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This paper represents the first result of an active collaboration between the University of Sannio and the San Pio Hospital (Benevento, Italy), started in the 2018, that aims to a detailed mineralogical investigation of urinary stones of patients from Campania region. Herein, selected human bladder stones have been deeply characterized for clinical purposes and environmental biomonitoring, focusing on the importance to evaluate the concentration and distribution of undesired trace elements by means of microscopic techniques in the place of conventional wet chemical analyses. A rare bladder stones with a sea-urchin appearance, known as jackstone calculus, were also investigated (along with bladder stones made of uric acid and brushite) by means a comprehensive analytical approach, including Synchrotron X-ray Diffraction and Simultaneous Thermal Analyses. Main clinical assumptions were inferred according to the morpho-constitutional classification of bladder stones and information about patient’s medical history and lifestyle. In most of the analyzed uroliths, undesired trace elements such as copper, cadmium, lead, chromium, mercury and arsenic have been detected and generally attributable to environmental pollution or contaminated food. Simultaneous occurrence of selenium and mercury should denote a methylmercury detoxification process, probably leading to the formation of a very rare HgSe compound known as tiemannite.

The present investigation exposes the main results raised from an active collaboration started in 2018 with the San Pio Hospital (Benevento, Southern Italy), aiming at a detailed mineralogical investigation of urinary stones of patients from the Campania region. Forty-nine uroliths (both bladder and kidney stones) have been surgically collected from patients admitted between 2018 and 2020 at the Department of Urology of the San Pio Hospital and characterized for clinical purposes and environmental biomonitoring from a mineralogical point of view. Possible causes and environmental implications were inferred according to the morpho-constitutional classification of the uroliths carried out by means of a conventional analytical approach. The mineralogical frequency distribution of uroliths from the Campanian region can be discussed as a function of dietary, socio-demographic, and environmental risk factors. Whewellite [CaC2O4·H2O] and weddellite [CaC2O4·(2+x)H2O], along with anhydrous calcium oxalate, represent the main mineralogical phases forming the biominerals examined here. Worth to note is that the percentage of oxalates in the Campanian region (ca. 51%) is quite comparable to those of other Mediterranean areas. Frequent uricite [C5H4N4O3] (ca. 33%), mainly observed in bladder stones of older male patients, could be related to an incorrect lifestyle and dietary habits. Occurrence of lower percentages of phosphate (i.e., brushite [CaHPO4·2(H2O)] and carbonated apatite [Ca10(PO4CO3)6(OH)8]) and mixed stones (such as, for example, a mixture of ammonium urate [NH4C5H3N4O3] and calcium oxalates) indicates specific etiopathogenetic mechanisms, suggesting proper therapeutical approaches.

In this study, we utilized a model of trout intestinal explants to investigate the uptake and the metabolic effects of Hydroxytyrosol (HT), a bioactive molecule belonging to polyphenols, present in the fruit and leaf of the olive (Olea europaea L.). Fragments of the pyloric caeca (PC), mid intestine (MI) and distal intestine (DI) of the rainbow trout Oncorhynchus mykiss, were incubated with varying concentrations (2.0–20–200 μM) of HT for 5, 15, 60, 120 and 360 min. HT was uptaken by PC and MI, but not DI. The HT concentration reached the highest value after 15 min of incubation. 2.0 and 20 μM HT were well tolerated by both PC and MI, 200 μM HT was cytotoxic, causing DNA damage in PC, but not in MI. 1H NMR analysis of PC and MI incubated with 2.0 and 20 μM HT revealed the increase in the essential amino acids and fatty acids, suggesting that HT slowed down the amino acid degradation while acting as lipolysis stimulators. The effect of HT on the intestinal macromolecular asset was also investigated by Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy (ATR-FTIR) in order to test the advantages of this method in the analysis of biological samples. HT caused macromolecular modifications coherent with the results of the HT uptake, cytotoxicity and 1H NMR analysis. In conclusions, the intestinal explants allow many experimental conditions to be tested simultaneously. Such ex vivo approach may be usefully employed to study the effects of additives or feed components in screening tests before designing in vivo trials. Thus, it may represent an alternative to reduce the number of animals employed. Finally, the present study confirms the validity of ATR-FTIR as a rapid, non-invasive, high-resolution analytical method of analysis for biological samples.

