxic/ischemic damage in the brain and its relationship with neuronal survival under hypoxic/ischemic conditions. Four types of NR2B tyrosine site mutants (Tyr → Phe at 1252, 1336, and 1472, and all three mutations together, named Y1252F, Y1336F, Y1472F, and Triple) and wild-type plasmids were transfected into HT22 cells. The cells were then exposed to oxygen-glucose deprivation and reoxygenation (OGD/R). NR2B, cell apoptosis-related molecules, and neuronal survival factor CREB-related signaling proteins (CaMKII, ERK, Akt) were measured. Cell viability was assessed using the CCK-8 assay. Cell apoptosis and cell cycle were evaluated using flow cytometry. The death ratio of HT22 cells under OGD conditions was further tested using a live cell analysis platform. The viability of HT22 cells in the Y1252F, Y1336F, Y1472F, Triple mutants, and wild-type groups was elevated. Compared to the wild-type, western blotting and real-time PCR showed that Y1252F, Y1336F, Y1472F, and Triple mutants downregulated the expression of apoptosis factors and upregulated anti-apoptosis factors in the OGD/R model. Flow cytometry and cell cycle analysis demonstrated that Y1252F, Y1336F, Y1472F, and Triple mutants reduced the apoptosis rate. The percentage of cells in the S phase decreased significantly. Live cell analysis illustrated that the Y1252F, Y1336F, Y1472F, and Triple mutants contributed to HT22 cell survival under OGD conditions. Additionally, the Y1252F, Y1336F, Y1472F, and Triple mutants activated the survival signaling pathway. Furthermore, compared to the control group (without plasmid), only the Y1336F, Y1472F, and Triple mutants groups showed significant differences in the above tests. The tyrosine phosphorylation of NR2B at Y1336 and Y1472 plays key roles in hypoxic/ischemic injury. These phosphorylation sites may be potential targets for hypoxic/ischemic neural protection.

Regulation of receptor function in NiCo2O4-SnO2 heterojunction for H2S detection at room temperature

In recent years, the World Health Organization has increasingly emphasized air quality in production environments, leading to heightened societal demands for monitoring of pollutant gases at room temperature. Traditional semiconductor gas-sensitive materials, such as tin oxide (SnO2), have their gas-sensing performance largely limited by their receptor functions, resulting in common issues like low response and poor recovery at room temperature. Spinel-type bimetallic oxides, such as nickel cobaltate (NiCo2O4), offer a unique solution due to their rich adsorption sites on the surface, which provide distinctive receptor functions for detecting toxic gases at room temperature. Herein, NiCo2O4 is synthesized via a one-step hydrothermal method, with a porous spherical cluster structure, and combined with SnO2 to form a heterojunction. The NiCo2O4-SnO2 heterojunction film gas sensor exhibits excellent gas-sensing performance for H2S at room temperature, including high response, short response time, good repeatability, and selectivity. Additionally, the unique receptor functions of the NiCo2O4 were analyzed through first-principles calculations, revealing a semiconductor p-n conversion phenomenon in the presence of H2S gas. The composite also demonstrates a conversion from p-n heterojunction to n-n homojunction during the sensing process, enhancing its gas-sensing performance. This work not only addresses the receptor function limitations of traditional gas-sensitive semiconductor but also provides a feasible approach for controlling carrier types in semiconductors.

The modulation of cholecystokinin receptor 1 in the NAc core input from VTA on METH-induced CPP acquisition

