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Following publication of the original article [1], the authors flagged that the ‘Availability of data and materials’ declaration is incomplete. Namely, it does not report the official accession number for the zebrafish PepT1a nucleotide sequence. With concern to this, please see the (correct) ‘Availability of data and materials’ declaration here: “Zebrafish pept1a (slc15a1a) nucleotide sequence has been submitted to GenBank (https://www.ncbi.nlm. nih.gov/nuccore/) and is available with the following accession number: GenBank Acc. No. MN723161” The authors apologize for any inconvenience caused.

POT transporters drive the concentrative uptake of their substrates by coupling to the transmembrane H+ electrochemical gradient. POTs recognize highly diverse di/tripeptides. Substrate extent expands with mammalian PepT1/PepT2, which also transport β-lactam antibiotics and peptide-based prodrugs. While substrate recognition changes, protonation sites seem conserved among members. In POTs, transport is achieved through the movement of the gating helices around the central binding site. The extracellular (EC) gate, formed by TM1,2 and TM7,8, serves to control access to the binding site from the EC side of the membrane. The intracellular (IC) gate, formed by TM4,5 and TM10,11, controls the release of peptide and protons on the inside of the cell. Two salt bridges coordinate these helices and control protein conformation. Whereas the IC gate contains a conserved Lys-Glu pair, the EC gate salt bridge is less conserved. In most of the bacterial POTs, the EC gate salt bridge is an Arg-Glu pair, while in mammalian PepT1, a conserved His on TM2 combined with an Arg-Asp salt bridge on TM1 and TM7 is seen. TM2 His is also found in “mammalian-like” bacterial members. In zebrafish PepT1b, the only vertebrate PepT1 known to work at alkaline pH, Lys replaces Arg on TM1. Likewise, PepT1 equally diverging from the “mammalian-like” transporters were retrieved (GenBank) from teleost fish (Cypriniformes, Cyprinodontiformes, Gymnotiformes, Gadiformes), birds (Apodiformes, Trochiliformes, Passeriformes, Piciformes), and even mammals (Chiroptera, Macroscelidea, Insectivora, Primates). Our findings extend the number of PepT1 prone to structure-function analyses and open to understand how their molecular diversity meets the physiology of the species and/or the environment where the species lives.

GI inflammation involves pathological processes affecting the gut epithelial barrier that are poorly reversible. A direct target of such processes is the enterocyte monolayer, which absorptive function is challenged by inflammation-induced cytoskeleton dynamics. Here, we evaluate the proinflammatory effects of PMA (phorbol-12-myristate-13-acetate) on Caco-2 intestinal cells at two different stages of spontaneous differentiation i.e. undifferentiated (7 dps, days post seeding) and differentiated enterocyte-like cells (>21 dps) in the presence/absence of the natural dipeptide carnosine (CAR) as potential anti-inflammatory molecule. By analysing actin cytoskeleton in 7 dps Caco-2 monolayers versus 21 dps monolayers, we revealed opposite effects of PMA which respectively induced disruption vs. intensification of actin rings and fibers; in both cases, simultaneous administration of PMA and CAR showed counteracting effects. Remarkably, the mRNA expression analysis of the ACTB (actin b) gene mirrored the morphological evidences and the counteraction of CAR. Moreover, the mRNA expression analysis of the AIF-1 gene (Allograft Inflammatory Factor 1) revealed opposite trends in 7 dps- versus 21 dps-grown Caco-2 with respect to PMA effects, and the same holds true for the SLC15A4/PHT1 gene involved both in intestinal peptide absorption and inflammatory responses; nevertheless, AIF-1 mRNA were not affected by CAR, whilst SLC15A4/PHT1 mRNA showed CAR-dependent up-regulation regardless of the differentiation stage. Overall, our results describe cytoskeletal and gene expression modulations which hint a model to distinguish physiological and inflammatory responses based on the differentiation stage of the Caco-2 monolayer and on the effects of CAR as differentially protective substrate.

