Expression of a glycosylated GFP as a bivalent reporter in exocytosis

The complex-type N-linked glycans of plants differ markedly in structure from those of animals. Like those of insects and mollusks they lack terminal sialic acid(s) and may contain an α-(1,3)-fucose (Fuc) linked to the proximal GlcNAc residue and/or a β-(1,2)-xylose (Xyl) residue attached to the proximal mannose (Man) of the glycan core. N-glycosylated GFPs were used in previous studies showing their effective use to report on membrane traffic between the ER and the Golgi apparatus in plant cells. In all these cases glycosylated tags were added at the GFP termini. Because of the position of the tag and depending on the sorting and accumulation site of these modified GFP, there is always a risk of processing and degradation, and this protein design cannot be considered ideal. Here, we describe the development of three different GFPs in which the glycosylation site is internally localized at positions 80, 133, or 172 in the internal sequence. The best glycosylation site was at position 133. This glycosylated GFPgl133 appears to be protected from undesired processing of the glycosylation site and represents a bivalent reporter for biochemical and microscopic studies. After experimental validation, we can conclude that amino acid 133 is an effective glycosylation site and that the GFPgl133 is a powerful tool for in vivo investigations in plant cell biology.     

Long-term within-basin isolation patterns,different conservation units,and interspecific mitochondrial DNA introgression in an amphipod endemic to the ancient Lake Skadar system,Balkan Peninsula

1. Investigation of biodiversity, including genetic diversity within species, is crucial for rational planning of nature conservation and species protection. Endemics or narrow-ranged taxa, sensitive to alteration of physical and chemical conditions, may serve as local bioindicators.
2. Homoeogammarus scutarensis is an endemic, cold-adapted amphipod crustacean, inhabiting fragmented spring habitats, within a relatively small area of the Lake Skadar basin. The basin is recently under heavy anthropogenic influence leading to habitat deterioration.
3. Genetic diversity within H. scutarensis was investigated using the mitochondrial cytochrome c oxidase subunit I and the nuclear 28S ribosomal RNA markers. Genetic diversity within the mitochondrial (mt)DNA appeared to be surprisingly high as for the limited spatial scale. The history of within-species divergence dates back at least to the Pliocene.
4. A case of interspecific mtDNA introgression from the endemic H. scutarensis to the widespread and partly sympatric Homoeogammarus thoni was observed in one of the studied populations.
5. Demographic analysis showed that the demography of H. scutarensis population has been stable, without evidence of expansion. Thus, we believe that H. scutarensis, responsive to environmental changes, is a proper model mirroring the conditions in sensitive habitats of the Lake Skadar basin. To protect ongoing evolutionary processes, the conservation measures for this species should take into account the complex fine-scale lineage endemism patterns. This case study may also provide a hint for planning conservation strategies in other geologically old spring systems.
6. Most of the localities where H. scutarensis occurs provide refugia for divergent mtDNA lineages, suggesting prolonged isolation between sites and independent evolutionary histories. We conclude that H. scutarensis is composed of two or three conservation units. One of them occupies the headwaters of the Crnojevića River and spring in its valley. The other is widespread throughout the sub-lacustrine springs and the Zeta Plain; however, a group of unique haplotypes is found in springs in the Podhum Bay. Their presence and distribution should be considered while planning any investments or conservation strategies.

     

The Proteomic Landscape of Centromeric Chromatin Reveals an Essential Role for the Ctf19CCAN Complex in Meiotic Kinetochore Assembly

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The microbial threat: Can rare earths help?

Despite an ever increasing demand for reliable and cheap methods in the detection and quantification of microbes, surprisingly few investigations have explored or utilized the luminescence of rare earths in the microbial context, neither in conventional, that is, plating and microscopic imaging techniques, nor in advanced methods like fluorescence flow cytometry. We have thus investigated the potential of some rare earth complexes and hybrid materials for microbiological analysis. We found fairly simple procedures for internal staining (dyes inside the bacterial cell) and external staining (dyes on the cell surface). The present paper is predominantly relying on microscopic imaging and luminescence spectroscopies (excitation, emission, decay times), but also evaluates model rare earth microspheres to estimate an eventual rare earth based stain for a fast and sensitive bacteria enumeration with luminescence flow cytometry.