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We aimed to evaluate the magnesium content in human cirrhotic liver and its correlation with serum AST levels, expression of hepatocellular injury, and MELDNa prognostic score. In liver biopsies obtained at liver transplantation, we measured the magnesium content in liver tissue in 27 cirrhotic patients (CIRs) and 16 deceased donors with healthy liver (CTRLs) by atomic absorption spectrometry and within hepatocytes of 15 CIRs using synchrotron-based X-ray fluorescence microscopy. In 31 CIRs and 10 CTRLs, we evaluated the immunohistochemical expression in hepatocytes of the transient receptor potential melastatin 7 (TRPM7), a magnesium influx chanzyme also involved in inflammation. CIRs showed a lower hepatic magnesium content (117.2 (IQR 110.5–132.9) vs. 162.8 (IQR 155.9–169.8) μg/g; p < 0.001) and a higher percentage of TRPM7 positive hepatocytes (53.0 (IQR 36.8–62.0) vs. 20.7 (10.7–32.8)%; p < 0.001) than CTRLs. In CIRs, MELDNa and serum AST at transplant correlated: (a) inversely with the magnesium content both in liver tissue and hepatocytes; and (b) directly with the percentage of hepatocytes stained intensely for TRPM7. The latter also directly correlated with the worsening of MELDNa at transplant compared to waitlisting. Magnesium depletion and overexpression of its influx chanzyme TRPM7 in hepatocytes are associated with severity of hepatocyte injury and prognosis in cirrhosis. These data represent the pathophysiological basis for a possible beneficial effect of magnesium supplementation in cirrhotic patients.

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Pyrrolizidine alkaloids (PAs) are secondary metabolites produced by plants as a self-defense against insects. After bioactivation in the liver, some PAs can cause acute or chronic toxicity in humans. The aim of this study was to determine the presence of PAs in 121 samples of monofloral and multifloral honey from three different Italian regions (Friuli-Venezia Giulia, Marche and Calabria) to meet the European Food Safety Authority (EFSA) suggestion. An in-house liquid chromatography with tandem mass spectrometry (LC-MS/MS) method was validated according to European Union Reference Laboratory (EURL) performance criteria. This method allowed the detection and quantification of 35 PAs. Of the 121 honey samples, 38 (31%), mostly from Calabria, contained PAs. The total content of the PAs ranged from 0.9 µg/kg to 33.1 µg/kg. In particular, echimidine was the most prevalent PA. A rapid human exposure assessment to PAs in honey and a risk characterization was performed using the EFSA RACE tool. The assessment highlighted a potential health concern only for toddlers who frequently consume elevated quantities of honey. This study showed a low presence of PAs in Italian honey; however, the importance of continuously monitoring these compounds is stressed, along with the suggestion that the relevant authorities establish maximum limits to guarantee support for producers and consumer safety.

The recent introduction of the Asian yellow-legged hornet, Vespa velutina, into Europe has raised concern regarding the threat to honeybees and the competition with the European hornet, Vespa crabro. The aim of this study was to investigated essential (Mg, Fe, Zn, Cu) and non-essential (Cd and Pb) elements in these two species. Element concentrations were determined in the whole body and separately in the head, thorax and abdomen using atomic absorption spectrometry (AAS). The changes in essential element concentration and speciation during metamorphosis were also studied using size exclusion chromatography followed by AAS and proteomic analysis. In both species, the essential elements were more concentrated in the abdomen due to the presence of fat bodies. Magnesium, Fe and Zn concentrations were significantly higher in V. crabro than in V. velutina and could have been related to the higher aerobic energy demand of the former species required to sustain foraging flight. Low concentrations of Cd and Pb were indicative of low environmental exposure. The concentration and speciation of essential elements, particularly Fe, varied among the developmental stages, indicating a modification of ligand preferences during metamorphosis. Overall, the results in the present study provide a better understanding of the hornet metal metabolism and a foundation for additional studies.

