First Insights into Body Localization of an Osmoregulation-Related Cotransporter in Estuarine Annelids.

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.

Altered glycosylation in secreting cells of the gastric glands of aquaporin-4-deficient mice.

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-µm 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- ß- 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.

Aluminum exposure alters the pedal mucous secretions of the chocolate-band snail, Eobania vermiculata (Gastropoda: Helicidae).

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 µM 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.

In vivo treatment with calcilytic of CaSR knock-in mice ameliorates renal phenotype reversing downregulation of the vasopressin-AQP2 pathway.

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.

The Effect of Calcium Ions on hIAPP Channel Activity: Possible Implications in T2DM.

The calcium ion (Ca2+) has been linked to type 2 diabetes mellitus (T2DM), although the role of Ca2+ in this disorder is the subject of intense investigation. Serum Ca2+ dyshomeostasis is associated with the development of insulin resistance, reduced insulin sensitivity, and impaired glucose tolerance. However, the molecular mechanisms involving Ca2+ ions in pancreatic ß-cell loss and subsequently in T2DM remain poorly understood. Implicated in the decline in ß-cell functions are aggregates of human islet amyloid polypeptide (hIAPP), a small peptide secreted by ß-cells that shows a strong tendency to self-aggregate into ß-sheet-rich aggregates that evolve toward the formation of amyloid deposits and mature fibrils. The soluble oligomers of hIAPP can permeabilize the cell membrane by interacting with bilayer lipids. Our study aimed to evaluate the effect of Ca2+ on the ability of the peptide to incorporate and form ion channels in zwitterionic planar lipid membranes (PLMs) composed of palmitoyl-oleoyl-phosphatidylcholine (POPC) and on the aggregation process of hIAPP molecules in solution. Our results may help to clarify the link between Ca2+ ions, hIAPP peptide, and consequently the pathophysiology of T2DM.

Evidence of the different effect of mercury and cadmium on the hIAPP aggregation process.

hIAPP is a hormone consisting of 37 aminoacids that shows a strong tendency to self-assemble into ß-sheet-rich aggregates, which evolve to form insoluble aggregates that seem to be associated with ß-cell degeneration in Type 2 Diabetes Mellitus. Numerous factors, intrinsic and extrinsic to the peptide molecule, appear to influence the hIAPP aggregation process. Different metal ions are able to interact with the hIAPP molecule, modulating its secondary structure and subsequently the peptide's capacity to aggregate. In this study, the effect of Hg2+ and Cd2+ on the hIAPP aggregation process was evaluated using direct and indirect methods. The kinetics and morphology of amyloid aggregate formation were respectively evaluated with Thioflavin T assays and electron microscopy, while the ability of the peptide to incorporate into POPC PLMs and form ion channels was monitored by single-channel current measurements. Hg2+ and Cd2+ each seem to modulate the peptide's ability to aggregate in a different way, suggesting a different mechanism of hIAPP toxicity.

Differential expression of glycans in the urothelial layers of horse urinary bladder.

BACKGROUND: Urothelium is a multilayer epithelium covering the inner surface of the urinary bladder that acts as a blood-urine barrier and is involved in maintaining the wellbeing of the whole organism. Glycans serve in the maturation and differentiation of cells and thus play a key role in the morphology and function of the multilayered epithelium. The aim of the present study was to examine the glycoprotein pattern of the horse urinary bladder urothelium by lectin histochemistry. METHODS: The study involved urinary bladders from four horse stallions. Tissue sections were stained with a panel of eleven lectins, in combination with saponification and sialidase digestion (Ks). RESULTS: Basal cells displayed high-mannose N-glycans (Con A), α2,6-linked sialic acid (SNA), and O-linked sialoglycans with sialic acids linked to Galßl,3GalNAc (T antigen) (KsPNA) and terminal N-acetylgalactosamine (Tn antigen) (KsSBA). The young intermediate cells expressed terminal N-acetylglucosamine (GlcNAc) (GSA II), galactose (GSA I-B4), T- and Tn antigens (PNA, SBA). The mature intermediate cells showed additional high-mannose N-glycans, O-linked sialoglycans (sialyl-T antigen, sialyl-Tn antigen), α2,6- and α2,3-linked sialic acid (MAL II), α1,2-linked fucose (UEA I), and GlcNAc (KsWGA). The latter residue marked the boundary with the overlying surface layer. Few Con A positive intermediate cells were seen to cross the entire urothelium thickness. The surface cells showed additional glycans such as T antigen and sialic acids linked to GalNAc binding DBA (KsDBA). Few surface cells contained α1,3-linked fucose (LTA), whereas some other cells displayed intraluminal secretion of mucin-type glycans terminating with GalNAcα1,3(LFucα1,2)Galß1,3/4GlcNAcß1 (DBA). The luminal surface expressed the most complex glycan pattern in the urothelium because only α1,3-linked fucose lacked among the demonstrated glycans. CONCLUSIONS: This study showed that the glycan pattern becomes more complex from the basal to surface layer of the urothelium and that surface cells could modify the composition of urine via the secretion of glycoproteins.

