High-affinity Receptor Research Articles

A Randomized, Controlled Trial of Palonosetron Versus Ondansetron for Nausea, Vomiting, and Pruritus in Cesarean Delivery with Intrathecal Morphine.

Spinal anesthesia is the preferred anesthetic technique for cesarean deliveries. Postoperative nausea and vomiting (PONV) and pruritus occur in up to 80% and 83% of patients, respectively, after cesarean delivery with intrathecal opioids. Ondansetron is the recommended medication for PONV prophylaxis, but palonosetron, a second-generation 5-HT3 receptor antagonist, has a higher receptor affinity and a longer half-life. However, studies on palonosetron use in cesarean deliveries are limited. This study aimed to determine whether palonosetron was more effective than ondansetron in preventing intrathecal morphine-induced PONV and pruritus in cesarean deliveries. Parturients who underwent cesarean delivery under spinal anesthesia were randomized into 3 groups: P (palonosetron 0.075 mg), O (ondansetron 4 mg), and N (normal saline). The study drug was intravenously administered after the umbilical cord was clamped. The primary outcome measures were the 48-hour incidence of PONV and pruritus. The secondary outcome measures were the PONV and pruritus scores at the postanesthesia care unit (PACU) and ward, rescue medications, satisfaction scores, and adverse events. Ordinal data were analyzed using the Kruskal-Wallis test. Continuous and categorical data were analyzed using a 1-way analysis of variance, Kruskal-Wallis test, and Pearson's χ2 test, respectively. A value of P < .05 was considered significant. Post hoc analysis pairwise comparisons with Bonferroni correction were also performed. Overall, 300 parturients were enrolled, and 297 parturients completed the study. One patient in the P group and 2 in the O group were excluded because of conversion to general anesthesia after failed spinal anesthesia. The baseline patient characteristics were comparable between the groups. The PONV incidence rates in the P, O, and N groups were 26.3% (95% confidence interval [CI], 17.4-35.1), 34.7% (95% CI, 25.1-44.3), and 50.0% (95% CI, 40.0-59.9), respectively (P = .002). The incidence rates of pruritus in the P, O, and N groups were 69.7% (95% CI, 60.5-78.9), 76.5% (95% CI, 67.9-85.1), and 87.0% (95% CI, 80.3-93.7), respectively (P = .013). Pairwise comparisons revealed significantly lower incidences of PONV and pruritus in the P group than in the N group (P < .001 and P = .003, respectively). However, no significant differences were observed between the P and O groups or between the O and N groups. Additionally, the P group required significantly less nalbuphine rescue for pruritus than the N group (P = .004 and P = .005 for the PACU and ward, respectively). PONV rescue, satisfaction scores, and adverse events were not significantly different among the 3 groups. Palonosetron effectively prevents intrathecal morphine-induced PONV and pruritus during cesarean delivery. However, the efficacy of palonosetron is not significantly different from that of ondansetron.

Melatonin as an inducer of Th17 cell differentiation: new mechanisms

Cells of the immune system are sensitive to the action of melatonin, and the most obvious target of the hormone is the Th17 T helper subset: in addition to two high-affinity membrane receptors for melatonin, MT1 and MT2, these cells express a nuclear receptor, RORα. Among the secondary messengers involved in the transmission of signals from melatonin receptors, proteins of the sirtuin family are currently of particular interest. It is known that in non-tumor cells, sirtuin 1 (SIRT1) not only increases its expression in response to melatonin, but is also involved in the implementation of melatonin effects, as indicated by inhibitory assays using a specific SIRT1 blocker or corresponding siRNA/shRNA. This relates to the regulation of circadian oscillators, as well as the anti-inflammatory, antioxidant and anti-apoptotic effects of melatonin. Further mechanisms of SIRT1 activity in the cell include transcriptional and posttranscriptional regulation of gene expression through deacetylation of histones and non-histone proteins, and the apparent target of SIRT1 is the transcription factor RORα. It is this factor that mediates classical melatonin/SIRT1-dependent transcriptional control of key circadian regulators, the genes of which have ROR-binding sequences in their promoters. These data raise questions about the functions of SIRT1 in other cells expressing RORα, in particular in Th17 T helper cells, for which it is one of two key differentiation factors, along with RORγt. And if for RORα such a connection still remains hypothetical, for RORγt it has been convincingly demonstrated in studies both in vivo and in vitro: it has been shown that SIRT1 directly binds to RORγt and stimulates the development of Th17 cells, and blockade of sirtuin suppresses the differentiation of these cells in normal and prevents the development of Th17-associated pathology in mice. Summarizing these data, we can confidently predict the existence of a new mechanism for the regulation of the T helper Th17 population by melatonin, through the activation of sirtuin SIRT1, and this mechanism must be taken into account when interpreting data on the immunoregulatory activity of melatonin.

