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Aim: Comparative analysis of clinical, anamnestic, and laboratory and instrumental data of patients with chronic heart failure (CHF) and iron deficiency (ID) diagnosed according to the AHA/ESC/RSC criteria, and CHF patients diagnosed with ID based on decreased transferrin saturation (TSAT) ≤19.8% or serum iron (Fe) ≤13 μmol/l., Material and Methods: An additional analysis of the ID-CHF-RF study was performed. The analyzed population included 498 patients (198 women) with CHF. In addition to the ID criteria provided by the protocol (AHA/ESC/RSC criteria: ferritin <100 μg/l or ferritin from 100 to 299 μg/l and TSAT<20%), concentrations of ID biomarkers were assessed, which showed high sensitivity and specificity for the diagnosis of ID compared to the morphological picture of the bone marrow (TSAT<19.8% or Fe ≤13 μmol/l). Subgroups of patients with ID determined only by the AHA/ESC/RSC criteria, only by the TSAT≤19.8% and Fe ≤13 μmol/l criteria, and by both were analyzed., Results: ID diagnosed by the AHA/ESC/RSC criteria was found in 83.1% of patients. The TSAT ≤19.8% and Fe ≤13 μmol/l criteria revealed ID in 74.5% of patients. In 341 patients (76.8%), ID was diagnosed using both criteria. Patients with ID diagnosed by the TSAT≤19.8% and Fe≤13 μmol/l criteria, compared with patients with ID diagnosed by the AHA/ESC/RKO criteria, had a 50% lower Fe (9.8 μmol/l vs. 19.4 μmol/l) and a higher incidence of anemia (43.3% vs. 23.3%) and diabetes mellitus (DM) (36.7% and 24.7%). Also, these patients had higher values of body mass index (BMI) and NT-proBNP concentration (2317 [1305;9092] vs. 1691 [709;3856] pg/ml), and lower LV EF values (41.5 [29.0;54.5]% vs. 45.0 [34.0;54.0]%), respectively. The most severe course of CHF and the greatest changes in laboratory tests associated with ID and anemia were observed in patients with ID determined by two criteria. Patients in this group were older, with a higher BMI, more frequent presence of atrial fibrillation, and higher NT-proBNP (4182 [1854;9341] pg/ml)., Conclusion: Patients with isolated low ferritin are characterized by less severe clinical and functional impairment compared to patients with low TSAT or Fe. At the same time, patients with ferritin higher than 300 μg/l and low TSAT and/or Fe were characterized by very severe CHF and a low functional status, although this may not be related with ID. Thus, the use of the ferritin-based criteria of ID may lead to overdiagnosis of ID in some patients and, at the same time, miss some of the most "severe" patients who likely require the ID correction. Patients with ID who show a decrease in all three parameters are likely to benefit most from Fe supplementation. It is advisable to perform additional studies on the effect of Fe supplements on the course and prognosis of the disease in this cohort of patients.

Objective: To evaluate the influence of disease activity, the degree of patient disability and pathogenetic therapy on the concentration of neurofilament light chain (NFL) in the blood serum of patients with multiple sclerosis (MS)., Material and Methods: One hundred and fourteen patients (84 women and 30 men) with definite MS were examined. The concentration of NFL in the blood serum of patients with MS was determined by enzyme immunoassay. The level of NFL was analyzed depending on the characteristics of the course and activity of the demyelinating process, the severity of neurological disorders as well as disease modifying drugs (DMDs)., Results: An NFL level equal to or greater than 4201 pg/ml was found to be associated with a higher risk of developing a clinical exacerbation in the next 4 months. In patients with progression of disability over the next 2 years, the initial concentration of NFL was significantly higher than in the group with a stable EDSS score. The NFL level (4943 pg/ml and higher), which may be a predictor of increased disability in the next two years, was established., Conclusion: The study demonstrates the possibility of using serum NFL levels as a diagnostic marker of possible exacerbation, as well as predicting disability in patients with MS.

Cohen syndrome is an autosomal recessive disorder caused by VPS13B ( COH1 ) gene mutations. This syndrome is significantly underdiagnosed and is characterized by intellectual disability, microcephaly, autistic symptoms, hypotension, myopia, retinal dystrophy, neutropenia, and obesity. VPS13B regulates intracellular membrane transport and supports the Golgi apparatus structure, which is critical for neuron formation. We generated induced pluripotent stem cells from two patients with pronounced manifestations of Cohen syndrome and differentiated them into neural stem cells and neurons. Using transmission electron microscopy, we documented multiple new ultrastructural changes associated with Cohen syndrome in the neuronal cells. We discovered considerable disturbances in the structure of some organelles: Golgi apparatus fragmentation and swelling, endoplasmic reticulum structural reorganization, mitochondrial defects, and the accumulation of large autophagosomes with undigested contents. These abnormalities underline the ultrastructural similarity of Cohen syndrome to many neurodegenerative diseases. The cell models that we developed based on patient-specific induced pluripotent stem cells can serve to uncover not only neurodegenerative processes, but the causes of intellectual disability in general.

Aim    To evaluate the incidence of iron deficiency (ID) in men and women with chronic heart failure (CHF) and to compare clinical and functional indexes in patient with and without ID depending on the gender.Material and methods    An additional analysis of the study "Prevalence of Iron Deficiency in Patients With Chronic Heart Failure in the Russian Federation (ID-CHF-RF)" was performed. The study included 498 (198 women, 300 men) patients with CHF, in whom, in addition to iron metabolism, the quality of life and exercise tolerance (ET) were studied. 97 % of patients were enrolled during their stay in a hospital. ID was defined in consistency with the European Society of Cardiology (ESC) Guidelines. Also, and additional analysis was performed according to ID criteria validated by the morphological picture of the bone marrow.Results    ID was detected in 174 (87.9 %) women and 239 (79.8 %) men (p=0.028) according to the ESC criteria, and in 154 (77.8 %) women and 217 (72.3 %) men (p=0.208) according to the criteria validated by the morphological picture of the bone marrow. Men with ID were older and had more severe CHF. They more frequently had HF functional class (FC) III and IV (63.4 % vs. 43.3 % in men without ID); higher concentrations of N-terminal pro-brain natriuretic peptide (NT-proBNP) and lower ET. HF FC III increased the probability of ID presence 3.4 times (p=0.02) and the probability of HF FC IV 13.7 times (p=0.003). This clinical picture was characteristic of men when either method of determining ID was used. In women, ID was not associated with more severe CHF.Conclusion    Based on the presented analysis, it is possible to characterize the male and female ID phenotypes. The male ID phenotype is associated with more severe CHF, low ET, and poor quality of life. In females of the study cohort, ID was not associated with either the severity of CHF or with ET.

