Analysis of knockout/knockin mice that express a mutant FasL lacking the intracellular domain

Fas ligand (FasL; CD178; CD95L) is a type II transmembrane protein belonging to the tumour necrosis factor family; its binding to the Fas receptor (CD95; APO-1) triggers apoptosis in the receptor-bearing cell. Signalling through this pathway plays a pivotal role during the immune response and in immune system homeostasis. Similar to other TNF family members, the intracellular domain has been reported to transmit signals to the inside of the FasL-bearing cell (reverse signalling). Recently, we identified the proteases ADAM10 and SPPL2a as molecules important for the processing of FasL. Protease cleavage releases the intracellular domain, which then is able to translocate to the nucleus and to repress reporter gene activity. To study the physiological importance of FasL reverse signalling in vivo, we established knockout/knockin mice with a FasL deletion mutant that lacks the intracellular portion (FasLDeltaIntra). Co-culture experiments confirmed that the truncated FasL protein is still capable of inducing apoptosis in Fas-sensitive cells. Preliminary immune histochemistry data suggest that, in contrast to published data, the absence of the intracellular FasL domain does not alter the intracellular FasL localization in activated T cells. We are currently investigating signalling and proliferative capacities of T cells derived from homozygous FasLDeltaIntra mice to validate a co-stimulatory role of FasL reverse signalling

Розробка агентно-орієнтованих компонентів програмного забезпечення для вилучення маркетингової інформації з web

The article is devoted to researching the processes of extracting marketing information from the Web space. Conclusions are drawn on the need to introduce an information marketing system into modern business activities. A decision has been taken to develop software for the collection and analysis of marketing information. Identified and analyzed the main problems of collecting marketing information in the Web space. External systems for extracting and processing marketing information from the Web space were considered. During the analysis of the subject area, functional and non-functional requirements for the software being developed were formulated. Requirements for the selection of technologies for the development of an information system were defined. The analysis of software development technologies is carried out and the approach to the development of a software component is chosen. Such approaches to software development as: object-oriented programming, service-oriented architecture, component-oriented programming, agent-oriented programming were analyzed. A decision has been made to use the agent three-tier architecture in software development. The most commonly used programming languages in programming systems were: Java, KIF, KQML, AgentSpeak, April, TeleScript, Tcl / Tk, Oz. Analyzed such popular agent platforms and their functions as: JADE, Cougaar, ZEUS, Jason. For the development of software, the JADE platform was chosen, its classes, methods and interfaces were examined. The advantages and peculiarities of the SOLID principle are analyzed. In detail, the levels of the CLEAN architecture are examined. And also explained the possibilities of software implementation of this architecture. A software architecture was developed for the data collection system. In accordance with the requirements, a selection of software development tools has been made. It was decided to use the programming language Java, Spring Framework, GoF design pattern, the template Dependency Injection, SOLID and CLEAN architectural principles. A software component was developed for marketing information gathering systems, which allows to optimize this process. The limitations and ways to improve the software system are analyzed.Статтю присвячено питанням дослідження процесів вилучення маркетингової інформації з Web-простору. Зроблено висновки про необхідність введення інформаційної маркетингової системи в сучасну підприємницьку діяльність. Прийнято рішення про розробку програмного забезпечення для збору та аналізу маркетингової інформації. Виявлено та проаналізовано основні проблеми збору маркетингової інформації у Web-просторі. Були розглянуті зовнішні системи по вилученню та обробці маркетингової інформації з Web-простору. В ході аналізу предметної області були сформульовані функціональні і нефункціональні вимоги до розроблюємого програмного забезпечення. Були визначені вимоги до вибору технологій для розробки інформаційної системи. Проведено аналіз технологій розробки програмного забезпечення та обрано підхід до розробки програмного компонента. Були проаналізовані такі підходи до розробки програмного забезпечення як: об’єктно-орієнтоване програмування, сервіс-орієнтована архітектура, компонентно-орієнтоване програмування, агентно-орієнтоване програмування. Прийнято рішення про використання агентної трирівневої архітектури в розробці програмного забезпечення. Були розглянуті найбільш часто використовувані в агентних системах мови програмування: Java, KIF, KQML, AgentSpeak, April, TeleScript, Tcl/Tk, Oz. Проаналізовано такі популярні агентні платформи і їх функції як: JADE, Cougaar, ZEUS, Jason. Для розробки програмного забезпечення була обрана платформа JADE, розглянуті її класи, методи і інтерфейси. Проаналізовано переваги та особливості принципу SOLID. В деталях розглянуті рівні архітектури CLEAN. А також зроблені пояснення можливостей програмної реалізації цієї архітектури. Була розроблена програмна архітектура для системи зі збору даних. Відповідно до вимог зроблений вибір інструментів розробки програмного продукту. Прийнято рішення про використання мови програмування Java, Spring Framework, GoF патерну проектування, шаблону Dependency Injection, SOLID і CLEAN архітектурних принципів. Був розроблений програмний компонент для систем збору маркетингової інформації, що дозволяє оптимізувати цей процес. Проаналізовано обмеження і шляхи поліпшення програмної систем

