[The Efficiency of Gene Activation Using CRISPR/dCas9-Based Transactivation Systems Depends on the System Run Time].

Transactivation systems are a promising application based on the CRISPR/Cas9 system and allow targeted control of gene expression levels in cell culture. However, their performance has been reported to vary considerably depending on the cell type and the activator system. Three activator systems (dCas9-VP160, dCas9-SunTag, and dCas9-VPR) were compared for the efficiency of activating expression of OCT4, NANOG, PDX1, FOXA2, NKX2-2, and NKX6-1 in an immortalized human skin fibroblast line. The activation efficiency was found to depend on the activation system type; the extent of activation depended on the system run time.

Pathological Features in 100 Deceased Patients With COVID-19 in Correlation With Clinical and Laboratory Data.

Background: Autopsies on COVID-19 deceased patients have many limitations due to necessary epidemiologic and preventative measures. The ongoing pandemic has caused a significant strain on healthcare systems and is being extensively studied around the world. Clinical data does not always corelate with post-mortem findings. The goal of our study was to find pathognomonic factors associated with COVID-19 mortality in 100 post-mortem full body autopsies. Materials and Methods: Following necessary safety protocol, we performed 100 autopsies on patients who were diagnosed with COVID-19 related death. The macroscopic and microscopic pathologies were evaluated along with clinical and laboratory findings. Results: Extensive coagulopathic changes are seen throughout the bodies of diseased patients. Diffuse alveolar damage is pathognomonic of COVID-19 viral pneumonia, and is the leading cause of lethal outcome in younger patients. Extrapulmonary pathology is predominantly seen in the liver and spleen. Intravascular thrombosis is often widespread and signs of septic shock are often present. Conclusion: The described pathological manifestations of COVID-19 in deceased patients are an insight into the main mechanisms of SARS-CoV-2 associated lethal outcome. The disease bears no obvious bias in severity, but seems to be more severe in some patients, hinting at genetic or epigenetic factors at play.

Analysis of Cytoprotective Properties of Afobazole in Streptozotocin Model of Diabetes.

Previous in vitro and in vivo studies revealed the neuroprotective effect of anxiolytic Afobazole. Based on similarities in the regulation of functions of neurons and ß cells, we studied the effect of Afobazole on streptozotocin (STZ) model of type 2 diabetes in Wistar rats. Immunohistochemical analysis showed that the decrease in the number of ß cells and a violation of their morphological structure caused by STZ were significantly alleviated by Afobazole administration (10 mg/kg orally for 28 days) to diabetic animals. A correlation between morphometric data and blood glucose level was revealed. A possible role of σ1-receptors in the cytoprotective effects of Afobazole in respect to pancreatic ß cells is discussed.

A Translational Model of Chronic Heart Failure.

We created a translational model of chronic heart failure in rats that developed in 3 months after reproducing experimental anterior transmural myocardial infarction. The model simulated the basic clinicodiagnostic criteria of this disease: impaired contractility and dilatation of heart ventricles, signs of venous congestion, elevated plasma content of biochemical markers, and abnormal overexpression of AT1aR and ß-adrenoceptors.

Alcoholic Cardiomyopathy: Translation Model.

We developed a translation model of alcoholic cardiomyopathy in rats. By the end of forced alcoholization (the rats received 10% ethanol solution as the only source of fluid for 24 weeks; mean daily ethanol consumption was 5.0-6.5 g/kg), the rats developed dilated heart failure. Echocardiography and morphometric study of the myocardium revealed a decrease in inotropic function of the heart and dilatation of the right and left ventricles. Fatty degeneration of the myocardium (pathognomonic sign of alcoholic cardiomyopathy) and decrease in electrical stability of cardiomyocytes reliably reproduce the clinical pattern of alcoholic cardiomyopathy.

Antiangiogenic Effects of Nerve Growth Factor Loop 4 Monomeric Dipeptide Mimetic.

