Extracellular Levels Research Articles

The Renin-Angiotensin-Aldosterone System Regulates Sarcoidosis Granulomatous Inflammation.

Rationale: Sarcoidosis is a granulomatous disorder of unclear cause notable for abnormal elevation of blood and tissue ACE1 (angiotensin converting enzyme 1) levels and activity. ACE1 regulates the renin-angiotensin-aldosterone system (RAAS), the terminal product of which is aldosterone, which selectively engages mineralocorticoid receptors to promote inflammation. Objectives: We sought to determine whether the RAAS promotes sarcoidosis granuloma formation and related inflammatory responses. Methods: Using an established ex vivo model, we first determined whether aldosterone was produced by sarcoidosis granulomas and verified the presence of CYP11B2, the enzyme required for its production. We then evaluated the effects of selective inhibitors of ACE1 (captopril), angiotensin type 1 receptor (losartan), and mineralocorticoid receptors (spironolactone, eplerenone) on granuloma formation, reflected by computer image analysis-generated granuloma area, and selected cytokines incriminated in sarcoidosis pathogenesis. Measurements and Main Results: Aldosterone was spontaneously produced by sarcoidosis peripheral blood mononuclear cells, and both intra- and extracellular levels steadily increased during granuloma formation. In parallel, peripheral blood mononuclear cells were shown to express more CYP11B2 during granuloma formation. Significant inhibition of sarcoidosis granulomas and related cytokines (TNFα, IL-1β, IFNγ, IL-10) was observed in response to pretreatments with captopril, losartan, spironolactone, or eplerenone, comparable to that of prednisone. Conclusions: The RAAS is intact in sarcoidosis granulomas and contributes significantly to early granuloma formation and to related inflammatory mediator responses, with important implications for clinical management.

Noradrenergic modulation of stress induced catecholamine release: Opposing influence of FG7142 and yohimbine.

Life stress modulates decision making, particularly in the face of risk, in some cases prompting vulnerable populations to make suboptimal, life-altering choices. In the brain, stress is known to alter the extracellular release of catecholamines in structures such as basolateral amygdala (BLA) and nucleus accumbens (NAc), but the relationship between catecholamines and decision-making behavior under stress has not been systemically explored. We developed an operant touchscreen decision-making task for rats comprising elements of loss aversion and risk seeking behavior. Rats were first injected systemically with an adrenergic -receptor agonist (guanfacine) and antagonist (yohimbine), as well as a partial inverse GABAA agonist, FG 7142, known to induce anxiety and stress related physiological responses in a variety of species, including humans. We then used fiber photometry to monitor NE in the basolateral amygdala (BLA), and DA activity in the nucleus accumbens (NAc) while animals engaged in decision-making and following systemic injections of FG 7142 and yohimbine. Neither yohimbine nor guanfacine had any impact on decision making strategy but altered motivational state with yohimbine making the animal almost insensitive to the reward outcome. The pharmacological induction of stress with FG 7142 biased the rats' decisions towards safety, but this bias shifted toward risk when co-treated with yohimbine. In the BLA and NAc, the FG 7142 altered catecholamine release, with systemic yohimbine producing opposing effects on NE and DA release. Stress induced changes in catecholamine release in the BLA and NAc can directly influence loss sensitivity, decisions and motivation, which can be modulated by the adrenoreceptor antagonist, yohimbine.

The cytoplasmic phosphate level has a central regulatory role in the phosphate starvation response of Caulobacter crescentus

In bacteria, the availability of environmental inorganic phosphate is typically sensed by the conserved PhoR-PhoB two-component signal transduction pathway, which uses the flux through the PstSCAB phosphate transporter as a readout of the extracellular phosphate level to control phosphate-responsive genes. While the sensing of environmental phosphate is well-investigated, the regulatory effects of cytoplasmic phosphate are unclear. Here, we disentangle the physiological and transcriptional responses of Caulobacter crescentus to changes in the environmental and cytoplasmic phosphate levels by uncoupling phosphate uptake from the activity of the PstSCAB system, using an additional, heterologously produced phosphate transporter. This approach reveals a two-pronged response of C. crescentus to phosphate limitation, in which PhoR-PhoB signaling mostly facilitates the utilization of alternative phosphate sources, whereas the cytoplasmic phosphate level controls the morphological and physiological adaptation of cells to growth under global phosphate limitation. These findings open the door to a comprehensive understanding of phosphate signaling in bacteria.