Aim    To evaluate the prevalence of iron deficiency (ID) in Russian patients with heart failure (HF).Material and methods    Iron metabolism variables were studied in 498 (198 women, 300 men) patients with HF. Data were evaluated at admission for HF (97 %) or during an outpatient visit (3 %). ID was determined according to the European Society of Cardiology Guidelines.Results    83.1 % of patients had ID; only 43.5 % of patients with ID had anemia. Patients with ID were older: 70.0 [63.0;79.0] vs. 66.0 years [57.0;75.2] (p=0.009). The number of patients with ID increased in parallel with the increase in HF functional class (FC). Among patients with ID, fewer people were past or current alcohol users (p=0.002), and a greater number of patients had atrial fibrillation (60.1 vs. 45.2 %, p=0.016). A multiple logistic regression showed that more severe HF (HF FC) was associated with a higher incidence of ID detection, whereas past alcohol use was associated with less pronounced ID. An increase in N-terminal pro-brain natriuretic peptide (NT-proBNP) by 100 pg/ml was associated with an increased likelihood of ID (odds ratio, 1.006, 95 % confidence interval: 1.002-1.011, p=0.0152).Conclusion    The incidence rate of HF patients is high in the Russian Federation (83.1 %). Only 43.5 % of these patients had anemia. The prevalence of ID in the study population increased with increases in HF FC and NT-proBNP.

Objective: To identify hyperhomocysteinemia and to assess its possible association with the course and other markers of endothelial damage in multiple sclerosis., Material and Methods: Analysis of blood serum for homocysteine, for the content of adhesion molecules sPECAM-1, matrix metalloproteinase 9, blood plasma test for von Willebrand factor antigen in patients with multiple sclerosis. The values of these indicators were analyzed depending on the course and activity of the demyelinating process, the severity of neurological disorders, as also depending on the therapy received., Results: Hyperhomocysteinemia was found in more than half of patients with multiple sclerosis. A significantly higher homocysteine level was found in male patients, and hyperhomocysteinemia was associated with the activity of the process in patients with highly active multiple sclerosis., Conclusion: The results of the study suggest a possible association of hyperhomocysteinemia with high process activity and disease progression, as well as with mechanisms of neurodegeneration. Determination of homocysteine concentration may be one potential marker for predicting the course of the disease.

The fundamental question about the functionality of in vitro derived human primordial germ cell-like cells remains unanswered, despite ongoing research in this area. Attempts have been made to imitate the differentiation of human primordial germ cells (hPGCs) and meiocytes in vitro from human pluripotent stem cells (hPSCs). A defined system for developing human haploid cells in vitro is the challenge that scientists face to advance the knowledge of human germ cell development. To develop human primordial germ cell-like cells (hPGCLCs) from human pluripotent stem cells (hPSCs) that are capable of giving rise to haploid cells, we applied a sequential induction protocol via the early mesodermal push of female human embryonic and induced pluripotent stem cells. BMP4-induced early mesoderm-like cells showed significant alterations in their expression profiles toward early (PRDM1 and NANOS3) and late (VASA and DAZL) germ cell markers. Furthermore, using retinoic acid (RA), we induced hPGCLCs in embryoid bodies and identified positive staining for the meiotic initiation marker STRA8. Efforts to find the cells exhibiting progression to meiosis were unsuccessful. The validation by the expression of SCP3 did not correspond to the natural pattern. Regarding the 20-day meiotic induction, the derived hPGCLCs containing two X-chromosomes were unable to complete the meiotic division. We observed the expression of the oocyte marker PIWIL1 and PIWIL4. RNAseq analysis and cluster dendrogram showed a similar clustering of hPGCLC groups and meiotic like cell groups as compared to previously published data. This reproducible in vitro model for deriving hPGCLCs provides opportunities for studying the molecular mechanisms involved in the specification of hPGCs. Moreover, our results will support a further elucidation of gametogenesis and meiosis of female hPGCs., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: The problem of differential diagnosis of constitutional delay of puberty/CDP and hypogonadotropic hypogonadism/HH in boys is discussed, as boys have similar genetic mechanisms and appearance., Aims: to determine accuracy of the criteria for the differential diagnosis of CDP and HH., Materials: The study included 56 boys 14.4±0.7 years old with delayed puberty (G1P1-3/testicular volume <3 сm3). We excluded patients with hypergonadotropic hypogonadism, treated with sex steroids or gonadotropins for 12 months, with endocrine/somatic diseases affecting puberty. At the first visit, we evaluated anthropometric data, bone age, testicular volume, hormones and the results of the gonadotropin-releasing hormone test (GnRH) agonist test and the human chorionic gonadotropin test (hCG) test. The HH was defined by a testicular volume <3 сm3 after 2 years follow-up. The patients were divided into two groups: the first group with CDP and testicles ≥3 cm3 (n=50) and the second group with HH and testicles <3 cm3 (n=6)., Results: At the first visit in boys with CDP corrected target height was less (Me SDS –1.8 vs –0,4, р=0.02), bone age was less (Ме SDS –2.5 vs –0.2 р=0.03), testicular volume was more (Ме 1.9 vs 0.5, p=0.0003), hormones were significantly higher, such as LH (Ме 1.1 vs 0.1 mIU/ml, p=0.0002), FSH (Ме 1.9 vs 0.2 IU/l, p=0.00007), inhibin B (Ме 142.3 vs 31.3 pg/ml, p=0.00009), max LH (Ме 18.9 vs 0.6 mIU/ml, p=0.00007), max LH/FSH (Ме 2.3 vs 0.4, p=0.0002) on the GnRH agonist test and Δ testosterone (Ме 14.4 vs 1.1 nmol/l, p=0.0001) on the hCG test than in boys with HH. The LH ≥0.3 mIU/ml had 86% sensitivity, 100% specificity; max LH/FSH ≥1 – 92% sensitivity, 100% specificity; Δ testosterone ≥2.7 nmol/l on the hCG test – 98% sensitivity, 100% specificity for differential diagnosis of CDP and HH in boys. However, max LH ≥3.5 mIU/ml on the GnRH agonist test, FSH ≥0.5 IU/l, inhibin B ≥58 pg/ml had 100% sensitivity and specificity for diagnosis of CDP., Conclusions: The inhibin B ≥58 pg/ml, LH ≥0.3 mIU/ml, FSH ≥0.5 IU/l or max LH ≥3.5 mIU/ml, max LH/FSH ≥1,0 on the GnRH agonist test, Δ testosterone ≥2.7 nmol/l on the hCG test have an excellent accuracy for the differential diagnosis of CDP and HH in prepubertal boys with delayed puberty.