Urinalysis could be helpful to investigate the health status of giraffes held in captivity using noninvasive methods to avoid animal handling or anesthesia. We collected 52 voided urine samples from 20 giraffes of different ages, sexes, and subspecies from the ground. To evaluate potential interference by soil contaminants, a pilot study was performed using 20 urine samples obtained from 10 cows. All bovine and 29 giraffe samples were subjected to routine urinalysis including urine specific gravity (USG). All samples were analyzed for urine total protein (uTP), urine creatinine (uCrea) concentration, and urine protein-to-urine creatinine ratio (UPC). Urinary proteins were separated by SDS-PAGE electrophoresis. No significant differences were determined between free-catch and urine sampled from the ground in cows. Giraffe urine was pale-yellow, with alkaline pH (>8.0) and a mean USG of 1.035 ± 0.013. The uTP, uCrea, and UPC expressed as median (range) were 0.20 (0.08–0.47) g/L, 2.36 (0.62–5.2) g/L, and 0.08 (0.05–0.15), respectively. SDS-PAGE allowed the separation of protein bands with different molecular masses, including putative uromodulin at 90 kD, putative albumin at 64 kD, and putative immunoglobulin heavy and light chains at 49 kD and 25 kD, respectively. Urine collection from the ground appears to be a reliable technique for urinalysis and urine electrophoresis in giraffes.

Simple Summary Honeybees are vital pollinators for the human food chain. Colony depopulation is a serious threat to Apis mellifera populations and unfortunately it is also one of the most elusive and difficult to study. This research deals with the problem at its foundation: population imbalances. The proposed method allows to discriminate, with remarkably good performances, precocious foragers from proper aged ones using SDS-PAGE patterns of haemolymph proteins. Implications and future perspectives are discussed. Abstract Honeybees, as social insects, live in highly organised colonies where tasks reflect the age of individuals. As is widely known, in this context, emergent properties arise from interactions between them. The accelerated maturation of nurses into foragers, stimulated by many negative factors, may disrupt this complex equilibrium. This complexity needs a paradigm shift: from the study of a single stressor to the study of the effects exerted by multiple stressors on colony homeostasis. The aim of this research is, therefore, to study colony population disturbances by discriminating overaged nurses from proper aged nurses and precocious foragers from proper aged foragers using SDS-PAGE patterns of haemolymph proteins and a machine-learning algorithm. The KNN (K Nearest Neighbours) model fitted on the forager dataset showed remarkably good performances (accuracy 0.93, sensitivity 0.88, specificity 1.00) in discriminating precocious foragers from proper aged ones. The main strength of this innovative approach lies in the possibility of it being deployed as a preventive tool. Depopulation is an elusive syndrome in bee pathology and early detection with the method described could shed more light on the phenomenon. In addition, it enables countermeasures to revert this vicious circle.

Wild animals have been used as food since ancient times and, currently, the consumption of unconventional animals is increasing worldwide. The process of cooking meat using traditional recipes includes a variety of ingredients, which can influence the total metal intake from the diet. In this study, the concentrations of eight essential (Fe, Zn, Cu, Mn, Se, Ni, Mo, and Co) and six non-essential (Pb, Cd, Hg, Al, As, and Cr) trace elements were determined in home-processed food obtained from snails and from three common species of game animals (woodcock, pheasant, and hare), seasoned with anchovies, mushrooms, and different vegetables using inductively coupled plasma mass spectrometry (ICP-MS). In general, Fe was the most abundant trace element, ranging from 18 &plusmn, 8 &micro, g/g in pheasant to 99 &plusmn, 76 &micro, g/g in snail, and Co was the least abundant, ranging from 0.007 &plusmn, 0.003 &micro, g/g in hare to 0.093 &plusmn, 0.048 &micro, g/g in snail. Regarding the non-essential trace elements, Pb concentrations showed wide variations, reaching a concentration of 17.30 &micro, g/g in hare, while Cd concentrations were higher in snail, ranging from 0.18 to 0.46 &micro, g/g. These alternative food sources can offer an important contribution to the human nutritional requirements of essential trace elements, in particular of Fe. The high concentrations of Pb and Cd present in some samples should be considered as potentially dangerous for the consumers.

Urinalysis is widely recognized to be a useful tool in routine health investigations, since it can diagnose numerous pathologies. Considering the paucity of knowledge concerning giraffes, urine from 44 giraffes (Giraffa camelopardalis) (18 males and 26 females, from 3 months of age to 21 years of age) underwent routine urinalysis, 1D-electrophoresis, and protein identification using mass spectrometry, with the aim of identifying the urinary reference values and the urine proteome. The urine specific gravity (USG), urine total proteins (uTP), urine creatinine (uCr), and urine protein:creatinine ratio (UPC) reference values, reported as the median, and lower limit (LL) and upper limit (UL), were 1.030 (1006&ndash, 1.049), 17.58 (4.54&ndash, 35.31) mg/dL, 154.62 (39.59&ndash, 357.95) mg/dL, and 0.11 (0.07&ndash, 0.16), respectively. Mass spectrometry, together with electrophoresis, revealed a pattern of common urinary proteins, albumin, lysozyme C, and ubiquitin were the most represented proteins in the giraffe urine. It has been hypothesized that these proteins could act as a defense against microbes. Moreover, in giraffes, urinalysis could be a valid tool for gauging renal function and physiological status changes.

