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AAES is a powdered form of Biostymina, herbal medicinal product of Phytopharm Klęka S.A., a water extract of Aloe arborescens Mill. leaves. Aloe arborescens Mill. (woody aloe, tree-like aloe) is known to have several traditional medicinal properties including anti-inflammatory, immunomodulatory, antiviral and antimicrobial activity.

Anticancer activity of many herbs was observed for hundreds of years. They act as modifiers of biologic response, and their effectiveness may be increased by combining multiple herbal extracts . PERVIVO, traditional digestive herbal remedy, contains some of them, and we previously described its antiangiogenic activity. Numerous studies documented anticancer effects of nonsteroidal anti-inflammatory drugs. We were the first to show that sulindac and its metab olites inhibit angiogenesis. In the present paper the combined in vivo effect of multicomponent herbal remedy PERVIVO and nonsteroidal anti-inflammatory drug sulindac on tumor growth, tumor angiogenesis, and tumor volume in Balb/c mice was studied. These effects were checked after grafting cells collected from syngeneic sarcoma L-1 tumors into mice skin. The strongest inhibitory effect was observed in experimental groups treated with PERVIVO and sulindac together. The results of our investigation showed that combined effect of examined drugs may be the best way to get the strongest antiangiogenic and antitumor effect. [ABSTRACT FROM AUTHOR]

Advancements in single-cell analyzis technologies, particularly single-cell RNA sequencing (scRNA-seq) and Fluorescence-Activated Cell Sorting (FACS), have enabled the analyzis of cellular diversity by providing resolutions that were not available previously. These methods enable the simultaneous analyzis of thousands of individual transcriptomes, facilitating the classification of cells into distinct subpopulations, based on transcriptomic differences, adding a new level of complexity to biomolecular and medical research. Fibroblasts, despite being one of the most abundant cell types in the human body and forming the structural backbone of tissues and organs, remained poorly characterized for a long time. This is largely due to the high morphological similarity between different types of fibroblasts and the lack of specific markers to identify distinct subpopulations. Once thought to be cells responsible solely for the synthesis of extracellular matrix (ECM) components, fibroblasts are now recognized as active participants in diverse physiological processes, including inflammation and antimicrobial responses. However, defining the molecular profile of fibroblast subpopulations remains a significant challenge. In this comprehensive review, which is based on over two thousand research articles, we focus on the identification and characterization of fibroblast subpopulations and their specific surface markers, with an emphasis on their potential as molecular targets for selective cell isolation. By analyzing surface markers, alongside intra- and extracellular protein profiles, we identified multiple fibroblast subtypes within the female reproductive system. These subtypes exhibit distinct molecular signatures and functional attributes, shaped by their anatomical localization and the surrounding physiological or pathological conditions. Our findings underscore the heterogeneity of fibroblasts and their diverse roles in various biological contexts. This improved understanding of fibroblast subpopulations paves the way for innovative diagnostic and therapeutic strategies, offering the potential for precision targeting of specific fibroblast subsets in clinical applications.

Adipocytes derived from 3T3-L1 cells are a gold standard for analyses of adipogenesis processes and the metabolism of fat cells. A widely used histological and immunohistochemical staining and mass spectrometry lipidomics are mainly aimed for examining lipid droplets (LDs). Visualizing other cellular compartments contributing to the cellular machinery requires additional cell culturing for multiple labeling. Here, we present the localization of the intracellular structure of the 3T3-L1-derived adipocytes utilizing vibrational spectromicroscopy, which simultaneously illustrates the cellular compartments and provides chemical composition without extensive sample preparation and in the naïve state. Both vibrational spectra (FTIR-Fourier transform infrared and RS-Raman scattering spectroscopy) extended the gathered chemical information. We proved that both IR and RS spectra provide distinct chemical information about lipid content and their structure. Despite the expected presence of triacylglycerols and cholesteryl esters in lipid droplets, we also estimated the length and unsaturation degree of the fatty acid acyl chains that were congruent with known MS lipidomics of these cells. In addition, the clustering of spectral images revealed that the direct surroundings around LDs attributed to lipid-associated proteins and a high abundance of mitochondria. Finally, by using quantified markers of biomolecules, we showed that the fixative agents, paraformaldehyde and glutaraldehyde, affected the cellular compartment differently. We concluded that PFA preserves LDs better, while GA fixation is better for cytochromes and unsaturated lipid analysis. The proposed analysis of the spectral images constitutes a complementary tool for investigations into the structural and molecular features of fat cells.

Fibroblasts are among the most abundant cell types in the human body, playing crucial roles in numerous physiological processes, including the structural maintenance of the dermis, production of extracellular matrix components, and mediation of inflammatory responses. Despite their importance, fibroblasts remain one of the least characterized cell populations. The advent of single-cell analysis techniques, particularly single-cell RNA sequencing (scRNA-seq) and fluorescence-activated cell sorting (FACS), has enabled detailed investigations into fibroblast biology. In this study, we present an extensive analysis of fibroblast surface markers suitable for cell sorting and subsequent functional studies. We reviewed over three thousand research articles describing fibroblast populations and their markers, characterizing and comparing subtypes based on their surface markers, as well as their intra- and extracellular proteins. Our detailed analysis identified a variety of distinct fibroblast subpopulations, each with unique markers, characteristics dependent on their location, and the physiological or pathophysiological environment. These findings underscore the diversity of fibroblasts as a cellular population and could lead to the development of novel diagnostic and therapeutic tools.

