Efficacy and safety of mitomycin C as an agent to treat corneal scarring in horses using an in vitro model

Objective Mitomycin C (MMC) is used clinically to treat corneal scarring in human patients. We investigated the safety and efficacy of MMC to treat corneal scarring in horses by examining its effects at the early and late stages of disease using an in vitro model. Procedure An in vitro model of equine corneal fibroblast (ECF) developed was used. The ECF or myofibroblast cultures were produced by growing primary ECF in the presence or absence of transforming growth factor beta‐1 (TGFβ1) under serum‐free conditions. The MMC dose for the equine cornea was defined with dose‐dependent trypan blue exclusion and (3‐4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assays after applying MMC to the cultures once for 2 min. The efficacy of MMC to control corneal scarring in horses was determined by measuring mRNA and protein expression of corneal scarring markers (alpha‐smooth muscle actin and F‐actin) with western blotting, immunocytochemistry and/or quantitative real‐time polymerase chain reactions. Results A single 2‐min treatment of 0.02% or less MMC did not alter ECF phenotype, viability, or cellular proliferation whereas 0.05% or higher MMC doses showed mild‐to‐moderate cellular toxicity. The TGFβ1 at 1 ng/mL showed significant myofibroblast formation in ECF under serum‐free conditions. A single 2‐min, 0.02% MMC treatment 24 h (early) after TGFβ1 stimulation significantly reduced conversion of ECF to myofibroblasts, however, a single 0.02% MMC treatment 11 days after TGFβ1 stimulation showed moderate myofibroblast inhibition. Conclusions That MMC safely and effectively reduced scarring in ECF by reducing the degree of transdifferentiation of corneal fibroblasts to myofibroblasts in vitro. Further clinical in vivo investigations are warranted using MMC in horses.     

Gene delivery in the equine cornea: a novel therapeutic strategy

Objective To determine if hybrid adeno‐associated virus serotype 2/5 (AAV5) vector can effectively deliver foreign genes into the equine cornea without causing adverse side effects. The aims of this study were to: (i) evaluate efficacy of AAV5 to deliver therapeutic genes into equine corneal fibroblasts (ECFs) using enhanced green fluorescent protein (EGFP) marker gene, and (ii) establish the safety of AAV5 vector for equine corneal gene therapy. Material Primary ECF cultures were harvested from healthy donor equine corneas. Cultures were maintained at 37°C in humidified atmosphere with 5% CO₂. Procedure AAV5 vector expressing EGFP under control of hybrid cytomegalovirus + chicken β‐actin promoter was applied topically to ECF. Expression of delivered EGFP gene in ECF was quantified using fluorescent microscopy. Using fluorescent staining, the total number of cells and transduction efficiency of tested AAV vector was determined. Phase contrast microscopy, trypan blue and TUNEL assays were used to determine toxicity and safety of AAV5 for ECFs. Results Topical AAV5 application successfully transduced significant numbers of ECFs. Transduction efficiency was 13.1%. Tested AAV5 vector did not cause phenotype change or significant cell death and cell viability was maintained. Conclusions Tested AAV5 vector is effective and safe for gene therapy in ECFs in vitro.     

Ruffed grouse-habitat preference in the central and southern Appalachians

Ruffed grouse (Bonasa umbellus; hereafter grouse) populations in the central and southern Appalachians (CSA) are declining due to widespread maturation of forest cover. Effective management of this species requires a sex- and age-specific understanding of habitat preferences at multiple temporal and spatial scales. We used multivariate logistic regression models to compare habitat within 1440 grouse home ranges and 1400 equally sized buffered random points across 7 CSA study areas. On most sites, grouse home ranges were positively associated with roads and young forest (<20 years old). Sex and age status affected habitat preference. In general, males used younger forest than females, likely because of differences in habitat use during reproductive periods. Juveniles had fewer vegetation types preferred by adult grouse and more of the avoided vegetation types within their home ranges, indicative of competitive exclusion. Adult females had the greatest specificity and selectivity of habitat conditions within their home ranges. Habitat selection varied among seasons and years on most sites. Winter habitat use reflected behavior that maximized energy conservation, with open vegetation types avoided in the winter on the northernmost study areas, and topography important on all areas. Summer habitat selection reflected vegetation types associated with reproductive activities. Scale influenced habitat preference as well. Although roads and forest age predominantly influenced grouse home range location within the landscape, mesic forest types were most important in determining core area use within the home range. This was likely a result of increased food availability and favorable microclimate. Habitat management efforts should attempt to maintain ∼3–4% of the landscape in young forest cover (<20 years old), evenly distributed across management areas. Roads into these areas should be seeded as appropriate to enhance brood habitat and provide travel corridors connecting suitable forest stands.     

