Exploring Galectin Interactions with Human Milk Oligosaccharides and Blood Group Antigens Identifies BGA6 as a Functional Galectin-4 Ligand.

Galectins (Gals), a family of multifunctional glycan-binding proteins, have been traditionally defined as ß-galactoside binding lectins. However, certain members of this family have shown selective affinity towards specific glycan structures including human milk oligosaccharides (HMOs) and blood group antigens. In this work, we explored the affinity of human galectins (particularly Gal-1, -3, -4, -7 and -12) towards a panel of oligosaccharides including HMOs and blood group antigens using a complementary approach based on both experimental and computational techniques. While prototype Gal-1 and Gal-7 exhibited differential affinity for type I vs. type II Lac/LacNAc residues and recognized fucosylated neutral glycans, chimera-type Gal-3 showed high binding affinity towards poly-LacNAc structures including LNnH and LNnO. Notably, the tandem-repeat human Gal-12 showed preferential recognition of 3-fucosylated glycans, a unique feature among members of the galectin family. Finally, Gal-4 presented a distinctive glycan-binding activity characterized by preferential recognition of specific blood group antigens, also validated by saturation transfer difference nuclear magnetic resonance (STD-NMR) experiments. Particularly, we identified oligosaccharide blood group A type 6 (BGA6) as a biologically relevant Gal-4 ligand, which specifically inhibited IL-6 secretion induced by this lectin on human peripheral blood mononuclear cells. These findings highlight unique determinants underlying specific recognition of HMOs and blood group antigens by human galectins, emphasizing the biological relevance of Gal-4-BGA6 interactions, with critical implications in the development and regulation of inflammatory responses.

Acute activation of hemichannels by ethanol leads to Ca2+-dependent gliotransmitter release in astrocytes.

Multiple studies have demonstrated that acute ethanol consumption alters brain function and cognition. Nevertheless, the mechanisms underlying this phenomenon remain poorly understood. Astrocyte-mediated gliotransmission is crucial for hippocampal plasticity, and recently, the opening of hemichannels has been found to play a relevant role in this process. Hemichannels are plasma membrane channels composed of six connexins or seven pannexins, respectively, that oligomerize around a central pore. They serve as ionic and molecular exchange conduits between the cytoplasm and extracellular milieu, allowing the release of various paracrine substances, such as ATP, D-serine, and glutamate, and the entry of ions and other substances, such as Ca2+ and glucose. The persistent and exacerbated opening of hemichannels has been associated with the pathogenesis and progression of several brain diseases for at least three mechanisms. The uncontrolled activity of these channels could favor the collapse of ionic gradients and osmotic balance, the release of toxic levels of ATP or glutamate, cell swelling and plasma membrane breakdown and intracellular Ca2+ overload. Here, we evaluated whether acute ethanol exposure affects the activity of astrocyte hemichannels and the possible repercussions of this phenomenon on cytoplasmatic Ca2+ signaling and gliotransmitter release. Acute ethanol exposure triggered the rapid activation of connexin43 and pannexin1 hemichannels in astrocytes, as measured by time-lapse recordings of ethidium uptake. This heightened activity derived from a rapid rise in [Ca2+]i linked to extracellular Ca2+ influx and IP3-evoked Ca2+ release from intracellular Ca2+ stores. Relevantly, the acute ethanol-induced activation of hemichannels contributed to a persistent secondary increase in [Ca2+]i. The [Ca2+]i-dependent activation of hemichannels elicited by ethanol caused the increased release of ATP and glutamate in astroglial cultures and brain slices. Our findings offer fresh perspectives on the potential mechanisms behind acute alcohol-induced brain abnormalities and propose targeting connexin43 and pannexin1 hemichannels in astrocytes as a promising avenue to prevent deleterious consequences of alcohol consumption.

The hepatocyte epidermal growth factor receptor (EGFR) pathway regulates the cellular interactome within the liver fibrotic niche.