Durum wheat is widespread cultivated in the Mediterranean basin, where it is used to produce high-quality semolina for pasta. Although over the years local and ancient wheat cultivars have been replaced by new ones, better suited to intensive cultivation, the increasing demand of consumers for nutritional and sensory qualities, as well as their attention to sustainable agronomic practices, renewed the interest toward traditional varieties. In order to fully exploit their agronomical and nutritional potential, a systematic analysis of molecular traits would be desirable. Nowadays, this examination is greatly facilitated by the current availability of high-throughput genomic and proteomic methods, which are integrated with classical measurements on plant physiology. To this purpose, we performed a comparative study on germination performances, hormone level variations, and differential protein representations of three-days germinated shoots of two traditional wheat cultivars from Southern Italy, namely Senatore Cappelli and Saragolla, and the commercial elite variety Svevo. Two-dimensional electrophoresis- and nanoLC-ESI-LIT-MS/MS-based proteomic analysis revealed 45 differentially represented spots, which were associated with 32 non-redundant protein species grouping into storage, stress/defense and metabolism/energy production functional categories. Major differences in the traditional varieties concerned over-representation of glutenins, gamma-gliadin and some enzymes of glycolysis and TCA cycle, as well as a down-representation of proteins involved in the response to stress conditions. These features were here discussed in relation to the hormone profile and the known agronomic features of traditional varieties, as compared to the commercial one.

In the present study, we aimed at assessing the influence of breed and feeding system on the bovine milk profile of betaines and carnitines and milk capacity in counteracting the inflammatory endothelial cell (EC) damage induced by interleukin (IL)-6. In the first experimental design, two breeds were chosen (Holstein vs. Modicana) to investigate the biomolecule content and antioxidant capacity in milk and dairy products. In the second experimental design, two feeding systems (pasture vs. total mixed ratio) were tested only in Holstein to evaluate the possible effect on the functional profile of milk and dairy products. Finally, the bulk milk from the two experimental designs was used to evaluate the efficacy of preventing IL-6-induced endothelial inflammatory damage. Results showed that Modicana milk and whey had higher biomolecule content and antioxidant activity compared to Holstein milk (p < 0.01). Milk from Holstein fed TMR showed higher concentration of γ-butyrobetaine, δ-valerobetaine (p < 0.01), and l-carnitine (p < 0.05). Similarly, whey from Holstein fed TMR also showed higher content of δ-valerobetaine, glycine betaine, l-carnitine, and acetyl-l-carnitine (p < 0.01) compared to the Holstein fed pasture. Conversely, the antioxidant activity of milk and dairy products was not affected by the feeding system. In ECs, all milk samples reduced the IL-6-induced cytokine release, as well as the accumulation of reactive oxygen species (ROS) and the induction of cell death, with the most robust effect elicited by Modicana milk (p < 0.01). Overall, Modicana milk showed a higher content of biomolecules and antioxidant activity compared to Holstein, suggesting that the breed, more than the feeding system, can positively affect the health-promoting profile of dairy cattle milk.

Simple Summary With the increase in global population the production of animal proteins becomes increasingly crucial. Aquaculture is the first animal protein supply industry for human consumption. Intensive farming techniques are employed to increase productivity, but these may cause stressful conditions for fish, resulting in impaired growth and poor health conditions. Intestinal inflammation is one of the most common diseases of fish in intensive farming. Intestinal inflammation is usually accompanied by an alteration of the microbiota or dysbiosis. Inflammation and dysbiosis are so tightly intertwined that inflammation may contribute to or result from dysregulation of gut microbiota. Natural substances of plant origin rich in bioactive molecules or more simply phytochemicals, have been proved to be able to reduce inflammation and improve the general health status in various commercially relevant species. In this study, we evaluated the effect of tannins, a class of polyphenols, the most abundant phytochemicals, on intestinal inflammation and microbiota in zebrafish (Danio rerio), a small freshwater fish become an attractive biomedicine and aquaculture animal model during the last decades. The zebrafish has been employed in a vast array of studies aiming at investigating the essential processes underlying intestinal inflammation and injury due to its conservative gut morphology and functions. In this study, we administered a diet enriched with chestnut shell extract rich in tannins to a zebrafish model of intestinal inflammation. The treatment ameliorated the damaged intestinal morphophysiology and the microbiota asset. Our results sustain that products of natural origin with low environmental impact and low cost, such as tannins, may help to ease some of the critical issues affecting the aquaculture sector. Abstract The aim of the present study was to test the possible ameliorative efficacy of phytochemicals such as tannins on intestinal inflammation and dysbiosis. The effect of a chestnut shell (Castanea sativa) extract (CSE) rich in polyphenols, mainly represented by tannins, on k-carrageenan-induced intestinal inflammation in adult zebrafish (Danio rerio) was tested in a feeding trial. Intestinal inflammation was induced by 0.1% k-carrageenan added to the diet for 10 days. CSE was administered for 10 days after k-carrageenan induced inflammation. The intestinal morphology and histopathology, cytokine expression, and microbiota were analyzed. The k-carrageenan treatment led to gut lumen expansion, reduction of intestinal folds, and increase of the goblet cells number, accompanied by the upregulation of pro-inflammatory factors (TNFα, COX2) and alteration in the number and ratio of taxonomic groups of bacteria. CSE counteracted the inflammatory status enhancing the growth of health helpful bacteria (Enterobacteriaceae and Pseudomonas), decreasing the pro-inflammatory factors, and activating the anti-inflammatory cytokine IL-10. In conclusion, CSE acted as a prebiotic on zebrafish gut microbiota, sustaining the use of tannins as food additives to ameliorate the intestinal inflammation. Our results may be relevant for both aquaculture and medical clinic fields.