The SoLute Carrier 15 (Slc15) family includes H+-dependent transporters that play a key role in the cellular uptake/reuptake of di/tripeptides and peptidomimetics. In mammals, in the epithelial cells of intestine and renal tubules two di/tripeptide transport systems have been characterized: the low-affinity/high-capacity system Slc15a1 (PepT1) and the high-affinity/low-capacity system Slc15a2 (PepT2). While PepT1 transporters have been studied in many teleost species, data on PepT2 is still lacking, except for zebrafish (Danio rerio). Here, we performed basic functional and expression analyses of a newly cloned Atlantic salmon (Salmo salar) PepT2. In Xenopus laevis oocytes, transient currents analysis showed that both total amount of charges moved (Q) and decay time (τ) vs membrane voltage shifted to more positive potential values when extracellular pH decreased, highlighting the role of H+ in the first step of transport cycle. Transport current vs voltage relations, as from Gly-L-Gln dose-response experiments, allowed kinetic parameters to be determined as a function of potential (from -140 to +20 mV) and external pH (5.5, 6.5, 7.6). Salmon PepT2 showed the higher apparent Gly-L-Gln affinity (K0.5) at pH 5.5 and 6.5 at the physiological membrane potential and an increase of maximal relative current (Imax) for more negative potentials and more acidic conditions. Notably, mRNA tissue expression analysis revealed that it is highly expressed in midgut and hindgut. Similar to zebrafish, this salmon PepT2 is a high-affinity/low-capacity transporter (K0.5 for Gly-Gln 4,4 μM, Imax -10 nA at -40 mV at pH 6.5), but its specific expression in the mid-to-distal portions of the gut opens to distinct and not yet known roles for a PepT2-type protein in fish physiology.

Di/tripeptides are key nutrients in animal diets, and fundamental for growth. They are transported across the membrane of enterocytes via the Slc15a1/PepT1 peptide transporter, that uses an inwardly-directed proton electrochemical gradient to drive the uptake. Due to a genome duplication event, PepT1a and PepT1b paralogues are found in teleost fish. Two PepT1a transporters, respectively cloned from the Atlantic salmon (Salmo salar) and zebrafish (Danio rerio), namely AsPepT1a and zfPepT1a, have been characterized. For both orthologs, function was verified by heterologous expression in Xenopus laevis oocytes, highlighting electrogenic, Na+-independent and pH-dependent transport similarly to the well-known PepT1b. The transient currents and the transport currents recorded from AsPept1a and zfPepT1a indicate significant functional differences with respect to PepT1b. PepT1a can be described as a low‐affinity/high‐capacity system, but its substrates preference profile is peculiar in the species, particularly for charged dipeptides. Moreover, the pre-steady state (PSS) currents, that reflect the first steps of transport cycle display in the charge/voltage relationship differences with respect to Pept1b in the voltage dependence. Considering that the PSS are consequence of the rearrangement of the protein in the membrane electric field, PepT1a transporters interact with the substrate differently from PepT1b. In addition, PepT1a and PepT1b have similar expression profile but different expression levels. These data together with a significant dissimilar substrate specificity, support the idea of distinct roles for these proteins in peptide recognition and transport.

SLC genes display different expression profiles along the rostral-caudal axis of the gut depending on species, digestive requirements and specialization of the sequential domains. Here, we report on the longitudinal expression profiles of SLC genes in the intestine of two simple-gutted teleost fish, zebrafish and Ballan wrasse (L. bergylta), using a subset of publicly available data from the GEO repository (zebrafish: [GSE20884]; Ballan wrasse: [GSE93191]). We explored intra-species/intra-dataset concordances/discordances in the expression profiles through Spearman simple correlation, which coefficients, even if heavily affected by the compositionality of the data, may be informative of which genes are co- or counter-expressed. Through hierarchical clustering on the correlation coefficients we defined groups of concordant and discordant profiles of SLC genes. An online app (freely available at https://gianmarcopiccinno.shinyapps.io/zebrafish_wrasse_v2/) was developed that allows rapid display of the longitudinal patterns of the genes differentially expressed in the two datasets. All the analyses were performed through R programming language. To define differentially expressed genes, we performed multiple One-Way ANOVA tests, whose p-values were subjected to Benjamini-Hochberg correction. Genes were considered differentially expressed if the corresponding corrected p-value from the ANOVA test was lower than 0.05. The plots were prepared using the R package ggplot2. The online app was produced through the R package shiny, and is hosted by free servers of RStudio. Our tools allow comparative analysis of the longitudinal profiles of the genes expressed in simple guts, and can be extended to implement any new similar datasets.