Liver fibrosis is the consequence of chronic liver injury in the presence of an inflammatory component. Although the main executors of this activation are known, the mechanisms that lead to the inflammatory process that mediates the production of pro-fibrotic factors are not well characterized. Epidermal growth factor receptor (EGFR) signaling in hepatocytes is essential for the regenerative processes of the liver; however, its potential role in regulating the fibrotic niche is not yet clear. Our group generated a mouse model that expresses an inactive truncated form of the EGFR specifically in hepatocytes (ΔEGFR mice). Here, we have analyzed the response of WT and ΔEGFR mice to chronic treatment with carbon tetrachloride (CCl4), which induces a pro-inflammatory and fibrotic process in the liver. The results indicated that the hallmarks of liver fibrosis were attenuated in CCl4-treated ΔEGFR mice when compared with CCl4-treated WT mice, coinciding with a faster resolution of the fibrotic process and ameliorated damage. The absence of EGFR activity in hepatocytes induced changes in the pattern of immune cells in the liver, with a notable increase in the population of M2 macrophages, more related to fibrosis resolution, as well as in the population of lymphocytes related to eradication of the damage. Transcriptome analysis of hepatocytes, and secretome studies of extracellular media from in vitro experiments, allowed us to elucidate the specific molecular mechanisms regulated by EGFR that mediate hepatocyte production of both pro-fibrotic and pro-inflammatory mediators; these have consequences for the deposition of extracellular matrix proteins, as well as for the immune microenvironment. Overall, our study uncovered novel mechanistic insights regarding EGFR kinase-dependent actions in hepatocytes that reveal its key role in chronic liver damage. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Ca2+ permeation through C-terminal cleaved, but not full-length human Pannexin1 hemichannels, mediates cell death.

Pannexin1 hemichannels (Panx1 HCs) are found in the membrane of most mammalian cells and communicate the intracellular and extracellular spaces, enabling the passive transfer of ions and small molecules. They are involved in physiological and pathophysiological conditions. During apoptosis, the C-terminal tail of Panx1 is proteolytically cleaved, but the permeability features of hemichannels and their role in cell death remain elusive. To address these topics, HeLa cells transfected with full-length human Panx1 (fl-hPanx1) or C-terminal truncated hPanx1 (Δ371hPanx1) were exposed to alkaline extracellular saline solution, increasing the activity of Panx1 HCs. The Δ371hPanx1 HC was permeable to DAPI and Etd+, but not to propidium iodide, whereas fl-hPanx1 HC was only permeable to DAPI. Furthermore, the cytoplasmic Ca2+ signal increased only in Δ371hPanx1 cells, which was supported by bioinformatics approaches. The influx of Ca2+ through Δ371hPanx1 HCs was necessary to promote cell death up to about 95% of cells, whereas the exposure to alkaline saline solution without Ca2+ failed to induce cell death, and the Ca2+ ionophore A23187 promoted more than 80% cell death even in fl-hPanx1 transfectants. Moreover, cell death was prevented with carbenoxolone or 10Panx1 in Δ371hPanx1 cells, whereas it was undetectable in HeLa Panx1-/- cells. Pretreatment with Ferrostatin-1 and necrostatin-1 did not prevent cell death, suggesting that ferroptosis or necroptosis was not involved. In comparison, zVAD-FMK, a pancaspase inhibitor, reduced death by ~60%, suggesting the involvement of apoptosis. Therefore, alkaline pH increases the activity of Δ371hPanx1HCs, leading to a critical intracellular free-Ca2+ overload that promotes cell death.

Artificial Intelligence in Eye Movements Analysis for Alzheimer's Disease Early Diagnosis.

As the world's population ages, Alzheimer's disease is currently the seventh most common cause of death globally; the burden is anticipated to increase, especially among middle-class and elderly persons. Artificial intelligence-based algorithms that work well in hospital environments can be used to identify Alzheimer's disease. A number of databases were searched for English-language articles published up until March 1, 2024, that examined the relationships between artificial intelligence techniques, eye movements, and Alzheimer's disease. A novel non-invasive method called eye movement analysis may be able to reflect cognitive processes and identify anomalies in Alzheimer's disease. Artificial intelligence, particularly deep learning, and machine learning, is required to enhance Alzheimer's disease detection using eye movement data. One sort of deep learning technique that shows promise is convolutional neural networks, which need further data for precise classification. Nonetheless, machine learning models showed a high degree of accuracy in this context. Artificial intelligence-driven eye movement analysis holds promise for enhancing clinical evaluations, enabling tailored treatment, and fostering the development of early and precise Alzheimer's disease diagnosis. A combination of artificial intelligence-based systems and eye movement analysis can provide a window for early and non-invasive diagnosis of Alzheimer's disease. Despite ongoing difficulties with early Alzheimer's disease detection, this presents a novel strategy that may have consequences for clinical evaluations and customized medication to improve early and accurate diagnosis.

