A rapid in vivo screen for pancreatic ductal adenocarcinoma therapeutics

Pancreatic ductal adenocarcinoma (PDA) is the fourth leading cause of cancer-related deaths in the United States, and is projected to be second by 2025. It has the worst survival rate among all major cancers. Two pressing needs for extending life expectancy of affected individuals are the development of new approaches to identify improved therapeutics, addressed herein, and the identification of early markers. PDA advances through a complex series of intercellular and physiological interactions that drive cancer progression in response to organ stress, organ failure, malnutrition, and infiltrating immune and stromal cells. Candidate drugs identified in organ culture or cell-based screens must be validated in preclinical models such as KIC (p48Cre;LSL-KrasG12D;Cdkn2af/f) mice, a genetically engineered model of PDA in which large aggressive tumors develop by 4 weeks of age. We report a rapid, systematic and robust in vivo screen for effective drug combinations to treat Kras-dependent PDA. Kras mutations occur early in tumor progression in over 90% of human PDA cases. Protein kinase and G-protein coupled receptor (GPCR) signaling activates Kras. Regulators of G-protein signaling (RGS) proteins are coincidence detectors that can be induced by multiple inputs to feedback-regulate GPCR signaling. We crossed Rgs16::GFP bacterial artificial chromosome (BAC) transgenic mice withKIC mice and show that the Rgs16::GFP transgene is a KrasG12D-dependent marker of all stages of PDA, and increases proportionally to tumor burden in KIC mice. RNA sequencing (RNA-Seq) analysis of cultured primary PDA cells reveals characteristics of embryonic progenitors of pancreatic ducts and endocrine cells, and extraordinarily high expression of the receptor tyrosine kinase Axl, an emerging cancer drug target. In proof-of-principle drug screens, we find that weanling KIC mice with PDA treated for 2 weeks with gemcitabine (with or without Abraxane) plus inhibitors of Axl signaling (warfarin and BGB324) have fewer tumor initiation sites and reduced tumor size compared with the standard-of-care treatment. Rgs16::GFP is therefore an in vivo reporter of PDA progression and sensitivity to new chemotherapeutic drug regimens such as Axl-targeted agents. This screening strategy can potentially be applied to identify improved therapeutics for other cancers

Нокдаун клеточных генов FLT4, Nup98 и Nup205 как супрессор вирусной активности гриппа А/WSN/33 (H1N1) в культуре клеток А549

Objectives. To evaluate the effect of cellular genes FLT4, Nup98, and Nup205 on the reproduction of the influenza A virus in A549 human lung cancer cell line.Methods. The work was carried out using the equipment of the center for collective use of the I.I. Mechnikov Research Institute of Vaccines and Sera (Russia). The virus-containing fluid was collected within three days from the moment of transfection and infection and the intensity of viral reproduction was assessed by viral titration and hemagglutination reaction. The viral RNA concentration was determined by real-time reverse-transcription polymerase chain reaction (RT-PCR). To calculate statistically significant differences between groups, the nonparametric Mann–Whitney test was used.Results. In cells treated with small interfering RNAs (siRNAs) targeted at FLT4, Nup98, and Nup205 genes, a significant decrease in their expression and indicators of viral reproduction (virus titer, hemagglutinating activity, viral RNA concentration) was observed at a multiplicity of infection (MOI) = 0.1. Additionally, it was found that a decrease in the expression of target genes using siRNA does not lead to a significant decrease in cell survival. The viral titer in cells treated with siRNA FLT4.2, Nup98.1, and Nup205 on the first day was lower by an average of 1.0 lg, and on the second and third days, by 2.2–2.3 lg, compared to cells treated with nonspecific siRNA. During real-time RT-PCR, a significant decrease in the concentration of viral RNA was observed with siRNA Nup98.1 (up to 190 times) and Nup205 (up to 30 times) on the first day, 26 and 29 times on the second day, and 6 and 30 times on the third day, respectively. For FLT4.2 siRNA, the number of viral RNA copies decreased by 23, 18, and 16 times on the first, second, and third days. Similar results were obtained when determining the hemagglutinating activity of the virus. The hemagglutinating activity on the third day most strongly decreased in cells treated with siRNA Nup205 and FLT4.2 (16 times). In cells treated with siRNA FLT4.1, Nup98.1, and Nup98.2, hemagglutinating activity decreased by 8 times.Conclusions. In the present study, three cellular genes (FLT4, Nup98, and Nup205) were identified—the decrease in the expression of which effectively suppresses viral reproduction— and the original siRNA sequences were obtained. The results obtained are important for creating therapeutic and prophylactic medication, whose action is based on the RNA interference mechanism.Цели. Оценка влияния подавления экспрессии клеточных генов FLT4, Nup98 и Nup205 на динамику репродукции вируса гриппа А в культуре легочных клеток человека А549.Методы. Работа выполнена с использованием оборудования центра коллективного пользования Научно-исследовательского института вакцин и сывороток им И.И. Мечникова (Россия). Вируссодержащую жидкость отбирали в течение трех дней с момента трансфекции и заражения и оценивали интенсивность вирусной репродукции методами титрования по цитопатическому действию и в реакции гемагглютинации. Концентрацию вирусной РНК определяли методом полимеразной цепной реакции (ПЦР) в реальном времени с обратной транскрипцией (ОТ-ПЦР-РВ). Для вычисления статистически значимых различий между группами использовали непараметрический критерий Манна–Уитни.Результаты. В клетках, обработанных малыми интерферирующими РНК (миРНК) к генам FLT4, Nup98 и Nup205, отмечалось достоверное подавление экспрессии целевых генов и показателей вирусной репродукции (титр вируса, гемагглютинирующая активность, концентрация вирусной РНК) при коэффициенте множественности заражения, равном 0.1. Дополнительно было установлено, что подавление экспрессии целевых генов с помощью миРНК не приводит к значительному снижению выживаемости клеток. Вирусный титр в клетках, обработанных миРНК FLT4.2, Nup98.1 и Nup205, на первые сутки был меньше в среднем на 1.0 lg, а на вторые и третьи – на 2.2–2.3 lg, по сравнению с клетками, обработанными неспецифической миРНК. При проведении ОТ-ПЦР-РВ отмечено достоверное уменьшение концентрации вирусной РНК с миРНК Nup98.1 (до 190 раз) и Nup205 (до 30 раз) на первые сутки, в 26 и в 29 раз на вторые и в 6 и 30 раз на третьи сутки, соответственно. Для миРНК FLT4.2 количество копий вирусной РНК уменьшилось в 23, 18 и 16 раз на первые, вторые и третьи сутки. Схожие результаты были получены при определении гемагглютинирующей активности вируса. Наиболее сильно, в 16 раз, гемагглютинирующая активность на третьи сутки снизилась в клетках, обработанных миРНК Nup205 и FLT4.2. В клетках, обработанных миРНК FLT4.1, Nup98.1 и Nup98.2, гемагглютинирующая активность уменьшилась в 8 раз.Выводы. В ходе исследования были выявлены три клеточных гена (FLT4, Nup98 и Nup205), подавление экспрессии которых позволяет эффективно уменьшить вирусную репродукцию, а также получены оригинальные последовательности миРНК. Полученные результаты имеют важное значение для создания терапевтических и профилактических препаратов, чье действие основано на механизме РНК-интерференции

