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Comparative landscape genetics studies provide insights on the impact of landscape elements on gene flow patterns of different species inhabiting the same geographic area. We investigated the population genetic structure of two sympatric ungulates, roe deer Capreolus capreolus and Northern chamois Rupicapra rupicapra, in a mountain area of the central Italian Alps (Trentino, northern Italy). A total of 122 chamois and 72 roe deer samples were genotyped by two species-specific panels of 11 polymorphic microsatellite loci and analyzed by aspatial and spatially explicit analyses. While the roe deer population resulted unstructured, a clear population structure was detected in chamois, with two main groups, one inhabiting the eastern and the other spread in the western part of the study area. Landscape genetics analysis confirmed these scenarios and revealed a different effect of landscape on gene flow. An IBD (Isolation-By-Distance) model best explained genetic variation in roe deer, while IBR (Isolation-By-Resistance) was found as the process underlying genetic variation patterns in chamois, suggesting arable lands, coniferous forests, watercourses, and main roads as potential barriers. Species distribution and landscape use might explain these results: roe deer mostly occupy valley floors relatively connected to each other, and their spatial behavior may promote gene flow across areas. On the other hand, chamois prefer higher elevations and their movements may be hindered by valleys, rivers, and road networks. This study highlights the different impacts of natural and anthropic landscape elements on gene flow in two sympatric species, resulting from their different ecological requirements.

Assessing the behavioural responses of floating wolves to human presence is crucial for investigating the chance of wolf populations expanding into urbanised landscapes. We studied the movement ecology of three rehabilitated wolves in a highly human-dominated landscape (Po Plain, Italy) to explore wolf’s plasticity amid widespread human pressure. To reach this aim, we estimated individual 95% utilisation distributions (UD) after the release and inspected both 95% UDs and net squared displacements to identify individual movement patterns; tested for differences in movement patterns during day and night; and analysed the selection of resting sites during dispersal movement in a highly human-altered environment. Both the 95% UDs and step lengths were smaller for wolves settling in suitable areas than for those settling in more urbanised areas. All wolves exhibited strong temporal segregation with humans during all movement phases, particularly while dispersing across highly urbanised areas. Main roads and proximity to built-up areas were shown to limit wolves’ dispersal, whereas small-wooded patches that provide shelter during rest facilitated long-distance movements. This study provides important insights into wolf movement and settling in urban and peri-urban areas, providing critical knowledge to promote human–carnivore coexistence.

Obesity is a chronic, complex pathology associated with a risk of developing secondary pathologies, including cardiovascular diseases, cancer, type 2 diabetes (T2DM) and musculoskeletal disorders. Since skeletal muscle accounts for more than 70% of total glucose disposal, metabolic alterations are strictly associated with the onset of insulin resistance and T2DM. The present study relies on the proteomic analysis of gastrocnemius muscle from 15 male and 15 female C56BL/J mice fed for 14 weeks with standard, 45% or 60% high-fat diets (HFD) adopting a label-free LC–MS/MS approach followed by bioinformatic pathway analysis. Results indicate changes in males due to HFD, with increased muscular stiffness (Col1a1, Col1a2, Actb), fiber-type switch from slow/oxida-tive to fast/glycolytic (decreased Myh7, Myl2, Myl3 and increased Myh2, Mylpf, Mybpc2, Myl1), increased oxidative stress and mitochondrial dysfunction (decreased respiratory chain complex I and V and increased complex III subunits). At variance, females show few alterations and activation of compensatory mechanisms to counteract the increase of fatty acids. Bioinformatics analysis allows identifying upstream molecules involved in regulating pathways identified at variance in our analysis (Ppargc1a, Pparg, Cpt1b, Clpp, Tp53, Kdm5a, Hif1a). These findings underline the presence of a gender-specific response to be considered when approaching obesity and related comor-bidities.

