Intracellular Signaling Pathways Research Articles

A Protein Kinase Cε/Protein Kinase D3 Signalling Axis Modulates RhoA Activity During Cytokinesis

Background: Protein kinase D3 (PKD3) is a member of the PKD family that has been implicated in many intracellular signalling pathways. However, defined statements regarding PKD isoform specificity and in vivo functions are rare. Methods: Here, we use PKD3-depleted mouse embryonic fibroblast cells and employ various cell culture-based assays and fluorescence microscopy. Results: We show that PKD3 is involved in the regulation of cytokinesis after immortalisation by modulating RhoA activity through a PKCε/PKD3 signalling axis. Conclusions: PKD3 depletion leads to prolonged RhoA activity during cytokinesis, resulting in failed abscission and an increase in the number of multinucleated cells. This identifies a novel, previously unrecognised PKCε/PKD3 pathway involved in the modulation of cytokinesis.

Targeting the hERG1/β1 integrin complex in lipid rafts potentiates statins anti-cancer activity in pancreatic cancer

Plasma membrane macromolecular complexes function as signaling hubs that regulate cell behavior, which is particularly relevant in cancer. Our study provides evidence that the complex formed by the hERG1 potassium channel and the β1 subunit of integrin receptors preferentially localizes in Lipid Rafts (LRs) in Pancreatic Ductal Adenocarcinoma (PDAC) cell lines and primary samples. The complex recruits the p85 subunit of phosphatidyl-inositol-3-kinase (PI3K), activating phosphoinositide metabolism and triggering an intracellular signaling pathway centered on Akt. This pathway ultimately affects cancer cell proliferation through cyclins and p21, and cell migration through the small GTPase Rac-1 and f-actin organization. The hERG1/β1 integrin complex in LRs can be dissociated and the downstream signaling pathway can be inhibited by either disrupting LRs through methyl-beta-cyclodextrin (MβCD) or inhibiting cholesterol synthesis by statins. Treatment with a single chain bispecific antibody—scDb-hERG1-β1—specifically targeting the complex significantly potentiates the effects of both MβCD and statins on intracellular signaling. Consequently, these treatments decrease PDAC cell proliferation and motility in vitro. From a pharmacological perspective, different statins produce anti-neoplastic effects in synergy with scDb-hERG1-β1. Such combination also enhances tumor sensitivity to chemotherapeutic drugs, such as gemcitabine and oxaliplatin. The efficacy of these combination treatments depends on the amount of the hERG1/β1 integrin complex present on the plasma membrane of cancer cells. Finally, the combined treatment with statins and scDb-hERG1-β1 significantly reduces tumor growth and improves survival in vivo, in a preclinical mouse model. These results suggest that the combination of scDb-hERG1-β1 and statins represent a potential novel strategy for treating PDAC patients.

Unveiling the interplay between soluble guanylate cyclase activation and redox signalling in stroke pathophysiology and treatment.

Soluble guanylate cyclase (sGC) stands as a pivotal regulatory element in intracellular signalling pathways, mediating the formation of cyclic guanosine monophosphate (cGMP) and impacting diverse physiological processes across tissues. Increased formation of reactive oxygen species (ROS) is widely recognized to modulate cGMP signalling. Indeed, oxidatively damaged, and therefore inactive sGC, contributes to poor vascular reactivity and more severe neurological damage upon stroke. However, the specific involvement of cGMP in redox signalling remains elusive. Here, we demonstrate a significant cGMP-dependent reduction of reactive oxygen and nitrogen species upon sGC activation under hypoxic conditions, independent of any potential scavenger effects. Importantly, this reduction is directly mediated by downregulating NADPH oxidase (NOX) 4 and 5 during reperfusion. Using an in silico simulation approach, we propose a mechanistic link between increased cGMP signalling and reduced ROS formation, pinpointing NF-κB1 and RELA/p65 as key transcription factors regulating NOX4/5 expression. In vitro studies revealed that p65 translocation to the nucleus was reduced in hypoxic human microvascular endothelial cells following sGC activation. Altogether, these findings unveil the intricate regulation and functional implications of sGC, providing valuable insights into its biological significance and ultimately therapeutic potential.