The full understanding of molecular mechanisms of cell differentiation requires a holistic view. Here we combine label-free FTIR and Raman hyperspectral imaging with data mining to detect the molecular cell composition enabling noninvasive monitoring of cell differentiation and identifying biochemical heterogeneity. Mouse adipose-derived mesenchymal stem cells (AD-MSCs) undergoing adipogenesis were followed by Raman and FT-IR imaging, Oil Red, and immunofluorescence. A workflow of the data analysis (IRRSmetrics4stem) was designed to identify spectral predictors of adipogenesis and test machine-learning (ML) methods (hierarchical clustering, PCA, PLSR) for the control of the AD-MSCs differentiation degree. IRRSmetrics4stem provided insights into the chemism of adipogenesis. With single-cell tracking, we established IRRS metrics for lipids, proteins, and DNA variations during AD-MSCs differentiation. The over 90% predictive efficiency of the selected ML methods proved the high sensitivity of the IRRS metrics. Importantly, the IRRS metrics unequivocally recognize a switch from proliferation to differentiation. This study introduced a new bioassay identifying molecular markers indicating molecular transformations and delivering rapid and machine learning-based monitoring of adipogenesis that can be relevant to other differentiation processes. Thus, we introduce a novel, rapid, machine learning-based bioassay to identify molecular markers of adipogenesis. It can be relevant to identification of differentiation-related molecular processes in other cell types, and beyond the cell differentiation including progression of different cellular pathophysiologies reconstituted in vitro., Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The blood-brain barrier (BBB) is responsible for maintaining homeostasis within the central nervous system (CNS). Depending on its permeability, certain substances can penetrate the brain, while others are restricted in their passage. Therefore, the knowledge about BBB structure and function is essential for understanding physiological and pathological brain processes. Consequently, the functional models can serve as a key to help reveal this unknown. There are many in vitro models available to study molecular mechanisms that occur in the barrier. Brain endothelial cells grown in culture are commonly used to modeling the BBB. Current BBB platforms include: monolayer platforms, transwell, matrigel, spheroidal, and tissue-on-chip models. In this paper, the BBB structure, molecular characteristic, as well as its dysfunctions as a consequence of aging, neurodegeneration, or under hypoxia and neurotoxic conditions are presented. Furthermore, the current modelling strategies that can be used to study BBB for the purpose of further drugs development that may reach CNS are also described., (© 2024. The Author(s).)

Circulating endothelial cell progenitors originating from the bone marrow are considered to be a powerful tool in the repair of endothelium damage. Due to their unique properties, endothelial progenitors are now broadly investigated to assess their clinical significance in diseases e.g., associated with brain endothelial dysfunction. However, their distinction in terms of the expression of specific markers remains ambiguous. Additionally, endothelial progenitor cells may change their repertoire of markers depending on the microenvironment of the tissue in which they are currently located. Here, we applied the label-free Raman and FTIR imaging to discriminate mice brain endothelium and endothelial progenitors. Cells cultured separately showed distinctly different spectral signatures extracted from the whole cellular interior as well as the detected intracellular compartments (nucleus, cytoplasm, perinuclear area, and lipid droplets). Then, we used these spectroscopic signals to examine the cells co-cultured for 24 h. Principal cluster analysis showed their grouping with the progenitor cells and segregation from brain endothelium at a level of the entire cell machinery (in FTIR images) which resulted from biochemical alternations in the cytoplasm and lipid droplets (in Raman images). The models included in partial least square regression indicated that lipid droplets are the key element for the classification of endothelial progenitor-brain endothelial cells interactions., (© 2023. The Author(s).)

Recent studies suggest that the content of immune components in milk is influenced by the mother's health and also by the infant she feeds. We aimed to evaluate the effect of a child's respiratory tract infection on the cellular composition of breast milk (neutrophils, monocytes, eosinophils, lymphocytes, and their subpopulations). Twenty-six breastfeeding mothers whose children were hospitalized for respiratory tract infections were enrolled in the study. The control group consisted of 23 mothers of healthy children. Regarding the children, baseline laboratory blood tests were performed, and nasal swabs were taken for the presence of RS virus. In the next step, milk samples were collected from the mothers to assess the cellular composition of the milk, including neutrophils, monocytes, eosinophils, lymphocytes, and their subpopulations. Significantly higher percentages of T lymphocytes (helper and cytotoxic lymphocytes) were observed in the milk of the studied mothers. There was a significantly higher percentage of milk lymphocytes in the group of affected children with confirmed RSV etiology than in children with excluded RSV etiology. A significant positive correlation was observed between the duration of infection and the percentage of milk NK cells and between milk CD19 lymphocytes and the child's serum leukocytosis. This study may provide evidence of a link between cells in breast milk and disease in the breastfed infant. The severity of the infection, its duration, and the etiological agent of the infection may affect the cellular composition of milk.

Maternal embryonic leucine-zipper kinase (MELK) plays a significant role in cell cycle progression, mitosis, cell migration, cell renewal, gene expression, embryogenesis, proliferation, apoptosis, and spliceosome assembly. In addition, MELK is known to be overexpressed in multiple types of cancer and is associated with cancer proliferation. Tumorigenesis shares many similarities with wound healing, in which the rate of cell proliferation is a critical factor. Therefore, this study aimed to determine the involvement of MELK in the regulation of cell division in two cell types involved in this process, namely fibroblasts and keratinocytes. We examined how temporal overexpression of wild-type and kinase-dead MELK kinase variants affect the rate of proliferation, viability, cell cycle, and phosphorylation state of other kinases involved in these processes, such as ERK1/2, AKT1, MAPK9, p38, and p53. We explored if MELK could be used as a therapeutic stimulator of accelerated wound healing via increased proliferation. We observed that aberrant expression of MELK results in abnormal proliferation, altered cell cycle distribution, and decreased viability of the cells, which challenge the utility of MELK in accelerated wound healing. Our results indicate that, at least in healthy cells, any deviation from precisely controlled MELK expression is harmful to fibroblasts and keratinocytes.

Immunological memory is a crucial part of the immune defense that allows organisms to respond against previously encountered pathogens or other harmful factors. Immunological memory is based on the establishment of epigenetic modifications of the genome. The ability to memorize encounters with pathogens and other harmful factors and mount enhanced defense upon subsequent encounters is an evolutionarily ancient mechanism operating in all animals and plants. However, the term immunological memory is usually restricted to the organisms (invertebrates and vertebrates) possessing the immune system. The mammalian immune system, with innate and adaptive branches, is the most sophisticated among vertebrates. The concept of innate memory and memory macrophages is relatively new and thus understudied. We introduce the concept of immunological memory and describe types of memory in different species and their evolutionary status. We discuss why the traditional view of innate immune cells as the first-line defenders is too restrictive and how the innate immune cells can accumulate and retain immunologic memory. We describe how the initial priming leads to chromatin remodeling and epigenetic changes, which allow memory macrophage formation. We also summarize what is currently known about the mechanisms underlying development of memory macrophages; their molecular and metabolic signature and surface markers; and how they may contribute to immune defense, diseases, and organ transplantation., Competing Interests: The authors declare no conflict of interest.