Endocrine Disruptors (EDs) are exogenous chemicals that interfere with the endocrine system and may cause adverse health effects. The difficulties encountered so far to identify EDs and define the risks to humans and wildlife strongly suggest that investigations must be performed with a new focus, based on main features of endocrine physiology: i) effects of hormones (Hs) are exerted at very low concentration through specific receptors and coupled pathways; ii) some Hs may act through more than one receptor; iii) Hs may act through different/multiple mechanisms in different physiological systems; iv) Hs produce different effects during animal life cycle. Same properties are shared by EDs and us such have to be investigated. Our laboratories demonstrated that natural and environmental estrogens affect embryo development in mussels and zebrafish, and impair cardiac activity leading to expression of cellular stress markers in seabass. BPA in particular is known to impair many steps of amphibians metamorphosis. We showed that it induces lipid accumulation in rat liver, and mimics the effect of E2 reducing ER intracellular levels and activating ER - dependent gene transcription in human cell lines; it also induces breast and trophoblast cell proliferation. Although the involvement of intracellular receptors was expected, the different models showed that membrane receptors, including Gprotein coupled receptors, are also targets, and MAPK, PI3K, calcium and cAMP – dependent pathways are involved. Data highlighted action mechanisms of EDs, suggested the taxonomic conservation of some routes, and provided the basis to establish key-events shared by Hs and EDs. Further work addressing comparative aspects from invertebrates to humans is needed to develop full understanding.

Programme and Abstracts of the 68th National Congress of the Italian Physiological Society (Società Italiana di Fisiologia) (Abstract) Carnosine (β-Ala-L-his, CAR) is an endogenous dipeptide acknowledged to contribute to maintenance of intracellular homeostasis in vertebrate excitable tissues. CAR biochemical properties (e.g. antioxidant and antiglycating activity) account for CAR homeostatic function(s) in many dysregulated states, such as hyperglycaemia upsetting diabetes-targeted tissue/organ districts. In this view, CAR hypoglycaemic effects have been shown to be physiologically regulated in the peripheral nervous system. Previously, we reported responsiveness of CAR-regulating genes in peripheral nerves of hyperglycaemic mice; remarkably, this responsiveness is strongly enhanced in the nerve’s fibroblast component. Here, we identify variations in expression of genes responsible for biosynthesis, degradation and transmembrane transport of CAR in the NIH/3T3 model of murine fibroblasts exposed to a hyperglycaemic stress, in the presence or absence of extracellular CAR. In parallel, we detect functional protection against the stress induced by glucose excess due to CAR, which inhibits ROS production and apoptotic events, and preserves fibroblast proliferation. Overall, the in vitro cellular model mirrors the ex vivo evidence in the peripheral nerves of hyperglycaemic mice, highlighting the (patho)physiological impact of CAR homeostatic variations in diabetes-targeted tissues and, remarkably, in fibroblasts, for which a functional role of ‘sensors’ of hyperglycaemic impairment in tissues could be reckoned.