The expression and secretion of human lactoferrin in Bacillus subtilis

Human lactoferrin (HLF), an essential nutrient found in breast milk, possesses antibacterial, anti-inflammatory, and immune-enhancing properties. In this study, the effects of three constitutive promoters (P21, P43, and Pveg) and three inducible promoters (Pgrac100, PxylA, and Ptet*) on the expression of HLF were compared using Bacillus subtilis G601 as the host strain. The results showed that the highest expression of HLF, reaching 651.57 μg/L, was achieved when regulated by the Ptet* promoter. Furthermore, the combinational optimization of ribosome binding site (RBS) and signal peptides was investigated, and the optimal combination of RBS6 and SPyycP resulted in increased HLF expression to 1 099.87 μg/L, with 498.68 μg/L being secreted extracellularly. To further enhance HLF secretion, the metal cations-related gene dltD was knocked out, leading to an extracellular HLF level of 637.28 μg/L. This study successfully demonstrated the secretory expression of HLF in B. subtilis through the selection and optimization of expression elements, laying the foundation for the development of efficient B. subtilis cell factories for lactoprotein synthesis.

All-Trans Retinoic Acid-Induced Cell Surface Heat Shock Protein 90 Mediates Tau Protein Internalization and Degradation in Human Microglia.

This study investigates the role of all-trans retinoic acid (ATRA) in modulating the expression of heat shock protein 90 (Hsp90) and its influence on the uptake and degradation of tau proteins in immortalized human microglia cells. We demonstrate that ATRA significantly upregulates Hsp90 expression in a concentration-dependent manner, enhancing both extracellular and intracellular Hsp90 levels. Our results show that ATRA-treated cells exhibit increased tau protein uptake via caveolae/raft-dependent endocytosis pathways. This uptake is mediated by surface Hsp90, as evidenced by the inhibition of tau internalization using an extracellular Hsp90-selective inhibitor. Further, we establish that the exogenously added full-sized monomeric tau proteins bind to Hsp90. The study also reveals that ATRA-enhanced tau uptake is followed by effective degradation through both lysosomal and proteasomal pathways. We observed a significant reduction in intracellular tau levels in ATRA-treated cells, which was reversed by lysosome or proteasome inhibitors, suggesting the involvement of both degradation pathways. Our findings highlight the potential therapeutic role of ATRA in Alzheimer's disease and related tauopathies. By enhancing Hsp90 expression and facilitating tau degradation, ATRA could contribute to the clearance of pathological tau proteins, offering a promising strategy for mitigating neurodegeneration. This research underscores the need for further exploration into the molecular mechanisms of tau protein internalization and degradation, which could provide valuable insights into the treatment of neurodegenerative diseases.

Virus-Induced MicroRNA Modulation and Systemic Sclerosis Disease.

MicroRNAs (miRNAs) are short noncoding RNA sequences that regulate gene expression at the post-transcriptional level. They are involved in the regulation of multiple pathways, related to both physiological and pathological conditions, including autoimmune diseases, such as Systemic Sclerosis (SSc). Specifically, SSc is recognized as a complex and multifactorial disease, characterized by vascular abnormalities, immune dysfunction, and progressive fibrosis, affecting skin and internal organs. Among predisposing environmental triggers, evidence supports the roles of oxidative stress, chemical agents, and viral infections, mostly related to those sustained by beta-herpesviruses such as HCMV and HHV-6. Dysregulated levels of miRNA expression have been found in SSc patients compared to healthy controls, at both the intra- and extracellular levels, providing a sort of miRNA signature of the SSc disease. Notably, HCMV/HHV-6 viral infections were shown to modulate the miRNA profile, often superposing that observed in SSc, potentially promoting pathological pathways associated with SSc development. This review summarizes the main data regarding miRNA alterations in SSc disease, highlighting their potential as prognostic or diagnostic markers for SSc disease, and the impact of the putative SSc etiological agents on miRNA modulation.

Glioprotective Effects of Sulforaphane in Hypothalamus: Focus on Aging Brain.

Sulforaphane is a natural compound with neuroprotective activity, but its effects on hypothalamus remain unknown. In line with this, astrocytes are critical cells to maintain brain homeostasis, and hypothalamic astrocytes are fundamental for sensing and responding to environmental changes involved in a variety of homeostatic functions. Changes in brain functionality, particularly associated with hypothalamic astrocytes, can contribute to age-related neurochemical alterations and, consequently, neurodegenerative diseases. Thus, here, we investigated the glioprotective effects of sulforaphane on hypothalamic astrocyte cultures and hypothalamic cell suspension obtained from aged Wistar rats (24months old). Sulforaphane showed anti-inflammatory and antioxidant properties, as well as modulated the mRNA expression of astroglial markers, such as aldehyde dehydrogenase 1 family member L1, aquaporin 4, and vascular endothelial growth factor. In addition, it increased the expression and extracellular levels of trophic factors, such as glia-derived neurotrophic factor and nerve growth factor, as well as the release of brain-derived neurotrophic factor and the mRNA of TrkA, which is a receptor associated with trophic factors. Sulforaphane also modulated the expression of classical pathways associated with glioprotection, including nuclear factor erythroid-derived 2-like 2, heme oxygenase-1, nuclear factor kappa B p65 subunit, and AMP-activated protein kinase. Finally, a cell suspension with neurons and glial cells was used to confirm the predominant effect of sulforaphane in glial cells. In summary, this study indicated the anti-aging and glioprotective activities of sulforaphane in aged astrocytes.

