Fluorescence Methods for the Analysis of Microtubule/Microfilament Involvement in the Regulation of Endothelial Barrier Function

The endothelial monolayer is located on the inner surface of blood vessels and provides an important barrier function controlling the transport of metabolites and nutrients through the vessel wall, both toward the circulating blood and in the direction of the underlying tissues. This function is provided by cytoskeletal structures that generate contractile and tensile forces, existing in equilibrium in the intact endothelium. In the case of cytoskeleton rearrangements, there are changes in the shape of cells and the formation of intercellular spaces, which lead to endothelial dysfunction. Deep understanding of endothelial barrier function maintaining is a crucial problem because this phenomenon is common for a number of pathological states and diseases (inflammation, asthma, sepsis, acute lung injury, diabetes, etc.) and can lead to severe organ dysfunction, as well as a complication upon the treatment by a number of anticancer pharmacological drugs. Microtubules and the actin cytoskeleton function in cooperation in normal endothelium and under conditions of the barrier loss. In this review, we describe the application of modern fluorescence methods for investigation and analysis of the individual characteristics of cytoskeletal elements whose reorganization affects endothelial permeability, to emphasize the role of microtubules/microfilament crosstalk in EC barrier regulation

Opera Software "Discover" service : Research on Relevancy and Freshness of the Suggested Content and Ways to Improve It

Opera Software ASA is a Norwegian company developing Internet browsers for a variety of devices. Starting from version 15 it includes a feature “Discover”, which is a news recommendation system created to suggest news articles right in the browser window. The purpose of this thesis work was to conduct a research on relevancy of content in Discover and suggest improvements for the service. First, a research on the main content filtering techniques in recommender systems was done. By analysing filtering methods, different advantages and drawbacks were revealed. After covering the theory on recommendation systems, currently used filtering techniques in Discover were presented. It was concluded that news recommendation systems gained considerable popularity over the recent years, with increasing amount of people preferring news aggregators to other traditional ways to retrieve news stories. As news aggregation is a competitive market, users have a wide choice of services to use. Considering this tendency, the importance of relevant content in Discover was considered to play a vital part in user engagement. Prior to suggesting improvements, different relevancy criteria were presented. Those factors help to determine the significance of a news story to the general type of reader. By evaluating the current situation with content relevancy in Discover, a number of disadvantages in the current system were presented. The final chapter of this thesis includes the suggestions for improving the relevancy of content in Discover, which are based on previously stated relevancy factors. The improvements are proposed for general recommendations, as well as for personalized recommendations.Opera Software ASA on norjalainen yritys, joka kehittää internet-selaimia ja erilaisia laitteita varten.Versiosta 15 alkaen se sisältää "Discover" ominaisuuden, joka on uutisten suositusjärjestelmä ja joka ehdottaa uutisartikkeleita selaimessa esitettäväksi. Tämän opinnäytetyön tarkoituksena oli toteuttaa tutkimus ”Discover” uutisten sisällön suosituspalvelun käyttökelpoisuudesta ja ehdottaa parannuksia toimintaan. Tutkimus aloitettiin suositusjärjestelmän uutisten pääsisällön suodatustekniikoista. Analysoimalla suodatusmenetelmiä saatiin erilaiset edut ja haitat paljastettua. Suositusjärjestelmien teorian esityksen jälkeen työssä esitellään nykyisin käytössä olevat suodatusmenetelmät. Todettiin, että uutisten suositusjärjestelmät ovat saavuttaneet huomattavaa suosiota viime vuosina ja yhä suurempi määrä käyttäjiä suosii mieluummin uutisten yhdistelypalvelua verrattuna perinteisiin tapoihin hakea uutisia. Uutisten yhdistely on kilpailtu ala markkinoilla ja käyttäjillä on valittavissaan monia vaihtoehtoja. Tälläisen suuntauksen ollessa vallalla katsottiin olevan tärkeää, että ”Discover” palvelun tarjoama uutissisältö on asiallista. Ennen parannusehdotusten tekemistä esitellään uutisten asiallisuuden eri kriteerit. Kriteerit auttavat määrittämään uutisten merkityksen tavalliselle lukijalle. Työssä arvioitiin ”Discover” palvelun tarjoama sisällön ajankohtaisuus ja saatiin samalla esille useita epäkohtia toiminnassa. Viimeinen työn luku sisältää parannusehdotuksia ”Discover” palvelun sisällön asiallisuuden parantamiseen ja jotka perustuvat aiemmin esitettyihin kriteereihin. Parannuksia ehdotetaan ”Discover” palvelun yleisiin suosituksiin ja henkilökohtaisiin suosituksiin

