Activity budget and movement patterns of Brown Swiss and Alpine Grey lactating cows during summer grazing in alpine pastures

We used GPS tracking to monitor the grazing patterns of Brown Swiss and Alpine grey lactating cows on an alpine summer pasture (2038 m a.s.l.; SD = 146) in the Dolomites. The pasture (171 ha) was managed with a continuous grazing system (0.52 LU/ha) with morning and evening milking in the barn, guided grazing during the ‘day’, and free grazing at ‘night’. GPS positions were collected from 8 Brown Swiss multiparous and 9 Alpine Grey (4 primiparous and 5 multiparous) cows every two minutes. We inferred behaviours (grazing, resting, walking) from movement metrics, activity sensors and direct behavioural observations. After excluding milking periods, the cows grazed for 8 h/d, rested 10–11 h/d, and walked for 1.5/d. Grazing extended into late evening after milking, and resting prevailed throughout the ‘night’ until the morning milking. When grazing and resting, cows mainly used grasslands as the preferred habitat, but forest and sparse shrub were also used remarkably without consistent negative or positive selection. The pasture use was highly heterogeneous, with higher animal loads close to the barn, especially at night, and in areas with gentler slopes. Alpine Grey primiparous cows were less limited by slope and distance from the barn in their movement but were more selective in habitat use than multiparous cows. Differences between multiparous cows of the two breeds were less marked. Further studies should help understand the internal and external drivers of cattle grazing patterns to devise management practices combining animals’ productivity and welfare with the conservation of the grassland ecosystem services

Folate-Functionalization Enhances Cytotoxicity of Multivalent DNA Nanocages on Triple-Negative Breast Cancer Cells

DNA is an excellent programmable polymer for the generation of self-assembled multivalent nanostructures useful for biomedical applications. Herein, we developed (i) folate-functionalized nanocages (Fol-NC), very efficiently internalized by tumor cells overexpressing the alpha isoform of the folate receptor; (ii) AS1411-linked nanocages (Apt-NC), internalized through nucleolin, a protein overexpressed in the cell surface of many types of cancers; and (iii) nanostructures that harbor both folate and AS1411 aptamer functionalization (Fol-Apt-NC). We analyzed the specific miRNA silencing activity of all types of nanostructures harboring miRNA sequestering sequences complementary to miR-21 and the cytotoxic effect when loaded with doxorubicin in a drug-resistant triple-negative breast cancer cell line. We demonstrate that the presence of folate as a targeting ligand increases the efficiency in miR-21 silencing compared to nanocages functionalized with AS1411. Double-functionalized nanocages (Fol-Apt-NC), loaded with doxorubicin, resulted in an increase of over 51% of the cytotoxic effect on MDA-MB-231 cells compared to free doxorubicin, demonstrating, besides selectivity, the ability of nanocages to overcome Dox chemoresistance. The higher efficiency of the folate-functionalized nanocages is due to the way of entrance, which induces more than four times higher intracellular stability and indicates that the folate-mediated route of cell entry is more efficient than the nucleolin-mediated one when both folate and AS1411 modifications are present

Membrane cholesterol modulates LOX-1 shedding in endothelial cells

The lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a scavenger receptor responsible for ox-LDL recognition, binding and internalization, which is up-regulated during atherogenesis. Its activation triggers endothelium dysfunction and induces inflammation. A soluble form of LOX-1 has been identified in the human blood and its presence considered a biomarker of cardiovascular diseases. We recently showed that cholesterol-lowering drugs inhibit ox-LDL binding and internalization, rescuing the ox-LDL induced apoptotic phenotype in primary endothelial cells. Here we have investigated the molecular bases of human LOX-1 shedding by metalloproteinases and the role of cell membrane cholesterol on the regulation of this event by modulating its level with MβCD and statins. We report that membrane cholesterol affects the release of different forms of LOX-1 in cells transiently and stably expressing human LOX-1 and in a human endothelial cell line (EA.hy926). In particular, our data show that i) cholesterol depletion triggers the release of LOX-1 in exosomes as a full-length transmembrane isoform and as a truncated ectodomain soluble fragment (sLOX-1); ii) endothelial cells secrete a soluble metalloproteinase which induces LOX-1 ectodomain shedding and iii) long term statins treatment enhances sLOX-1 proteolytic shedding

Rational Design of Antiangiogenic Helical Oligopeptides Targeting the Vascular Endothelial Growth Factor Receptors

Tumor angiogenesis, essential for cancer development, is regulated mainly by vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs), which are overexpressed in cancer cells. Therefore, the VEGF/VEGFR interaction represents a promising pharmaceutical target to fight cancer progression. The VEGF surface interacting with VEGFRs comprises a short α-helix. In this work, helical oligopeptides mimicking the VEGF-C helix were rationally designed based on structural analyses and computational studies. The helical conformation was stabilized by optimizing intramolecular interactions and by introducing helix-inducing Cα,α-disubstituted amino acids. The conformational features of the synthetic peptides were characterized by circular dichroism and nuclear magnetic resonance, and their receptor binding properties and antiangiogenic activity were determined. The best hits exhibited antiangiogenic activity in vitro at nanomolar concentrations and were resistant to proteolytic degradation