BackgroundMethamphetamine (METH) is a potent psychostimulant that interferes the functionality of various brain regions and nervous connections, leading to addiction. The nucleus accumbens core (NAcC), primarily composed of gamma-aminobutyric acid (GABAergic) neurons, serves as a critical nucleus intimately related to addictive behavior. Previous research has indicated the involvement of cholecystokinin (CCK) receptors in drug addiction, yet the precise function of CCK receptors within the neural circuitry mediating METH-induced addiction remains elusive. MethodsMETH-induced conditioned place preference (CPP) model was established in mice. In CCK receptor 1 conditional knockout (CCK1Rflox/flox) or CCK receptor 2 conditional knockout (CCK2Rflox/flox) mice, we then utilized the adeno-associated virus (AAV) transfection system to knock out the specific CCK receptor subtype and explore the function of the CCK receptors in the ventral tegmental area (VTA) to NAcC circuit during METH-induced CPP acquisition. ResultsDuring the acquisition of METH-induced CPP, the expression of CCK1R, but not CCK2R, was upregulated specifically in NAcC. Genetic disruption of either CCK1R in the NAcC effectively hindered METH-induced CPP acquisition and prevented the hyper-excitability of neurons triggered by METH. Furthermore, CCK is released by dopaminergic neurons in the VTA, projecting to the NAcC. Notably, specifically knocking out CCK1R in the VTADA → NAcCGABA circuit blocked the presynaptic release and synaptic plasticity enhancement induced by METH. ConclusionsThese discoveries highlight the critical effect of CCK1R in the VTADA → NAcCGABA circuit on METH-induced CPP acquisition and provide a more comprehensive understanding of the mechanisms underlying CCK receptors contributing to the METH-induced addictive behavior.

Cooperative tumor inhibition by CpG-oligodeoxynucleotide and cyclic dinucleotide in head and neck cancer involves T helper cytokine and macrophage phenotype reprogramming

Head and neck cancer ranks as the sixth most common cancer worldwide, highlighting the critical need for the development of new therapies to enhance treatment efficacy. The activation of innate immune receptors given their potent immune stimulatory properties aid in the eradication of cancer cells. In this study, we investigated the immune mechanism and anti-tumor function of a Toll-like receptor 9 (TLR9) agonist, CpG-oligodeoxynucleotide-2722 (CpG-2722), in combination with cyclic dinucleotides, which are agonists of stimulator of interferon genes (STING). Our results revealed that CpG-2722 stimulation increased the expression of Th1 pro-inflammatory cytokines. Stimulation by STING agonists exhibited lower expression of Th1 cytokines but higher expression of Th2 cytokines compared to CpG-2722. However, the combination of these two agonists significantly enhanced Th1 cytokines while reducing Th2 cytokines. Moreover, in vivo experiment showed that both CpG-2722 and 2'3'-c-di-AMP suppressed head and neck tumor growth, with their combination proving more effective than the use of these agonists alone. The combined treatment cooperatively promoted the production of Th1 cytokines and type I interferons, while suppressing Th2 cytokines in the tumors as observed in vitro. Additionally, it led to the accumulation of M1 macrophages, dendritic cells, and T cells, shaping a favorable tumor microenvironment for T cell-mediated tumor killing. The anti-tumor activity of the CpG-2722 and 2'3'-c-di-AMP combination depends on the macrophage presence but does not directly activate M1 macrophage polarization, instead working through a reprogrammed cytokine profile. Furthermore, this combination shows a cooperative anti-tumor activity with anti-PD-1 in treating head and neck tumors. Overall, these findings highlight a Th response and macrophage phenotype reprograming involved functional mechanism underlying the cooperative activity of the combination of TLR9 and STING agonists in the immunotherapy of head and neck cancer.

Expression of concern regarding “The effect of moderate intensity aerobic exercise on cardiovascular function, cardiorespiratory fitness and estrogen receptor alpha gene in overweight/obese postmenopausal women: A randomized controlled trial”

Expression of concern regarding “The effect of moderate intensity aerobic exercise on cardiovascular function, cardiorespiratory fitness and estrogen receptor alpha gene in overweight/obese postmenopausal women: A randomized controlled trial”

Inhibition of lysophosphatidic acid receptor 2 attenuates neonatal chronic lung disease in mice by preserving vascular and alveolar development

