ERIL1, the plant homologue of ERI‐1, is involved in the processing of chloroplastic rRNAs

Proteins belonging to the enhancer of RNA interference‐1 subfamily of 3′–5′ exoribonucleases participate in divergent RNA pathways. They degrade small interfering RNAs (siRNAs), thus suppressing RNA interference, and are involved in the maturation of ribosomal RNAs and the degradation of histone messenger RNAs (mRNAs). Here, we report evidence for the role of the plant homologue of these proteins, which we termed ENHANCED RNA INTERFERENCE‐1‐LIKE‐1 (ERIL1), in chloroplast function. In vitro assays with AtERIL1 proved that the conserved 3′–5′ exonuclease activity is shared among all homologues studied. Confocal microscopy revealed that ERL1, a nucleus‐encoded protein, is targeted to the chloroplast. To gain insight into its role in plants, we used Nicotiana benthamiana and Arabidopsis thaliana plants that constitutively overexpress or suppress ERIL1. In the mutant lines of both species we observed malfunctions in photosynthetic ability. Molecular analysis showed that ERIL1 participates in the processing of chloroplastic ribosomal RNAs (rRNAs). Lastly, our results suggest that the missexpression of ERIL1 may have an indirect effect on the microRNA (miRNA) pathway. Altogether our data point to an additional piece of the puzzle in the complex RNA metabolism of chloroplasts.     

Multifunctionality of prostatic acid phosphatase in prostate cancer pathogenesis

The role of human prostatic acid phosphatase (PAcP, {"type":"entrez-protein","attrs":{"text":"P15309","term_id":"130730"}}P15309|PPAP_HUMAN) in prostate cancer was investigated using a new proteomics tool termed signal sequence swapping (replacement of domains from the native cleaved amino terminal signal sequence of secretory/membrane proteins with corresponding regions of functionally distinct signal sequence subtypes). This manipulation preferentially redirects proteins to different pathways of biogenesis at the endoplasmic reticulum (ER), magnifying normally difficult to detect subsets of the protein of interest. For PAcP, this technique reveals three forms identical in amino acid sequence but profoundly different in physiological functions, subcellular location, and biochemical properties. These three forms of PAcP can also occur with the wildtype PAcP signal sequence. Clinical specimens from patients with prostate cancer demonstrate that one form, termed ^PLPAcP, correlates with early prostate cancer. These findings confirm the analytical power of this method, implicate ^PLPAcP in prostate cancer pathogenesis, and suggest novel anticancer therapeutic strategies.     

A simple, versatile method for GFP-based super-resolution microscopy via nanobodies

We developed a method to use any GFP-tagged construct in single-molecule super-resolution microscopy. By targeting GFP with small, high-affinity antibodies coupled to organic dyes, we achieved nanometer spatial resolution and minimal linkage error when analyzing microtubules, living neurons and yeast cells. We show that in combination with libraries encoding GFP-tagged proteins, virtually any known protein can immediately be used in super-resolution microscopy and that simplified labeling schemes allow high-throughput super-resolution imaging.     

Dimorphic DNA variation in the anionic peroxidase gene AtPrx53 of Arabidopsis thaliana

The DNA polymorphism in the AtPrx53 gene which encodes anionic peroxidase was analyzed in 20 Arabidopsis thaliana accessions. There are two divergent sequence types (Col and Dj-like haplotypes) in the AtPrx53 gene that differ by 2 indel and 16 non-singleton nucleotide polymorphisms including 5 nucleotide polymorphic sites responsible for 4 deduced amino acid replacements. Two of the amino acid substitutions (Phe/Ser180and Asp/Asn270) could be responsible for the difference in electrophoretic mobility of AtPrx53 allozymes. One of them (Phe/Ser180) lies within the hypervariable region, indicating that this amino acid polymorphism is subjected to balancing selection. The revealed difference between deduced allozymes is related to the dimorphism in mobility of three major anionic peroxidase isoforms which according to previously established data encoded by AtPrx53 gene. The haplotype Col which included 12 accessions from three different continents is characterized by faster mobility of three isoforms in comparison with the Dj haplotype represented by eight accessions. There is a significant association between the haplotype and several developmental traits: leaf number, flowering time, main stem height etc. Lines of the Dj haplotype have shorter duration of vegetative stages and flower earlier than most of Col haplotype accessions. The reasons of this association are discussed.     