A C-type lectin from Bothrops jararacussu venom reprograms endothelial cell biology.

Snake venoms are intricate mixtures of enzymes and bioactive factors that induce a range of detrimental effects in afflicted hosts. Certain Viperids, including Bothrops jararacussu, harbor C-type lectins (CTLs) known for their modulation of a variety of host cellular responses. In this study, we isolated and purified BjcuL, a CTL from B. jararacussu venom and investigated its impact on endothelial cell behavior, contrasting it with human galectin-1 (Gal-1), a prototype member of the galectin family with shared ß-galactoside-binding activity. We found that BjcuL binds to human dermal microvascular endothelial cells (HMECs) in a concentration- and carbohydrate-dependent fashion and reprograms the function of these cells, favoring a pro-inflammatory and pro-coagulant endothelial phenotype. In light of the quest for universal antagonists capable of mitigating the harmful consequences of snake venoms, BjcuL emerges as a promising target to be blocked in order to regulate pathological endothelial cell responses.

Inhibition of astroglial hemichannels prevents synaptic transmission decline during spreading depression.

BACKGROUND: Spreading depression (SD) is an intriguing phenomenon characterized by massive slow brain depolarizations that affect neurons and glial cells. This phenomenon is repetitive and produces a metabolic overload that increases secondary damage. However, the mechanisms associated with the initiation and propagation of SD are unknown. Multiple lines of evidence indicate that persistent and uncontrolled opening of hemichannels could participate in the pathogenesis and progression of several neurological disorders including acute brain injuries. Here, we explored the contribution of astroglial hemichannels composed of connexin-43 (Cx43) or pannexin-1 (Panx1) to SD evoked by high-K+ stimulation in brain slices. RESULTS: Focal high-K+ stimulation rapidly evoked a wave of SD linked to increased activity of the Cx43 and Panx1 hemichannels in the brain cortex, as measured by light transmittance and dye uptake analysis, respectively. The activation of these channels occurs mainly in astrocytes but also in neurons. More importantly, the inhibition of both the Cx43 and Panx1 hemichannels completely prevented high K+-induced SD in the brain cortex. Electrophysiological recordings also revealed that Cx43 and Panx1 hemichannels critically contribute to the SD-induced decrease in synaptic transmission in the brain cortex and hippocampus. CONCLUSIONS: Targeting Cx43 and Panx1 hemichannels could serve as a new therapeutic strategy to prevent the initiation and propagation of SD in several acute brain injuries.

CD74 supports accumulation and function of regulatory T cells in tumors.

Regulatory T cells (Tregs) are plastic cells playing a pivotal role in the maintenance of immune homeostasis. Tregs actively adapt to the microenvironment where they reside; as a consequence, their molecular and functional profiles differ among tissues and pathologies. In tumors, the features acquired by Tregs remains poorly characterized. Here, we observe that human tumor-infiltrating Tregs selectively overexpress CD74, the MHC class II invariant chain. CD74 has been previously described as a regulator of antigen-presenting cell biology, however its function in Tregs remains unknown. CD74 genetic deletion in human primary Tregs reveals that CD74KO Tregs exhibit major defects in the organization of their actin cytoskeleton and intracellular organelles. Additionally, intratumoral CD74KO Tregs show a decreased activation, a drop in Foxp3 expression, a low accumulation in the tumor, and consistently, they are associated with accelerated tumor rejection in preclinical models in female mice. These observations are unique to tumor conditions as, at steady state, CD74KO-Treg phenotype, survival, and suppressive capacity are unaffected in vitro and in vivo. CD74 therefore emerges as a specific regulator of tumor-infiltrating Tregs and as a target to interfere with Treg anti-tumor activity.

Uncovering a Novel Functional Interaction Between Adult Hepatic Progenitor Cells, Inflammation and EGFR Signaling During Bile Acids-Induced Injury.