User voice overview: topic recognition and sentiment analysis of customer feedback in the B2C sector

With deep customer knowledge being a competitive advantage for a lot of companies, especially those in the B2C field, integration of Support teams into Product organization is considered essential nowadays. However, a proper feedback loop between those teams is usually a common pitfall, especially for companies that have a lot of customer contacts. Consolidating feedback and getting an overview of the users’ voice is a challenging task, and this research study investigates whether using Natural Language Processing (NLP) can help to automate this manual process. This thesis describes the implementation of the proof-of-concept prototype for conducting practical research focused around topic recognition and sentiment analysis domains. After introducing the main concepts and concluding, which NLP methods would suit best in the customer support setting, the research environment gets appointed. Namely, work is conducted using Zendesk for corpus extraction and Python for the prototype implementation. In turn, GuidedLDA package gets selected for topic recognition, VADER — for sentiment analysis, and PyInquirer for the CLI implementation. To evaluate the performance of the prototype, it was tested on real customer requests data. In order to closely supervise the results, the tests were first carried out on a small data-set using a supervised approach. Afterwards, tests on larger datasets were performed and the accuracy rate got evaluated using random samples. These experiments showed satisfactory results for both of the evaluation methods. Supervised tests managed to reach the accuracy rate of 94% for topic recognition and 93% for sentiment analysis. With the larger data sets, random sample evaluation showed 75% success rate for topic assignments, and 80% success rate for sentiment recognition. Aforementioned evaluations helped to conclude that the chosen methods for automating feedback analysis can, in fact, be utilised in the “real world” scenario. While occasional human input and supervision is still required, possible inaccuracies are outbalanced by the advantages of applying the NLP algorithms. With the achieved accuracy rates, both GuidedLDA and VADER can be considered suitable for the purpose of analysing the sentiment of customer feedback and providing an overview of the users’ voice

The “Third Violin” in the Cytoskeleton Orchestra—The Role of Intermediate Filaments in the Endothelial Cell’s Life

The endothelium plays an important role in the transcytosis of lipoproteins. According to one of the theories, endothelial injury is a triggering factor for the development of atherosclerosis, and intracellular structures, including components of the endotheliocyte cytoskeleton (microtubules, actin, and intermediate filaments), are involved in its development. In contrast to the proteins of tubulin-based microtubules and actin microfilaments, intermediate filaments are comprised of various tissue-specific protein members. Vimentin, the main protein of endothelial intermediate filaments, is one of the most well-studied of these and belongs to type-III intermediate filaments, commonly found in cells of mesenchymal origin. Vimentin filaments are linked mechanically or by signaling molecules to microfilaments and microtubules by which coordinated cell polarisation and migration are carried out, as well as control over several endotheliocyte functions. Moreover, the soluble vimentin acts as an indicator of the state of the cardiovascular system, and the involvement of vimentin in the development and course of atherosclerosis has been demonstrated. Here we discuss current concepts of the participation of vimentin filaments in the vital activity and functioning of endothelial cells, as well as the role of vimentin in the development of inflammatory processes and atherosclerosis

A tissue-mimicking phantom with flexible optical properties for studying photoacoustic response of nanoparticles