Antioxidant and Neuroprotective Effects of Fucoxanthin and Its Metabolite Fucoxanthinol: A Comparative In Vitro Study.

Fucoxanthin is the most abundant carotenoid found in marine brown algae that exhibits several healthy properties. Dietary fucoxanthin is metabolized in the intestine, plasma, and other tissues to various metabolites, including fucoxanthinol. In this regard, the contribution of fucoxanthinol to the healthy properties of its precursor, fucoxanthin, against pathogenetic events associated with neurodegenerative diseases remains unexplored. Here, we evaluated and compared the antioxidant and neuroprotective effects of the carotenoids fucoxanthin and fucoxanthinol in in vitro models of Alzheimer's (AD) and Parkinson's (PD) disease. Neuronal SH-SY5Y cells were used to evaluate the antioxidant properties of the carotenoids against ABTS radical in the membrane and cytoplasm and oxidative stress elicited by tert-butyl hydroperoxide using the 2',7'-dichlorodihydrofluorescein diacetate probe. We also assessed the ability of the carotenoids to increase the glutathione (GSH) and activate the Nrf2/Keap1/ARE pathway using the monochlorobimane probe and western blotting method, respectively. The neuroprotective effects of the carotenoids against the neurotoxicity generated by oligomers of Beta-Amyloid (1-42) peptide (OAβ) and 6-hydroxydopamine (6-OHDA), which are neurotoxins of AD and PD, respectively, were finally evaluated in the same neuronal cells using the thiazolyl blue tetrazolium bromide assay. Both carotenoids could reach the cytoplasm, which explains the mainly free radical scavenging activity at this level. Notably, fucoxanthinol had higher and lower antioxidant activity than fucoxanthin at extracellular and cellular levels. Although studied carotenoids exerted the ability to activate the Nrf2/Keap1/ARE pathway, leading to an increase of intracellular GSH, our results suggested that the antioxidant activity of the carotenoids could be mainly attributed to their radical scavenging activity in neuronal membrane and cytoplasm, where they accumulate. Fucoxanthinol also shared similar neuroprotective effects as fucoxanthin against the neurotoxicity generated by OAβ and 6-OHDA, suggesting a potential neuroprotective contribution to the action of fucoxanthin administered as a food supplement in in vivo experimental models. These results encourage further research to evaluate the bioavailability of fucoxanthinol and other metabolites of fucoxanthin at the brain level to elucidate the dietary neuroprotective potential of fucoxanthin.

Mechanisms Underlying the Effects of Chloroquine on Red Blood Cells Metabolism.

Chloroquine (CQ) is a 4-aminoquinoline derivative largely employed in the management of malaria. CQ treatment exploits the drug's ability to cross the erythrocyte membrane, inhibiting heme polymerase in malarial trophozoites. Accumulation of CQ prevents the conversion of heme to hemozoin, causing its toxic buildup, thus blocking the survival of Plasmodium parasites. Recently, it has been reported that CQ is able to exert antiviral properties, mainly against HIV and SARS-CoV-2. This renewed interest in CQ treatment has led to the development of new studies which aim to explore its side effects and long-term outcome. Our study focuses on the effects of CQ in non-parasitized red blood cells (RBCs), investigating hemoglobin (Hb) functionality, the anion exchanger 1 (AE1) or band 3 protein, caspase 3 and protein tyrosine phosphatase 1B (PTP-1B) activity, intra and extracellular ATP levels, and the oxidative state of RBCs. Interestingly, CQ influences the functionality of both Hb and AE1, the main RBC proteins, affecting the properties of Hb oxygen affinity by shifting the conformational structure of the molecule towards the R state. The influence of CQ on AE1 flux leads to a rate variation of anion exchange, which begins at a concentration of 2.5 μM and reaches its maximum effect at 20 µM. Moreover, a significant decrease in intra and extracellular ATP levels was observed in RBCs pre-treated with 10 µM CQ vs. erythrocytes under normal conditions. This effect is related to the PTP-1B activity which is reduced in RBCs incubated with CQ. Despite these metabolic alterations to RBCs caused by exposure to CQ, no signs of variations in oxidative state or caspase 3 activation were recorded. Our results highlight the antithetical effects of CQ on the functionality and metabolism of RBCs, and encourage the development of new research to better understand the multiple potentiality of the drug.