Chronic kidney disease (CKD) is a progressive and irreversible disease. Although urine is an ideal biological sample for proteomics and metabolomics studies, sensitive and specific biomarkers are currently lacking in dogs. This study characterised dog urine proteome and metabolome aiming to identify and possibly quantify putative biomarkers of CKD in dogs. Twenty-two healthy dogs and 28 dogs with spontaneous CKD were selected and urine samples were collected. Urinary proteome was separated by SDS-PAGE and analysed by mass spectrometry, while urinary metabolome was analysed in protein-depleted samples by 1D 1H NMR spectra. The most abundant proteins in urine samples from healthy dogs were uromodulin, albumin and, in entire male dogs, arginine esterase. In urine samples from CKD dogs, the concentrations of uromodulin and albumin were significantly lower and higher, respectively, than in healthy dogs. In addition, these samples were characterised by a more complex protein pattern indicating mixed glomerular (protein bands ≥65 kDa) and tubular (protein bands

Urinalysis offers important clinical information regarding not only the kidney function but also the general health status of an organism.The aims of this research were to obtain preliminary data on urine chemistry and electrophoretic protein profile from cows and heifers,to compare electrophoretic profiles of not pregnantwith pregnant animals and to evaluate their changes as the pregnancy progresses. Eight heifers and six cows were included in the study and 97 urine samples were collected. Complete urinalysis was performed and urinary proteins were separated by electrophoresis. Considering the pregnancy as a source of variability, significant differences were reported between pregnant and not pregnantheifers for the urine specific gravity (P=0.005), urine total proteins (P=0.009) and urine total proteins to urine creatinine ratio (UPC) (P=0.008). The majority of urine samples analysed in this study showed common protein bands. A mean of protein bands of 17±3 was detected in heifers, while a mean of 13±3 protein bands was recorded in cows. The putative proteins were uromodulin, transferrin, albumin, heavy and light chains of immunoglobulins. The comparison between pregnant and not pregnantanimals showed qualitative differences, with the absence of threebands in not pregnantcows including the putative alpha-fetoprotein. In conclusion, urinalysis is an economical and a non-invasive diagnostic protocol, which should be routinely used for the clinical evaluation of large animals. The data reported in the present study could be considered suggestive of healthy animals and they confirmed those previously reported in the literature.

Spontaneous mammary tumors are very common in bitches. The involvement of oxidative stress and the function of antioxidant enzymes in cancerogenesis have been studied in depth in human medicine, while data in veterinary medicine are still fragmentary. The main aim of this study was to evaluate the activity and the expression of superoxide dismutases (Cu-ZnSOD and MnSOD) and the activity of catalase (CAT) in canine mammary tumors in comparison with the adjacent healthy tissue. Six female dogs (mean age 10.4 years) were included in this study. After surgery, fresh tumor and healthy tissue samples were immediately frozen in dry ice and stored at −80°C for biochemical analyses, while the remaining parts were used for histopathological analysis. Enzyme activity was measured by spectrophotometric assays and protein expression by western blotting. In canine mammary tumors, Cu-ZnSOD activity and expression increased significantly compared with healthy control tissues (p=0.03). MnSOD showed a significantly lower activity in tumoral tissues at stage 2 (p

Artemisia annua has been used for centuries in Traditional Chinese Medicine. Although used as an antimalarial drug, its active compound artemisinin and the semisynthetic derivatives have also been investigated for their anticancer properties, with interesting and promising results. The aims of this research were to evaluate (i) the cytotoxicity and the antiproliferative effect of pure artemisinin and a hydroalcoholic extract obtained from A. annua on the D-17 canine osteosarcoma cell line and (ii) the intracellular iron concentration and its correlation with the cytotoxic effects. Both artemisinin and hydroalcoholic extract induced a cytotoxic effect in a dose-dependent manner. Pure artemisinin caused an increase of cells in the S phase, whereas the hydroalcoholic extract induced an evident increase in the G2/M phase. A significant decrease of iron concentration was measured in D-17 cells treated with pure artemisinin and hydroalcoholic extract compared to untreated cells. In conclusion, although preliminary, the data obtained in this study are indicative of a more potent cytotoxic activity of the hydroalcoholic extract than pure artemisinin, indicating a possible synergistic effect of the phytocomplex and a mechanism of action involving iron and possibly ferroptosis. Considering the similarities between human and canine osteosarcomas, progress in deepening knowledge and improving therapeutic protocols will probably be relevant for both species, in a model of reciprocal translational medicine.

