Search Results (97)

Dictyostelium is a unique model used to study the complex and interactive cyclic nucleotide signaling pathways that regulate multicellular development. Dictyostelium grow as individual single cells, but in the absence of nutrients, they initiate a multicellular developmental program. Central to this is secreted cAMP, a primary GPCR-response signal. Activated cAMP receptors at the cell surface direct a number of downstream signaling pathways, including synthesis of the intracellular second messengers cAMP and cGMP. These, in turn, activate a series of downstream targets that direct chemotaxis within extracellular cAMP gradients, multicellular aggregation, and, ultimately, cell-specific gene expression, morphogenesis, and cytodifferentiation. Extracellular cAMP and intracellular cAMP and cGMP exhibit rapid fluctuations in concentrations and are, thus, subject to exquisite regulation by both synthesis and degradation. The Dictyostelium genome encodes seven phosphodiesterases (PDEs) that degrade cyclic nucleotides to nucleotide 5’-monophosphates. Each PDE has a distinct structure, substrate specificity, regulatory input, cellular localization, and developmentally regulated expression pattern. The intra- or extra-cellular localizations and enzymatic specificities for cAMP or cGMP are essential for degradative precision at different developmental stages. We discuss the diverse PDEs, the nucleotide cyclases, and the target proteins for cAMP and cGMP in Dictyostelium. We further outline the major molecular, cellular, and developmental events regulated by cyclic nucleotide signaling, with emphasis on the input of each PDE and consequence of loss-of-function mutations. Finally, we relate the structures and functions of the Dictyostelium PDEs with those of humans and in the context of potential therapeutic understandings. Full article

Cystic Fibrosis (CF) is a life-shortening, multi-organ disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. Prominent clinical features of CF take place in the lung, hallmarked by cycles of bacterial infection and a dysfunctional inflammatory airway response, leading to eventual respiratory failure. Bidirectional crosstalk between epithelial cells, leukocytes (e.g., neutrophils, macrophages) and bacteria via release of intra-cellular mediators is key to driving inflammation in CF airways. In recent years, a highly effective combination of therapeutics targeting the CFTR defect have revolutionized treatment in CF. Despite these advancements and due to the complexity of the immune response in the CF airway, the full impact of highly effective modulator therapy (HEMT) on airway inflammation is not fully determined. This review provides the evidence to date on crosstalk mechanisms between host epithelium, leukocytes and bacteria and examines the effect of HEMT on both soluble and membrane-derived immune mediators in clinical samples. The varied effects of HEMT on expression of key proteases, cytokines and extracellular vesicles (EVs) in relation to clinical parameters is assessed. Advances in treatment with HEMT have shown potential in dampening the chronic inflammatory response in CF airways. However, to fully quell inflammation and maximize lung tissue resilience, further interventions may be necessary. Exploring the effects of HEMT on key immune mediators paves the way for identifying new anti-inflammatory approaches targeting host immune cell interactions, such as EV-directed lung therapies. Full article

