The Expression of Cell Cycle Cyclins in a Human Megakaryoblast Cell Line Exposed to Simulated Microgravity.

The study of the physiological and pathophysiological processes under extreme conditions facilitates a better understanding of the state of a healthy organism and can also shed light on the pathogenesis of diseases. In recent years, it has become evident that gravitational stress affects both the whole organism and individual cells. We have previously demonstrated that simulated microgravity inhibits proliferation, induces apoptosis, changes morphology, and alters the surface marker expression of megakaryoblast cell line MEG-01. In the present work, we investigate the expression of cell cycle cyclins in MEG-01 cells. We performed several experiments for 24 h, 72 h, 96 h and 168 h. Flow cytometry and Western blot analysis demonstrated that the main change in the levels of cyclins expression occurs under conditions of simulated microgravity after 96 h. Thus, the level of cyclin A expression showed an increase in the RPM group during the first 4 days, followed by a decrease, which, together with the peak of cyclin D, may indicate inhibition of the cell cycle in the G2 phase, before mitosis. In addition, based on the data obtained by PCR analysis, we were also able to see that both cyclin A and cyclin B expression showed a peak at 72 h, followed by a gradual decrease at 96 h. STED microscopy data also confirmed that the main change in cyclin expression of MEG-01 cells occurs at 96 h, under simulated microgravity conditions, compared to static control. These results suggested that the cell cycle disruption induced by RPM-simulated microgravity in MEG-01 cells may be associated with the altered expression of the main regulators of the cell cycle. Thus, these data implicate the development of cellular stress in MEG-01 cells, which may be important for proliferating human cells exposed to microgravity in real space.

iPSC-Derived Endothelial Cells Reveal LDLR Dysfunction and Dysregulated Gene Expression Profiles in Familial Hypercholesterolemia.

Defects in the low-density lipoprotein receptor (LDLR) are associated with familial hypercholesterolemia (FH), manifested by atherosclerosis and cardiovascular disease. LDLR deficiency in hepatocytes leads to elevated blood cholesterol levels, which damage vascular cells, especially endothelial cells, through oxidative stress and inflammation. However, the distinctions between endothelial cells from individuals with normal and defective LDLR are not yet fully understood. In this study, we obtained and examined endothelial derivatives of induced pluripotent stem cells (iPSCs) generated previously from conditionally healthy donors and compound heterozygous FH patients carrying pathogenic LDLR alleles. In normal iPSC-derived endothelial cells (iPSC-ECs), we detected the LDLR protein predominantly in its mature form, whereas iPSC-ECs from FH patients have reduced levels of mature LDLR and show abolished low-density lipoprotein uptake. RNA-seq of mutant LDLR iPSC-ECs revealed a unique transcriptome profile with downregulated genes related to monocarboxylic acid transport, exocytosis, and cell adhesion, whereas upregulated signaling pathways were involved in cell secretion and leukocyte activation. Overall, these findings suggest that LDLR defects increase the susceptibility of endothelial cells to inflammation and oxidative stress. In combination with elevated extrinsic cholesterol levels, this may result in accelerated endothelial dysfunction, contributing to early progression of atherosclerosis and other cardiovascular pathologies associated with FH.

Förster-free Effect Fluorescence Quenching of 5,10,15,20-tetraphenylporphyrin in Solution.

The absorption of exciting light by an inner filter may be the cause of large errors in estimating the efficiency of fluorescence quenching. We performed fluorescence measurements of 5,10,15,20-tetraphenylporphyrin toluene solution in a wide concentration range. We have shown a Förster-free fluorescence quenching in a right-angle geometry experiment, caused by second order inner filter. We proposed to perform measurements in a front-surface geometry to investigate the nature of quenching not related to the inner filters. We have shown that concentration effects in tetraphenylporphyrin solutions in toluene are not accompanied by a decrease in the fluorescence intensity in a front-surface geometry at concentrations of 10- 3-10- 7 mol/l. It was possible to separate the phenomena of the internal filters from the processes occurring in a liquid medium. Our results are of great importance due to the widespread fundamental research of porphyrin-based dyes properties.

Prospects for the Use of Intranasally Administered Insulin and Insulin-Like Growth Factor-1 in Cerebral Ischemia.