Limited Cheese Intake Paradigm Replaces Patterns of Behavioral Disorders in Experimental PTSD: Focus on Resveratrol Supplementation

Currently, the efficacy of drug therapy for post-traumatic stress disorder or PTSD leaves much to be desired, making nutraceutical support a promising avenue for treatment. Recent research has identified the protective effects of resveratrol in PTSD. Here, we tested the behavioral and neurobiological effects of combining cheese consumption with resveratrol supplements in an experimental PTSD model. Using the elevated plus maze test, we observed that cheese intake resulted in a shift from anxiety-like behavior to depressive behavior, evident in increased freezing acts. However, no significant changes in the anxiety index value were observed. Interestingly, supplementation with cheese and resveratrol only led to the elimination of freezing behavior in half of the PTSD rats. We further segregated the rats into two groups based on freezing behavior: Freezing+ and Freezing0 phenotypes. Resveratrol ameliorated the abnormalities in Monoamine Oxidize -A and Brain-Derived Neurotrophic Factor gene expression in the hippocampus, but only in the Freezing0 rats. Moreover, a negative correlation was found between the number of freezing acts and the levels of Monoamine Oxidize-A and Brain-Derived Neurotrophic Factor mRNAs in the hippocampus. The study results show promise for resveratrol supplementation in PTSD treatment. Further research is warranted to better understand the underlying mechanisms and optimize the potential benefits of resveratrol supplementation for PTSD. © 2023 by the authors.23-15-20040; Russian Science Foundation, RSFThis work was supported by the Russian Scientific Foundation, Regional grant, Chelyabinsk Region (#23-15-20040)

Simulation modeling based method for choosing an effective set of fault tolerance mechanisms for real-time avionics systems

In this paper, the reliability allocation problem (RAP) for real-time avionics systems (RTAS) is considered. The proposed method for solving this problem consists of two steps: (i) creation of an RTAS simulation model at the necessary level of abstraction and (ii) application of metaheuristic algorithm to find an optimal solution (i. e., to choose an optimal set of fault tolerance techniques). When during the algorithm execution it is necessary to measure the execution time of some software components, the simulation modeling is applied. The procedure of simulation modeling also consists of the following steps: automatic construction of simulation model of the RTAS configuration and running this model in a simulation environment to measure the required time. This method was implemented as an experimental software tool. The tool works in cooperation with DYANA simulation environment. The results of experiments with the implemented method are presented. Finally, future plans for development of the presented method and tool are briefly described