The Origin of Phenotypic Heterogeneity in a Clonal Cell Population In Vitro

BACKGROUND: The spontaneous emergence of phenotypic heterogeneity in clonal populations of mammalian cells in vitro is a rule rather than an exception. We consider two simple, mutually non-exclusive models that explain the generation of diverse cell types in a homogeneous population. In the first model, the phenotypic switch is the consequence of extrinsic factors. Initially identical cells may become different because they encounter different local environments that induce adaptive responses. According to the second model, the phenotypic switch is intrinsic to the cells that may occur even in homogeneous environments. PRINCIPAL FINDINGS: We have investigated the “extrinsic” and the “intrinsic” mechanisms using computer simulations and experimentation. First, we simulated in silico the emergence of two cell types in a clonal cell population using a multiagent model. Both mechanisms produced stable phenotypic heterogeneity, but the distribution of the cell types was different. The “intrinsic” model predicted an even distribution of the rare phenotype cells, while in the “extrinsic” model these cells formed small clusters. The key predictions of the two models were confronted with the results obtained experimentally using a myogenic cell line. CONCLUSIONS: The observations emphasize the importance of the “ecological” context and suggest that, consistently with the “extrinsic” model, local stochastic interactions between phenotypically identical cells play a key role in the initiation of phenotypic switch. Nevertheless, the “intrinsic” model also shows some other aspects of reality: The phenotypic switch is not triggered exclusively by the local environmental variations, but also depends to some extent on the phenotypic intrinsic robustness of the cells

Preclinical studies of antiviral activity of the RPH-137 fusion protein and molnupiravir against COVID-19

Finding effective and safe medicines to fight SARS-CoV-2 infection is an urgent task. RPH-137 is an original trap fusion protein against SARS-CoV-2 virus. It comprises the angiotensin-converting enzyme type 2 extracellular domain and the human IgG1 Fc fragment.The aim of the study was to carry out a preclinical evaluation of the efficacy of RPH-137 and molnupiravir against SARS-CoV-2 infection.Materials and methods: the authors analysed RPH-137 expressed in a stable CHO cell line and molnupiravir used as an active pharmaceutical ingredient. Drug-mediated inhibition of virus-induced cytotoxicity was assessed in Vero cell culture. In vivo efficacy assessments were performed in Syrian hamsters. The animals were infected intranasally with SARS-CoV-2 (PIK35 clinical isolate) in the dose of 5 log TCID50. The authors evaluated body weight measurements, lung–body weight ratios, and lung histopathology findings and determined viral RNA levels in oropharyngeal swabs by RT-PCR using the amplification cycle threshold (Ct). The statistical analyses involved one- and two-way ANOVA, Student's t-test, and Mann–Whitney test.Results: RPH-137 and molnupiravir inhibited the cytopathic effect of SARS-CoV-2 in Vero cells; the EC50 values of RPH-137 amounted to 4.69 μg/mL (21.3 nM) and 16.24 μg/mL (73.8 nM) for 50 TCID50 and 200 TCID50, respectively, whereas the EC50 values of molnupiravir were 0.63 μg/mL (1900 nM) for both doses. Intramuscular RPH-137 (30 and 80 mg/kg) had no effect on the infection process in Syrian hamsters. The comparison with the challenge control group showed that intraperitoneal RPH-137 (100 mg/kg) had statistically significant effects on a number of parameters, including a 27% reduction in inflammation and a 30% reduction in the total lesion area of the lungs by Day 7. Intragastric molnupiravir (300 mg/kg twice daily) significantly inhibited SARS-CoV-2 infection.Conclusions: both RPH-137 and molnupiravir inhibited the cytopathic effect of SARS-CoV-2 in Vero cells. In Syrian hamsters, molnupiravir demonstrated a more pronounced inhibition of SARS-CoV-2 infection than RPH-137. However, RPH-137 had statistically significant effects on a range of parameters. This offers additional perspectives for further research