Current approaches to the treatment of stroke have significant limitations, and neuroprotective therapy is ineffective. In view of this, searching for effective neuroprotectors and developing new neuroprotective strategies remain a pressing topic in research of cerebral ischemia. Insulin and insulin-like growth factor-1 (IGF-1) play a key role in the brain functioning by regulating the growth, differentiation, and survival of neurons, neuronal plasticity, food intake, peripheral metabolism, and endocrine functions. Insulin and IGF-1 produce multiple effects in the brain, including neuroprotective action in cerebral ischemia and stroke. Experiments in animals and cell cultures have shown that under hypoxic conditions, insulin and IGF-1 improve energy metabolism in neurons and glial cells, promote blood microcirculation in the brain, restore nerve cell functions and neurotransmission, and produce the anti-inflammatory and antiapoptotic effects on brain cells. The intranasal route of insulin and IGF-1 administration is of particular interest in the clinical practice, since it allows controlled delivery of these hormones directly to the brain, bypassing the blood-brain barrier. Intranasally administered insulin alleviated cognitive impairments in elderly people with neurodegenerative and metabolic disorders; intranasally administered insulin and IGF-1 promoted survival of animals with ischemic stroke. The review discusses the published data and results of our own studies on the mechanisms of neuroprotective action of intranasally administered insulin and IGF-1 in cerebral ischemia, as well as the prospects of using these hormones for normalization of CNS functions and reduction of neurodegenerative changes in this pathology.

Calling and Phasing of Single-Nucleotide and Structural Variants of the LDLR Gene Using Oxford Nanopore MinION.

The LDLR locus has clinical significance for lipid metabolism, Mendelian familial hypercholesterolemia (FH), and common lipid metabolism-related diseases (coronary artery disease and Alzheimer's disease), but its intronic and structural variants are underinvestigated. The aim of this study was to design and validate a method for nearly complete sequencing of the LDLR gene using long-read Oxford Nanopore sequencing technology (ONT). Five PCR amplicons from LDLR of three patients with compound heterozygous FH were analyzed. We used standard workflows of EPI2ME Labs for variant calling. All rare missense and small deletion variants detected previously by massively parallel sequencing and Sanger sequencing were identified using ONT. One patient had a 6976 bp deletion (exons 15 and 16) that was detected by ONT with precisely located breakpoints between AluY and AluSx1. Trans-heterozygous associations between mutation c.530C>T and c.1054T>C, c.2141-966_2390-330del, and c.1327T>C, and between mutations c.1246C>T and c.940+3_940+6del of LDLR, were confirmed. We demonstrated the ability of ONT to phase variants, thereby enabling haplotype assignment for LDLR with personalized resolution. The ONT-based method was able to detect exonic variants with the additional benefit of intronic analysis in one run. This method can serve as an efficient and cost-effective tool for diagnosing FH and conducting research on extended LDLR haplotype reconstruction.

Dual PTP1B/TC-PTP Inhibitors: Biological Evaluation of 3-(Hydroxymethyl)cinnoline-4(1H)-Ones.

Dual inhibitors of protein phosphotyrosine phosphatase 1B (PTP1B)/T-cell protein phosphotyrosine phosphatase (TC-PTP) based on the 3-(hydroxymethyl)-4-oxo-1,4-dihydrocinnoline scaffold have been identified. Their dual affinity to both enzymes has been thoroughly corroborated by in silico modeling experiments. The compounds have been profiled in vivo for their effects on body weight and food intake in obese rats. Likewise, the effects of the compounds on glucose tolerance, insulin resistance, as well as insulin and leptin levels, have been evaluated. In addition, the effects on PTP1B, TC-PTP, and Src homology region 2 domain-containing phosphatase-1 (SHP1), as well as the insulin and leptin receptors gene expressions, have been assessed. In obese male Wistar rats, a five-day administration of all studied compounds led to a decrease in body weight and food intake, improved glucose tolerance, attenuated hyperinsulinemia, hyperleptinemia and insulin resistance, and also compensatory increased expression of the PTP1B and TC-PTP genes in the liver. The highest activity was demonstrated by 6-Chloro-3-(hydroxymethyl)cinnolin-4(1H)-one (compound 3) and 6-Bromo-3-(hydroxymethyl)cinnolin-4(1H)-one (compound 4) with mixed PTP1B/TC-PTP inhibitory activity. Taken together, these data shed light on the pharmacological implications of PTP1B/TC-PTP dual inhibition, and on the promise of using mixed PTP1B/TC-PTP inhibitors to correct metabolic disorders.