The 3,5-diiodo-L-thyronine (3,5-T2) is an endogenous metabolite of thyroid hormones, whose administration to rodents fed high-fat diet (HFD) prevents body weight increase and reverts the expression pattern of pro-inflammatory factors associated to HFD. The diet-induced obese (D.I.O.) zebrafish (Danio rerio) has been recently used as an experimental model to investigate fundamental processes underlying central and peripheral obesity-driven inflammation. Herein, we aim to understand the role of 3,5-T2 in regulating central and peripheral inflammation in D.I.O. model of zebrafish. 3,5-T2 (10 nM and 100 nM) was administered with the obesity-inducing diet (D.I.O. with 3,5-T2) or after 4 weeks of obesity-inducing diet (D.I.O. flw 3,5-T2). 3,5-T2 significantly increased the body weight and serum triglyceride levels in D.I.O. zebrafish in both conditions. Moreover, 3,5-T2 sustained or increased inflammation in the anterior (AI) and mid (MI) intestine when administered with the obesity-inducing diet, as indicated by the immunoexpression of the inflammatory markers tumor-necrosis factor-&alpha, (TNF&alpha, ), cyclooxygenase 2 (COX2), calnexin, caspase 3, and proliferating cell nuclear antigen (PCNA). On the contrary, when 3,5-T2 was administered after the obesity-inducing diet, partly reverted the intestinal alteration induced by D.I.O. In addition, brain inflammation, as indicated by the increase in the activation of microglia, was detected in D.I.O. zebrafish and D.I.O. treated with 3,5-T2. These findings reveal that the effects of 3,5-T2 on fish intestine and brain can deviate from those shown in obese mammals, opening new avenues to the investigation of the potential impact of this thyroid metabolite in different diseases including obesity.

This study purpose was to evaluate the in vitro inhibitory properties of Italian acacia honey extracts against pathogenic aquatic oomycete/fungal isolates that cause different diseases in crayfish, resulting in an elevated mortality rate. The antimycotic activity of acacia honey aqueous extracts was evaluated against the strain UEF88662 of Aphanomyces astaci (oomycete) and the strain SMM2 of Fusarium avenaceum (fungus). The extracts preparation was carried out with water by a cheap, not complex and organic solvent-free procedure, with low environmental impact and the higher possibility of large-scale reproducibility. The anti-oomycete and antifungal activities were quantitatively evaluated by growth, survival and sporulation microbiological assays. The extracts displayed a dose-dependent inhibitory efficacy on oomycete and fungal growth and survival, as well as on the production of oomycete and fungal spores. Supported by future in vivo studies, our results encourage the use of natural extracts like honey as innovative tools to counteract mycotic infections. SIGNIFICANCE AND IMPACT OF THE STUDY: The continuous spread of aquatic fungal disease as the 'crayfish plague' and the 'burn spot disease' has severe ecological and commercial repercussions. Critical factor to prevent further spread is the availability of effective antifungals possibility derived from local natural resources to use in innovative strategies of control and eradication of these diseases. This study provides relevant information about the in vitro anti-oomycete and antifungal activity of Italian acacia honey aqueous extracts against two highly infectious and dangerous pathogenic species, Aphanomyces astaci and Fusarium avenaceum, that are responsible for important crayfish diseases.

In this study, evidence for leptin receptor (LR) and gastric leptin immunoreactivity along the digestive tract of the rainbow trout (Oncorhynchus mykiss), is reported. Besides this, the regulation of gastric leptin and its transcript by fatty acids was analyzed in vitro. LR was detected mainly in the cells of the stomach gastric glands and in the brush border of the epithelium of the anterior, middle and distal intestine. In the stomach LR was co-distributed with leptin. The regulation of gastric leptin and its transcript by fatty acids was analyzed by in vitro incubations. Rabbit polyclonal antibodies anti rainbow trout leptin were developed and employed to detect leptin concentration in the stomach and in the incubation medium. Stomach slices were incubated with butyric (4:0), oleic (18:1n-9), α-linolenic (18:3n-3) and arachidonic fatty acids (20:4n-6). All fatty acids caused an increase in the protein in both the stomach and culture medium, while leptin transcript was not modified. Overall, the results confirm the gastric leptin release upon nutritional modulation.

