Reconstitution of Homomeric GluA2flop Receptors in Supported Lipid Membranes: FUNCTIONAL AND STRUCTURAL PROPERTIES*

AMPA receptors (AMPARs) are glutamate-gated ion channels ubiquitous in the vertebrate central nervous system, where they mediate fast excitatory neurotransmission and act as molecular determinants of memory formation and learning. Together with detailed analyses of individual AMPAR domains, structural studies of full-length AMPARs by electron microscopy and x-ray crystallography have provided important insights into channel assembly and function. However, the correlation between the structure and functional states of the channel remains ambiguous particularly because these functional states can be assessed only with the receptor bound within an intact lipid bilayer. To provide a basis for investigating AMPAR structure in a membrane environment, we developed an optimized reconstitution protocol using a receptor whose structure has previously been characterized by electron microscopy. Single-channel recordings of reconstituted homomeric GluA2_flop receptors recapitulate key electrophysiological parameters of the channels expressed in native cellular membranes. Atomic force microscopy studies of the reconstituted samples provide high-resolution images of membrane-embedded full-length AMPARs at densities comparable to those in postsynaptic membranes. The data demonstrate the effect of protein density on conformational flexibility and dimensions of the receptors and provide the first structural characterization of functional membrane-embedded AMPARs, thus laying the foundation for correlated structure-function analyses of the predominant mediators of excitatory synaptic signals in the brain.     

The Effects of CX3CR1 Deficiency and Irradiation on the Homing of Monocyte-Derived Cell Populations in the Mouse Eye

This study examined whether CX₃CR1 deficiency altered monocytic cell replenishment dynamics in ocular tissues in the context of radiation chimeras. Long-term effects of irradiation and effects of sublethal irradiation on ocular macrophages were also assessed. Bone marrow from BALB/c Cx ₃ cr1 ^+/gfp or Cx ₃ cr1 ^gfp/gfp mice was used to reconstitute full body irradiated WT mice and donor cell densities in the uveal tract were compared at 4 and 8 weeks post-transplantation. BALB/c and C57BL/6J chimeric mice were examined at 6 months of age to determine strain-related differences in microglial replenishment and radiation sensitivity. A separate cohort of mice were sublethally irradiated (5.5 Gy) and retinal tissue assessed 8 and 12 weeks later. CX₃CR1 deficiency altered the early replenishment of monocytes in the posterior iris but not in the iris stroma, choroid or retina. In six month old chimeric mice, there were significantly higher GFP⁺ cell densities in the uveal tract when compared to non-irradiated 8-12 week old Cx ₃ cr1 ^+/gfp mice. Additionally, MHC Class II expression was upregulated on hyalocytes and GFP⁺ cells in the peripheral retina and the repopulation of microglia appeared to be more rapid in C57BL/6J mice compared to BALB/c mice. Transient expression of MHC Class II was observed on retinal vasculature in sublethally irradiated mice. These data indicate CX₃CR1-deficiency only slightly alters monocyte-derived cell replenishment in the murine uveal tract. Lethal irradiation leads to long-term increase in monocytic cell density in the uveal tract and retinal microglial activation, possibly as a sequelae to local irradiation induced injury. Microglial replenishment in this model appears to be strain dependent.     

Changes in L-phenylalanine ammonia-lyase activity and isoflavone phytoalexins accumulation in soybean seedlings infected with Sclerotinia sclerotiorum

Soybean [Glycine max (L.) Merr.] cultivars (Meli, Alisa, Sava and 1511/99) were grown up to V1 phase (first trifoliate and one node above unifoliate) and then inoculated with Sclerotinia sclerotiorum (Lib.) de Bary under controlled conditions. Changes in L-phenylalanine ammonia-lyase (PAL) activity and isoflavone phytoalexins were recorded 12, 24, 48 and 72 h after the inoculation. Results showed an increase in PAL activity in all four examined soybean cultivars 48 h after the inoculation, being the highest in Alisa (2-fold higher). Different contents of total daidzein, genistein, glycitein and coumestrol were detected in all samples. Alisa and Sava increased their total isoflavone content (33.9% and 6.2% higher than control, respectively) as well as 1511/99, although 48 h after the inoculation its content decreased significantly. Meli exhibited the highest rate of coumestrol biosynthesis (72 h after the inoculation) and PAL activity (48 h after the inoculation). All investigated cultivars are invariably susceptible to this pathogen. Recorded changes could point to possible differences in mechanisms of tolerance among them.     