Early-Life Gut Health Indicators and Reported Prevalence of Infant Functional Constipation by Healthcare Professionals.

A healthy gut during early childhood is important. However, it seems that there are no standard indicators used to assess it. Healthcare professionals (HCPs) were asked via an electronic survey question about gut health indicators (GHIs) for infants and toddlers, in addition to an estimated prevalence of infant's functional constipation (FC) and its management. HCPs from eight countries participated in the survey (Russia (66.0%, 1449), Indonesia (11.0%, 242), Malaysia (6.0%, 132), Mexico (5.7%, 125), KSA (5.1%, 113), Turkey (3.0%, 66), Hong Kong (2.2%, 49), and Singapore (1.0%, 23)). The 2199 participating respondents were further classified into three continents (Asia (20.2%), Europe (68.8%), and others (11.0%)). Most of them were pediatricians (80.3%), followed by pediatric gastroenterologists (7.0%), general practitioners (6.4%), and others (6.3%). The top three preferred GHIs were similar for infants and toddlers: an absence of gastrointestinal (GI) symptoms, effective digestion/absorption as assessed by normal growth, and a general feeling of well-being. The absence of GI-related infection was the least preferred indicator. Most of the respondents reported the prevalence of FC among infants was less than 5%, with the peak incidence between the ages of 3 and 6 months. The reported choices of intervention to manage FC in infants were a change to a specific nutritional solution from a standard formula (40.2%), parental reassurance (31.7%), and lactulose (17.0%). Conclusion: The HCPs in the eight countries preferred the absence of GI symptoms, normal growth for effective digestion and absorption, and general well-being as the gut health indicators in infants and toddlers. The reported prevalence of FC in infants was less than 5%.

Letterer-Siwe disease presenting with gastrointestinal and cutaneous manifestations.

Histiocytosis is a set of distinct proliferative illnesses defined by the proliferation and infiltration of varied numbers of dendritic cells, macrophages, and monocytes in the afflicted tissues. The skin and other organs may be impacted by the inflammatory infiltration. It can occur at any age. The severity of the symptoms can range from mild to severe, depending on the degree and type of organ involvement. Although certain forms of histiocytosis can be fatal, others can be treated successfully without sequelae. Langerhans cell histiocytosis manifests itself clinically in both children and adults. A combination of clinical, histological, and radiological tests is required to achieve a diagnosis. A severe, multisystemic, acute form of Langerhans cell histiocytosis is called Letterer-Siewe illness, which usually affects infants in their first year of life. In this article, we provide a brief literature review and a case study of a 9-month-old girl who presented with recurring gastrointestinal problems as the first sign of Letterer-Siwe disease.

Reported Prevalence and Nutritional Management of Functional Constipation among Young Children from Healthcare Professionals in Eight Countries across Asia, Europe and Latin America.

Background: The prevalence of functional constipation (FC) among children varies widely. A survey among healthcare professionals (HCPs) was conducted to better understand the HCP-reported prevalence and (nutritional) management of FC in children 12−36 months old. Methods: An anonymous e-survey using SurveyMonkey was disseminated via emails or WhatsApp among HCPs in eight countries/regions. Results: Data from 2199 respondents were analyzed. The majority of the respondents (65.9%) were from Russia, followed by other countries (Indonesia (11.0%), Malaysia (6.0%)), Mexico, KSA (5.1% (5.7%), Turkey (3.0%), Hong Kong (2.2%), Singapore (1.1%)). In total, 80% of the respondents (n = 1759) were pediatricians. The prevalence of FC in toddlers was reported at less than 5% by 43% of the respondents. Overall, 40% of the respondents reported using ROME IV criteria in > 70% of the cases to diagnose FC, while 11% never uses Rome IV. History of painful defecation and defecations < 2 x/week are the two most important criteria for diagnosing FC. In total, 33% of the respondents reported changing the standard formula to a specific nutritional solution, accompanied by parental reassurance. Conclusion: The most reported prevalence of FC in toddlers in this survey was less than five percent. ROME IV criteria are frequently used for establishing the diagnosis. Nutritional management is preferred over pharmacological treatment in managing FC.

Constitutive heterochromatin propagation contributes to the X chromosome inactivation.