The effects of GK-1, a monomeric dipeptide mimetic of nerve growth factor (NGF) loop 4, on angiogenesis were studied in vitro and in vivo. Experiments on human umbilical vein endothelial cells HUVEC showed that the test compound did not affect tubulogenesis (initial stage of angiogenesis) and prevented realization of the angiogenic effect of NGF and its dimeric dipeptide mimetic GK-2. Experiments on rat hind limb ischemia model demonstrated that GK-1 (1 mg/kg/day intraperitoneally over 14 days) significantly reduced the density of the capillary network in ischemic tissue and increased the number and area of Zenker necrosis in comparison with the control. These data suggest that GK-1 exhibits a pronounced antiangiogenic activity.

Angiogenic Effects of Dimeric Dipeptide Mimetic of Loop 4 of Nerve Growth Factor.

Angiogenic action of compound GK-2, a dimeric dipeptide mimetic of loop 4 of nerve growth factor (NGF), was studied in in vitro and in vivo experiments. Experiments on human endothelial cell culture HUVEC showed that compound GK-2 significantly (p<0.05) stimulated the initial stage of angiogenesis, and its angiogenic activity was not inferior to the reference neurotrophin NGF. In experiments with hindlimb ischemia modeled in rats, GK-2 (1 mg/kg intraperitoneally for 14 days) significantly increased the total length of capillary vessels (p<0.003) and the number of vessels per 1 mm2 ischemic tissue (p<0.001) in comparison with the control. Our findings indicate that under experimental conditions compound GK-2 exhibits not only angiogenic, but also anti-ischemic activity.

Evidence of Echocardiography Validity in Model Experiments on Small Animals.

Dynamic echocardiographic monitoring in rats subjected to forced alcoholization showed the formation of disorders in intracardiac hemodynamics characteristic of ethanol cardiomyopathy formed by the end of 24-week continuous ethanol consumption. The results of echocardiographic monitoring were confirmed by histological and morphometric studies demonstrating fatty infiltration of the myocardium pathognomonic for this condition and bifocal dilatation of cardiac ventricles. These results persuasively demonstrate that echocardiographic studies on small animals are valid and can be used for search for cardiotropic drugs and studies of the mechanisms of their activities.

[Role of octreotide and prednisolone in prophylactic of poshepatectomy liver failure. Experimental study].

AIM: Comparative morphologic assessment of the liver tissue response to the preoperative infusion of octreotide and prednisolon after the major hepatic resection was studied in rats. MATERIAL AND METHODS: 25 male Wistar rats weighing 230--280 g were used. All rats underwent 70--80% hepatectomy. The rats were divided into three groups according to the infusions before hepatectomy: group 1 (n=7) -- received octreotide, group 2 (n=8) -- prednisolone, group 3 (n=10) -- 0.9% saline solution as the control. Histologic features of the remnant liver were evaluated in the sacrificied rats after 72 hours post-hepatectomy. RESULTS: In the group 1 we observed more rapid decrease of edema and tendency to the accelerated regeneration process of hepatocytes. CONCLUSION: Octreotide infusion before the major hepatic resection may have protective effect on hepatocytes and accelerate the regeneration in the remnant liver.

[Cognitive impairments in depression: neuropsychological and MRI-studies]. / Kognitivnye rasstroistva pri depressiiakh: neiropsikhologicheskoe i MRT-issledovanie.

OBJECTIVE: A complex neuropsychological and neuroimaging study of deep brain structures in depression with cognitive impairment. MATERIAL AND METHODS: We studied 73 patients with endogenous depression and 86 patients with depressive syndrome in temporal epilepsy. MRI and neuropsychological methods were used to study brain structures. Results and сonclusion. Neurocognitive impairment was more severe in patients with depressive syndrome in the structure of temporal epilepsy. The differences between the patients with endogenous and organic (temporal epilepsy) affective disturbances were determined by the more marked memory and spatial-constructive impairments in patients with organic disorders. Deficit of executive functions, planning functions and cognitive organization were more typical for the patients with endogenous affective disorders. The MRI study revealed the decrease in the left hippocampus due to sclerotic processes in patients with temporal epilepsy and the increase in the right amygdale in patients with endogenous depressive disorders. The results demonstrate the significant similarity between characteristics of the cognitive profile in patients with endo- and exogenous depressions.