Sexual and asexual reproduction in a Mediterranean Tethya (Porifera, Demospongiae) species.

BACKGROUND: The reproductive cycle of the recently described sponge Tethya meloni was investigated for a period of 15 months (September 2018 - November 2019) in the Mar Piccolo of Taranto (Southern Italy) and was compared with data previously collected for the other two sympatric species of the same genus known for Mediterranean Sea, T. citrina and T. aurantium. RESULTS: T. meloni is a gonochoric species with a sex ratio strongly shifted towards females. Asexual budding was a seasonal process, limited to few specimens. In a specimen collected in September 2018 both oocytes and buds occurred, suggesting that in T. meloni the sexual and asexual phases may coexist both at the population and individual levels. CONCLUSIONS: The data obtained from this research compared with the available literature confirm the high temporal variability of the reproductive cycles in the Mediterranean species of Tethya, but with common general characteristics. In sexual reproduction, the oocyte production period lasts several months, with a peak between summer and autumn while spermatogenesis, shorter but with greater reproductive effort, follows the onset of oogenesis. The asexual reproduction phase of T. meloni, on the other hand, occurs in a short period and seems to have less importance in the overall reproductive process.

Semi-interpenetrating polymer network cryogels based on poly(ethylene glycol) diacrylate and collagen as potential off-the-shelf platforms for cancer cell research.

In the present work, we investigated the potential of novel semi-interpenetrating polymer network (semi-IPN) cryogels, obtained through ultraviolet exposure of aqueous mixtures of poly(ethylene glycol) diacrylate and type I collagen, as tunable off-the-shelf platforms for 3D cancer cell research. We synthesized semi-IPN cryogels with variable collagen amounts (0.1% and 1% w/v) and assessed the effect of collagen on key cryogel properties for cell culture, for example, porosity, degradation rate and mechanical stiffness. Then, we investigated the ability of the cryogels to sustain the long-term growth of two pancreatic ductal adenocarcinoma (PDAC) cell populations, the parenchymal Panc1 cells and their derived cancer stem cells. Results revealed that both cell lines efficiently infiltrated, attached and expanded in the cryogels over a period of 14 days. However, only when grown in the cryogels with the highest collagen concentration, both cell lines reproduced their characteristic growth pattern previously observed in collagen-enriched organotypic cultures, biomimetic of the highly fibrotic PDAC stroma. Cellular preembedding in Matrigel, that is, the classical approach to develop/grow organoids, interfered with an efficient intra-scaffold migration and growth. Although preliminary, these findings highlight the potential of the proposed cryogels as reproducible and tunable cancer cell research platforms.

Orthogonal arrays of particle assembly are essential for normal aquaporin-4 expression level in the brain.

Astrocyte endfeet are endowed with aquaporin-4 (AQP4)-based assemblies called orthogonal arrays of particles (OAPs) whose function is still unclear. To investigate the function of OAPs and of AQP4 tetramers, we have generated a novel "OAP-null" mouse model selectively lacking the OAP forming M23-AQP4 isoform. We demonstrated that AQP4 transcript levels were not reduced by using qPCR. Blue native (BN)/SDS-PAGE and Western blot performed on OAP-null brain and primary astrocyte cultures showed the complete depletion of AQP4 assemblies, the selective expression of M1-AQP4-based tetramers, and a substantial reduction in AQP4 total expression level. Fluorescence quenching and super-resolution microscopy experiments showed that AQP4 tetramers were functionally expressed in astrocyte plasma membrane and their dimensions were reduced compared to wild-type assemblies. Finally, as shown by light and electron microscopy, OAP depletion resulted in a massive reduction in AQP4 expression and a loss of perivascular AQP4 staining at astrocyte endfeet, with only sparse labeling throughout the brain areas analyzed. Our study relies on the unique property of AQP4 to form OAPs, using a novel OAP-null mouse model for the first time, to show that (a) AQP4 assembly is essential for normal AQP4 expression level in the brain and (b) most of AQP4 is organized into OAPs under physiological conditions. Therefore, AQP4 tetramers cannot be used by astrocytes as an alternative to OAPs without affecting AQP4 expression levels, which is important in the physiological and pathological conditions in which OAP aggregation/disaggregation dynamics have been implicated.