Light Controls in the Regulation of Carotenoid Biosynthesis in Leafy Vegetables: A Review

Leafy vegetables are excellent dietary sources of carotenoids, offering various nutritional benefits to human health. With the growing interest in health, the enhancement of functional compounds in crops through environmental control is emerging as an important topic in the field of agricultural research. Light serves as a major environmental signal regulating carotenoid levels. Light-activated photoreceptor proteins initiate intracellular signaling pathways that regulate carotenoid metabolism in response to environmental changes. Recent studies have shown that blue and ultraviolet (UV) light wavelengths are particularly effective in accumulation of foliar carotenoids, as they trigger photo-oxidative stress while activating defense mechanisms to mitigate it. Light intensity and exposure time, as quantitative aspects of light, are also involved in carotenoid biosynthesis in a similar manner. However, although previous studies provide valuable insights into light-mediated carotenoid accumulation, the interplay of light parameters (e.g., spectrum, intensity, exposure) with crop species and growth stages remains unclear due to the lack of well-organized data. In this review, we summarize recent advanced information about light-mediated carotenoid biosynthesis in leafy vegetables and highlight an integrated experimental approach to explore optimal light conditions for maximizing carotenoid accumulation in commercial production systems.

Transcriptomic landscapes of STING-mediated DNA-sensing reveal cellular response heterogeneity.

Transfection of plasmid DNA (pDNA) encoding target genes is a routine tool in gene function studies and therapeutic applications. However, nucleic acid-sensing-mediated innate immune responses influence multiple intracellular signaling pathways. The stimulator of interferon genes (STING) is a crucial adapter protein for DNA sensors in mammalian cells. In this study, we explored the molecular mechanisms underlying DNA sensing by investigating the relationship between mRNA and protein expression levels and the STING pathway using single-cell analysis. We observed that reporter gene expression was dose-nonlinear after transfection of pDNA in cells with intact DNA-sensing pathways. Moreover, blocking the STING pathway in THP-1 cells significantly downregulated innate immune responses, upregulated exogenous gene expression, and mitigated the effects of innate immune responses on cell and gene function, but did not affect the proportion of reporter protein-positive cells. We elucidated the mechanisms of DNA sensing-induced innate immune response and cell death by analyzing heterozygous cellular responses to DNA transfection and transcriptome changes in positive cells. These findings suggest that the regulation of STING-mediated nucleic acid-sensing pathways is crucial for the accuracy of gene function studies and could enhance the efficacy of gene therapy.

Exploring the immunomodulatory effects of sertraline: Cytokine modulation and signaling pathway dynamics.

This study explores the nuanced immunomodulatory effects of sertraline, which is widely used in the treatment of major depression, obsessive-compulsive disorder, and anxiety in adults and children. Recent investigations have emphasized the intricate interplay between depression and the body's inflammatory response. This has sparked an exploration into the impact of sertraline on the immune system, an area that still awaits comprehensive exploration. Our research methodically examines the influence of sertraline on the levels of cytokines (TNF-a, IL-6, IL-12p40, GM-CSF) in the macrophage cell line J774.2. This analysis is conducted under conditions with and without the lipopolysaccharide (LPS) danger signal. To enhance specificity, sertraline's effects are juxtaposed with those of salicylic acid, a known anti-inflammatory agent. Furthermore, a comprehensive exploration of sertraline's impact on the intracellular signaling pathways regulated phosphoinositide-3-kinase (PI3K) and the p38 pathway is the third major signaling cassettes of the mitogen-activated protein kinase (MAPK) signaling is presented. The outcomes of our study unveil distinctive patterns in sertraline's modulation of cytokine levels within macrophage cells. Under the influence of the LPS danger signal, sertraline exhibits immunostimulatory characteristics, contrasting with its ability to suppress GM-CSF cytokine levels, even in the presence of LPS. Notably, the p38 pathway portrays a pro-inflammatory role for sertraline, while inhibiting the PI3K signaling pathway highlights its anti-inflammatory attributes. These findings contribute novel insights into the intricate interplay between sertraline and the immune system.