Human choices, slope, and vegetation productivity determine pattern of traditional Alpine summer grazing

Grazing patterns in extensive pastures influence both animal productivity and conservation of grasslands ecosystem services. This study used Global Positioning Systems (GPS) tracking to describe the daily movement patterns and habitat selection of Simmental and Alpine grey lactating cows in the summer pastures of \u2018Malga Ombretta\u2019, located at 1900 m a.s.l. in the Dolomites, eastern Italian Alps (46.424549; 11.880871). During the study period (5 July\u20135 August 2018), the pastures (approximately 35 ha) were grazed by 14 Simmental and 7 Alpine grey cows (stocking rate =0.6 LU/ha). Each morning (8.00\u20139.00 am) the farmer-led the cows to graze in a different section of the pasture until 12.30\u20131.00 p.m. when cows were left free until they returned spontaneously to the barn (5.30\u20136.30 p.m), where they spent the night. Nine Simmental and 4 Alpine grey cows were equipped with GPS collars collecting one position/minute. Recorded positions were edited to exclude the in-barn night periods (final database =174,208 records) and used to describe grazing patterns at two temporal scales. At the daily scale, total distances walked varied from 2.0 to 8.9 km (mean: 4.7) across dates and increased with longer daily outdoor periods, reflecting the farmer\u2019s choices of when/where to move the herd, but showed also a remarkable variation across individual cows, possibly because of individual features. No differences were found between breeds. At the step scale (i.e. for each interval between subsequent locations), slopes and altitudes used were higher, and walking speed was lower, in morning hours when cows were guided by the farmer than in afternoon hours when they were free. Slopes steeper than 30\ub0 were very seldom used, possibly indicating a threshold that cows are unwilling to trespass. Alpine grey cows used slightly higher slopes and altitudes and moved slightly faster than Simmental cows in the afternoon, suggesting a better adaption to difficult terrain conditions. Finally, in morning hours cows of both breeds used grassland patches with lower vegetation abundance, as indicated by the satellite-derived Normalized Difference Vegetation Index, than in afternoon hours. In this study, using high-frequency GPS tracking outlined the great variability of grazing patterns, and helped to understand how they can be influenced by human (farmer\u2019s decisions) and animal (breed, but also individual) choices

Ligand binding free-energy calculations with funnel metadynamics

The accurate resolution of the binding mechanism of a ligand to its molecular target is fundamental to develop a successful drug design campaign. Free-energy calculations, which provide the energy value of the ligand–protein binding complex, are essential for resolving the binding mode of the ligand. The accuracy of free-energy calculation methods is counteracted by their poor user-friendliness, which hampers their broad application. Here we present the Funnel-Metadynamics Advanced Protocol (FMAP), which is a flexible and user-friendly graphical user interface (GUI)-based protocol to perform funnel metadynamics, a binding free-energy method that employs a funnel-shape restraint potential to reveal the ligand binding mode and accurately calculate the absolute ligand–protein binding free energy. FMAP guides the user through all phases of the free-energy calculation process, from preparation of the input files, to production simulation, to analysis of the results. FMAP delivers the ligand binding mode and the absolute protein–ligand binding free energy as outputs. Alternative binding modes and the role of waters are also elucidated, providing a detailed description of the ligand binding mechanism. The entire protocol on the paradigmatic system benzamidine–trypsin, composed of ~105 k atoms, took ~2.8 d using the Cray XC50 piz Daint cluster at the Swiss National Supercomputing Centre

Cholesterol level regulates lectin-like oxidized low-density lipoprotein receptor-1 function

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), the primary receptor for ox-LDL in endothelial cells, is a multi-ligand scavenger receptor that plays a crucial role in the pathogenesis of atherosclerosis and cardiovascular disorders and recently identified as a tumor marker. LOX-1 is naturally present in caveolae/lipid rafts in plasma membranes and disruption of these membrane domains by cholesterol-lowering drugs leads to a spatial disorganization of LOX-1 and a marked loss of specific LOX-1 function in terms of ox-LDL binding and internalization. Moreover, cholesterol depletion triggers the release of LOX-1 in exosomes and enhances shedding of LOX-1 ectodomain. We here provide an overview of the involvement of membrane and circulating cholesterol in LOX-1 function and shedding and its impact on cardiovascular pathologies and cancer. In particular, we consider the available biological and molecular evidence indicating LOX-1 as a potential therapeutic target for atherosclerosis, inflammation processes, myocardial infarction and cholesterol-lowering drugs as specific inhibitors of LOX-1 function

GPS tracking indicates high variability in grazing patterns of lactating cows in Alpine summer farms