In Vitro and In Vivo Nutraceutical Characterization of Two Chickpea Accessions: Differential Effects on Hepatic Lipid Over-Accumulation.

Dietary habits are crucially important to prevent the development of lifestyle-associated diseases. Diets supplemented with chickpeas have numerous benefits and are known to improve body fat composition. The present study was undertaken to characterize two genetically and phenotypically distinct accessions, MG_13 and PI358934, selected from a global chickpea collection. Rat hepatoma FaO cells treated with a mixture of free fatty acids (FFAs) (O/P) were used as an in vitro model of hepatic steatosis. In parallel, a high-fat diet (HFD) animal model was also established. In vitro and in vivo studies revealed that both chickpea accessions showed a significant antioxidant ability. However, only MG_13 reduced the lipid over-accumulation in steatotic FaO cells and in the liver of HFD fed mice. Moreover, mice fed with HFD + MG_13 displayed a lower level of glycemia and aspartate aminotransferase (AST) than HFD mice. Interestingly, exposure to MG_13 prevented the phosphorylation of the inflammatory nuclear factor kappa beta (NF-kB) which is upregulated during HFD and known to be linked to obesity. To conclude, the comparison of the two distinct chickpea accessions revealed a beneficial effect only for the MG_13. These findings highlight the importance of studies addressing the functional characterization of chickpea biodiversity and nutraceutical properties.

High-fat Diet Alters the Glycosylation Patterns of Duodenal Mucins in a Murine Model.

High-fat diet (HFD) alters the glycosylation patterns of intestinal mucins leading to several health problems. We studied by histochemical and lectin-binding methods mucin alterations in the duodenum of mice fed a HFD for 25 weeks. Histochemical methods included periodic acid-Schiff, alcian blue pH 2.5, and high-iron diamine. Lectin-binding experiments were performed with SBA, PNA, WGA, MAA-II, SNA, ConA, UEA-I, LTA, and AAA. SBA, PNA, WGA, MAA-II, and SNA were tested also after desulfation and ConA after periodate-sodium borohydrate treatments (paradoxical ConA). Duodenal mucins are secreted by Brunner's glands and goblet cells in the villi. Brunner's glands of HFD mice showed increased secreting activity and a general reduction of glycosylated residuals, such as fucose and terminal α1,4-linked GlcNAc. Moreover, a general reduction of glycosylated residuals in the goblet cells of villi such as the fucosylated and sulfated ones was observed. Since the cited residuals are involved in cytoprotective and cytostatic functions, as well as in interactions with the intestinal microbiota and protection against parasites and inflammatory disorders, we conclude that HFD can predispose duodenum to several possible health disorders.

AQP1-Containing Exosomes in Peritoneal Dialysis Effluent As Biomarker of Dialysis Efficiency.

The water channel Aquaporin 1 (AQP1) plays a fundamental role in water ultrafiltration during peritoneal dialysis (PD) and its reduced expression or function may be responsible for ultrafiltration failure (UFF). In humans, AQP1 is expressed in the endothelium of the peritoneal capillaries but its expression in mesothelial cells (MC) and its functional role in PD is still being debated. Here, we studied a cohort of 30 patients using PD in order to determine the presence of AQP1 in peritoneal biopsies, AQP1 release in the PD effluent through exosomes and the correlation of AQP1 abundance with the efficiency of peritoneal ultrafiltration. The experiments using immunofluorescence showed a strong expression of AQP1 in MCs. Immunoblotting analysis on vesicles isolated from PD effluents showed a consistent presence of AQP1, mesothelin and Alix and the absence of the CD31. Thus, this suggests that they have an exclusive mesothelial origin. The immunoTEM analysis showed a homogeneous population of nanovesicles and confirmed the immunoblotting results. Interestingly, the quantitative analysis by ELISA showed a positive correlation between AQP1 in the PD effluent and ultrafiltration (UF), free water transport (FWT) and Na-sieving. This evidence opens the discussion on the functional role of mesothelial AQP1 during PD and suggests that it may represent a potential non-invasive biomarker of peritoneal barrier integrity, with predictive potential of UFF in PD patients.

Effects of a probiotic on the morphology and mucin composition of pig intestine.