Ecophysiological properties of cultivable heterotrophic bacteria and yeasts dominating in phytocenoses of Galindez Island,maritime Antarctica

Antarctic plants are stable specific microenvironments for microbial colonization that are still less explored. In this study, we investigated cultivable heterotrophic bacteria and yeasts dominating in plant samples collected from different terrestrial biotopes near Ukrainian Antarctic Base on Galindez Island, maritime Antarctica. Phylogenetic analysis revealed affiliation of the bacterial isolates to genera Pseudomonas, Stenotrophomonas, Brevundimonas, Sporosarcina, Dermacoccus, Microbacterium, Rothia and Frondihabitans, and the yeast isolates to genera Rhodosporidium, Cryptococcus, Leucosporidiella, Candida and Exophiala. Some ecophysiological properties of isolated strains were determined that are important in response to different stresses such as psychro- and halotolerance, UV-resistance and production of hydrolytic enzymes. The majority of isolates (88 %) was found to be psychrotolerant; all are halotolerant. Significant differences in survival subsequent to UV-C radiation were observed among the isolates, as measured by culturable counts. For the bacterial isolates, lethal doses in the range 80–600 J m^?2 were determined, and for the yeast isolates—in the range 300–1,000 J m^?2. Dermacoccus profundi U9 and Candida davisiana U6 were found as most UV resistant among the bacterial and yeast isolates, respectively. Producers of caseinase, gelatinase, β-glucosidase, and cellulase were detected. To the best of our knowledge, this is the first report on isolation of UV resistant strain D. profundi, and Frondihabitans strain from Antarctica, and on detection of cellulase activity in Antarctic yeast strain C. davisiana. The results obtained contribute to clarifying adaptation strategies of Antarctic microbiota and its possible role in functional stability of Antarctic biocenoses. Stress tolerant strains were detected that are valuable for ecological and applied studies.     

Membrane interactions of the hydrophobic segment of diacylglycerol kinase epsilon

Diacylglycerol kinase epsilon (DGKε) is unique among mammalian DGK isoforms in having a segment of hydrophobic amino acids as a putative membrane anchor. To model the conformation, and stoichiometry of this segment in membrane-mimetic environments, we have prepared a peptide corresponding to this hydrophobic segment of DGKε of sequence KKKKLILWTLCSVLLPVFITFWKKKKK-NH₂. Flanking Lys residues mimic the natural setting of this peptide in DGKε, while facilitating peptide synthesis and characterization. Circular dichroism and fluorescence spectroscopic analysis demonstrated that the peptide has increased helical content and significant blue shifts in the presence of anionic - but not zwitterionic - bilayer membranes. When labeled with fluorophores that can undergo fluorescence resonance energy transfer, the peptide was found to dimerize - a result also observed from migration rates on SDS-PAGE gels under both reducing and non-reducing disulfide bridge conditions. The peptide was shown to preferentially interact with cholesterol in lipid films comprised of homogeneous mixtures of cholesterol and phosphatidylcholine, yet the presence of cholesterol in hydrated vesicle bilayers decreases its helical content. The peptide was also able to inhibit the activity of DGKε protein in vitro. Our overall findings suggest that the peptide ultimately cannot leave the bulk water for attachment/insertion into the outer leaflet of an erythrocyte-like bilayer, yet its core sequence is sufficiently hydrophobic to insert into membrane core regions when membrane attachment is promoted by electrostatic attraction to anionic lipid head groups of the inner leaflet of an erythrocyte-like bilayer.     