Long-lasting brain fatigue is a consequence of stroke or traumatic brain injury associated with emotional, psychological, and physical overload, distress in hypertension, atherosclerosis, viral infection, and aging-related chronic low-grade inflammatory disorders. The pathogenesis of brain fatigue is linked to disrupted neurotransmission, the glutamate-glutamine cycle imbalance, glucose metabolism, and ATP energy supply, which are associated with multiple molecular targets and signaling pathways in neuroendocrine-immune and blood circulation systems. Regeneration of damaged brain tissue is a long-lasting multistage process, including spontaneously regulating hypothalamus-pituitary (HPA) axis-controlled anabolic–catabolic homeostasis to recover harmonized sympathoadrenal system (SAS)-mediated function, brain energy supply, and deregulated gene expression in rehabilitation. The driving mechanism of spontaneous recovery and regeneration of brain tissue is a cross-talk of mediators of neuronal, microglia, immunocompetent, and endothelial cells collectively involved in neurogenesis and angiogenesis, which plant adaptogens can target. Adaptogens are small molecules of plant origin that increase the adaptability of cells and organisms to stress by interaction with the HPA axis and SAS of the stress system (neuroendocrine-immune and cardiovascular complex), targeting multiple mediators of adaptive GPCR signaling pathways. Two major groups of adaptogens comprise (i) phenolic phenethyl and phenylpropanoid derivatives and (ii) tetracyclic and pentacyclic glycosides, whose chemical structure can be distinguished as related correspondingly to (i) monoamine neurotransmitters of SAS (epinephrine, norepinephrine, and dopamine) and (ii) steroid hormones (cortisol, testosterone, and estradiol). In this narrative review, we discuss (i) the multitarget mechanism of integrated pharmacological activity of botanical adaptogens in stress overload, ischemic stroke, and long-lasting brain fatigue; (ii) the time-dependent dual response of physiological regulatory systems to adaptogens to support homeostasis in chronic stress and overload; and (iii) the dual dose-dependent reversal (hormetic) effect of botanical adaptogens. This narrative review shows that the adaptogenic concept cannot be reduced and rectified to the various effects of adaptogens on selected molecular targets or specific modes of action without estimating their interactions within the networks of mediators of the neuroendocrine-immune complex that, in turn, regulates other pharmacological systems (cardiovascular, gastrointestinal, reproductive systems) due to numerous intra- and extracellular communications and feedback regulations. These interactions result in polyvalent action and the pleiotropic pharmacological activity of adaptogens, which is essential for characterizing adaptogens as distinct types of botanicals. They trigger the defense adaptive stress response that leads to the extension of the limits of resilience to overload, inducing brain fatigue and mental disorders. For the first time, this review justifies the neurogenesis potential of adaptogens, particularly the botanical hybrid preparation (BHP) of Arctic Root and Ashwagandha, providing a rationale for potential use in individuals experiencing long-lasting brain fatigue. The review provided insight into future research on the network pharmacology of adaptogens in preventing and rehabilitating long-lasting brain fatigue following stroke, trauma, and viral infections. Full article

Transferrin Receptor 2 (TfR2) is a homolog of Transferrin Receptor 1 (TfR1), involved in regulating intra and extracellular iron levels. Altered iron pathways have been associated with cancer onset and progression; however, their role in canine tumors remains poorly explored. This study investigated TfR2 immunohistochemical expression in non-neoplastic canine testis for the first time and in the most common types of canine testicular tumors: intratubular seminomas (ITSEMs), diffuse seminomas (DSEMs), Leydig cell tumors (LCTs), and Sertoli cell tumors (SCTs). Immunohistochemical analysis revealed a differential pattern of TfR2 expression according to tumor type, with high expression observed in ITSEMs and DSEMs, occasional expression in LCTs, and absence in SCTs. These results suggest that TfR2 may play a relevant role in canine seminoma development. Furthermore, the specific expression of TfR2 in seminomas highlights its potential as a therapeutic target, where its role in iron regulation and possible compensatory mechanisms warrant further investigation. Full article

Advancements in single-cell analyzis technologies, particularly single-cell RNA sequencing (scRNA-seq) and Fluorescence-Activated Cell Sorting (FACS), have enabled the analyzis of cellular diversity by providing resolutions that were not available previously. These methods enable the simultaneous analyzis of thousands of individual transcriptomes, facilitating the classification of cells into distinct subpopulations, based on transcriptomic differences, adding a new level of complexity to biomolecular and medical research. Fibroblasts, despite being one of the most abundant cell types in the human body and forming the structural backbone of tissues and organs, remained poorly characterized for a long time. This is largely due to the high morphological similarity between different types of fibroblasts and the lack of specific markers to identify distinct subpopulations. Once thought to be cells responsible solely for the synthesis of extracellular matrix (ECM) components, fibroblasts are now recognized as active participants in diverse physiological processes, including inflammation and antimicrobial responses. However, defining the molecular profile of fibroblast subpopulations remains a significant challenge. In this comprehensive review, which is based on over two thousand research articles, we focus on the identification and characterization of fibroblast subpopulations and their specific surface markers, with an emphasis on their potential as molecular targets for selective cell isolation. By analyzing surface markers, alongside intra- and extracellular protein profiles, we identified multiple fibroblast subtypes within the female reproductive system. These subtypes exhibit distinct molecular signatures and functional attributes, shaped by their anatomical localization and the surrounding physiological or pathological conditions. Our findings underscore the heterogeneity of fibroblasts and their diverse roles in various biological contexts. This improved understanding of fibroblast subpopulations paves the way for innovative diagnostic and therapeutic strategies, offering the potential for precision targeting of specific fibroblast subsets in clinical applications. Full article