Background Diet supplementation with polyphenols is a novel strategy to improve meat quality in livestock, by preventing oxidative deterioration of lipids and protein. Polyphenols have beneficial effects on both human and animal health and can be obtained from several sources, such as olive mill wastewaters (OMWW). These are severe environmental pollutants and therefore may be recycled and utilized in other sectors. The aim of this study was to evaluate growth performance, meat characteristics, fatty acid composition, antioxidant status, different forms of myoglobin and malondialdehyde formation in kids who received a diet supplemented with polyphenols obtained from OMWW. Weaned goat kids (n = 18) were divided into two homogenous groups: control (C) group (n = 9) received a fattening standard diet while the other group (n = 9) received the same diet, supplemented with 3.2 mg/day of polyphenols powder extract (PE group). Average daily gain (ADG) was calculated 10 days apart throughout the study. After 78 days, the kids were slaughtered and pH and carcass yield were evaluated. Longissimus thoracis et lumborum muscle was collected and utilized for chemical analysis, meat quality evaluation and oxidative stability. Results No differences were recorded in ADG, carcass weight, pH and dressing between the two groups. Furthermore a similar meat proximate composition, texture and color was observed. Dietary polyphenols supplementation significantly (P

The existence of nutritional and energy reserves is fundamental for fish female fertility, so that the existence of a correlation between metabolic reserves and reproductive capacity is suggested. Leptin regulates body weight and energy homeostasis. Estradiol induces the synthesis of vitellogenin, a phospholipoglycoprotein produced by the liver and taken up by the growing oocytes. The objective of this study was to investigate the possible existence of a crosstalk between 17β-estradiol (E2) and leptin in the modulation of E2-induced vtg in the rainbow trout Oncorhynchus mykiss. Liver slices were incubated with recombinant trout leptin (rt-lep) at three different concentrations (1-10-100 ng/ml). rt-lep brought about the decrease of E2-induced vtg secretion in the medium and the down-regulation of vtg mRNA expression. Moreover, rt-lep stimulated the lipase activity and diminished the liver fatty acid content. The combined employment of signal transduction inhibitors and the analysis of signal transduction phosphorylated factors revealed that rt-lep effect on E2-induced vtg occurred through the activation of phosphodiesterase, protein kinase C, MAP kinases, and protein kinase A. In conclusion, our study suggests that leptin influences E2-induced vtg synthesis in the rainbow trout Oncorhynchus mykiss by modifying both the protein and the lipid components.

In the last years, great importance has been given to the beneficial effects of polyphenols [1]. Agricultural by-products are a rich source of polyphenols and have a potential suitable for development into dietary supplements and various food additives [2]. The objective of the present research was to investigate gut morphology, inflammation and immune response of adult pigs (Casertana strain) fed with polyphenols extracted from olive mill wastewater added to the standard diet, by in vivo and in vitro analysis. The ‘Casertana’ pig is an ancient autochthon genetic type and represents an experimental model suitable for semi-wild controlled breeding technique. During the finishing period, pigs were randomly assigned in two groups: control group fed with a standard diet and treated group fed with standard diet supplemented with polyphenols extracted from olive mill wastewater (OMWW) using capsules (0.03 g/kg of feed per pig per day) [3]. The trial lasted for 120days. After slaughter, alveolar macrophages were extracted from pig lungs [4] and the superoxide anion assay was performed to test the anti-oxidant effects of polyphenols extracted from OMWW. Gastrointestinal tracts (stomach, duodenum, jejunum, ileum, caecum and colon) were collected from all animals, embedded in paraffin wax, serially cut in transversal sections and stained with hematoxylin– eosin for histomorphometric analysis. No significant differences in the length of the jejunum-ileum villi and the depth of the colon crypts were detected between control and polyphenols fed pigs. COX-2 immunoreactivity in the gastrointestinal tract was more intense in the control group. The low level of expression of COX-2 in immunoreactive cells in the intestine of treated pigs could suggests a protective role of polyphenols, modulating and reducing the inflammatory response [5]. Superoxide anion production in alveolar macrophages was lower in pigs fed polyphenols (p

Orexin A and B peptides and the receptor OX2R were studied in sea bass and goldfish gastroenteric tract by immunoblotting combined with densitometric analysis using NIH Image J software and immunohistochemical techniques. These teleost species present a different gut organization and diverse feeding habits. Immunoblotting experiments showed one band of 16 kDa corresponding to prepro-orexin, and one band of 38 kDa corresponding to the OX2R receptor. Immunohistochemical localization of OXA and OXB was observed in the enteric nervous system throughout the gastroenteric tract of both species. OXA and OXB immunoreactive cells were found in the gastric and intestinal regions of sea bass, and were mainly found in the basal region of folds in intestinal bulb, and in the midgut and hindgut of goldfish. The distribution of OX2R was mainly detected in the mucosa of the gastroenteric tract of sea bass and goldfish. This distribution suggests an endocrine action of OXA and OXB in the gastrointestinal tract as well as involvement in the peripheral control of food intake and digestive processes in both species. This study might also serve to determine the productive factors in breeding and as a baseline for future experimental studies on the regulation of the gastroenteric functions in non-mammalian vertebrates. Anat Rec, 299:1121-1129, 2016. © 2016 Wiley Periodicals, Inc.