Presented herein is a clinical case report regarding successful treatment of a female patient with a relapsing malignant retroperitoneal tumour complicated by disseminated thrombosis of the inferior vena cava, right atrium and right ventricle of the heart. A relapse of the malignant uterine tumour had developed 3 years after the primary operation and was represented by a large-size mass ingrowing into the infrarenal segment of the inferior vena cava, aorta, as well as jejunum. Additional examination revealed the presence of a tumorous thrombus extending from the primary tumorous node in the lumen of the inferior vena cava to the right atrium and ventricle. The procedures performed consisted in removal of the tumour with resection of the inferior vena cava, aorta, and jejunum, followed by thrombectomy from the right portions of the heart under extracorporeal circulation. The postoperative period turned out uneventful, with no complications observed. The woman was discharged on POD 15. Twelve-month postoperative follow up revealed neither relapse nor progression of the disease. Currently, the patient continues undergoing specific treatment (second-line chemotherapy). Also discussed in the article are current challenges concerning both the classification of tumour thrombosis of the inferior vena cava in retroperitoneal sarcomas, and the choice of optimal strategy and policy of treatment.

New microporous homopolymers were readily prepared from norbornadiene-2,5, its dimer and trimer by addition (vinyl) polymerization of the corresponding monomers with 60⁻98% yields. As a catalyst Pd- N -heterocyclic carbene complex or Ni(II) 2-ethylhexanoate activated with Na⁺[B(3,5-(CF₃)₂C₆H₃)₄] - or methylaluminoxane was used. The synthesized polynorbornenes are cross-linked and insoluble. They are glassy and amorphous polymers. Depending on the nature of the catalyst applied, BET surface areas were in the range of 420⁻970 m²/g. The polymers with the highest surface area were obtained in the presence of Pd-catalysts from the trimer of norbornadiene-2,5. The total pore volume of the polymers varies from 0.39 to 0.79 cm³/g, while the true volume of micropores was 0.14⁻0.16 cm³/g according to t-plot. These polymers gave CO₂ uptake from 1.2 to 1.9 mmol/g at 273 K and 1 atm. The porous structure of new polymers was also studied by means of wide-angle X-ray diffraction and positron annihilation lifetime spectroscopy.

Despite recent advances in bioengineered therapies, wound healing remains a serious clinical problem. In acute full-thickness wounds, it is desirable to replace both the damaged dermis and epidermis in a single procedure. This approach requires appropriate properties of tissue-engineered dressings to support simultaneous regenerative processes in the dermis and epidermis while they are temporally separated in the natural wound healing process. In this study, a collagen-based scaffold inhabited by skin cells was employed. Its ability to stimulate the skin repair of full-thickness excisional splinting wounds in a murine model was evaluated in comparison with that of acellular collagen and commercially available gelatin porous sponge Spongostan®. The study showed that cell-based skin equivalent promoted the immediate filling of the wound bed and provided simultaneous reorganization of the dermal component into highly vascularized granulation-like tissue and rapid epithelialization, thus improving the quality of healing. Inflammation was delayed and less pronounced. In contrast, acellular collagen and especially Spongostan® failed to demonstrate similar results. The porous structure of Spongostan® prevented effective long-term epithelialization and impeded the formation of an adequate connective tissue at the wound bed.

Growing evidence suggests that mitochondrial dysfunction is closely linked to the pathogenesis of sporadic Alzheimer's disease (AD). One of the key contributors to various aspects of AD pathogenesis, along with metabolic dysfunction, is mitochondrial dynamics, involving balance between fusion and fission, which regulates mitochondrial number and morphology in response to changes in cellular energy demand. Recently, Zhang et al. ((2016) Sci. Rep., 6, 18725) described a previously unknown mitochondrial phenotype manifesting as elongated chain-linked mitochondria termed "mitochondria-on-a-string" (MOAS) in brain tissue from AD patients and mouse models of AD. The authors associated this phenotype with fission arrest, but implications of MOAS formation in AD pathogenesis remain to be understood. Here we analyze the presence and number of MOAS in the brain of OXYS rats simulating key signs of sporadic AD. Using electron microscopy, we found MOAS in OXYS prefrontal cortex neuropil in all stages of AD-like pathology, including manifestation (5-month-old rats) and progression (12-18-month-old rats). The most pronounced elevation of MOAS content (8-fold) in OXYS rats compared to Wistar controls was found at the preclinical stage (20 days) on the background of decreased numbers of non-MOAS elongated mitochondria. From the age of 20 days through 18 months, the percentage of MOAS-containing neuronal processes increased from 1.7 to 8.3% in Wistar and from 13.9 to 16% in OXYS rats. Our results support the importance of the disruption of mitochondrial dynamics in AD pathogenesis and corroborate the existence of a causal link between impaired mitochondrial dynamics and formation of the distinctive phenotype of "mitochondria-on-a-sting".

Idiopathic scoliosis is one of the most common disabling pathologies of children and adolescents. Etiology and pathogenesis of idiopathic scoliosis remain unknown. To study the etiology of this disease we identified the cells' phenotypes in the vertebral body growth plates in patients with idiopathic scoliosis. Materials and methods: The cells were isolated from vertebral body growth plates of the convex and concave sides of the deformity harvested intraoperatively in 50 patients with scoliosis. Cells were cultured and identified by methods of common morphology, neuromorphology, electron microscopy, immunohistochemistry and PCR analysis. Results: Cultured cells of convex side of deformation were identified as chondroblasts. Cells isolated from the growth plates of the concave side of the deformation showed numerous features of neuro- and glioblasts. These cells formed synapses, contain neurofilaments, and expressed neural and glial proteins. Conclusion: For the first time we demonstrated the presence of cells with neural/glial phenotype in the concave side of the vertebral body growth plate in scoliotic deformity. We hypothesized that neural and glial cells observed in the growth plates of the vertebral bodies represent derivatives of neural crest cells deposited in somites due to alterations in their migratory pathway during embryogenesis. We also propose that ectopic localization of cells derived from neural crest in the growth plate of the vertebral bodies is the main etiological factor of the scoliotic disease., Competing Interests: Conflict of Interest: The authors declare that they have no conflict of interest.

