Новый подход к моделированию генных сетей

The article is devoted to the mathematical modeling of artificial genetic networks. A phenomenological model of the simplest genetic network called repressilator is considered. This network contains three elements unidirectionally coupled into a ring. More specifically, the first of them inhibits the synthesis of the second, the second inhibits the synthesis of the third, and the third, which closes the cycle, inhibits the synthesis of the first one. The interaction of the protein concentrations and of mRNA (message RNA) concentration is surprisingly similar to the interaction of six ecological populations — three predators and three preys. This allows us to propose a new phenomenological model, which is represented by a system of unidirectionally coupled ordinary differential equations. We study the existence and stability problem of a relaxation periodic solution that is invariant with respect to cyclic permutations of coordinates. To find the asymptotics of this solution, a special relay system is constructed. It is proved in the paper that the periodic solution of the relay system gives the asymptotic approximation of the orbitally asymptotically stable relaxation cycle of the problem under consideration.Статья посвящена математическому моделированию искусственных генных сетей. Рассматривается феноменологическая модель простейшей трехзвенной осцилляторной генной сети — так называемого репрессилятора. Эта сеть содержит три элемента, однонаправленно связанных в кольцо. Первый из них ингибирует синтез второго, второй ингибирует синтез третьего, а третий, который замыкает цикл, ингибирует синтез первого. Взаимодействие концентраций белка и концентрации мРНК удивительно похоже на функционирование биоценоза, состоящего из шести экологических популяций — трех хищников и трех жертв. Это позволяет предложить новую феноменологическую модель, которая представлена системой однонаправленно связанных обыкновенных дифференциальных уравнений. В работе изучена задача существования и устойчивости у этой системы релаксационного периодического решения, инвариантного по отношению к циклическим перестановкам координат. Для нахождения асимптотики этого решения строится специальная релейная система. В статье доказывается, что периодическое решение релейной системы дает асимптотическое приближение орбитально асимптотически устойчивого релаксационного цикла рассматриваемой задачи

Temporal Logic for Programmable Logic Controllers

We address the formal verification of the control software of critical systems, i.e., ensuring the absence of design errors in a system with respect to requirements. Control systems are usually based on industrial controllers, also known as Programmable Logic Controllers (PLCs). A specific feature of a PLC is a scan cycle: 1) the inputs are read, 2) the PLC states change, and 3) the outputs are written. Therefore, in order to formally verify PLC, e.g., by model checking, it is necessary to describe the transition system taking into account this specificity and reason both in terms of state transitions within a cycle and in terms of larger state transitions according to the scan-cyclic semantics. We propose a formal PLC model as a hyperprocess transition system and temporal cycle-LTL logic based on LTL logic for formulating PLC property. A feature of the cycle-LTL logic is the possibility of viewing the scan cycle in two ways: as the effect of the environment (in particular, the control object) on the control system and as the effect of the control system on the environment. For both cases we introduce modified LTL temporal operators. We also define special modified LTL temporal operators to specify inside properties of scan cycles. We describe the translation of formulas of cycle-LTL into formulas of LTL, and prove its correctness. This implies the possibility ofmodel checking requirements expressed in logic cycle-LTL, by using well-known model checking tools with LTL as specification logic, e.g., Spin. We give the illustrative examples of requirements expressed in the cycle-LTL logic

The recombinant fusion protein CFP10–ESAT6–dIFN has protective effect against tuberculosis in guinea pigs

Development of effective vaccine candidates against tuberculosis (TB) is currently the most important challenge in the prevention of this disease since the BCG vaccine fails to guarantee a lifelong protection, while any other approved vaccine with better efficiency is still absent. The protective effect of the recombinant fusion protein CFP10–ESAT6–dIFN produced in a prokaryotic expression system (Escherichia coli) has been assessed in a guinea pig model of acute TB. The tested antigen comprises the Mycobacterium tuberculosis (Mtb) proteins ESAT6 and CFP10 as well as modified human γ-interferon (dIFN) for boosting the immune response. Double intradermal immunization of guinea pigs with the tested fusion protein (2 × 0.5 µg) induces a protective effect against subsequent Mtb infection. The immunized guinea pigs do not develop the symptoms of acute TB and their body weight gain was five times more as compared with the non-immunized infected guinea pigs. The animal group immunized with this dose of antigen displays the minimum morphological changes in the internal organs and insignificant inflammatory lesions in the liver tissue, which complies with a decrease in the bacterial load in the spleen and average Mtb counts in macrophages