Photoacoustic imaging (PAI) is a rapidly growing imaging modality, which combines high contrast of optical absorption with deep penetration depth of ultrasound. When combined with endogenous contrast agents based on light-absorbing nanoparticles (NPs), PAI can visualize various biological processes and tissues. Here, we describe a simple experimental setup based on a tissue-mimicking phantom with flexible optical properties for studying photoacoustic (PA) response of NPs. Our approach is based on a polyacrylamide gel phantom with independently variable optical absorption and scattering. The phantom allows one to model and study PA response of contrast agents with diverse spatial distributions and concentrations. To demonstrate high potential of the developed experimental setup, we prepared a phantom with optical properties matching human prostate tissue and performed a PAI of laser-synthesized titanium nitride (TiN) NPs, distributed in a disk-shaped area, located 10 mm under the phantom surface. We believe that our approach will contribute the successful development of clinical PAI with NPs-based contrast agents

Huntingtin and Other Neurodegeneration-Associated Proteins in the Development of Intracellular Pathologies: Potential Target Search for Therapeutic Intervention

Neurodegenerative diseases are currently incurable. Numerous experimental data accumulated over the past fifty years have brought us closer to understanding the molecular and cell mechanisms responsible for their development. However, these data are not enough for a complete understanding of the genesis of these diseases, nor to suggest treatment methods. It turns out that many cellular pathologies developing during neurodegeneration coincide from disease to disease. These observations give hope to finding a common intracellular target(s) and to offering a universal method of treatment. In this review, we attempt to analyze data on similar cellular disorders among neurodegenerative diseases in general, and polyglutamine neurodegenerative diseases in particular, focusing on the interaction of various proteins involved in the development of neurodegenerative diseases with various cellular organelles. The main purposes of this review are: (1) to outline the spectrum of common intracellular pathologies and to answer the question of whether it is possible to find potential universal target(s) for therapeutic intervention; (2) to identify specific intracellular pathologies and to speculate about a possible general approach for their treatment

Colocalization Analysis of Cytoplasmic Actin Isoforms Distribution in Endothelial Cells

Actin cytoskeleton is an essential component of living cells and plays a decisive role in many cellular processes. In mammals, β- and γ-actin are cytoplasmic actin isoforms in non-muscle cells. Despite minor differences in the amino acid sequence, β- and γ-actin localize in different cell structures and perform different functions. While cytoplasmic β-actin is involved in many intracellular processes including cell contraction, γ-actin is responsible for cell mobility and promotes tumor transformation. Numerous studies demonstrate that β- and γ-actin are spatially separated in the cytoplasm of fibroblasts and epithelial cells; this separation is functionally determined. The spatial location of β/γ-actin in endothelial cells is still a subject for discussion. Using super-resolution microscopy, we investigated the β/γ-actin colocalization in endotheliocytes and showed that the β/γ-actin colocalization degree varies widely between different parts of the marginal regions and near the cell nucleus. In the basal cytoplasm, β-actin predominates, while the ratio of isoforms evens out as it moves to the apical cytoplasm. Thus, our colocalization analysis suggests that β- and γ-actin are segregated in the endotheliocyte cytoplasm. The segregation is greatly enhanced during cell lamella activation in the nocodazole-induced endothelial barrier dysfunction, reflecting a different functional role of cytoplasmic actin isoforms in endothelial cells

Modeling of Neurodegenerative Diseases: ‘Step by Step’ and ‘Network’ Organization of the Complexes of Model Systems

Neurodegenerative diseases have acquired the status of one of the leading causes of death in developed countries, which requires creating new model systems capable of accurately reproducing the mechanisms underlying these pathologies. Here we analyzed modern model systems and their contribution to the solution of unexplored manifestations of neuropathological processes. Each model has unique properties that make it the optimal tool for modeling certain aspects of neurodegenerative disorders. We concluded that to optimize research, it is necessary to combine models into complexes that include organisms and artificial systems of different organizational levels. Such complexes can be organized in two ways. The first method can be described as “step by step”, where each model for studying a certain characteristic is a separate step that allows using the information obtained in the modeling process for the gradual study of increasingly complex processes in subsequent models. The second way is a ‘network’ approach. Studies are carried out with several types of models simultaneously, and experiments with each specific type are adjusted in conformity with the data obtained from other models. In our opinion, the ‘network‘ approach to combining individual model systems seems more promising for fundamental biology as well as diagnostics and therapy

