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Abstract Mesenchymal stem cells (MSCs) interact with their surroundings via integrins, which link to the actin cytoskeleton and translate physical cues into biochemical signals through mechanotransduction. N-cadherins enable cell-cell communication and are also linked to the cytoskeleton. This crosstalk between integrins and cadherins modulates MSC mechanotransduction and fate. Here we show the role of this crosstalk in the mechanosensing of viscosity using supported lipid bilayers as substrates of varying viscosity. We functionalize these lipid bilayers with adhesion peptides for integrins (RGD) and N-cadherins (HAVDI), to demonstrate that integrins and cadherins compete for the actin cytoskeleton, leading to an altered MSC mechanosensing response. This response is characterised by a weaker integrin adhesion to the environment when cadherin ligation occurs. We model this competition via a modified molecular clutch model, which drives the integrin/cadherin crosstalk in response to surface viscosity, ultimately controlling MSC lineage commitment.

Summary: Background: Genetic variants in COL4A1 and COL4A2 (encoding collagen IV alpha chain 1/2) occur in genetic and sporadic forms of cerebral small vessel disease (CSVD), a leading cause of stroke, dementia and intracerebral haemorrhage (ICH). However, the molecular mechanisms of CSVD with ICH and COL4A1/COL4A2 variants remain obscure. Methods: Vascular function and molecular investigations in mice with a Col4a1 missense mutation and heterozygous Col4a2 knock-out mice were combined with analysis of human brain endothelial cells harboring COL4A1/COL4A2 mutations, and brain tissue of patients with sporadic CSVD with ICH. Findings: Col4a1 missense mutations cause early-onset CSVD independent of hypertension, with enhanced vasodilation of small arteries due to endothelial dysfunction, vascular wall thickening and reduced stiffness. Mechanistically, the early-onset dysregulated endothelium-dependent hyperpolarization (EDH) is due to reduced collagen IV levels with elevated activity and levels of endothelial Ca2+-sensitive K+ channels. This results in vasodilation via the Na/K pump in vascular smooth muscle cells. Our data support this endothelial dysfunction preceding development of CSVD-associated ICH is due to increased cytoplasmic Ca2+ levels in endothelial cells. Moreover, cerebral blood vessels of patients with sporadic CSVD show genotype-dependent mechanisms with wall thickening and lower collagen IV levels in those harboring common non-coding COL4A1/COL4A2 risk alleles. Interpretation: COL4A1/COL4A2 variants act in genetic and sporadic CSVD with ICH via dysregulated EDH, and altered vascular wall thickness and biomechanics due to lower collagen IV levels and/or mutant collagen IV secretion. These data highlight EDH and collagen IV levels as potential treatment targets. Funding: MRC, Wellcome Trust, BHF.

The extracellular matrix (ECM) is a complex fibrillar network that couples a cell with its environment and directly regulates cells’ functions via structural, mechanical, and biochemical signals. The goal of this study was to engineer and characterize ECM-mimicking protein platforms with material properties covering both physiological and pathological (tumorous) tissues. We designed and fabricated three-dimensional (3D) fibrillar scaffolds comprising the two major components of the ECM, namely collagen (Col) and fibronectin (Fn), using a previously developed freeze-drying method. While scaffolds porous architecture and mechanics were controlled by varying Col I concentration, Fn deposition and conformation were tuned using varied immersion temperature and assessed via intramolecular Förster Resonance Energy Transfer (FRET). Our data indicate that all scaffolds were able to support various crucial cellular functions such as adhesion, proliferation and matrix deposition. Additionally, we show that, keeping the stiffness constant and tuning the conformation of the Fn layer used to coat the Col scaffolds, we were able to control not only the invasion of cells but also the conformation of the matrix they would deposit, from a compact to an unfolded structure (as observed in the breast tumor microenvironment). Therefore, these tunable scaffolds could be used as 3D cell culture models, in which ECM microarchitecture, mechanics and protein conformation are controlled over large volumes to investigate long-term mechanisms such as wound healing phases and/or vascularization mechanisms in both physiological and pathological (tumorous) microenvironments. These findings have implications for tissue engineering and regenerative medicine.

Bone marrow and adipose tissue human mesenchymal stem cells were seeded in highly performing 3D gelatin–chitosan hybrid hydrogels of varying chitosan content in the presence of human platelet lysate and evaluated for their proliferation and osteogenic differentiation. Both bone marrow and adipose tissue human mesenchymal stem cells in gelatin–chitosan hybrid hydrogel 1 (chitosan content 8.1%) or gelatin–chitosan hybrid hydrogel 2 (chitosan 14.9%) showed high levels of viability (80%–90%), and their proliferation and osteogenic differentiation was significantly higher with human platelet lysate compared to fetal bovine serum, particularly in gelatin–chitosan hybrid hydrogel 1. Mineralization was detected early, after 21 days of culture, when human platelet lysate was used in the presence of osteogenic stimuli. Proteomic characterization of human platelet lysate highlighted 59 proteins mainly involved in functions related to cell adhesion, cellular repairing mechanisms, and regulation of cell differentiation. In conclusion, the combination of our gelatin–chitosan hybrid hydrogels with hPL represents a promising strategy for bone regenerative medicine using human mesenchymal stem cells.