Role of the AKT signaling pathway in regulating tumor-associated macrophage polarization and in the tumor microenvironment: A review.

Tumor-associated macrophages (TAMs) are present in and are important components of the tumor microenvironment (TME). TAMs differentiate into 2 functionally distinct morphologies, classically activated (M1)-type TAMs and alternatively activated (M2)-type TAMs, when stimulated by different cytokines. The 2 types of TAMs exhibit distinct properties and functions. M1 TAMs secrete high levels of pro-inflammatory and chemotactic factors, exerting proinflammatory, antitumor effects. Conversely, M2 TAMs alter the extracellular matrix, facilitate cellular immune escape, and stimulate tumor angiogenesis, thereby promoting anti-inflammatory responses and tumor growth. The ratio of M1 TAMs to M2 TAMs in the TME is closely related to the prognosis of the tumor. Tumor cells and other cells in the TME can regulate the polarization of TAMs and thus promote tumor progression through the secretion of various substances; however, polarized TAMs can also act on various cells in the TME through the secretion of exosomes, thus forming a positive feedback loop. Therefore, modulating the phenotype of TAMs in the TME or blocking the polarization of M2 TAMs might be a new approach for cancer treatment. However, the intracellular signaling pathways involved in the polarization of TAMs are poorly understood. The AKT signaling pathway is an important signaling pathway involved in the polarization, growth, proliferation, recruitment, and apoptosis of TAMs, as well as the action of TAMs on other cells within the TME. This paper reviews the AKT signaling pathway in the polarization of TAMs and the regulation of the TME and provides new ideas for tumor immunotherapy.

Rescue of in vitro models of CSF1R-related adult-onset leukodystrophy by iluzanebart: mechanisms and therapeutic implications of TREM2 agonism.

Microglia dysfunction is implicated in several neurodegenerative disorders, including a rare microgliopathy; CSF1R-related adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (CSF1R-ALSP). CSF1R-ALSP is caused by heterozygous loss-of-function mutations in the colony stimulating factor 1 receptor (CSF1R) gene, which encodes a receptor required for the differentiation of myeloid cells, as well as for microglial survival and proliferation. Similar functions have also been ascribed to triggering receptor expressed on myeloid cells 2 (TREM2), which shares an analogous microglia enrichment profile and converging intracellular signaling pathway mediated by spleen associated tyrosine kinase (SYK) and phosphoinositide-3-kinase (PI3K). Iluzanebart is a human monoclonal IgG1, human TREM2 (hTREM2) agonist antibody under development for the treatment of CSF1R-ALSP. To explore the therapeutic hypothesis that loss of CSF1R signaling and related microglial hypofunction can be circumvented via activation of TREM2, we evaluated the potential of iluzanebart to compensate for CSF1R loss-of-function. Herein, we demonstrate that iluzanebart is a potent, dose-dependent, and specific activator of TREM2 signaling in human primary cells. Iluzanebart treatment rescued viability of human monocyte-derived macrophages (hMDM) and induced pluripotent stem cell-derived human microglia (iMGL) in multiple in vitro models of CSF1R-ALSP, including in induced pluripotent stem cell (iPSC) differentiated microglia carrying the heterozygous I794T mutation found in CSF1R-ALSP patients. Additionally, iluzanebart treatment in microglia modulated surface levels of CSF1R, resulting in increased receptor activation as measured by phosphorylation of CSF1R. Differentially expressed genes identified in the hippocampus of mice treated with iluzanebart were exemplary of TREM2 activation and were related to cell proliferation, regulation of inflammatory processes, and innate immune response pathways. Proliferation of microglia, changes in protein levels of specific chemokines identified by gene expression analysis, and increased CSF1R levels were also confirmed in vivo. These findings demonstrate that iluzanebart is a potent and selective TREM2 agonistic antibody, with pharmacology that supports the hypothesis that TREM2 activation can compensate for CSF1R dysfunction and its continued clinical development for individuals with CSF1R-ALSP.