Imprinted X chromosome inactivation (iXCI) balances the expression of X-linked genes in preimplantation embryos and extraembryonic tissues in rodents. Long noncoding Xist RNA drives iXCI, silencing genes and recruiting Xist-dependent chromatin repressors. Some domains on the inactive X chromosome include repressive modifications specific to constitutive heterochromatin, which show no direct link to Xist RNA. We explored the relationship between Xist RNA and chromatin silencing during iXCI in vole Microtus levis. We performed locus-specific activation of Xist transcription on the only active X chromosome using the dCas9-SAM system in XO vole trophoblast stem cells (TSCs), which allow modeling iXCI events to some extent. The artificially activated endogenous vole Xist transcript is truncated and restricted ~ 6.6 kb of the exon 1. Ectopic Xist RNA accumulates on the X chromosome and recruits Xist-dependent modifications during TSC differentiation, yet is incapable by itself repressing X-linked genes. Transcriptional silencing occurs upon ectopic Xist upregulation only when repressive marks spread from the massive telomeric constitutive heterochromatin to the X chromosome region containing genes. We hypothesize that the Xist RNA-induced propagation of repressive marks from the constitutive heterochromatin could be a mechanism involved in X chromosome inactivation.

Mitomycin-Treated Endothelial and Smooth Muscle Cells Suitable for Safe Tissue Engineering Approaches.

In our previous study, we showed that discarded cardiac tissue from the right atrial appendage and right ventricular myocardium is an available source of functional endothelial and smooth muscle cells for regenerative medicine and tissue engineering. In the study, we aimed to find out what benefits are given by vascular cells from cardiac explants used for seeding on vascular patches engrafted to repair vascular defects in vivo. Additionally, to make the application of these cells safer in regenerative medicine we tested an in vitro approach that arrested mitotic division to avoid the potential tumorigenic effect of dividing cells. A tissue-engineered construction in the form of a patch based on a polycaprolactone-gelatin scaffold and seeded with endothelial and smooth muscle cells was implanted into the abdominal aorta of immunodeficient SCID mice. Aortic patency was assessed using ultrasound, MRI, immunohistochemical and histological staining. Endothelial and smooth muscle cells were treated with mitomycin C at a therapeutic concentration of 10 µg/ml for 2 h with subsequent analysis of cell proliferation and function. The absence of the tumorigenic effect of mitomycin C-treated cells, as well as their angiogenic potential, was examined by injecting them into immunodeficient mice. Cell-containing patches engrafted in the abdominal aorta of immunodeficient mice form the vessel wall loaded with the appropriate cells and extracellular matrix, and do not interfere with normal patency. Endothelial and smooth muscle cells treated with mitomycin C show no tumorigenic effect in the SCID immunodeficient mouse model. During in vitro experiments, we have shown that treatment with mitomycin C does not lead to a decrease in cell viability. Despite the absence of proliferation, mitomycin C-treated vascular cells retain specific cell markers, produce specific extracellular matrix, and demonstrate the ability to stimulate angiogenesis in vivo. We pioneered an approach to arresting cell division with mitomycin C in endothelial and smooth muscle cells from cardiac explant, which prevents the risk of malignancy from dividing cells in vascular surgery. We believe that this approach to the fabrication of tissue-engineered constructs based on mitotically inactivated cells from waste postoperative material may be valuable to bring closer the development of safe cell products for regenerative medicine.

Induced pluripotent stem cell line ICGi038-A, obtained by reprogramming peripheral blood mononuclear cells from a patient with familial hypercholesterolemia due to compound heterozygous c.1246C > T/c.940 + 3_940 + 6del mutations in LDLR.

The development of cellular models for familial hypercholesterolemia (FH) is an important direction for creating new approaches to atherosclerosis treatment. Pathogenic mutations in the LDLR gene are the main FH source. We generated an iPSC line from peripheral blood mononuclear cells of the patient with compound heterozygous c.1246C > T/c.940 + 3_940 + 6del LDLR mutation. The resulting iPSC line with confirmed patient-specific mutations maintains a normal karyotype and a typical undifferentiated state, including morphology, pluripotent gene expression, and in vitro differentiation potential. This iPSC line can be further differentiated toward relevant cells to better understand FH pathogenesis.

Induced pluripotent stem cell line ICGi037-A, obtained by reprogramming peripheral blood mononuclear cells from a patient with familial hypercholesterolemia due to heterozygous p.Trp443Arg mutations in LDLR.

