Stimulated Emission Depletion (STED) microscopy enables superresolution imaging of fluorescently marked nano-structures in vivo. Biological investigations are often hindered by the difficulty of relating super-resolved structures to other non-labeled features. Here we demonstrate that the similarity in optical design of Spiral Phase Contrast (SPC) and STED microscopes allows straightforward implementation of a phase contrast channel into a STED microscope in widefield and scanning modes. This method allows dual imaging and overlay in two contrast modes in fixed and in living specimens, in which double labeling is especially challenging. Living GFP- and YPF-stained neurons are imaged in one label-free phase contrast and one high-resolution STED channel. Furthermore, we implement SPC in widefield and scanning modes demonstrating that scanning confocal SPC yields the highest optical contrast. The latter configuration can provide contour detection or highlights and shadows reminiscent of differential interference contrast.
Engrailed 1 (En1) and 2 (En2) code for closely related homeoproteins acting as transcription factors and as signaling molecules that contribute to midbrain and hindbrain patterning, to development and maintenance of monoaminergic pathways, and to retinotectal wiring. En2 has been suggested to be an autism susceptibility gene and individuals with autism display an overexpression of this homeogene but the mechanisms remain unclear. We addressed in the present study the effect of exogenously added En2 on the morphology of hippocampal cells that normally express only low levels of Engrailed proteins. By means of RT-qPCR, we confirmed that En1 and En2 were expressed at low levels in hippocampus and hippocampal neurons, and observed a pronounced decrease in En2 expression at birth and during the first postnatal week, a period characterized by intense synaptogenesis. To address a putative effect of Engrailed in dendritogenesis or synaptogenesis, we added recombinant En1 or En2 proteins to hippocampal cell cultures. Both En1 and En2 treatment increased the complexity of the dendritic tree of glutamatergic neurons, but only En2 increased that of GABAergic cells. En1 increased the density of dendritic spines both in vitro and in vivo. En2 had similar but less pronounced effect on spine density. The number of mature synapses remained unchanged upon En1 treatment but was reduced by En2 treatment, as well as the area of post-synaptic densities. Finally, both En1 and En2 elevated mTORC1 activity and protein synthesis in hippocampal cells, suggesting that some effects of Engrailed proteins may require mRNA translation. Our results indicate that Engrailed proteins can play, even at low concentrations, an active role in the morphogenesis of hippocampal cells. Further, they emphasize the over-regulation of GABA cell morphology and the vulnerability of excitatory synapses in a pathological context of En2 overexpression.
Background: Methicillin-resistant Staphylococcus aureus is a resistant bacterium responsible for food poisoning. The present survey was done to assess the genotyping and antibiotic resistance of MRSA strains isolated from raw and frozen meat samples and assessment of the antimicrobial effects of Origanum vulgare against MRSA isolates. Methods: Two-hundred raw and frozen meat samples were collected and presence of MRSA was assessed using the culture. Disk diffusion was used to assess the antibiotic resistance of isolates. Isolates were subjected to PCR to obtain the virulence characters. Gas chromatography was used to determine chemical components of O. vulgare. Minimum Inhibitory Concentration and Minimum Bacterial Concentration of O. vulgare essential oil was also assessed. Results: Thirty two out of 200 meat samples (16%) were positive for MRSA. MRSA isolates had the highest resistance toward penicillin (100%), ceftaroline (100%), tetracycline (87.50%), and erythromycin (68.75%). Distribution of seA and eta genes amongst the raw and frozen meat samples were 75% and 58.33% and 50% and 33.33%, respectively. Β-Caryophyllene (31.10%), 1-Octen-3-ol (18.41%), 1,8-Cineole (15.19%), and Thujopsene (8.20%) were the most commonly identified chemical components.
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