This paper presents a literature review considering the role and mechanism of apoptosis in the pathogenesis of ischemic stroke (IS). The authors introduce a new concept: the functional request of the patient as a set of external (the nature and intensity of rehabilitation measures, characteristics of everyday life, diet, etc.) and internal (genetic factors, internal picture of the disease, availability of rental and other psychological facilities and etc.) attributes. This concept allows a new angle in understanding the pathogenesis of IS and creates fundamental and clinical potential for more successful approaches to therapy and rehabilitation after IS.

Key hemostatic serine proteases such as thrombin and activated protein C (APC) are signaling molecules controlling blood coagulation and inflammation, tissue regeneration, neurodegeneration, and some other processes. By interacting with protease-activated receptors (PARs), these enzymes cleave a receptor exodomain and liberate new amino acid sequence known as a tethered ligand, which then activates the initial receptor and induces multiple signaling pathways and cell responses. Among four PAR family members, APC and thrombin mainly act via PAR1, and they trigger divergent effects. APC is an anticoagulant with antiinflammatory and cytoprotective activity, whereas thrombin is a protease with procoagulant and proinflammatory effects. Hallmark features of APC-induced effects result from acting via different pathways: limited proteolysis of PAR1 localized in membrane caveolae with coreceptor (endothelial protein C receptor) as well as its targeted proteolytic action at a receptor exodomain site differing from the canonical thrombin cleavage site. Hence, a new noncanonical tethered PAR1 agonist peptide (PAR1-AP) is formed, whose effects are poorly investigated in inflammation, tissue regeneration, and neurotoxicity. In this review, a concept about a role of biased agonism in effects exerted by APC and PAR1-AP via PAR1 on cells involved in inflammation and related processes is developed. New evidence showing a role for a biased agonism in activating PAR1 both by APC and PAR1-AP as well as induction of antiinflammatory and cytoprotective cellular responses in experimental inflammation, wound healing, and excitotoxicity is presented. It seems that synthetic PAR1 peptide-agonists may compete with APC in controlling some inflammatory and neurodegenerative diseases.

Electrospinning is a convenient and promising manufacturing method a variety of materials for tissue engineering. 3D matrices fabricated by electrospinning from solutions of polycaprolactone with human serum albumin or gelatin in 1,1,1,3,3,3-hexafluoroisopropanol were studied. The microstructure of the 3D matrices and surface of the fibers were investigated using scanning electron microscopy. Protein distribution in the surface layer was studied by modification of protein amino groups with N-(2-hydroxyethyl)phenazine and X-ray photoelectron spectroscopy. It was shown, that concentration of the proteins in the surface layer of fibers exceeded their concentration in the initial electrospun solution up to 12 times and the surface layer was enriched in the protein inversely to the concentration of the protein in solution. The minor part of the proteins was released from fibers during first 30-60 min after swelling in water. Treatment of matrices with proteinase K hydrolyzed about 1/3 of the surface exposed human serum albumin. Thus, both methods can be used to study the surface content of the materials produced by electrospinning from blends of synthetic and natural polymers, however X-ray photoelectron spectroscopy appears to be more convenient and informative.

Heat shock proteins are universal agents protecting all known organisms from environmental stresses. These proteins are classified into six families differing in their structure and function, yet having a common purpose of maintaining cellular proteins in operating condition. The small heat shock protein family has the most diversified set of effects on the organism’s vital activity with a great number of its members’ features studied thoroughly in a fruit fly Drosophila melanogaster, which is regarded as a convenient model object. In this review, we represent and discuss data on the role of Drosophila melanogaster Hsp67Bc protein, which was earlier identified as the closest functional ortholog of human HSPB8 small heat shock protein.

Background: Extracellular double-stranded DNA participates in various processes in an organism. Here we report the suppressive effects of fragmented human double-stranded DNA along or in combination with cyclophosphamide on solid and ascites grafts of mouse Krebs-2 tumor cells and DNA preparation on human breast adenocarcinoma cell line MCF-7., Methods: Apoptosis and necrosis were assayed by electrophoretic analysis (DNA nucleosomal fragmentation) and by measurements of LDH levels in ascitic fluid, respectively. DNA internalization into MCF-7 was analyzed by flow cytometry and fluorescence microscopy., Results: Direct cytotoxic activity of double-stranded DNA (along or in combination with cyclophosphamide) on a solid transplant was demonstrated. This resulted in delayed solid tumor proliferation and partial tumor lysis due to necrosis of the tumor and adjacent tissues. In the case of ascites form of tumor, extensive apoptosis and secondary necrosis were observed. Similarly, MCF-7 cells showed induction of massive apoptosis (up to 45%) as a result of treatments with double-stranded DNA preparation., Conclusions: Double-stranded DNA (along or in combination with cyclophosphamide) induces massive apoptosis of Krebs-2 ascite cells and MCF-7 cell line (DNA only). In treated mice it reduces the integrity of gut wall cells and contributes to the development of systemic inflammatory reaction.