NGF in Neuropathic Pain: Understanding Its Role and Therapeutic Opportunities

Nerve growth factor (NGF) is one of the essential components that have been implicated in the pathophysiology of neuropathic pain, a condition that develops following nerve injury or dysfunction. This neurotrophin is critical for the survival and maintenance of sensory neurons, and its dysregulation has been implicated in the sensitization of pain pathways. NGF interacts with its receptor TrkA and p75NTR to activate intracellular signaling pathways associated with nociception and the emergence of allodynia and hyperalgesia. Therapeutic approaches employing neutralizing antibodies and molecule inhibitors have been highly effective at both preclinical and clinical levels, hence giving hope again for the use of NGF as an important biomarker and therapeutic target in the management of neuropathic pain. By exploiting the unique properties of NGF and its interactions within the nervous system, new therapeutic modalities could be designed to enhance efficacy while minimizing side effects. In conclusion, taking advantage of the multifaceted dynamics of NGF could provide effective pain management therapies to finally respond to the unmet needs of patients experiencing neuropathic pain.

Bistable Functions and Signaling Motifs in Systems Chemistry: Taking the Next Step Toward Synthetic Cells.

ConspectusA key challenge in modern chemistry research is to mimic life-like functions using simple molecular networks and the integration of such networks into the first functional artificial cell. Central to this endeavor is the development of signaling elements that can regulate the cell function in time and space by producing entities of code with specific information to induce downstream activity. Such artificial signaling motifs can emerge in nonequilibrium systems, exhibiting complex dynamic behavior like bistability, multistability, oscillations, and chaos. However, the de novo, bottom-up design of such systems remains challenging, primarily because the kinetic characteristics and energy aspects yielding bifurcation have not yet been globally defined. We herein review our recent work that focuses on the design and functional analysis of peptide-based networks, propelled by replication reactions and exhibiting bistable behavior. Furthermore, we rationalize and discuss their exploitation and implementation as variable signaling motifs in homogeneous and heterogeneous environments.The bistable reactions constitute reversible second-order autocatalysis as positive feedback to generate two distinct product distributions at steady state (SS), the low-SS and high-SS. Quantitative analyses reveal that a phase transition from simple reversible equilibration dynamics into bistability takes place when the system is continuously fueled, using a reducing agent, to keep it far from equilibrium. In addition, an extensive set of experimental, theoretical, and simulation studies highlight a defined parameter space where bistability operates.Analogous to the arrangement of protein-based bistable motifs in intracellular signaling pathways, sequential concatenation of the synthetic bistable networks is used for signal processing in homogeneous media. The cascaded network output signals are switched and erased or transduced by manipulating the order of addition of the components, allowing it to reach "on demand" either the low-SS or high-SS. The pre-encoded bistable networks are also useful as a programming tool for the downstream regulation of nanoscale materials properties, bridging together the Systems Chemistry and Nanotechnology fields. In such heterogeneous cascade pathways, the outputs of the bistable network serve as input signals for consecutive nanoparticle formation reaction and growth processes, which-depending on the applied conditions-regulate various features of (Au) nanoparticle shape and assembly.Our work enables the design and production of various signaling apparatus that feature higher complexity than previously observed in chemical networks. Future studies, briefly discussed at the end of the Account, will be directed at the design and analysis of more elaborate functionality, such as bistability under flow conditions, multistability, and oscillations. We propose that a profound understanding of the design principles facilitating the replication-based bistability and related functions bear implications for exploring the origin of protein functionality prior to the highly evolved replication-translation-transcription machinery. The integration of our peptide-based signaling motifs within future synthetic cells seems to be a straightforward development of the two alternating states as memory and switch elements for controlling cell growth and division and even communication among different cells. We furthermore suggest that such systems can be introduced into living cells for various biotechnology applications, such as switches for cell temporal and spatial manipulations.

