Plasticity of the Nuclear Pore Complex Revealed With Proteomics

Cellular functions are performed by the concerted action of macromolecular assemblies. These protein machines rarely have permanent, invariable structures. Instead, the subunits forming the macromolecular assemblies exchange between free and bound states, changing the composition, conformation and function of the assembly. Characterizing the function of macromolecular assemblies, therefore, requires the study of their dynamics. I have developed a strategy for in vivo assessment of macromolecular plasticity and have successfully applied it to the yeast nuclear pore complex (NPC), one of the largest and most elaborate protein machines in the cell. NPC is a massive protein assembly situated in the nuclear envelope and mediating macromolecular transport between the nucleus and cytoplasm. To study the NPC plasticity I have combined multiple methodologies: affinity capture, metabolic labeling and mass spectrometry. Moreover, to identify the required affinity capture conditions I have developed a high-throughput screen, which has successfully worked with numerous protein complexes, in addition to the NPC, to reveal novel interactions and arrangements of proteins within a complex. My study on NPC plasticity has produced the first comprehensive description of NPC subunit dynamics, although for the most dynamic subunits I can only approximately estimate the exchange rate. I have also mapped the subunit dynamics on the architecture of the NPC. The central core of the NPC was revealed to be very stable, while the peripherally associated subunits exchanged with varying rates. Moreover, the rate of exchange appeared well correlated with the strength of the interaction that the NPC subunit forms with the scaffold. Notably, NPC subunits directly involved in the transport function of the NPC were among the most dynamic ones, implying that modulating the association dynamics of those subunits might alter the nuclear transport function. The dynamics of NPC modules, as well as the plasticity changes in NPC subunit mutants have suggested that the core of the complex is extremely stable because of the additive effect of numerous weak interactions formed between NPC subunits and also the surrounding nuclear envelope membrane. The individual weak interactions may be a mechanism to prevent off target NPC assembly. Lastly, I propose a study to directly test the existence of a potential NPC repair mechanism, which is a topic of heated debate and has direct implication for aging in all eukaryotes

Characterization of Cuzd1 expression and development of in vivo CRISPR technology in Atlantic salmon (Salmo salar L.)

The Atlantic salmon (Salmo salar L.) is a commercially important species that exhibits an anadromous life cycle involving migratory movements between freshwater and marine environments. Understanding the fundamental biology of the Atlantic salmon, particularly during key life history transitions such as smoltification, is therefore of great importance. In light of modern biotechnology and the completed reference genome for Atlantic Salmon, we can now examine the parr-smolt transition using a hypothesis-free characterization of gene expression analyses. We hypothesized that CUB and Zona Pellucida-like Domain 1 (Cuzd1) gene played a critical role in changing gill physiology during smoltification. To test this hypothesis, the study had two major aims: 1) to assess Cuzd1 expression in the whole fish through ontogeny and in various tissues during smoltification; 2) to develop in vivo CRISPR/Cas9 tools in Atlantic salmon, including the design of novel guides to enable functional characterization of Cuzd1. There was a significant difference in gene expression of Cuzd1 between the organ development stage and eyed stage, hatching, fin development, mid- point sampling, and approaching external feeding. This could provide valuable insight into the gene's developmental patterns. The gene expression during smoltification in various tissues during smoltification also varied with statistically significant differences in the muscle, gill, and brain. This finding implies that the role of Cuzd1 during smoltification is not limited to the gill. Thus, Cuzd1 exhibited tissue-specific expression patterns and could shed light on the mechanisms involved in the physiological changes during smoltification. A successful knockout of the positive control Slc45a2, a gene that affects pigmentation, was achieved. However, no evidence of Cuzd1 knockout could be observed, and further investigation is needed to enable a proper assessment. The result of this study demonstrated strong potential of the tested methodology for improving our understanding of Atlantic salmon biology and contributing to the development of novel methods for genetic characterization. Future work could be aimed at improving the methodology, including the use of promiscuous gRNAs and more comprehensive methods for genetic characterization. This work represents a significant step towards understanding the complex developmental transition of smoltification in Atlantic salmon. Overall, this study expands our characterization of Cuzd1 expression in Atlantic salmon and reports the first successful in vivo CRISPR knockouts at UiT, The Arctic University of Norway