Mutations in the acidic alpha-glucosidase (GAA) coding gene cause Pompe disease. Late-onset Pompe disease (LOPD) is characterized by progressive proximal and axial muscle weakness and atrophy, causing respiratory failure. Enzyme replacement therapy (ERT), based on recombinant human GAA infusions, is the only available treatment; however, the efficacy of ERT is variable. Here we address the question whether proteins at variance in LOPD muscle of patients before and after 1 year of ERT, compared withhealthy age-matched subjects (CTR), reveal a specific signature. Proteins extracted from skeletal muscle of LOPD patients and CTR were analyzed by combining gel based (two-dimensional difference gel electrophoresis) and label-free (liquid chromatography-mass spectrometry) proteomic approaches, and ingenuity pathway analysis. Upstream regulators targeting autophagy and lysosomal tethering were assessed by immunoblotting. 178 proteins were changed in abundance in LOPD patients, 47 of them recovered normal level after ERT. Defects in oxidative metabolism, muscle contractile protein regulation, cytoskeletal rearrangement, and membrane reorganization persisted. Metabolic changes, ER stress and UPR (unfolded protein response) contribute to muscle proteostasis dysregulation with active membrane remodeling (high levels of LC3BII/LC3BI) and accumulation of p62, suggesting imbalance in the autophagic process. Active lysosome biogenesis characterizes both LOPD PRE and POST, unparalleled by molecules involved in lysosome tethering (VAMP8, SNAP29, STX17, and GORASP2) and BNIP3. In conclusion this study reveals a specific signature that suggests ERT prolongation and molecular targets to ameliorate patient’s outcome.

The crested porcupine (Hystrix cristata) underwent a rapid and widespread range expansion in Italy. Nowadays the species is moving towards the northernmost regions of the country and its occurrence is increasing in the highly anthropized Po Plain. Our objectives were to evaluate the suitability of the Po Plain for the species, as well as to identify dispersal corridors connecting the northern Apennines occurrence areas and the Prealps. We modelled the species home-range scale habitat suitability based on an ensemble modelling approach. Additionally, a habitat suitability prediction carried out at a finer scale was used to parametrize the landscape resistance, based on which we modelled the potential dispersal corridors for the species using a factorial least-cost path approach. The ensemble prediction estimated a potential occurrence of the crested porcupine in 27.4% of the study area. The species occurrence probability was mainly driven by the distribution of extensive cultivations, woodlands and shrublands, and water courses and by the annual mean temperature. Conversely, the movements of the species resulted mainly sustained by woodlands and shrublands and highly hindered by simple arable lands and rice paddies. The connectivity prediction showed that three main dispersal routes are likely to connect crested porcupine occurrence areas in the northern Apennines to currently unoccupied but highly suitable areas in the Prealps. The study allowed us to identify the areas in the Prealps with the highest probability to be colonized by the crested porcupine in the near future and provided important insights for the conservation of a strictly protected species in a human-dominated landscape.

The Italian wolf (Canis lupus italicus) population has remained isolated South of the Alps for the last few thousand years. After a strong decline, the species has recolonized the Apennines and the Western Alps, while it is currently struggling to colonize the Eastern Alps. Recently, the species was detected in a lowland park connecting the Northern Apennines to the Central Alps. If the park was able to sustain a net wolf dispersal flow, this could significantly boost the connection with the Eastern Alps and the Dinaric-Balkan population. We investigated the suitability of the park as a functional ecological corridor for the wolf through the unhospitable lowland of Northern Italy. We collected wolf occurrence data in two study areas. We modeled species distribution running a separate ensemble model for each study area and then merging the output of the models to obtain an integrated suitability map. We used this map to identify corridors for the wolf adopting a factorial least-cost path and a cumulative resistant kernel approach. The connectivity models showed that only two corridors exist in the lowland areas between the Northern Apennines and the Central Alps. The Western corridor is a blind route, while the eastern corridor passes through the park and has a continuous course. However, the models also revealed a scarce resilience of corridor connectivity in the passageways between the park and the Apennines and the Prealps, which suggests that urgent management actions are necessary to ensure the future functionality of this important corridor.

Compared with the rapid expansion across Europe, the golden jackal colonization of Italy is still limited and slow. No study focused on the habitat selection or landscape connectivity for this species was performed in Italy; thus, the potential distribution and dispersal patterns in the country remain unknown. Our objectives were to evaluate the suitability of the Friuli-Venezia Giulia region (north-eastern Italy) for the golden jackal, as well as to identify the ecological corridors connecting the areas currently occupied by the species. Corridors modelling allowed us both to hypothesize the dispersal dynamics occurring in the study region and to identify possible obstacles to future range expansion. We surveyed golden jackal presence in two study areas, covering an area of 500 km2, from March 2017 to February 2018. Using collected data, we modelled the species home-range scale habitat suitability based on an ensemble modelling approach. Subsequently, a habitat suitability prediction at a finer scale was used to estimate landscape resistance, starting from which, we modelled dispersal corridors among areas currently occupied by the species using a factorial least cost path and a cumulative resistant kernel approach. Our results indicated a moderate potential for large parts of the study region to support the occurrence of golden jackal family groups, whose presence seems to be mainly driven by the presence of wide areas covered by broadleaved forests and shrublands and by the absence of wide intensive agricultural areas. The predicted connectivity networks showed that three main permeable corridors are likely to connect golden jackal occurrence areas within the study region, while all the other corridors are characterized by a very low path density. Both the habitat selection and connectivity analyses showed a strong negative impact of the intensive cultivated plain on species stable presence and movement providing critical information for the conservation of