Nesfatin-1 (Nesf-1) is an anorexigenic peptide involved in the regulation of homeostatic feeding. Nesf-1 is expressed in the central nervous system and other organs, including pancreas, where it promotes the release of insulin from β-cells. This raises the possibility that Nesf-1 dysfunction could be involved in metabolic disorders, particularly in type 2 diabetes mellitus (T2D). Recently, it has been discovered that dolphins can be a natural animal model that fully replicates human T2D, due to its prolonged glucose tolerance curve and maintenance of a state of hyperglycemia similar to human T2D during fasting. This correspondence suggests that dolphins may be a suitable model for investigating physiological and pathological metabolic disorders. Here, we have characterized Nesf-1 distribution in the pancreas of the common bottlenose dolphin (Tursiops truncatus) and measured plasmatic levels of Nesf-1 and glucose during fasting and post-prandial states. The Mediterranean Marine Mammal Tissue Bank (MMMTB) of the University of Padova provided us with pancreas samples, derived from four animals, and plasma samples, collected before and after the main meal. Interestingly, our results showed that Nesf-1-immunoreactive cells were distributed in Langerhans islets, co-localized with glucagon in α-cells. Similar to humans, dolphin plasma Nesf-1 concentration doesn’t show a statistically significant difference when comparing fasting and post-prandial states. On the other hand, blood glucose levels were significantly higher before than after the main meal. Our data provide a comparative analysis for further studies on the involvement of Nesf-1 in mammalian metabolic disorders.

CARD14/CARMA2sh (CARMA2sh) is a scaffold protein whose mutations are associated with the onset of human genetic psoriasis and other inflammatory skin disorders. Here we show that the immunomodulatory adapter protein TRAF family member-associated NF-κB activator (TANK) forms a complex with CARMA2sh and MALT1 in a human keratinocytic cell line. We also show that CARMA2 and TANK are individually required to activate the nuclear factor κB (NF-κB) response following exposure to polyinosinic-polycytidylic (poly [I:C]), an agonist of toll-like receptor 3. Finally, we present data indicating that TANK is essential for activation of the TBK1/IRF3 pathway following poly (I:C) stimulation, whereas CARMA2sh functions as a repressor of it. More important, we report that two CARMA2sh mutants associated with psoriasis bind less efficiently to TANK and are therefore less effective in suppressing the TBK1/IRF3 pathway. Overall, our data indicate that TANK and CARMA2sh regulate TLR3 signaling in human keratinocytes, which could play a role in the pathophysiology of psoriasis.

The aims of this study were to assess the efficacy of alphacypermethrin (ACYP) on pediculosis due to Haematopinus tuberculatus and to evaluate the influence of the treatment on productive and reproductive performance in buffaloes (Bubalus bubalis) reared in an intensive system. The trial was performed on 56 pluriparous buffaloes at 86.8 ± 8.1 d in milk. The animals underwent individual louse count and were divided into 2 homogenous groups according to louse count, age, number of lactations, days in milk, live BW, BCS, pregnancy status, and milk yield. Group A (n = 28) was treated by a pour-on formulation of ACYP, and Group S (n = 28) was treated by pour-on saline solution. Individual louse counts were performed weekly on 10 buffaloes in each group. Feed intake was recorded daily and the total mixed ration, individual ingredients, and orts were analyzed to calculate DM ingestion. Individual milk yield was recorded daily and milk samples were analyzed at the beginning of the trial, after 4 wk, and at the end of the trial to assess milk composition. Individual BCS was also evaluated simultaneously. Finally, the animals underwent synchronization of ovulation starting 4 wk after treatment and the pregnancy rate and the calving-conception interval were evaluated. Data were analyzed by the Mann-Whitney test and ANOVA for repeated measures. The infestation was constant in Group S, whereas no lice were present in Group A throughout the study. Daily DMI was similar in the 2 groups (16.7 ± 0.4 vs. 16.3 ± 0.3 kg/d in Group A vs. Group S, respectively), although buffaloes in Group A showed higher (P < 0.05) BCS score at the end of the trial (7.39 ± 0.1 vs. 7.14 ± 0.1 in Group A vs. Group S, respectively). The average milk yield/buffalo was higher (P < 0.05) in Group A compared to Group S (10.58 ± 0.1 vs. 10.39 ± 0.1 kg in Group A vs. Group S, respectively) and this was mainly due to the higher milk production recorded in buffaloes at less than 75 d in milk (11.81 ± 0.1 vs. 11.45 ± 0.1 kg in Group A vs. Group S, respectively). Despite of a similar fertility rate (90.5 vs. 80.9% in Group A vs. Group S, respectively), a lower (P < 0.05) calving-conception interval was recorded in Group A compared to Group S (118 ± 16 vs. 177 ± 16 d in Group A vs. Group S, respectively). In addition to the pour-on treatment against pediculosis, productive and reproductive performance were also improved. This represents a significant improvement in dairy buffalo herd management.

