Külakiige ehitamine Puskusse

Loov-praktiline lõputöö käsitleb külakiige valmimist Puskusse, Läänemaale. Töös andis autor põgusa ülevaate külakiige ajaloost ja kaardistas olemasolevad neljavõlliga kiiged Hiiumaal. Hiiumaal kogutud teabe põhjal loodi ja täiustati püstitatud kiige konstruktsiooni, arvestades ka kliendi soove ja mõtteid. Leiti konstruktsioonile sobivad materjalid ning külakiige ehituseks optimaalne ehitustehnoloogia ja -teostus. Käesoleva lõputöö esimene osa kirjeldas uurimistöö kulgu, kus autor käsitles eelinfo hankimist tööde teostamiseks. See hõlmas kogutud taustinformatsiooni, olemasolevate kiikede kaardistamist, kliendiga suhtlust ja materjali valikut. Teises osas tehti ülevaade kiige ehitusprotsessist, vundamendist-katuseni. Selles osas kajastus ehitustehnoloogia valik, kiige detailide valmistamine, ühenduste arendamine, paigaldamine ja katuse loomine. Külakiikesid iseloomustab eriilmelisus ja erinevad mahud, küll on neil kõigil läbivaks tunnuseks nende võimekus suurt seltskonda/kogukonda kiigutada ning ikka ja jälle inimesi kokku kutsuda. Töö raames sai luua uue külakiige, vaadeldes teiste kiige ehitajate töid ning loodetavasti õpitud nende vigadest. Uut külakiike rajades õnnestub ehk elus hoida kiikumise ja kiige paigaga seonduvat kombestikku. Ehitustraditsioonid on ajas muutuvad, autor pakub oma lõputöös ühte paljudest võimalikest lahendustest külakiige ehituseks

Cutaneous T-Cell Lymphoma (CTCL) Cell Line-Derived Extracellular Vesicles Contain HERV-W-Encoded Fusogenic Syncytin-1

Non peer reviewe

Discovery of small molecules that activate RNA-methylation through cooperative binding to the METTL3/14/WTAP complex active site

Chemical modifications of RNA provide an additional, epitranscriptomic, level of control over cellular functions. N-6-methylated adenosines (m6As) are found in several types of RNA, and their amounts are regulated by methyltransferases and demethylases. One of the most important enzymes catalyzing generation of m6A on mRNA is the trimer N-6-methyltransferase METTL3-14-WTAP complex. Its activity has been linked to such critical biological processes as cell differentiation, proliferation, and death. We used in silico-based discovery to identify small-molecule ligands that bind to METTL3-14-WTAP and determined experimentally their binding affinity and kinetics, as well as their effect on enzymatic function. We show that these ligands serve as activators of the METTL3-14-WTAP complex

Metabolomic Profiling of Extracellular Vesicles and Alternative Normalization Methods Reveal Enriched Metabolites and Strategies to Study Prostate Cancer-Related Changes

Body fluids are a rich source of extracellular vesicles (EVs), which carry cargo derived from the secreting cells. So far, biomarkers for pathological conditions have been mainly searched from their protein, (mi) RNA, DNA and lipid cargo. Here, we explored the small molecule metabolites from urinary and platelet EVs relative to their matched source samples. As a proof-of-concept study of intra-EV metabolites, we compared alternative normalization methods to profile urinary EVs from prostate cancer patients before and after prostatectomy and from healthy controls. Methods: We employed targeted ultra-performance liquid chromatography-tandem mass spectrometry to profile over 100 metabolites in the isolated EVs, original urine samples and platelets. We determined the enrichment of the metabolites in the EVs and analyzed their subcellular origin, pathways and relevant enzymes or transporters through data base searches. EV-and urine-derived factors and ratios between metabolites were tested for normalization of the metabolomics data. Results: Approximately 1 x 10(10) EVs were sufficient for detection of metabolite profiles from EVs. The profiles of the urinary and platelet EVs overlapped with each other and with those of the source materials, but they also contained unique metabolites. The EVs enriched a selection of cytosolic metabolites including members from the nucleotide and spermidine pathways, which linked to a number of EV-resident enzymes or transporters. Analysis of the urinary EVs from the patients indicated that the levels of glucuronate, D-ribose 5-phosphate and isobutyryl-L-carnitine were 2-26-fold lower in all pre-prostatectomy samples compared to the healthy control and post-prostatectomy samples (p <0.05). These changes were only detected from EVs by normalization to EV-derived factors or with metabolite ratios, and not from the original urine samples. Conclusions: Our results suggest that metabolite analysis of EVs from different samples is feasible using a high-throughput platform and relatively small amount of sample material. With the knowledge about the specific enrichment of metabolites and normalization methods, EV metabolomics could be used to gain novel biomarker data not revealed by the analysis of the original EV source materials.Peer reviewe

Improved Characterization of EV Preparations Based on Protein to Lipid Ratio and Lipid Properties.