Introduction: In recent years, mesenchymal stem cells (MSCs) have been demonstrated to play an important role in carcinogenesis. However, the effect of MSCs on tumor and metastasis development and the mechanisms underlying the interaction of cancer and stem cells are not completely understood. This study investigated the effect of MSCs on breast cancer metastasis formation by using the methods of in vivo fluorescence and luminescence imaging., Methods: MSCs were isolated from bone marrow of normal donors, characterized, and genetically labeled with luciferase (luc2). The effects of MSCs on MDA-MB-231 cancer cell proliferation were evaluated in conditioned medium from MSCs. To generate lung metastases, MDA-MB-231 cells stably expressing red fluorescent protein Turbo FP650 were injected intravenously into nude mice. On day 10 after the cancer cell injection, mice were injected via the tail vein with MSCs-luc2 cells (the MET+MSCs group). Animals that received the injection of MDA-MB-231-Turbo FP650 alone (the MET group) and no injections (the intact control group) served as controls. Fluorescence and bioluminescence imaging was performed for monitoring of the metastasis formation and MSC distribution in the recipient's body., Results: We found that the proliferative activity of the cancer cells in the presence of MSC conditioned medium was lower than that of the cells grown in conventional culture medium. The metastasis formation in the MET+MSCs group was delayed in time as compared with the MET group. Macroscopic and histological examination of isolated lungs 8 weeks after cancer cell injection showed that the total number of metastases in animals of the MET+MSCs group was significantly lower. Using bioluminescence imaging in vivo, we found that MSCs-luc2 cells survived in the host animal for at least 7 weeks and re-migrated to the lung 6 to 7 weeks after injection. Immunohistochemical analysis revealed the presence of MSCs-luc2 in metastases and lung tissue., Conclusions: Long-term in vivo bioluminescence imaging of intravenously injected MSCs-luc2 cells showed distribution of MSCs to the lungs and abdominal organs within the first 2 to 3 weeks and re-migration to the lungs in weeks 6 to 7. It was found that MSCs reduced the proliferative activity of cancer cells in vitro and lung metastasis formation in mice.

Activated protein C (APC) is serine protease hemostasis, independent of its anticoagulant activity, exhibits anti-inflammatory and anti-apoptotic properties that determine the possibility of the protective effects of APC in different diseases, including sepsis and chronic wound healing. APC, binding of endothelial protein C receptor (EPCR) and specifically cleaving PAR1 receptor and releasing peptide agonist PAR1 stabilizes not only endothelial cells, but also many others, including epidermal keratinocytes of the skin. We develop the hypothesis that the cytoprotective effect of APC on the cells, involved in wound healing, seem to imitate peptide - analogous of PAR1 "tethered ligand" that activate PAR1. In our work, we synthesized a peptide (AP9) - analogue of PAR1 tethered ligand, released by APC, and firstly showed that peptide AP9 (0.1-10 мM), like to APC (0.01-100 nM), stimulates the proliferative activity of human primary keratinocytes. Using a model of the formation of epithelial wounds in vitro we found that peptide AP9, as well as protease APC, accelerates wound healing. Using specific antibodies to the receptor PAR1 and EPCR was studied the receptor mechanism of AP9 action in wound healing compared with the action of APС. The necessity of both receptors - PAR1 and EPСR, for proliferative activity of agonists was revealed. Identified in our work imitation by peptide AP9 - PAR1 ligand, APC acts on keratinocytes suggests the possibility of using a peptide AP9 to stimulate tissue repair.

This review describes the nesprins (nuclear envelope spectrin-repeat proteins), which are recently discovered family of nuclear envelope proteins. These proteins play an important role in maintaining the cellular architecture and establish the link between the nucleus and other sub-cellular compartments. Many tissue-specific diseases including lipodystrophies, hearing loss, cardiac and skeletal myopathies are associated with nesprins mutations. These proteins comprise of multiple tissue specific isoforms which contain spectrin repeats providing interaction of nesprins with other nuclear membrane proteins, cytoskeleton and intranuclear matrix. We summarize recent findings and suggestions about nesprins structural organization and function inside the cell. Human diseases caused by abnormal nesprins expression are also described.

A comparative study was performed of dense 5-hour cultures of rat hepatocytes and equal-density cultures of mesenchymal stromal cells (MSC) isolated from human adipose tissue of rat bone marrow. The cells were grown on collagen-coated class slides in serum-free medium. Unlike in hepatocytes, no rhythm of protein synthesis was initially revealed in MSC, but such a rhythm manifested itself when the culture medium was supplemented with melatonin (2 nM, 5 min). The results of experiments with cytoplasmic calcium chelator BAPTA-AM and protein kinase inhibitor H7 indicate that the mechanism of protein synthesis synchronization in MSC consists in calcium-dependent phosphorylation of cell proteins.

Two dozen hybrid clones were produced by fusion of diploid embryonic stem (ES) cells positive for green fluorescent protein (GFP) with tetraploid fibroblasts derived from DD/c and C57BL-I(I)1RK mice. Cytogenetic analysis demonstrated that most cells from these hybrid clones contained near-hexaploid chromosome sets. Additionally, the presence of chromosomes derived from both parental cells was confirmed by polymerase chain reaction (PCR) analysis of polymorphic microsatellites. All hybrid cells were positive for GFP and demonstrated growth characteristics and fibroblast-like morphology. In addition, most hybrid cells were positive for collagen type I, fibronectin, and lamin A/C but were negative for Oct4 and Nanog proteins. Methylation status of the Oct4 and Nanog gene promoters was evaluated by bisulfite genomic sequencing analysis. The methylation sites (CpG-sites) of the Oct4 and Nanog gene promoters were highly methylated in hybrid cells, whereas the CpG-sites were unmethylated in the parental ES cells. Thus, the fibroblast genome dominated the ES genome in the diploid ES cell/tetraploid fibroblast hybrid cells. Immunofluorescent analysis of the pluripotent and fibroblast markers demonstrated that establishment of the fibroblast phenotype occurred shortly after fusion and that the fibroblast phenotype was further maintained in the hybrid cells. Fusion of karyoplasts and cytoplast derived from tetraploid fibroblasts with whole ES cells demonstrated that karyoplasts were able to establish the fibroblast phenotype of the reconstructed cells but not fibroblast cytoplasts. Thus, these data suggest that the dominance of parental genomes in hybrid cells of ES cell/somatic cell type depends on the ploidy of the somatic partner.

Exogenous allogenic DNA as nucleosome-free fragments reaches main cellular compartments (cytoplasm, nucleus) of human dendritic cells and deposits in the nuclear interchromosomal space without visibly changing in linear size. The presence of such allogenic fragmented DNA in medium in which human dendritic cells are cultured produces an enhancement of their allostimulatory activity. This enhancement is comparable to that produced by the standard maturation stimulus lipopolysaccharide Escherichia coli., (Copyright 2010 Elsevier Inc. All rights reserved.)