Polylaminin (polyLM) is a non-covalent acid-induced nano- and micro-structured polymer of the protein laminin displaying distinguished biological properties. Polylaminin stimulates neuritogenesis beyond the levels achieved by ordinary laminin and has been shown to promote axonal regeneration in animal models of spinal cord injury. Here we used confocal fluorescence microscopy (CFM), scanning electron microscopy (SEM) and atomic force microscopy (AFM) to characterize its three-dimensional structure. Renderization of confocal optical slices of immunostained polyLM revealed the aspect of a loose flocculated meshwork, which was homogeneously stained by the antibody. On the other hand, an ordinary matrix obtained upon adsorption of laminin in neutral pH (LM) was constituted of bulky protein aggregates whose interior was not accessible to the same anti-laminin antibody. SEM and AFM analyses revealed that the seed unit of polyLM was a flat polygon formed in solution whereas the seed structure of LM was highly heterogeneous, intercalating rod-like, spherical and thin spread lamellar deposits. As polyLM was visualized at progressively increasing magnifications, we observed that the morphology of the polymer was alike independently of the magnification used for the observation. A search for the Hausdorff dimension in images of the two matrices showed that polyLM, but not LM, presented fractal dimensions of 1.55, 1.62 and 1.70 after 1, 8 and 12 hours of adsorption, respectively. Data in the present work suggest that the intrinsic fractal nature of polymerized laminin can be the structural basis for the fractal-like organization of basement membranes in the neurogenic niches of the central nervous system.

Perfusion culture systems are widely used in tissue engineering applications for enhancing cell culture viability in the core of three-dimensional scaffolds. In this work, we present a multichamber confined-flow perfusion system, designed to provide a straightforward platform for three-dimensional dynamic cell cultures. The device comprises 6 culture chambers allowing independent and simultaneous experiments in controlled conditions. Each chamber consists of three parts: a housing, a deformable scaffold-holder cartridge, and a 7 mL reservoir, which couples water-tightly with the housing compressing the cartridge. Short-term dynamic cell seeding experiments were carried out with MC3T3-E1 cells seeded into polycaprolactone porous scaffolds. Preliminary results revealed that the application of flow perfusion through the scaffold favored the penetration of the cells to its interior, producing a more homogeneous distribution of cells with respect to dropwise or injection seeding methods. The culture chamber layout was conceived with the aim of simplifying the user operations under laminar flow hood and minimizing the risks for contamination during handling and operation. Furthermore, a compact size, a small number of components, and the use of bayonet couplings ensured a simple, fast, and sterility-promoting assembling. Finally, preliminary in vitro tests proved the efficacy of the system in enhancing cell seeding efficiency, opening the way for further studies addressing long-term scaffold colonization.

A multispecies GIS-based approach has been used to evaluate species richness from potential distribution maps, calculated for 58 bird species present in the study area. Species-level predictions agree with the recent Varese Province Bird Georeferenced Atlas data, not used in modeling, whereas biodiversity hotspots identified allowed a first evaluation of the efficiency of Natura 2000 sites in the area.

Abstract The following study contains and analyses data about the wild boar (Sus scrofa) hunted in the Como and Varese provinces from October 1993 to January 1994. These populations have been formed from individuals which have been illegally introduced continuously since 1978 in the Varese province and since the mid 80s in the Como province. Some of them have also come from the neighbouring Switzerland. Biometric analyses were carried out on 189 specimens, and were divided into age and sex classes. We considered 8 measurements for some external morphological characteristics and 28 for the skull structure. For each specimen the age class was evaluated by tooth growth analysis. The possible presence of fetuses in every female was also checked. The data collected by the Veterinary Service (U.S.S.L.) enabled us to identify the main diseases of the studied populations. Frequent cases of tuberculosis were observed.

Abstract The environment found in the upper and lower Padane Plain and the adjoining hills isn't very homogeneous. In fact it is impossible to find biotopes extended enough to satisfy the necessary criteria for density estimate of small mammals based on the Removal method. This limitation has been partially overcome by adopting a reduced grid, counting 39 traps whose spacing depends on the studied species. Aim of this work was to verify - and eventually measure - the efficiency of a sampling method based on a "reduced" number of catch points. The efficiency of 18 trapping cycles, realized from 1991 to 1993, was evaluated as percent bias. For each of the trapping cycles, 100 computer simulations were performed, so obtaining a Monte-Carlo estimate of bias in density values. Then later, the efficiency of different trap arrangements was examined by varying the criteria. The numbers of traps ranged from 9 to 49, with trap spacing varying from 5 to 15 m and a trapping period duration from 5 to 9 nights. In this way an optimal grid system was found both for dimensions and time duration. The simulation processes involved, as a whole, 1511 different grid types, for 11347 virtual trapping cycles. Our results indicate that density estimates based on "reduced" grids are affected by an average -16% bias, that is an underestimate, and that an optimally sized grid must consist of 6x6 traps square, with about 8.7 m spacing. and be in operation for 7 nights.