Molecular mechanism of IgE-mediated FcεRI activation.

Allergic diseases, affecting over a quarter of individuals in industrialized countries, have become significant public health concerns1,2. The high-affinity Fc receptor for IgE (FcεRI), mainly present on mast cells and basophils, plays a crucial role in allergic diseases3-5. Monomeric IgE binding to FcεRI regulates mast cell survival, differentiation, and maturation6-8. However, the underlying molecular mechanism remains unclear. Here we demonstrate that, prior to IgE binding, FcεRI mostly exists as a homo-dimer on human mast cell membrane. The structure of human FcεRI confirms the dimeric organization, with each promoter comprising one α subunit, one β subunit, and two γ subunits. The transmembrane helices of the α subunits form a layered arrangement with those of the γ and β subunits. The dimeric interface is mediated by a four-helix bundle of the α and γ subunits at the intracellular juxtamembrane region. Cholesterol-like molecules embedded within the transmembrane domain may stabilize the dimeric assembly. Upon IgE binding, the dimeric FcεRI dissociates into two protomers, each binding to an IgE molecule. Importantly, this process elicits transcriptional activation of Egr1/3 and Ccl2 in rat basophils, which can be attenuated by inhibiting the FcεRI dimer-to-monomer transition. Collectively, our study unveils the mechanism of antigen-independent, IgE-mediated FcεRI activation.

Targeting overexpressed antigens in glioblastoma via CAR T cells with computationally designed high-affinity protein binders.

Chimeric antigen receptor (CAR) T cells targeting receptors on tumour cells have had limited success in patients with glioblastoma. Here we report the development and therapeutic performance of CAR constructs leveraging protein binders computationally designed de novo to have high affinity for the epidermal growth factor receptor (EGFR) or the tumour-associated antigen CD276, which are overexpressed in glioblastoma. With respect to T cells with a CAR using an antibody-derived single-chain variable fragment as antigen-binding domain, the designed binders on CAR T cells promoted the proliferation of the cells, the secretion of cytotoxic cytokines and their resistance to cell exhaustion, and improved antitumour performance in vitro and in vivo. Moreover, CARs with the binders exhibited higher surface expression and greater resistance to degradation, as indicated by bulk and single-cell transcriptional profiling of the cells. The de novo design of binding domains for specific tumour antigens may potentiate the antitumour efficacy of CAR T cell therapies for other solid cancers.