Reconstructive surgery often collides with complicated task of defect recovery in the situation of deficit of maxillofacial skeleton tissues, that leads to necessary search of accessible cells source with osteogenic potentials and proper substrate for cell transplantation. In the study the regeneratory potential of stromal cells of adipose tissue was tested in the rabbit model of mandible through angle defect. Cells of allogenic and autologic nature stimulated reparative osteogenesis but their influence upon bone matrix maturation was different. When the construction with allogenic cells was transplanted reaction of its rejection was not detected on histological level.

In experiment on 12 Chinchilla rabbits dynamics of reparative regeneration was studied at the terms 2 and 4 months. Bone defect in mandible corner was closed by osteoplastic material Gapkol which was covered from inside by allogenic or autologic stem cells received from rabbit adipose tissue. The results of the ray tracing methods of study were verified by SEM and histological methods.

In experiment on 16 grown-up chinchilla rabbits the dynamic of reparative regeneration was evaluated by digital microfocal rontgenography in the terms of 1, 2 and 4 months. Bone defect of the 8capital CHE, Cyrillic8 mm size in the region of mandible angle was caused by surgical laser Smart 2940 D+ on the right side and by physiodespenser Surgec XT on the left side. Surgical laser use let to reduce intact mother bone traumatisation and to improve remote results of bone tissue regeneration. After bone defect creation bone tissue regeneration was put into effect by all 3 callus types - endosteal, periosteal and intermediary.

Comparative analyses of symbiotic bacteria Wolbachia (stamm wMelPop reducing lifespan of flies) morphology in normal and mutant strains of Drosophila melanogaster as well as the influence of Wolbachia on the host cell ultrastructure have been done. Wolbachia infected D. melanogaster mutation strains Trithorax-like -- Trl362/TM3, Sb1 Ser y+ and Trlen82/TM3, Sb1 Ser y+ have been received by special flies crossing. Uninfected strain D. melanogaster white-1118 (w1118) have been obtained by antibiotic treatment of initially infected strain D. melanogaster [w]w1118. Complex of different methods and approaches let to investigate for the first time the morphology of cell structure before and after bacterial infection of insects and to value the bacterial presence effect on flies viability and reproduction of normal and mutant flies. Morphology af cytoplasmic compartments in early embryos and eggs layed by infected and uninfecyed females Trl362/TM3, Sb1 Ser y+ and Trlen82/TM3, Sb1 Ser y+ have been analyzed. Electron microscopy has shown that D. melanogaster embryos contain typical Wolbachia contacting with different host organelles that verify preservation of their functional activity. Atificial mitochondria and Wolbachia (wMelPop) of unusual morphology with defective bacterial membranes have been visualised in D. melanogaster [w]Trl362/TM3, Sb1 Ser y+. Wolbachia presence in ovarium cells from strains [w]Trl362/TM3, Sb1 Ser y+ and [w]Trlen82/TM3, Sb1 Ser y+ did not influence on eggs quantity layed by females. We have demonstrated for the first time that lifespan of infected and uninfected strains: D. melanogaster Trl362/TM3, Sb1 Ser y+ and Trlen82/TM3, Sb1 Ser y+ were similar. However the lifespan of imago from strain [w]w1118 was lower in comparison to those from strains Trl362/TM3, Sb1 Ser y+ and Trlen82/TM3, Sb1 Ser y+. It suggests that either chromosomal balancer TM3 or Trl mutation play an importance role in host-symbiotic relationship. Next experiments have revealed that lifespan of homozygotic flies decreased essentially and was close to lifespan of strain [w]w1118. Data obtained confirm that chromosomal balancer TM3 can affect on symbiont-host relationship.

We compared the morphology and differentiation characteristics of the human cells from bone marrow, adipose tissue, hair papilla and skin dermis. All cell types showed fibroblastic morphology. Immunofluorescent analysis showed that adipose tissue derived stem cells (ADAS) and hair papilla cells (HPC) expressed CD105, CD49d and STRO-1, bone marrow mesenchymal stem cells (BMSC) were absent for CD49, dermal fibroblasts (DFb) expressed CD49 and STRO-1 at low level. Populations of ADAS, BMSC and HPC had similar capacities to lipid and bone differentiation. Following exposure to appropriate induction stimuli, these cells changed phenotype and expressed specific cell markers. However, the rate and extent of HPC differentiation were lower in comparison with populations of ADAS and BMSC. We propose that all investigated cell populations contain primitive progenitor cells with mesenchymal stem cell properties.

Digital microfocal x-ray study was experimentally studied in animals to examine the time course of changes in their bone regeneration. Sixteen Chinchila rabbits whose bone defect in the angle of the mandibular ramus had been closed with the osteoplastic material Gapcol with the applied allogeneic, autologous stem cells isolated from rabbit adipose tissue and human plasma enriched with thrombocytic growth factors were examined. The capabilities of digital microfocal x-ray study versus x-ray computed tomography were compared in the evaluation of reparative regeneration of bone tissue. The results of radiation studies were verified with the data of scanning electron microscopy.

Actin-containing filaments have been visualized inside the Xenopus oocyte nuclei due to combination of fluorescence and transmission electron microscopy. It has been shown that these filaments contact with nucleoli, spherical bodies and nuclear pore complexes. The incubation of oocytes with actin-depolymerizing latrunculin causes membrane vesiculation in the cytoplasm, and disruption of the nucleoplasm and nuclear envelope integrity. We suppose that actin-containing filaments belong to crucial cell components which are involved in coordination of nuclear-cytoplasmic interactions as well as distribution and transport of intranuclear components in growing Xenopus oocytes.

In eukaryotic cells, mitotic events are controlled by evolutionarily conserved cyclin-dependent kinases (cdk): these kinases phosphorylate cell proteins, which causes structural reorganization of the entire cell. Our recent studies of Drosophila syncytial embryos have demonstrated that cdk1 activity is a key factor that controls nuclear pore complex assembly/disassembly and affects the structure of cytoplasmic pores in the annulate. In this paper, we report a comparative analysis of these cytoplasmic organelles throughout the cell-cycle and throughout the development of Drosophila syncytial embryos. Based on the results obtained, it was presupposed that distribution of annulate lamellae containing cytoplasmic pores could reflect the inactivation of the mitotic kinase cdk1 in Drosophila syncytial embryos.