Artemisia annua has been used for centuries in Traditional Chinese Medicine. Although used as an antimalarial drug, its active compound artemisinin and the semisynthetic derivatives have also been investigated for their anticancer properties, with interesting and promising results. The aims of this research were to evaluate (i) the cytotoxicity and the antiproliferative effect of pure artemisinin and a hydroalcoholic extract obtained from A. annua on the D-17 canine osteosarcoma cell line and (ii) the intracellular iron concentration and its correlation with the cytotoxic effects. Both artemisinin and hydroalcoholic extract induced a cytotoxic effect in a dose-dependent manner. Pure artemisinin caused an increase of cells in the S phase, whereas the hydroalcoholic extract induced an evident increase in the G2/M phase. A significant decrease of iron concentration was measured in D-17 cells treated with pure artemisinin and hydroalcoholic extract compared to untreated cells. In conclusion, although preliminary, the data obtained in this study are indicative of a more potent cytotoxic activity of the hydroalcoholic extract than pure artemisinin, indicating a possible synergistic effect of the phytocomplex and a mechanism of action involving iron and possibly ferroptosis. Considering the similarities between human and canine osteosarcomas, progress in deepening knowledge and improving therapeutic protocols will probably be relevant for both species, in a model of reciprocal translational medicine.

Bats are particularly suited as bioindicators of trace element pollution due to their longevity and their position in the trophic chain. In this study, the concentrations of ten non-essential trace elements (Al, As, Ba, Cd, Hg, Pb, Sb, Sr, Th, Tl) were determined in the tissues (whole body, skin-fur, skinned body, liver, kidney and bone) of lactant Tadarida teniotis from a nursery colony in Rome. A large number of bats from this nursery died before fledging and had bone deformities and fractures. The concentrations of non-essential trace elements in bone and whole body were also analysed in adult specimens of Miniopterus schreibersii from a colony located in a natural park in Northern Italy. In lactant T.teniotis, the Pb concentration decreased in the following order: bone>liver>skinned body>whole body>skin-fur>kidney, and exceeded the toxic threshold associated with negative effects reported for different mammalian species. The levels of the other non-essential trace elements were within a range indicative of low environmental contamination in both species. Significant interspecies differences (P

Metallothioneins (MTs) are ubiquitous low molecular weight cysteine-rich proteins characterized by high affinity for d10 electron configuration metals, including essential (Zn and Cu) and non-essential (Cd and Hg) trace elements. The biological role of these ancient and well-conserved multifunctional proteins has been debated since MTs were first discovered in 1957. Their main hypothesized functions are: (1) homeostasis of Zn and Cu; (2) detoxification of Cd, and Hg; and (3) free radical scavenging. This review will focus on MTs in unconventional animals, those not traditionally studied in veterinary medicine but of increasing interest in this field of research. Living in different environments, these animals represent an incredible source of physiological and biochemical adaptations still partly unexplored. The study of metal-MT interactions is of great interest for clinicians and researchers working in veterinary medicine, food quality and endangered species conservation.

The urine from 35 giraffes was studied by untargeted 1H-NMR, with the purpose of obtaining, for the first time, a fingerprint of its metabolome. The metabolome, as downstream of the transcriptome and proteome, has been considered as the most representative approach to monitor the relationships between animal physiological features and environment. Thirty-nine molecules were unambiguously quantified, able to give information about diet, proteins digestion, energy generation, and gut-microbial co-metabolism. The samples collected allowed study of the effects of age and sex on the giraffe urinary metabolome. In addition, preliminary information about how sampling procedure and pregnancy could affect a giraffe’s urinary metabolome was obtained. Such work could trigger the setting up of methods to non-invasively study the health status of giraffes, which is utterly needed, considering that anesthetic-related complications make their immobilization a very risky practice.

