Fructose, a key component of modern diets, is closely linked to the growing prevalence of pediatric obesity and metabolic alterations. Although numerous studies highlight its systemic consequences, including altered carbohydrate and lipid metabolism and increased cardiovascular risk, the direct impact of fructose, particularly its role in modulating mucin composition, a key determinant of the mucosal barrier, remains poorly explored. This study investigated whether fructose supplementation modifies high-fat diet (HFD)-induced changes in duodenal mucin production and whether these effects vary depending on age in animals. To this end, young and adult mice were fed a normal diet (ND), HFD, or an HFD supplemented with 30% fructose (w/v) in drinking water (HFD+Fru) for 16 weeks. Brunner's glands and villus goblet cells were then analyzed using conventional histochemistry and a panel of lectins to evaluate possible alterations in intestinal mucus glycosylation. Results showed that both HFD and HFD+Fru significantly increased body weight. In young mice, HFD+Fru induced a compensatory mucosal phenotype characterized by increased villus PAS (Periodic Acid-Schiff) reactivity (2% vs. ND), elevated sialylated mucin secretion rate (SSR) in Brunner's glands (25% vs. ND) and villi (17% vs. ND), and higher SNA (up to 46% vs. ND) and PNA (up to 39% vs. ND) in villus goblet cells. In contrast, adult mice receiving HFD+Fru exhibited a maladaptive response, characterized by a reduction in villus PAS-positive mucins (6% vs. ND), decreased villus SSR (5% vs. ND), diminished sialylation (up to 43% SNA vs. ND) and GlcNAc (up to 50% reduction in WGA vs. ND) in villus goblet cells, and marked loss of fucosylation in Brunner's glands (81% vs. ND) and villus goblet cells (66% vs. ND). These results reveal that fructose-enriched HFD remodels duodenal mucin O-glycosylation in an age-dependent manner, suggesting that while young mice exhibit transient adaptive responses, prolonged exposure can deplete these mechanisms, leading to a compromised adult epithelial barrier. This age-specific vulnerability may significantly contribute to the pathogenesis of diet-related intestinal disorders and obesity-related complications in later life, highlighting the need for early dietary interventions.

The hormone vasopressin (AVP) controls renal water reabsorption by modulating the expression and trafficking of the water channel aquaporin-2 (AQP2) through the activation of the cAMP/PKA signal transduction pathway. Previous studies revealed that Olive Leaf Extract (OLE) counteracts the vasopressin-dependent AQP2 functions by stimulating the calcium-sensing receptor (CaSR). Here, the biological activities of p-Coumaric acid, a selective polyphenol in OLE, were investigated. Stimulation of renal collecting duct MCD4 cells with p-Coumaric acid at a concentration of 1 nM caused a significant intracellular calcium release. NPS-2143, a selective CaSR antagonist, abolished this increase. Molecular docking analysis revealed that p-Coumaric acid can form binding interactions with the binding pocket of Tecalcet, a known CaSR activator, likely suggesting that p-Coumaric acid may stimulate the CaSR. Confocal analysis and immunoblotting experiments showed that p-Coumaric acid impaired the DDAVP-dependent membrane expression of AQP2 and the consequent increase of the osmotic water permeability (Pf). Additionally, Fluorescence Resonance Energy Transfer (FRET) experiments demonstrated that p-Coumaric acid prevented the DDAVP-induced cAMP generation, consequently attenuating the AQP2 phosphorylation at serine 256. Together, these findings suggest that p-Coumaric acid may antagonize the effects of vasopressin, possibly by binding to and stimulating the CaSR.