Despite the generally positive trend of European populations, the wolf (Canis lupus) is still today a challenging species to conserve, particularly in the most anthropogenic southern European countries, because of its conflict with humans. In this chapter we summarize the dynamics of wolf distribution in Italy, one of the most densely populated European countries, over the last 50 years. We track changes in the wolf’s diet by comparing its change in Italy with other countries, with the aim of understanding how these changes may have affected the evolution of the human-predator conflict in Italy. In particular, we summarize the results of studies both in Italy and in other European countries to clarify the true impact of wolf predation on both livestock and wild ungulates, which represent the two main causes of predator-human conflict. In order to provide specific insight about the past and the current distribution and feeding habits of the wolf in Italy, and to take stock of the conflict between wolves and humans, we present three case studies. All were carried out over recent decades in northern Italy, i.e. in the area where wolf packs, and particularly their ability to produce dispersing individuals, could affect the future of the entire Italian population. Finally, we consider how to mitigate wolf-human conflict and suggest effective management of wolf populations.

Most studies focused on species coexistence have been directed at the differential use of habitat and food resources; nonetheless, the differential use of the diel cycle may enhance the coexistence of same-sized species. We investigated the activity patterns of mesocarnivores (red fox (Vulpes vulpes), European badger (Meles meles), pine marten (Martes martes), stone marten (M. foina)) in NW Italy via camera-trapping. We hypothesized that the smallest species would tend to avoid competition by selecting time periods when larger species were less active. Foxes, badgers, and stone martens were mainly nocturnal. In lowland areas overlap between coexisting species was generally low, while in Mediterranean habitats all activity patterns tended to be unimodal and overlap was generally high. The pine marten showed a cathemeral pattern. We suggest that the lower ability of the stone marten to avoid interference competition at community-level may play a major role in determining its widespread exclusion from forested areas by the pine marten.

Collagen VI is an extracellular matrix (ECM) protein playing a key role in skeletal muscles and whose deficiency leads to connective tissue diseases in humans and in animal models. However, most studies have been focused on skeletal muscle features. We performed an extensive proteomic profiling in two skeletal muscles (diaphragm and gastrocnemius) of wild-type and collagen VI null (Col6a1−/−) mice at different ages, from 6- (adult) to 12- (aged) month-old to 24 (old) month-old. While in wild-type animals the number of proteins and the level of modification occurring during aging were comparable in the two analyzed muscles, Col6a1−/− mice displayed a number of muscle-type specific variations. In particular, gastrocnemius displayed a limited number of dysregulated proteins in adult mice, while in aged muscles the modifications were more pronounced in terms of number and level. In diaphragm, the differences displayed by 6-month-old Col6a1−/− mice were more pronounced compared to wild-type mice and persisted at 12 months of age. In adult Col6a1−/− mice, the major variations were found in the enzymes belonging to the glycolytic pathway and the tricarboxylic acid (TCA) cycle, as well as in autophagy-related proteins. When compared to wild-type animals Col6a1−/− mice displayed a general metabolic rewiring which was particularly prominent the diaphragm at 6 months of age. Comparison of the proteomic features and the molecular analysis of metabolic and autophagic pathways in adult and aged Col6a1−/− diaphragm indicated that the effects of aging, culminating in lipotoxicity and autophagic impairment, were already present at 6 months of age. Conversely, the effects of aging in Col6a1−/−gastrocnemius were similar but delayed becoming apparent at 12 months of age. A similar metabolic rewiring and autophagic impairment was found in the diaphragm of 24-month-old wild-type mice, confirming that fatty acid synthase (FASN) increment and decreased microtubule-associated proteins 1A/1B

