Symbolic Partial-Order Execution for Testing Multi-Threaded Programs

We describe a technique for systematic testing of multi-threaded programs. We combine Quasi-Optimal Partial-Order Reduction, a state-of-the-art technique that tackles path explosion due to interleaving non-determinism, with symbolic execution to handle data non-determinism. Our technique iteratively and exhaustively finds all executions of the program. It represents program executions using partial orders and finds the next execution using an underlying unfolding semantics. We avoid the exploration of redundant program traces using cutoff events. We implemented our technique as an extension of KLEE and evaluated it on a set of large multi-threaded C programs. Our experiments found several previously undiscovered bugs and undefined behaviors in memcached and GNU sort, showing that the new method is capable of finding bugs in industrial-size benchmarks.Comment: Extended version of a paper presented at CAV'2

Different distribution of CD4 and CD8 T cells in synovial membrane and peripheral blood of rheumatoid arthritis and osteoarthritis patients.

Rheumatoid arthritis (RA) and osteoarthritis (OA) are chronic diseases associated with morphological joint changes. Synovial membrane (SM) involvement was established for RA, but the data for OA are limited, because OA is usually regarded as noninflammatory disease. Changes in immune system in RA are not limited to joints, and the significant role of T cells of peripheral blood (PB) is not disputable. However, there is still an open debate about PB immunological profile in OA. Therefore, we decided to measure the distribution of CD4+ and CD8+ T cells, regarding CD28 expression, both in PB and SM of RA and OA patients, on the same day. Altogether, eleven RA patients, 11 OA patients and similar numbers of age-matched healthy controls were included into the study. Flow cytometry was used for T cells subpopulation distinguishing and quantification; monoclonal antibodies against CD3, CD4, CD8 and CD28 with different fluorochromes were used for stainings. The RA patients had significantly higher percentage of CD3+4+ cells in PB as compared to OA patients and relevant control group. Both within the CD4+ and CD8+ compartments, significantly lower percentages of cells bearing the CD28 marker were found in the PB of OA as compared to RA patients. The proportion of CD3+CD4+ cells in SM was dependent on age of OA patients, older OA patients had significantly higher value of their SM/blood ratio than RA patients. Older OA subjects were also characterized by higher values of the SM/blood ratio of both CD4+CD28+ and CD8+CD28+ subpopulations than RA or younger OA patients. In conclusion, in contrast to the traditional view of OA disease, our results give support to the hypothesis that OA may also (like RA) be a disease with a local immunological involvement

Virulence of Oomycete Pathogens from \u3cem\u3ePhragmites australis\u3c/em\u3e-Invaded and Noninvaded Soils to Seedlings of Wetland Plant Species

Soil pathogens affect plant community structure and function through negative plant-soil feedbacks that may contribute to the invasiveness of non-native plant species. Our understanding of these pathogen-induced soil feedbacks has relied largely on observations of the collective impact of the soil biota on plant populations, with few observations of accompanying changes in populations of specific soil pathogens and their impacts on invasive and noninvasive species. As a result, the roles of specific soil pathogens in plant invasions remain unknown. In this study, we examine the diversity and virulence of soil oomycete pathogens in freshwater wetland soils invaded by non-native Phragmites australis (European common reed) to better understand the potential for soil pathogen communities to impact a range of native and non-native species and influence invasiveness. We isolated oomycetes from four sites over a 2-year period, collecting nearly 500 isolates belonging to 36 different species. These sites were dominated by species of Pythium, many of which decreased seedling survival of a range of native and invasive plants. Despite any clear host specialization, many of the Pythium species were differentially virulent to the native and non-native plant species tested. Isolates from invaded and noninvaded soils were equally virulent to given individual plant species, and no apparent differences in susceptibility were observed between the collective groups of native and non-native plant species