Programme and Abstracts of the 68th National Congress of the Italian Physiological Society (Società Italiana di Fisiologia)

Programme and Abstracts of the 68th National Congress of the Italian Physiological Society (Società Italiana di Fisiologia)

Allograft Inflammatory Factor1 (AIF1), alias ionized calcium-binding adapter molecule 1 (IBA1), is a highly conserved Ca2+-binding cytokine that has been identified as a key regulator of the immune response in vertebrates. AIF1 is highly expressed in activated macrophages during inflammatory responses, thus representing an accurate indicator of macrophage activation in the body and a pathogenic factor in several inflammatory diseases. Proteins of the AIF1 superfamily are also present in invertebrates, from sponges to echinoderms. Here, we describe the Paracentrotus lividus Aif-1, which encodes a predicted protein of 151 amino acids with high similarity to vertebrate AIF1. In the common sea urchin, molecular and immunocytochemical analyses showed the constitutive expression of Aif-1 in the coelomocytes. Aif-1 localizes in the perinuclear area of amoebocytes and inside the granules of red cells, but it is not present in vibratile cells and colorless spherula cells. Moreover, significant increase of P. lividus Aif-1 expression, at both mRNA and protein level, are observed in coelomocytes after Gram+ bacterial challenge. BLAST searches across Echinoderm databases resulted in identification of orthologous proteins from 24 species (8 sea urchins, 1 brittle star, 12 starfishes and 3 sea cucumbers). Among these, P. lividus Aif-1 shared a high identity with several species, e.g., 85.4% with the sea urchin Strongylocentrotus purpuratus, 60.9% with the brittle star Ophiocoma echinata, 59.6% with the starfish Achantaster planci, and 52.3% with the sea cucumber Apostichopus japonicus. Our study on P. lividus Aif-1 will contribute to elucidate AIF1 function along the evolutionary scale and to consolidate the key evolutionary position of echinoderms throughout metazoans with respect to the common immune paths.

Programme and Abstracts of the 68th National Congress of the Italian Physiological Society (Società Italiana di Fisiologia) (Abstract) The SLC15A4 gene codes for the peptide/histidine transporter 1 (PHT1), a carrier of di/tripeptides and histidine. In vertebrates, the SLC15A4 genes are mainly expressed by the immune and nervous systems, in which they are acknowledged to undergo splicing events of unknown physiological meaning. After the early generic detection of the canonical SLC15A4/PHT1 isoforms on plasma and subcellular membranes, recent studies have pointed out that the human SLC15A4 gene codes for a late endosome/lysosome transmembrane carrier taking part in TLR7-, TLR9-, and NOD1-mediated pathways, thus suggesting that its gene products may act as regulators of inflammatory/immune/autoimmune onsets and diseases. Accordingly, SLC15A4/PHT1 altered activity has been associated to conditions such as type 2 diabetes, inflammatory bowel diseases (IBD) and systemic lupus erythematosus. Here, we report the description of a unique alternative splicing-mediated regulation of the SLC15A4 gene, conserved from zebrafish to human. We demonstrate that this regulation is physiologically facilitated by the Nonsense-Mediated mRNA Decay (NMD) surveillance system, in human immune-derived cells too, and that it is sensitive to inflammatory molecular triggers. Furthermore, in an IBD murine model induced with the inflammatory challenge by dextran sulfate sodium, we describe its impact on the expression of the alternative SLC15A4 gene products, revealing their differential modulations in different GI tracts.

In agreement with all authors of this paper the order of the authors and the contribution “Paola Lunetti[+], Alessandro Romano[+], Chiara Carrisi, Daniela Antonucci, Tiziano Verri, Giuseppe E. De Benedetto, Vincenza Dolce, Francesco P. Fanizzi, Michele Benedetti,* and Loredana Capobianco.* [+] These authors contributed equally to this paper. * Corresponding authors: Michele Benedetti and Loredana Capobianco, Department of Biological and Environmental Sciences and Technologies, University of Salento, 73100 Lecce.” is corrected to read the original order of the first submitted version of the paper: “Chiara Carrisi[+], Alessandro Romano[+], Paola Lunetti, Daniela Antonucci, Tiziano Verri, Giuseppe E. De Benedetto, Vincenza Dolce, Francesco P. Fanizzi, Michele Benedetti,* and Loredana Capobianco.* [+] These authors contributed equally to this paper. * Corresponding authors: Loredana Capobianco and Michele Benedetti, Department of Biological and Environmental Sciences and Technologies, University of Salento, 73100 Lecce.” For completeness the following individual contributions of the authors were added in the Supporting Information: “Author contributions: L.C., M.B., C.C., A.R. designed research; C.C., A.R., P.L. and D.A. performed research; L.C., M.B., C.C., A.R., V.D. and P.L. analysed data; F.P.F., A.R., L.C. and M.B. wrote the paper; L.C., M.B., T.V., G.D.B. and F.P.F. active discussion paper revision.”.