Understanding grazing patterns is necessary to manage semi-natural grasslands for both productivity and conservation of biodiversity with the associated Ecosystem Services. This study used GPS tracking to characterize the daily movement patterns and use of Alpine pastures by lactating Brown Swiss cows in three summer farms located at an average elevation of 1,900 m a.s.l. (SD: 90) in the eastern Italian Alps. Herd management was similar for all summer farms. Each day, after the morning milking in the barn, cows were driven to graze in different areas of the pastures, where they were then left free until the return to the barn for the evening milking, after which they were again released free to spend the night outdoor. From July 5th to September 5th in 2019 and 2020, 12 and 10 lactating cows per each farm, respectively, were equipped with GPS \u2018collars\u2019 scheduled to collect a position every 2 minutes. After editing data to exclude individual milking periods in the barns and outlier positions, the total daily distances walked by cows were on average close to 7 km but ranged from less than 2 km to around 15 km, varying greatly between dates, but also farms and years, mostly in relation the farmers daily decisions for grazing areas. Slopes steeper than 30\ub0 were very seldom used, most likely reflecting the cows choices of single movement steps. The total surface used by the cows of each summer farm (average: 211 ha; SD: 29 ha) varied by 4 to 30% between years, because of pasture patches being used only in one year. Internal use of such surface was highly heterogeneous: the quartile of surface with the lowest locations density contained <1% of total locations, while that with the highest density contained around 80% of the locations and was concentrated in the proximity of the barns. Use of high-frequency GPS tracking outlined the great variability of movement patterns and pasture use of the cows in the extensive conditions of Alpine summer farms, and helped to outline the role of farmers and animals choices. This knowledge is necessary to assess movement costs for the animals, and the fine scale spatial distribution of animals load and potential impacts on the grazed area

Human choices, slope and vegetation productivity determine patterns of traditional alpine summer grazing

Grazing behaviour influences animal productivity and the conservation of grassland ecosystem services. We used GPS tracking and remote sensing (NDVI index) to monitor the grazing patterns of lactating cows on the 'Malga Ombretta' summer farm (1,957 m asl) in the Dolomites, eastern Italian Alps, from 5th July to 5th August 2018. The pasture area (35 ha) was grazed by a mixed herd of Simmental and Alpine grey cows (stocking density = 0.6 LU/ha) under traditional management: each morning the farmer led the cows to graze in a selected sub-area of pasture, and during the afternoon he left them free to graze unrestricted until they returned to the barn for the night. GPS positions were collected every minute from 9 Simmental and 4 Alpine Grey cows with low milk production during the time they were outdoors. The farmer's choice of where to drive the herd to graze in the morning determined the distances the cows walked/day, which varied from 2.0 to 8.9 km, and favoured the use of higher and steeper areas that the cows tended otherwise to avoid. When free in the afternoon, the cows selected areas with higher NDVI values than those selected by the farmer in the morning, and Alpine Grey cows used slightly higher slopes and altitudes than Simmental cows, suggesting better adaptation to mountain pastures. The study revealed highly heterogenous grazing patterns dependent on multiple factors that can be assessed at fine temporal and spatial scales using GPS and remote sensing technologies to improve grazing management

Relationships between cattle grazing, soil microbiology and nutrients cycle in highland pastures

Highland pastures are particular agro-ecosystems with a high level of plant and animal biodiversity, and for this reason semi-natural grassland are classified as High Nature Value Farmland. This study aims at characterizing the agroecological relationships among grazing animals-plants-microorganisms in alpine summer pastures. The study area included two summer farms in the Dolomites, Eastern Italian Alps. Both summer farms were located at an elevation around 2,000 m asl, and were grazed by dairy cows from mid-June to mid-September. In order to assess the impact of dairy cattle grazing on the functional microbial biodiversity, surveys were carried out using topsoil cores before (\u2018point zero\u2019) and during the grazing period. The distribution of cattle stocking rate at temporal and spatial scale was analysed by using GPS tracking of grazing animals and farmer\u2019s interviews. The samples of soils were collected in patches representative of different vegetation types and stocking rates, with three repetitions for each sampling points. A molecular ecological approach was applied, analysing microbial communities in terms of abundances relative to copies of target genes through the application of real-time PCR. The studied genes were nosZ for denitrification, amoA archea and bacteria for nitrification. The analysis highlighted different outcomes between the considered areas, showing a likely impact of grazing on microbial communities but in different terms depending on the local pedoclimatic and vegetational conditions and on the cattle stocking rates. This kind of approach will be integrated with indicators on carbon sink, in order to have an in-depth view of the ecosystem support services, such as nutrient cycle and soil formation regulation, carbon storage and supply in soil, and forage production. These preliminary results could be used to develop specific and minimally-invasive biophysical indicators of supporting (and regulating) ecosystem services. These tools will assist the definition of strategies aiming at enhancing the sustainability of grazing livestock systems in compliance to agroecology principles