INFLUENCE ALLOGENEIC MESENCHYMAL STEM CELLS IN PERITONEAL MACROPHAGES OKSYHENZALEZHNYY METABOLISM MICE S57BL / 6

Дослідження проводили на самцях мишей C57BL/6 масою 20–22 г віком 2–3 місяці. Алогенні МСК отримували культивуванням первинного матеріалу, що був виділений з кісткового мозку мишей С57BL/6. Культивування клітин проводили  у середовищі DMEM з додаванням 20 % фетальної сироватки бичків (FBS) та 1 % суміші антибіотика-антимікотика (Sigma, USA) за 37°С, 100 % вологості і 5 % СО2.Мишам С57BL/6 внутрішньом’язово інокулювали клітинну суспензію метастатичної карциноми легень Льюїс (LLC) у концентрації 1×106/0,1 мл розчину Хенкса. На 8-му добу після інокуляції пухлинних клітин групі тварин вводили  внутрішньовенно алогенні МСК в концентрації 1,25×104 на тварину. Після цього було сформовано такі групи тварин: 1-ша включала інтактних тварин (контроль), 2-га включала тварин, яким вводили тільки  алогенні мезенхімальні стовбурові клітини (MSC), 3-тя включала тварин, яким вводили суспензію метастатичної карциноми легень Льюїс (LLC), 4-та – тварини, яким вводили суспензію метастатичної карциноми легень Льюїс  і  алогенні МСК (LLC+ MSC).Для визначення оксигензалежної біоцидності перитонеальних макрофагів  застосовували спонтанний та стимулювальний НСТ-тест.Встановлено, що введення  алогенних МСК чинить вплив на оксигензалежний метаболізм перитонеальних макрофагів у мишей С57BL/6. Застосування алогенних МСК забезпечує вірогідне зниження метаболічної активності перитонеальних макрофагів  у мишей С57BL/6 з показником  стимуляції  -12% , що вказує  на відсутність функціонального резерву клітин. Встановлено вірогідне зниження метаболічної активності перитонеальних макрофагів  у мишей С57BL/6 з перещепленою карциномою легень Льюїс з показником  стимуляції  -30% , що вказує  на відсутність функціонального резерву клітин.  Введення алогенних МСК призводить до  вірогідного  незначного підвищення метаболічної активності перитонеальних макрофагів  у мишей С57BL/6 з перещепленою карциномою легень Льюїс із показником  стимуляції –  8% , що вказує  на наявність функціонального резерву клітин.Исследования проводили на самцах мышей C57BL/6 массой 20–22 г в возрасте 2–3 месяца. Аллогенные МСК получали культивированием первичного материала, который был выделен из костного мозга мышей С57BL/6. Культивирование клеток проводили в среде DMEM с добавлением 20% фетальной сыворотки бычков (FBS) и 1% смеси антибиотика-антимикотика (Sigma, USA) при 37 °С, 100% влажности и 5% СО2.Мышам С57BL/6 внутримышечно вводили клеточную суспензию метастатической карциномы легких Льюис (LLC) в концентрации 1×106/0,1 мл раствора Хэнкса. На 8-е сутки после инокуляции опухолевых клеток группе животных вводили внутривенно аллогенные МСК в концентрации 1,25×104 на животное. После этого было сформировано следующие группы животных: 1-я включала интактных животных (контроль), 2-я включала животных, которым вводили только аллогенные мезенхимальные стволовые клетки (МСК), 3-я включала животных, которым вводили суспензию метастатической карциномы легких Льюис (LLC), 4-я – животные, которым вводили суспензию метастатической карциномы легких Льюис и аллогенные МСК (LLC + MSC).Для определения оксигензависимой биоцидности перитонеальных макрофагов применяли спонтанный и стимулированный НСТ-тест.Установлено, что введение аллогенных МСК оказывает влияние на оксигензависимый метаболизм перитонеальных макрофагов у мышей С57BL/6. Применение аллогенных МСК обеспечивает достоверное снижение метаболической активности перитонеальных макрофагов у мышей С57BL/6 с показателем стимуляции -12%, что указывает на отсутствие функционального резерва клеток. Установлено достоверное снижение метаболической активности перитонеальных макрофагов у мышей С57BL/6 с первитой карциномой легких Льюис с показателем стимуляции  -30%, что указывает на отсутствие функционального резерва клеток. Введение аллогенных МСК приводит к достоверному незначительного повышению метаболической активности перитонеальных макрофагов у мышей С57BL / 6 с перевитой карциномой легких Льюис с показателем стимуляции – 8%, что указывает на наличие функционального резерва клеток.The study was conducted on male mice C57BL/6 weighing 20-22 g aged 2-3 months. Receiving allogeneic MSCs cultivation of primary material that was isolated from the bone marrow of mice C57BL/6. Cultivation of cells was carried out in DMEM medium with addition of 20% fetal bovis serum (FBS) and 1% antibiotic-antimycotics (Sigma, USA) at 37 °C, 100% humidity and 5% CO2.It was inoculated intramuscularly cell suspension metastatic Lewis lung carcinoma (LLC) in a concentration 1×106/0.1 ml Hanks to mice C57BL/6. On the 8th day after tumor cell inoculation it was administered intravenously allogeneic MSCs in a concentration 1,25×104  to 4th  group of animals. After that was formed following groups of animals: 1st included intact animals (control), 2nd – included animals which was administered only allogeneic mesenchymal stem cells (MSC), 3rd  – included animals which was administered suspension metastatic Lewis lung carcinoma (LLC ),  4th  group  – which was administered suspension metastatic Lewis lung carcinoma and allogeneic MSCs (LLC + MSC).Spontaneous and stimulated oxidative metabolism of peritoneal macrophages were established  in NBT-test.It was established that administration  allogeneic MSCs   influence on  oxidative metabolism of peritoneal macrophages in mice C57BL/6. The use of allogeneic MSCs provides a probable decrease metabolic activity of peritoneal macrophages in miceC57BL/6 with stimulation  index -12%. It was indicate  that  cells lose of functional reserve. In mice with  Lewis lung carcinoma ( 3rd  group of animals)  metabolic activity of peritoneal macrophages in mice C57BL/6   was decreased  with stimulation  index  -30% like  indicating a losek of cells. functional reserve.Allogeneic MSCs application in mice C57BL/6 perescheplenoyu Lewis lung carcinoma is lead to a slight increase metabolic activity of peritoneal macrophages   with stimulation  index  8%, which indicates the presence of cells functional reserve

