Regulated expression of human beta-defensin-2 leads to altered phenotype and growth patterns of cultured human embryonal kidney cells

Aim: To create cell line with regulated expression of human beta-defensin-2 (hBD-2) and evaluate the influence of expressed peptide on its phenotypic and growth patterns. Materials and Methods: Using cloning techniques, on the base of human embryonic kidney cells of HEK293T line, stable T-rex HEK-hBD2-m cell subline expressing mature biologically active hBD-2 molecule upon the presence of tetracycline in culture medium was generated. The morphological patterns, growth characteristics and colony forming activity of these cells were studied using routine techniques. Results: T-rex HEK-HBD2-m cell subline was shown to express both mRNA and hBD-2m protein upon the presence of 1 µg/ml tetracycline in culture medium as it was demonstrated by RT-PCR and immunocytochemical approach. Upon prolonged expression of hBD-2, the cells acquired special features: they lost ability to grow in monolayer in vitro and to form colonies in soft agar, characteristic to parental HEK293T cells, but possess higher growth rate and longer survival in FBS-free medium than wild type cells. Conclusion: Expression of hBD-2 in T-rex HEK-HBD2-m cell subline results in specific biological consequences that favor cell survival.Цель: создать линию клеток с регулируемой экспрессией бета-дефенсина-2 человека (hBD-2) и проанализировать влияние экспрессии этого пептида на особенности фенотипа и рост клеток. Материалы и методы: клеточная сублиния T-rex HEK hBD2-m, экспрессирующая биологически активную зрелую форму hBD-2 при индукции клеток тетрациклином, получена путем клонирования на основе эмбриональных клеток почки человека линии HEK293T. Морфологические особенности, характеристики роста и показатели колониеобразования в полужидкой среде исследовали стандартными методами. Результаты: с помощью методов РТ-ПЦР и иммуноцитохимии показано, что сублиния клеток T-rex HEK-HBD2- экспрессирует hBD2 в присутствии 1 µг/мл тетрациклина в среде инкубации. Продолжительная экспрессия hBD-2 приводила к тому, что клетки утрачивали способность образовывать монослой при культивировании in vitro и образовывать колонии в полужидкой среде, но характеризовались более высокой скоростью роста и способностью к более продолжительному выживанию в бессывороточной среде, чем исходная линия клеток HEK293T. Выводы: экспрессия hBD-2 в сублинии клеток T-rex HEK-HBD2- cell обусловливает специфические биологические эфеекты, способствующие выживанию клеток

Remarks on the "non-canonicity puzzle": Lagrangian symmetries of the Einstein-Hilbert action

Given the non-canonical relationship between variables used in the Hamiltonian formulations of the Einstein-Hilbert action (due to Pirani, Schild, Skinner (PSS) and Dirac) and the Arnowitt-Deser-Misner (ADM) action, and the consequent difference in the gauge transformations generated by the first-class constraints of these two formulations, the assumption that the Lagrangians from which they were derived are equivalent leads to an apparent contradiction that has been called "the non-canonicity puzzle". In this work we shall investigate the group properties of two symmetries derived for the Einstein-Hilbert action: diffeomorphism, which follows from the PSS and Dirac formulations, and the one that arises from the ADM formulation. We demonstrate that unlike the diffeomorphism transformations, the ADM transformations (as well as others, which can be constructed for the Einstein-Hilbert Lagrangian using Noether's identities) do not form a group. This makes diffeomorphism transformations unique (the term "canonical" symmetry might be suggested). If the two Lagrangians are to be called equivalent, canonical symmetry must be preserved. The interplay between general covariance and the canonicity of the variables used is discussed.Comment: 23 page

Quantum Cosmology Aspects Of D3 Branes and Tachyon Dynamics

We investigate aspects of quantum cosmology in relation to string cosmology systems that are described in terms of the Dirac-Born-Infeld action. Using the Silverstein-Tong model, we analyze the Wheeler-DeWitt equation for the rolling scalar and gravity as well for $R\times{S^3}$ universe, by obtaining the wave functions for all dynamical degrees of freedom of the system. We show, that in some cases one can construct a time dependent version of the Wheeler-DeWitt (WDW) equation for the moduli field $\phi$. We also explore in detail the minisuperspace description of the rolling tachyon when non-minimal gravity tachyon couplings are inserted into the tachyon action.Comment: 29 pages, 3 figures, REVTeX 4; v2 clarifications, comments and references added; v3 more typos corrected, additional comments on the minisuperspace description of unstable universes, version published in JHE

The interactions between inflammation and insulin resistance: molecular mechanisms in insulin-producing and insulin-dependent tissues

In the modern world the prevalence of obesity and type 2 diabetes mellitus (T2DM) significantly increases. In this light the risks of obesity-associated complications also grow up. The crucial linkage between obesity and its metabolic and cardiovascular complications is inflammatory process. The mechanism of this linkage is similar in pancreas and insulin-dependent tissues both on cells, cell-to-cell communication and signaling pathway levels: the catalysts are different lipids (cholesterol, free fatty acids, triglycerides), which are able to activate Toll-like receptors of innate immunity and inflammation. Nextly, IKK- and JNK-dependent cascades activate the secretion of inflammatory cytokines TNFa, IL-1b, IL-6 and others, which act by paracrine and autocrine manner and support inflammation both in local and systemic levels. Thus, insulin-producing and insulin-dependent tissues, which are involved in T2DM pathogenesis, through the inflammatory process integrate in pathogenic and self-maintaining cycle, which leads to the suppression of insulin secretion, pancreatic β-cell failure and the development of insulin-dependent tissues insulin resistance

The interactions between inflammation and insulin resistance: prospects of immunoregulation as a potential approach for the type 2 diabetes mellitus treatment