Exhaled carbon monoxide in asthmatics: a meta-analysis

<p>Abstract</p> <p>Background</p> <p>The non-invasive assessment of airway inflammation is potentially advantageous in asthma management. Exhaled carbon monoxide (eCO) measurement is cheap and has been proposed to reflect airway inflammation and oxidative stress but current data are conflicting. The purpose of this meta-analysis is to determine whether eCO is elevated in asthmatics, is regulated by steroid treatment and reflects disease severity and control.</p> <p>Methods</p> <p>A systematic search for English language articles published between 1997 and 2009 was performed using Medline, Embase and Cochrane databases. Observational studies comparing eCO in non-smoking asthmatics and healthy subjects or asthmatics before and after steroid treatment were included. Data were independently extracted by two investigators and analyzed to generate weighted mean differences using either a fixed or random effects meta-analysis depending upon the degree of heterogeneity.</p> <p>Results</p> <p>18 studies were included in the meta-analysis. The eCO level was significantly higher in asthmatics as compared to healthy subjects and in intermittent asthma as compared to persistent asthma. However, eCO could not distinguish between steroid-treated asthmatics and steroid-free patients nor separate controlled and partly-controlled asthma from uncontrolled asthma in cross-sectional studies. In contrast, eCO was significantly reduced following a course of corticosteroid treatment.</p> <p>Conclusions</p> <p>eCO is elevated in asthmatics but levels only partially reflect disease severity and control. eCO might be a potentially useful non-invasive biomarker of airway inflammation and oxidative stress in nonsmoking asthmatics.</p

Heme oxygenase-1 and carbon monoxide in pulmonary medicine

Heme oxygenase-1 (HO-1), an inducible stress protein, confers cytoprotection against oxidative stress in vitro and in vivo. In addition to its physiological role in heme degradation, HO-1 may influence a number of cellular processes, including growth, inflammation, and apoptosis. By virtue of anti-inflammatory effects, HO-1 limits tissue damage in response to proinflammatory stimuli and prevents allograft rejection after transplantation. The transcriptional upregulation of HO-1 responds to many agents, such as hypoxia, bacterial lipopolysaccharide, and reactive oxygen/nitrogen species. HO-1 and its constitutively expressed isozyme, heme oxygenase-2, catalyze the rate-limiting step in the conversion of heme to its metabolites, bilirubin IXα, ferrous iron, and carbon monoxide (CO). The mechanisms by which HO-1 provides protection most likely involve its enzymatic reaction products. Remarkably, administration of CO at low concentrations can substitute for HO-1 with respect to anti-inflammatory and anti-apoptotic effects, suggesting a role for CO as a key mediator of HO-1 function. Chronic, low-level, exogenous exposure to CO from cigarette smoking contributes to the importance of CO in pulmonary medicine. The implications of the HO-1/CO system in pulmonary diseases will be discussed in this review, with an emphasis on inflammatory states

The integration of lipid-sensing and anti-inflammatory effects: how the PPARs play a role in metabolic balance

The peroxisomal proliferating-activated receptors (PPARs) are lipid-sensing transcription factors that have a role in embryonic development, but are primarily known for modulating energy metabolism, lipid storage, and transport, as well as inflammation and wound healing. Currently, there is no consensus as to the overall combined function of PPARs and why they evolved. We hypothesize that the PPARs had to evolve to integrate lipid storage and burning with the ability to reduce oxidative stress, as energy storage is essential for survival and resistance to injury/infection, but the latter increases oxidative stress and may reduce median survival (functional longevity). In a sense, PPARs may be an evolutionary solution to something we call the 'hypoxia-lipid' conundrum, where the ability to store and burn fat is essential for survival, but is a 'double-edged sword', as fats are potentially highly toxic. Ways in which PPARs may reduce oxidative stress involve modulation of mitochondrial uncoupling protein (UCP) expression (thus reducing reactive oxygen species, ROS), optimising forkhead box class O factor (FOXO) activity (by improving whole body insulin sensitivity) and suppressing NFkB (at the transcriptional level). In light of this, we therefore postulate that inflammation-induced PPAR downregulation engenders many of the signs and symptoms of the metabolic syndrome, which shares many features with the acute phase response (APR) and is the opposite of the phenotype associated with calorie restriction and high FOXO activity. In genetically susceptible individuals (displaying the naturally mildly insulin resistant 'thrifty genotype'), suboptimal PPAR activity may follow an exaggerated but natural adipose tissue-related inflammatory signal induced by excessive calories and reduced physical activity, which normally couples energy storage with the ability to mount an immune response. This is further worsened when pancreatic decompensation occurs, resulting in gluco-oxidative stress and lipotoxicity, increased inflammatory insulin resistance and oxidative stress. Reactivating PPARs may restore a metabolic balance and help to adapt the phenotype to a modern lifestyle

