Pretubulysin derived probes as novel tools for monitoring the microtubule network via activity-based protein profiling and fluorescence microscopy

Microtubules (mt) are highly dynamic polymers composed of alpha- and beta-tubulin monomers that are present in all dividing and non-dividing cells. A broad variety of natural products exists that are known to interfere with the microtubule network, by either stabilizing or de-stabilizing these rope-like polymers. Among those tubulysins represent a new and potent class of cytostatic tetrapeptides originating from myxobacteria. Early studies suggested that tubulysins interact with the eukaryotic cytoskeleton by inhibition of tubulin polymerization with EC50 values in the picomolar range. Recently, pretubulysins have been described to retain the high tubulindegradation activity of their more complex tubulysin relatives and represent an easier synthetic target with an efficient synthesis already in place. Although tubulin has been suggested as the dedicated target of tubulysin a comprehensive molecular target analysis of pretubulysin in the context of the whole proteome has not been carried out so far. Here we utilize synthetic chemistry to develop two pretubulysin photoaffinity probes which were applied in cellular activity-based protein profiling and imaging studies in order to unravel and visualize dedicated targets. Our results clearly show a remarkable selectivity of pretubulysin for beta-tubulin which we independently confirmed by a mass-spectrometry based proteomic profiling platform as well as by tubulin antibody based co-staining on intact cells

Evaluation studies of robotic rollators by the user perspective: A systematic review

Background: Robotic rollators enhance the basic functions of established devices by technically advanced physical, cognitive, or sensory support to increase autonomy in persons with severe impairment. In the evaluation of such Ambient Assisted Living solutions, both the technical and user perspectives are important to prove usability, effectiveness, and safety, and to ensure adequate device application.Objective: The aim of this systematic review is to summarize the methodology of studies evaluating robotic rollators with focus on the user perspective and to give recommendations for future evaluation studies.Methods: A systematic literature search up to December 31, 2014 was conducted based on the Cochrane Review methodology using the electronic databases PubMed and IEEE Xplore. Articles were selected according to the following inclusion criteria: Evaluation studies of robotic rollators documenting human-robot interaction, no case reports, published in English language.Results: Twenty-eight studies were identified that met the predefined inclusion criteria. Large heterogeneity in the definitions of the target user group, study populations, study designs, and assessment methods was found across the included studies. No generic methodology to evaluate robotic rollators could be identified. We found major methodological shortcomings related to insufficient sample descriptions and sample sizes, and lack of appropriate, standardized and validated assessment methods. Long-term use in habitual environment was also not evaluated.Conclusions: Apart from the heterogeneity, methodological deficits in most of the identified studies became apparent. Recommendations for future evaluation studies include: clear definition of target user group, adequate selection of subjects, inclusion of other assistive mobility devices for comparison, evaluation of the habitual use of advanced prototypes, adequate assessment strategy with established, standardized and validated methods, and statistical analysis of study results. Assessment strategies may additionally focus on specific functionalities of the robotic rollators allowing an individually tailored assessment of innovative features to document their added value

Фізичні властивості магнітних наночастинок упровідній матриці

Дисертацію присвячено комплексному дослідженню магніторезистивних і оптичних властивостей та газової чутливості приладових систем на основі масивів магнітних наночастинок (НЧ) NiFe2O4, СоFe2O4, Fe3O4 у провідній немагнітній матриці Ag або мультишарового графену. У роботі проаналізовано зв'язок між структурним станом НЧ і магніторезистивними, магнітооптичними та оптичними властивостями наноприладових систем. Встановлено механізми формування масивів спін-вентильних переходів при збільшенні товщини провідної матриці Ag від 5 до 20 нм і умов її температурної обробки (Тв = 600 К) та їх внесок у величину магнітоопору. Експериментально вивчено характер зміни електричного опору наноструктурованих шарів від умов температурної обробки (Тв = 1100 К) та досліджено вплив декорування поверхні мультишарового графену масивами НЧ NiFe2O4 для покращення чутливості до шкідливого газу NO2 на 40 %. Результати досліджень можуть бути використані як практичні рекомендації при побудові наноструктурованих чутливих елементів датчиків різного функціонального призначення.Диссертация посвящена комплексному исследованию магниторезистивных и оптических свойств, газовой чувствительности приборных систем на основе массивов магнитных наночастиц (НЧ) NiFe2O4, СоFe2O4, Fe3O4 в проводящей немагнитной матрице Ag или мультислойного графена (МСГ). В работе изучены особенности и условия формирования двухмерных наноструктур из наночастиц и мультислойного графена на подложках SiO2 (500 нм) / Si (001) с использованием методик спин-коатинга и Ленгмюра – Блоджетт. Проанализирована связь между структурным состоянием НЧ и магниторезистивными, магнитооптическими и оптическими свойствами наноприборных систем. Установлены зависимости оптических параметров от характера распределения массивов наночастиц или фрагментов МСГ на подложке. Установлены механизмы формирования массивов спин-вентильных переходов при увеличении толщины проводящей матрицы Ag от 5 до 20 нм и условий ее температурной обработки (То = 600 К) и их вклад в величину магнитосопротивления. Разработана теоретическая модель, позволяющая оценить величину вклада рассеивания электронов на ферромагнитных частицах в электрическое сопротивление массивов магнитных наночастиц в проводящей матрице Ag до и после еѐ термообработки (То = 600 К). Экспериментально изучен характер изменения электрического сопротивления наноструктурированных слоев от условий температурной обработки (То = 1100 К) и исследовано влияние декорирования поверхности мультислойного графена массивами наночастиц NiFe2O4 для улучшения чувствительности к вредному газу NO2 на 40 %, а также предложен механизм взаимодействия чувствительного материала приборных структур и газа NO2. Исследовано влияние температуры (То = 1100 К) на величину газовой чувствительности МСГ и установлены оптимальные условия термообработки для получения наиболее эффективных чувствительных элементов с минимальным уровнем шума. Изучено влияние размеров фрагментов МСГ на газовую чувствительность датчиков на их основе. Результаты исследований могут быть использованы как практические рекомендации при построении наноструктурированных чувствительных элементов датчиков разного функционального назначения.The thesis is devoted to the complex investigation of magnetoresistive and optical properties and sensitivity to gases of instrumentation systems based on arrays of nanoparticles (NP) NiFe2O4, СоFe2O4, Fe3O4 in Ag conductive matrix or multilayered graphene. In this work an interconnection between the structural features of the NP and magnetoresistive, magnetooptical and optical properties of instrumentation systems was analyzed. The mechanisms of formation of arrays of spin-valve junctions while increasing the thickness of Ag conductive matrix from 5 to 20 nm, conditions of it temperature treatment and their influence on the value of magnetoresistance were established. Electrical resistance changes of nanostructured layers depends on conditions of thermal treatment (Tt = 1100 K) were experimentally studied and an effect of decoration of multilayered graphene by arrays of NiFe2O4 NPs for increasing of sensitivity to NO2 gas by 40% was investigated. The results of research can be utilized as practical recommendation while develop the nanostructured sensitive elements of the sensors with different functional purposes

