Novel approach to cell sampling from preimplantation ovine embryos and its potential use in embryonic genome analysis

The major obstacle in the extensive analysis of the embryonic genome is the small number of cells typically obtained after the embryo biopsy. The object of the present study was to develop a simple approach that would allow the collection of a sufficient number of cells from a single embryo for use in further analyses. A micromanipulator was used to make a hole in the zona pellucida of 28 compacted morulae, 27 early blastocysts and 31 expanded blastocysts. After further culture, the trophoblastic cells, which herniated through this hole, were cut and cultured in vitro for different periods and used for embryo sexing. The results showed that biopsies can be taken successfully from 96.3% of early blastocysts, compared with 67.7% of expanded blastocysts and 71.4% of compacted morulae. The trophoblastic vesicles contained 20.8 +/- 6.7 cells (mean +/- SEM) and, when cultured, formed a confluent monolayer. The sex of cells cultured was assayed by PCR and the 12 lambs born after transfer of biopsied embryos confirmed its 100% accuracy. Moreover, no significant differences were found in the viability rates in vitro among blastocysts vitrified immediately after biopsy (77.8%), blastocysts biopsied and vitrified after 24 h culture (76.9%) and blastocysts vitrified without manipulation (88.5%). In experiments in vivo, the lambing rate of biopsied and vitrified blastocysts was significantly (P < 0.05) lower (40.0%) compared with vitrified control embryos (68.7%). This new approach to the biopsy of preimplantation embryos is a useful good model in the assisted reproductive technologies of domestic, wild and human species.     

Seasonal succession of Cladocerans in a ricefield in Italy

A biological ricefield in northern Italy, without periodic dry spells in its growing cycle and therefore more familiar to naturally humid zones, was studied for its heleoplankton community. The biocoenosis reached a greater level of complexity than reported in literature. In particular, the seasonal succession of Cladocerans, the dominant group throughout the study period, was analyzed. Wlassicsia pannonica (Daday, 1904; Anomopoda Macrothricidae), is new to Italy; its morphology is compared to that of other populations of the same species and its biological cycle is compared with that of other dominant Cladocerans.     

Activation of GPR55 Receptors Exacerbates oxLDL-Induced Lipid Accumulation and Inflammatory Responses,while Reducing Cholesterol Efflux from Human Macrophages

The G protein-coupled receptor GPR55 has been proposed as a new cannabinoid receptor associated with bone remodelling, nervous system excitability, vascular homeostasis as well as in several pathophysiological conditions including obesity and cancer. However, its physiological role and underlying mechanism remain unclear. In the present work, we demonstrate for the first time its presence in human macrophages and its increased expression in ox-LDL-induced foam cells. In addition, pharmacological activation of GPR55 by its selective agonist O-1602 increased CD36- and SRB-I-mediated lipid accumulation and blocked cholesterol efflux by downregulating ATP-binding cassette (ABC) transporters ABCA1 and ABCG1, as well as enhanced cytokine- and pro-metalloprotease-9 (pro-MMP-9)-induced proinflammatory responses in foam cells. Treatment with cannabidiol, a selective antagonist of GPR55, counteracted these pro-atherogenic and proinflammatory O-1602-mediated effects. Our data suggest that GPR55 could play deleterious role in ox-LDL-induced foam cells and could be a novel pharmacological target to manage atherosclerosis and other related cardiovascular diseases.     

Macular Microcysts in Mitochondrial Optic Neuropathies: Prevalence and Retinal Layer Thickness Measurements

Purpose

To investigate the thickness of the retinal layers and to assess the prevalence of macular microcysts (MM) in the inner nuclear layer (INL) of patients with mitochondrial optic neuropathies (MON).

Methods

All patients with molecularly confirmed MON, i.e. Leber’s Hereditary Optic Neuropathy (LHON) and Dominant Optic Atrophy (DOA), referred between 2010 and 2012 were enrolled. Eight patients with MM were compared with two control groups: MON patients without MM matched by age, peripapillary retinal nerve fiber layer (RNFL) thickness, and visual acuity, as well as age-matched controls. Retinal segmentation was performed using specific Optical coherence tomography (OCT) software (Carl Zeiss Meditec). Macular segmentation thickness values of the three groups were compared by one-way analysis of variance with Bonferroni post hoc corrections.

Results

MM were identified in 5/90 (5.6%) patients with LHON and 3/58 (5.2%) with DOA. The INL was thicker in patients with MON compared to controls regardless of the presence of MM [133.1±7μm vs 122.3±9μm in MM patients (p<0.01) and 128.5±8μm vs. 122.3±9μm in no-MM patients (p<0.05)], however the outer nuclear layer (ONL) was thicker in patients with MM (101.4±1mμ) compared to patients without MM [77.5±8mμ (p<0.001)] and controls [78.4±7mμ (p<0.001)]. ONL thickness did not significantly differ between patients without MM and controls.