Chronic cholestatic damage is associated to both accumulation of cytotoxic levels of bile acids and expansion of adult hepatic progenitor cells (HPC) as part of the ductular reaction contributing to the regenerative response. Here, we report a bile acid-specific cytotoxic response in mouse HPC, which is partially impaired by EGF signaling. Additionally, we show that EGF synergizes with bile acids to trigger inflammatory signaling and NLRP3 inflammasome activation in HPC. Aiming at understanding the impact of this HPC specific response on the liver microenvironment we run a proteomic analysis of HPC secretome. Data show an enrichment in immune and TGF-ß regulators, ECM components and remodeling proteins in HPC secretome. Consistently, HPC-derived conditioned medium promotes hepatic stellate cell (HSC) activation and macrophage M1-like polarization. Strikingly, EGF and bile acids co-treatment leads to profound changes in the secretome composition, illustrated by an abolishment of HSC activating effect and by promoting macrophage M2-like polarization. Collectively, we provide new specific mechanisms behind HPC regulatory action during cholestatic liver injury, with an active role in cellular interactome and inflammatory response regulation. Moreover, findings prove a key contribution for EGFR signaling jointly with bile acids in HPC-mediated actions.

Autocrine activation of P2X7 receptors mediates catecholamine secretion in chromaffin cells.

BACKGROUND AND PURPOSE: ATP is highly accumulated in secretory vesicles and secreted upon exocytosis from neurons and endocrine cells. In adrenal chromaffin granules, intraluminal ATP reaches concentrations over 100 mM. However, how these large amounts of ATP contribute to exocytosis has not been investigated. EXPERIMENTAL APPROACH: Exocytotic events in bovine and mouse adrenal chromaffin cells were measured with single cell amperometry. Cytosolic Ca2+ measurements were carried out in Fluo-4 loaded cells. Submembrane Ca2+ was examined in PC12 cells transfected with a membrane-tethered Ca2+ indicator Lck-GCaMP3. ATP release was measured using the luciferin/luciferase assay. Knockdown of P2X7 receptors was induced with short interfering RNA (siRNA). Direct Ca2+ influx through this receptor was measured using a P2X7 receptor-GCamp6 construct. KEY RESULTS: ATP induced exocytosis in chromaffin cells, whereas the ectonucleotidase apyrase reduced the release events induced by the nicotinic agonist dimethylphenylpiperazinium (DMPP), high KCl, or ionomycin. The purinergic agonist BzATP also promoted a secretory response that was dependent on extracellular Ca2+. A740003, a P2X7 receptor antagonist, abolished secretory responses of these secretagogues. Exocytosis was also diminished in chromaffin cells when P2X7 receptors were silenced using siRNAs and in cells of P2X7 receptor knockout mice. In PC12 cells, DMPP induced ATP release, triggering Ca2+ influx through P2X7 receptors. Furthermore, BzATP, DMPP, and KCl allowed the formation of submembrane Ca2+ microdomains inhibited by A740003. CONCLUSION AND IMPLICATIONS: Autocrine activation of P2X7 receptors constitutes a crucial feedback system that amplifies the secretion of catecholamines in chromaffin cells by favouring submembrane Ca2+ microdomains.

Cx43 hemichannels and panx1 channels contribute to ethanol-induced astrocyte dysfunction and damage.

BACKGROUND: Alcohol, a widely abused drug, significantly diminishes life quality, causing chronic diseases and psychiatric issues, with severe health, societal, and economic repercussions. Previously, we demonstrated that non-voluntary alcohol consumption increases the opening of Cx43 hemichannels and Panx1 channels in astrocytes from adolescent rats. However, whether ethanol directly affects astroglial hemichannels and, if so, how this impacts the function and survival of astrocytes remains to be elucidated. RESULTS: Clinically relevant concentrations of ethanol boost the opening of Cx43 hemichannels and Panx1 channels in mouse cortical astrocytes, resulting in the release of ATP and glutamate. The activation of these large-pore channels is dependent on Toll-like receptor 4, P2X7 receptors, IL-1ß and TNF-α signaling, p38 mitogen-activated protein kinase, and inducible nitric oxide (NO) synthase. Notably, the ethanol-induced opening of Cx43 hemichannels and Panx1 channels leads to alterations in cytokine secretion, NO production, gliotransmitter release, and astrocyte reactivity, ultimately impacting survival. CONCLUSION: Our study reveals a new mechanism by which ethanol impairs astrocyte function, involving the sequential stimulation of inflammatory pathways that further increase the opening of Cx43 hemichannels and Panx1 channels. We hypothesize that targeting astroglial hemichannels could be a promising pharmacological approach to preserve astrocyte function and synaptic plasticity during the progression of various alcohol use disorders.