Background/Objectives: Osteoarthritis (OA) is the most common joint disease in the adult population. OA is the result of multiple mechanisms leading to inflammation and the degradation of the cartilage. A complex series of etiological actors have been identified so far, including extracellular vesicles (EVs). The EV content of the synovial fluid (SF) can release inflammatory mediators that enhance OA progression. An intra-articular viscosupplementation of high-MW hyaluronic acid (HyA) constitutes the first-line conservative treatment for OA. Although attractive for the potential pharmacological implications, the possibility that HyA may interact with EVs in the context of OA has not yet been specifically investigated; therefore, the present study aimed to fill this gap. Methods: We studied the effect of a HyA preparation (a blend of crosslinked and linear polymers, CLHyA) on the relevant inflammatory markers in chondrocytes (HC cells or primary chondrocytes isolated from patients with advanced OA) exposed to the EVs collected from IL-1β-stimulated THP-1 human monocytes (EVs+). Results: EVs+ caused specific inflammatory responses in chondrocytes that could be prevented by coincubation with CLHyA. This anti-inflammatory activity is likely dependent on the direct binding of CLHyA to CD44 receptors highly expressed in EVs+ and on the subsequent hindrance to EVs+ diffusion and docking to target cells. Conclusions: On the whole, the tight interactions identified herein between HMW HyA and EVs+ represent a novel, pharmacologically exploitable mechanism potentially relevant in the context of OA treatment. Full article

Phospholipids are the main building components of cell membranes and are also used for cell signaling and as energy storages. Cancer cells alter their lipid metabolism, which ultimately leads to an increase in phospholipids in cancer tissue. Surgical energy instruments use electrical or vibrational energy to heat tissues, which causes intra- and extracellular water to expand rapidly and degrade cell structures, bursting the cells, which causes the formation of a tissue aerosol or smoke depending on the amount of energy used. This gas phase analyte can then be analyzed via gas analysis methods. Differential mobility spectrometry (DMS) is a method that can be used to differentiate malignant tissue from benign tissues in real time via the analysis of surgical smoke produced by energy instruments. Previously, the DMS identification of cancer tissue was based on a ‘black box method’ by differentiating the 2D dispersion plots of samples. This study sets out to find datapoints from the DMS dispersion plots that represent relevant target molecules. We studied the ability of DMS to differentiate three subclasses of phospholipids (phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine) from a control sample using a bovine skeletal muscle matrix with a 5 mg addition of each phospholipid subclass to the sample matrix. We trained binary classifiers using linear discriminant analysis (LDA) and support vector machines (SVM) for sample classification. We were able to identify phosphatidylcholine, -inositol, and -ethanolamine with SVM binary classification accuracies of 91%, 73%, and 66% and with LDA binary classification accuracies of 82%, 74%, and 72%, respectively. Phosphatidylcholine was detected with a reliable classification accuracy, but ion separation setups should be adjusted in future studies to reliably detect other relevant phospholipids such as phosphatidylinositol and phosphatidylethanolamine and improve DMS as a microanalysis method and identify other phospholipids relevant to cancer tissue. Full article