Familial hypercholesterolemia (FH) is an autosomal dominant disorder increasing premature cardiovascular diseases risk due to atherosclerosis. Pathogenic mutations in the LDLR gene cause most FH cases. Available treatments are effective not for all LDLR mutations. Testing drugs on FH cell models help develop new efficient treatments. We obtained an iPSC line from peripheral blood mononuclear cells of the patient with heterozygous p.Trp443Arg LDLR mutation. The iPSCs with confirmed patient-specific mutations express pluripotency markers, spontaneously differentiate into three germ layers and demonstrate normal karyotype. Patient-specific iPSCs-derived hepatocyte-like and endothelial cells are promising to develop new targeted therapies for FH.

Induced pluripotent stem cell line ICGi036-A generated by reprogramming peripheral blood mononuclear cells from a patient with familial hypercholesterolemia caused due to compound heterozygous p.Ser177Leu/p.Cys352Arg mutations in LDLR.

Familial hypercholesterolemia (FH) is a monogenic disease, leading to atherosclerosis due to a high level of low-density lipoprotein cholesterol. Most cases of the disease are based on pathological variants in the LDLR gene. Hepatocyte-like and endothelial cells derived from individual iPSCs are a good model for developing new approaches to therapy. We obtained an iPSC line from peripheral blood mononuclear cells of the patient with compound heterozygous p.Ser177Leu/p.Cys352Arg mutation in LDLR using non-integrating vectors. The iPSCs with a confirmed patient-specific mutation demonstrate pluripotency markers, normal karyotype, and the ability to differentiate into derivatives of three germ layers.

Insulin and α-Tocopherol Enhance the Protective Effect of Each Other on Brain Cortical Neurons under Oxidative Stress Conditions and in Rat Two-Vessel Forebrain Ischemia/Reperfusion Injury.

Clinical trials show that insulin administered intranasally is a promising drug to treat neurodegenerative diseases, but at high doses its use may result in cerebral insulin resistance. Identifying compounds which could enhance the protective effects of insulin, may be helpful to reduce its effective dose. Our aim was thus to study the efficiency of combined use of insulin and α-tocopherol (α-T) to increase the viability of cultured cortical neurons under oxidative stress conditions and to normalize the metabolic disturbances caused by free radical reaction activation in brain cortex of rats with two-vessel forebrain ischemia/reperfusion injury. Immunoblotting, flow cytometry, colorimetric, and fluorometric techniques were used. α-T enhanced the protective and antioxidative effects of insulin on neurons in oxidative stress, their effects were additive. At the late stages of oxidative stress, the combined action of insulin and α-T increased Akt-kinase activity, inactivated GSK-3beta and normalized ERK1/2 activity in cortical neurons, it was more effective than either drug action. In the brain cortex, ischemia/reperfusion increased the lipid peroxidation product content and caused Na+,K+-ATPase oxidative inactivation. Co-administration of insulin (intranasally, 0.25 IU/rat) and α-T (orally, 50 mg/kg) led to a more pronounced normalization of the levels of Schiff bases, conjugated dienes and trienes and Na+,K+-ATPase activity than administration of each drug alone. Thus, α-T enhances the protective effects of insulin on cultured cortical neurons in oxidative stress and in the brain cortex of rats with cerebral ischemia/reperfusion injury.

The Effects of Separate and Combined Treatment of Male Rats with Type 2 Diabetes with Metformin and Orthosteric and Allosteric Agonists of Luteinizing Hormone Receptor on Steroidogenesis and Spermatogenesis.