Molecular biological characterization of avian poxvirus strains isolated from different avian species

Fifteen strains of Avipoxvirus from different avian species were isolated and molecular biologically characterized. Most strains did not produce evident pocks on the chorioallantoic membranes of commercial and specific-pathogen free embryonated chicken eggs where, on the contrary, microscopic signs of viral growth were always detected. Polymerase chain reaction of highly conserved P4b gene was positive for all cases confirming to be a reliable diagnostic method for Avipoxvirus. Sequencing of these amplicons confirmed most strains clustered either with Fowlpox virus or with Canarypox virus whereas a possible new clade could be hypothesized for one strain from Japanese quail. Classification of Avipoxvirus strains by amplification of the newly identified locus fpv140 revealed major limitations as only five samples were positive. These results underline the importance to undertake similar studies on higher numbers of Avipoxvirus isolates and on wider genomic regions of this large viral group.     

Effect of thinning on above-ground biomass accumulation in a Douglas-fir plantation in southern Italy

We investigated the effects of a long-term thinning experiment on the distribution of above-ground biomass of Douglas-fir(Pseudotsuga menziesii [Mirb.]Franco var. menziesii) in a plantation in southern Italy.Allometric equations were used to estimate biomass and partitioning to stem and crown compartments. Variation in biomass stock estimated with allometric equations were evaluated according to seven thinning treatments: geometric-systematic(1 row every 3), selective(light-moderate-heavy), mixed systematic-selective(1 row every 4, 1 row every 5), unthinned(control). Over the experimental period of 13 years, current annual increments of carbon were lower(3.4 Mg ha~(-1)year~(-1)) in control plots than in treated plots. At age 30, plots subjected to light selective thinning showed higher values of above-ground biomass(249.7 Mg ha~(-1)). The biomass harvested with this treatment was 29.3 Mg ha~(-1), and the mean annual increment of carbon over 13 years was 4.8 Mg ha~(-1). Our results showed that light thinning stimulated increase in carbon stock, with a minimal loss of carbon during the treatment and a current annual increment of carbon higher than in control sub-plots and sub-plots thinned using systematic methods. This treatment yielded least carbon emissions and we affirm it has discrete global warming mitigation potential.     

Selective and Apoptotic Action of Ethanol Extract of Annona cherimola Seeds against Human Stomach Gastric Adenocarcinoma Cell Line AGS

Plant Foods for Human Nutrition - Annona cherimola is a tree belonging to the family Annonacea, whose fruit (cherimoya) is very desirable, but its seeds are considered waste. Present in these seeds...     

Comparative expression of liver cytochrome P450-dependent monooxygenases in the horse and in other agricultural and laboratory species

The apoprotein expression and the catalytic activities of cytochrome P450s involved in the biotransformation of xenobiotics were investigated in horse liver microsomes and compared with those of food producing (cattle, pigs, broiler chicks, and rabbits) and laboratory species (rats). Western blot analysis revealed the presence of proteins immunorelated to rat CYP 1A, CYP 2B, CYP 2E, and CYP 3A subfamilies in hepatic microsomes from horses and from any other examined species. With the exception of the N-demethylation of N-nitrosodimethylamine in broiler chicks, all the recorded interspecies differences were quantitative in nature. Equine preparations proved the most active in the biotransformation of the CYP 1A substrates ethoxy- and methoxyresorufin and the least active in the metabolism of aminopyrine and ethoxycoumarin. On a comparative basis, large differences were observed in the rate of the in vitro metabolism of model substrates between "minor" (rabbits, horses) and "major" food producing species. Taken in due consideration the limitations of the in vitro approach, results from this study reinforce the conclusion that studies on drug efficacy and residue depletion should be performed in each target species.     

Two new O-geranyl coumarins from the resinous exudate of Haplopappus multifolius

From the resinous exudate of leaves of Haplopappus multifolius two new coumarins were isolated and assigned the structures 6-hydroxy-7-(5'-hydroxy-3',7'-dimethylocta-2',6'-dien)-oxycoumarin (1) and 6-hydroxy-7-(7'-hydroxy-3',7'-dimethylocta-2',5'-dien)-oxy coumarin (2).     

Xenotransplantation of cryopreserved equine squamous cell carcinoma to athymic nude and SCID mice

Cryopreserved equine ocular squamous cell carcinoma (SCC) was inoculated subcutaneously into 15 athymic nude and 15 SCID mice. Xenotransplantation resulted in tumor growth in two athymic nude mice and 1 SCID mouse. Histological appearance and immunohistochemical characterization using cytokeratin 5/6 markers and p53 markers of the tumor grown in mice was in full accord with the original equine tumors. No evidence of metastasis was noted in any mouse. This model may serve as a relevant in vivo model for studying the biology of equine ocular SCC and for the testing of new therapeutic modalities.