Polysaccharides with antioxidant activity: Extraction, beneficial roles, biological mechanisms, structure-function relationships, and future perspectives: A review.

Polysaccharides are valuable macromolecules due to their multiple bioactivities, safety, and a wide range of sources. Recently, a series of polysaccharides with antioxidant activity have been intensively reported. In this review, the latest advances in polysaccharides with antioxidant activity have been reviewed, primarily based on the investigations of polysaccharides regarding advanced extraction methods, roles in oxidative stress-related diseases, intracellular signaling pathways associated with antioxidant responses, activating pathways in the gut, structure-function relationships, and methods to improve antioxidant activity. The summarized information highlighted that much work needs to be conducted, from laboratory to industry, to understand and fully utilize the antioxidant potential of polysaccharides. Finally, future perspectives, including scaling-up of advanced extraction methods, standardizing the protocols for assessing and screening polysaccharides, bridging gaps on the biological mechanisms underlying antioxidant activity, performing clinical trials, and elucidating structure-antioxidant relationships, have been addressed. The information present in this review will be helpful to the scientific community when studying on polysaccharides with antioxidant potential and provides research directions for a better understanding of the polysaccharides and promotes their successful applications in functional foods and nutraceuticals.

Insights from animal studies exploring the efficacy and safety of topical losartan, in prophylaxis and treatment of corneal scarring fibrosis.

Several studies in rabbits demonstrated the efficacy and safety of topical losartan, an angiotensin II receptor blockers (ARB) that modulates the TGF-β intracellular signaling pathways by inhibiting the activation of Extracellular Signal-regulated Kinase (ERK), in preventing or treating stromal fibrosis after a range of injuries such as Descemetorhexis, alkali burns, incisions, and photorefractive keratectomy (PRK). Several case reports have shown that topical losartan treatment is also efficacious and safe in humans to prevent or treat stromal fibrosis after many different injuries or diseases. Topical losartan penetrates the full thickness of the cornea and, therefore, can treat both anterior and posterior stromal fibrosis. These rabbit studies have demonstrated that there can be epithelial and stromal toxicity to losartan at dosages greater than 0.8 mg/ml and that higher dosages will not accelerate the return to transparency of fibrotic corneas. In corneas with an epithelial defect, it is likely safer to use 0.2 mg/ml losartan six times a day until the epithelium closes to further decrease the risk of epithelial toxicity before going to the 0.8 mg/ml six times a day dosage. Future clinical studies will explore additional questions, such as whether four times a day dosing is less effective than six times a day dosing in the treatment of stromal fibrosis.

Endothelial Damage in JAK2V617F Myeloproliferative Neoplasms with Splanchnic Vein Thrombosis.

JAK2V617F-mutated myeloproliferative neoplasms (MPN) exhibit abnormal proliferation of bone marrow progenitors and increased risk of thrombosis, specifically in splanchnic veins (SVT). The contribution of the endothelium to the development of the prothrombotic phenotype was explored. Plasma and serum samples from JAK2V617F MPN patients with (n=26) or without (n=7) thrombotic debut and different treatments, were obtained (n=33). Cultured endothelial cells (ECs) were exposed to serum samples from these patients and from healthy donors as controls. Changes in markers of inflammation (VCAM-1, ICAM-1), cell permeability (VE-cadherin), production of VWF, extracellular matrix (ECM) reactivity, and activation of intracellular signaling pathways related to stress, proliferation, inflammation (Akt, p44/42, IkBa), and JAK2/STAT3 pathway, were assessed by immunofluorescence, flow adhesion, SDS-PAGE and immunoblot. Additionally, circulating markers of endothelial activation and damage (VWF, sVCAM-1, sTNFRI, thrombomodulin, angiopoietin-2, a2-antiplasmin activity, PAI-1) were evaluated in Patients' plasma. The in vitro studies showed that EC exposure to MPN thrombotic patients' sera resulted in increased VCAM-1 and ICAM-1, and reduced VE-cadherin expression (p<0.05) at the cell surface. Production and release of VWF to the ECM were higher (p<0.05), with increased platelet adhesion after perfusing whole blood, being more noticeable in response to sera from non-treated patients. Furthermore, intracellular activation of Akt, p44/42, IkBa and JAK2/STAT3 was observed. Moreover, plasma levels of VWF, TNF-R1, VCAM-1, thrombomodulin, and angiopoietin-2 were higher in JAK2V617F+ MPN patients with thrombosis. The present findings suggest that circulating factors in MPNs with SVT debut induce endothelial proinflammatory and prothrombotic phenotypes, which are modulated in vitro with MPN treatment.