Local bone-marrow exposure: how to interpret the data on stable chromosome aberrations in circulating lymphocytes? (some comments on the use of FISH method for dose reconstruction for Techa riverside Residents)

The method of fluorescence in situ hybridization (FISH) applied to peripheral blood T lymphocytes is used for retrospective dose estimation, and the results obtained from the analysis of stable chromosomal aberrations are usually interpreted as a dose accumulated in the red bone marrow (RBM). However, after local internal exposure of the RBM, doses derived from FISH were found to be lower than those derived from direct measurements of radionuclides accumulated in the bodies of exposed persons. These results were obtained for people residing near the Techa River contaminated by ^89,90Sr (beta-emitters) in 1949–1956 (Chelyabinsk Oblast, Russia). A new analysis has been performed of the combined results of FISH studies (n = 178) undertaken during 1994–2012 for persons living on the Techa Riverside. Analysis confirms the lower slope of the translocation yield per Gy (8.0 ± 0.7 × 10^?3) for Techa residents in comparison with FISH data for donors with external exposures (11.6 ± 1.6 × 10^?3, Tawn et al., Radiat Res 184(3):296–303, 2015). It was suggested that some portion of T cells remained unexposed, because they represented the descendants of T cell progenitors, which had migrated to the thymus before the start of ^89,90Sr intakes. To clarify this problem, the dynamics of T-cell Genera (TG), combining all descendants of specific T-cell progenitor reaching the thymus, was considered. Rates of TGs produced by RBM over different age periods of human life were estimated with the use of the mathematic model of T-cell homeostasis (Bains, Mathematical modeling of T-cell homeostasis. A thesis submitted for the degree of Doctor of Philosophy of the University College London. http://discovery.ucl.ac.uk/20159/1/20159.pdf, 2010). The rate of TG loss during the lifetime was assumed to be very small in comparison with production rate. The recirculation of mature T lymphocytes in contaminated RBM was taken into account. According to our model estimates, at the time of blood sampling, the fraction of exposed T lymphocytes (whose progenitors were irradiated) ranged from 20 to 80% depending on the donors’ age at the start of exposure to ^89,90Sr. Dose to T lymphocytes, estimated from FISH studies, should be about 0.6–0.9 of RBM dose for residents of the upper Techa region and about 0.4–0.8 in the middle Techa region. Our results could explain the lower value of translocation yield per Gy obtained for Techa residents. The approaches for further model improvement and validation are discussed in this paper.     

Metal-enhanced fluorescence immunoassays using total internal reflection and silver island-coated surfaces

We present a generic immunoassay platform that uses enhanced total internal reflection fluorescence in the proximity of silver island films (SIFs), a surface coating consisting of metal (silver) particles. This platform is used with a model immunoassay where a protein antigen, rabbit immunoglobulin G, was immobilized on the SIF-coated glass surface. The signal from a fluorescent dye-labeled anti-rabbit antibody binding to the surface antigen was detected; different color dyes have been tested. Close placement of the fluorophore to surface-bound silver nanostructures results in dramatic signal enhancement (up to 40-fold) on the SIFs as compared with the glass slides. Use of the total internal reflection mode of excitation has significant advantages (over classic front-face excitation) for practical assay development. The limited evanescent wave excitation volume makes it possible to minimize the background signal and use the immunoassay with no need for any washing steps.     

Regulation of telomere length and suppression of genomic instability in human somatic cells by Ku86

Ku86 plays a key role in nonhomologous end joining in organisms as evolutionarily disparate as bacteria and humans. In eukaryotic cells, Ku86 has also been implicated in the regulation of telomere length although the effect of Ku86 mutations varies considerably between species. Indeed, telomeres either shorten significantly, shorten slightly, remain unchanged, or lengthen significantly in budding yeast, fission yeast, chicken cells, or plants, respectively, that are null for Ku86 expression. Thus, it has been unclear which model system is most relevant for humans. We demonstrate here that the functional inactivation of even a single allele of Ku86 in human somatic cells results in profound telomere loss, which is accompanied by an increase in chromosomal fusions, translocations, and genomic instability. Together, these experiments demonstrate that Ku86, separate from its role in nonhomologous end joining, performs the additional function in human somatic cells of suppressing genomic instability through the regulation of telomere length.