The failures of anti-β-amyloid immunotherapies suggested that the very low fraction of injected antibodies reaching the brain parenchyma due to the filtering effect of the BBB may be a reason for the lack of therapeutic effect. However, there is no treatment, as yet, for the amyotrophic lateral sclerosis (ALS) despite substantial evidence existing of the involvement of TDP-43 protein in the evolution of ALS. To circumvent this filtering effect, we have developed a novel approach to facilitate the penetration of antibody fragments (Fabs) into the brain parenchyma. Leveraging the homing properties of endothelial progenitor cells (EPCs), we transfected, ex vivo, such cells with vectors encoding anti-β-amyloid and anti-TDP43 Fabs turning them into an "antibody fragment factory". When injected these cells integrate into the BBB, where they secrete anti-TDP43 Fabs. The results showed the formation of tight junctions between the injected engineered EPCs and the unlabeled resident endothelial cells. When the EPCs were further modified to express the anti-TDP43 Fab, we could observe integration of these cells into the vasculature and the secretion of Fabs. Results confirm that production and secretion of Fabs at the BBB level leads to their migration to the brain parenchyma where they might exert a therapeutic effect.

Cell to cell interactions are crucial for morphogenesis and tissue formation. Desmoplakin (encoded by the Dsp gene) is a component of desmosomes and anchors the transmembrane adhesion proteins to the cytoskeleton. Its role in gonad development remains vague. To study the role of desmoplakin in gonad development, we used a tissue-specific knockout of the Dsp gene in the NR5A1+ somatic cells of the gonads. We show here that desmoplakin is necessary for the survival of germ cells in fetal testes and ovaries. The Dspknockout in NR5A1+ somatic cells in testes decreased the number of germ cells, and thus the size of the testes, but did not affect the Sertoli cells or the structure of testis cords and interstitium. The Dspknockout in NR5A1+ somatic cells in ovaries decreased the number of female germ cells and drastically reduced the formation of ovarian follicles. Dsp knockout in NR5A1+ somatic cells did not affect the sex determination and sexual differentiation of the gonads, as judged from an unchanged expression of genes essential for these processes. We conclude that mediation by desmoplakin cell adhesion between the gonadal cells is necessary for germ cell survival.

Vitamin D regulates homeostasis, anti-microbial response, and inflammation. The vitamin D receptors are expressed in the macrophages and other immune cells, regulating the transcription of many different genes, including those coding the anti-microbial peptides. One of the most severe complications of the SARS-CoV-2 infection is the acute respiratory distress syndrome (ARDS) caused by the hyperinflammatory response (commonly called cytokine storm) of the lung macrophages. Studies showed that Vitamin D deficiency increases the severity of the ARDS in COVID-19 infection. We discuss here how the vitamin D supplementation may influence macrophage and myeloid-derived suppressor cells (MDSCs) inflammatory response, subdue the hyperinflammatory response, and lessen the ARDS in COVID-19 patients., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The small GTPase RhoA, and its down-stream effector ROCK kinase, and the interacting Rac1and and mTORC2 pathways, are the principal regulators of the actin cytoskeleton and actin-related functions in all eukaryotic cells, including the immune cells. As such, they also regulate the phenotypes and functions of macrophages in the immune response and beyond. Here, we review the results of our and other's studies on the role of the actin and RhoA pathway in shaping the macrophage functions in general and macrophage immune response during the development of chronic (long term) rejection of allografts in the rodent cardiac transplantation model. We focus on the importance of timing of the macrophage functions in chronic rejection and how the circadian rhythm may affect the anti-chronic rejection therapies.

The timing of the M-phase is precisely controlled by a CDC6-dependent mechanism inhibiting the mitotic histone H1 kinase. Here, we describe the differential regulation of the dynamics of this mitotic kinase activity by exogenous cyclin A or cyclin B in the Xenopus laevis cycling extracts. We show that the experimental increase in cyclin A modifies only the level of histone H1 kinase activity, while the cyclin B increase modifies two parameters: histone H1 kinase activity and the timing of its full activation, which is accelerated. On the other hand, the cyclin A depletion significantly delays full activation of histone H1 kinase. However, when CDC6 is added to such an extract, it inhibits cyclin B-associated histone H1 kinase, but does not modify the mitotic timing in the absence of cyclin A. Further, we show via p9 co-precipitation with Cyclin-Dependent Kinases (CDKs), that both CDC6 and the bona fide CDK1 inhibitor Xic1 associate with the mitotic CDKs. Finally, we show that the Xic1 temporarily separates from the mitotic CDKs complexes during the peak of histone H1 kinase activity. These data show the differential coordination of the M-phase progression by cyclin A- and cyclin B-dependent CDKs, confirm the critical role of the CDC6-dependent histone H1 kinase inhibition in this process, and show that CDC6 acts differentially through the cyclin B- and cyclin A-associated CDKs. This CDC6- and cyclins-dependent mechanism likely depends on the precisely regulated association of Xic1 with the mitotic CDKs complexes. We postulate that: i. the dissociation of Xic1 from the CDKs complexes allows the maximal activation of CDK1 during the M-phase, ii. the switch between cyclin A- and cyclin B-CDK inhibition upon M-phase initiation may be responsible for the diauxic growth of mitotic histone H1 kinase activity.

A pregnant woman's diet consists of many products, such as fruits, vegetables, cocoa, tea, chocolate, coffee, herbal and fruit teas, and various commercially available dietary supplements, which contain a high number of biological active plant-derived compounds. Generally, these compounds play beneficial roles in women's health and the development of fetus health. There are, however, some authors who report that consuming excessive amounts of plants that contain high concentrations of polyphenols may negatively affect the development of the fetus and the offspring's health. Important and problematic issues during pregnancy and lactation are bacterial infections treatment. In the treatment are proposals to use plant immunomodulators, which are generally considered safe for women and their offspring. Additional consumption of biologically active compounds from plants, however, may increase the risk of occurrences to irreversible changes in the offspring's health. Therefore, it is necessary to carry out safety tests for immunomodulators before introducing them into a maternal diet. Here, we present data from animal experiments for the four most-studied plants immunomodulators genus: Rhodiola , Echinacea , Panax , and Camellia , which were used in maternal nutrition.