7940 Is Urocortin 2 A Mediator Of Stress-induced Suppression Of The Luteinizing Hormone Surge?

Abstract Disclosure: H.F. Bell: None. R.A. Carrasco: None. M.J. Kreisman: None. R.B. McCosh: None. K.M. Breen Church: None. Stress is thought to be a contributing factor to the reduction of reproductive function and infertility observed in many different species. Although this is a well-documented phenomenon, the neural mechanisms mediating the suppression of reproductive neuroendocrine activity in response to stress are not yet fully understood. The paraventricular nucleus (PVN) of the hypothalamus is an integral part of the neural response to stress. The PVN initiates activation of Corticotropin Releasing Hormone (CRH) neurons and, in addition to CRH, the PVN contains a population of neurons expressing Urocortin 2 (UCN2), a CRH-like family member, with high affinity for CRH receptor 2. In females, stress has been shown to inhibit both the pulse and surge modes of Luteinizing Hormone (LH) secretion. We have preliminary evidence that kisspeptin neurons, controlling either type of LH secretion, contain CRH receptor 2. In this study we tested the sufficiency of UCN2 to disrupt the estradiol-induced LH surge. Female mice were ovariectomized and given a high estradiol treatment to induce a circadian-timed LH surge. Two days later, UCN2 or Vehicle was administered into the lateral ventricle via ICV injection on the morning of the expected surge (10 AM). Blood and brains were collected in the evening (6 PM), at the time of the expected surge. Whole blood from the tail tip was measured by LH ELISA and a surge was defined as &amp;gt; 0.5 ng/mL. A majority of saline-treated animals expressed an LH surge (8 of 9 females). In contrast, 0% of UCN2-treated animals surged (n=7), suggesting that UCN2 impaired the positive feedback response to estradiol. Additionally, immunohistochemistry was performed to observe colocalization of c-Fos within Kisspeptin neurons in the rostral periventricular region of the third ventricle (RP3V) which are responsible for surge generation. Lower Kisspeptin/C-Fos co-expression in animals treated with UCN2 demonstrated the efficacy of UCN2 to impair estradiol-induced activation of RP3V Kisspeptin neurons which are critical for LH surge generation. These results support the hypothesis that UCN2 signaling plays a mediatory role in the stress-induced reduction of ovulatory function in the female mouse. Presentation: 6/1/2024

Acetaminophen Overdose Reveals PAR4 as a Low-Expressing but Potent Receptor on the Hepatic Endothelium in Mice.

The protease thrombin, which elicits multiple physiological and pathological effects on vascular endothelial cells (ECs), can signal through PARs (protease-activated receptors) 1 and 4. PAR1 is a high-affinity thrombin receptor known to signal on ECs, whereas PAR4 is a low-affinity thrombin receptor, and evidence for its expression and function on ECs is mixed. This study aims to exploit the high levels of thrombin generation and hepatic vascular dysfunction that occur during acetaminophen (APAP) overdose to determine (1) whether hepatic endothelial PAR4 is a functional receptor, and (2) the endothelial-specific functions for PAR1 and PAR4 in a high thrombin and pathological setting. We generated mice with conditional deletion of Par1/Par4 in ECs and overdosed them with APAP. Hepatic vascular permeability, erythrocyte accumulation in the liver, thrombin generation, and liver function were assessed following overdose. Additionally, we investigated the expression levels of endothelial PARs and how they influence transcription in APAP-overdosed liver ECs using endothelial translating ribosome affinity purification followed by next-generation sequencing. We found that mice deficient in high-expressing endothelial Par1 or low-expressing Par4 had equivalent reductions in APAP-induced hepatic vascular instability, although mice deficient for both receptors had lower vascular permeability at an earlier timepoint after APAP overdose than either of the single mutants. Additionally, mice with loss of both endothelial Par1 and Par4 had reduced thrombin generation after APAP overdose, suggesting decreased hypercoagulability. Last, we found that endothelial PAR1-but not PAR4-can regulate transcription in hepatic ECs. Low-expressing PAR4 can react similarly to high-expressing PAR1 in APAP-overdosed hepatic ECs, demonstrating that PAR4 is a potent thrombin receptor. Additionally, these receptors are functionally redundant but act divergently in their expression and ability to influence transcription in hepatic ECs.

A1 adenosine receptor evoked Ca2+ signals in astrocytes

Abstract In addition to neurons, glial cells make up the nervous system. These glial cells are divided into macroglial cells - including astrocytes - and microglial cells. Astrocytes have several important functions in the central nervous system, such as synthesizing transmitters and releasing them, thereby activating the surrounding neurons and astrocytes. Astrocytes do not generate action potentials, but they can transmit information to neighboring cells and react to external stimuli by employing Ca2+ dynamics. Increased neuronal activity can lead to an increased extracellular level of adenosine, reflected by a change in the metabolic state. Adenosine is a naturally occurring nucleoside distributed throughout the body as a metabolic intermediary. Its action is mediated by binding to adenosine receptors, such as the G-protein coupled receptors (GPCRs) A1 adenosine receptor. The binding of adenosine to the specific receptor type activates different signaling pathways in astrocytes. In our work, we studied Ca2+ signals generated upon adenosine A1 receptor activation by adding the pathway to the computational astrocyte model by Oschmann et.al (2017). We investigated the influence of the high-affinity A1 adenosine receptor in various astrocyte domains on the regulated Ca2+ release from the endoplasmic reticulum (ER) of astrocytes and concluded that the receptor-dependent pathway contributes to Ca2+ signals mainly in the soma.