Carrageenan-based biocomposites have significant potential to develop packaging films and coatings for shelf-life extension of food products. In this study trout fresh fillets were separated into three groups: uncoated trout fillet (UTF), coated with carrageenan-based materials (coated trout fillet, CTF), and coated with carrageenan-based materials containing 1 g/100 mL essential lemon oil (active coated trout fillet, ACTF). The effect of carrageenan coating enriched with essential lemon oil on the quality of rainbow trout ( Oncorhynchusmykiss ) fillets during refrigerated storage (4 ± 1 °C) over a period of 15 days was evaluated. Trout fillets were analyzed for microbiological (total viable count, Enterobacteriacea counts, lactic acid bacteria, H 2 S-producing bacteria), chemical (TVB-N, moisture, pH), and biochemical (fatty acids content) characteristics. This study demonstrates the effectiveness of an edible active carrageenan coating in preserving fresh trout fillets from lipid oxidation and microbial growth.

Highlights • The complexes formed by BCL10, MALT1 and CARMA proteins are key regulators of NF-κB activation. • We report the functional characterization of tBCL10, a BCL10 isoform from the trout Oncorhynchus mykiss. • tBCL10 can functionally replace the human protein. • The rainbow trout Oncorhynchus mykiss can serve as a model organism to study this pathway., The complexes formed by BCL10, MALT1 and members of the family of CARMA proteins have recently been the focus of much attention because they represent a key mechanism for regulating activation of the transcription factor NF-κB. Here, we report the functional characterization of a novel isoform of BCL10 in the trout Oncorhynchus mykiss, which we named tBCL10. tBCL10 dimerizes, binds to components of the CBM complex and forms cytoplasmic filaments. Functionally, tBCL10 activates NF-κB transcription factor and is inhibited by the deubiquitinating enzyme A20. Finally, depletion experiments indicate that tBCL10 can functionally replace the human protein. This work demonstrates the evolutionary conservation of the mechanism of NF-κB activation through the CBM complex, and indicates that the rainbow trout O.mykiss can serve as a model organism to study this pathway.

Human BCL10 (hBCL10) protein is a signal transduction molecule originally identified because of its direct involvement in a subset of mucosa-associated lymphoid tissue (MALT) lymphomas, and later recognized as a crucial factor in regulating activation of NF-kB transcription factor following antigen receptor stimulation on lymphocytes. In this study, we characterized the NF-kB inducing activity of porcine BCL10 (pBCL10). pBCL10 oligimerizes, binds to components of the CARMA/ BCL10/MALT1 complex and forms cytoplasmic filaments. Functionally, in human cells pBCL10 is more effective in activating NF-kB compared to hBCL10, possibly due to the lack of carboxy-terminal inhibitory serine residues present in the human protein. Also, depletion experiments carried out through expression of short hairpin RNAs targeting hBCL10 indicate that pBcl10 can functionally replace the human protein and retains its higher NF-kB-inducing property in the absence of hBCL10. Our results contribute useful information on BCL10 protein in pigs, and may help the development of strategies based on the control of the immune response in pigs.

Orexin A and B peptides and the receptor OX2R were studied in sea bass and goldfish gastroenteric tract by immunoblotting combined with densitometric analysis using NIH Image J software and immunohistochemical techniques. These teleost species present a different gut organization and diverse feeding habits. Immunoblotting experiments showed one band of 16 kDa corresponding to prepro-orexin, and one band of 38 kDa corresponding to the OX2R receptor. Immunohistochemical localization of OXA and OXB was observed in the enteric nervous system throughout the gastroenteric tract of both species. OXA and OXB immunoreactive cells were found in the gastric and intestinal regions of sea bass, and were mainly found in the basal region of folds in intestinal bulb, and in the midgut and hindgut of goldfish. The distribution of OX2R was mainly detected in the mucosa of the gastroenteric tract of sea bass and goldfish. This distribution suggests an endocrine action of OXA and OXB in the gastrointestinal tract as well as involvement in the peripheral control of food intake and digestive processes in both species. This study might also serve to determine the productive factors in breeding and as a baseline for future experimental studies on the regulation of the gastroenteric functions in non-mammalian vertebrates.