Combined targeting of MEK and the glucocorticoid receptor for the treatment of RAS-mutant multiple myeloma

Background Multiple myeloma (MM) remains incurable despite recent therapeutic advances. RAS mutations are frequently associated with relapsed/refractory disease. Efforts to target the mitogen-activated protein kinase (MAPK) pathway with the MEK inhibitor, trametinib (Tra) have been limited by toxicities and the development of resistance. Dexamethasone (Dex) is a corticosteroid commonly used in clinical practice, to enhance efficacy of anti-myeloma therapy. Therefore, we hypothesised that the combination of Tra and Dex would yield synergistic activity in RAS-mutant MM. Methods The response of human MM cell lines to drug treatment was analysed using cell proliferation assays, Western blotting, Annexin V and propidium iodide staining by flow cytometry and reverse phase protein arrays. The efficacy of trametinib and dexamethasone treatment in the MM.1S xenograft model was assessed by measuring tumor volume over time. Results The Tra/Dex combination demonstrated synergistic cytotoxicity in KRAS G12A mutant lines MM.1S and RPMI-8226. The induction of apoptosis was associated with decreased MCL-1 expression and increased BIM expression. Reverse phase proteomic arrays revealed suppression of FAK, PYK2, FLT3, NDRG1 and 4EBP1 phosphorylation with the Tra/Dex combination. Notably, NDRG1 expression was associated with the synergistic response to Tra/Dex. MM cells were sensitive to PDK1 inhibition and IGF1-induced signalling partially protected from Tra/Dex treatment, highlighting the importance of this pathway. In the MM.1S tumor xenograft model, only the combination of Tra/Dex resulted in a significant inhibition of tumor growth. Conclusions Overall Tra/Dex demonstrates antiproliferative activity in RAS-mutant MM cell lines associated with suppression of pro-survival PDK1 signalling and engagement of apoptotic pathways. Our data support further investigation of this combination in RAS-mutant MM

Therapeutic Dose Response of Acoustic Cluster Therapy in Combination With Irinotecan for the Treatment of Human Colon Cancer in Mice.

