Alessio Soggiu

Farm animal milk proteomics

Milk is one of the most important nutrients for humans during lifetime. Farm animal milk in all its products like cheese and other fermentation and transformation products is a widespread nutrient for the entire life of humans. Proteins are key molecules of the milk functional component repertoire and their investigation represents a major challenge. Proteins in milk, such as caseins, contribute to the formation of micelles that are different from species to species in dimension and casein-type composition; they are an integral part of the MFGM (Milk Fat Globule Membrane) that has being exhaustively studied in recent years. Milk proteins can act as enzymes or have an antimicrobial activity; they could act as hormones and, last but not least, they have a latent physiological activity encoded in their primary structure that turns active when the protein is cleaved by fermentation or digestion processes. In this review we report the last progress in proteomics, peptidomics and bioinformatics. These new approaches allow us to better characterize the milk proteome of farm animal species, to highlight specific PTMs, the peptidomic profile and even to predict the potential nutraceutical properties of the analyzed proteins.

Proteomics of inflammatory and oxidative stress response in cows with subclinical and clinical mastitis

Cow serum proteome was evaluated by three different complementary approaches in the control group, subclinical and clinical mastitis in order to possibly find differential protein expression useful for a better understanding of the pathophysiology of mastitis as well as for an early diagnosis of the disease. The systemic inflammatory and oxidative stress response in cows with subclinical and clinical mastitis were observed. The collected evidence shows a differential protein expression of serpin A3-1, vitronectin-like protein and complement factor H in subclinical mastitis in comparison with the control. It was also found a differential protein expression of inter-alpha-trypsin inhibitor heavy chain H4, serpin A3-1, C4b-binding protein alpha chain, haptoglobin and apolipoprotein A-I in clinical mastitis compared to the control. Among the inflammatory proteins up-regulated in clinical mastitis, vitronectin is over-expressed in both subclinical and clinical mastitis indicating a strong bacterial infection. This suggests vitronectin as an important mediator in the pathogenesis of the onset of mastitis as well as a valuable marker for diagnosis of the subclinical form of the disease. Obtained data could be useful for the detection of mastitis during the subclinical phase and for a better comprehension of the pathophysiological mechanisms involved in the onset of the disease.

NMDARs Mediate the Role of Monoamine Oxidase A in Pathological Aggression

Converging evidence shows that monoamine oxidase A (MAO A), the key enzyme catalyzing serotonin (5-hydroxytryptamine; 5-HT) and norepinephrine (NE) degradation, is a primary factor in the pathophysiology of antisocial and aggressive behavior. Accordingly, male MAO A-deficient humans and mice exhibit an extreme predisposition to aggressive outbursts in response to stress. As NMDARs regulate the emotional reactivity to social and environmental stimuli, we hypothesized their involvement in the modulation of aggression mediated by MAO A. In comparison with WT male mice, MAO A KO counterparts exhibited increases in 5-HT and NE levels across all brain regions, but no difference in glutamate concentrations and NMDAR binding. Notably, the prefrontal cortex (PFC) of MAO A KO mice exhibited higher expression of NR2A and NR2B, as well as lower levels of glycosylated NR1 subunits. In line with these changes, the current amplitude and decay time of NMDARs in PFC was significantly reduced. Furthermore, the currents of these receptors were hypersensitive to the action of the antagonists of the NMDAR complex (dizocilpine), as well as NR2A (PEAQX) and NR2B (Ro 25-6981) subunits. Notably, systemic administration of these agents selectively countered the enhanced aggression in MAO A KO mice, at doses that did not inherently affect motor activity. Our findings suggest that the role of MAO A in pathological aggression may be mediated by changes in NMDAR subunit composition in the PFC, and point to a critical function of this receptor in the molecular bases of antisocial personality.

Proteomics to investigate fertility in bulls

In dairy cattle breeding, herd reproductive management is the primary focus, affecting a large part of the general costs. A negative association was observed between the level of milk production and fertility. Some studies have shown that a significant percentage of reproductive failure is attributable to semen quality; therefore, if reproduction management is based on artificial insemination, then it is important to assess the fertility level of the sires. In this study, proteomic analysis was used to compare the protein expression profiles from sperm of high- and low-fertility bulls. Comparative proteomic analysis showed that expression of several proteins [nine different two-dimensional electrophoresis (2-DE) spots] is related to fertility level (p ≤ 0.05). These proteins are involved in sperm-egg interactions and cell cycle regulation. Differences in protein expression levels might explain reductions in fertility due to mistakes in sperm-oocyte communication or in cell cycle regulation. Proteomics of sperm can be a valuable tool to identify protein expression changes related to fertility; in particular, 2-DE-based proteome analysis is very useful for the characterization of spermatozoa protein expression related to high- and low-fertility rates. Furthermore, analysis of expression profiles could be critical to the identification of protein biomarkers of bull fertility.

