An ovine preimmune foetal model to study the effect of cellular therapies for myocardic diseases

The ovine foetus is an ideal model for preclinical medical studies of cell therapies. It allows us to follow the behaviour of repairing cells inserted into a favourable physiological microenvironment in an animal species more closely related to humans than the rat or rabbit. In addition, the preimmune foetus is able to support cell engraftment by eliminating the problems of tissue rejection. Labelled fibroblasts were transplanted into the myocardium of preimmune foetuses, injecting through a disposable bowed mouth pipette into the left ventricular apex. Two weeks later, foetuses were isolated by laparotomy and each heart was collected and morphologically analyzed. No cases of abortion or postoperative complications in mothers or foetuses occurred. Macroscopically, the isolated hearts did not display any abnormality apart from a small petechia at the injection site. Tissue sections did not show any sign of acute tissue inflammation and viable labelled cells were easily identified among myocardiocytes. This model system guarantees a reduced damage in the engrafted tissue, a high survival and easy retrieval of the injected cells. The direct injection of cells into the preimmune ovine foetus myocardium can be satisfactorily performed to control tissue delivery and to reduce the risk of cell loss and dispersion.     

Differentiation and characterization of rat adipose tissue mesenchymal stem cells into endothelial‐like cells

In this study, mesenchymal stem cells were isolated from rat adipose tissue (AD‐MSCs) to characterize and differentiate them into endothelial‐like cells. AD‐MSCs were isolated by mechanical and enzymatic treatments, and their identity was verified by colony‐forming units (CFU) test and by differentiation into cells of mesodermal lineages. The endothelial differentiation was induced by plating another aliquot of cells in EGM‐2 medium, enriched with specific endothelial growth factors. Five subcultures were performed. The expression of stemness genes (OCT4, SOX2 and NANOG) was investigated. The presence of CD90 and the absence of the CD45 were evaluated by flow cytometry. The endothelial‐like cells were characterized by the evaluation of morphological changes and gene expression analysis for endothelial markers (CD31, CD144, CD146). Characterization of AD‐MSCs showed their ability to form clones, to differentiate in vitro and the OCT‐4, SOX‐2, NANOG genes expression. Immunophenotypic characterization showed the CD90 presence and the CD45 absence. The endothelial‐like cells showed morphological changes, the expression of CD31, CD144, CD146 genes and the presence of CD31 membrane receptor. Matrigel assay showed their ability to form network and vessels‐like structures. This study lays the foundations for future evaluation of the potential AD‐MSCs pro‐angiogenic and therapeutic role.     

Prevalence and epidemiological features of ovine enzootic abortion in Lithuania

The aim of performed study was to determine the level of enzootic abortion (EA) in sheep breeding farms in different districts of Lithuania, to determine differences in clinical signs and infection frequency between various age groups, and to evaluate the sensitivity and specificity of complement fixation test for antibodies detection and indirect immunofluorescence for antigen detection in sheep chlamydiosis. The clinical, serological and immunological tests in sheep farms were performed in 2004 and 2005. Comparing different age groups of sheep revealed that the lowest number of infected sheep was registered in animals younger than 18 months (23.1%, antibodies titre 3.191 log2, P<0.05) and highest in animals aged 18 to 24 months (53.8%, antibodies titre 4.224 log2, P<0.001). In sheep aged more than 3 years, titre of antibodies was significantly reduced. The majority of infected sheep which aborted (86.4%) was registered in 18-24 month age group. Furthermore, in sheep which aborted the infection level was 2.5-fold higher as compared to sheep which didn't abort. Analysis of smears from patological material by indirect FAT revealed that 54.5% of animals were positive to Chlamydophila abortus infection. The highest prevalence of chlamydia (66.7%) was registered in placentas of sheep which aborted.     

Demographic variation and habitat specialization of tree species in a diverse tropical forest of Cameroon