Bone is a dynamic specialized connective tissue that undergoes continuous remodeling to preserve its health. Bone health is influenced throughout life by a combination of genetic, hormonal, and environmental factors, as well as physical activity and diet. This study aims to evaluate the effects of diets with different fat content on the femurs of mice fed for 16 or 20 weeks on a normal diet (ND16w and ND20w) or a Western diet (WD16w and WD20w) and for 20 weeks with their combinations on a ketogenic diet (KD) (WD + ND20w, ND + KD20w, and WD + KD20w). Micro-CT analysis on femoral cancellous bone revealed a non-significant trend toward decreased bone volume fraction (BV/TV) and trabecular thickness in mice fed a combined WD + KD20w compared to WD20w. Cortical bone thickness was significantly lower in mice fed WD16w and WD20w compared to those fed ND16w and ND20w (p = 0.049 and p = 0.039, respectively), in mice fed WD20w Ct.Th increased compared to WD + ND20w (p = 0.024) and a strong decrease is evident when comparing WD + ND20w to WD + KD20w (p < 0.0004). Consistently, histological analysis revealed that the number of osteoclasts per bone perimeter on cancellous bone increases compared with ND20w with ND + KD20w (p = 0.007) and WD + ND20w with ND + KD20w (p = 0.0006). In addition, a decrease in osteoblasts was observed (p < 0.041) in cortical bone, comparing ND20w with ND + KD20w; this suggests that the KD may have differential effects depending on the baseline condition. Osteocyte numbers did not significantly change when comparing the different treatments. Masson staining supports micro-CT results on both cortical and cancellous bone. In conclusion, transition from a high-fat diet to a normal diet may partially restore cortical bone health, whereas transition to a ketogenic diet exerts a trend toward additional detrimental effects on trabecular bone.

The integrity of esophageal epithelial cells in patients with gastroesophageal reflux disease (GERD) or GERD-like symptoms is the first mechanism of protection to decrease the sensitivity to gastric reflux and heartburn symptoms. We investigated the protective effects of Poliprotect® (PPRO), a CE-marked medical device, on esophageal epithelial integrity using in vitro and ex vivo models. In vitro, the protective effects of PPRO were tested on Caco-2 cells. PPRO demonstrated safety and protection against oxidative damage induced by hydrogen peroxide. It also preserved epithelial integrity by maintaining transepithelial electrical resistance (TEER) against damage from calcium removal or bile acid exposure (taurodeoxycholic acid, TDCA). Ex vivo, esophageal biopsies from patients subjected to endoscopy were mounted in Ussing chambers and exposed to damaging agents (HCl or HCl + TDCA). Untreated biopsies (control) showed significant loss of epithelial resistance (up to −33%). In contrast, low concentrations of PPRO (50–100 µg/mL) provided strong protection against these damages (p < 0.001), even after 60 min of washing. Histological analysis confirmed the barrier-enhancing effect of PPRO. Overall, PPRO effectively protected the esophageal epithelium from damage in both models, suggesting its potential role in alleviating GERD or GERD-like symptoms by strengthening mucosal barriers and reducing epithelial sensitivity to reflux.

Background/Objectives: Unhealthy nutrition and lifestyles contribute to the development of chronic diseases such as cardiovascular disease, type 2 diabetes, and colorectal cancer. The Western diet can impair gastrointestinal motility and function. The underlying mechanisms that lead to changes in the mucus barrier and mucin profiles in response to these dietary patterns are still being studied. In mice, dietary fiber intake can improve the intestinal mucosal barrier function, enhance the differentiation process of goblet cells, and increase acidic mucin production. Our study aimed to investigate the effects of a high-fat diet (HFD) on colonic mucin expression and to assess whether chickpea accessions, known for their nutritional benefits, can mitigate these adverse effects. Methods: We investigated the effects of an HFD and an HFD associated with two accessions of chickpeas (HFD + MG_13; HFD + PI358934) on the mucin expression in murine colons of mice by conventional histochemistry and lectin-histochemistry, combined with chemical treatment and enzymatic digestion and immunohistochemistry. We evaluated possible alterations of Muc2, the main mucin secreted by the mucous cells of the colon. Results: HFD significantly reduced the expression of the mucin Muc2 and altered its composition in the colon. Compared to the CTRL group, distal and proximal measurements for HFD + PI, respectively, showed reductions of 78% and 36%; for the distal colon, a reduction of 34% was also observed for both the HFD and HFD + MG_13 diets. Changes in mucin glycosylation, including sialylation and sulfation, as well as residues such as N-acetylglucosamine, GalNAc, Mannose, and Galactose, were observed, suggesting a beneficial influence of chickpeas on mucosal integrity. In HFD + MG_13 these effects were reduced and resulted similar to the control. Conclusions: HFD reduces Muc2 expression in the colon and alters mucin composition: chickpea accessions, particularly MG_13, partially restore Muc2 levels and mucin oligosaccharide profiles, suggesting protective effects on the intestinal mucosal barrier.