Antibiotics treatment during pregnancy and lactation is problematic. The alternative to the antibiotic treatment is the use of plant-derived supplements, which stimulate immune system to prevent and eliminate bacterial infection. Here, we evaluated the effect of long-term use of Rhodiola kirilowii on the health of mouse mothers. Pregnant mice were fed daily, for whole pregnancy and for 28 days after giving birth, with Rhodiola kirilowii water (RKW) or hydroalcoholic extract (RKW-A) (at 20 mg of extracts/kg). The control group received sterile water. There was no significant change in the total body weight and selected organs weight and in the status of macroscopically evaluated liver, spleen, kidney, brain, and eyes, between the Rhodiola kirilowii groups and the control group. There was also no change in hematological parameters and components of adaptive immunity (level of CD3 + , CD4 + , CD8 + , CD19 + , CD335 + cells). Mice fed with RKW extracts exhibited lower percentage of oxidative burst in the granulocytes. In contrast, the supplementation with RKW-A extract caused increase in the percentage of granulocytes in the blood and the percentage of monocytes with oxidative burst. Other studied components of innate immunity were unaffected. Minor effect on the innate immunity and lack of side effects on hematological parameters and components of immunological system of mouse mothers indicates that both water and 50% hydroalcoholic extracts of Rhodiola kirilowii (in concentration 20 mg/kg per day) could be used as an immunostimulators during pregnancy and nursing. However, to fully assess the effects of Rhodiola kirilowii extracts on the mother and offspring health, further studies in mouse and large animal models and clinical studies in humans are necessary.

Purpose: The aim of the study was assessment of four selected macromolecules level: osteopontin, calgranulin, uromodulin and bikunin in fresh morning urine sample in children with nephrolithiasis in the course of idiopathic hypercalciuria., Materials and Methods: The study included 90 subjects aged from 12 months to 18 years. The study group comprised 57 subjects- children with urinary tract lithiasis in the course of idiopathic hypercalciuria and the control group - 33 healthy children with no history of urolithiasis. Determinations of osteopontin, calgranulin, uromodulin and bikunin levels in the first morning urine were performed., Results: The study group had a significantly decreased osteopontin excretion and significantly increased bikuninexcretion, and increased, however statistically nonsignificant, calgranulin excretion in comparison with the control group. Uromodulin excretion did not differ between groups. In both groups a statistically significant positive correlation was observed between uromodulin and bikunin levels., Conclusion: Children with urinary tract lithiasis in the course of idiopathic hypercalciuria reveal a different distribution of the study proteins than a healthy population.

Tritium is a potentially significant source of internal radiation exposure which, at high levels, can be carcinogenic. We evaluated whether single intraperitoneal injection of BALB/c and C57BL/6 mice with tritiated water (HTO) leading to exposure to low (0.01 or 0.1 Gy) and intermediate (1.0 Gy) cumulative whole-body doses of β radiation is immunosuppressive, as judged by enhancement of artificial tumour metastases, functioning of NK lymphocytes and macrophages, circulating cytokine's levels, and numbers of bone marrow, spleen, and peripheral blood cells. We demonstrate that internal contamination of radiosensitive BALB/c and radioresistant C57BL/6 mice with HTO at all the absorbed doses tested did not affect the development of neoplastic colonies in the lungs caused by intravenous injection of syngeneic cancer cells. However, internal exposure of BALB/c and C57BL/6 mice to 0.1 and 0.01 Gy of β radiation, respectively, up-regulated cytotoxic activity of and IFN-γ synthesis in NK lymphocytes and boosted macrophage secretion of nitric oxide. Internal contamination with HTO did not affect the serum levels of pro- (IL-1β, IL-2, IL-6, TNF-α,) and anti-inflammatory (IL-1Ra, IL-4, IL-10) cytokines. In addition, exposure of mice of both strains to low and intermediate doses from the tritium-emitted β-particles did not result in any significant changes in the numbers of bone marrow, spleen, and peripheral blood cells. Overall, our data indicate that internal tritium contamination of both radiosensitive and radioresistant mice leading to low and intermediate absorbed β-radiation doses is not immunosuppressive but may enhance some but not all components of anticancer immunity.

BACKGROUND Acetylcholinesterase (AChE) and cholinergic receptors have an important role in the immune system and angiogenesis. This work evaluated the effects of isopropyl methylphosphonofluoridate (IMPF), an irreversible inhibitor of AChE, on tumor growth and selected parameters associated with tumor angiogenesis. MATERIAL AND METHODS Experiments were performed on male BALB/c mice exposed to IMPF (study group) or saline buffer (control group) and inoculated with L-1 sarcoma; the number of new blood vessels (TIA test) and the level of αvβ3 integrin (131I-MAb-antiβ3 assay) were analyzed at seven, 14, or 21 days after implantation of the tumor cells. RESULTS The IMPF poisoning affected tumor angiogenesis (TIA test). There was a statistically significant increase in the number of newly forming blood vessels in the group subjected to IMPF and inoculated with tumor cells. CONCLUSIONS This study showed that IMPF had a significant effect on the regulation of lymphocyte-induced angiogenesis and the modulation of angiogenic and pro-inflammatory cytokines secretion. The observed effects suggest involvement of neuronal and/or non-neuronal cholinergic signaling pathway.