Endarachne binghamiae Extract Ameliorates Inflammatory Responses in Macrophages Through Regulation of MAPK, NF-kB and PI3K/AKT Pathways, and Prevents Acute Lung Injury in Mice.

In this study, the anti-inflammatory effect of the hot water extract of Endarachne binghamiae (EB-WE), a type of marine brown algae, was investigated in LPS-stimulated RAW 264.7 cells and an acute lung injury (ALI) mouse model induced by intranasal LPS administration. Treatment with EB-WE significantly inhibited NO and pro-inflammatory cytokine (TNF-a and IL-6) production in LPS-stimulated RAW 264.7 cells. In mRNA analysis, the expression of pro-inflammatory cytokines, COX-2, and iNOS mRNAs, was down-regulated by EB-WE treatment. The phosphorylation of MAPK, IkB, and PI3K/AKT molecules responsible for signal pathways during inflammation in LPS-stimulated macrophages was also significantly inhibited by EB-WE. In an in vivo model for ALI, oral administration of EB-WE significantly reduced the level of pro-inflammatory cytokines (TNF-a, IL-1b, and IL-6) and chemokines (MCP-1, CXC-16, CXCL1, and TARC) in serum or bronchoalveolar lavage fluid (BALF) of mice. Similarly to the results in LPS-stimulated RAW 264.7 cells, treatment with EB-WE significantly inhibited intracellular signal pathways mediated by MAPK, IkB, and PI3K/AKT in lung tissues of mice with ALI, and also decreased the expression of mRNAs of inflammatory mediators such as TNF-a, IL-6, iNOS, and COX-2. Furthermore, the inhibitory effect of EB-WE on ALI was apparently confirmed in histological examination through lung tissue staining. Taken together, it is clear that EB-WE has potential activity to effectively ameliorate the inflammatory responses in macrophages through down-regulation of MAPK, NF-kB, and PI3K/AKT activation, and suppress acute lung injury induced by LPS. These findings strongly suggest that EB-WE is a promising natural product beneficial for developing preventive treatments and cures of inflammation-related diseases.

Associations Between Diabetes Mellitus and Neurodegenerative Diseases.

Diabetes mellitus (DM) and neurodegenerative diseases/disturbances are worldwide health problems. The most common chronic conditions diagnosed in persons 60 years and older are type 2 diabetes mellitus (T2DM) and cognitive impairment. It was found that diabetes mellitus is a major risk for cognitive decline, dementia, Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS) and other neurodegenerative disorders. Different mechanisms of associations between these diseases and diabetes mellitus have been suggested. For example, it is postulated that an impaired intracellular insulin signaling pathway, together with hyperglycemia and hyperinsulinemia, may cause pathological changes, such as dysfunction of the mitochondria, oxidative stress inflammatory responses, etc. The association between diabetes mellitus and neurodegenerative diseases, as well as the mechanisms of these associations, needs further investigation. The aim of this review is to describe the associations between diabetes mellitus, especially type 1 (T1DM) and type 2 diabetes mellitus, and selected neurodegenerative diseases, i.e., Alzheimer's disease, Parkinson's disease, Huntington's disease and amyotrophic lateral sclerosis. Suggested mechanisms of these associations are also described.