Sea pollution caused by anthropological activities represents a risk both for the organisms that inhabit it and for humans themselves. Great attention is paid to plastic waste because it takes decades to decompose and fragments into microscopic pieces that can be easily dispersed and ingested by marine fauna. Polymeric materials, in general, are rich in plasticizers (phthalates, PAEs; and bisphenol A, BPA), substances recognized as toxic both for aquatic organisms and for humans who could ingest them once contaminated marine organisms were to enter their diet. In this work, effective analytical protocols based on the use of solid phase microextraction (SPME) coupled with chromatography techniques were employed to evaluate the presence of PAEs and BPA in the extracted pulp of shrimps of the commercial species Aristaemorpha foliacea from four different fishing stations in the Mediterranean Sea. In addition to chemical analysis, a comprehensive microbiological characterization was carried out to assess microbiological risk due to shrimps’ consumption. This dual approach provides a more complete evaluation of the impact of human pollution on these crustaceans, revealing both chemical contamination and potential biological disruptions that could pose a danger to food safety.

Mucomics is the study of mucus and its biochemical properties. This discipline has gained increasing attention due to the critical roles mucus plays in protection, adhesion, and communication across species. Ethical restrictions on vertebrate research have driven the interest in invertebrate models such as mollusks. Mollusks produce large amounts of mucus which has several functions, including immune defense, digestion, and environmental adaptation. Mollusks—terrestrial, freshwater, or marine—are valuable models for investigating mucus composition and its responses to environmental stressors, including heavy metal contamination. Histochemical and glycomic techniques have revealed variations in mucin glycosylation patterns that influence mucus functionality, such as its viscoelastic and adhesive properties. Bivalves and gastropods, widely used as bioindicators and generally not subject to regulatory constraints in experimental use, accumulate pollutants in their mucus, reflecting environmental health. Investigative techniques such as lectin histochemistry, proteomic, and glycomic analyses provide insights into the impact of contaminants on mucus composition. Further research on molluscan mucins can enhance understanding of their physiological roles, environmental interactions, and potential biomedical applications. By integrating molecular and histochemical approaches, mucomic studies offer a comprehensive perspective on mucus function, advancing both ecological monitoring and biotechnological applications.

The effects of treatments of pool frog embryos of Pelophylax lessonae with engineered zerovalent nanoparticles of Fe Co, and Ni at increasing concentrations (½ LC50, LC50, and 2 × LC50) were studied at macroscopical and microscopical levels, focusing on the epidermis that is often subject to physiological changes in response to environmental factors. Total body length and eye diameter were statistically higher in the control groups. A significantly higher percentage of embryos in the controls reached later Gosner developmental stages (19-23) than the treatments. Malformations such as ventral blister, microcephalia, bent body axis, and microphthalmia were significantly more frequent in the Co and Ni treatments than in the controls. In treated epidermis, ciliated, goblet, and small secretory cells resulted in a significantly larger volume than the same cell types in control embryos. At the ultrastructural level, ciliated cells showed stuck cilia and mitochondrial swelling, as well as cytoplasmic inclusions. Small secretory cells exhibited a significantly higher number of secreting vacuoles, suggesting an increased secretion. In conclusion, nanoparticles affect the development of frog embryos in multiple ways, even if the mechanisms are still mostly unknown.