Expression of Neurotrophic Factors and Their Receptors in the Rat Spinal Trigeminal Nucleus

The spinal trigeminal nucleus as part of the trigeminal sensory nuclear complex is associated with the transmission of discriminative tactile sensations, primarily pain, and temperature, from the orofacial region. It is divided into three parts – caudal, interpolar, and oral subnucleus which process pain and temperature stimulate. Our previous experiments have revealed the presence of certain neurotrophic factors such as the nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) in this nucleus. These neurotrophins facilitate neuronal differentiation, survival, and plasticity by signalling through high-affinity transmembrane receptors. By using primary antibodies against NGF, BDNF, NT-3, and glial-derived neurotrophic factor (GDNF), and their corresponding receptors of the tyrosine receptor kinase (Trk) proto-oncogene family and GFRα1, we found immunoreactive cells scattered along the whole length of the nucleus in rats in all three subnuclei. In particular, we observed that the majority of spinal trigeminal neurons were intensely immunostained for all the neurotrophic factors examined and that they were richly endowed with their Trk receptors. Our results also showed that NGF, NT-3, TrkC and GFRα1 expression did not differ between different topographic regions of the nucleus, while BDNF, GDNF, and TrkA expression was highest in the SpVo, and TrkB expression was highest in the SpVi and SpVc. Given that these neurotrophic factors are involved in mechanisms of central sensitization in trigeminal nociceptive pathways, it can be inferred that neurotrophins may contribute to a better understanding of the fundamental mechanisms of orofacial pain.

DRUG PRECURSOR TARGETING THE BOMBESIN RECEPTOR FOR PEPTIDE-RECEPTOR RADIONUCLIDE THERAPY

Cancer is a leading cause of death worldwide. A promising modality for cancer treatment is peptide receptor radionuclide therapy. Therapeutic radionuclide is delivered using peptide-based vectors, which can bind to specific receptors on the cancer cell surface. Bombesin receptors are one of the receptors peculiar to many types of cancer, which can be targeted by peptide vectors. Peptides have a number of advantages, but they also have one serious drawback: low stability in the internal environment. To solve the problem, it is possible to the include a therapeutic peptide in the structure of a highly stable knottin peptide. Objective. The aim of the study is to examine the stability of BBN/C1-C2 structure, created on the basis of U5-scytotoxinSth1a knottin and bombesin tropic to bombesin receptor, and the ability of this structure to bind to target receptors on the cancer cell surface. Materials and Methods. BBN/C1-C2 peptide was obtained by solid-phase peptide synthesis. Then, is underwent chromatography purification under analytical chromatography and mass spectrometry control. Stability was studied by analytical chromatography. Competitive inhibition analysis was carried out using a fluorescently labeled GRP peptide with excess BBN/C1-C2 and fluorescently labeled BBN/C1-C2 with GRP bombesin receptor inhibitor. Cancer cell line PC-3 expressing bombesin receptors and normal cell line CHO-K1 not expressing bombesin receptors were used in the work. Results. The conducted studies have shown that hybrid BBN/C1-C2 peptide based on bombesin peptide inserted into the U5-scytotoxinSth1a knottin framework between the first and second cysteine residues has a greater stability compared to the commercial radiopharmaceutical PSMA-617. BBN/C1-C2 peptide is specific to bombesin receptor: it binds to PC-3 cancer cell line with a target bombesin receptor on its surface, and does not bind to the healthy CHO-K1 cell line, without a target receptor. BBN/C1-C2 peptide shows high affinity for the bombesin receptor, since GRP prevents its binding to the PC-3 cell line.

Targeting nerve growth factor for pain relief: pros and cons.