Foxo1 is an iron-responsive transcriptional factor regulating systemic iron homeostasis

AbstractThe liver plays a crucial role in maintaining systemic iron homeostasis by secreting hepcidin, which is essential for coordinating iron levels in the body. Imbalances in iron homeostasis are associated with various clinical disorders related to iron deficiency or iron overload. Despite the clinical significance, the mechanisms underlying how hepatocytes sense extracellular iron levels to regulate hepcidin synthesis and iron storage are not fully understood. In this study, we identified Foxo1, a well-known regulator of macronutrient metabolism, which translocates to the nucleus of hepatocytes in response to high-iron feeding, holo-transferrin, and bone morphogenetic protein 6 (BMP6) treatment. Furthermore, Foxo1 plays a crucial role in mediating hepcidin induction in response to both iron and BMP signals by directly interacting with evolutionally conserved Foxo binding sites within the hepcidin promoter region. These binding sites were found to colocalize with Smad-binding sites. To investigate the physiological relevance of Foxo1 in iron metabolism, we generated mice with hepatocyte-specific deletion of Foxo1. These mice exhibited reduced hepatic hepcidin expression and serum hepcidin levels, accompanied by elevated serum iron and liver nonheme iron concentrations. Moreover, high-iron diet further exacerbated these abnormalities in iron metabolism in mice lacking hepatic Foxo1. Conversely, hepatocyte-specific Foxo1 overexpression increased hepatic hepcidin expression and serum hepcidin levels, thereby ameliorating iron overload in a murine model of hereditary hemochromatosis (Hfe−/− mice). In summary, our study identifies Foxo1 as a critical regulator of hepcidin and systemic iron homeostasis. Targeting Foxo1 may offer therapeutic opportunities for managing conditions associated with aberrant iron metabolism.

Adrenalectomy exacerbates stress-induced impairment in fear discrimination: A causal role for kynurenic acid?

Impaired activity of the hypothalamic-pituitary axis and reduced blood levels of glucocorticoids (GCs) are signature features of stress-related maladies. Recent evidence suggests a possible role of the tryptophan metabolite kynurenic acid (KYNA) in this context. Here we investigated possible causal relationships in adult male rats, using stress-induced fear discrimination as a translationally relevant behavioral outcome measure. One week following adrenalectomy (ADX) or sham surgery, animals were for 2 h either physically restrained or exposed to a predator odor, which caused a much milder stress response. Extracellular KYNA levels were determined before, during and after stress by in vivo microdialysis in the prefrontal cortex. Separate cohorts underwent a fear discrimination procedure starting immediately after stress termination. Different auditory conditioned stimuli (CS) were either paired with a foot shock (CS+) or non-reinforced (CS−). One week later, fear was assessed by re-exposing the animals to each CS. Separate groups of rats were treated with the KYNA synthesis inhibitor BFF-816 prior to stress initiation to test a causal role of KYNA in fear discrimination. Restraint stress raised extracellular KYNA levels by ∼85 % in ADX rats for several hours, and these animals were unable to discriminate between CS+ and CS−. Both effects were prevented by BFF-816 and were not observed after exposure to predator odor or in sham-operated rats. These findings suggest that a causal connection exists between adrenal function, stress-induced KYNA increases, and behavioral deficits. Pharmacological inhibition of KYNA synthesis may therefore be an attractive, novel option for the treatment of stress-related disorders.

A Mesoporous Gold Sensor Unveils Phospho PD-L1 in Extracellular Vesicles as a Proxy for PD-L1 Expression in Lung Cancer Tissue.

