Atomic-level characterization of the structural dynamics of proteins

Molecular dynamics (MD) simulations are widely used to study protein motions at an atomic level of detail, but they have been limited to time scales shorter than those of many biologically critical conformational changes. We examined two fundamental processes in protein dynamics--protein folding and conformational change within the folded state--by means of extremely long all-atom MD simulations conducted on a special-purpose machine. Equilibrium simulations of a WW protein domain captured multiple folding and unfolding events that consistently follow a well-defined folding pathway; separate simulations of the protein's constituent substructures shed light on possible determinants of this pathway. A 1-millisecond simulation of the folded protein BPTI reveals a small number of structurally distinct conformational states whose reversible interconversion is slower than local relaxations within those states by a factor of more than 1000.     

Anaplasma phagocytophilum in horses and ticks: a preliminary survey of Central Italy

Anaplasma phagocytophilum, the causative agent of granulocytic ehrlichiosis, affects several species of wild and domesticated mammals, including horses. In this work we compared direct and indirect methods to evaluate A. phagocytophilum presence in Central Italy: 135 sera were screened by IFA for A. phagocytophilum and other haemopathogens (Theileria equi and Babesia caballi). Each horse was also tested for A. phagocytophilum 16S rRNA with a nested-PCR technique. In order to examine the risk of A. phagocytophilum transmission, 114 ticks were examined for the presence of A. phagocytophilum by PCR targeting the 16S rRNA. The seroprevalence against A. phagocytophilum was 17.03% and 11 horses (8.14%) showed positive PCR results. The concordance rate of A. phagocytophilum detection between IFAT and PCR had a K value of 0.34.     

Ellagic Acid and Polyhydroxylated Urolithins Are Potent Catalytic Inhibitors of Human Topoisomerase II: An in Vitro Study

Ellagic acid (EA), a natural polyphenol abundant in fruits and common in our diet, is under intense investigation for its chemopreventive activity resulting from multiple effects. EA inhibits topoisomerase II, but the effects on the human enzyme of urolithins, its monolactone metabolites, are not known. Therefore, the action of several synthetic urolithins toward topoisomerases II was evaluated, showing that polyhydroxylated urolithins, EA, and EA-related compounds are potent inhibitors of the α and β isoforms of human topoisomerase II at submicromolar concentrations. Competition tests demonstrate a dose-dependent relationship between ATP and the inhibition of the enzyme. Docking experiments show that the active compounds bind the ATP pocket of the human enzyme, thus supporting the hypothesis that EA and polyhydroxylated urolithins act as ATP-competitive inhibitors of human topoisomerase II.     

Experimentally induced melanin-like pigmentation (thesaurismosis) related to acorn ingestion in Nero Siciliano pigs

In this study, an experimental challenge was carried out by feeding Nero Siciliano pigs with acorn to evoke melanin-like pigmentation and support the hypothesis that it is caused by ingested material. Twelve pigs were studied, 6 fed with acorns and 6 fed commercial feed. At slaughter, all the animals fed on acorns showed black discolouration of almost all lymph nodes. The lymph nodes were normal in size and shape. Histochemical tests performed on tissues allowed us to identify and differentiate the pigment. Immunohistochemical staining for macrophage markers showed macrophages containing a variable amount of melanin-like granules. Our data, and the well-known steps of melanin formation, confirm that swine enzymes could act on phenolic substances found in acorns.     

One dose of a porcine circovirus 2 subunit vaccine induces humoral and cell-mediated immunity and protects against porcine circovirus-associated disease under field conditions

