Tracing of cell lineage in embryonic development of Phallusia mammillata (ascidia) by vital staining of mitochondria

Vital staining of mitochondria with a fluorescent dye 3,3′-diethyloxacarbocyanine was used to follow cell lineage in embryos of Phallusia mammillata. The results agree in general with the plan established by Conklin in 1905. Strong fluorescence migrated after fertilization similarly to the pigment of the “yellow crescent” in Styela. Later, fluorescence segregated into muscle cell primordia, but not into mesenchyme cells. An animal hemisphere cell, b 8.17 also exhibited strong fluorescence and joined a group of muscle primordia, very likely becoming a muscle cell itself. In the tadpole, all the tail muscle cells were fluorescent. Fluorescence was also noticed in nerve cell primordia of the vegetal hemisphere, particularly in the cell A 8.16 whose descendants appeared to become part of the sensory vesicle which was strongly fluorescent in the tadpole. The usefulness of this type of vital staining in following cell lineage of colorless embryos is stressed.     

Cardiac mitochondrial ATP-sensitive potassium channel is activated by nitric oxide in vitro

Previous observations on the activation of the mitochondrial ATP-sensitive potassium channel (mitoK(ATP)) by nitric oxide (NO) in myocardial preconditioning were based on indirect evidence. In this study, we have investigated the direct effect of NO on the rat cardiac mitoK(ATP) after reconstitution of the inner mitochondrial membranes into lipid bilayers. We found that the mitoK(ATP) was activated by exogenous NO donor S-nitroso-N-acetyl penicillamine or PAPA NONOate. This activation was inhibited by mitoK(ATP) blockers 5-hydroxydecanoate or glibenclamide. Our observations confirm that NO can directly activate the cardiac mitoK(ATP), which may underlie its contribution to myocardial preconditioning.     

Differential Regulation and Function of CD73, a Glycosyl-Phosphatidylinositol–linked 70-kD Adhesion Molecule, on Lymphocytes and Endothelial Cells

CD73, otherwise known as ecto-5′-nucleotidase, is a glycosyl-phosphatidylinositol–linked 70-kD molecule expressed on different cell types, including vascular endothelial cells (EC) and certain subtypes of lymphocytes. There is strong evidence for lymphocyte CD73 having a role in several immunological phenomena such as lymphocyte activation, proliferation, and adhesion to endothelium, but the physiological role of CD73 in other cell types is less clear. To compare the biological characteristics of CD73 in different cell types, we have studied the structure, function, and surface modulation of CD73 on lymphocytes and EC. CD73 molecules on lymphocytes are shed from the cell surface as a consequence of triggering with an antiCD73 mAb, mimicking ligand binding. In contrast, triggering of endothelial CD73 does not have any effect on its expression. Lymphocyte CD73 is susceptible to phosphatidylinositol phospholipase, whereas only a small portion of CD73 on EC could be removed by this enzyme. Furthermore, CD73 on EC was unable to deliver a tyrosine phosphorylation inducing signal upon mAb triggering, whereas triggering of lymphocyte CD73 can induce tyrosine phosphorylation. Despite the functional differences, CD73 molecules on lymphocytes and EC were practically identical structurally, when studied at the protein, mRNA, and cDNA level. Thus, CD73 is an interesting example of a molecule which lacks structural variants but yet has a wide diversity of biological functions. We suggest that the ligand- induced shedding of lymphocyte CD73 represents an important and novel means of controlling lymphocyte– EC interactions.     

Improving Education in Medical Statistics: Implementing a Blended Learning Model in the Existing Curriculum

Background

Although recent studies report on the benefits of blended learning in improving medical student education, there is still no empirical evidence on the relative effectiveness of blended over traditional learning approaches in medical statistics. We implemented blended along with on-site (i.e. face-to-face) learning to further assess the potential value of web-based learning in medical statistics.

Methods

This was a prospective study conducted with third year medical undergraduate students attending the Faculty of Medicine, University of Belgrade, who passed (440 of 545) the final exam of the obligatory introductory statistics course during 2013–14. Student statistics achievements were stratified based on the two methods of education delivery: blended learning and on-site learning. Blended learning included a combination of face-to-face and distance learning methodologies integrated into a single course.

Results

Mean exam scores for the blended learning student group were higher than for the on-site student group for both final statistics score (89.36±6.60 vs. 86.06±8.48; p = 0.001) and knowledge test score (7.88±1.30 vs. 7.51±1.36; p = 0.023) with a medium effect size. There were no differences in sex or study duration between the groups. Current grade point average (GPA) was higher in the blended group. In a multivariable regression model, current GPA and knowledge test scores were associated with the final statistics score after adjusting for study duration and learning modality (p<0.001).

Conclusion

This study provides empirical evidence to support educator decisions to implement different learning environments for teaching medical statistics to undergraduate medical students. Blended and on-site training formats led to similar knowledge acquisition; however, students with higher GPA preferred the technology assisted learning format. Implementation of blended learning approaches can be considered an attractive, cost-effective, and efficient alternative to traditional classroom training in medical statistics.     