Marfan syndrome (MFS) is a hereditary condition accompanied by disorders in the structural and regulatory properties of connective tissue, including elastic fibers, due to a mutation in the gene encodes for fibrillin-1 protein (FBN1 gene) and the synthesis of abnormal fibrillin-1 glycoprotein. Despite the high potential of mast cells (MCs) to remodel the extracellular matrix (ECM), their pathogenetic significance in MFS has not been considered yet. The group of patients with Marfan syndrome included two mothers and five children (three girls aged 4, 11, and 11 and two boys aged 12 and 13). Normal skin was examined in two children aged 11 and 12. Histochemical, monoplex, and multiplex immunohistochemical techniques; combined protocols of simultaneous histochemical and immunohistochemical staining (the results of staining were assessed using light, epifluorescence, and confocal microscopy); and bioinformatics algorithms for the quantitative analysis of detected targets were used to evaluate mast cells and their relationship with other cells from extracellular structures in the skin dermis. Analysis of the skin MC population in children with Marfan syndrome revealed a considerably increased number of intra-organic populations with the preservation of the specific Tryptase⁺Chymase⁺CPA3⁺ protease profile typical of the skin. The features of the MC histotopography phenotype in MFS consisted of closer colocalization with elastic fibers, smooth muscle cells, and fibroblasts. MCs formed many intradermal clusters that synchronized the activity of cell functions in the stromal landscape of the tissue microenvironment with the help of spatial architectonics, including the formation of cell chains and the creation of fibrous niches. In MCs, the expression of specific proteases, TGF-β, and heparin increased, with targeted secretion of biologically active substances relative to the dermal elastic fibers, which had specific structural features in MFS, including abnormal variability in thickness along their entire length, alternating thickened and thinned areas, and uneven surface topography. This paper discusses the potential role of MCs in strain analysis (tensometry) of the tissue microenvironment in MFS. Thus, the quantitative and qualitative rearrangements of the skin MC population in MFS are aimed at altering the stromal landscape of the connective tissue. The results obtained should be taken into account when managing clinical signs of MFS manifested in other pathogenetically critical structures of internal organs, including the aorta, tendons, cartilage, and parenchymal organs. Full article

Colorectal cancer (CRC) is a frequent, worldwide tumor described for its huge complexity, including inter-/intra-heterogeneity and tumor microenvironment (TME) variability. Intra-tumor heterogeneity and its connections with metabolic reprogramming and epithelial–mesenchymal transition (EMT) were investigated with explorative shotgun proteomics complemented by a Random Forest (RF) machine-learning approach. Deep and superficial tumor regions and distant-site non-tumor samples from the same patients (n = 16) were analyzed. Among the 2009 proteins analyzed, 91 proteins, including 23 novel potential CRC hallmarks, showed significant quantitative changes. In addition, a 98.4% accurate classification of the three analyzed tissues was obtained by RF using a set of 21 proteins. Subunit E1 of 2-oxoglutarate dehydrogenase (OGDH-E1) was the best classifying factor for the superficial tumor region, while sorting nexin-18 and coatomer-beta protein (beta-COP), implicated in protein trafficking, classified the deep region. Down- and up-regulations of metabolic checkpoints involved different proteins in superficial and deep tumors. Analogously to immune checkpoints affecting the TME, cytoskeleton and extracellular matrix (ECM) dynamics were crucial for EMT. Galectin-3, basigin, S100A9, and fibronectin involved in TME–CRC–ECM crosstalk were found to be differently variated in both tumor regions. Different metabolic strategies appeared to be adopted by the two CRC regions to uncouple the Krebs cycle and cytosolic glucose metabolism, promote lipogenesis, promote amino acid synthesis, down-regulate bioenergetics in mitochondria, and up-regulate oxidative stress. Finally, correlations with the Dukes stage and budding supported the finding of novel potential CRC hallmarks and therapeutic targets. Full article