Immune checkpoint inhibitors (ICIs) targeting programmed cell death ligand 1 (PD-L1), or its receptor, PD-1 have improved survival in patients with non-small-cell lung cancer (NSCLC). Assessment of PD-L1 expression requires tissue biopsy or fine needle aspiration that are currently used to identify patients most likely to respond to single agent anti-PD-1/PD-L1 therapy. However, obtaining sufficient tissue to generate a PD-L1 tissue proportion score (TPS) ≥ 50% using immunohistochemistry remains a challenge that potentially may be overcome by liquid biopsies. This study utilized a mesoporous gold sensor (MGS) assay to examine the phosphorylation status of PD-L1 in plasma extracellular vesicles (EV pPD-L1) and PD-L1 levels in plasma from NSCLC patient samples and their association with tumor PD-L1 TPS. The 3-dimensional mesoporous network of the electrodes provides a large surface area, high signal-to-noise ratio, and a superior electro-conductive framework, thereby significantly improving the detection sensitivity of PD-L1 nanosensing. Test (n = 20) (Pearson's r = 0.99) and validation (n = 45) (Pearson's r = 0.99) cohorts show that EV pPD-L1 status correlates linearly with the tumor PD-L1 TPS assessed by immunohistochemistry irrespective of the tumor stage, with 64% of patients overall showing detectable EV pPD-L1 levels in plasma. In contrast to the EV pPD-L1 results, plasma PD-L1 levels did not correlate with the tumor PD-L1 TPS score or EV pPD-L1 levels. These data demonstrate that EV pPD-L1 levels may be used to select patients for appropriate PD-1 and PD-L1 ICI therapy regimens in early, locally advanced, and advanced NSCLC and should be tested further in randomized controlled trials. Most importantly, the assay used has a less than 24h turnaround time, facilitating adoption of the test into the routine diagnostic evaluation of patients prior to therapy.

Elevated neutrophil extracellular trap levels in periodontitis: Implications for keratinization and barrier function in gingival epithelium.

To explore the levels of neutrophil extracellular traps (NETs) in patients with periodontitis and examine their effects on keratinization, barrier function of human gingival keratinocytes (HGKs) and the associated mechanisms. Saliva, gingival crevicular fluid (GCF), clinical periodontal parameters and gingival specimens were collected from 10 healthy control subjects and 10 patients with stage II-IV periodontitis to measure the NET levels. Subsequently, mRNA and protein levels of keratinization and barrier indicators, as well as intracellular calcium and epithelial barrier permeability, were analysed in HGKs after NET stimulation. The study showed that NET levels significantly elevated in patients with periodontitis, across multiple specimens including saliva, GCF and gingival tissues. Stimulation of HGKs with NETs resulted in a decrease in the expressions of involucrin, cytokeratin 10, zonula occludens 1 and E-cadherin, along with decreased intracellular calcium levels and increased epithelial barrier permeability. Furthermore, the inhibition of keratinization by NETs is ERK-KLF4-dependent. This study indicates that NETs impair the barrier function of HGKs and suppress keratinization through ERK/KLF4 axis. These findings provide potential targets for therapeutic approaches in periodontitis to address impaired gingival keratinization.

Chronic Lead Exposure in Adult Mice: Associations with miR-671/CDR1as Regulation, NF-κB Signaling, and Alzheimer's Disease-like Pathology.

Long-term exposure to lead (Pb) can result in chronic damage to the body through accumulation in the central nervous system (CNS) leading to neurodegenerative diseases, such as Alzheimer's disease (AD). This study delves into the intricate role of miR-671/CDR1as regulation in the etiology of AD-like lesions triggered by chronic Pb exposure in adult mice. To emulate the chronic effects of Pb, we established a rodent model spanning 10 months of controlled Pb administration, dividing 52 C57BL/6J mice into groups receiving varying concentrations of Pb (1, 2, or 4 g/L) alongside an unexposed control. Blood Pb levels were monitored using serum samples to ensure accurate dosing and to correlate with observed toxicological outcomes. Utilizing the Morris water maze, a robust behavioral assay for assessing cognitive functions, we documented a dose-dependent decline in learning and memory capabilities among the Pb-exposed mice. Histopathological examination of the hippocampal tissue revealed tell-tale signs of AD-like neurodegeneration, characterized by the accumulation of amyloid plaques and neurofibrillary tangles. At the molecular level, a significant upregulation of AD-associated genes, namely amyloid precursor protein (APP), β-secretase 1 (BACE1), and tau, was observed in the hippocampal tissue of Pb-exposed mice. This was accompanied by a corresponding surge in the protein levels of APP, BACE1, amyloid-β (Aβ), and phosphorylated tau (p-tau), further implicating Pb in the dysregulation of these key AD markers. The expression of CDR1as, a long non-coding RNA implicated in AD pathogenesis, was found to be suppressed in Pb-exposed mice. This observation suggests a potential mechanistic link between Pb-induced neurotoxicity and the dysregulation of the CDR1as/miR-671 axis, which warrants further investigation. Moreover, our study identified a dose-dependent alteration in the intracellular and extracellular levels of the transcription factor nuclear factor-kappa B (NF-κB). This finding implicates Pb in the modulation of NF-κB signaling, a pathway that plays a pivotal role in neuroinflammation and neurodegeneration. In conclusion, our findings underscored the deleterious effects of Pb exposure on the CNS, leading to the development of AD-like pathology. The observed modulation of NF-κB signaling and miR-671/CDR1as regulation provides a plausible mechanistic framework for understanding the neurotoxic effects of Pb and its potential contribution to AD pathogenesis.