Although the use of probiotics in human and animal medicine is growing, their mode of action remains poorly understood. This study examined the effects of a multi-strain probiotic (SLAB51™) on the morphology and carbohydrate composition of mucins secreted by goblet cells of intestinal crypts in growing-finishing pigs. Sections of duodenum, caecum and colon from pigs fed for 12 weeks with an orally administered control basal diet (No-Pro) or one with a probiotic blend (Pro) were processed for microscopic analysis and stained with (1) haematoxylin-eosin for structural and morphometrical investigation; (2) conventional histochemistry (periodic acid-Schiff, Alcian Blue pH 2.5, high iron diamine staining) for neutral, acidic non-sulphated, and sulphated mucin analysis; and (3) FITC-labelled MAA-II and SNA lectins for α2,3- and α2,6-sialomucin identification. Compared with No-Pro samples, Pro samples displayed (1) increased goblet cell numbers in all investigated tract crypts; (2) an increase in acidic non-sulphomucins but a decrease in neutral, sulphated and α2,6-sialomucin-secreting goblet cells in the duodenum; (3) decreased crypt depth, an increase in α2,6-sialomucin secretory goblet cells, and a loss of goblet cell-secreting α2,3-sialomucins, which appeared on the apical surface of crypt fundus epithelial cells in the caecum; and (4) an increase in α2,6-sialomucin-producing goblet cells in the colon. Results suggest that treatment with SLAB51™ induces region-specific changes in the morphology and carbohydrate composition of mucins secreted along intestinal tracts of growing-finishing pigs. These changes could ameliorate the health status of the animals, which displayed higher growth performance and meat quality than controls (Tufarelli et al., 2017).

Green olive leaf extract (OLE) provides cytoprotection in renal cells exposed to low doses of cadmium.

Cadmium (Cd) is a heavy and highly toxic metal that contaminates air, food and water. Cadmium accumulates in several organs altering normal functions. The kidney is the major organ at risk of damage from chronic exposure to cadmium as a contaminant in food and water. This study aims to investigate the beneficial effects of OLE in renal collecting duct MCD4 cells exposed to a low dose cadmium (1 µM). In MCD4 cells cadmium caused an increase in ROS production, as well as generation of lipid droplets and reduced cell viability. Moreover, cadmium exposure led to a remarkable increase in the frequency of micronuclei and DNA double-strand breaks, assessed using the alkaline comet assay. In addition, cadmium dramatically altered cell cytoskeleton architecture and caused S-glutathionylation of actin. Notably, all cadmium-induced cellular deregulations were prevented by co-treatment with OLE, possibly due to its antioxidant action and to the presence of bioactive phytocompounds. Indeed, OLE treatment attenuated Cd-induced actin S-glutathionylation, thereby stabilizing actin filaments. Taken together, these observations provide a novel insight into the biological action of OLE in renal cells and support the notion that OLE may serve as a potential adjuvant against cadmium-induced nephrotoxicity.

A Mediterranean mesophotic coral reef built by non-symbiotic scleractinians.

This is the first description of a Mediterranean mesophotic coral reef. The bioconstruction extended for 2.5 km along the Italian Adriatic coast in the bathymetric range -30/-55 m. It appeared as a framework of coral blocks mostly built by two scleractinians, Phyllangia americana mouchezii (Lacaze-Duthiers, 1897) and Polycyathus muellerae (Abel, 1959), which were able to edify a secondary substrate with high structural complexity. Scleractinian corallites were cemented by calcified polychaete tubes and organized into an interlocking meshwork that provided the reef stiffness. Aggregates of several individuals of the bivalve Neopycnodonte cochlear (Poli, 1795) contributed to the compactness of the structure. The species composition of the benthic community showed a marked similarity with those described for Mediterranean coralligenous communities and it appeared to be dominated by invertebrates, while calcareous algae, which are usually considered the main coralligenous reef-builders, were poorly represented. Overall, the studied reef can be considered a unique environment, to be included in the wide and diversified category of Mediterranean bioconstructions. The main reef-building scleractinians lacked algal symbionts, suggesting that heterotrophy had a major role in the metabolic processes that supported the production of calcium carbonate. The large amount of available suspended organic matter in the area could be the main nutritional source for these species, as already suggested in the literature referred to Mediterranean cold-water corals.

CSRP3 mediates polyphenols-induced cardioprotection in hypertension.