Monotonicity of Fitness Landscapes and Mutation Rate Control

A common view in evolutionary biology is that mutation rates are minimised. However, studies in combinatorial optimisation and search have shown a clear advantage of using variable mutation rates as a control parameter to optimise the performance of evolutionary algorithms. Much biological theory in this area is based on Ronald Fisher's work, who used Euclidean geometry to study the relation between mutation size and expected fitness of the offspring in infinite phenotypic spaces. Here we reconsider this theory based on the alternative geometry of discrete and finite spaces of DNA sequences. First, we consider the geometric case of fitness being isomorphic to distance from an optimum, and show how problems of optimal mutation rate control can be solved exactly or approximately depending on additional constraints of the problem. Then we consider the general case of fitness communicating only partial information about the distance. We define weak monotonicity of fitness landscapes and prove that this property holds in all landscapes that are continuous and open at the optimum. This theoretical result motivates our hypothesis that optimal mutation rate functions in such landscapes will increase when fitness decreases in some neighbourhood of an optimum, resembling the control functions derived in the geometric case. We test this hypothesis experimentally by analysing approximately optimal mutation rate control functions in 115 complete landscapes of binding scores between DNA sequences and transcription factors. Our findings support the hypothesis and find that the increase of mutation rate is more rapid in landscapes that are less monotonic (more rugged). We discuss the relevance of these findings to living organisms

Effective local connectivity properties

We investigate, and prove equivalent, effective versions of local connectivity and uniformly local arcwise connectivity for connected and computably compact subspaces of Euclidean space. We also prove that Euclidean continua that are computably compact and effectively locally connected are computably arcwise connected.Comment: Final versio

Proton Motive Force-Dependent Hoechst 33342 Transport by the ABC Transporter LmrA of Lactococcus lactis

The fluorescent compound Hoechst 33342 is a substrate for many multidrug resistance (MDR) transporters and is widely used to characterize their transport activity. We have constructed mutants of the adenosine triphosphate (ATP) binding cassette (ABC)-type MDR transporter LmrA of Lactococcus lactis that are defective in ATP hydrolysis. These mutants and wild-type LmrA exhibited an atypical behavior in the Hoechst 33342 transport assay. In membrane vesicles, Hoechst 33342 transport was shown to be independent of the ATPase activity of LmrA, and it was not inhibited by orthovanadate but sensitive to uncouplers that collapse the proton gradient and to N,N'-dicyclohexylcarbodiimide, an inhibitor of the F0F1-ATPase. In contrast, transport of Hoechst 33342 by the homologous, heterodimeric MDR transporter LmrCD showed a normal ATP dependence and was insensitive to uncouplers of the proton gradient. With intact cells, expression of LmrA resulted in an increased rate of Hoechst 33342 influx while LmrCD caused a decrease in the rate of Hoechst 33342 influx. Cellular toxicity assays using a triple knockout strain, i.e., L. lactis ΔlmrA ΔlmrCD, demonstrate that expression of LmrCD protects cells against the growth inhibitory effects of Hoechst 33342, while in the presence of LmrA, cells are more susceptible to Hoechst 33342. Our data demonstrate that the LmrA-mediated Hoechst 33342 transport in membrane vesicles is influenced by the transmembrane pH gradient due to a pH-dependent partitioning of Hoechst 33342 into the membrane.

High-throughput, quantitative analyses of genetic interactions in E. coli.

Large-scale genetic interaction studies provide the basis for defining gene function and pathway architecture. Recent advances in the ability to generate double mutants en masse in Saccharomyces cerevisiae have dramatically accelerated the acquisition of genetic interaction information and the biological inferences that follow. Here we describe a method based on F factor-driven conjugation, which allows for high-throughput generation of double mutants in Escherichia coli. This method, termed genetic interaction analysis technology for E. coli (GIANT-coli), permits us to systematically generate and array double-mutant cells on solid media in high-density arrays. We show that colony size provides a robust and quantitative output of cellular fitness and that GIANT-coli can recapitulate known synthetic interactions and identify previously unidentified negative (synthetic sickness or lethality) and positive (suppressive or epistatic) relationships. Finally, we describe a complementary strategy for genome-wide suppressor-mutant identification. Together, these methods permit rapid, large-scale genetic interaction studies in E. coli