In men with type 2 diabetes mellitus (T2DM), steroidogenesis and spermatogenesis are impaired. Metformin and the agonists of luteinizing hormone/human chorionic gonadotropin(hCG)-receptor (LH/hCG-R) (hCG, low-molecular-weight allosteric LH/hCG-R-agonists) can be used to restore them. The aim was to study effectiveness of separate and combined administration of metformin, hCG and 5-amino-N-tert-butyl-2-(methylsulfanyl)-4-(3-(nicotinamido)phenyl)thieno[2,3-d]pyrimidine-6-carboxamide (TP3) on steroidogenesis and spermatogenesis in male rats with T2DM. hCG (15 IU/rat/day) and TP3 (15 mg/kg/day) were injected in the last five days of five-week metformin treatment (120 mg/kg/day). Metformin improved testicular steroidogenesis and spermatogenesis and restored LH/hCG-R-expression. Compared to control, in T2DM, hCG stimulated steroidogenesis and StAR-gene expression less effectively and, after five-day administration, reduced LH/hCG-R-expression, while TP3 effects changed weaker. In co-administration of metformin and LH/hCG-R-agonists, on the first day, stimulating effects of LH/hCG-R-agonists on testosterone levels and hCG-stimulated expression of StAR- and CYP17A1-genes were increased, but on the 3-5th day, they disappeared. This was due to reduced LH/hCG-R-gene expression and increased aromatase-catalyzed estradiol production. With co-administration, LH/hCG-R-agonists did not contribute to improving spermatogenesis, induced by metformin. Thus, in T2DM, metformin and LH/hCG-R-agonists restore steroidogenesis and spermatogenesis, with metformin being more effective in restoring spermatogenesis, and their co-administration improves LH/hCG-R-agonist-stimulating testicular steroidogenesis in acute but not chronic administration.

Vitamin D Status in Russian Children and Adolescents: Contribution of Genetic and Exogenous Factors.

Background: The problem of vitamin D deficiency is particularly relevant for the entire territory of Russia, since most parts of the country are located above the 42nd geographical latitude and the residents are therefore at risk of vitamin D deficiency. Despite the urgency of the problem, a comprehensive study of the molecular and genetic mechanisms and exogenous factors of vitamin D deficiency in children living in various geographical areas of the Russian Federation has not been conducted. Different variants in the loci of the genes responsible for the synthesis, hydroxylation, and transport of vitamin D (such as DHCR7, CYP2R1, CYP24A1, and GC), as well as VDR gene polymorphisms may also be associated with the risk of vitamin D deficiency. The aim of this study was to analyze the influence of exogenous factors on the blood levels of 25-hydroxyvitamin D (25(OH)D) in children of three regions of the Russian Federation, as well as the relationship of blood 25(OH)D levels with polymorphic variants of cytochrome P450 genes and VDR gene. Methods: We conducted blood 25(OH)D level analysis in 333 healthy children and adolescents in three regions located in different geographical zones of the Russian Federation. We studied the polymorphic variants c.1075A>C (I359L, rs1057910, CYP2C9 * 3) and c.430C>T (R144C, rs1799853, CYP2C9 * 2) in the CYP2C9 gene, c.1334T>C (M445T, rs4986910, CYP3A4 * 3), and CYP3A4 * 1B (c.-392C>T, rs2740574) in the CYP3A4 gene, 1846G>A, (rs3892097, CYP2D6 * 4) in the CYP2D6gene, TaqI (NM_000376.2: c.1056T>C; rs731236), FokI (NM_000376.2:c.2T>C; (rs2228570), and BsmI (NM_000376.2: c.1024+283G>A; rs1544410) in the VDR gene. We also analyzed the influence of exogenous factors on the level of 25(OH)D in children of the three study regions, as well as the relationship of the level of 25(OH)D with variants CYP2C9 * 2 (c.430C>T; R144C), CYP2C9 * 3 (c,1075A>C; I359L), CYP2D6 * 4 (1846G>A), CYP3A4 * 3 (c.1334T>C), and CYP3A4 * 1B (c.-392C>T) and rs731236, rs2228570 and rs1544410 in the VDR gene. Results: We found that the blood level of 25(OH)D depended on the geographical location and the number of sunny days per year. The average blood level of 25(OH)D in adolescent boys was statistically significantly lower than in girls of this age group. The level of 25(OH)D also significantly depended on the prophylactic dose of cholecalciferol administered to the subjects. In the study, it was shown that a dose of cholecalciferol ≥1,000 IU per day can achieve a normal level of 25(OH)D in healthy children. We found no statistically significant association between single-nucleotide polymorphic variants of cytochrome P450 genes (CYP2C9 * 3, CYP3A4 * 3, CYP2C9 * 2, CYP2D6 * 4, and CYP3A4 * 1B) and blood level of 25(OH)D in the subjects. We also did not find a relationship between the TaqI, FokI, and BsmI polymorphisms of the VDR gene and serum 25(OH)D concentration. Conclusion: Exogenous factors (time of year, place of residence, and prophylactic administration of cholecalciferol), as well as endogenous factors (age and sex), play a determining role in the development of vitamin D deficiency and insufficiency; in contrast to genetic factors-polymorphic variants of the genes of xenobiotic phase 1 enzymes (CYP2C9, CYP2C19, CYP2D6, and CYP3A4) and the VDR gene-which do not play such role. This study shows the need to create a diagnostic algorithm for Vitamin D deficiency based on the age, season of the year, and prophylactic dose of cholecalciferol.