Comparison of the efficiency of transplantation of bone marrow multipotent mesenchymal stromal cells cultured under normoxic and hypoxic conditions and their conditioned media on the model of acute lung injury.

The therapeutic efficiency of intravenous injection of rat bone marrow multipotent mesenchymal stromal cells grown under conditions of normoxia and hypoxia (3% O2) and conditioned media from these cultures were compared on the rat model of acute lung injury induced by intraperitoneal injection of lipopolysaccharide. The best therapeutic efficiency was demonstrated by cells grown under hypoxic conditions. The effect of conditioned media was less pronounced and did not depend on the culturing conditions.

[Pseudogene PTENP1 5'-region methylation in endometrial cancer and hyperplasias].

Genetic mutations in tumor suppressor gene PTEN are often detected in malignant human cells and these genomic changes are especially characteristic ofendometrial cancer. In our previous researches we assumed that alternative epigenetic mechanism of PTEN inactivation trough promoter methylation may exist in endometrial cancer. Moreover, PTENP1 pseudogene has recently been shown to play a role in positive regulation of PTENgene expression. Taking into account these facts, we analyzed PTEN and PTENP1 methylation status in endometrial hyperplasia and cancer. It was demonstrated that PTENgene promoter was not methylated but PTENP1 was methylated in 11 of 18 endometrial cancers and in 5 of9 endometrial hyperplasias. We can assume that PTENP1 methylation may inhibit transcription of this gene and also PTEN gene transcription trough RNA interference in accordance with ceRNA theory. Thus, aberrant suppression of PTENP1 transcription can play a role in endometrial cancer pathogenesis.

Study of anti-ischemic effect of afobazole in experimental myocardial infarction.

Seven-day treatment of rats with experimental myocardial infarction with afobazole (5-ethoxy-2-[2-morpholino)-ethylthio] benzimidasole dihydrochloride) resulted in shrinkage of the ischemic damage area in the heart, stimulation of reparative processes in the myocardium, and prevention of postinfarction remodeling of the left ventricle. Anti-ischemic effect of afobazole in experimental myocardial infarction is presumably due to its interactions with σ(1) receptors.

[The role of innate immunity system in the course of adenomyosis].

The investigation of leukocytic elastase (LE) and alpha1-proteinase inhibitor (alpha1-PI) from patients with different stage adenomyosis and in control group was found activation innate immunity system in all the patients with adenomyosis. The degree of LE activity is a prevalence rate of adenomyosis. The degree of alpha1-PI activity is correlated with antiproteolytic potential that blocks the effects shown by LE. It can lead the prognose of disease and timely treatment.

Study of anti-inflammatory effects of GB-115, a glycine-containing retropeptide cholecystokinin analog.

Anti-inflammatory effects of GB-115 compound (N-phenylhexanoyl-glycyl-L-tryptophan amide) injected intraperitoneally in doses of 0.1, 1, and 10 mg/kg were demonstrated on the model of ConA- and carrageenan-induced inflammation. Intraperitoneal injection of GB-115 in a dose of 1 mg/kg to C57Bl/6 female mice with experimental autoimmune encephalomyelitis significantly alleviated the pathological symptoms, improved spontaneous locomotor activity, promoted recovery of thymus weight, and reduced edema and neutrophil infiltration of the perivascular space of the brain tissue. Intraperitoneal injection of GB-115 in a dose of 1 mg/kg suppressed generation of active oxygen forms by neutrophils in the chemiluminescence test.

[Preclinical safety investigation of GB-115 dipeptide].

Preclinical safety investigations of newly synthesized dipeptide compound GB-115 (amide N-phenylhexanoyl-glycyl-L-tryptophan), an antagonist of cholecystokinin receptors, were performed. No animals were lost after GB-115 acute oral administration at a maximum dose of 6000 mg/kg in mice and at 3500 mg/kg in rats. GB-115 administered per os during 6 months in rabbits and rats (both males and females) at the doses of 0.1 and 10 mg/kg induced no irreversible pathological changes in organs and systems studied. The tested dipeptide exhibited no allergenic, immunotoxic and mutagenic activity, and did not affect generative function and the antenatal and postnatal development of progeny. GB-115 at a dose of 10 mg/kg produced suppression of the inflammatory reaction to concanavalin A.