The dinoflagellates of the genus Ostreopsis Schmidt are toxic species involved in the occurrence of massive blooms. These dinoflagellates are known to produce palytoxin and ovatoxins, which are considered amongst the most poisonous phycotoxins in the world. In this work, aqueous and methanolic extracts of one O. cf. ovata Ionian strain were tested on the human-derived HeLa cell line, and cytotoxicity, cytoskeletal rearrangment and apoptosis induction were recorded by morphological and molecular analysis. The MTT test revealed high toxicity for both extracts, which activity was inhibited by ouabain, which suggests the main involvement of palytoxin-like molecules in the observed toxic effects of this strain. Overall, the effects induced by O. cf. ovata on HeLa cells were dependent on the type of adopted extracts, being both toxic, but with the methanolic extract more toxic than the aqueous one and able to induce cell death by apoptosis. This process was evidenced by both phosphatidylserine exposition and up regulation of caspase-3 gene expression. The different effect of the two extracts on cell death is indicative of their different composition and/or activity. Our findings represent the starting step for the characterization of novel bioactive molecules.

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Information regarding cationic amino acid transport systems in thyroid is limited to Northern blot detection of y(+)LAT1 mRNA in the mouse. This study investigated cationic amino acid transport in PC cell line clone 3 (PC Cl3 cells), a thyroid follicular cell line derived from a normal Fisher rat retaining many features of normal differentiated follicular thyroid cells. We provide evidence that in PC Cl3 cells plasmalemmal transport of cationic amino acids is Na+ independent and occurs, besides diffusion, with the contribution of high-affinity, carrier-mediated processes. Carrier-mediated transport is via y+, y(+)L, and b(0,+) systems, as assessed by L-arginine uptake and kinetics, inhibition of L-arginine transport by N-ethylmaleimide and neutral amino acids, and L-cystine transport studies. y(+)L and y(+) systems account for the highest transport rate (with y(+)L > y+) and b(0,+) for a residual fraction of the transport. Uptake data correlate to expression of the genes encoding for CAT-1, CAT-2B, 4F2hc, y(+)LAT1, y(+)LAT2, rBAT, and b(0,+)AT, an expression profile that is also shown by the rat thyroid gland. In PC Cl3 cells cationic amino acid uptake is under TSH and/or cAMP control (with transport increasing with increasing TSH concentration), and upregulation of CAT-1, CAT-2B, 4F2hc/y(+)LAT1, and rBAT/b(0,+)AT occurs at the mRNA level under TSH stimulation. Our results provide the first description of an expression pattern of cationic amino acid transport systems in thyroid cells. Furthermore, we provide evidence that extracellular L-arginine is a crucial requirement for normal PC Cl3 cell growth and that long-term L-arginine deprivation negatively influences CAT-2B expression, as it correlates to reduction of CAT-2B mRNA levels.

The aim of this work was to study both growth and biochemical composition of M. barbatus juveniles feeding monospecific cultures of Isochrysis galbana, Tetrasemis suecica, Dunaliella tertiolecta and Phaeodactylum tricornutum. The results obtained suggest that M. barbatus juveniles respond differently to monospecific algal administration, with T. suecica and I. galbana being the most interesting species under the nutritional point of view as they support the highest increase in growth.