Electron microscopic analysis of Drosophila melanogaster (w1118) ovarian cells has demonstrated that stressful heat treatment of flies results in the appearance of electron dense granules and large lysosomes in the cytoplasm of ovarian cells, which is not related with the presence of Wolbachia, as these changes are observed in both the infected and uninfected flies. High temperature initiates essential envelope defects and other structural changes of symbiotic bacteria in the cytoplasm of ovarian cells. Some embryos developing from eggs of heat shocked flies die, however, bacteria in the survival embryos retain their typical morphology. Endosymbionts do not change their localization and their contacts with the mitochondria and endoplasmic reticulum in the ovarian cells and early embryos after heat shock treatment of the flies. The results obtained show that high temperature influences on both the host and the endosymbiont, but does not change their structural mutual interactions.

This review describes and discusses new data about the structure and function of proteins which contain ankyrin-like repeats in their structure. These proteins have been found in cells of different organisms but they are not belonging to the cytoskeletal proteins. Many important functions of such proteins are provided by ankyrin repeats which maintain protein-protein interactions involved in the formation of transcription complexes, initiation of immuno-responses, biogenesis and assembly of cation channels in the membranes, regulation of some cell cycle stages, symbiotic interactions and many other processes. Mutations in genes encoding ankyrin-like proteins can cause defects in gene expression leading to diseases onset and progression in animals and humans. Therefore, the structure, dynamics and function of these proteins is an area of extensive research in modern biology.

Higher-eukaryotic membrane compartmentalization of different processes provides the control of functional activity in cells. The nuclear envelope that consists of outer and inner nuclear membranes, lamina, and nuclear pore complexes is one of the most significant and complex cell compartments. It separates DNA replication and transcription in the nucleus from RNA translation in the cytoplasm and regulates the nuclear-cytoplasmic transport of different molecules. This review describes and discusses the structural organization and biochemical composition of different nuclear envelope components (except nuclear pore complexes, which were discussed in our previous review) as well as its dynamics during mitosis in vitro and in vivo. Special attention is given to the role of lamina in a group of human genetic diseases, collectively referred to as laminopathies.

This review summarized data on the morphological and biochemical analysis of nuclear pore complexes, which are complex organelles providing the route of passive and active nuclear-cytoplasmic transport to different molecules in the eukaryotic cell. The morphology and functional role of nuclear pores in higher and lower eukaryotes, and molecular aspects of the import and export of molecules from the nucleus are described in addition to factors involved in the regulation of these process. Special attention has been paid to sequential steps of the nuclear pore assembly in vitro and in vivo.

Morphological analysis of endoplasmic reticulum (ER) dynamics was carried out in early amphybian oocytes at different stages of their development. Comparative morphometric analysis of ER membrane relative surface area and relative volume has shown that at the 1st-2nd stages of oogenesis the accumulation of ER membranes occurred mainly in the peripheral region of the cell cytoplasm. At the 3rd-4th stages of oocyte growth characterized by active transcription, the increase in the relative volume of ER and relative surface area of ER membranes occurs in the middle and near-nuclear region of cytoplasm, and reduction of these parameters takes place in the peripheral part of the cell. Simultaneously observed fusion of numerous ER vesicles with the outer nuclear membrane may serve as an evidence of active formation of new fragments of nuclear envelope. The 5th-6th stages of oocyte development are characterized by an increase in the relative volume of ER in the whole oocyte. It has been supposed that the revealed reorganization of endoplasmic reticulum in growing amphibians oocytes might be due to yolk accumulation (vitellogenesis), redistribution of calcium ions, and synthesis and storage of lipids and proteins. These processes are necessary for the future embryo development, and also for assembly of new fragments of growing nuclear envelope.

Disassembly and reassembly of the nuclear pore complexes (NPCs) is one of the major events during open mitosis in higher eukaryotes. However, how this process is controlled by the mitotic machinery is not clear. To investigate this we developed a novel in vivo model system based on syncytial Drosophila embryos. We microinjected different mitotic effectors into the embryonic cytoplasm and monitored the dynamics of disassembly/reassembly of NPCs in live embryos using fluorescently labeled wheat germ agglutinin (WGA) or in fixed embryos using electron microscopy and immunostaining techniques. We found that in live embryos Cdk1 activity was necessary and sufficient to induce disassembly of NPCs as well as their cytoplasmic mimics: annulate lamellae pore complexes (ALPCs). Cdk1 activity was also required for keeping NPCs and ALPCs disassembled during mitosis. In agreement recombinant Cdk1/cyclin B was able to induce phosphorylation and dissociation of nucleoporins from the NPCs in vitro. Conversely, reassembly of NPCs and ALPCs was dependent on the activity of protein phosphatases, sensitive to okadaic acid (OA). Our findings suggest a model where mitotic disassembly/reassembly of the NPCs is regulated by a dynamic equilibrium of Cdk1 and OA-sensitive phosphatase activities and provide evidence that mitotic phosphorylation mediates disassembly of the NPC.

Survival of cultured rat hippocampal neurons was estimated 4, 24, and 48 h after 15-min exposure to the toxic effect of glutamate under conditions of pre- or coincubation with 10 nM thrombin. Thrombin inhibited glutamate-induced apoptosis in neurons 24 and 48 h after treatment, but had no effect on necrosis. Selective peptide agonist of proteinase-activated type 1 receptors simulated, but receptor antagonist suppressed the neuroprotective effect of thrombin. Our results suggest that peptide antagonist of type 1 receptors play a role in the mechanisms of neuronal protection from glutamate toxicity.

The effect of thrombin on the rat hippocampal neurons death in model of neurotoxicity induced by hemoglobin or glutamate, was studied. Thrombin (10 nM) was shown to inhibit 100-mkM glutamate--or 10-mkM hemoglobin-induced apoptosis of the rat hippocampal neurons. With the aid of PAR1 (protease-activated receptor1) agonist peptide and PAR1 antagonist, the PAR1 was found to be necessary for protective action of thrombin in hippocampal neurons in models of neurotoxicity induced by hemoglobin or glutamate. Because the prolonged elevation [Ca2+] ib neurons is a critical part of neurodestructive processes in CNS, the effect of thrombin on Ca2+-homeostatis of neurons after its injury by the inducer of neuronal apoptosis: a synthetic agonist of the NMDA receptors N-methyl-D-aspartate (NMDA), was studied. We hypothesized that thrombin via receptors PAR may prove to be neuroprotective for the hippocampus. Thrombin was shown to stimulate via PAR1 a transient increase in [Ca2+] in neurons in a concentration-dependent manner. Thrombin (1 nM) decreased the [Ca2+] signal induced by activation of the NMDA-subtype of glutamate receptors. This thrombin effect may be one of the reasons of the protective action of thrombin in hippocampal neurons.