A discovery-phase urine proteomics investigation in type 1 diabetes

Diabetes is a chronic metabolic disease which can lead to serious health problems particularly in and to the development of cardiovascular and renal complications. The aim of this study is to possibly identify distinctive molecular features in urine samples which might correlate to the progression and complications of type 1 diabetes. Diabetic patients with normo- and micro-albuminuria have been analyzed and compared to a group of control subjects. Urine proteins of control and type 1 diabetes subjects were investigated in their proteome profiles, using high-resolution two-dimensional gel electrophoresis separation and protein identifications by MALDI–TOF–MS and LC–MS/MS analysis. Proteomics analysis highlighted differential expression of several proteins between control and type 1 diabetes subjects. In particular, five proteins were found to be down-regulated and four proteins up-regulated. Lower protein representations in diabetic subjects were associated with Tamm–Horsfall urinary glycoprotein, apolipoprotein A-I, apolipoprotein E, α2-thiol proteinase inhibitor, and human complement regulatory protein CD59, while higher protein representations were found for α-1-microglobulin, zinc-α2 glycoprotein, α-1B glycoprotein, and retinol-binding protein 4. These differences were maintained comparing control subjects with type 1 diabetes normo-albuminuric and micro-albuminuric subjects. Furthermore, these proteins are correlated to glycosylated hemoglobin and microalbuminuria, confirming their role in diabetic pathology. This study gives new insights on potential molecular mechanisms associated with the complications of type 1 diabetic disease providing evidences of urine proteins potentially exploitable as putative prognostic biomarkers.

Mechanisms of antibiotic resistance to enrofloxacin in uropathogenic Escherichia coli in dog

Escherichia coli (E. coli) urinary tract infections (UTIs) are becoming a serious problem both for pets and humans (zoonosis) due to the close contact and to the increasing resistance to antibiotics. This study has been performed in order to unravel the mechanism of induced enrofloxacin resistance in canine E. coli isolates that represent a good tool to study this pathology. The isolated E. coli has been induced with enrofloxacin and studied through 2D DIGE and shotgun MS. Discovered differentially expressed proteins are principally involved in antibiotic resistance and linked to oxidative stress response, to DNA protection and to membrane permeability. Moreover, since enrofloxacin is an inhibitor of DNA gyrase, the overexpression of DNA starvation/stationary phase protection protein (Dsp) could be a central point to discover the mechanism of this clone to counteract the effects of enrofloxacin. In parallel, the dramatic decrease of the synthesis of the outer membrane protein W, which represents one of the main gates for enrofloxacin entrance, could explain additional mechanism of E. coli defense against this antibiotic. All 2D DIGE and MS data have been deposited into the ProteomeXchange Consortium with identifier PXD002000 and DOI http://dx.doi.org/10.6019/PXD002000. This article is part of a Special Issue entitled: HUPO 2014.

Differential protein profile in sexed bovine semen: shotgun proteomics investigation

The preparation of sexed semen is based on the differential DNA content between the X and Y chromosome bearing sperm cells determined by fluorescence-activated cell sorting. In spite of its intrinsic limitations this represents the only effective method. However, the employment of sexed sperm for breeding food producing animals on a large scale requires additional knowledge in the protein repertoire for the development of improved methods to differentiate X and Y sperm cells maintaining high vitality. In order to address this issue, we performed a comparative shotgun proteomic investigation by nUPLC-MS/MS to characterize sexed bovine semen. The protein profiles of these two types of sperm cells have shown differential expression of proteins that may be directly associated with the main components of cytoskeletal structures of flagellum, as the axoneme, outer dense fibers and fibrous sheath, as well as glycolytic enzymes and calmodulin, involved in the energetic metabolism regulation. Overall these results may provide a base to a better comprehension of the biological features of sperm cells and may be useful to the development of alternative methods of separation.