Microplastics (MPs) pose an increasing and significant threat to marine biodiversity and there is a current need to determine the effects of exposure on benthic sessile invertebrates. This study examines the filtration capacity and retention of MP particles, as well as their physiological impacts in the marine sponge Spongia officinalis, a bioindicator species. The findings revealed a very high filtration capacity for MPs within the size range of 1-5 μm, along with a rapid turnover rate, as a large portion of particles were expelled within 48 h of exposure. Histological analyses detected MP particles within the cellular structures of the analyzed tissues, indicating that MPs of this size can penetrate cellular barriers. In terms of physiological effects, detoxification activity was activated during the depuration phase, and lipid peroxidation was observed during both the exposure and depuration phases. Overall, this study provides critical insights into the filtration and retention capacity, intercellular integration of MP particles, and the physiological effects of MP exposure in S. officinalis, providing a baseline for future research.

The expression of the Na+-K+-2Cl− cotransporter (NKCC), widely associated with cell volume regulation, has never been directly demonstrated in annelids. Its putative presence was firstly recovered in silico, and then using immunofluorescence, its signal was retrieved for the first time in different tissues of four species of estuarine annelids from southern Brazil that are regularly subjected to salinity fluctuations. We tested two euryhaline species (wide salinity tolerance), the nereidids Alitta yarae and Laeonereis acuta (habitat salinity: ~10–28 psu), and two stenohaline species (restricted salinity tolerance), the nephtyid Nephtys fluviatilis (habitat salinity: ~6–10 psu), and the melinnid Isolda pulchella (habitat salinity: ~28–35 psu). All four species showed specific immunofluorescent labelling for NKCC-like expression. However, the expression of an NKCC-like protein was not homogeneous among them. The free-living/burrowers (both euryhaline nereidids and the stenohaline nephtyid) displayed a widespread signal for an NKCC-like protein along their bodies, in contrast to the stenohaline sedentary melinnid, in which the signal was restricted to the branchiae and the internal tissues of the body. The results are compatible with NKCC involvement in cell volume, especially in annelids that face wide variations in salinity in their habitats.

Lanthanum (La), a rare earth element, has unique properties that make it valuable in various industries. Extraction activities pose environmental and health risks due to pollution, habitat disruption, and human exposure to hazardous chemicals. Animal models are needed to evaluate La effects on biological systems. We focused on the Manila clam, Ruditapes philippinarum, a common bivalve introduced in the Mediterranean Sea for commercial purposes. This study evaluates La-induced alterations in the Manila clam using an integrated method. Specimens were exposed to varying concentrations of lanthanum chloride, and subsequent changes in mucus secretion and microbial communities were observed. Inductively coupled plasma mass spectrometry analyses supported the bioaccumulation data. Histochemical and lectin-histochemical analyses revealed modifications in glycosaminoglycan composition and secretion patterns, indicating the clam's defensive response to La exposure. Bacterial counts in intervalvar water increased with higher La concentrations. These findings highlight the Manila clam's potential use as a bioindicator for La pollution and underline the importance of responsible management in La extraction to minimize environmental impact.

Aquaporins (AQPs) are important for water transport in the gastrointestinal tract. Changes in their expression and/or localization could cause in disorders and be used as therapeutic targets. Aquaporin-4 (AQP4) is expressed predominantly on the basolateral membrane of the parietal cells in the corpus of the murine gastric glands. Although the secretion of gastric juice is not affected in AQP4-deficient knockout, we evaluated by light microscopy whether the lack of AQP4 affects the glycopatterns of secreting gastric cells. Wild type (WT) and AQP4-deficient knockout mice (KO) were fed a standard diet ad libitum before sacrifice. Segments of stomach corpus were collected, fixed in buffered formalin, and embedded in paraffin wax. Sections, 5-mu m thick, were analyzed by histochemical methods (Periodic acid-Schiff, Alcian Blue pH 2.5), and binding of lectins specific to GalNAc (SBA, DBA), Gal (PNA) GlcNAc (WGA, GSAII) mannose and/or glucose (ConA), and fucose (UEA-I, AAA, LTA). Immunohistochemical methods such as anti-Muc6 for neck cells and anti- beta- H+/K+-ATPase for parietal cells were also performed. Compared to WT mice, in the mucous cells of KO lower amounts of glycans with galactosyl/galactosaminylated, glycosyl/glycosaminylated, and fucosylated residues were observed; lower fucosylation resulted also in the parietal cells. The observed differences of KO in respect to WT could lead to severer pathological conditions.Research Highlights Glycopatterns in gastric glands were compared between wild type (WT) and AQP4-deficient knockout (KO) mice by histochemical and lectin-binding methods. In the mucous cells of KO lower amounts of glycans with galactosyl/galactosaminylated, glycosyl/glycosaminylated and fucosylated residues were observed. In the parietal cells lower fucosylation also resulted. AQP4-deficiency affects glycosylation and could result in altered functionality and pathological conditions. Altered glycosylation patterns in both mucous and parietal gastric cells were evidenced by histochemistry, lectin-histochemistry, and immunohistochemistry in AQP4-deficient KO mice, with possible physio-pathological consequences. image