Pollinators in agroecosystems are often exposed to pesticide mixtures. Even at low concentrations, the effects of these mixtures on bee populations are difficult to predict due to potential synergistic interactions. In this paper, we orally exposed newly emerged females of the solitary bee Osmia bicornis to environmentally realistic levels of clothianidin (neonicotinoid insecticide) and propiconazole (fungicide), singly and in combination. The amount of feeding solution consumed was highest in bees exposed to the neonicotinoid, and lowest in bees exposed to the pesticide mixture. Ovary maturation and longevity of bees of the neonicotinoid and the fungicide treatments did not differ from those of control bees. By contrast, bees exposed to the pesticide mixture showed slow ovary maturation and decreased longevity. We found a synergistic interaction between the neonicotinoid and the fungicide on survival probability. We also found an interaction between treatment and emergence time (an indicator of physiological condition) on longevity. Longevity was negatively correlated to physiological condition only in the fungicide and the mixture treatments. Delayed ovary maturation and premature death imply a shortened nesting period (highly correlated to fecundity in Osmia ). Our findings provide a mechanism to explain the observed dynamics of solitary bee populations exposed to multiple chemical residues in agricultural environments.

Objective To assess the agreement between point-of-care and laboratory analysers in measuring biochemical and blood gas analytes in venous samples from tortoises and to define preliminary reference intervals for venous blood gas analysis in Hermann's tortoises (Testudo hermanni). Materials and methods Jugular venous blood samples from 47 Hermann's tortoises underwent paired analysis with a portable gas analyser (i-STAT 1, Abaxis), a portable chemical analyser (VetScan VS2, Abaxis), and with the respective reference analysers. Agarose gel electrophoresis was used to determine albumin concentrations on 12 specimens. Agreement was evaluated with Bland-Altman plots and regression analysis using the Passing-Bablok method. Results Point-of-care analysers had variable agreement with the reference analysers, presenting constant or proportional bias depending on the analyte. Relevant analytes in reptiles, such as ionised and total calcium, had acceptable agreement. The method for determining albumin concentration currently available in both point-of-care and laboratory analysers significantly overestimated albumin concentrations as compared to protein electrophoresis. Clinical significance While the use of POC analysers is extremely advantageous in small animal primary care facilities, agreement between point-of-care and laboratory analysers varies depending on the analyte. For certain analytes, interchangeability of results is limited and specific reference intervals for point-of-care analysers are required. Veterinarians should be aware of the size and the direction of the bias of each analyte.

This study is aimed at investigating the cytotoxicity, anti-inflammatory, and angiogenic activities of two Boswellia serrata extracts on primary culture of porcine aortic endothelial cells (pAECs). Chemical characterization of a dry extract (extract A) and a hydroenzymatic extract (extract G) of B. serrata was performed by HPLC using pure boswellic acids (BAs) as standard. In cultured pAECs, extract G improved cell viability, following LPS challenge, in a dose-dependent manner and did not show any toxic effect. On the other hand, extract A was toxic at higher doses and restored pAEC viability after LPS challenge only at lower doses. Pure BAs, used at the same concentrations as those determined in the phytoextracts, did not contrast LPS-induced cytotoxicity. Extract A showed proangiogenic properties at the lowest dose, and the same result was observed using pure AKBA at the corresponding concentration, whereas extract G did not show any effect on the migration capacity of endothelial cells. In conclusion, an anti-inflammatory activity of B. serrata extracts on endothelial cells was reported, though cytotoxicity or proliferative stimulation can occur instead of a protective effect, depending on the dose and the formulation.