A systematic review of study results reported for the evaluation of robotic rollators from the perspective of users

© 2017 Informa UK Limited, trading as Taylor & Francis Group. Purpose: To evaluate the effectiveness and perception of robotic rollators (RRs) from the perspective of users. Methods: Studies identified in a previous systematic review published on 2016 on the methodology of studies evaluating RRs by the user perspective were re-screened for eligibility based on the following inclusion criteria: evaluation of the human–robot interaction from the user perspective, use of standardized outcome measurements, and quantitative presentation of study results. Results: Seventeen studies were eligible for inclusion. Due to the clinical and methodological heterogeneity across studies, a narrative synthesis of study results was conducted. We found conflicting results concerning the effectiveness of the robotic functionalities of the RRs. Only a few studies reported superior user performance or reduced physical demands with the RRs compared to unassisted conditions or conventional assistive mobility devices; however, without providing statistical evidence. The user perception of the RRs was found to be generally positive. Conclusions: There is still no sufficient evidence on the effectiveness of RRs from the user perspective. More well-designed, high-quality studies with adequate study populations, larger sample sizes, appropriate assessment strategies with outcomes specifically tailored to the robotic functionalities, and statistical analyses of results are required to evaluate RRs at a higher level of evidence.Implications for Rehabilitation RRs cover intelligent functionalities that focus on gait assistance, obstacle avoidance, navigation assistance, sit-to-stand transfer, body weight support or fall prevention. The evaluation from the user perspective is essential to ensure that RRs effectively address users’ needs, requirements and preferences. The evidence on the effectiveness of RRs is severely hampered by the low methodological quality of most of the available studies. RRs seem generally to be perceived as positive by the users. There is very limited evidence on the effectiveness and benefits of RRs compared to conventional assistive mobility devices. Further research with high methodological quality needs to be conducted to reach more robust conclusions about the effectiveness of RRs

Qualitative Analyse von Chats und anderer usergenerierter Kommunikation

Der Text bietet einen Überblick über qualitative Forschung usergenerierter Inhalte, die unverzichtbar ist, um Prozesse der Sinngenerierung und der Herstellung von Sinnzusammenhängen im Internet zu verstehen. Mit der fortschreitenden Verbreitung des Web 2.0 wächst auch die Möglichkeit für Internetnutzer, eigene Beiträge auf Webseiten mit unterschiedlichsten Kommunikationsgeräten (PC, Tablet PC, Mobiltelefon etc.) zu publizieren und über diese Beiträge miteinander in Austausch zu treten. Weblogs und YouTube, Facebook oder Online-Foren sind Beispiele für Internetplattformen, die kollektiv produzierte Informationen, einzelne Berichte und Kommentare oder Diskussionsbeiträge von Nutzern enthalten. Mit dem Ausbau von Instant-Messaging-Diensten über Mobiltelefone breitet sich zudem seit einiger Zeit rasch eine annähernd synchrone Chat-Kommunikation unter den Nutzern aus. Internetplattformen wie WhatsApp, TikTok, Instagram oder Snapchat ermöglichen das Erstellen nutzergenerierter Chats im Kreis von Einzelpersonen oder größeren Gruppen. Im ersten Teil des vorliegenden Beitrags werden soziotechnische und sprachliche Merkmale usergenerierter Kommunikation diskutiert und im zweiten Teil wird auf den qualitativen Forschungsprozess eingegangen