High concentrations of urinary calcium counteract vasopressin action via the activation of the Calcium-Sensing Receptor (CaSR) expressed in the luminal membrane of the collecting duct cells, which impairs the trafficking of aquaporin-2 (AQP2). In line with these findings, we provide evidence that, with respect to wild-type mice, CaSR knock-in (KI) mice mimicking autosomal dominant hypocalcaemia, display a significant decrease in the total content of AQP2 associated with significantly higher levels of AQP2 phosphorylation at Ser261, a phosphorylation site involved in AQP2 degradation. Interestingly, KI mice also had significantly higher levels of phosphorylated p38MAPK, a downstream effector of CaSR and known to phosphorylate AQP2 at Ser261. Moreover, ATF1 phosphorylated at Ser63, a transcription factor downstream of p38MAPK, was significantly higher in KI. In addition, KI mice had significantly higher levels of AQP2-targeting miRNA137 consistent with a post-transcriptional downregulation of AQP2. In vivo treatment of KI mice with the calcilytic JTT-305, a CaSR antagonist, increased AQP2 expression and reduced AQP2-targeting miRNA137 levels in KI mice. Together, these results provide direct evidence for a critical role of CaSR in impairing both short-term vasopressin response by increasing AQP2-pS261, as well as AQP2 abundance, via the p38MAPK-ATF1-miR137 pathway. KEY POINTS: Calcium-Sensing Receptor (CaSR) activating mutations are the main cause of autosomal dominant hypocalcaemia (ADH) characterized by inappropriate renal calcium excretion leading to hypocalcaemia and hypercalciuria. Current treatments of ADH patients with parathyroid hormone, although improving hypocalcaemia, do not improve hypercalciuria or nephrocalcinosis. In vivo treatment with calcilytic JTT-305/MK-5442 ameliorates most of the ADH phenotypes of the CaSR knock-in mice including hypercalciuria or nephrocalcinosis and reverses the downregulation of the vasopressin-sensitive aquaporin-2 (AQP2) expression, providing direct evidence for a critical role of CaSR in impairing vasopressin response. The beneficial effect of calcilytic in reducing the risk of renal calcification may occur in a parathyroid hormone-independent action through vasopressin-dependent inhibition of cAMP synthesis in the thick ascending limb and in the collecting duct. The amelioration of most of the abnormalities in calcium metabolism including hypercalciuria, renal calcification, and AQP2-mediated osmotic water reabsorption makes calcilytic a good candidate as a novel therapeutic agent for ADH.

Antipathella subpinnata is a black coral species with Atlantic-Mediterranean distribution, capable of forming dense forests at mesophotic depths, thereby providing essential ecological goods and services. Limited data are available on its reproductive biology and ecology. Understanding the reproductive patterns of this species is pivotal for effective conservation strategies. This study proposes a histological protocol for the treatment of A. subpinnata samples previously preserved in ethanol, aiming to restore the internal morphology of the polyps and consequently improve the detection of internal structures and the investigation of the reproductive traits of each polyp. The success of this protocol in A. subpinnata suggests its potential applicability to other black corals, providing a valuable tool for ecological and biological research.