The phenomenon of antibiotic resistance, that appears increasingly alarming worldwide, has renewed the interest in the study of many natural extracts with antimicrobial properties, such as honey. Even if several honeys have been approved for clinical using, there are some obstacles for therapeutic applicability of honey in medicine, and not only. The variability of antimicrobial activity of different honeys and the incomplete knowledge of the involved antimicrobial compounds are the major ones. The aim of this study has been to define the antimicrobial profile of hydro-alcoholic extracts prepared from Italian unifloral honeys, particularly acacia, chestnut and sulla. The antimicrobial capability of hydro-alcoholic extracts was evaluated against clinical isolates of different bacterial species involved in human pathology, such as Staphylococcus aureus, Staphylococcus epidermidis and Escherichia coli. In addition, the antifungal effect of honeys extracts was verified against the UEF88662-Aphanomyces astaci and SMM2-Fusarium avenaceum strains, both fungal species responsible of different diseases in crayfish and plants. In vitro preliminary microbiological results demonstrated that unifloral honeys hydro-alcoholic extracts from Campania are able to effectively counteract the growth and survival of different pathogenic microrganisms.

In this study, chestnut (Castanea sativa) shell was extracted with different solvents, and immunomodulatory activity was investigated in an in vitro model system using blood and intestinal leukocytes of Oncorhynchus mykiss. Gallic acid (GA) was used as a standard. Chestnut shell extract (CSE) and GA readily entered both blood and intestinal leukocytes. Superoxide anion production and phagocytosis were decreased by low doses of CSE and increased with high doses. CSE and GA differently regulated cytokine expression in blood and intestinal leukocytes. High doses of CSE upregulated IL-1β, TNF-α, and IL-10 in intestinal leukocytes and IL-10 in blood leukocytes. Low doses of CSE upregulated IL-1β and TNF-α in blood leukocytes. GA appeared to be effective only in blood leukocytes. The effects of CSE on pro- and anti-inflammatory cytokines seemed to suggest an alert effect of the immune defense system against a possible infectious agent. The less evident effect of GA in comparison to CSE could have been attributable to the synergistic and/or additive effects of polyphenols in the latter. The immune-stimulating activity of CSE reported here could be useful for future practical applications in fish health.

Studies in mammals indicate that leptin is a multifunctional cytokine involved in regulation of energy metabolism and the modulation of the immune function. However, evidence for an immunomodulatory effect of leptin in fish is still missing. At least in part, this lack of knowledge is due to the absence of materials and models. In this study, we produced trout recombinant leptin (rt-lep) and tested its capacity to trigger cellular pathways, usually active in mammal immune system cells. STAT3, NF-κB, and the three major MAPK cascades (JNK, p38 and ERK), were activated by rt-lep in in vitro incubations with blood leucocytes of the rainbow trout Oncorhynchus mykiss. We also showed that rt-lep causes a decrease in superoxide anion production in trout blood leucocytes. Thus our data indicate that as in mammals also in teleosts leptin plays pleiotropic activities. Importantly, its actions in fishes do not always conform to the picture emerging for mammals.

This study provides a general approach to the presence and possible role of orexins and their receptors in the gut (three gastric chambers and intestine) of confined environment bottlenose dolphin. The expression of prepro-orexin, orexin A and B and orexin 1 and 2 receptors were investigated by single immunostaining and western blot analysis. The co-localization of vasoactive intestinal peptide and orexin 1 receptor in the enteric nervous system was examined by double immunostaining. Also, orexin A concentration were measured in plasma samples to assess the possible diurnal variation of the plasma level of peptide in this species. Our results showed that the orexin system is widely distributed in bottlenose dolphin enteric nervous system of the all gastrointestinal tract examined. They are very peculiar and partially differs from that of terrestrial mammals. Orexin peptides and prepro-orexin were expressed in the main stomach, pyloric stomach and proximal intestine; while orexin receptors were expressed in the all examined tracts, with the exception of main stomach where found no evidence of orexin 2 receptor. Co-localization of vasoactive intestinal peptide and orexin 1 receptor were more evident in the pyloric stomach and proximal intestine. These data could suggest a possible role of orexin system on the contractility of bottlenose dolphin gastrointestinal districts. Finally, in agreement with several reports, bottlenose dolphin orexin A plasma level was higher in the morning during fasting. Our results emphasize some common features between bottlenose dolphin and terrestrial mammals. Certainly, further functional investigations may help to better explain the role of the orexin system in the energy balance of bottlenose dolphin and the complex interaction between feeding and digestive physiology.