Gating deficits in isolation‐reared rats are correlated with alterations in protein expression in nucleus accumbens

The isolation‐rearing (IR) paradigm, consisting of the social deprivation for 6–9 weeks after weaning, induces a spectrum of aberrant behaviors in adult rats. Some of these alterations such as sensorimotor gating deficits are reminiscent of the dysfunctions observed in schizophrenia patients. Although gating impairments in IR rats have been linked to impairments in the cortico‐mesolimbic system, the specific molecular mechanisms underlying this relation are unclear. To elucidate the neurochemical modifications underlying the gating disturbances exhibited by IR rats, we compared their pre‐pulse inhibition (PPI) of the acoustic startle reflex with that of socially reared (SR) controls, and correlated this index to the results of proteomic analyses in prefrontal cortex and nucleus accumbens from both groups. As expected, IR rats exhibited significantly lower startle amplitude and PPI than their SR counterparts. Following behavioral testing, IR and SR rats were killed and protein expression profiles of their brain regions were examined using two‐dimensional electrophoresis based proteomics. Image analysis in the Coomassie blue‐stained gel revealed that three protein spots were differentially expressed in the nucleus accumbens of IR and SR rats. Mass spectrometry (matrix‐assisted laser desorption ionization‐time of flight and MS/MS) identified these spots as heat shock protein 60 (HSP60), α‐synuclein (α‐syn), and 14‐3‐3 protein ζ/δ. While accumbal levels of HSP60 was decreased in IR rats, α‐syn and 14‐3‐3 proteins were significantly increased in IR in comparison with SR controls. Notably, these two last alterations were significantly correlated with different loudness intensity‐specific PPI deficits in IR rats. In view of the role of these proteins in synaptic trafficking and dopaminergic regulation, these findings might provide a neurochemical foundation for the gating alterations and psychotic‐like behaviors in IR rats.

Proteomics in food: Quality, safety, microbes, and allergens.

Food safety and quality and their associated risks pose a major concern worldwide regarding not only the relative economical losses but also the potential danger to consumers health. Customers confidence in the integrity of the food supply could be hampered by inappropriate food safety measures. A lack of measures and reliable assays to evaluate and maintain a good control of food characteristics may affect the food industry economy and shatter consumer confidence. It is imperative to create and to establish fast and reliable analytical methods that allow a good and rapid analysis of food products during the whole food chain. Proteomics can represent a powerful tool to address this issue, due to its proven excellent quantitative and qualitative drawbacks in protein analysis. This review illustrates the applications of proteomics in the past few years in food science focusing on food of animal origin with some brief hints on other types. Aim of this review is to highlight the importance of this science as a valuable tool to assess food quality and safety. Emphasis is also posed in food processing, allergies, and possible contaminants like bacteria, fungi, and other pathogens.

Propofol protects against opioid-induced hyperresponsiveness of airway smooth muscle in a horse model of target-controlled infusion anaesthesia

General anaesthesia in horses is associated with elevated mortality rate in subjects suffering of heaves. Target-controlled infusion (TCI) of sedative-hypnotic medications and opioids represents a total intravenous anaesthesia (TIVA) method validated in veterinary medicine. Since there are no data concerning the impact of these classes of drugs in inducing bronchial hyperresponsiveness (BHR) in horses, the aim of this study was to investigate the effect propofol and remifentanil on the contractile response of equine airway smooth muscle. The influence of propofol and remifentanil on the contractile response of equine isolated bronchi to electrical field stimulation (EFS) was assessed. The role of capsaicin-sensitive sensory nerves, inducible nitric oxide synthase (iNOS) and neurokinin 2 (NK2) receptor was also assessed. The interaction analysis was performed by Bliss Independence theory. Experiments were repeated in desensitized and passively sensitized airways. Remifentanil induced BHR in both non-sensitized and passively sensitized bronchi, (+56.33±8.01% and +99.10±14.52%, respectively; P<0.01 vs. control) and propofol significantly prevented this effect (P>0.05 vs. remifentanil). The inactivation of capsaicin-sensitive sensory nerves via desensitization and blocking NK2 receptor inhibited the BHR remifentanil-induced (P>0.05 vs. controls). The inhibition of iNOS reverted the protective effect of propofol on the BHR induced by remifentanil (non-sensitized: +47.11±7.70%; passively sensitized: +70.51±11.39%; P<0.05 vs. control). Propofol synergistically interacted (overall ≈40%) in preventing the remifentanil-induced BHR. Remifentanil induces BHR via stimulating capsaicin-sensitive sensory nerves that facilitate the cholinergic neurotransmission through the activation of NK2 receptor. The propofol/remifentanil combination may be safely administered in course of TCI-TIVA procedures also in heaves affected horses.