Nerve growth factor (NGF) is a neurotrophic protein that has crucial roles in survival, growth and differentiation. It is expressed in neuronal and non-neuronal tissues. NGF exerts its effects via two types of receptors including the high affinity receptor, tropomyosin receptor kinase A and the low affinity receptor p75 neurotrophin receptor highlighting the complex signaling pathways that underlie the roles of NGF. In pain perception and transmission, multiple studies shed light on the effects of NGF on different types of pain including inflammatory, neuropathic, cancer and visceral pain. Also, the binding of NGF to its receptors increases the availability of many nociceptive receptors such as transient receptor potential vanilloid 1, transient receptor potential ankyrin 1, N-methyl-D-aspartic acid, and P2X purinoceptor 3 as well as nociceptive transmitters such as substance P and calcitonin gene-related peptide. The role of NGF in pain has been documented in pre-clinical and clinical studies. This review aims to shed light on the role of NGF and its signaling in different types of pain.

Mechanisms of Cinnamomi Cortex against Diabetes Mellitus Explored by Network Pharmacology combined with Molecular Docking and Experimental Validation.

Cinnamomi cortex (CC), a traditional Chinese herbal medicine, exhibits antidiabetic properties, yet the underlying mechanisms are not fully understood. Our study combined network pharmacology, molecular docking, and experimental validation to elucidate the antidiabetic mechanisms of CC. Active components of CC and their potential antidiabetic targets were identified through TCMSP, DisGeNET, and GeneCards. The PPI networks were constructed with STRING and analyzed with Cytoscape, while GO and KEGG analyses utilized the DAVID database. Molecular docking with core targets was performed using Autodock Vina. The efficacy of CC in diabetes mellitus was evaluated through H&E staining, qPCR, and Western blot in the T2DM mouse. Eleven active components and sixty-six potential antidiabetic targets of CC were identified. The enrichment analysis revealed 288 GO terms and 37 pathways. The molecular docking showed high affinity for PPAR-γ and IL-6 receptors. In vivo studies further confirmed CC's ability to modulate PPAR-γ and IL-6, contributing to its antidiabetic effects. CC manages diabetes by regulating the PPAR-γ pathway and suppressing associated inflammation, providing a multi-pathway therapeutic approach.

12-HETE/GPR31 induces endothelial dysfunction in diabetic retinopathy.

12-hydroxyeicosatetraenoic acid (12-HETE), a major metabolite of arachidonic acid, is converted by 12/15-lipoxygenase and implicated in diabetic retinopathy (DR). Our previous study demonstrated a positive correlation between 12-HETE and the prevalence of DR. However, reasons for the increased production of 12-HETE are unclear, and the underlying mechanisms through which 12-HETE promotes DR are unknown. This study aimed to elucidate the correlation between 12-HETE and DR onset, investigate potential mechanisms through which 12-HETE promotes DR, and seek explanations for the increased production of 12-HETE in diabetes. We conducted a prospective cohort study, which revealed that higher serum 12-HETE levels could induce DR. Additionally, G protein-coupled receptor 31 (GPR31), a high-affinity receptor for 12-HETE, was expressed in human retinal microvascular endothelial cells (HRMECs). 12-HETE/GPR31-mediated HRMEC inflammation occurred via the p38 MAPK pathway. 12-HETE levels were significantly higher in the retina of mice with high-fat diet (HFD)- and streptozotocin (STZ)-induced diabetes than in those with only STZ-induced diabetes and healthy controls. They were positively correlated with the levels of inflammatory cytokines in the retina, indicating that HFD could induce increased 12-HETE synthesis in patients with diabetes in addition to hyperglycemia. Conclusively, 12-HETE is a potential risk factor for DR. The 12-HETE/GPR31 axis plays a crucial role in HRMEC dysfunction and could be a novel target for DR prevention and control. Nevertheless, further research is warranted to provide comprehensive insights into the complex underlying mechanisms of 12-HETE in DR.