Electron microscopy and morphometric analysis have shown that the symbiotic bacteria Wolbachia occur the testis cells during spermatogenesis and are absent in mature spermatids. Bacteria did not affect the structural organization of testis cells, which have a typical morphology during morphogenesis. Bacteria were distributed along the meiotic spindle microtubules near the mitochondria. They increased in number in spermatids at the stage of elongation. Endosymbionts aggregated at the spermatid distal end and contained many vacuoles but were absent at the spermatid proximal end near the nuclei. It was shown for the first time that the diameter of spermatids in a strongly infected line was two of three times that in a noninfected line. We hypothesize that the increase in the number of endosymbionts during spermatid elongation can affect the chromatin condensation in the spermatozoon.

This review is devoted to annulate lamellae, a specific compartment of endoplasmic reticulum that occurs, presumably, in actively growing and rapidly dividing cells (oocytes, embryonic and tumor cells). We summarized both earlier and recent data on the dustribution of annulate lamellae in various cell types, on their morphology, and the distribution of interaction with intracellular structures at various treatments. As the annulate lamellae contain cytoplasmic pore complexes, a special attention was paid to their relation with nuclear pores. Possible functions of the annulate lamellae in intracellular processes and, particularly, in nuclear envelope assembly, are discussed.

PAR1 peptide thrombin receptor agonist (PAR1-AP) was encapsulated in microcorpuscles based on lactic and glycolic acid copolymer. The desorption profile of the preparation was studied in vitro and its wound-healing effects were studied on a model of cut skin wound in mice. The study showed that 90% PAR1-AP was desorbed over 6 h, but the peptide was detected in eluates from the microparticle surface after 23 h. The desorbed peptide retained its physiological activity and was capable of activating PAR1 receptors on human platelets. The study of the dynamics of experimental skin wound healing in mice showed lower number of macrophages in the wounds treated with PAR1-AP microparticles compared to the control (open wounds and wounds covered with microparticles) and higher number of fibroblasts on day 3 of tissue reparation. Hence, PAR1-AP desorbed from microparticles shortened the inflammation phase in the wound. On day 7 the best healing parameters were also observed in wounds treated with PAR1-AP microparticles, which attests to shortening of the proliferation phase and acceleration of wound healing.

The effect of thrombin, an agonist of proteinase-activated receptor (PAR) family, was studied on cultured rat hippocampal neurons. Thrombin in a concentration range of 1 pM - 10 nM induced a transitory dose-dependent increase in intracellular free calcium concentration. Involvement of PAR1 in neural response to thrombin was corroborated in experiments with TFLLRN, a selective synthetic peptide agonist of these receptors. In a calcium-free medium and after treatment with cyclopiazonic acid (inhibitor of Ca(2+)-ATPase in the endoplasmic reticulum) activation of PAR not only mobilized Ca(2+) from intracellular stores, but also induced Ca(2+) entry into the cells. Thrombin decreased Ca(2+) signal triggered by activation of NMDA-subtype glutamate receptors.

The nuclear pore complexes (NPCs), multiprotein assemblies embedded in the nuclear envelope, conduct nucleo-cytoplasmic traffic of macromolecules. Mimics of NPCs, called annulate lamellae pore complexes (ALPCs), are usually found in cytoplasmic membranous stacks in oocytes and early embryonic cells. They are believed to constitute storage compartments for excess premade nucleoporins. To evaluate the extent to which ALPCs store nucleoporins in early embryonic cells we took advantage of syncytial Drosophila embryos, containing both AL and rapidly proliferating nuclei in the common cytoplasm. Electron microscopic morphometric analysis showed that the number of ALPCs did not decrease to compensate for the growing number of NPCs during syncytial development. We performed Western blot analysis to quantify seven different nucleoporins and analyzed their intraembryonal distribution by confocal microscopy and subcellular fractionation. Syncytial embryos contained a large maternally contributed stockpile of nucleoporins. However, even during interphases, only a small fraction of the excess nucleoporins was assembled into ALPCs, whereas the major fraction was soluble and contained at least one phosphorylated nucleoporin. We conclude that in Drosophila embryos ALPCs play only a minor role in storing the excess maternally contributed nucleoporins. Factors that may prevent nucleoporins from assembly into ALPCs are discussed.

Electron microscopic and morphometric analyses of Wolbachia distribution in early embryos of Drosophila flies have demonstrated that the number of bacteria in the embryo remains constant from fertilization to blastoderm, and that afterwards the symbionts could be observed only in the polar cells. Each bacterium has a three-layer envelope, makes contacts with microtubules and moves through the cytoplasm following the actively dividing nuclei. It has been found for the first time that Wolbachia could produce secretory vacuoles in the cytoplasm of early embryos. The relative volume of Wolbachia was five times as much in the embryos of Drosophila simulans as in those of D. melanogaster (Canton S), while the survival rate of D. simulans was half as much as that of D. melanogaster. It was shown that Wolbachia could form spore-like structures in D. simulans embryos. Ultrastructural investigations of Drosophila ovaries suggest that the bacteria may be present in all ovariol cells, including the oocyte, within whose cytoplasm they are delivered to the host. The highest number of symbionts was observed in germarium cells. In ovariol cells, the bacteria gradually decrease in number as oogenesis progresses. It has been determined for the first time that the symbionts are located closely to membranes of rough endoplasmatic reticulum in follicular and nurse cells of D. melanogaster. The data obtained suggest that Wolbachia may be involved in the regulation of oocyte maturation.

We consider the potential of applying wavelet analysis to fluctuations found in physiological systems. We focus on cardiac interbeat interval time series (RR-intervals) from a group of young adults and children and fetus heart beat rate time series (antepartum cardiotocography), because the wavelet analysis of these heart rate dynamics may provide important practical diagnostic and prognostic information not obtainable with current approaches. We show that all the signals analyzed are self-similar and propose a method for estimating this scaling feature.