This study investigated the efficacy of a one-dose porcine circovirus 2 (PCV2) subunit vaccine based on the PCV2 Cap protein expressed in a baculovirus system on two different farms at which a history of porcine circovirus-associated disease (PCVD) was present. Morbidity, mortality, average daily weight gain, carcass weight, PCV2 load in serum and vaccine immunogenicity were assessed. Serology to porcine reproductive and respiratory syndrome virus (PRRSV) and Mycoplasma hyopneumoniae was performed. A double-blind, randomised, and controlled field trial was performed distributing 818 piglets between two treatment groups. At inclusion (weaning at 21 ± 3 days of age), 408 animals (group B) received a 2-mL intramuscular dose of Porcilis PCV(?) (vaccinated group). Controls (group A, 410 pigs) received 2 mL of the adjuvant Diluvac Forte(?) intramuscularly. Weights were recorded at inclusion and at 12 and 26 weeks of age, and the average daily weight gain (ADWG) was calculated. The carcass weights of the pigs from farm 2 were recorded at slaughter (274 days old). All dead animals (died or culled) underwent autopsy to classify them as PMWS-affected or not. At each farm, blood samples were taken from 22 pigs/group for serologic studies. A beneficial effect was found after vaccination with a single dose of a PCV2 Cap vaccine against PCVD. The vaccination reduced the mortality rate and morbidity, reduced PCV2 viremia and viral load, improved productive performances (e.g. ADWG: +70 g/day between 12 and 26 weeks of age when viremia and the specific disease occurred) as well as carcass weight at slaughter age (+4.5 kg). These effects were associated with virologic and clinical protection from the immunogenicity of the vaccine measured as activation of both a humoral and a cellular immune response.     

Soil organic C variability and microbial functions in a Mediterranean agro-forest ecosystem

Five soils characterised by different agro-forest managements, typical of Mediterranean environment and with increasing human impact were chosen in Sardinia (Italy): two vineyards with different management systems, a rotation hay crop-pasture and a forest (Quercus suber L.). The study aimed to investigate the relationships between C storage and microbial functionality in soil under different managements. Pools of total organic C and microbial biomass C were determined, as well as the loss of organic C due to microbial respiration (basal and cumulative) and several microbial indices (metabolic, mineralization, and microbial quotient) as indicators of the microbial efficiency in the use of energy and the degree of substrate limitation for soil microbes. Enzymes were chosen on their relevance in the C (β-cellobiohydrolase, N-acetyl-β-glucosaminidase, β-glucosidase, α-glucosidase), N (leucine aminopeptidase), S (arylsulphatase) and P (acid phosphatase) cycling and were used as indicators of functional diversity in soil. Organic C pools and enzyme activities on average increased noticeably in soils with a lower human impact showing the highest values in forest and the lowest in the vineyards, following the trend of organic matter availability. The trend in functional diversity reflected the increase of microbial pool and organic C availability: the vineyards showed a lower Shannon’s diversity index, whilst pasture and forest sites reached the maximum levels of functional diversity. These soils showed an increase of microbial efficiency in the use of available resources and the decrease of substrate limitation for soil microbes.     

Post-mortem recovery,in vitro maturation and fertilization of fallow deer (Dama dama,Linnaeus 1758) oocytes collected during reproductive and no reproductive season

Habitat degradation leads to small and fragmented populations, lower genetic variability and fertility overtime. Assisted reproductive techniques represent important tools to cope with the dramatic loss of biodiversity. Fallow deer (Dama dama), beyond its high commercial value and wide distribution, may represent the most suitable model to study endangered cervids. In this study, oocytes were recovered post-mortem from fallow deer during the breeding and no breeding seasons and were in vitro matured (IVM). The ability of cryopreserved thawed sperm samples recovered by electroejaculation from four adult males was tested by in vitro fertilization of IVM oocytes. The number of oocytes collected per ovary did significantly vary across seasons from 6.2 ± 0.92 during breeding season to 10.4 ± 1.26 during no breeding season (p = .006). Oocytes collected during the breeding season showed higher in vitro fertilization rate compared to the no breeding season (p = .045). However, no embryos reached the blastocyst stage. Semen samples obtained by electroejaculation were successfully cryopreserved, although the cryopreservation process negatively affected most kinetic parameters, mainly at 2 hr post-thawing. Moreover, the percentage of rapid spermatozoa significantly decreased between fresh samples and at 2 hr post-thawing, whereas the percentage of slow spermatozoa increased across the same period (p < .05). Our study provides the logistic steps for the application of assisted reproductive techniques in fallow deer and might be of great interest for genetic resource bank planning.