[Effects of zatebradine on the course of experimental myocardial infarction under long-term treatment conditions in rats].

A long-term (21 days) administration of the specific bradycardic agent zatebradine to rats with experimental myocardial infarction led to a decrease in the intensity of necrotic changes in the cardiac muscle as evaluated from the ECG-recorded QS complex incidence rate. Morphological studies provided evidence for reduced intensity of the dystrophic processes in myocardium. Under these conditions, the drug did not affect the pump and contractile heart functions. At the same time, zatebradine normalized the reaction of mean aorta blood flow acceleration to volume load (which was inhibited at myocardial infarction), that is, prevented the development of latent heart failure. Zatebradine restored the infarction-decreased norepinephrine (NE) level in cardiac muscle and increased NE content in hypothalamus and brainstem. Along with that, the ratios of deoxyphenylacetic acid/dopamine and homovanillic acid/dopamine were reduced.

[Promoters of genes MTHFR from patients with hyperhomocysteinemia and PTEN from patients with malignant and benign endometrial and ovarian tumors].

Mutational changes in the promoter regions of MTHFR genes from patients with hyperhomocysteinemia and PTEN genes from patients with endometrial and ovarian tumors were studied. An increased level of homocysteine was found in a part of the patients with a heterozygous C677T mutation in the MTHFR gene, although a moderate hyperhomocysteinemia is usually associated with homozygous mutation. We hypothesized that, in this case, the allele lacking the C677T mutation may be inactivated by the promoter mutation. The sequencing of both DNA strands of the minimal promoter region of the MTHFR gene in ten patients did not reveal any mutation, which implied another mechanism of the development of hyperhomocysteinemia in these patients. A PCR analysis of the minimal promoter region of the tumor suppressor PTEN in the presence of 2-pyrrolidone in 101 patients from Moscow clinics revealed changes in it in patients with endometrial (56%) or ovarian (29%) cancer, as well as in patients with endometrial hyperplasia and benign ovarian tumors (34.6 and 29%, respectively). It was presumed that the found PTEN gene promoters may arise from epigenetic alterations (erroneous methylation) or may (more rarely) be induced by mutations. As a result of the studies, new molecular markers associated with endometrial and ovarian tumors were revealed and a simple and effective method of detection of these markers was developed.

[The effect of hemantane on the generative function and gonad morphology in rats]. / Vliianie Gimantana na generativnuiu funktsiiu i morfologiiu gonad.

Effect of the new potential antiparkinsonian drug hemantane (N-(adamant-2-yl)hexamethyleneimine hydrochloride) on the generative function and gonad morphology was studied in a group of male and female mongrel rats. The generative function was studied after peroral drug administration in a dose of 10 mg/kg (ED50) and 50 mg/kg (5 ED50): males were treated over a 60-day period of spermatogenesis, while females received the drug in the same doses over 15 days (three estrous cycles). The gonad morphology was studied after a 6-month treatment of experimental animals with hemantane in the same doses. It was established that the administration hemantane in indicated doses did not influence the generative function and gonad morphology in male and female rats.

[Preclinical study of noopept toxicity]. / Doklinicheskoe izuchenie toksichnisti noopepta.

Within the framework of a preclinical investigation, the new nootrope drug noopept (N-phenyl-acetyl-L-propyl-glycine ethylate) was tested for chronic toxicity upon peroral administration in a dose of 10 or 100 mg/kg over 6 months in both male and female rabbits. The results of observations showed that noopept administered in this dose range induced no irreversible pathologic changes in the organs and systems studied and exhibited no allergenic, immunotoxic, and mutagen activity. The drug affected neither the generative function nor the antenatal or postnatal progeny development. Noopept produced a dose-dependent suppression of inflammation reaction to concanavalin A and stimulated the cellular and humoral immune response in mice.