Background： Many tree species in tropical forests have distributions tracking local ridge-slope-valley topography. Previous work in a 50-ha plot in Korup National Park, Cameroon, demonstrated that 272 species, or 63% of those tested, were significantly associated with topography. Methods： We used two censuses of 329,000 trees ≥1 cm dbh to examine demographic variation at this site that would account for those observed habitat preferences. We tested two predictions. First, within a given topographic habitat, species specializing on that habitat （＇residents＇） should outperform species that are specialists of other habitats （＇foreigners＇）. Second, across different topographic habitats, species should perform best in the habitat on which they specialize （＇home＇） compared to other habitats （＇away＇）. Species＇ performance was estimated using growth and mortality rates. Results： In hierarchical models with species identity as a random effect, we found no evidence of a demographic advantage to resident species. Indeed, growth rates were most often higher for foreign species. Similarly, comparisons of species on their home vs. away habitats revealed no sign of a performance advantage on the home habitat. Conclusions＂ We reject the hypothesis that species distributions along a ridge-valley catena at Korup are caused by species differences in trees _〉1 cm dbh. Since there must be a demographic cause for habitat specialization, we offer three alternatives. First, the demographic advantage specialists have at home occurs at the reproductive or seedling stage, in sizes smaller than we census in the forest plot. Second, species may have higher performance on their preferred habitat when density is low, but when population builds up, there are negative density-dependent feedbacks that reduce performance. Third, demographic filtering may be produced by extreme environmental conditions that we did not observe during the census interval.     

Analysis of some Italian lemon liquors (limoncello)

The chemical composition of several commercial Italian Limoncellos, lemon-peel-based alcoholic beverages, was studied by chromatographic techniques. These methods allowed a rapid monitoring of Limoncello, giving information on quality markers and possible adulteration of the product. Quantitative data for more than 60 compounds are reported. Limoncellos were characterized by the presence of selected volatile (terpenes, aldehydes, alcohols) and nonvolatile compounds (psoralens, coumarins, phenolics, carbohydrates and acids). On the basis of their composition, the samples were grouped by PCA analysis in two sets; the first group showed a composition similar to lemon essential oils, with a high content of b-pinene, myrcene, trans-a-bergamottene, and b-bisabolene, and a low content in neral and geranial. The composition of the second group suggested the occurrence of oxidative phenomena and/or the addition of flavors. The presence of ethyl acetate, acetaldehyde, 2-methyl-1-propanol and glycerol showed that a fermentation probably occurred in the sugar syrup used to dilute the Limoncello after the extraction process.     

Fate of prions in soil: Interaction of a recombinant ovine prion protein with synthetic humic-like mineral complexes

Prion proteins are regarded as the main agents of transmissible spongiform encephalopathies. Understanding their fate in soil may be crucial to elucidate the dissemination of the prion in the environment, associated with a possible transmission of infectivity. Studies were performed with simplified model systems, derived by the birnessite-mediated oxidative polymerization of catechol, which simulate processes naturally occurring in soil. A benign full-length recombinant purified ovine protein (PrP) (residues 23-234), as well as a truncated-form ovPrP (tPrP) (103-234) were utilized. Catechol and prion protein interacted under experimental conditions that reproduced the interaction of PrP with soluble organic matter or with insoluble organic matter during or after formation of catechol polymers. PrP stability in all buffers and chemicals was preliminarily monitored by circular dicroism (CD) measurements. The disappearance of protein molecules from the solution, the decrease of UV-Visible absorbance of supernatants, and the FT-IR spectra and the elemental analyses of solid-phase residues indicated that both PrP and tPrP were involved in catechol polymerization by birnessite. Furthermore, a clear flocculation of soluble catechol-protein polymeric products in solid aggregates was observed when PrP was added to the supernatants. Different kinds of extracting agents were not able to desorb/extract PrP as well as tPrP from the formed solid aggregates, thereby highlighting the high stability of protein-organic and -organo-mineral complexes.     

A simple and selective method for the measurement of azadirachtin and related azadirachtoid levels in fruits and vegetables using liquid chromatography electrospray ionization tandem mass spectrometry

Neem-based insecticides containing azadirachtin and related azadirachtoids are widely used in agriculture. Here, we report an analytical method for the rapid and accurate quantification of the insecticide azadirachtin A and B and other azadirachtoids such as salannin, nimbin, and their deacetylated analogues on tomatoes and peaches. Azadirachtoids were extracted from fruits and vegetables with acetonitrile. Using high-performance liquid chromatography/electrospray ionization tandem mass spectrometer, azadirachtoids were selectively detected monitoring the multiple reaction transitions of sodium adduct precursor ions. For azadirachtin A, calibration was linear over a working range of 1-1000 microg/L with r > 0.996. The limit of detection and limit of quantification for azadirachtin A were 0.4 and 0.8 microg/kg, respectively. The presence of interfering compounds in the peach and tomato extracts was evaluated and found to be minimal. Because of the linear behavior, it was concluded that the multiple reaction transitions of sodium adduct ions can be used for analytical purposes, that is, for the identification and quantification of azadirachtin A and B and related azadirachtoids in fruit and vegetable extracts at trace levels.