In this study, we used natural polysaccharides (pectin, alginate and chitosan) as binders to generate pellets for crayfish feeding. Pellets were produced by cold extrusion in order to preserve nutrients from degradation and reduce energy consumption. Thereafter, pellets were submitted to a coating procedure, with the aim of improving pellet stability in water. Pellet water stability was analyzed by monitoring the diameter of the released particles in water over progressive time intervals up to 24 h, employing a Low Angle Laser Light Scattering Technique. Alginate containing pellets released particles with a smaller diameter than chitosan and pectin containing pellets, indicating that alginate containing pellets disaggregated more and therefore were less stable in water than the other pellet types. The effects of the different polysaccharide containing pellets were evaluated on crayfish feeding response employing juvenile Cherax albidus . The feeding experiment was carried out for 12 weeks, at the end of which growth parameters and the activity of amylase, lipases and proteases in the gastric juice, hepatopancreas and intestine were recorded. Crayfish fed pectin containing pellets exhibited a significant weight gain. Digestive enzyme activities did not statistically show significant differences in the digestive tract except for amylase that was significantly higher in the intestine of animals fed pectin containing pellets. Our data indicate that pectin and chitosan pellets showed the best water stability performances, moreover pectin pellets brought about the highest body weight gain and affected the amylase profile in the intestine of juvenile Cherax albidus .

Orexin receptor 1 (OX₁R) and orexin receptor 2 (OX₂R) are two G-protein-coupled receptors that bind their ligands, orexin A (OXA) and B (OXB), with different affinities. The male genital system represents an important target for OXA, which appears to play a role in the control of steroidogenesis and germ cell development in the testis. It is known that among domestic breeding animals, in the boar the number of Leydig cells is very high and OXA appears to have stimulatory activity on testosterone production. In this study, we aimed to evaluate the presence of OX₁R in the boar testis in order to extend our knowledge concerning the distribution and a potential functional role of the orexinergic system in the male reproductive tract of farm animals. The presence of OX₁R immunopositive cells in seminiferous tubules of the boar testis enables us to hypothesize a possible role of OXA on male germ cells cycle in pig. Further investigations, involving functional and ultrastructural analysis, may contribute to our understanding of the role of orexins in the boar genital system.

The aim of this trial was to evaluate ghrelin response to milk administration in 20 days old buffalo calves. The trial was carried out on 5 female buffalo calves with a mean age of 21.2±2.8 days. Five blood samples were collected from each animal into EDTA tubes, starting at 07.00 until 15.00, at 2-h intervals. At 09.00, after the second blood sample, replaced milk was administered to the calves. Blood samples were immediately placed at 4°C until processing, which was performed on the same day. We used real-time reverse transcription PCR system to detect the expression of ghrelin mRNA levels in blood of buffalo calves. Two calves showed a low ghrelin concentration at the start of the trial (Group A = low ghrelin concentration) and three calves a high ghrelin concentration (Group B = high ghrelin concentration). Ghrelin expression was significantly higher either two hours (P

In this study we assessed the effect of leptin treatment on testicular morphology, spermatogenesis, Peroxisome Proliferator Activated Receptor (PPAR) alpha, 17beta-hydroxysteroide dehydrogenase, 17beta-estradiol and testosterone levels in the testis and blood of the lizard Podarcis sicula at the beginning of summer regression before entering the refractory period, when lizards no longer respond to hormonal and environmental stimuli. Lizards treated with five injections of leptin showed seminiferous tubules with germinal cells at all stages and wider lumina with respect to the controls. After 10 injections, the diameter of the lumina increased compared to the controls and 5 injection-group. After 10 injections plus 20 days before the sacrifice, the seminiferous tubules with open lumina and germinal cells were less abundant than in the 5 and 10 injection-groups. In all groups, the epididymis epithelium was higher than in the controls, with mitosis and binucleated cells. In both the control and treated animals secondary spermatocytes and spermatids were immunoreactive to leptin receptor and PPARalpha. In treated animals the interstitial cells and peritubular fibrocytes were also leptin receptor immunoreactive, while PPARalpha immunoreactivity translocated from the cytoplasm to the nucleus. 17beta-HSD immunoreactivity was present in the spermatids and interstitial cells of control lizards and in secondary spermatocytes and spermatids of treated lizards. Leptin treatment had no statistically significant effect on testicular and circulating 17beta-estradiol and testosterone levels. These observations indicate that leptin brings about a delay in testis summer regression in Podarcis sicula, playing a regulatory role in reproduction in this species as already hypothesized for mammals.