Diabetes mellitus (DM) is a common disorder in middle-aged to older dogs. Treatment options, similar to those for humans, include insulin injection, dietary changes and exercise. Since some diabetic dogs may develop humoral immune responses to exogenous insulin resulting in treatment failure, the use of alternative medicine could represent an interesting new therapeutic strategy for DM in addition to the traditional insulin therapy. The main objective of this report was to evaluate a new therapeutic strategy for DM, based on the association of insulin injections with an orally administered extract of Boswellia serrata to improve glycemic control in a diabetic dog. A nine year old female neutered mixed breed dog diagnosed with DM was treated with increasing doses of a porcine insulin zinc suspension starting from 0.2 U/kg up to 0.6 U/kg q 12 hours. Twenty weeks after the start of insulin therapy the duration of effect and glucose nadir were ideal and clinical symptoms had disappeared but hyperglycemia, although less severe, persisted. Supplementation with a dry extract from the gum- resin of Boswellia serrata was initiated, at a dosage of 15 mg/kg q 12 hours, to improve insulin sensitivity or possible remaining endogenous insulin secretion. B. serrata supplementation led to good glycemic control. No side-effect or adverse reaction were observed during the study. The present case report provides the first evidence in veterinary medicine of a positive effect of dietary supplementation with boswellic acids associated with traditional insulin therapy on glycemic control in a diabetic dog. A major pitfall of the study is the lack of a control. More extensive clinical trials are required to provide definitive evidence of B. serrata efficacy. Key words: diabetes mellitus; dog; boswellic acids; glycemic control  VPLIV DODAJANJA BOSVELIJE ( Boswellia serrata ) OB ZDRAVLJENJU Z INZULINOM NA UREJANJE RAVNI GLUKOZE V KRVI PRI PSU S SLADKORNO BOLEZNIJO Sladkorna bolezen oz. diabetes mellitus (DM) je pogosta motnja pri psih v srednjih letih in starejAiih psih. MoA¾nosti zdravljenja, ki so podobne kot pri ljudeh, vkljuAujejo dodajanje inzulina, spremembe v prehrani in telesno aktivnost. Ker lahko nekateri psi s sladkorno boleznijo razvijejo imunski odziv na dodajanje inzulina, kar lahko vodi do neuspeAinega zdravljenja, lahko uporaba alternativne medicine predstavlja zanimivo novo metodo zdravljenja DM poleg tradicionalnega zdravljenja z inzulinom. Glavni cilj raziskave je bil ovrednotiti novo metodo zdravljenja DM, ki temelji na hkratnem dodajanju inzulina in peroralnem dodajanju izvleAka bosvelije ( Boswellia serrata ) za izboljAianje urejanja ravni glukoze v krvi pri psu s sladkorno boleznijo. Devet let stara sterilizirana samica meAiane pasme, ki je bila diagnosticirana s sladkorno boleznijo, je bila zdravljena z naraAiAajoAimi odmerki suspenzije praAiiAjega inzulina in cinka (od 0,2 E/kg do 0,6 U/kg), ki jih je dobivala vsakih 12 ur. Dvajset tednov po zaAetku zdravljenja z inzulinom so bili uAinki in najniA¾ja vrednost izmerjene glukoze v normalnih mejah vrednosti, kliniAni simptomi so izginili, le hiperglikemija je bila Aie vedno prisotna, vendar pa je bila manj resna. Dodatek suhega izvleAka iz gumijeve smole bosvelije Boswellia serrata v odmerku 15 mg/kg vsakih 12 ur je poveAal obAutljivost na inzulin oz. morebitne preostanke endogenega izloAanja inzulina. Dodatek B. serrata je pozitivno vplival na nadzorovanje sladkorne bolezni. Med zdravljenjem niso bili opaA¾eni stranski ali nezaA¾eleni uAinki. PoroAilo o opisanem primeru vsebuje prve dokaze o pozitivnem uAinku prehranskega dopolnila z bosveliAnimi kislinami v veterinarski medicini, povezanimi z obiAajnim zdravljenjem z inzulinom, pri nadzoru glukoze v krvi pri psu s sladkorno boleznijo. Najbolj neugoden del te Aitudije je odsotnost kontrole. Za zagotovitev dokonAnega dokaza o uAinkovitosti B. serrata so potrebni obseA¾nejAii kliniAni poskusi. KljuAne besede: diabetes mellitus ; pes; bosveliAna kislina; nadzor sladkorne bolezn

The role of trace elements in animal health has attracted increasing interest in recent years. The essentiality and toxicity of these elements have been extensively investigated in humans, laboratory animal models and partially in domestic animals, whereas little is known about trace element metabolism in most living organisms. Forty years ago our research started on Cd metabolism in molluscs, thereafter expanding to Zn, Cu, and Fe metabolism in many unconventional animal species of veterinary interest. This review summarizes the main results obtained over this long period of time: some of the findings are original and have not been published to date. They are discussed in more detail and compared with data obtained in conventional animals, including man.

Boswellia serrata(BS) is an important traditional medicinal plant that currently represents an interesting topic for pharmaceutical research since it possesses several pharmacological properties (e.g., anti-inflammatory, antimicrobial, and antitumour). The safety and versatility of this dietary supplement should allow for its use in numerous pathological conditions; however the quality of the extracts needs to be standardized to increase the clinical success rate resulting from its use. In the present study, different commercially availableB. serrataextracts were employed to compare their AKBA content and in vitro antioxidant power. Furthermore, their ability to modulate the immune system regulatory properties was investigated. Our results showed that the AKBA content varied from3.83±0.10to0.03±0.004%, with one sample in which it was not detectable. The highest antioxidant power and phenolic content were shown by the same extract, which also exhibited the highest AKBA concentration. Finally, the BS extracts showed the ability to influence the regulatory and effector T-cell compartments. Our results suggest that frankincense should be further investigated for its promising potentiality to modulate not only inflammation/oxidative stress but also immune dysregulation, but attention should be paid to the composition of the commercial extracts.