A Dye Uptake Assay to Measure Large-Pore Channel Activity in Trypanosoma cruzi.

Large-pore channels allow the exchange of ions and molecules between the intra- and extracellular compartments. These channels are structures formed by several protein families with little or no evolutionary linkages that include connexins (Cxs), pannexins (Panxs), innexins (Inxs), CALHM1, and LRRC8 proteins. Recently, we have described the unnexins (Unxs) proteins expressed in Trypanosoma cruzi (T. cruzi) that also is like to form large-pore channels at the plasma membrane. In this chapter, we describe a dye uptake method for evaluating the unnexin-formed channel function in T. cruzi, as well as the methods for evaluating their participation in the transformation of trypomastigotes into amastigotes. These methods can facilitate understanding the role of large-pore channels in the parasite's biology.

Enhanced Methodologies for Investigating the Electrophysiological Characteristics of Endogenous Pannexin 1 Intercellular Cell-Cell Channels.

Gap junctions, pivotal intercellular conduits, serve as communication channels between adjacent cells, playing a critical role in modulating membrane potential distribution across cellular networks. The family of Pannexin (Panx) proteins, in particular Pannexin1 (Panx1), are widely expressed in vertebrate cells and exhibit sequence homology with innexins, the invertebrate gap junction channel constituents. Despite being ubiquitously expressed, detailed functional and pharmacological properties of Panx1 intercellular cell-cell channels require further investigation. In this chapter, we introduce optimized cell culture methodologies and electrophysiology protocols to expedite the exploration of endogenous Panx1 cell-cell channels in TC620 cells, a human oligodendroglioma cell line that naturally expresses Panx1. We anticipate these refined protocols will significantly contribute to future characterizations of Panx1-based intercellular cell-cell channels across diverse cell types and offer valuable insights into both normal cellular physiology and pathophysiology.

Sex-dependent expression of galectin-1, a cardioprotective ß-galactoside-binding lectin, in human calcific aortic stenosis.

We aimed to analyze sex-related differences in galectin-1 (Gal-1), a ß-galactoside-binding lectin, in aortic stenosis (AS) and its association with the inflammatory and fibrocalcific progression of AS. Gal-1 was determined in serum and aortic valves (AVs) from control and AS donors by western blot and immunohistochemistry. Differences were validated by ELISA and qPCR in AS samples. In vitro experiments were conducted in primary cultured valve interstitial cells (VICs). Serum Gal-1 was not different neither between control and AS nor between men and women. There was no association between circulating and valvular Gal-1 levels. The expression of Gal-1 in stenotic AVs was higher in men than women, even after adjusting for confounding factors, and was associated with inflammation, oxidative stress, extracellular matrix remodeling, fibrosis, and osteogenesis. Gal-1 (LGALS1) mRNA was enhanced within fibrocalcific areas of stenotic AVs, especially in men. Secretion of Gal-1 was up-regulated over a time course of 2, 4, and 8 days in men's calcifying VICs, only peaking at day 4 in women's VICs. In vitro, Gal-1 was associated with similar mechanisms to those in our clinical cohort. ß-estradiol significantly up-regulated the activity of an LGALS1 promoter vector and the secretion of Gal-1, only in women's VICs. Supplementation with rGal-1 prevented the effects elicited by calcific challenge including the metabolic shift to glycolysis. In conclusion, Gal-1 is up-regulated in stenotic AVs and VICs from men in association with inflammation, oxidative stress, matrix remodeling, and osteogenesis. Estrogens can regulate Gal-1 expression with potential implications in post-menopause women. Exogenous rGal-1 can diminish calcific phenotypes in both women and men.

A Global Positioning System Used to Monitor the Physical Performance of Elite Beach Handball Referees in a Spanish Championship.