Prophylactic nicotinamide mononucleotide (NMN) mitigates CSDS-induced depressive-like behaviors in mice via preserving of ATP level in the mPFC

Depression is a prevalent psychiatric disorder with accumulating evidence implicating dysregulation of extracellular adenosine triphosphate (ATP) levels in the medial prefrontal cortex (mPFC). It remains unclear whether facilitating endogenous ATP production and subsequently increasing extracellular ATP level in the mPFC can exert a prophylactic effect against chronic social defeat stress (CSDS)-induced depressive-like behaviors and enhance stress resilience. Here, we found that nicotinamide mononucleotide (NMN) treatment effectively elevated nicotinamide adenine dinucleotide (NAD+) biosynthesis and extracellular ATP levels in the mPFC. Moreover, both the 2-week intraperitoneal (i.p.) injection and 3-week oral gavage of NMN prior to exposure to CSDS effectively prevented the development of depressive-like behavior in mice. These protective effects were accompanied with the preservation of both NAD+ biosynthesis and extracellular ATP level in the mPFC. Furthermore, catalyzing ATP hydrolysis by mPFC injection of the ATPase apyrase negated the prophylactic effects of NMN on CSDS-induced depressive-like behaviors. Prophylactic NMN treatment also prevented the reduction in GABAergic inhibition and the increase in excitability in mPFC neurons projecting to the lateral habenula (LHb). Collectively, these findings demonstrate that the prophylactic effects of NMN on depressive-like behaviors are mediated by preventing extracellular ATP loss in the mPFC, which highlights the potential of NMN supplementation as a novel approach for protecting and preventing stress-induced depression in susceptible individuals.

Evaluation of the potential role of glutamatergic, cholinergic, and nitrergic systems in the dopamine release induced by the pesticide glyphosate in rat striatum.

Glyphosate (GLY) is a pesticide that severely alters nigrostriatal dopaminergic neurotransmission, inducing great increases in dopamine release from rat dorsal striatum. This GLY-induced striatal dopamine overflow occurs through mechanisms not yet fully understood, hence the interest in evaluating the role of other neurotransmitter systems in such effects. So, the main objective of this mechanistic study was to evaluate the possible mediation of the glutamatergic, cholinergic, and nitrergic systems in the GLY-induced in vivo dopamine release from rat dorsal striatum. The extracellular dopamine levels were measured by cerebral microdialysis and HPLC with electrochemical detection. Intrastriatal administration of GLY (5mmol/L) significantly increased the dopamine release (1102%). Pretreatment with MK-801 (50 or 400 μmol/L), a non-competitive antagonist of NMDA receptors, significantly decreased the effect of GLY (by 70% and 74%, respectively), whereas AP-5 (400 μmol/L), a competitive antagonist of NMDA receptors, or CNQX (500 μmol/L), an AMPA/kainate receptor antagonist, had no significant effect. Administration of the nitric oxide synthase inhibitors, L-nitroarginine (L-NAME, 100 μmol/L) or 7-nitroindazole (7-NI, 100 μmol/L), also did not alter the effect of GLY on dopamine release. Finally, pretreatment of the animals with mecamylamine, an antagonist of nicotinic receptors, decreased the effect of GLY on dopamine release by 49%, whereas atropine, a muscarinic antagonist, had no significant effect. These results indicate that GLY-induced dopamine release largely depends on the activation of NMDA and nicotinic receptors in rat dorsal striatum. Future research is needed to determine the effects of this pesticide at environmentally relevant concentrations.

Curcumin modulates purinergic signaling and inflammatory response in cutaneous metastatic melanoma cells.