The hexachlorophene (HCP) is a highly lipophilic chlorinated bisphenol present in hygienic and dermatological products. The HCP accumulates preferentially in adipose tissue that is a privileged source of mesenchymal stem cells (MSCs). The evaluation of the potential effects of HCP on MSCs is important for their medical application. Here we examined the effects of HCP on murine adipose tissue-derived stem cells (ADSCs) and human umbilical cord-derived stem cells (UCSCs) in cell culture. We found that 10 -4 and 10 -5  M HCP inhibits proliferation, osteogenesis and increases apoptosis of ADSCs and UCSCs. While the effect of HCP on proliferation and differentiation potential of these two cell lines was similar, the UCSCs appeared much more resistant to HCP-induced apoptosis than ADSCs. These results suggest that the adipose tissue-derived ADSCs have higher sensitive for HCP than umbilical cord-derived UCSCs and indicate that the umbilical cord can be a preferable source of MSCs for prospective medical applications in the future., Competing Interests: The authors declare no conflict of interest.Protocol for isolation of fat tissue was approved by IV Local Ethics Committee (IV) on Animal Testing, National Medicines Institute, Warsaw, Poland (Permission No. 79/2012). Protocol for use human tissues in research was approved by Bioethical Commission, Medical University of Warsaw, Poland (Permission No. 70/2012).

Histamine intolerance (pseudoallergy) is a poorly investigated type of food hypersensitivity. The main enzyme responsible for histamine degradation in the extracellular matrix is diamine oxidase (DAO). Disturbances in the concentration or activity of DAO may lead to the development of clinical signs of allergy. The aim of the present work was to assess the DAO concentration, peripheral blood morphology, lymphocytes phenotyping (CD3+, CD4+, CD8+, CD19+, NK cells, NKT cells, and activated T-cells), and natural regulatory Treg (nTregs) cell population (CD4+, CD25+, CD127low, and FoxP3) in 34 pediatric patients with histamine-dependent syndromes. Patients were divided into two groups: classical allergy and pseudoallergy on the basis of IgE concentration. The investigation was based on the analysis of peripheral blood samples. A significantly lower serum DAO, both total and specific IgE, concentration was found in the pseudoallergy group compared with the allergy group. There were no significant differences in blood morphology or lymphocyte populations. A similar level of nTreg lymphocytes was also found in both groups, although it was lower than that present in healthy individuals. The findings suggest that the serum DAO is responsible for the symptoms of histamine intolerance. Moreover, a general decrease in nTreg cells in comparison with healthy individuals may lead to symptom aggravation.

Introduction: Genistein (plant isoflavone) is a well-known anti-cancer drug with estrogenic-like properties. Genistein also regulates sugar and lipid metabolism; thus, it has anti-diabetic properties. The aim of the study was to evaluate in vitro effects of genistein on glucose transport, fatty acids oxidation, activation of PKB, and expression of genes related to insulin pathway in differentiated myoblast C2C12 mouse cell line., Material and Methods: Differentiated myoblast C2C12 mouse cell line was used to assess the effects of different genistein concentrations on glucose transport and fatty acids oxidation measured by radioactivity technique, activation of PKB, and expression of selected genes related to insulin signaling pathway (IR-a, IR-b, IRS-1, PKB, GLUT-4, PP2A, SH-PTP2) at the mRNA and protein levels. Cells were incubated with various concentrations of genistein under standard conditions for 0-48 hours., Results: Genistein in low concentrations (0.1-1 μM) significantly increased glucose transport and decreased fatty acids oxidation in C2C12 cells after 48 h of incubation. High concentration of genistein (50 μM) had the opposite effect. Genistein stimulated PKB phosphorylation during the first 5-10 minutes of incubation. There was no significant impact on the protein expression of selected genes (IR-a, IR-b, IRS-1, PKB, GLUT-4, PP2A-Ca, ER-a and ER-b) after 48 h treatment. We observed inverse correlation between genistein concentration and the expression of SH-PTP2 protein. Genistein affected the expression pattern of mRNAs for genes related to the insulin pathway, however, not the expression of the encoded proteins., Conclusions: The results of this study showed that depending on the concentration and time of incubation genistein significantly affects glucose and lipid metabolism and at low concentration modifies expression pattern of a few genes in C2C12 cells.

BACKGROUND Understanding the mechanisms conditioning development of chronic kidney disease (CKD) is still a challenge. The aim of this study was to evaluate the activity of the intrarenal nitric oxide (NO) pathway in the context of sensitivity or resistance of different animal strains to the development and degree of renal failure. MATERIAL AND METHODS Two rat strains were used: Wistar (WR) and Sprague-Dawley rats (SDR) in a model of CKD - 5/6 nephrectomy. We assessed parameters of renal failure and expression of nitric oxide synthase (NOS) isoforms in renal cortex and medulla. RESULTS We did not observe renal failure in WR, and CKD developed in SDR with increase of creatinine and urea concentration as well as decrease of diuresis and glomerular filtration. In the renal cortex, baseline expression of NOS2 was higher in WR than in SDR. 5/6 nephrectomy resulted in reduction of NOS2 in both strains and NOS3 in WR. In the renal medulla, baseline NOS2 expression was higher in SDR, and nephrectomy resulted in its decrease only in SDR. Although baseline NOS3 expression was higher in SDR, the NOS3 expression after nephrectomy was higher in WR rats. CONCLUSIONS In model of CKD - 5/6 nephrectomy, SDR proved to be sensitive and WR resistant to development of CKD. The intrarenal activity of the nitric oxide pathway was the factor that differentiated both strains. This mechanism may be responsible for insensitivity of WR to development of renal failure in this model of CKD.

Background: CD105 was postulated as a renal cell carcinoma (RCC) stem cell marker, and CD133 as a putative RCC progenitor. Hypoxia, a natural microenvironment that prevails in tumors, was also incorporated into the study, especially in terms of the promotion of hypothetical stem-like cell properties., Methods: Within this study, we verify the existence of CD105+ and CD133+ populations in selected papillary subtype RCC (pRCC) cell lines. Both populations were analyzed for correlation with stem-like cell properties, such as stemness gene expression, and sphere and colony formation. For the preliminary analysis, several RCC cell lines were chosen (786-O, SMKT-R2, Caki-2, 796-P, ACHN, RCC6) and the control was human kidney cancer stem cells (HKCSC) and renal cells of embryonic origin (ASE-5063). Four cell lines were chosen for further investigation: Caki-2 (one of the highest numbers of CD105+ cells; primary origin), ACHN (a low number of CD105+ cells; metastatic origin), HKCSC (putative positive control), and ASE-5063 (additional control)., Results: In 769-P and RCC6, we could not detect a CD105+ population. Hypoxia variously affects pRCC cell growth, and mainly diminishes the stem-like properties of cells. Furthermore, we could not observe the correlation of CD105 and/or CD133 expression with the enhancement of stem-like properties., Conclusions: Based on this analysis, CD105/CD133 cannot be validated as cancer stem cell markers of pRCC cell lines.