Objectives The aims of this study were to validate a semi-automated high-resolution electrophoretic technique to quantify urinary albumin in healthy and diseased cats, and to evaluate its diagnostic performance in cases of proteinuria and renal diseases. Methods Urine samples were collected from 88 cats (healthy; chronic kidney disease [CKD]; lower urinary tract disease [LUTD]; non-urinary tract diseases [OTHER]). Urine samples were routinely analysed and high-resolution electrophoresis (HRE) was performed. Within-assay and between-assay variability, linearity, accuracy, recovery and the lowest detectable and quantifiable bands were calculated. Receiver operating curve (ROC) analysis was also performed. Results All coefficients of variation were Conclusions and relevance HRE is an accurate and precise method that could be used to measure albuminuria in cats. UAC was useful to correctly classify proteinuria and to discriminate between healthy and diseased cats. HRE might also provide additional information on urine proteins with a profile of all proteins (albumin and globulins) to aid clinicians in the diagnosis of diseases characterised by proteinuria.

Characterisation of the physiologic equine amniotic fluid (AF) proteome is a prerequisite to study its changes during diseases and discover new biomarkers. The aim of this study was to identify by a proteomic approach the most abundant proteins of equine AF. AF samples were collected at parturition from 24 healthy mares that delivered healthy foals. All samples were subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) on 4–12% gels. A pool of the 24 samples, after SDS-PAGE, was cut in 25 slices, trypsin-digested and analysed by mass spectrometry (MS) for protein identification. Mean AF protein concentration was 1.96±1.12g/L. Thirty-four proteins were successfully identified by MS and subsequently categorised according to Gene Ontology (GO). Twelve proteins (e.g. fibronectin, lumican, thrombospondin and fibulin) belonged to or interacted with the extracellular matrix (ECM) playing an important role in the development of foetal tissues. Most of the remaining proteins were classified as transport (e.g. albumin, major allergen Equ c1 and alpha-fetoprotein) delivering nutrients, ions and lipids essential for foetal growth and development. Among these proteins, major allergen Equ c1 is widely studied in human medicine because it induces Ig-E mediated type I allergic reaction. The absence of immunoglobulins in equine AF was also confirmed.

This study was conducted to determine heavy metals concentrations in tissues of 94 birds belonging to different species from coastal areas of Naples and Salerno (Southern Italy) in order to provide baseline data concerning trace element levels in wild birds living in or close to an area characterized by high anthropogenic impact. Additional aim of this study was to verify if diet influenced metal accumulation, so birds were classified as birds of prey, fish eating birds and insectivorous birds. Kidney was the primary organ for Cd accumulation in all groups considered, Pb was accumulated preferentially in bone, whereas Hg showed high values in liver of fish eating birds. Zn showed the highest mean concentrations, while Cu levels were one order of magnitude less. The variance analysis with respect to feeding habits disclosed only a lower accumulation of Hg in insectivores with respect to the other groups. For all metals, the concentrations measured in tissues should be considered indicative of chronic exposure to low, “background” environmental levels and/or to the presence of low bioavailable metals in the environment. In addition, metal levels were not of toxicological concern, being always well below the toxic thresholds defined for each metal.

This study compared the toxicity and accumulation of two different Cu compounds, CuO nanoparticles (NPs) and soluble CuSO4, in erythrocytes and different tissues in rainbow trout (Oncorhynchus mykiss). The crystal structure of CuO NP analysed by XRD indicates that the NP are Tenorite, a monoclinic CuO. The in vitro toxicity results indicate that both Cu compounds increase the haemolysis rate in a dose-dependent way, but the effect was reduced treating cells with CuO NP. Moreover, both Cu compounds induce DNA damage and the entity of the damage, similarly to haemolysis, was more marked in cells treated with CuSO4. In vivo results, obtained after intraperitoneal injection, showed that Cu concentrations were significantly higher in gills (p