In recent years the study of extracellular vesicles has gathered much scientific and clinical interest. As the field is expanding, it is becoming clear that better methods for characterization and quantification of extracellular vesicles as well as better standards to compare studies are warranted. The goal of the present work was to find improved parameters to characterize extracellular vesicle preparations. Here we introduce a simple 96 well plate-based total lipid assay for determination of lipid content and protein to lipid ratios of extracellular vesicle preparations from various myeloid and lymphoid cell lines as well as blood plasma. These preparations included apoptotic bodies, microvesicles/microparticles, and exosomes isolated by size-based fractionation. We also investigated lipid bilayer order of extracellular vesicle subpopulations using Di-4-ANEPPDHQ lipid probe, and lipid composition using affinity reagents to clustered cholesterol (monoclonal anti-cholesterol antibody) and ganglioside GM1 (cholera toxin subunit B). We have consistently found different protein to lipid ratios characteristic for the investigated extracellular vesicle subpopulations which were substantially altered in the case of vesicular damage or protein contamination. Spectral ratiometric imaging and flow cytometric analysis also revealed marked differences between the various vesicle populations in their lipid order and their clustered membrane cholesterol and GM1 content. Our study introduces for the first time a simple and readily available lipid assay to complement the widely used protein assays in order to better characterize extracellular vesicle preparations. Besides differentiating extracellular vesicle subpopulations, the novel parameters introduced in this work (protein to lipid ratio, lipid bilayer order, and lipid composition), may prove useful for quality control of extracellular vesicle related basic and clinical studies

Isolation of High-Purity Extracellular Vesicles by the Combination of Iodixanol Density Gradient Ultracentrifugation and Bind-Elute Chromatography From Blood Plasma

Background: Extracellular vesicles (EVs) (isolated from blood plasma) are currently being extensively researched, both as biomarkers and for their therapeutic possibilities. One challenging aspect to this research is the efficient isolation of high-purity EVs from blood plasma in quantities sufficient for in vivo experiments. In accordance with this challenge, the aim of this study was to develop an isolation method in which to separate the majority of EVs from major impurities such as lipoprotein particles and the abundant plasma proteins albumin and fibrinogen. Methods: Samples of rat blood were centrifuged to remove cells, platelets, large EVs and protein aggregates without prior filtration. Density gradient ultracentrifugation was performed by loading plasma sample onto 50, 30, and 10% iodixanol layers and then centrifuged at 120,000 ×g for 24 h. Ten fractions (F1-10) were collected from top to bottom. Fractions with the highest EV content were further purified by ultracentrifugation, size exclusion, or bind-elute chromatography. Efficiency and purity were assessed by Western blots. Morphology and size distribution of particles were examined by dynamic light scattering and electron microscopy (EM). Results: The highest band intensities of EV markers Alix, Tsg101 and CD81 were detected by Western blot in F6 of small-scale DGUC (61.5 ± 10.4%; 48.1 ± 5.8%; 41.9 ± 3.8%, respectively) at a density of 1.128-1.174 g/mL, where the presence of vesicles with a mean diameter of 38 ± 2 nm was confirmed by EM and DLS. Only 1.4 ± 0.5% of LDL and chylomicron marker, 3.0 ± 1.3% of HDL marker, and 9.9 ± 0.4% of albumin remained in the EV-rich F6. However, 32.8 ± 1.5% of the total fibrinogen beta was found in this fraction. Second-step purification by UC or SEC did not improve EV separation, while after BEC on HiScreen Capto Core 700 albumin and lipoprotein contamination were below detection limit in EV-rich fractions. However, BEC decreased efficiency of EV isolation, and fibrinogen was still present in EV-rich fractions. Conclusion: This is the first demonstration that DGUC is able to markedly reduce the lipoprotein content of EV isolates while it separates EVs with high efficiency. Moreover, isolation of lipoprotein- and albumin-free EVs from blood plasma can be achieved by DGUC followed by BEC, however, on the expense of reduced EV yield

Nelja võlli ja katusega külakiik Läänemaale Pusku külla / Constructing a four-shaft swing in Pusku, Läänemaa

The use and construction of village swings, including the corresponding know-how, has not disappeared from Estonia during the last couple of decades. The common construction solution for the village swing has a traditional outer shape, based on the constructor’s/developer’s intuition and visual images. Usually a swing located nearby and possessing good proportions is taken as an example. Also new materials or construction details can be tried out. In the area of technology and due to the safety requirements, the more modern and familiar connections of details are used as they are considered to be more reliable. Fundamental change in the overall shape of the swing is very rare. This article focuses on the four-shaft village swing, which was introduced to Hiiumaa at the beginning of the 20th century. The peculiarity of the swing is a base platform with benches on which swingers sit and which remains in a horizontal position to the ground during swinging. For that reason the swing has two parallel shafts supported on the upper part of the posts of the construction; two more shafts are fixed to the thills hanging from the shafts on which the platform of the swing is supported. In order to become familiar with the topic, the author mapped four-shaft swings in Hiiumaa and used the models for planning and constructing a new swing. The common features of swings in Hiiumaa included diagonals placed inbetween the poles of the swing and the thills fixed to the upper shafts. The proportions of swings observed varied; however, the structural connections were solved similarly. It is more common for swings to have benches than not. According to the photos of the Museum of Hiiumaa it can be concluded that four-shaft swings have been known here for more than a hundred years, which might reflect the influence of seaman who after travelling in the wide world came back home and tried to copy something they had seen in practice. The builder gained a new experience when he used pitched timber in the construction and planned a low slanting roof. The article describes the construction process of the swing, and photos enable to gain an overview of the work stages. The swing built in Pusku village in 2018–2019 follows the proportions of earlier swings but some constructional improvements have been made. First the pier foundation was constructed to establish the swing on stable ground so if any details breaks, it will be easy to replace it. Poles were made of pitched pine, which ensures a longer service life for the construction&nbsp; in exterior conditions (pine trees were barked three years before felling to let the timber saturate with resin). As an idiosyncratic experiment a low slanting shingle roof was built on the swing, to protect the connections of details and to extend the service life of the swing and prolong the swinging time. The metal parts of the swing were made by masters of the trade; wooden details were made in the workshop and installed on site so that the building process did not depend on weather conditions and this also saved money and time otherwise needed on the construction site. Keywords: village swings, swinging, log construction, facilitie