Angiogenesis plays an important role in many physiological processes, among them the formation of tissues and organs during embryogenesis. A lot of medicinal plants exhibit angiomodulatory properties. This creates the need for a thorough check of whether the plant extracts that we would like to give to pregnant women in order to increase their resistance to bacterial or viral infection will have negative effects on angiogenesis, and consequently on fetal development. This paper seeks to investigate the effect of serum of pregnant and nursing Balb/c mice that received aqueous (RKW) or hydro-alcoholic (RKW-A) R. kirilowii extracts (20 mg/kg), or epigallocatechin (0.2 mg/kg), on the in vitro proliferation and migration of mouse endothelial cell line Heca10. Of the 15 identified polyphenols in the extracts by HPLC, 8 were present in the sera. Chemical analysis revealed higher salidroside, kaempferol, chlorogenic acid, bFGF and VEGF concentration in RKW-A sera than in the sera of RKW group of mice. RKW-A and EGC sera did not affect migration of endothelial cells, however we noted some increase of migrating cells after RKW-sera treatment. RKW and EGC sera did not affect proliferation of endothelial cells. Sera of mother s from RKW-A group impaired the proliferation of endothelial cells in comparison to other groups. These data allow us to assume that Rhodiola kirilowii hydro-alcoholic extract (RKW-A) is potentially able to modulate pre- and post- natal angiogenesis what might influence the development of organs in progeny. Sera of RKW mothers have not harm the proliferation of endothelial cells, despite they also contain antiangiogenic catechins and salidroside. This suggests the existence in RKW-A extract and in RKW-A sera of some other, as yet unidentified substances influencing endothelial cells proliferation., Competing Interests: The authors declare no conflict of interest.

Treating infections in pregnant patients is potentially dangerous even when herbal medicines are used. Many herbal medicines, among them extracts from plants of Rhodiola genus, have antimicrobial, anti-inflammatory, and immunostimulatory properties owing to their polyphenol content; they may, however, affect fetal development due to their antiangiogenic properties. The aim of this study was to explain whether daily feeding pregnant and lactating mice with 20 mg/kg Rhodiola kirilowii aqueous (RKW) or 50% hydro-alcoholic (RKW-A) extracts, or 0.2 mg/kg epigallocatechin (EGC, antiangiogenic compound of Rhodiola extracts), may lead to abnormalities in morphology and function of the kidneys of adult progeny. Such abnormalities were not observed in the kidneys of 6-week-old offspring, neither in RKW nor in the control group. However, the progeny of RKW-A- or EGC-fed mothers presented morphometric abnormalities in the kidney structure, with a significantly higher number of glomeruli/mm 2 and a lower diameter of glomeruli (RKW-A group) or a significantly higher glomeruli diameter (EGC), than in the control and RKW groups. Abnormalities in serum vascular endothelial growth factor, tumor necrosis factor (TNF)-alpha, urea, creatinine, and cystatin C levels were also found. We recommend caution in long-term use of RKW-A extract and EGC-rich foods during pregnancy and lactation.

Methyloxantines are present in many herbs and vegetal foods, among them in tea, coffee and chocolate. Previous studies revealed that theophylline and theobromine have anti-angiogenic properties. Anti-tumor properties of theobromine were also described. Pentoxifylline (3,7-dimethyl-1-(5-oxohexyl)xanthine, PTX) is a synthetic xanthine derivative. It is a phosphodiesterase inhibitor and has various anti-inflammatory abilities. Pentoxifylline is widely used in therapy of inflammatory arterial diseases such as intermittent claudication of upper and lower limbs as well as in coronary heart disease. The aim of our research was to evaluate the effect of pentoxifylline (individually and in combination with non-steroidal anti-inflammatory drug sulindac), on L-1 sarcoma angiogenic activity and tumor formation in syngeneic Balb/c mice. Pre-incubation of tumor cells for 90 min with various PTX concentrations resulted in dose-dependent decrease of their ability to induce newly-formed blood vessels after transplantation into the skin of recipient mice. Administration of PTX to mice, recipients of tumor cells, slows tumor growth and reduces its volume. Synergistic inhibitory effect of PTX and sulindac, expressed as % of tumors sixth and thirteen day after subcutaneous grafting of L-1 sarcoma into syngeneic Balb/c mice, was observed., Competing Interests: The authors declare no conflicts of interest.

Asthma is a common chronic respiratory diseases in children. Understanding the immune mechanisms of epigenetic factors may contribute to a better control of asthma. This study seeks to determine the effects of serum vitamin D and urine cotinine on asthma severity and on T regulatory cells (Tregs) and other immune-related factors such as CD3, CD4, CD8, CD19, CD16/56, and anti-CD3 HLA-DR3. The study involved 34 children with asthma. Disease severity was assessed with the Asthma Control Test, spirometry, and the fractional exhaled nitric oxide (FeNO). The control group consisted of 18 healthy children. We found a significantly lower proportion of Tregs in the serum of asthmatic children compared with the control group (p < 0.002). There were no significant differences in the other immunological factors investigated. Nor was there any appreciable association between vitamin D or cotinine and the course of asthma, FeNO, Tregs, and the other immune factors. However, the percentage of Tregs was positively associated with the level of FeNO (p < 0.02). In conclusion, the study shows a role of T regulatory cells in the pathogenesis of asthma in children, but fails to show any influence of serum vitamin D or urine cotinine on disease course.