Brain tumors, and, in particular, glioblastoma (GBM), are among the most aggressive forms of cancer. In spite of the advancement in the available therapies, both diagnosis and treatments are still unable to ensure pathology-free survival of the GBM patients for more than 12–15 months. At the basis of the still poor ability to cope with brain tumors, we can consider: (i) intra-tumor heterogeneity; (ii) heterogeneity of the tumor properties when we compare different patients; (iii) the blood–brain barrier (BBB), which makes difficult both isolation of tumor-specific biomarkers and delivering of therapeutic drugs to the brain. Recently, it is becoming increasingly clear that cancer cells release large amounts of extracellular vesicles (EVs) that transport metabolites, proteins, different classes of RNAs, DNA, and lipids. These structures are involved in the pathological process and characterize any particular form of cancer. Moreover, EVs are able to cross the BBB in both directions. Starting from these observations, researchers are now evaluating the possibility to use EVs purified from organic fluids (first of all, blood and saliva), in order to obtain, through non-invasive methods (liquid biopsy), tumor biomarkers, and, perhaps, also for obtaining nanocarriers for the targeted delivering of drugs. Full article

Neutrophil elastase (HNE), like other members of the so-called GASPIDs (Granule-Associated Serine Peptidases of Immune Defense), is activated during protein biosynthesis in myeloid precursors and stored enzymatically active in cytoplasmic granules of resting neutrophils until secreted at sites of host defense and inflammation. Inhibitors thus could bind to the fully formed active site of the protease intracellularly in immature progenitors, in circulating neutrophils, or to HNE secreted into the extracellular space. Here, we have compared the ability of a panel of diverse inhibitors to inhibit HNE in the U937 progenitor cell line, in human blood-derived neutrophils, and in solution. Most synthetic inhibitors and, surprisingly, even a small naturally occurring proteinaceous inhibitor inhibit HNE intracellularly, but the extent and dynamics differ markedly from classical enzyme kinetics describing extracellular inhibition. Intracellular inhibition of HNE potentially affects neutrophil functions and has side effects, but it avoids competition of inhibitors with extracellular substrates that limit its efficacy. As both intra- and extracellular inhibition have advantages and disadvantages, the quantification of intracellular inhibition, in addition to classical enzyme kinetics, will aid the design of novel, clinically applicable HNE inhibitors with targeted sites of action. Full article

Metabolic alterations are increasingly recognized as important aspects of colorectal cancer (CRC), offering potential avenues for identifying therapeutic targets. Previous studies have demonstrated the cytotoxic potential of bamboo leaf extract obtained from Guadua incana (BLEGI) against HCT-116 colon cancer cells. However, the altered metabolic pathways in these tumor cells remain unknown. Therefore, this study aimed to employ an untargeted metabolomic approach to reveal the metabolic alterations of the endometabolome and exometabolome of HCT-116 cells upon exposure to BLEGI treatment. First, a chemical characterization of the BLEGI was conducted through liquid chromatography coupled with mass spectrometry (LC-MS). Next, we assessed cell viability via MTT and morphological analysis using an immunofluorescence assay against colon cancer cells, and anti-inflammatory activity using an LPS-stimulated macrophage model. Subsequently, we employed LC-MS and proton nuclear magnetic resonance (¹H-NMR) to investigate intra- and extracellular changes. Chemical characterization primarily revealed the presence of compounds with a flavone glycoside scaffold. Immunofluorescence analysis showed condensed chromatin and subsequent formation of apoptotic bodies, suggesting cell death by apoptosis. The results of the metabolomic analysis showed 98 differential metabolites, involved in glutathione, tricarboxylic acid cycle, and lipoic acid metabolism, among others. Additionally, BLEGI demonstrated significant nitric oxide (NO) inhibitory capacity in macrophage cells. This study enhances our understanding of BLEGI’s possible mechanism of action and provides fresh insights into therapeutic targets for treating this disease. Full article