Темпоральная логика для программируемых логических контроллеров

We address the formal verification of the control software of critical systems, i.e., ensuring the absence of design errors in a system with respect to requirements. Control systems are usually based on industrial controllers, also known as Programmable Logic Controllers (PLCs). A specific feature of a PLC is a scan cycle: 1) the inputs are read, 2) the PLC states change, and 3) the outputs are written. Therefore, in order to formally verify PLC, e.g., by model checking, it is necessary to describe the transition system taking into account this specificity and reason both in terms of state transitions within a cycle and in terms of larger state transitions according to the scan-cyclic semantics. We propose a formal PLC model as a hyperprocess transition system and temporal cycle-LTL logic based on LTL logic for formulating PLC property. A feature of the cycle-LTL logic is the possibility of viewing the scan cycle in two ways: as the effect of the environment (in particular, the control object) on the control system and as the effect of the control system on the environment. For both cases we introduce modified LTL temporal operators. We also define special modified LTL temporal operators to specify inside properties of scan cycles. We describe the translation of formulas of cycle-LTL into formulas of LTL, and prove its correctness. This implies the possibility ofmodel checking requirements expressed in logic cycle-LTL, by using well-known model checking tools with LTL as specification logic, e.g., Spin. We give the illustrative examples of requirements expressed in the cycle-LTL logic.Мы исследуем формальную верификацию управляющего программного обеспечения критических систем, т. е. проверку соответствия функционирования проектируемой системы предъявленным требованиям. Важнейший класс управляющего программного обеспечения составляют программы для программируемых логических контроллеров (ПЛК). Особенностью программ ПЛК является цикл управления: 1) считываются входы, 2) изменяются состояния ПЛК и 3) записываются выходы. Поэтому для формальной верификации программ ПЛК нужна возможность описывать учитывающие эту специфику системы переходов, а также определять свойства систем, моделирующих программы ПЛК, как относительно переходов внутри цикла, так и относительно более крупных переходов в соответствии с семантикой цикла управления. Мы предлагаем формальную модель программы ПЛК как систему переходов гиперпроцессов и темпоральную логику cycle-LTL для формализации свойств ПЛК. Особенностью логики cycle-LTL является возможность рассматривать свойства систем управления двояким образом: воздействие окружения на систему управления и воздействие системы управления на окружение. Мы определяем модификации стандартных темпоральных операторов логики LTL для каждого из этих случаев, а также для свойств внутри цикла управления. Рассмотрены примеры требований, определенных в нашей логике. Описана трансляция формул cycle-LTL в формулы LTL и показана её корректность. Доказана возможность сведения задачи верификации методом проверки моделей для требований, определенных в логике cycle-LTL, к задаче верификации требований, определенных в стандартной логике LTL

Optimization of Genome Knock-In Method: Search for the Most Efficient Genome Regions for Transgene Expression in Plants

Plant expression systems are currently regarded as promising alternative platforms for the production of recombinant proteins, including the proteins for biopharmaceutical purposes. However, the accumulation level of a target protein in plant expression systems is still rather low compared with the other existing systems, namely, mammalian, yeast, and E. coli cells. To solve this problem, numerous methods and approaches have been designed and developed. At the same time, the random nature of the distribution of transgenes over the genome can lead to gene silencing, variability in the accumulation of recombinant protein, and also to various insertional mutations. The current research study considered inserting target genes into pre-selected regions of the plant genome (genomic “safe harbors”) using the CRISPR/Cas system. Regions of genes expressed constitutively and at a high transcriptional level in plant cells (housekeeping genes) that are of interest as attractive targets for the delivery of target genes were characterized. The results of the first attempts to deliver target genes to the regions of housekeeping genes are discussed. The approach of “euchromatization” of the transgene integration region using the modified dCas9 associated with transcription factors is considered. A number of the specific features in the spatial chromatin organization allowing individual genes to efficiently transcribe are discussed

Strategies for designing novel tuberculosis vaccines

More than one third of the global population are Mycobacterium tuberculosis carriers. Nevertheless, no adequate vaccine against tuberculosis has been developed. The article reviews the limitations of BCG, the only tuberculosis vaccine in current use, the main difficulties in the development of tuberculosis vaccines, and novel strategies in the development of next-generation vaccines. All types of new vaccines in the pipeline are considered. Special attention is paid to DNA- and protein subunit vaccines. The most efficient delivery methods for tuberculosis vaccines are discussed. Long-run prospects in designing novel tuberculosis vaccines are outlined

Strategies for designing novel tuberculosis vaccines

More than one third of the global population are Mycobacterium tuberculosis carriers. Nevertheless, no adequate vaccine against tuberculosis has been developed. The article reviews the limitations of BCG, the only tuberculosis vaccine in current use, the main difficulties in the development of tuberculosis vaccines, and novel strategies in the development of next-generation vaccines. All types of new vaccines in the pipeline are considered. Special attention is paid to DNA- and protein subunit vaccines. The most efficient delivery methods for tuberculosis vaccines are discussed. Long-run prospects in designing novel tuberculosis vaccines are outlined

Increasing the Efficiency of the Accumulation of Recombinant Proteins in Plant Cells: The Role of Transport Signal Peptides

The problem with increasing the yield of recombinant proteins is resolvable using different approaches, including the transport of a target protein to cell compartments with a low protease activity. In the cell, protein targeting involves short-signal peptide sequences recognized by intracellular protein transport systems. The main systems of the protein transport across membranes of the endoplasmic reticulum and endosymbiotic organelles are reviewed here, as are the major types and structure of the signal sequences targeting proteins to the endoplasmic reticulum and its derivatives, to plastids, and to mitochondria. The role of protein targeting to certain cell organelles depending on specific features of recombinant proteins and the effect of this targeting on the protein yield are discussed, in addition to the main directions of the search for signal sequences based on their primary structure. This knowledge makes it possible not only to predict a protein localization in the cell but also to reveal the most efficient sequences with potential biotechnological utility

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Main conclusio

Main strategies of plant expression system glycoengineering for producing humanized recombinant pharmaceutical proteins

Most the pharmaceutical proteins are derived not from their natural sources, rather their recombinant analogs are synthesized in various expression systems. Plant expression systems, unlike mammalian cell cultures, combine simplicity and low cost of procaryotic systems and the ability for posttranslational modifications inherent in eucaryotes. More than 50% of all human proteins and more than 40% of the currently used pharmaceutical proteins are glycosylated, that is, they are glycoproteins, and their biological activity, pharmacodynamics, and immunogenicity depend on the correct glycosylation pattern. This review examines in detail the similarities and differences between N- and O-glycosylation in plant and mammalian cells, as well as the effect of plant glycans on the activity, pharmacokinetics, immunity, and intensity of biosynthesis of pharmaceutical proteins. The main current strategies of glycoengineering of plant expression systems aimed at obtaining fully humanized proteins for pharmaceutical application are summarized