The influence of vitamin D on allergic diseases, including atopic dermatitis, is linked to the presence of vitamin D nuclear receptors in immune cells. The present study seeks to determine the possible relationship between serum vitamin D content and immune indices in children with atopic dermatitis. The study was conducted in 19 children with atopic dermatitis. The control consisted of 17 age-matched healthy children. A single significant finding was a distinctly lower number of serum regulatory T cells in atopic dermatitis compared with controls (p < 0.00001). There were no appreciable differences between the two groups concerning the immunological indices such as the phenotypes: CD3, CD4, CD8, CD4/CD8, CD19, CD16/56, natural killer T cells, and anti-CD3 human leukocyte antigen - antigen D related cell surface receptor (HLA-DR3), or the percentage of lymphocytes, eosinophils, and the IgE level. We also revealed an inverse association between the serum vitamin D and the percentage of CD8+ cells (p < 0.05; r = 0.62) in atopic dermatitis. In conclusion, the results point to a regulatory role of T cells in the pathogenesis of atopic dermatitis, but fail to substantiate the influence of vitamin D on the course of the disease.

Due to the increased incidence of allergic diseases and emerging effects of unsatisfactory control of asthma, new mechanisms for supervising the immune system should be searched. The aim of the study was to analyze the percentage of CD3, CD4, CD8, CD19, CD16/56, NKT, CD3 anti-HLADR3 and Foxp3 regulatory lymphocytes in patients with asthma. Additionally the correlation between immune parameters, severity of asthma and serum concentration of vitamin D was performed. 25 children diagnosed with asthma were enrolled. Disease severity was assessed with the Asthma Control Test (ACT) and spirometry. The control group consisted of 15 healthy children. Venous blood from each patient was collected on EDTA or on "clott". Phenotypes of lymphocytes were evaluated by flow cytometry. Vitamin D concentration was assessed by chemiluminescent immunoassay (CLIA) technology. There was a significant decrease in the percentage of T regulatory cells (p < 0.006) in children with asthma compared to the control group. There were no significant differences in the other investigated immunological parameters. In addition, in asthma group statistically significant decreased of vitamin D concentration (p < 0.04) was observed. There were also no significant correlations between vitamin D3 concentration and the course of asthma or percentage of regulatory cells. The results confirmed the role of regulatory T cells in the pathogenesis of asthma. Effects of vitamin D on the severity of the disease has not been proven., Competing Interests: The authors declare no conflict of interests.

Tumor angiogenesis is an important link in the process of tumor growth and metastasis. A number of substances with an anti-angiogenic activity has been described, but their efficiency remains low. Many researchers believe that a better therapeutic effect could be achieved using a cocktail of several anti-angiogenic agents, having different points of action. A lot of synthetic and natural products of plant and animal origin have anti-tumor and anti-angiogenic properties. The aim of the present study was to evaluate the effect of some combinations of angiogenesis inhibitors on the growth and neovascularization of murine sarcoma L-1 , and on angiogenesis induced in the mouse skin by grafting of human renal cancer. The influence of theobromine, sulindac and its metabolite sulindac sulfone, chlorogenic acid, and shark liver oil on the afferent and efferent angiogenesis pathways was tested. Individually, all of these substances suppressed tumor growth and angiogenesis. Synergy was found for a combination of theobromine, sulindac, and chlorogenic acid (L-1 sarcoma tumor growth), and for theobromine with sulindac sulfone or with shark liver oil, which were given to the mice grafted with human renal cancer cells (angiogenesis). No synergistic effects were shown after preincubation with tumor cells and inhibitors.

Rhodiola kirilowii, a member of Crassulaceae family, grows wildly in Asiatic mountains and is also cultivated in some European countries. Its underground parts traditionally are used for enhance physical and mental performance of the body. In our previous papers we reported immuno- and angio-modulatory effects of aqueous and hydro-alcoholic extracts of radix and rhizome of this plant in mice. In the present work we evaluated the effect of Rhodiola kirilowii water- (RKW) or hydro-alcoholic (RKW-A) extracts and epigallocatechin (one of the polyphenols present in these extracts) given to mice, during pregnancy and nursing period, on the number and localization of CD4+ and CD8+ cells in spleens of adult progeny mice. Previously, we observed several abnormalities in functionality of spleen cells collected from these mice. No differences in CD4+ T cells localisation or numbers were found between all tested mice groups. In contrast, CD8+ T cells localisation and staining were altered in progeny of water or alcohol extract-fed mice. CD8+ T cells were found not only in the PALS but also in the B cell follicle and in the red pulp. Furthermore, CD8+ T cells from T cell zones in the progeny of extract-fed mice showed much intensive staining for CD8 antigen and significantly higher numbers per area in comparison to control mice., Competing Interests: The authors declare no conflict of interest.

In previous work we described the decline in the number of splenocytes of mice which during pregnancy and lactation were fed Rhodiola kirilowii. In this work we present the size of individual subpopulations of splenic lymphocytes in these mice. Experiments were performed on adult inbred female Balb/c mice, 8-9 weeks old, 20-22 g b.m., mated with adult males from the same strain. Females, from when the copulatory plug was noted up to the 28 th day after delivery, were supplemented daily with lyophilized aqueous (RKW) or 50% hydro-ethanolic (RKW-A) extract (20 mg/kg b.m.) dissolved in distilled water. Then, mice were euthanized, spleens dissected, cells counted and the total numbers of CD3+, CD19+, CD4+, CD8+ and CD335+ splenic lymphocytes were evaluated by cytometry. The number of CD3+ lymphocytes per 1 g of splenic tissue was higher in RKW-A than in RKW spleens and did not differ from the control. The number of CD3+ lymphocytes in RKW spleens was lower than in the controls. The number of CD19+ and CD4+ cells was lower in both experimental groups than in the controls. The number of CD335+(NK) cells was lower in RKW spleens than in the control., Competing Interests: The authors declare no conflict of interest.

The use of antibiotics during pregnancy and lactation is associated with an increased risk of developmental disorders. One of the natural medicinal plants- Rhodiola kirilowii , widely used as an immunostimulant in adults-might be a good alternative to antibiotic treatment. The aim of present study was to assess whether daily oral administration of 20 mg/kg of Rhodiola kirilowii aqueous (RKW) or 50% hydroalcoholic (RKW-A) extracts affected hematological and immunological parameters of 6-week-old mouse progeny. There was no significant change in hematological parameters of blood with the exception of hemoglobin, which was significantly higher (about 4%) in RKW group. Offspring of mothers fed Rhodiola kirilowii extracts had increased percentage of granulocytes and decreased percentage of lymphocytes. These changes correlated with decreased percentage of CD3 + /CD4 + T-cells (RKW and RKW-A), decrease of CD8 + cells, and increase percentage of NK cells in RKW group. In addition, both types of Rhodiola kirilowii extracts stimulated granulocyte phagocytosis and increased level of respiratory burst. In conclusion, the long-term supplementation of mouse mothers during pregnancy and lactation with RKW or RKW-A extracts affects the immune system of their progeny. These results should be taken into consideration before administration of Rhodiola kirilowii to pregnant and lactating women.