Cutaneous melanoma (CM) poses a therapeutic challenge due to its aggressive nature and often limited response to conventional treatments. Exploring novel therapeutic targets is essential, and natural compounds have emerged as potential candidates. This study aimed to elucidate the impact of curcumin, a natural compound known for its anti-inflammatory, antioxidant, and anti-tumor properties, on metastatic melanoma cells, focusing on the purinergic system and immune responses. Human melanoma cell line SK-Mel-28 were exposed to different curcumin concentrations for either 6 or 24h, after which we assessed components related to the purinergic system and the inflammatory cascade. Using RT-qPCR, we assessed the gene expression of CD39 and CD73 ectonucleotidases, as well as adenosine deaminase (ADA). Curcumin effectively downregulated CD39, CD73, and ADA gene expression. Flow cytometry analysis revealed that curcumin significantly reduced CD39 and CD73 protein expression at specific concentrations. Moreover, the A2A receptor's protein expression decreased across all concentrations. Enzymatic activity assays demonstrated that curcumin modulated CD39, CD73, and ADA activities, with effects dependent on concentration and duration of treatment. Extracellular ATP levels increased after 24h of curcumin treatment, emphasizing its role in modulating hydrolytic activity. Curcumin also displayed anti-inflammatory properties by reducing NLRP3 gene expression and impacting the levels of key inflammatory cytokines. In conclusion, this study unveils the potential of curcumin as a promising adjuvant in CM treatment. Curcumin modulates the expression and activity of crucial components of the purinergic system and exhibits anti-inflammatory effects, indicating its potential therapeutic role in combating CM. These findings underscore curcumin's promise and warrant further investigation in preclinical and clinical settings for melanoma management.

Cartilage-Specific Gene Expression and Extracellular Matrix Deposition in the Course of Mesenchymal Stromal Cell Chondrogenic Differentiation in 3D Spheroid Culture.

Articular cartilage damage still remains a major problem in orthopedical surgery. The development of tissue engineering techniques such as autologous chondrocyte implantation is a promising way to improve clinical outcomes. On the other hand, the clinical application of autologous chondrocytes has considerable limitations. Mesenchymal stromal cells (MSCs) from various tissues have been shown to possess chondrogenic differentiation potential, although to different degrees. In the present study, we assessed the alterations in chondrogenesis-related gene transcription rates and extracellular matrix deposition levels before and after the chondrogenic differentiation of MSCs in a 3D spheroid culture. MSCs were obtained from three different tissues: umbilical cord Wharton's jelly (WJMSC-Wharton's jelly mesenchymal stromal cells), adipose tissue (ATMSC-adipose tissue mesenchymal stromal cells), and the dental pulp of deciduous teeth (SHEDs-stem cells from human exfoliated deciduous teeth). Monolayer MSC cultures served as baseline controls. Newly formed 3D spheroids composed of MSCs previously grown in 2D cultures were precultured for 2 days in growth medium, and then, chondrogenic differentiation was induced by maintaining them in the TGF-β1-containing medium for 21 days. Among the MSC types studied, WJMSCs showed the most similarities with primary chondrocytes in terms of the upregulation of cartilage-specific gene expression. Interestingly, such upregulation occurred to some extent in all 3D spheroids, even prior to the addition of TGF-β1. These results confirm that the potential of Wharton's jelly is on par with adipose tissue as a valuable cell source for cartilage engineering applications as well as for the treatment of osteoarthritis. The 3D spheroid environment on its own acts as a trigger for the chondrogenic differentiation of MSCs.

Agrin-Lrp4 pathway in hippocampal astrocytes restrains development of temporal lobe epilepsy through adenosine signaling

BackgroundHuman patients often experience an episode of serious seizure activity, such as status epilepticus (SE), prior to the onset of temporal lobe epilepsy (TLE), suggesting that SE can trigger the development of epilepsy. Yet, the underlying mechanisms are not fully understood. The low-density lipoprotein receptor related protein (Lrp4), a receptor for proteoglycan-agrin, has been indicated to modulate seizure susceptibility. However, whether agrin-Lrp4 pathway also plays a role in the development of SE-induced TLE is not clear.MethodsLrp4f/f mice were crossed with hGFAP-Cre and Nex-Cre mice to generate brain conditional Lrp4 knockout mice (hGFAP-Lrp4−/−) and pyramidal neuron specific knockout mice (Nex-Lrp4−/−). Lrp4 was specifically knocked down in hippocampal astrocytes by injecting AAV virus carrying hGFAP-Cre into the hippocampus. The effects of agrin-Lrp4 pathway on the development of SE-induced TLE were evaluated on the chronic seizure model generated by injecting kainic acid (KA) into the amygdala. The spontaneous recurrent seizures (SRS) in mice were video monitored.ResultsWe found that Lrp4 deletion from the brain but not from the pyramidal neurons elevated the seizure threshold and reduced SRS numbers, with no change in the stage or duration of SRS. More importantly, knockdown of Lrp4 in the hippocampal astrocytes after SE induction decreased SRS numbers. In accord, direct injection of agrin into the lateral ventricle of control mice but not mice with Lrp4 deletion in hippocampal astrocytes also increased the SRS numbers. These results indicate a promoting effect of agrin-Lrp4 signaling in hippocampal astrocytes on the development of SE-induced TLE. Last, we observed that knockdown of Lrp4 in hippocampal astrocytes increased the extracellular adenosine levels in the hippocampus 2 weeks after SE induction. Blockade of adenosine A1 receptor in the hippocampus by DPCPX after SE induction diminished the effects of Lrp4 on the development of SE-induced TLE.ConclusionThese results demonstrate a promoting role of agrin-Lrp4 signaling in hippocampal astrocytes in the development of SE-induced development of epilepsy through elevating adenosine levels. Targeting agrin-Lrp4 signaling may serve as a potential therapeutic intervention strategy to treat TLE.