Synthesis of Titanium Nitride Nanoparticles by Pulsed Laser Ablation in Different Aqueous and Organic Solutions

International audienceOwing to a strong photothermal response in the near-IR spectral range and very low toxicity, titanium nitride (TiN) nanoparticles (NPs) synthesized by pulsed laser ablation in liquids (PLAL) present a novel appealing object for photo-induced therapy of cancer, but the properties of these NPs still require detailed investigation. Here, we have elaborated methods of femtosecond laser ablation from the TiN target in a variety of liquid solutions, including acetonitrile, dimethylformamide, acetone, water, and H2O2, to synthesize TiN NPs and clarify the effect of liquid type on the composition and properties of the formed NPs. The ablation in all solvents led to the formation of spherical NPs with a mean size depending on the liquid type, while the composition of the NPs ranged from partly oxidized TiN to almost pure TiO2, which conditioned variations of plasmonic peak in the region of relative tissue transparency (670–700 nm). The degree of NP oxidation depended on the solvent, with much stronger oxidation for NPs prepared in aqueous solutions (especially in H2O2), while the ablation in organic solvents resulted in a partial formation of titanium carbides as by-products. The obtained results contribute to better understanding of the processes in reactive PLAL and can be used to design TiN NPs with desired properties for biomedical applications

Long-term live cells observation of internalized fluorescent Fe@C nanoparticles in constant magnetic field

International audienceBackground: Theranostics application of superparamagnetic nanoparticles based on magnetite and maghemite isimpeded by their toxicity. The use of additional protective shells significantly reduced the magnetic properties of thenanoparticles. Therefore, iron carbides and pure iron nanoparticles coated with multiple layers of onion-like carbonsheath seem to be optimal for biomedicine. Fluorescent markers associated with magnetic nanoparticles provide reliablemeans for their multimodal visualization. Here, biocompatibility of iron nanoparticles coated with graphite-likeshell and labeled with Alexa 647 fluorescent marker has been investigated.Methods: Iron core nanoparticles with intact carbon shells were purified by magnetoseparation after hydrochloricacid treatment. The structure of the NPs (nanoparticles) was examined with a high resolution electron microscopy.The surface of the NPs was alkylcarboxylated and further aminated for covalent linking with Alexa Fluor 647 fluorochrometo produce modified fluorescent magnetic nanoparticles (MFMNPs). Live fluorescent imaging and correlativelight-electron microscopy were used to study the NPs intracellular distribution and the effects of constant magneticfield on internalized NPs in the cell culture were analyzed. Cell viability was assayed by measuring a proliferative poolwith Click-IT labeling.Results: The microstructure and magnetic properties of superparamagnetic Fe@C core–shell NPs as well as theirendocytosis by living tumor cells, and behavior inside the cells in constant magnetic field (150 mT) were studied.Correlative light-electron microscopy demonstrated that NPs retained their microstructure after internalization bythe living cells. Application of constant magnetic field caused orientation of internalized NPs along power lines thusdemonstrating their magnetocontrollability. Carbon onion-like shells make these NPs biocompatible and enablelong-term observation with confocal microscope. It was found that iron core of NPs shows no toxic effect on the cellphysiology, does not inhibit the cell proliferation and also does not induce apoptosis.Conclusions: Non-toxic, biologically compatible superparamagnetic fluorescent MFMNPs can be further used forbiological application such as delivery of biologically active compounds both inside the cell and inside the wholeorganism, magnetic separation, and magnetic resonance imaging (MRI) diagnostics