Omalizumab-Induced Arthralgias in a Patient with Chronic Idiopathic Urticaria

Background: Omalizumab (Xolair) is a recombinant DNA-derived humanized IgG1 κ monoclonal antibody that selectively binds to the high-affinity IgE receptor on the surface of mast cells and basophils, limiting release of mediators of the allergic response. It is the first monoclonal antibody approved for the treatment of refractory chronic idiopathic urticaria in patients over the age of 12 and has been shown to result in clinically significant improvement of itching and wheal formation in these patients. Rarely, inflammatory arthralgias can be a severe side effect of omalizumab. Clinical Case: We report a case of a 38-year-old female patient with chronic idiopathic urticaria who responded well on a therapeutic dosage of omalizumab, but subsequently developed severe inflammatory arthralgias. We decreased her dosage and added methotrexate for complete resolution of her urticarial and arthritic symptoms. Conclusion: In patients with concern for medication-induced inflammatory arthralgias, physicians should consider reducing or discontinuing omalizumab with consideration of patient goals for the treatment of chronic idiopathic urticaria. This article highlights the occurrence of inflammatory arthralgias as a side effect of omalizumab, the importance of eliciting patient goals and preferences for treatment, and proposes a solution to manage these arthralgias while appropriately treating symptoms in patients with chronic idiopathic urticaria.

Protection against DSS-induced colitis in mice through FcεRIα deficiency: the role of altered Lactobacillus

The role of mast cells (MCs) in ulcerative colitis (UC) development is controversial. FcεRI, the IgE high-affinity receptor, is known to activate MCs. However, its role in UC remains unclear. In our study, Anti-FcεRI showed highly diagnostic value for UC. FcεRIα knockout in mice ameliorated DSS-induced colitis in a gut microbiota-dependent manner. Increased Lactobacillus abundance in FcεRIα deficient mice showed strongly correlation with the remission of colitis. RNA sequencing indicated activation of the NLRP6 inflammasome pathway in FcεRIα knockout mice. Additionally, Lactobacillus plantarum supplementation protected against inflammatory injury and goblet cell loss, with activation of the NLRP6 inflammasome during colitis. Notably, this effect was absent when the strain is unable to produce lactic acid. In summary, colitis was mitigated in FcεRIα deficient mice, which may be attributed to the increased abundance of Lactobacillus. These findings contribute to a better understanding of the relationship between allergic reactions, microbiota, and colitis.

Acute stress and blockade of mineralocorticoid or glucocorticoid receptors: Effects on working memory

Although early studies were able to demonstrate a negative impact of stress on working memory performance, present research findings are heterogeneous. Numerous further studies found no effects or even improved performance, with the direction of these stress effects likely depending on the underlying biological mechanisms. The aim of this study was to investigate receptor-specific effects, as part of the stress-induced cortisol response, on working memory performance. Healthy, male participants (N=318, mean age 25.4 ± 5.1y) were exposed to the Trier Social Stress Test (TSST), a social-evaluative stress manipulation, or a non-stress control condition after they had received either spironolactone (blockade of the mineralocorticoid receptor, MR) or mifepristone (blockade of the glucocorticoid receptor, GR) or a placebo. Both substances are potent antagonists with high affinity for the respective receptors. To assess working memory, we implemented the n-back task subsequent to stress exposure, number of correct responses and reaction times served as outcome measures. We did not find effects of stress on working memory for any outcome measure, i.e. correct responses and reaction times. Yet, post hoc tests revealed that the group that received mifepristone exhibited longer reaction times under medium load conditions when compared to the placebo group, which might be an indication of the GR’s involvement in task performance. We conclude that working memory performance is not affected by acute stress, at least under these prevalent conditions.

Exploring Radioiodinated Anastrozole and Epirubicin as AKT1-Targeted Radiopharmaceuticals in Breast Cancer: In Silico Analysis and Potential Therapeutic Effect with Functional Nuclear Imagining Implications.