The Microtubule-Targeting Agent Pretubulysin Impairs the Inflammatory Response in Endothelial Cells by a JNK-Dependent Deregulation of the Histone Acetyltransferase Brd4

The anti-inflammatory effects of depolymerizing microtubule-targeting agents on leukocytes are known for a long time, but the potential involvement of the vascular endothelium and the underlying mechanistic basis is still largely unclear. Using the recently synthesized depolymerizing microtubule-targeting agent pretubulysin, we investigated the antiinflammatory potential of pretubulysin and other microtubule-targeting agents with respect to the TNF-induced leukocyte adhesion cascade in endothelial cells, to improve our understanding of the underlying biomolecular background. We found that treatment with pretubulysin reduces inflammation in vivo and in vitro via inhibition of the TNF-induced adhesion of leukocytes to the vascular endothelium by down-regulation of the pro-inflammatory cell adhesion molecules ICAM-1 and VCAM-1 in a JNK-dependent manner. The underlying mechanism includes JNK-induced deregulation and degradation of the histone acetyltransferase Bromodomaincontaining protein 4. This study shows that depolymerizing microtubule-targeting agents, in addition to their established effects on leukocytes, also significantly decrease the inflammatory activation of vascular endothelial cells. These effects are not based on altered pro-inflammatory signaling cascades, but require deregulation of the capability of cells to enter constructive transcription for some genes, setting a baseline for further research on the prominent antiinflammatory effects of depolymerizing microtubule-targeting agents

Pretubulysin: From Hypothetical Biosynthetic Intermediate to Potential Lead in Tumor Therapy

Pretubulysin is a natural product that is found in strains of myxobacteria in only minute amounts. It represents the first enzyme-free intermediate in the biosynthesis of tubulysins and undergoes post-assembly acylation and oxidation reactions. Pretubulysin inhibits the growth of cultured mammalian cells, as do tubulysins, which are already in advanced preclinical development as anticancer and antiangiogenic agents. The mechanism of action of this highly potent compound class involves the depolymerization of microtubules, thereby inducing mitotic arrest. Supply issues with naturally occurring derivatives can now be circumvented by the total synthesis of pretubulysin, which, in contrast to tubulysin, is synthetically accessible in gram-scale quantities. We show that the simplified precursor is nearly equally potent to the parent compound. Pretubulysin induces apoptosis and inhibits cancer cell migration and tubulin assembly in vitro. Consequently, pretubulysin appears to be an ideal candidate for future development in preclinical trials and is a very promising early lead structure in cancer therapy

Dynamics of Transcription Regulation in Human Bone Marrow Myeloid Differentiation to Mature Blood Neutrophils.

Neutrophils are short-lived blood cells that play a critical role in host defense against infections. To better comprehend neutrophil functions and their regulation, we provide a complete epigenetic overview, assessing important functional features of their differentiation stages from bone marrow-residing progenitors to mature circulating cells. Integration of chromatin modifications, methylation, and transcriptome dynamics reveals an enforced regulation of differentiation, for cellular functions such as release of proteases, respiratory burst, cell cycle regulation, and apoptosis. We observe an early establishment of the cytotoxic capability, while the signaling components that activate these antimicrobial mechanisms are transcribed at later stages, outside the bone marrow, thus preventing toxic effects in the bone marrow niche. Altogether, these data reveal how the developmental dynamics of the chromatin landscape orchestrate the daily production of a large number of neutrophils required for innate host defense and provide a comprehensive overview of differentiating human neutrophils

Protein tyrosine phosphatases expression during development of mouse superior colliculus

Protein tyrosine phosphatases (PTPs) are key regulators of different processes during development of the central nervous system. However, expression patterns and potential roles of PTPs in the developing superior colliculus remain poorly investigated. In this study, a degenerate primer-based reverse transcription-polymerase chain reaction (RT-PCR) approach was used to isolate seven different intracellular PTPs and nine different receptor-type PTPs (RPTPs) from embryonic E15 mouse superior colliculus. Subsequently, the expression patterns of 11 PTPs (TC-PTP, PTP1C, PTP1D, PTP-MEG2, PTP-PEST, RPTPJ, RPTPε, RPTPRR, RPTPσ, RPTPκ and RPTPγ) were further analyzed in detail in superior colliculus from embryonic E13 to postnatal P20 stages by quantitative real-time RT-PCR, Western blotting and immunohistochemistry. Each of the 11 PTPs exhibits distinct spatiotemporal regulation of mRNAs and proteins in the developing superior colliculus suggesting their versatile roles in genesis of neuronal and glial cells and retinocollicular topographic mapping. At E13, additional double-immunohistochemical analysis revealed the expression of PTPs in collicular nestin-positive neural progenitor cells and RC-2-immunoreactive radial glia cells, indicating the potential functional importance of PTPs in neurogenesis and gliogenesis