Introduction: Acoustic Cluster Therapy (ACT) comprises coadministration of a formulation containing microbubble-microdroplet clusters (PS101) together with a regular medicinal drug and local ultrasound (US) insonation of the targeted pathological tissue. PS101 is confined to the vascular compartment and when the clusters are exposed to regular diagnostic imaging US fields, the microdroplets undergo a phase shift to produce bubbles with a median diameter of 22 µm. Low frequency, low mechanical index US is then applied to drive oscillations of the deposited ACT bubbles to induce biomechanical effects that locally enhance extravasation, distribution, and uptake of the coadministered drug, significantly increasing its therapeutic efficacy. Methods: The therapeutic efficacy of ACT with irinotecan (60 mg/kg i.p.) was investigated using three treatment sessions given on day 0, 7, and 14 on subcutaneous human colorectal adenocarcinoma xenografts in mice. Treatment was performed with three back-to-back PS101+US administrations per session with PS101 doses ranging from 0.40-2.00 ml PS101/kg body weight (n = 8-15). To induce the phase shift, 45 s of US at 8 MHz at an MI of 0.30 was applied using a diagnostic US system; low frequency exposure consisted of 1 or 5 min at 500 kHz with an MI of 0.20. Results: ACT with irinotecan induced a strong, dose dependent increase in the therapeutic effect (R2 = 0.95). When compared to irinotecan alone, at the highest dose investigated, combination treatment induced a reduction in average normalized tumour volume from 14.6 (irinotecan), to 5.4 (ACT with irinotecan, p = 0.002) on day 27. Median survival increased from 34 days (irinotecan) to 54 (ACT with irinotecan, p = 0.002). Additionally, ACT with irinotecan induced an increase in the fraction of complete responders; from 7% to 26%. There was no significant difference in the therapeutic efficacy whether the low frequency US lasted 1 or 5 min. Furthermore, there was no significant difference between the enhancement observed in the efficacy of ACT with irinotecan when PS101+US was administered before or after irinotecan. An increase in early dropouts was observed at higher PS101 doses. Both mean tumour volume (on day 27) and median survival indicate that the PS101 dose response was linear in the range investigated

Assays to monitor aggrephagy in Drosophila brain

Accumulation of ubiquitinated protein aggregates is a hallmark of most ageingrelated neurodegenerative disorders. Autophagy has been found to be involved in the selective clearance of these protein aggregates, and this process is called aggrephagy. Here we provide two protocols for the investigation of protein aggregation and their removal by autophagy using western blotting and immunofluorescence techniques in Drosophila brain. Investigating the role of aggrephagy at the cellular and organismal level is important for the development of therapeutic interventions against ageing-related diseases

Evaluation of deformation characteristics of brittle rocks beyond the limit of strength in the mode of uniaxial servohydraulic loading

One of the most reliable methods for assessing the physical and mechanical properties of rocks as a result of their destruction are laboratory tests using hard or servo-driven test presses. They allow to obtain reliable information about changes in these properties beyond the limit of compressive strength. The results of laboratory tests of rich sulfide ore samples are presented, which made it possible to obtain graphs of their extreme deformation. Both monolithic samples and samples with stress concentrators in the form of circular holes with a diameter of 3, 5 and 10 mm were tested. It was revealed that during the destruction of the samples, the modules of elasticity and deformation decrease by 1.5-2 times, and in the zone of residual strength – by 5-7 times

Regulation of Translesion Synthesis DNA Polymerase η by Monoubiquitination

DNA polymerase eta is a Y family polymerase involved in translesion synthesis (TLS). Its action is initiated by simultaneous interaction between the PIP box in pol eta and PCNA and between the UBZ in pol eta and monoubiquitin attached to PCNA. Whereas monoubiquitination of PCNA is required for its interaction with pol eta during TLS, we now show that monoubiquitination of pol eta inhibits this interaction, preventing its functions in undamaged cells. Identification of monoubiquitination sites within pol eta nuclear localization signal (NLS) led to the discovery that pol eta NLS directly contacts PCNA, forming an extended pol eta-PCNA interaction surface. We name this the PCNA-interacting region (PIR) and show that its monoubiquitination is downregulated by various DNA-damaging agents. We propose that this mechanism ensures optimal availability of nonubiquitinated, TLS-competent pol eta after DNA damage. Our work shows how monoubiquitination can either positively or negatively regulate the assembly of a protein complex, depending on which substrates are targeted by ubiquitin