Intestinal microbiome analysis demonstrates azithromycin post-treatment effects improve when combined with lactulose.

BACKGROUND: Next-generation sequencing has revolutionized our perspective on the gut microbiome composition, revealing the true extent of the adverse effects of antibiotics. The impact of antibiotic treatment on gut microbiota must be considered and researched to provide grounds for establishing new treatment strategies that are less devastating on commensal bacteria. This study investigates the impact on gut microbiome when a commonly used antibiotic, azithromycin is administered, as well as uncovers the benefits induced when it is used in combination with lactulose, a prebiotic known to enhance the proliferation of commensal microbes. METHODS: 16S rRNA gene sequencing analysis of stool samples obtained from 87 children treated with azithromycin in combination with or without lactulose have been determined. Children's gut microbial profile was established at the pre- and post-treatment stage. RESULTS: Azithromycin caused an increase in the relative abundance of opportunistic pathogens such as Streptococcus that was evident 60 days after treatment. While few days after treatment, children who also received lactulose started to show a higher relative abundance of saccharolytic bacteria such as Lactobacillus, Enterococcus, Anaerostipes, Blautia and Roseburia, providing a protective role against opportunistic pathogens. In addition, azithromycin-prebiotic combination was able to provide a phylogenetic profile more similar to the pre-treatment stage. CONCLUSION: It is suggested that during azithromycin treatment, lactulose is able to reinstate the microbiome equilibrium much faster as it promotes saccharolytic microbes and provides a homeostatic effect that minimizes the opportunistic pathogen colonization.

The Protective Effect of Insulin on Rat Cortical Neurons in Oxidative Stress and Its Dependence on the Modulation of Akt, GSK-3beta, ERK1/2, and AMPK Activities.

Insulin is a promising drug for the treatment of diseases associated with brain damage. However, the mechanism of its neuroprotective action is far from being understood. Our aim was to study the insulin-induced protection of cortical neurons in oxidative stress and its mechanism. Immunoblotting, flow cytometry, colorimetric, and fluorometric techniques were used. The insulin neuroprotection was shown to depend on insulin concentration in the nanomolar range. Insulin decreased the reactive oxygen species formation in neurons. The insulin-induced modulation of various protein kinase activities was studied at eight time-points after neuronal exposure to prooxidant (hydrogen peroxide). In prooxidant-exposed neurons, insulin increased the phosphorylation of GSK-3beta at Ser9 (thus inactivating it), which resulted from Akt activation. Insulin activated ERK1/2 in neurons 5-30 min after cell exposure to prooxidant. Hydrogen peroxide markedly activated AMPK, while it was for the first time shown that insulin inhibited it in neurons at periods of the most pronounced activation by prooxidant. Insulin normalized Bax/Bcl-2 ratio and mitochondrial membrane potential in neurons in oxidative stress. The inhibitors of the PI3K/Akt and MEK1/2/ERK1/2 signaling pathways and the AMPK activator reduced the neuroprotective effect of insulin. Thus, the protective action of insulin on cortical neurons in oxidative stress appear to be realized to a large extent through activation of Akt and ERK1/2, GSK-3beta inactivation, and inhibition of AMPK activity increased by neuronal exposure to prooxidant.

Trapping of two-component light bullets in a gradient waveguide with normal group dispersion.

In this paper we consider the process of the second harmonic generation in a gradient waveguide, taking into account diffraction and relatively weak temporal dispersion. Using the slowly varying envelope approximation and neglecting the dispersion of the nonlinear part of the response of the medium we obtain the system of parabolic equations for the envelopes of both harmonics. We also derive integrals of motion of this system. To solve it numerically we construct a nonlinear finite-difference scheme based on the Crank-Nicolson method preserving the integrals. Primarily, we focus our investigations on the processes of a two-component light bullets generation. We demonstrate that the generation of a coupled pair is possible in a planar waveguide even at normal group velocity dispersion.