Aluminum (Al) is used in everyday life and present in food drugs, packaging, industry, and agriculture. Although it is the most common metal in the Earth crust, a correlation has been demonstrated between its presence and various pathologies, even serious ones, especially of a neurological type. However, there is a histological gap regarding the role Al can have in contact with the covering and secreting epithelia. The alterations of the ventral and dorsal foot mucocytes and their secretions of the snail Eobania vermiculata caused by Al were investigated in situ by histochemical and lectin-histochemical techniques. Administration to different experimental groups took place for 3 and 9 days with 50 and 200 mu M of AlCl3. Several types of mucocytes were detected with a prevalent secretion of acid glycans in the foot of E. vermiculata. Sulfated glycans prevail in the dorsal region, with one type showing only fucosylated residues and another also having galactosaminylated and glycosaminylated residues. Carboxylated glycans prevail in the ventral region, with presence of galactosaminylated, glycosaminylated, and fucosylated residuals in both cells. Snails treated presented a general decrease of mucin amount in the secreting cells and affected the mucus composition. These changes could alter the rheological and functional properties of the mucus with possible implications for the health of the treated animals. Research Highlights Snails were fed with Al-contaminated lettuce at different concentrations. In the foot mucocytes produced mucus with prevailing acidic glycans. In the treated resulted a reduction in the amount of mucus and an alteration of glycan composition.

We analysed the embryonic epidermis of the pool frog, Pelophylax lessonae, by light and electron microscopy techniques, and characterized preliminarily its mucous secretions with conventional histochemistry (Toluidine Blue, Periodic Acid-Schiff, Alcian blue pH 2.5 and Alcian blue pH 1.0). At Gosner’s developmental stage 21, the epidermis consisted of a bilayered epithelium with five cell types. Basal cells (BCs) constituted the basal layer, presented a large nucleus and were sometimes observed in mitosis. Ciliated cells (CCs) had a ciliated apical membrane, with carboxylated glycans in the sub-apical portion. TEM analysis revealed small vesicles just below the surface, a central nucleus, and evident yolk plates. Goblet cells (GCs) and small secretory cells (SSCs) showed secreting vesicles and vacuoles, respectively. In the mucus secretion of GCs sialosulphomucins appeared, while in that of SSCs sulphation was not observed. Under TEM, GCs had irregular shaped nuclei and a less electron-dense cytoplasm with respect to SSCs. Ionocytes (INs) presented apical microvilli and a high electron-dense cytoplasm with vacuoles. SEM analysis revealed that GCs outnumbered the other cell types and that SSCs and INs were usually located together near to GCs. The relatively simple structure of the epidermis and its direct interfacing with the environment renders it a good model for the study of structure and development of mucociliary epithelia, as well as of its alterations in the presence of toxicants.

An in situ analysis of mucin secretions along the annual cycle was performed on the striped Venus clam, Chamelea gallina. Individuals of both sexes from an exploited stock of Margherita di Savoia (Southern Adriatic Sea, Central Mediterranean Sea) were made in June 2020 and 2021, and in January 2021 and 2022, representative of summer and winter seasons, respectively. Tissues from the foot and the gills were analyzed for histochemical and lectin-histochemical analyses. Staining with Periodic Acid-Schiff, Alcian Blue pH 2.5 and High-Iron-Diamine indicated that mucins from both the foot and the gills were acidic, mostly sulfated. Lectin-binding analyses with PNA, SBA, WGA, LTA, UEA-I, AAA, SNA, MAA-II and ConA indicated the presence of N-acetyl-glycosaminylated, mannosylated and fucosylated residuals in the saccharidic chains. In the gills, the amount of acidic and glycosaminylated residuals was higher in summer in both sexes, whereas fucosylation was similar along the sampling period and mannosylation was never observed. In the foot, both surface epithelial and subepidermal secreting cells increased sulfation in winter in males, but not in females. Glycosaminylation and fucosylation were observed only in the surface cells of males. Mannosylated residuals were observed in all the foot cell types in both sexes. It is hypothesized that the observed qualitative and quantitative variations in mucin secretion is linked to the reproductive cycle.