In the modern world the prevalence of obesity and type 2 diabetes mellitus (T2DM) significantly increases. In this light the risks of obesity-associated complications also grow up. The crucial linkage between obesity and its complications is inflammation, which is a convenient target for potential anti-diabetic therapy. There are some anti-inflammatory therapy strategies: action on secreted cytokines, circulating lipids or intracellular signaling cascades. Canakinumab (antibody to IL-1b receptor) and colchicine (IL-6 secretion blocker) have the most balanced anti-diabetic and cardioprotective action among cytokine anti-inflammatory therapy. Lipid-lowering therapy is very diverse, but bempedoic acid nowadays has the best combination of anti-inflammatory and cardioprotective effects. Salicylate is an inhibitor of IKK-dependent inflammatory signaling cascade and significantly lowers glycated hemoglobin and C-reactive protein levels among obese patients. The future of anti-inflammatory T2DM therapy can be related with anti-inflammatory cytokines (IL-4, IL-37), chimeric engineered cytokines (IC7Fc), novel inhibitors of inflammatory and cytokines signaling cascades (imatinib, CC90001) and cell-based therapy (mesenchymal stem cells). In summary, despite on the limitations of current clinical trials, anti-inflammatory drugs have a potential to become a part of modern combined T2DM therapy with anti-diabetic and cardioprotective properties. Novel findings in potential anti-inflammatory T2DM therapy have great perspectives in protection against T2DM and related complication prevention

Creation of Technology for Microbial Protein Obtaining on the Basis of the Yeast Fungi Saccharomyces Cerevisiae

Микробная биомасса может быть использована для производства продуктов питания и кормов для животных благодаря высокому содержанию белков. В результате обзора технологии получения микробного белка за основу субстрата были выбраны дрожжевые грибы Saccharomyces cerevisiae. Разработана технологическая схема по получению микробного белка из дрожжевых грибов Saccharomyces cerevisiae. При разработке технологической схемы учитывались ранее разработанные технологии получения микробного белка. В результате проведенного анализа литературных данных для получения высокой массовой доли белка были использованы протеолитические ферменты протосубтилин, папаин и бромелайн. Высокое содержание белка в полученном белковом препарате отмечается при использовании фермента бромелайн.Microbial proteins, that is, single-celled proteins or microbial biomass, can be grown for the production of food and animal feed due to the high level of protein. As a result of a review of the technology for obtaining microbial protein, the yeast fungi Saccharomyces cerevisiae were chosen as the basis for the substrate. The technological scheme for obtaining microbial protein from yeast fungi Saccharomyces cerevisiae was developed. At development of the technological scheme previously developed technologies of reception of microbial protein were taken into account. As a result of the analysis of literature data, proteolytic enzymes protosubtilin, papain and bromelain were used to obtain a high mass fraction of protein. The high protein content in the obtained protein preparation is observed when using the bromelain enzyme

Quantum geometrodynamics creates new problems

The development of last years in quantum geometrodynamics highlights new problems which were not obvious in its first formulation proposed by Wheeler and DeWitt. At the first stage the main task was to apply known quantization schemes to gravitational field or a certain cosmological model. This way has led to the realization of the fact that a quantum description of the Universe is impossible without implicit or explicit indication to a reference frame presented by some medium, filling the whole Universe, with its own equation of state and thermodynamical properties. Thus the questions arise, should one seek for a "privileged" reference frame or consider all the variety of gauge conditions and appropriate solutions for the wave function? It is worth noting that thermodynamical properties of such a quantum Universe would also depend on a chosen reference frame to some extent. So, we need a self-consistent quantum theoretical and thermodynamical description of the Universe.Comment: 5 pages, talk presented at the XIII Russian Gravitational Conference, Moscow, June 2008, a reference correcte

Feсal microbiota transplantation in the format of complex therapy in obesive siblings: clinical case

Obesity and associated metabolic diseases are often accompanied by changes in the gut microbiota leading to metagenome gene diversity decrease. Fecal microbiota transplantation (FMT) is one of the most effective methods for correcting the  intestinal microflora. FMT obtained from healthy donors has been proven to be an effective treatment of infections caused by Clostridium difficile. The use of FMT for correction of metabolic disorders is promising, however, data on its application is limited and has contradictory results. In our work, two patients (siblings) presented with obesity grade II and various types of diabetes mellitus (DM): the older brother (44 years old) with diabetes mellitus type 2 (DM 2), a younger brother (39 years old) with diabetes mellitus type 1 (DM 1). Both patients underwent FMT as part of complex antidiabetic therapy. During the course of treatment, a decrease in body weight was noted in both patients (4–5 kg for the first month of observation, then -1–2 kg per month). One year after FMT, a patient with type 2 diabetes showed a decrease in the severity of insulin resistance (IR), measured by the hyperinsulinemic euglycemic clamp test (initial M-index 2.42 mg/kg*min, after 1 year — 3.83 mg/kg* min) as well as the maintenance of satisfactory carbohydrate metabolism compensation against the diminishing the hypoglycemic therapy. In a patient with DM 1, no significant dynamics of carbohydrate exchange indices, including detected glycated hemoglobin (HbA1c), insulin dose and IR were during the observation period. Metagenomic sequencing of stool samples (n = 20) collected from both patients before and within 1 year after FMT showed no significant changes in the taxonomic profile of the microbiota at the level of microbial families. Metabolomic analysis of the composition of feces showed no directed changes in the composition of metabolites after the FMT procedure, the nature of changes within the samples from each patient during the entire study period was random. Thus, FMT had no effect on the course of DM1, but served as a starting point for weight loss and improvement glucose profile in DM2. However, convincing data confirming a causal correlation between FMT and improvement in the course of T2DM have not been obtained