#2903 Comprehensive lipidomic analysis reveals further insights into the molecular mechanism of podocyte lipotoxicity in Alport syndrome

Abstract Background and Aims The accumulation of lipids in podocytes has been shown to be a mediator of kidney injury in diabetic kidney disease (DKD). Recent animal studies have shown evidence of podocyte lipotoxicity in Alport syndrome (AS). First, discoidin domain receptor 1 (DDR1) is overactivated by excessive accumulation of α1 chain type 1 collagen in AS resulting in CD36 activation, producing direct podocyte injury by increasing reuptake of free fatty acids (FFAs). Furthermore, local overexpression of glomerular tumor necrosis factor has been observed in AS, which produces suppression of ABCA1, which affects mitochondrial function and decreases cholesterol efflux from podocytes, causing an accumulation of cholesterol and podocyte lipotoxicity. This study aimed to compare serum and urine lipidome variation between patients with AS and patients with DKD compared to healthy controls to explore the underlying molecular mechanisms of podocyte lipotoxicity in AS. Method A total of 63 samples from AS patients (COL4A3/4, n = 50, COL4A5, n = 13) were employed for this study, and compared to 15 samples from DKD patients and 20 samples from healthy volunteers considered as controls. We performed an untargeted lipidomics analysis to cover the broader spectrum of plasma and urine lipidome. Samples were analyzed by using an Agilent 1290 Infinity II Ultra-High-Performance Liquid-Chromatography system coupled to an Agilent 6546 Quadrupole Time-of-Flight Mass Spectrometer equipped with dual Agilent Jet Stream Electrospray ion source. For plasma samples, raw data matrix underwent normalization for sequence intensity correction based on the QC samples by using the support vector regression. For urine samples, normalization was performed using the Probabilistic Quotient Normalization to account for unwanted variance due to sample preparation and the analytical run. Results We generated a heatmap employing hierarchical clustering to visually represent the distinctions in statistically significant lipids across AS COL4A3/4, AS COL4A5, and DKD groups compared to the healthy control group, categorized by classes. Figure 1 summarizes the results of our study. In urine samples, a distinct decrease in acylcarnitines is evident in AS COL4A3/4 and AS COL4A5. Regarding the fatty acid (FA) lipid class, there is an increased general trend. FA 20:3 shows a meaningful increase in AS COL4A3/4 and DKD compared to the healthy control group, whereas in AS COL4A5, the trend of this lipid species is to decline when compared with COL4A3/4 and DKD levels. Concerning plasma samples, different trends were observed in AS COL4A3/4 and DKD depending on specific lipid species, while general reduced levels were reported in AS COL4A5. The phosphatidylcholine (PC) lipid species, implicated in various biological functions, display a drastic increase in the DKD urine group compared to the healthy control. The same trend is followed by AS COL4A3/4 and AS COL4A5 urine groups, with relatively similar data. Among the sphingolipids class, in urine, the overall trend of sphingomyelins (SM) for both AS COL4A3/4 and AS COL4A5 indicates a decrease in the levels of the statistically significant lipid species, with relatively similar levels between them. The neutral sphingolipids (HexCer) experience a general decrease in AS COL4A3/4, AS COL4A5 and DKD urine samples compared to the healthy control group. Lastly, the ceramides (Cer) follow a decreasing trend, where the most remarkable differences could be observed in the DKD urine group compared to the healthy controls. Conclusion Overall, the present study provided new insights into the mechanism of podocyte lipotoxicity in patients with AS. Increased specific plasma Cer and SM that are responsible for correct COL4A1 and COL4A2 formation might be responsible for aberrant COL1 expression DDR1 activation. Furthermore, specific essential FFAs that are involved in collagen synthesis are increased in AS, and might be indicating aberrant CD36 levels increasing their levels and enhancing their accumulation. Increased ABCA1 efflux activity is evident in higher extracellular levels of PC observed in urine samples from patients with AS.