AimBronchopulmonary dysplasia (BPD) is a common morbidity in extremely premature infants. Previous studies demonstrated the important role of lysophosphatidic acid (LPA) in inflammation in BPD. However, the role of LPA and its receptors in hyperoxia-induced vascular malformations in BPD remains to be elucidated. Methods and ResultsElevated plasma LPA levels were observed in mice with BPD compared to controls (792 vs. 607 ng/mL, p < 0.05). Inhibition of LPA signaling protected against hyperoxia-induced lung injury in neonatal mice, demonstrated by a 2.8-fold increase in pulmonary vascular density and a 14% reduction in alveolar enlargement. In vitro studies showed that LPA suppressed tube formation in human umbilical vein endothelial cells (HUVECs) by approximately 50%. LPA receptor 2 (LPA2) was identified as a functional LPA receptor in primary endothelial cells from the lungs of hyperoxic mice and in HUVECs under hyperoxic conditions. The LPA2 antagonist H2L5186303 enhanced the tube formation ability of HUVECs exposed to LPA, both under normoxia (4-fold) and hyperoxia (5-fold). Moreover, H2L5186303 significantly protected against hyperoxia-induced vascular malformation (2-fold) and improved alveolarization in neonatal mice (12% decrease in mean linear intercept, MLI). Early growth response 1 (EGR1) was characterized as a downstream target of LPA2, silencing EGR1 restored tube formation in HUVECs exposed to LPA and hyperoxia. ConclusionsOur in vitro and in vivo findings demonstrate that the inhibition of LPA/LPA2 signaling mitigates hyperoxia-induced pulmonary vascular malformations, suggesting the LPA/LPA2-dependent signaling pathway has therapeutic potential for extremely premature infants with BPD.

NcBRI1 positively regulate vascular development and promote biomass production in Neolamarckia cadamba

Brassinosteroids (BRs) are essential phytohormones that play a crucial role in plant growth and development. However, our understanding of BR receptors and their functions in tree species is currently limited. In this study, we looked for potential BR receptor genes in the burflower-tree (Neolamarckia cadamba) genome. We identified five candidate gene from sequence analysis and phylogenetic reconstruction. Among these genes, Neolamarckia cadamba BRASSINOSTEROID-INSENSITIVE 1 (NcBRI1) is ubiquitously expressed in all tested tissues and encodes a functional BR receptor localized to the plasma membrane. Ectopic expression of NcBRI1 in the Arabidopsis (Arabidopsis thaliana) loss-of-function BRI1 mutant bri1-5 not only rescued its growth retardation phenotype but also facilitated vascular development by reactivating BR signal transduction. Furthermore, overexpression of NcBRI1 promoted vascular formation and cell elongation in transgenic hairy roots of Neolamarckia cadamba. By contrast, microRNA-mediated knockdown of NcBRI1 resulted in delayed vascular development and smaller cells. Importantly, we found that manipulation of NcBRI1 in Neolamarckia cadamba can enhance the biomass of hairy roots. These findings highlight the critical role of NcBRI1 in BR signaling and its significant influence on vascular development and rapid growth in Neolamarckia cadamba.

Ligand Binding Kinetics to Evaluate Function and Stability of A2AR in Nanodiscs.

G-Protein coupled receptors (GPCRs) represent one of the largest classes of therapeutic targets. However, developing successful therapeutics to target GPCRs is a challenging endeavor with many molecules failing during in vivo clinical trials due to a lack of efficacy. The in vitro identification of drug targeted residence time (1/koff) has been suggested to improve prediction of in vivo success. Here, a ligand binding assay using fluorescence anisotropy was implemented to successfully determine on-rates (kon) and off-rates (koff) of labeled and unlabeled ligands binding to the adenosine A2A receptor (A2AR) purified into nanodiscs (A2AR-NDs). The kinetic assay was used to determine the optimal storage conditions of A2AR-NDs, where they were found to be stable for more than six months at -80oC. The binding assay was implemented to further understand receptor function by determining the effects of charged lipids on agonist binding kinetics, how sodium levels allosterically modulate A2AR function, and how A2AR protonation affects agonist binding.