Conclusion

The prevalence of MM in MON is low (5-6%), but associated with ONL thickening. We speculate that in MON patients with MM, vitreo-retinal traction contributes to the thickening of ONL as well as to the production of cystic spaces.     

A New 4D Trajectory-Based Approach Unveils Abnormal LV Revolution Dynamics in Hypertrophic Cardiomyopathy

The assessment of left ventricular shape changes during cardiac revolution may be a new step in clinical cardiology to ease early diagnosis and treatment. To quantify these changes, only point registration was adopted and neither Generalized Procrustes Analysis nor Principal Component Analysis were applied as we did previously to study a group of healthy subjects. Here, we extend to patients affected by hypertrophic cardiomyopathy the original approach and preliminarily include genotype positive/phenotype negative individuals to explore the potential that incumbent pathology might also be detected. Using 3D Speckle Tracking Echocardiography, we recorded left ventricular shape of 48 healthy subjects, 24 patients affected by hypertrophic cardiomyopathy and 3 genotype positive/phenotype negative individuals. We then applied Generalized Procrustes Analysis and Principal Component Analysis and inter-individual differences were cleaned by Parallel Transport performed on the tangent space, along the horizontal geodesic, between the per-subject consensuses and the grand mean. Endocardial and epicardial layers were evaluated separately, different from many ecocardiographic applications. Under a common Principal Component Analysis, we then evaluated left ventricle morphological changes (at both layers) explained by first Principal Component scores. Trajectories’ shape and orientation were investigated and contrasted. Logistic regression and Receiver Operating Characteristic curves were used to compare these morphometric indicators with traditional 3D Speckle Tracking Echocardiography global parameters. Geometric morphometrics indicators performed better than 3D Speckle Tracking Echocardiography global parameters in recognizing pathology both in systole and diastole. Genotype positive/phenotype negative individuals clustered with patients affected by hypertrophic cardiomyopathy during diastole, suggesting that incumbent pathology may indeed be foreseen by these methods. Left ventricle deformation in patients affected by hypertrophic cardiomyopathy compared to healthy subjects may be assessed by modern shape analysis better than by traditional 3D Speckle Tracking Echocardiography global parameters. Hypertrophic cardiomyopathy pathophysiology was unveiled in a new manner whereby also diastolic phase abnormalities are evident which is more difficult to investigate by traditional ecocardiographic techniques.     

PCR-based methods for <Emphasis Type="Italic">CDA</Emphasis> K27Q and A70T genotyping: genotypes and alleles distribution in a central Italy population

Cytidine deaminase (CDA) is a pyrimidine salvage pathway enzyme that catalyzes the hydrolytic deamination of cytidine and deoxycytidine to their corresponding uracil nucleosides. CDA also catalyzes the inactivation of some chemotherapeutic nucleoside analogues such as cytosine arabinoside and gemcitabine. CDA 79A > C (K27Q, rs2072671) and 208G > A (A70T, rs60369023) were found to be associated either with clinical outcomes as well as with pharmacokinetics and toxicity of drugs administered to different subsets of patients. In this paper we reported two PCR-based methods for CDA 79A > C (K27Q) and 208G > A (A70T) genotyping and tested their feasibility using DNA extracted from whole blood as well as from buccal swabs. The aim of this study was also to assess the distribution of genotypic variants in a central Italy population. The allele frequencies were 56.3% (K*) and 43.7% (Q*) for K27Q and 100% (A*) and 0% (T*) for A70T. The genotype frequencies were 32.8% (K*/K*), 46.9% (K*/Q*) and 20.3% (Q*/Q*) for K27Q. The genotype frequencies did not deviate from Hardy–Weinberg equilibrium. The results were compared with those of other reported populations. They showed marked ethnic group differences.     

Relevance of disease- and organ-specific endothelial cells for in vitro research

The endothelium is a dynamic, heterogeneous, disseminated organ that possesses vital secretory, synthetic, metabolic and immunological functions. Endothelial dysfunction has been implicated as a key factor in the development of organ-specific vascular diseases. This minireview gives a brief overview on EC (endothelial cell) biomarkers in arterial and venous endothelium and critically discusses the different sources of ECs that are most frequently applied in in vitro assays and research. The relevance of organ- and disease-specific endothelial cell cultures for studying cellular responses as a basis for improving therapeutic interventions is highlighted with particular emphasis on endothelial dysfunction in transplant-associated coronary artery disease, in atherosclerotic lesions and in response to diabetes mellitus.