Obesity is a pathological condition caused by genetic and environmental factors, including vitamin D deficiency, which increases the risk of developing cardiovascular disorders and diabetes. This case-control study was designed to verify whether serum profiles could be identified differentiating obese and non-obese Saudis characterized by vitamin D deficiency and pathological levels of triglycerides, high-density lipoprotein cholesterol and high total cholesterol levels. The serum protein profiles of 64 vitamin D deficient (serum 25(OH)D < 50nmol/L) individuals with metabolic syndrome and with (n = 31; BMI ≥ 30) or without (n = 33; BMI < 30) obesity were analyzed by a quantitative label-free mass spectrometry approach (MALDI-profiling), combined with different serum immunodepletion strategies (Human7 and Human14 immuno-chromatographies), to analyze the intermediate- and low-abundant protein components. The analysis of intermediate-abundant proteins (Human7) in obese vs. non-obese subjects identified 14 changed peaks (p < 0.05) in the m/z range 1500-35000. Furthermore, the Human14 depletion provided new profiles related to obesity (121 changed peaks). Among changed peaks, 11 were identified in the m/z range 1500-4000 Da by high-resolution tandem mass spectrometry, belonging to apolipoprotein CIII, apolipoprotein B100, alpha-1-antichymotrypsin and complement C3. Data herein show that distinct protein profiles identify specific peptides belonging to lipid metabolism and inflammation processes that are associated with obesity and vitamin D deficiency.

NEU3 sialidase has been shown to be a key player in many physio- and pathological processes, including cell differentiation, cellular response to hypoxic stress, and carcinogenesis. The enzyme, peculiarly localized on the outer leaflet of the plasma membrane, has been shown to be able to remove sialic acid residues from the gangliosides present on adjacent cells, thus creating cell to cell interactions. Nonetheless, herein we report that the enzyme localization is dynamically regulated between the plasma membrane and the endosomes, where a substantial amount of NEU3 is stored with low enzymatic activity. However, under opportune stimuli,NEU3is shifted from the endosomes to the plasma membrane, where it greatly increases the sialidase activity. Finally, we found that NEU3 possesses also the ability to interact with specific proteins, many of which are different in each cell compartment. They were identified by mass spectrometry, and some selected ones were also confirmed by cross-immunoprecipitation with the enzyme, supporting NEU3 involvement in the cell stress response, protein folding, and intracellular trafficking.

In the skeletal muscle, the ageing process is characterized by a loss of muscle mass and strength, coupled with a decline of mitochondrial function and a decrease of satellite cells. This profile is more pronounced in hindlimb than in forelimb muscles, both in humans and in rodents. Utilizing light and electron microscopy, myosin heavy chain isoform distribution, proteomic analysis by 2D-DIGE, MALDI-TOF MS and quantitative immunoblotting, this study analyzes the protein levels and the nuclear localization of specific molecules, which can contribute to a preferential muscle loss. Our results identify the molecular changes in the hindlimb (gastrocnemius) and forelimb (triceps) muscles during ageing in rats (3- and 22-month-old). Specifically, the oxidative metabolism contributes to tissue homeostasis in triceps, whereas respiratory chain disruption and oxidative-stress-induced damage imbalance the homeostasis in gastrocnemius muscle. High levels of dihydrolipoyllysine-residue acetyltransferase (Dlat) and ATP synthase subunit alpha (Atp5a1) are detected in triceps and gastrocnemius, respectively. Interestingly, in triceps, both molecules are increased in the nucleus in aged rats and are associated to an increased protein acetylation and myoglobin availability. Furthermore, autophagy is retained in triceps whereas an enhanced fusion, decrement of mitophagy and of regenerative potential is observed in aged gastrocnemius muscle.

Sphingolipids have hydrophilic and hydrophobic properties, different saturation and combination of the oligosaccharide chains and mass homology of species located in a narrow m/z region hampering their recognition. To target sphingolipids for diagnostic purposes, standardized methods for lipid extraction, quali- and quantitative assessments are required. In this study, HPTLC-MALDI MS was adopted to establish sphingolipid and glycosphingolipid profiles in muscle, brain and serum to create a database of molecules to be searched in the preclinical and clinical investigations. Specific protocols for lipid extraction were set up based on the characteristics of the tissue or/and fluids; this approach maximizes the HPTLC-MALDI MS analytical throughput both for lipids extracted in organic and aqueous phase. This study indicates that alkaline hydrolysis is necessary for the detection of low abundant species such as Gb3Cer and ceramides in serum and Gb4Cer, CerP and HexCer in muscle tissue. The high hydrophobicity of ceramides has been overcome by the development of HPTLC plate in chloroform:methanol/50:3.5, which increases the number and the intensity of low abundant Cer species. MS/MS analysis has been conducted directly on HPTLC plate allowing the molecular recognition; furthermore a dataset of spectra was acquired to create a database for future profiling of these molecules.