Phagocytosis (and endocytosis) is an unusual cellular process that results in the formation of a novel subcellular organelle, the phagosome. This phagosome contains not only the internalised target of phagocytosis but also the external medium, creating a new border between extracellular and intracellular environments. The boundary at the plasma membrane is, of course, tightly controlled and exploited in ionic cell signalling events. Although there has been much work on the control of phagocytosis by ions, notably, Ca²⁺ ions influxing across the plasma membrane, increasing our understanding of the mechanism enormously, very little work has been done exploring the phagosome/cytosol boundary. In this paper, we explored the changes in the intra-phagosomal Ca²⁺ ion content that occur during phagocytosis and phagosome formation in human neutrophils. Measuring Ca²⁺ ion concentration in the phagosome is potentially prone to artefacts as the intra-phagosomal environment experiences changes in pH and oxidation. However, by excluding such artefacts, we conclude that there are open Ca²⁺ channels on the phagosome that allow Ca²⁺ ions to “drain” into the surrounding cytosol. This conclusion was confirmed by monitoring the translocation of the intracellularly expressed YFP-tagged C2 domain of PKC-γ. This approach marked regions of membrane at which Ca²⁺ influx occurred, the earliest being the phagocytic cup, and then the whole cell. This paper therefore presents data that have novel implications for understanding phagocytic Ca²⁺ signalling events, such as peri-phagosomal Ca²⁺ hotspots, and other phenomena. Full article

Extracellular vesicles produced by tumor cells (TEVs) influence all stages of cancer development and spread, including tumorigenesis, cancer progression, and metastasis. TEVs can trigger profound phenotypic and functional changes in target cells through three main general mechanisms: (i) docking of TEVs on target cells and triggering of intra-cellular signaling; (ii) fusion of TEVs and target cell membranes with release of TEVs molecular cargo in the cytoplasm of recipient cell; and (iii) uptake of TEVs by recipient cells. Though the overall tumor-promoting effects of TEVs as well as the general mechanisms involved in TEVs interactions with, and uptake by, recipient cells are relatively well established, current knowledge about the molecular determinants that mediate the docking and uptake of tumor-derived EVs by specific target cells is still rather deficient. These molecular determinants dictate the cell and organ tropism of TEVs and ultimately control the specificity of TEVs-promoted metastases. Here, we will review current knowledge on selected specific molecules that mediate the tropism of TEVs towards specific target cells and organs, including the integrins, ICAM-1 Inter-Cellular Adhesion Molecule), ALCAM (Activated Leukocyte Cell Adhesion Molecule), CD44, the metalloproteinases ADAM17 (A Disintegrin And Metalloproteinase member 17) and ADAM10 (A Disintegrin And Metalloproteinase member 10), and the tetraspanin CD9. Full article

Leptomeningeal metastasis (LM) is a common and fatal complication of advanced non-small cell lung cancer (NSCLC) caused by the spread of malignant cells to the leptomeninges and cerebrospinal fluid (CSF). While intra-CSF methotrexate (MTX) chemotherapy can improve prognosis, eventual MTX resistance deters continued chemotherapy. Recent studies have shown that increased miRNA-21 (miR-21) expression in the CSF of patients with LM after intraventricular MTX-chemotherapy is associated with poor overall survival; however, the molecular mechanisms underlying this resistance are poorly understood. Here, we confirm, in 36 patients with NSCLC-LM, that elevated miR-21 expression prior to treatment correlates with poor prognosis. MiR-21 overexpression or sponging results in a corresponding increase or decrease in MTX resistance, demonstrating that cellular miR-21 expression correlates with drug resistance. MiR-21-monitoring sensor and fluorescent extracellular vesicle (EV) staining revealed that EV-mediated delivery of miR-21 could modulate MTX resistance. Moreover, EVs isolated from the CSF of LM patients containing miR-21 could enhance the cell proliferation and MTX resistance of recipient cells. These results indicate that miR-21 can be transferred from cell-to-cell via EVs and potentially modulate MTX sensitivity, suggesting that miR-21 in CSF EVs may be a prognostic and therapeutic target for overcoming MTX resistance in patients with NSCLC-LM. Full article