Abstract The Alpine Marmot (Marmota marmota marmota) is widespread all over the eastern side of Como's province (NW Italy), with the exception of the Grigne massif, Mount Resegone and Mount Due Mani. These are three geographically isolated mountain ranges. For this reason the Faunal Service of the Provincial Administration of Como has undertaken a project aimed at releasing individuals in the Grigne area. In this early stage a suitability analysis was performed, using a Geographical Information System (GIS). Field data were collected concerning colonies distribution and spatial structure, inside a sample area where stable populations are present. This area is quite similar to the release area, and covers the whole part of Prealpi Comasche between Sponda Biandino and the Artavaggio plains. Using GIS data analysis techniques a base thematic cartography was set up, which was based on informational strata such as altitude. slope and aspect. This information helped us to calculate optimum values of environmental ranges to look for within the release area. Finally, GIS siting operations highlighted zones suitable for releasing marmots. The use of a GIS instrument helped in the definition of colonisation-suitable sites on the basis of several parameters, optimising the costs-benefits ratio with the further advantage of being able to keep the topographical database up to date.

Abstract This paper points out the relationships occurring between small mammal populations studied in some areas of the central-Western italian Alpine Arch, and some environmental parameters that characterize the Alpine habitat. The fauna1 data were collected with traps randomly placed except for Alpe Devero, where a trapping grid has been utilized.

Abstract 189 bottles and 6 cans, containing the remains of 904 small mammals were collected in alpine and pre-alpine areas of Piedmont, Aosta Valley and Lombardy. The following taxa were recorded: Sorex alpinus, Sorex araneus, Sorex minutus, Neomys fodiens, Crocidura leucodon, Crocidura suaveolens, Eliomys quercinus, Clethrionomys glareolus, Microtus arvalis, Microtus multiplex/subterraneus, Microtus savii, Chionomys nivalis, Apodemus spp., Mus domesticus, Soricidae was the best represented family in the sample (73.5% of the specimens found), with a different occurrence of Crocidurinae and Soricinae in xerothermic and non-xerothermic areas.

Abstract With reference to the literature and original data, a description is given of the present distribution and status of the Dormouse species found in Italy (Dryomys nitedula, Eliomys quercinus, Muscardinus avellanarius and Myoxus glis). Possible causes of reduction in numbers and distribution of the four Dormouse species are also discussed. Riassunto Distribuzione e conservazione dei Mioxidi in Italia - È riportata su dati bibliografici ed originali la distribuzione del Driomio (Dryomys nitedula), del Quercino (Eliomys quercinus), del Moscardino (Muscardinus avellanarius), e del Ghiro (Myoxus glis) in Italia. Inoltre vengono sinteticamente discusse, per ogni specie, le possibili cause di rarefazione e riduzione degli areali delle popolazioni.

Abstract The diet of the fox was investigated by analysis of 273 scats, collected along standard trails from April to November 1987 and 1988. Food habits of foxes were described for three altitudinal ranges. Mammals, mainly Clethrionomys glareolus and Microtus multiplex, were the staple food (percentage of frequency 42.8%), followed by fruits and other vegetables (26.7% and 37.3% respectively). Birds, Invertebrates (mainly Insects) and garbage were little eaten. The game species (ungulates, hares, pheasants) occurred with a low frequency (8.4%) in the diet. The trophic niche breadth varied little through the altitudinal ranges and the seasons. The trophic niche overlap between the fox and the genus Martes (190 scats of M. martes and M. foina were examined) is relatively wide (O=0.868). Riassunto La dieta della Volpe (Vulpes vulpes) in aree boscate delle Alpi Orobie (Val Lesina) è stata indagata nel periodo aprile-novembre 1987 e 1988 mediante l'analisi di 273 feci, raccolte lungo percorsi-campione ricadenti in tre piani vegetazionali. I Mammiferi, in particolare Clethrionomys glareolus e Microtus multiplex, sono la componente principale della dieta (frequenza percentuale 42,8%). Rilevante è anche il consumo di frutti (soprattutto in estate e autunno) e di altri vegetali (26,7% e 37,3% rispettivamente), mentre poco frequente è quello di Uccelli, Invertebrati e rifiuti. Complessivamente ridotta è l'azione predatoria della Volpe nei confronti delle specie di interesse venatorio (Ungulati, lepri, Galliformi). L'ampiezza della nicchia trofica mostra modeste variazioni stagionali e altitudinali. I1 grado di sovrapposizione tra la nicchia trofica della Volpe e quella del genere Martes, quest'ultima ricavata dall'analisi di 190 feci di Martora (M. martes) e Faina (M. foina), è elevato (O=0,868). Tuttavia, poiché in condizioni di simpatria le due specie di Mustelidi tendono a ridurre la competizione interspecifica differenziando le relative nicchie trofiche, tale sovrapposizione può essere solo apparente.