Доклинические исследования противовирусной активности гибридного белка RPH-137 и молнупиравира в отношении COVID-19

Finding effective and safe medicines to fight SARS-CoV-2 infection is an urgent task. RPH-137 is an original trap fusion protein against SARS-CoV-2 virus. It comprises the angiotensin-converting enzyme type 2 extracellular domain and the human IgG1 Fc fragment.The aim of the study was to carry out a preclinical evaluation of the efficacy of RPH-137 and molnupiravir against SARS-CoV-2 infection.Materials and methods: the authors analysed RPH-137 expressed in a stable CHO cell line and molnupiravir used as an active pharmaceutical ingredient. Drug-mediated inhibition of virus-induced cytotoxicity was assessed in Vero cell culture. In vivo efficacy assessments were performed in Syrian hamsters. The animals were infected intranasally with SARS-CoV-2 (PIK35 clinical isolate) in the dose of 5 log TCID50. The authors evaluated body weight measurements, lung–body weight ratios, and lung histopathology findings and determined viral RNA levels in oropharyngeal swabs by RT-PCR using the amplification cycle threshold (Ct). The statistical analyses involved one- and two-way ANOVA, Student's t-test, and Mann–Whitney test.Results: RPH-137 and molnupiravir inhibited the cytopathic effect of SARS-CoV-2 in Vero cells; the EC50 values of RPH-137 amounted to 4.69 μg/mL (21.3 nM) and 16.24 μg/mL (73.8 nM) for 50 TCID50 and 200 TCID50, respectively, whereas the EC50 values of molnupiravir were 0.63 μg/mL (1900 nM) for both doses. Intramuscular RPH-137 (30 and 80 mg/kg) had no effect on the infection process in Syrian hamsters. The comparison with the challenge control group showed that intraperitoneal RPH-137 (100 mg/kg) had statistically significant effects on a number of parameters, including a 27% reduction in inflammation and a 30% reduction in the total lesion area of the lungs by Day 7. Intragastric molnupiravir (300 mg/kg twice daily) significantly inhibited SARS-CoV-2 infection.Conclusions: both RPH-137 and molnupiravir inhibited the cytopathic effect of SARS-CoV-2 in Vero cells. In Syrian hamsters, molnupiravir demonstrated a more pronounced inhibition of SARS-CoV-2 infection than RPH-137. However, RPH-137 had statistically significant effects on a range of parameters. This offers additional perspectives for further research.Поиск эффективных и безопасных лекарственных средств для борьбы с коронавирусной инфекцией, вызванной вирусом SARS-CoV-2, является актуальной задачей. RPH-137 – оригинальный гибридный белок-ловушка вируса SARS-CoV-2, состоящий из внеклеточного домена ангиотензинпревращающего фермента 2 типа и Fc-фрагмента IgG1 человека.Цель работы: доклиническая оценка эффективности RPH-137 и молнупиравира в отношении инфекции, вызванной SARS-CoV-2.Материалы и методы: RPH-137 получали в стабильной линии клеток китайского хомячка. В работе использовали субстанцию молнупиравира. Изучение ингибирования вирус-индуцированной цитотоксичности проводили в культуре клеток Vero. В исследовании эффективности in vivo сирийских хомячков заражали интраназально SARS-CoV-2 (вариант ПИК35) в дозе 5 lg ТЦД50. Оценивали массу тела, массовый коэффициент и гистологическую картину легких. В орофарингеальных мазках измеряли содержание вирусной РНК методом ОТ-ПЦР по показателю порогового цикла амплификации Ct. Статистическая обработка: однофакторный и двухфакторный дисперсионный анализ (ANOVA), t-тест Стьюдента, критерий Манна–Уитни.Результаты: RPH-137 и молнупиравир ингибировали цитопатическое действие вируса SARS-CoV-2 в культуре клеток Vero: для RPH-137 EC50=4,69 мкг/мл (21,3 нМ) и 16,24 мкг/мл (73,8 нМ) для доз 50 ТЦД50 и 200 ТЦД50 соответственно, для молнупиравира EC50=0,63 мкг/мл (1900 нМ) для обеих доз вируса. RPH-137 при внутримышечном введении в дозах 30 и 80 мг/кг не оказывал влияния на развитие инфекции у сирийских хомячков. RPH-137 при внутрибрюшинном введении в дозе 100 мг/кг показал статистически значимый эффект по ряду параметров по сравнению с животными контрольной группы (контроль заражения), в том числе вызывая снижение воспалительного процесса и общей площади поражения легких на 7 сут на 27 и 30% соответственно. Молнупиравир при пероральном введении в дозе 300 мг/кг 2 раза в сутки значимо подавлял развитие инфекции, вызванной SARS-CoV-2.Выводы: RPH-137 и  молнупиравир ингибируют цитопатическое действие вируса SARS-CoV-2 в культуре клеток Vero. У сирийских хомячков введение молнупиравира демонстрировало более выраженное подавление инфекции, вызванной SARS-CoV-2, по сравнению с RPH-137. Однако RPH-137 проявлял статистически значимое действие по ряду параметров, что открывает перспективы для его дальнейшего изучения.