Berries contain bioactive polyphenols, whose capacity to prevent cardiovascular diseases has been established recently in animal models as well in human clinical trials. However, cellular processes and molecular targets of berries polyphenols remain to be identified. The capacity of a polyphenol-enriched diet (i.e., blueberries, blackberries, raspberries, strawberry tree fruits and Portuguese crowberries berries mixture) to promote animal survival and protect cardiovascular function from salt-induced hypertension was evaluated in a chronic salt-sensitive Dahl rat model. The daily consumption of berries improved survival of Dahl/salt-sensitive rats submitted to high-salt diet and normalized their body weight, renal function and blood pressure. In addition, a prophylactic effect was observed at the level of cardiac hypertrophy and dysfunction, tissue cohesion and cardiomyocyte hypertrophy. Berries also protected the aorta from fibrosis and modulated the expression of aquaporin-1, a channel involved in endothelial water and nitric oxide permeability. Left ventricle proteomics analysis led to the identification of berries and salt metabolites targets, including cystein and glycin-rich protein 3 (CSRP3), a protein involved in myocyte cytoarchitecture. In neonatal rat ventricular cardiomyocytes, CSRP3 was validated as a target of a berries-derived polyphenol metabolite, 4-methylcatechol sulfate, at micromolar concentrations, mimicking physiological conditions of human plasma circulation. Accordingly, siRNA silencing of CSRP3 and 4-methylcatechol sulfate pretreatment reversed cardiomyocyte hypertrophy and CSRP3 overexpression induced by phenylephrine. Our systemic study clearly supports the modulation of CSRP3 by a polyphenol-rich berries diet as an efficient cardioprotective strategy in hypertension-induced heart failure.

Gi Protein Modulation of the Potassium Channel TASK-2 Mediates Vesicle Osmotic Swelling to Facilitate the Fusion of Aquaporin-2 Water Channel Containing Vesicles.

Vesicle fusion is a fundamental cell biological process similar from yeasts to humans. For secretory vesicles, swelling is considered a step required for the expulsion of intravesicular content. Here this concept is revisited providing evidence that it may instead represent a general mechanism. We report the first example that non-secretory vesicles, committed to insert the Aquaporin-2 water channel into the plasma membrane, swell and this phenomenon is required for fusion to plasma membrane. Through an interdisciplinary approach, using atomic force microscope (AFM), a fluorescence-based assay of vesicle volume changes and NMR spectroscopy to measure water self-diffusion coefficient, we provide evidence that Gi protein modulation of potassium channel TASK-2 localized in AQP2 vesicles, is required for vesicle swelling. Estimated intravesicular K⁺ concentration in AQP2 vesicles, as measured by inductively coupled plasma mass spectrometry, was 5.3 mM, demonstrating the existence of an inwardly K⁺ chemical gradient likely generating an osmotic gradient causing vesicle swelling upon TASK-2 gating. Of note, abrogation of K⁺ gradient significantly impaired fusion between vesicles and plasma membrane. We conclude that vesicle swelling is a potentially important prerequisite for vesicle fusion to the plasma membrane and may be required also for other non-secretory vesicles, depicting a general mechanism for vesicle fusion.

Probiotic supplementation affects the glycan composition of mucins secreted by Brunner's glands of the pig duodenum.

The effect of a dietary probiotic blend on the carbohydrate composition of mucins secreted by the Brunner's glands in the duodenum of growing-finishing pigs was investigated by means of conventional (periodic acid-Schiff, Alcian Blue pH 2.5, high iron diamine staining) and lectin (15 lectins) histochemistry. Pigs were assigned to two dietary treatments: a control basal diet without the probiotic blend (No-Pro) and a test diet that included the probiotic blend (Pro). Duodenal tissue fragments were fixed in 4% phosphate-buffered-saline-buffered paraformaldehyde, dehydrated through a graded alcohol series, and embedded in paraffin wax. The secretory cells of the Brunner's glands from No-Pro pigs primarily produced neutral glycoproteins and a small amount of acidic non-sulphated mucins. This glycan pattern was opposite that of the Brunner's glands from Pro animals. A comparison of lectin-binding profiles of the secretory cells of Brunner's glands in these two groups showed that in Pro pigs, there was (i) a decrease in N-linked glycans containing α1,2-linked fucose (Con A, UEA I); (ii) a loss of complex types of N-glycans (PHA-L, PHA-E) terminating with lactosamine (RCA120), α1,6- and α1,3-linked fucose (LTA), and α-galactose (GSA I-B4), as well as of O-glycans with terminal Galß1,3GalNAc (PNA); and (iii) an increase in O-glycans containing GalNAc HPA. No-Pro and Pro samples showed no change in the expression of α2,6 sialoglycans and terminal GlcNAc residues and no affinity for MAL II, DBA, and SBA. These results indicate that probiotic supplementation affects the glycan composition of mucins produced in the Brunner's glands of growing-finishing pigs. These changes could effectively act on the gastrointestinal function and health status of these animals because the probiotic blend induced higher growth performance and meat quality in the test probiotic group than it did in the control basal diet group (Tufarelli et al., 2017).