Effects of bovine spermatozoa preparation on embryonic development in vitro

The aim of our research was to examine the ability of density gradient preparation BoviPure^? and swim up method on bull sperm separation and in vitro embryo production (IVP) systems. Frozen/thawed semen from six Simmental bulls was pooled and treated using both methods. The sperm motility, concentration, membrane activity, membrane integrity and acrosomal status were evaluated and compared before and after sperm processing using BoviPure^? and swim up methods. We also evaluated and compared cleavage rates, embryo yield and quality between the methods. There were significant differences (P < 0.05) between the sperm characteristics before and after BoviPure^?, but not after swim up method. However, there were significant differences for sperm results among those two mentioned methods. A total of 641 oocytes were matured and fertilized in vitro and cultured in SOFaaBSA. The percentage of cleavage (Day 2) and the percentage of hatched embryos (Day 9) were similar for both methods. However, embryo production rate (Day 7) was significantly higher using BoviPure^? method (P < 0.05). Also, total cell number and embryo differential staining (inner cell mass and trophectoderm cells) of Day 7 morulas and blastocysts showed that BoviPure^? treated sperm displayed higher quality embryos compared to swim up method (P < 0.05). Our results indicate that BoviPure^? method has an enhanced capacity in sperm selection for in vitro embryo production when compared with swim up method. So, we concluded that BoviPure^? could be considered as a better alternative to swim up method for separating bull spermatozoa from frozen/thawed semen for IVP of bovine embryos.     

Mammalian p55CDC Mediates Association of the Spindle Checkpoint Protein Mad2 with the Cyclosome/Anaphase-promoting Complex, and is Involved in Regulating Anaphase Onset and Late Mitotic Events

We have investigated the function of p55CDC, a mammalian protein related to Cdc20 and Hct1/Cdh1 in Saccharomyces cerevisiae, and Fizzy and Fizzy-related in Drosophila. Immunofluorescence studies and expression of a p55CDC-GFP chimera demonstrate that p55CDC is concentrated at the kinetochores in M phase cells from late prophase to telophase. Some p55CDC is also associated with the spindle microtubules and spindle poles, and some is diffuse in the cytoplasm. At anaphase, the concentration of p55CDC at the kinetochores gradually diminishes, and is gone by late telophase. In extracts prepared from M phase, but not from interphase HeLa cells, p55CDC coimmunoprecipitates with three important elements of the M phase checkpoint machinery: Cdc27, Cdc16, and Mad2. p55CDC is required for binding Mad2 with the Cdc27 and Cdc16. Thus, it is likely that p55CDC mediates the association of Mad2 with the cyclosome/anaphase-promoting complex. Microinjection of anti-p55CDC antibody into mitotic mammalian cells induces arrest or delay at metaphase, and impairs progression of late mitotic events. These studies suggest that mammalian p55CDC may be part of a regulatory and targeting complex for the anaphase-promoting complex.     

Mouse genetics identifies unique and overlapping functions of fibroblast growth factor receptors in keratinocytes

Fibroblast growth factors (FGFs) are key regulators of tissue development, homeostasis and repair, and abnormal FGF signalling is associated with various human diseases. In human and murine epidermis, FGF receptor 3 (FGFR3) activation causes benign skin tumours, but the consequences of FGFR3 deficiency in this tissue have not been determined. Here, we show that FGFR3 in keratinocytes is dispensable for mouse skin development, homeostasis and wound repair. However, the defect in the epidermal barrier and the resulting inflammatory skin disease that develops in mice lacking FGFR1 and FGFR2 in keratinocytes were further aggravated upon additional loss of FGFR3. This caused fibroblast activation and fibrosis in the FGFR1/FGFR2 double‐knockout mice and even more in mice lacking all three FGFRs, revealing functional redundancy of FGFR3 with FGFR1 and FGFR2 for maintaining the epidermal barrier. Taken together, our study demonstrates that FGFR1, FGFR2 and FGFR3 act together to maintain epidermal integrity and cutaneous homeostasis, with FGFR2 being the dominant receptor.     

Cellulose structure and lignin distribution in normal and compression wood of the Maidenhair tree(Ginkgo biloba L.)

We studied in detail the mean micro fibril angle and the width of cellulose crystals from the pith to the bark of a 15-year-old Maidenhair tree(Ginkgo biloba L.). The orientation of cellulose micro fibrils with respect to the cell axis and the width and length of cellulose crystallites were determined using Xray diffraction. Raman microscopy was used to compare the lignin distribution in the cell wall of normal/opposite and compression wood, which was found near the pith. Ginkgo biloba showed a relatively large mean micro fibril angle,varying between 19° and 39° in the S2 layer, and the average width of cellulose crystallites was 3.1–3.2 nm. Mild compression wood without any intercellular spaces or helical cavities was observed near the pith. Slit-like bordered pit openings and a heavily lignified S2 L layer con firmed the presence of compression wood. Ginkgo biloba showed typical features present in the juvenile wood of conifers. The micro fibril angle remained large over the 14 annual rings. The entire stem disc,with a diameter of 18 cm, was considered to consist of juvenile wood. The properties of juvenile and compression wood as well as the cellulose orientation and crystalline width indicate that the wood formation of G. biloba is similar to that of modern conifers.