Modulation of Second Messenger Signaling in the Brain Through PDE4 and PDE5 Inhibition: Therapeutic Implications for Neurological Disorders.

Phosphodiesterase (PDE) enzymes regulate intracellular signaling pathways crucial for brain development and the pathophysiology of neurological disorders. Among the 11 PDE subtypes, PDE4 and PDE5 are particularly significant due to their regulation of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) signaling, respectively, which are vital for learning, memory, and neuroprotection. This review synthesizes current evidence on the roles of PDE4 and PDE5 in neurological health and disease, focusing on their regulation of second messenger pathways and their implications for brain function. Elevated PDE4 activity impairs synaptic plasticity by reducing cAMP levels and protein kinase A (PKA) activity, contributing to cognitive decline, acute brain injuries, and neuropsychiatric conditions such as bipolar disorder and schizophrenia. Similarly, PDE5 dysregulation disrupts nitric oxide (NO) signaling and protein kinase G (PKG) pathways, which are involved in cerebrovascular homeostasis, recovery after ischemic events, and neurodegenerative processes in Alzheimer's, Parkinson's, and Huntington's diseases. PDE4 and PDE5 are promising therapeutic targets for neurological disorders. Pharmacological modulation of these enzymes offers potential to enhance cognitive function and mitigate pathological mechanisms underlying brain injuries, neurodegenerative diseases, and psychiatric disorders. Further research into the regulation of PDE4 and PDE5 will advance therapeutic strategies for these conditions.

Pre-Clinical Rationale for Amcenestrant Combinations in HER2+/ER+ Breast Cancer.

HER2-positive/oestrogen receptor-positive (HER2+/ER+) represents a unique breast cancer subtype. The use of individual HER2- or ER-targeting agents can lead to the acquisition of therapeutic resistance due to compensatory receptor crosstalk. New drug combinations targeting HER2 and ER could improve outcomes for patients with HER2+/ER+ breast cancer. In this study, the pre-clinical rationale is explored for combining amcenestrant (Amc), a selective oestrogen receptor degrader (SERD), with HER2-targeted therapies including trastuzumab, trastuzumab-emtansine (T-DM1) and tyrosine kinase inhibitors (TKIs). The combination of Amc and anti-HER2 therapies was investigated in a panel of four HER2+/ER+ cell lines: BT-474, MDA-MB-361, EFM-192a and a trastuzumab-resistant variant BT-474-T. Proliferation (IC50 and matrix combination assays) was determined using acid phosphatase assays. HER2/ER and intracellular signalling pathway protein levels/activity were investigated by western blot. Apoptosis was assessed using caspase 3/7 assays. Additivity and synergy were observed between Amc and the TKIs neratinib, lapatinib and tucatinib in all cell lines. Amc increased the anti-proliferative effect of trastuzumab in MDA-MB-361 and BT-474-T. Addition of Amc also increased anti-proliferative efficacy of T-DM1 in BT-474-T. TKI/Amc combinations reduced p-HER2 and ER levels and resulted in increased apoptosis. Higher ER expression in MDA-MB-361 and BT-474-T was associated with greater potential for synergy. In conclusion, the combination of Amc- and HER2-targeted treatments has potential as a therapeutic strategy for the treatment of HER2+/ER+ breast cancer and warrants further clinical investigation to validate safety and efficacy in patients.

Cloud-based adolescent physical health research incorporating cell molecular biomechanics insights into predictive modeling and biometric algorithms