This study evaluates radio-iodinated anastrozole ([125I]anastrozole) and epirubicin ([125I]epirubicin) for AKT1-targeted breast cancer therapy, utilizing radiopharmaceutical therapy (RPT) for personalized treatment. Through molecular docking and dynamics simulations (200 ns), it investigates these compounds' binding affinities and mechanisms to the AKT1 enzyme, compared to the co-crystallized ligand, a known AKT1 inhibitor. Molecular docking results show that [125I]epirubicin has the highest ΔGbind (-11.84 kcal/mol), indicating a superior binding affinity compared to [125I] anastrozole (-10.68 kcal/mol) and the co-crystallized ligand (-9.53 kcal/mol). Molecular dynamics (MD) simulations confirmed a stable interaction with the AKT1 enzyme, with [125I]anastrozole and [125I]epirubicin reaching stability after approximately 68 ns with an average RMSD of around 2.2 Å, while the co-crystallized ligand stabilized at approximately 2.69 Å after 87 ns. RMSF analysis showed no significant shifts in residues or segments, with consistent patterns and differences of less than 2 Å, maintaining enzyme stability. The [125I]epirubicin complex maintained an average of four H-bonds, indicating strong and stable interactions, while [125I]anastrozole consistently formed three H-bonds. The average Rg values for both complexes were ~16.8 ± 0.1 Å, indicating no significant changes in the enzyme's compactness, thus preserving structural integrity. These analyses reveal stable binding and minimal structural perturbations, suggesting the high potential for AKT1 inhibition. MM-PBSA calculations confirm the potential of these radio-iodinated compounds as AKT1 inhibitors, with [125I]epirubicin exhibiting the most favorable binding energy (-23.57 ± 0.14 kcal/mol) compared to [125I]anastrozole (-20.03 ± 0.15 kcal/mol) and the co-crystallized ligand (-16.38 ± 0.14 kcal/mol), highlighting the significant role of electrostatic interactions in stabilizing the complex. The computational analysis shows [125I]anastrozole and [125I]epirubicin may play promising roles as AKT1 inhibitors, especially [125I]epirubicin for its high binding affinity and dynamic receptor interactions. These findings, supported by molecular docking scores and MM-PBSA binding energies, advocate for their potential superior inhibitory capability against the AKT1 enzyme. Nevertheless, it is crucial to validate these computational predictions through in vitro and in vivo studies to thoroughly evaluate the therapeutic potential and viability of these compounds for AKT1-targeted breast cancer treatment.

Discovery of antitumor diterpenoids from Casearia graveolens targeting VEGFR-2 to inhibit angiogenesis

Eight novel clerodane diterpenoids (1−8) were isolated from the twigs of Casearia graveolens. Their structures were elucidated through comprehensive nuclear magnetic resonance (NMR), high-resolution electrospray ionization mass spectrometry (HR-ESI-MS), and electronic circular dichroism (ECD) analyses. In addition to structural determination, surface plasmon resonance (SPR) assays were conducted to investigate molecular interactions, revealing that compound 8 exhibited high affinity for vascular endothelial growth factor receptor 2 (VEGFR2), a key regulator of tumor angiogenesis. Subsequent in vivo experiments demonstrated that compound 8 effectively inhibited angiogenesis and displayed significant antitumor activity by suppressing tumor proliferation and metastasis in zebrafish xenograft models. These findings suggest that compound 8 holds promise as an anticancer lead compound targeting VEGFR-2 to obstruct tumor angiogenesis.

IL-18 primes T cells with an antigen-inexperienced memory phenotype for proliferation and differentiation into effector cells through Notch signaling.

Recent studies have revealed that a subset of CD8+ T cells exhibit innate features and can be activated by cytokines. However, the precise mechanisms underlying the proliferation and differentiation of these cells remain unclear. Here, we demonstrated that CD44highCD8+ T cells in the mouse spleen express functional interleukin-18 (IL-18) receptors, whereas CD44lowCD8+ T cells do not. In response to IL-18 stimulation, these cells activated various metabolic pathways, upregulated the expression of surface molecules, such as c-Kit (CD117), CD25, and PD-1, and induced progression through the G1/S phase in the cell cycle. IL-18-primed cells, expressing a high-affinity receptor for IL-2, exhibited robust proliferation in response to IL-2 and underwent differentiation into effector cells. The splenic CD44highCD8+ T cells exhibited high expression levels of CD122, CD62L, CCR7, and CXCR3, along with CD5, indicating their potential for migration to the lymph nodes, where they could undergo expansion and terminal differentiation into effector cells. Additionally, in a tumor model, administration of IL-18 increased the accumulation of CD8+ T cells in both the lymph nodes and tumors. It is noteworthy that stimulation of CD44highCD8+ T cells with IL-18 upregulated the Notch-1 receptor and c-Myc. Moreover, inclusion of γ-secretase inhibitors attenuated the effect of IL-18 on both proliferation and interferon-γ production in the cells. These results demonstrate that IL-18 primes CD44highCD122highCXCR3highCD62LhighCD8+ T cells for expansion and differentiation into effector cells in a Notch signaling-dependent manner.