Alkaline phosphatase (AP) has been studied in several situations to elucidate its role in reproductive biology of the male from different mammalian species; at present, its role in horse sperm physiology is not clear. The aim of the present work was to measure AP activity in seminal plasma and sperm extracts from freshly ejaculated as well as in frozen-thawed stallion spermatozoa and to verify whether relationship exists between AP activity and sperm quality parameters. Our data on 40 freshly ejaculated samples from 10 different stallions demonstrate that the main source of AP activity is seminal plasma, whereas sperm extracts contribution is very low. In addition, we found that AP activity at physiological pH (7.0) is significantly lower than that observed at pH 8.0, including the optimal AP pH (pH 10.0). Alkaline phosphatase did not exert any effect on sperm-oocyte interaction assessed by heterologous oocyte binding assay. Additionally, we observed a thermal stability of seminal plasma AP, concluding that it is similar to that of bone isoforms. Positive correlations were found between seminal plasma AP activity and sperm concentration, whereas a negative correlation was present between both spermatozoa extracts and seminal plasma AP activity and seminal plasma protein content. A significant decrease in sperm extract AP activity was found in frozen-thawed samples compared with freshly ejaculated ones (n = 21), concomitantly with the decrease in sperm quality parameters. The positive correlation between seminal plasma AP activity measured at pH 10 and viability of frozen-thawed spermatozoa suggests that seminal plasma AP activity could be used as an additional predictive parameter for stallion sperm freezability. In conclusion, we provide some insights into AP activity in both seminal plasma and sperm extracts and describe a decrease in AP after freezing and thawing.

Chronic kidney disease (CKD) is a major cause of mortality in cats, but sensitive and specific biomarkers for early prediction and monitoring of CKD are currently lacking. The present study aimed to apply proteomic techniques to map the urine proteome of the healthy cat and compare it with the proteome of cats with CKD. Urine samples were collected by cystocentesis from 23 healthy young cats and 17 cats with CKD. One-dimensional sodium-dodecyl-sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE) was conducted on 4-12% gels. Two-dimensional electrophoresis (2DE) was applied to pooled urine samples from healthy cats (n = 4) and cats with CKD (n = 4), respectively. Sixteen protein bands and 36 spots were cut, trypsin-digested and identified by mass spectrometry. 1D-SDS-PAGE yielded an overall view of the protein profile and the separation of 32 ± 6 protein bands in the urine of healthy cats, while CKD cats showed significantly fewer bands (P

The relationship between a supposed effect of molluscan extracts on bioluminescent bacteria and metal concentrations in the extracts was investigated. For this purpose a biotoxicological assay based on bioluminescent bacteria (BLB) and extracts from metal exposed molluscs, Scapharca inaequivalvis, was optimized to monitor Cd and Cu marine pollution. Cu and Cd concentrations increased in tissues of experimentally exposed molluscs. Molluscan extracts inhibited the bacterial luminescence, the inhibition decreasing as the time of mollusc exposure to metals increased, suggesting a reduction of the “bioactive” metals. In regard to the use of BLB test in environmental monitoring, the analysis of Cu, Cd, and metallothionein (MT) was first performed in tissues from molluscs collected in three different areas of Northern Adriatic Sea. Metal concentrations reached maximum values in the gills, while Cd was mostly bound to MT in the kidney. Significant differences in metals and MT concentrations were found depending on the sampling sites. The biotoxicological assay resulted slightly correlated with the biochemical parameters.

Summary Alkaline phosphatase (AP) catalyses the detachment of phosphate residues from different substrates. Its activity has been demonstrated in seminal plasma and spermatozoa from porcine and other mammalian species; anyway, the role of AP in male reproduction has not been clarified yet and the aim of this study was to determine AP function in boar sperm capacitation and in vitro fertilization (IVF). AP activity was assayed in seminal plasma and in uncapacitated and in vitro capacitated (IVC) spermatozoa; in addition, capacitation was studied in presence of different doses of AP (1.2 and 2.5 IU/mL). The effect of different doses of AP (1.2 and 2.5 IU/mL) on several sperm parameters after IVC (viability, acrosome integrity with FITC-PSA, capacitation status with CTC staining, tyrosine phosphorylation) and on fertilizing ability during IVF were also evaluated. High AP activity was detected in seminal plasma, in particular in sperm-rich fraction; a lower activity was detected in uncapacitated spermatozoa while a significant decrease was evidenced after IVC. Viability was not changed by AP supplementation of the capacitating medium, whereas acrosome integrity and capacitation status were significantly affected by 1.2 and 2.5 doses, with a dose-dependent decrease in acrosome-reacted cells as well as in CTC B pattern displaying cells. As for sperm head protein phosphorylation, a decrease in relative fluorescence was detected in AP 2.5 group, if compared with capacitated one. After IVF, a dose-dependent decrease in penetrated oocytes was recorded, with an increase in monospermic zygote rate. In conclusion, we demonstrated that AP activity decreases under capacitating condition and that addition of AP to spermatozoa during capacitation results in a depression of the capacitating process and IVF. We can infer that AP plays a role in keeping spermatozoa quiescent until they are ejaculated and in modulating the acquisition of the fertilizing ability.