A critical analysis of employee motivation in the spa and wellness industry

http://www.ester.ee/record=b4743075*es

ASSESSING DISPROPORTIONATE TERRITORIAL DEVELOPMENT: INSIGHTS FROM 10 COUNTRIES

Territorial development disparities are an undeniable reality for all the countries of the world, which implies that no country can practically avoid them. However, how different countries respond to these disparities is another matter. The effectiveness of policies in overcoming territorial development disparities depends significantly on how deeply these disparities are recognized and studied. In this context, assessing disparities in territorial development is necessary, and the article proposes a methodology for its implementation. The methodology examines territorial development indexes and their relative standard deviation. In the article, the developed methodology was also applied to 10 countries, as a result of which the levels of territorial development disparities in Canada, Poland, Bulgaria, Hungary, Finland, Serbia, Georgia, Moldova, Kazakhstan, and China were evaluated. Based on the assessments, general conclusions are also presented for each country in the article

Luminescence and radiation defects in irradiated ruby

The excitations of luminescence in irradiated and non-irradiated ruby crystals are investigated by means of highly polarized synchrotron radiation. In the VUV luminescence spectra the existence of quick and slow emission was observed in irradiated and nonirradiated crystals. The luminescence bands with maximum at 3.8 eV are produced by F+ centers. A new type of quick luminescence was established for the band at 4.6 eV. It is called cross-luminescence and is connected with the recombination of valence band electrons with holes in low-lying core levels. It is shown that the band at 3.0 eV is not due to anionic centers (F-centers), but is determined by a short lifetime emission center

Metagenomics-based proficiency test of smoked salmon spiked with a mock community

An inter-laboratory proficiency test was organized to assess the ability of participants to perform shotgun metagenomic sequencing of cold smoked salmon, experimentally spiked with a mock community composed of six bacteria, one parasite, one yeast, one DNA, and two RNA viruses. Each participant applied its in-house wet-lab workflow(s) to obtain the metagenomic dataset(s), which were then collected and analyzed using MG-RAST. A total of 27 datasets were analyzed. Sample pre-processing, DNA extraction protocol, library preparation kit, and sequencing platform, influenced the abundance of specific microorganisms of the mock community. Our results highlight that despite differences in wet-lab protocols, the reads corresponding to the mock community members spiked in the cold smoked salmon, were both detected and quantified in terms of relative abundance, in the metagenomic datasets, proving the suitability of shotgun metagenomic sequencing as a genomic tool to detect microorganisms belonging to different domains in the same food matrix. The implementation of standardized wet-lab protocols would highly facilitate the comparability of shotgun metagenomic sequencing dataset across laboratories and sectors. Moreover, there is a need for clearly defining a sequencing reads threshold, to consider pathogens as detected or undetected in a food sample

Proficiency Testing of Metagenomics-Based Detection of Food-Borne Pathogens Using a Complex Artificial Sequencing Dataset

Metagenomics-based high-throughput sequencing (HTS) enables comprehensive detection of all species comprised in a sample with a single assay and is becoming a standard method for outbreak investigation. However, unlike real-time PCR or serological assays, HTS datasets generated for pathogen detection do not easily provide yes/no answers. Rather, results of the taxonomic read assignment need to be assessed by trained personnel to gain information thereof. Proficiency tests are important instruments of validation, harmonization, and standardization. Within the European Union funded project COMPARE [COllaborative Management Platform for detection and Analyses of (Re-) emerging and foodborne outbreaks in Europe], we conducted a proficiency test to scrutinize the ability to assess diagnostic metagenomics data. An artificial dataset resembling shotgun sequencing of RNA from a sample of contaminated trout was provided to 12 participants with the request to provide a table with per-read taxonomic assignments at species level and a report with a summary and assessment of their findings, considering different categories like pathogen, background, or contaminations. Analysis of the read assignment tables showed that the software used reliably classified the reads taxonomically overall. However, usage of incomplete reference databases or inappropriate data pre-processing caused difficulties. From the combination of the participants\u2019 reports with their read assignments, we conclude that, although most species were detected, a number of important taxa were not or not correctly categorized. This implies that knowledge of and awareness for potentially dangerous species and contaminations need to be improved, hence, capacity building for the interpretation of diagnostic metagenomics datasets is necessary