Aging is a universal phenomenon involving the whole body and is characterized by metabolic and physiological decline, leading to cardiovascular defects and heart failure. To characterize the molecular basis of physiological cardiac aging, the proteomic profiles of Sprague Dawley rat hearts of 6, 22 and 30 months were analysed by DIGE and immunoblotting. Results indicate changes in myosin binding protein C, aldehyde dehydrogenase, serpins and sirtuin-3 which protects from the opening of the mitochondrial permeability transition pore induced by cyclophilin D increment. Conversely, an increase of fusion, a decrease of mitochondrial fission and the activation of the non-canonical autophagy pathway were observed. These results support the hypothesis of successful aging in this rat model.

Background: Prostate cancer (PCa) is the second cause of mortality in men worldwide. The prostate-specific antigen (PSA) test is routinely adopted in diagnosis; nevertheless more reliable biomarkers are continuously under investigation by monitoring the release of molecules into the bloodstream. The serum protein profiles appear to provide cancer-specific fingerprints that help to discriminate patients (especially with low PSA level) from controls, improving the performance of existing clinical tests. Methods: Samples from healthy controls and PCa patients with low (≤. 4. ng/mL) and high PSA (>. 4. ng/mL) levels were analyzed by MALDI profiling, and by a multi fractionation approach coupled to ESI-MS for peaks identification. Results: MALDI profiling achieved to detect 10 and 14 changed peaks (p-value <. 0.05), respectively, in PCa patients with low and high PSA versus controls. In particular, a peak identified as C3f fragment, resulted overexpressed in low PSA PCa patients. Conclusions: PSA test, coupled to MALDI profiling, is able to detect changes, specifically related to PCa, in low molecular weight protein range. Furthermore, for the first time in prostate cancer research, the identification and quantification of the small peptide C3f appears to be relevant for the detection of false negatives, providing an additive diagnostic power to PSA (p<. 0.01) and suggesting its use in clinical tests.

Owing to their exposure on the cell surface and the possibility of being directly recognized with specific antibodies, glycosphingolipids have aroused great interest in the field of stem cell biology. In the search for specific markers of the differentiation of human bone marrow mesenchymal stem cells (hBMSCs ) toward osteoblasts, we studied their glycosphingolipid pattern, with particular attention to gangliosides. After lipid extraction and fractionation, gangliosides, metabolically 3H-labeled in the sphingosine moiety, were separated by high-performance TLC and chemically characterized by MALDI MS. Upon induction of osteogenic differentiation, a 3-fold increase of ganglioside GD1a was observed. Therefore, the hypothesis of GD1a involvement in hBMSCs commitment toward the osteogenic phenotype was tested by comparison of the osteogenic propensity of GD1a-highly expressing versus GD1a-low expressing hBMSCs and direct addition of GD1a in the differentiation medium. It was found that either the high expression of GD1a in hBMSCs or the addition of GD1a in the differentiation medium favored osteogenesis, providing a remarkable increase of alkaline phosphatase. It was also observed that ganglioside GD2, although detectable in hBMSCs by immunohistochemistry with an anti-GD2 antibody, could not be recognized by chemical analysis, likely reflecting a case, not uncommon, of molecular mimicry

Glycosphingolipids (GSLs) are a class of ubiquitous lipids characterized by a wide structural repertoire and a variety of functional implications. Importantly, altered levels have been correlated with different diseases, suggesting their crucial role in health. Conventional methods for the characterization and quantification are based on high-performance TLC (HPTLC) separation and comparison with the migration distance of standard samples or on MS. We set up and herein report the application of an ImagePrep method for glycosphingolipids qualitative and quantitative profiling through direct HPTLC-MALDI with particular application to wild-type and NEU3 sialidase-overexpressing C2C12 myoblasts. Lipids were analyzed by HPTLC, coupled with MALDI-TOF, and the resulting GSLs profiles were compared to the [3H]sphingolipids HPTLC patterns obtained after metabolic radiolabeling. GSLs detection by HPTLC-MALDI was optimized by testing different methods for matrix delivery and by performing quantitative analyses using serial dilutions of GSLs standards. Through this approach an accurate analysis of each variant of neutral and acidic GSLs, including the detection of different fatty-acid chain variants for each GSL, was provided and these results demonstrated that HPTLC-MALDI is an easy and high-throughput analytical method for GSLs profiling, suggesting its use for an early detection of markers in different diseases, including cancer and heart ischemia