Creation Problems of Control System of the Lower Limbs Exoskeleton with Hydraulic Cylinders

The paper considers a problem of bio-mechanism control i.e. control of integrated human - exoskeleton, with its walking on the level horizontal surface on the flat regular single support foot.The exoskeleton is a metal rod structure having the same length as a human, but with different mass inertial characteristics. The exoskeleton is able to repeat the human-driven movement. There is a point load in the "backpack" behind human’s back. Elastic straps support the contact between the human body and the exoskeleton in eight points.The article explores the behavior of the exoskeleton in two modes: passive and active. The passive mode characterizes the behavior of the exoskeleton only under the action of forces generated by the human movement in the "comfortable" walking gait. As to the active mode, a difference from the passive one is that in the knee joints of the exoskeleton there are locomotors (propulsions) capable of developing the desired torques.Active mode investigation was based on the full dynamics equations. Exoskeleton control provided the desired movements in the knee joints with the required quality of transients. In the case of an ideal engine, simulation has shown the correctness of these algorithms and high accuracy of realization of defined gait (including also the angular position of the body exoskeleton) as well as has revealed a significant impact of damping coefficients and elastic straps. The study has demonstrated the possibility for selection factors to ensure a comfortable movement for the human - operator due to abrupt reduction of vibrations in the system. At the same time, decreasing human’s energy expenditures calculated by criteria of biomechanical functionality were observed. A model of the exoskeleton was also studied taking into account the dynamic properties of the hydraulic cylinders and the geometry of their position.The numerical studies allow us to make a sound choice of material for fastening straps of exoskeleton for the human body, to obtain desired parameters for selection of engines for the active option as well as to synthesize a control law that satisfies requirements for quality parameters.</p

Differential Regulation of Hepatic Heme Oxygenase-1 Protein With Aging and Heat Stress

Increased expression of heme oxygenase-1 (HO-1) in response to physiological stress is considered to be a protective response, which may be altered with aging. In this study, HO-1 expression was assessed following heat stress by immunoblotting of liver homogenates and isolated hepatocytes from young (6 months) and old (24 months) Fischer 344 rats and by immunohistochemistry. Livers of old rats showed higher baseline levels of HO-1, which was predominately localized to Kupffer cells. After heat stress, young animals showed a greater relative increase in hepatic HO-1, part of which was caused by increased numbers of nonparenchymal cells that were immunoreactive to HO-1. Consistent with these data, HO-1 was significantly upregulated after hyperthermia in vitro only in hepatocytes from young rats. Hence, aging alters stress-induced expression of HO-1 in a cell-specific manner, which may contribute to the diminished stress tolerance observed in older organisms