Evaluation of a Novel Detection Method for Allergen-Specific IgE Antibodies with IgE Receptor Crosslinking Using Rat Food Allergy Model.

The specific detection of serum IgE antibodies specific to allergens (sIgE Abs) that can crosslink the plural high-affinity IgE receptor (FcεRIα) molecules on the surface of mast cells or basophils with a multivalent allergen can reduce the false-positive diagnoses observed in chemiluminescent and fluorescence enzyme immunoassays for type-I allergic patients. In this study, we detected sIgE Abs to the egg-allergen ovalbumin (OVA) and the wheat-allergen gluten in the sera of rats sensitized with each allergen using an amplified luminescence proximity homogeneous assay by crosslinking (AlphaCL). OVA and gluten were reacted with each sIgE Ab in the sera. Then, acceptor and donor beads labeled with the human FcεRIα were added to the reacted solution. The luminescence intensity for anti-OVA IgE Abs in the sera with the removal of IgG Abs was observed in five of seven (71.4%) of the sensitized rats, whereas no signals were observed in any of the unsensitized rats. The AlphaCL could also detect anti-gluten sIgE Abs in the sera of sensitized rats, but not of unsensitized rats. In conclusion, we successfully detected sIgE Abs in the sera of rats sensitized to two allergens using the AlphaCL. This detection method has the potential to be used as a new diagnostic tool for type-I allergic patients.

Ligand-independent function of β2-adrenergic receptor affects IgE-mediated Ca2+ influx in mast cells

BackgroundMast cells are key effector cells that elicit immunoglobulin E (IgE)-mediated allergic inflammations. Allergen cross-linking of IgE bound to the high-affinity IgE receptor, FcεRI, on mast cells triggers signaling cascades that activate signal proteins and evoke extracellular Ca2+ influx, which are crucial for cytokine production. The β2-adrenergic receptor (Adrb2) on mast cells negatively regulates FcεRI signaling, as demonstrated by the inhibition of IgE/antigen (Ag)-induced activation by Adrb2 agonists. ObjectiveAlthough β2-adrenergic-related reagents are known to influence mast cell functions, the specific intrinsic role of Adrb2 in these cells is not fully understood, potentially because of off-target effects. In this study, the additional roles of Adrb2 in mast cells were investigated, specifically the involvement of Adrb2 in FcεRI signaling, using Adrb2−/− mice. MethodsAdrb2−/− mice were used to investigate the roles of Adrb2 in mast cells by examining bone marrow-derived mast cells (BMMCs) for surface expression of mast cell markers, granule numbers, and gene expression of mast cell proteases. Cytokine production, Ca2+ influx, and nuclear factor of activated T cells (NFAT) nuclear translocation were measured in Adrb2−/− and Adrb2+/+ BMMCs upon IgE/Ag stimulation. ResultsAdrb2−/− did not affect the generation of BMMCs, their surface expression of mast cell markers, granule numbers, or gene expression of mast cell proteases, indicating that the absence of Adrb2 had no adverse effect on mast cell development. However, Adrb2−/− BMMCs exhibited reduced tumor necrosis factor α (TNFα) production and diminished Ca2⁺ influx upon IgE/Ag stimulation, which correlated with decreased NFAT translocation. Restoration of Adrb2 in Adrb2−/− BMMCs rescued cytokine production. Notably, FcεRI-mediated phosphorylation of the phospholipase PLCγ1 and mitogen-activated protein kinases (MAPKs) remained unchanged in the absence of Adrb2. ConclusionThese results suggest that Adrb2 has a novel ligand-independent function, increasing Ca2+ entry in mast cells when stimulated with IgE/Ag.