The physical health of adolescents is critical to their future development and quality of life. However, there is still much room for improvement in monitoring and intervention of adolescent physical health in colleges and universities. Based on the National Standard for Student Physical Fitness and Health, this study proposes a physical health management framework for adolescents that combines a cloud platform and real-time data analysis. Through an innovative health monitoring and early warning system and feedback mechanism, the effectiveness of health intervention was significantly improved. Body mass index is related to the balance between energy intake and expenditure at the cellular level. Excessive calorie intake can lead to an increase in adipose cells, which secrete molecules that can affect overall metabolism and biomechanical stress on tissues. Lung capacity is linked to the elasticity and strength of the alveolar cells and the connective tissue in the lungs. he proper functioning of these cells, regulated by intracellular signaling pathways and molecular interactions, determines the efficiency of gas exchange. The 50-meter run, seated forward bending, standing long jump, pull-ups/sit-ups, and 800-meter/1000-meter run all involve muscle contractions. Muscle cells contain actin and myosin filaments, and the sliding of these filaments, regulated by calcium ions and other molecular signals, generates the force required for movement. The graded early warning parameters established by the system can thus be seen as indicators of potential disruptions in these cell molecular biomechanical processes. The innovative health monitoring and early warning system and feedback mechanism not only help students recognize the weaknesses in their physical functions from a macroscopic level but also potentially identify areas where cell molecular biomechanical imbalances may exist. This enables physical education teachers to formulate personalized training plans that can target and correct these imbalances, as experimentally validated by the system's ability to accurately identify health risks and enhance the overall health of students.

Chemogenetic engagement of different GPCR signaling pathways segregates the orexigenic activity from the control of whole-body glucose metabolism by AGRP Neurons.

BackgroundThe control of energy balance involves neural circuits in the central nervous system, including AGRP neurons in the arcuate nucleus of the hypothalamus (ARC). AGRP neurons are crucial for energy balance and their increased activity during fasting is critical to promote feeding behavior. The activity of these neurons is influenced by multiple signals including those acting on G-protein coupled receptors (GPCR) activating different intracellular signaling pathways. We sought to determine whether discrete G-protein mediated signaling in AGRP neurons, promotes differential regulation of feeding and whole-body glucose homeostasis. Methodology: To test the contribution of Gαq/11 or Gαs signaling, we developed congenital mouse lines expressing the different DREADD receptors (i.e., hM3q and rM3s), in AGRP neurons. Then we elicited chemogenetic activation of AGRP neurons in these mice during the postprandial state to determine the impact on feeding and glucose homeostasis. ResultsActivation of AGRP neurons via hM3q and rM3s promoted hyperphagia. In contrast, only hM3q activation of AGRP neurons of the hypothalamic arcuate nucleus during the postprandial state enhanced whole-body glucose disposal by reducing sympathetic nervous system activity to the pancreas and liver, promoting glucose-stimulated insulin secretion, glycogen deposition and improving glucose tolerance. ConclusionThese data indicate that AGRP neurons regulate food intake and glucose homeostasis through distinct GPCR-dependent signaling pathways and suggest that the transient increase in AGRP neuron activity may contribute to the beneficial effects of fasting on glycemic control.

Evaluation of mechanisms involved in regulation of intrinsic excitability by extracellular calcium in CA1 pyramidal neurons of rat.

It is well recognized that changes in the extracellular concentration of calcium ions influence the excitability of neurons, yet what mechanism(s) mediate these effects is still a matter of debate. Using patch-clamp recordings from rat hippocampal CA1 pyramidal neurons, we examined the contribution of G-proteins and intracellular calcium-dependent signaling mechanisms to changes in intrinsic excitability evoked by altering the extracellular calcium concentration from physiological (1.2 mM) to a commonly used experimental (2 mM) level. We find that the inhibitory effect on intrinsic excitability of calcium ions is mainly expressed as an increased threshold for action potential firing (with no significant effect on resting membrane potential) that is not blocked by either the G-protein inhibitor GDPβS or the calcium chelator BAPTA. Our results therefore argue that in the concentration range studied, G-protein coupled calcium-sensing receptors, non-selective cation conductances, and intracellular calcium signaling pathways are not involved in mediating the effect of extracellular calcium ions on intrinsic excitability. Analysis of the derivative of the action potential, dV/dt versus membrane potential, indicates a current shift towards more depolarized membrane potentials at the higher calcium concentration. Our results are thus consistent with a mechanism in which extracellular calcium ions act directly on the voltage-gated sodium channels by neutralizing negative charges on the extracellular surface of these channels to modulate the threshold for action potential activation.