Drug-herb Synergistic Interactions between Clopidogrel and Natural Medicine.

Natural medicine (NM) has been used since ancient times for therapeutic purposes worldwide. Presently, the combination of clopidogrel and NM with a reasonable synergistic effect has gained increasing acceptance in clinical therapeutics. Here, we have performed a comprehensive retrieval of literature published in both English and Chinese databases until August 1, 2022, studying the synergistic interactions of clopidogrel and NM through pharmacokinetic/pharmacodynamic (PK-PD) analyses. We retrieved 7, 3, and 5 studies on PK analysis and 3, 3, and 8 studies on PD analysis for the interaction of clopidogrel with single herbal medicines, bioactive compounds, and herbal prescriptions, respectively. Most studies on NM have been found to mainly focus on preclinical observations, and there have been fewer clinical PK analyses. A potential drug-herb interaction has been observed to occur when clopidogrel and NM were metabolized by an enzyme network comprising P-gp, CES1, and CYP450. In contrast, most PD studies have focused on clinical observations, and few preclinical findings have been reported. Some cases have suggested that the combination of the two types of drugs would alter the antiplatelet efficacy and adverse effects. Studies on PK, however, have shown significant or slightly varying results for the drug prototype and its metabolites. In the combination therapies, the interaction between clopidogrel and NM was found to alter antiplatelet aggregation pathways and P2Y12 receptor function.

Neuritogenesis and protective effects activated by Angiotensin 1–7 in astrocytes-neuron interaction

The renin angiotensin system (RAS) has been studied for its effects on various neurological disorders. The identification of functional receptors for Ang-(1–7) and Ang II peptides in astrocytes highlights the physiological modulation and the important role of these cells in the central nervous system. The present study aims to understand the role of RAS peptides, particularly Ang-(1–7) and Ang II, in the secretion of trophic factors by astrocytes and their effects on hippocampal neurons. We used primary cultures of astrocytes and neurons from the hippocampus of either sex neonate of Wistar strain rats. In the present study, we demonstrated that the treatment of astrocytes with Ang-(1–7) acts on the modulation of these cells, inducing reactive astrogliosis, identified through the increase in the expression of GFAP. Furthermore, we obtained a conditioned medium from astrocytes treated with Ang-(1–7), which in addition to promoting the secretion of neurotrophic factors essential for neuronal-glial interactions that are fundamental for neuritogenesis and neuronal survival, showed a neuroprotective effect against glutamatergic excitotoxicity. In turn, Ang II does not exhibit the same effects on astrocyte modulation, exacerbating deleterious effects on brain RAS. Neuron-astrocyte interactions have been shown to be an integral part of the central effects mediated by RAS, and this study has significantly contributed to the understanding of the biochemical mechanisms involved in the functioning of this system.

Receptor interactions of protoxin and activated Vip3Aa structural conformations in Spodoptera exigua.

The Vip3Aa insecticidal protein, produced by Bacillus thuringiensis, has been effectively used in commercial Bt-crops to manage lepidopteran pests. Upon ingestion by larvae, the protoxin is processed by midgut proteases into the activated protein and binds specifically to its receptors in the midgut, leading to insect mortality. Cryo-EM resolution of the trypsin-processed Vip3Aa protein unveiled structural remodelling of the N-terminal region during the transition from protoxin to activated protein. This conformational change has been demonstrated to be crucial for toxicity against Spodoptera exigua larvae, a major global lepidopteran pest. In this study, we investigated the relevance of the structural remodelling for the specific binding to midgut receptors. We conducted in vitro binding assays with radiolabelled proteins and brush border membrane vesicles (BBMV) from S. exigua, employing structural mutants that lock the protein in either its protoxin or its activated conformation. Our results indicate that both structural stages of the protein share binding sites in the midgut epithelium. Moreover, in vivo competition assays revealed that Vip3Aa is able to bind to functional receptors in S. exigua larvae both as protoxin and as activated protein. Altogether, our findings point to both structural conformations contributing to receptor binding. In vivo, either spontaneous structural shift upon proteolytic cleavage or receptor-mediated remodelling could be occurring. However, the timing and context in which the conformational change occurs could influence membrane insertion and toxicity. Our results show the complex interplay between proteolytic processing, protein structure and receptor interactions in Vip3Aa's toxicity. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