Beach handball is a fully developed sporting discipline on all five continents which has attracted the attention of researchers in the last decade, resulting in a proliferation of different studies focusing on players but not on referees. The main objective of this cross-sectional research was to determine the physical demands on elite male beach handball referees in four different competitions: U18 male; U18 female; senior male; and senior female. Twelve elite federated male referees (age: 30.86 ± 8 years; body height: 175.72 ± 4.51 cm; body weight: 80.18 ± 17.99 kg; fat percentage: 20.1 ± 4.41%; national or international experience) belonging to the Technical Committee of the Royal Spanish Handball Federation were recruited for this the study. The physical demands required of referees in official matches were measured by installing a GPS device. The sampling frequency used to record their speed and distance was 15 Hz. A triaxial accelerometer (100 Hz) was used to determine their acceleration. An analysis of variance (ANOVA) between competitions with post hoc comparisons using the Bonferroni adjustment was used to compare among categories. A higher distance covered in zone 1 and speeds of 0 to 6 km-h-1 were recorded. Most accelerations and decelerations occurred in zones 0 and 1 (zone 0: 0 to 1 m·s-2; zone 1: 1 to 2 m·s-2). The lack of differences (p > 0.05) between most analysed variables suggest quite similar physical demands of the four analysed competitions. These results provide relevant information to design optimal training plans oriented to the real physical demands on referees in an official competition.

The antiseizure medication valproate increases hemichannel activity found in brain cells, which could worsen disease outcomes.

Glial cells play relevant roles in neuroinflammation caused by epilepsy. Elevated hemichannel (HC) activity formed by connexins (Cxs) or pannexin1 (Panx1) largely explains brain dysfunctions commonly caused by neuroinflammation. Glia express HCs formed by Cxs 43, 30, or 26, while glia and neurons both express HCs formed by Panx1. Cx43 HCs allow for the influx of Ca2+, which promotes glial reactivity, enabling the release of the gliotransmitters that contribute to neuronal over-stimulation. Valproate (VPA), an antiseizure medication, has pleiotropic actions on neuronal molecular targets, and their action on glial cell HCs remains elusive. We used HeLa cells transfected with Cx43, Cx30, Cx26, or Panx1 to determine the effect of VPA on HC activity in the brain. VPA slightly increased HC activity under basal conditions, but significantly enhanced it in cells pre-exposed to conditions that promoted HC activity. Furthermore, VPA increased ATP release through Cx43 HCs. The increased HC activity caused by VPA was resistant to washout, being consistent with in silico studies, which predicted the binding site for VPA and Cx43, as well as for Panx1 HCs on the intracellular side, suggesting that VPA first enters through HCs, after which their activity increases.

Symptom-related screening programme for early detection of chronic thromboembolic pulmonary hypertension after acute pulmonary embolism: the SYSPPE study.

BACKGROUND: Chronic thromboembolic pulmonary hypertension (CTEPH) is the most severe long-term complication of acute pulmonary embolism (PE). We aimed to evaluate the impact of a symptom screening programme to detect CTEPH in PE survivors. METHODS: This was a multicentre cohort study of patients diagnosed with acute symptomatic PE between January 2017 and December 2018 in 16 centres in Spain. Patients were contacted by phone 2 years after the index PE diagnosis. Those with dyspnoea corresponding to a New York Heart Association (NYHA)/WHO scale≥II, visited the outpatient clinic for echocardiography and further diagnostic tests including right heart catheterisation (RHC). The primary outcome was the new diagnosis of CTEPH confirmed by RHC. RESULTS: Out of 1077 patients with acute PE, 646 were included in the symptom screening. At 2 years, 21.8% (n=141) reported dyspnoea NYHA/WHO scale≥II. Before symptom screening protocol, five patients were diagnosed with CTEPH following routine care. In patients with NYHA/WHO scale≥II, after symptom screening protocol, the echocardiographic probability of pulmonary hypertension (PH) was low, intermediate and high in 76.6% (n=95), 21.8% (n=27) and 1.6% (n=2), respectively. After performing additional diagnostic test in the latter 2 groups, 12 additional CTEPH cases were confirmed. CONCLUSIONS: The implementation of this simple strategy based on symptom evaluation by phone diagnosed more than doubled the number of CTEPH cases. Dedicated follow-up algorithms for PE survivors help diagnosing CTEPH earlier. TRIAL REGISTRATION NUMBER: NCT03953560.