Morphology versus DNA barcoding: two sides of the same coin. A case study of Ceutorhynchus erysimi and C. contractus identification

Genotyping of 2 well‐known weevil species from the genus Ceutorhynchus (Coleoptera: Curculionidae) distributed in west Palearctic, C. erysimi and C. contractus, revealed phenotype versus genotype inconsistencies in a set of 56 specimens (25 C. erysimi and 31 C. contractus) collected from 25 locations in Serbia and Montenegro. An analysis of the mitochondrial cytochrome oxidase subunit I gene (COI), widely used as a barcoding region, and a nuclear gene, elongation factor‐1α (EF‐1α), revealed stable genetic divergence among these species. The average uncorrected pairwise distances for the COI and EF‐1α genes were 3.8%, and 1.3%, respectively, indicating 2 genetically well‐segregated species. However, the genetic data were not congruent with the phenotypic characteristics of the studied specimens. In the first place, C. erysimi genotypes were attached to specimens with phenotypic characteristics of C. contractus. Species‐specific PCR‐RFLP assays for the barcoding gene COI were applied for the molecular identification of 101 additional specimens of both morphospecies (33 C. erysimi and 68 C. contractus) and were found to confirm this incongruity. The discrepancy between the genetic and morphological data raises the question of the accuracy of using a barcoding approach, as it may result in misleading conclusions about the taxonomic position of the studied organism. Additionally, the typological species concept shows considerable weakness when genetic data are not supported with phenotypic characteristics as in case of asymmetric introgression, which may cause certain problems, especially in applied studies such as biological control programs in which the biological properties of the studied organisms are the main focus.     

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.     

Effect of Cultivation Time on Production of Antifungal Metabolite(s) by Streptomyces hygroscopicus in Laboratory‐Scale Bioreactor

Biological control agents offer one of the best alternatives to reduce the use of pesticides . Fungi from the genera Alternaria, Colletotrichum and Fusarium are listed among the most important storage pathogens of apple fruits. During storage, transport and marketing, pathogenic fungi can cause significant losses of apple fruits. This investigation studied the potential of Streptomyces hygroscopicus as a biocontrol agent against pathogenic fungi obtained from apple fruit samples expressing rot symptoms. Production of antifungal metabolites by S. hygroscopicus was carried out in 3‐l bench‐scale bioreactor (Biostat^® Aplus, Sartorius AG, Germany) during 7 days. Fermentation was carried out at 27°C with aeration rate of 0.5 vvm and agitation rate of 200 rpm. The aim was to analyse bioprocess parameters of batch biofungicide production in medium containing glucose as a carbon source and to examine at which stage of bioprocess production of antifungal metabolite(s) against six phytopathogenic fungi occurs. In vitro antifungal activity of the produced metabolites against six fungi of the genera Colletotrichum, Fusarium and Alternaria grown on potato dextrose agar were determined every 24 h using wells technique. Antifungal activity of cell‐free culture filtrate and filtrate treated with high temperature were tested. The filtrate treated with high temperature did not show any antifungal activity suggesting that active components are thermo unstable. Stationary phase of growth occurred between the third and fourth day of cultivation when production of secondary metabolites begins. Obtained results showed that maximal antifungal activity is achieved on fifth and sixth day of S. hygroscopicus cultivation under defined conditions (inhibition zone diameter higher than 30 mm for all test fungi).