A dendritic cell vaccine for both vaccination and neoantigen-reactive T cell preparation for cancer immunotherapy in mice

Adoptive cell transfer (ACT) using neoantigen-specific T cells is an effective immunotherapeutic strategy. However, the difficult isolation of neoantigen-specific T cells limits the clinical application of ACT. Here, we propose a method to prepare neoantigen-reactive T cells (NRT) for ACT following immunization with a tumor lysate-loaded dendritic cell (DC) vaccine. We show that the DC vaccine not only induces a neoantigen-reactive immune response in lung cancer-bearing mice in vivo, but also facilitate NRT cell preparation in vitro. Adoptive transfer of the NRTs as combinatorial therapy into DC vaccine-immunized, LL/2 tumor-bearing mice allows infiltration of the infused NRTs, as well as the enrichment of neoantigen reactive, non-ACT/NRT T cells into the tumor microenvironment with the function of these neoantigen-reactive T-cell receptors validated in vitro. In summary, we propose a method for preparing NRTs that increases ACT efficacy and paves the way to the design of personalized immunotherapies.

Inositol 1,4,5-Trisphosphate Receptor Mutations Associated with Human Disease: Insights into Receptor Function and Dysfunction.

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are ubiquitous intracellular Ca2+ release channels. Their activation, subcellular localization, abundance, and regulation play major roles in defining the spatiotemporal characteristics of intracellular Ca2+ signals, which are in turn fundamental to the appropriate activation of effectors that control a myriad of cellular events. Over the past decade, ∼100 mutations in ITPRs associated with human diseases have been documented. Mutations have been detailed in all three IP3R subtypes and all functional domains of the protein, resulting in both gain and loss of receptor function. IP3R mutations are associated with a diverse array of pathology including spinocerebellar ataxia, peripheral neuropathy, immunopathy, anhidrosis, hyperparathyroidism, and squamous cell carcinoma. This review focuses on how studying the altered activity of these mutations provides information relating to IP3R structure and function, the physiology underpinned by specific IP3R subtypes, and the pathological consequences of dysregulated Ca2+ signaling in human disease.

Effects of a protease inhibitor camostat mesilate on gut microbial function in patients with irritable bowel syndrome: A pilot randomized placebo-controlled study

Introduction: Increased fecal protease activity which may induce visceral hypersensitivity has been observed in patients with irritable bowel syndrome (IBS). Serine proteases modulate FK506 binding protein (FKBP)-type peptidylprolyl cis-trans isomerase (PPIase) activity associated with immune and glucocorticoid receptor functions. The aims were to investigate whether camostat mesilate (CM), a serine protease inhibitor, modifies fecal bacterial function associated with FKBP-type PPIases ameliorates in patients with IBS. Methods: Randomly assigned 16 patients with IBS received 200 mg po tid of CM and 16 patients received placebo for 14 days. Self-reported adequate relief (AR) as a primary endpoint, IBS Symptom Severity Scale (IBS-SSS) and colonic motor and pain thresholds to colorectal distention were assessed before and after treatment. The fecal bacterial content was inferred from 16S rRNA gene sequence data using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Results: CM significantly increased the relative abundance of Streptococcus and the functional abundances of serine protease and FKBP-type PPIase FklB and SlyD more than placebo after treatment. CM treatment was not superior to placebo in proportion of AR although colonic motor response partially changed. Conclusion: CM modulated the fecal microbiome composition and functional potentials that are related to FKBP-type PPIase activity in IBS patients. These findings suggest that protease inhibitors may modify gut microbial function along with abnormal immunological and/or stress responses that underlie pathophysiology of IBS.