Background: Recent advancement in cancer research has shown that tumors are highly heterogeneous, and multiple phenotypically different cell populations are found in a single tumor. Cancer development and tumor growth are driven by specific types of cells-stem cell-like cancer cells (SCLCCs)-which are also responsible for metastatic spread and drug resistance. This research was designed to verify the presence of SCLCCs in renal cell cancer cell lines. Subsequently, we aimed to characterize phenotype and cell biology of CD105+ cells, defined previously as renal cell carcinoma tumor-initiating cells. The main goal of the project was to describe the gene-expression profile of stem cell-like cancer cells of primary tumor and metastatic origin., Materials and Methods: Real-time PCR analysis of stemness genes (Oct-4, Nanog and Ncam) and soft agar colony formation assay were conducted to check the stemness properties of renal cell carcinoma (RCC) cell lines. FACS analysis of CD105+ and CD133+ cells was performed on RCC cells. Isolated CD105+ cells were verified for expression of mesenchymal markers-CD24, CD146, CD90, CD73, CD44, CD11b, CD19, CD34, CD45, HLA-DR and alkaline phosphatase. Hanging drop assay was used to investigate CD105+ cell-cell cohesion. Analysis of free-floating 3D spheres formed by isolated CD105+ was verified, as spheres have been hypothesized to contain undifferentiated multipotent progenitor cells. Finally, CD105+ cells were sorted from primary (Caki-2) and metastatic (ACHN) renal cell cancer cell lines. Gene-expression profiling of sorted CD105+ cells was performed with Agilent's human GE 4x44K v2 microarrays. Differentially expressed genes were further categorized into canonical pathways. Network analysis and downstream analysis were performed with Ingenuity Pathway Analysis., Results: Metastatic RCC cell lines (ACHN and Caki-1) demonstrated higher colony-forming ability in comparison to primary RCC cell lines. Metastatic RCC cell lines harbor numerous CD105+ cell subpopulations and have higher expression of stemness genes (Oct-4 and Nanog). CD105+ cells adopt 3D grape-like floating structures under handing drop conditions. Sorted CD105+ cells are positive for human mesenchymal stem cell (MSC) markers CD90, CD73, CD44, CD146, and alkaline phosphatase activity, but not for CD24 and hematopoietic lineage markers CD34, CD11b, CD19, CD45, and HLA-DR. 1411 genes are commonly differentially expressed in CD105+ cells (both from primary [Caki-2] and metastatic RCC [ACHN] cells) in comparison to a healthy kidney epithelial cell line (ASE-5063). TGF-β, Wnt/β-catenine, epithelial-mesenchymal transition (EMT), Rap1 signaling, PI3K-Akt signaling, and Hippo signaling pathway are deregulated in CD105+ cells. TGFB1, ERBB2, and TNF are the most significant transcriptional regulators activated in these cells., Conclusions: All together, RCC-CD105+ cells present stemlike properties. These stem cell-like cancer cells may represent a novel target for therapy. A unique gene-expression profile of CD105+ cells could be used as initial data for subsequent functional studies and drug design., Competing Interests: All authors declare they have no competing commercial interests in relation to the submitted work.

Plants of Rhodiola genus are medicinal herbs that have a number of therapeutic properties, including anti-inflammatory and immunomodulatory activity. The present study aimed to determine whether the use Rhodiola kirilowii as an immunostimulant during pregnancy has an adverse effect on the development of the offspring immune system. Following mating, pregnant mice were placed in three groups that were fed during pregnancy and lactation with R. kirilowii aqueous extract (RKW; 20 mg/kg), R. kirilowii 50% hydro-alcoholic extract (RKW-A; 20 mg/kg) or water (control group), receiving water. Following birth, offspring were given six weeks to develop prior to evaluation of their immune system. Morphometric and morphological examination of the spleen did not reveal any abnormalities or differences between the experimental and control groups. However, both RKW and RKW-A splenic lymphocytes presented a diminished proliferative response to concanavalin A. RKW spleen lymphocytes demonstrated increased metabolic activity following phytohaemagglutinin (PHA) stimulation, which was associated with a higher percentage of cluster of differentiation 4 positive spleen cells and lower interleukin-17a (IL-17a) serum concentration. The RKW-A group exhibited a diminished proliferative response of spleen lymphocytes to PHA and lipopolysaccharide (LPS), and increased serum concentrations of IL-10 and tumor necrosis factor-α (TNF-α). The progeny of mice fed with RKW-A extract demonstrated a significantly lower level of anti-SRBC antibody following immunization compared with progeny of the control (P=0.0305) and RKW (P=0.0331) groups. In conclusion, caution is recommended in the use of RKW and RKW-A extracts as immunostimulants in pregnancy.

Triiodothyronine plays an important role in the regulation of kidney cell growth, differentiation and metabolism. Patients with renal cell cancer who develop hypothyreosis during tyrosine kinase inhibitor (TKI) treatment have statistically longer survival. In this study, we developed cell based model of triiodothyronine (T3) analysis in RCC and we show the different effects of T3 on renal cell cancer (RCC) cell growth response and expression of the thyroid hormone receptor in human renal cell cancer cell lines from primary and metastatic tumors along with human kidney cancer stem cells. Wild-type thyroid hormone receptor is ubiquitously expressed in human renal cancer cell lines, but normalized against healthy renal proximal tube cell expression its level is upregulated in Caki-2, RCC6, SKRC-42, SKRC-45 cell lines. On the contrary the mRNA level in the 769-P, ACHN, HKCSC, and HEK293 cells is significantly decreased. The TRβ protein was abundant in the cytoplasm of the 786-O, Caki-2, RCC6, and SKRC-45 cells and in the nucleus of SKRC-42, ACHN, 769-P and cancer stem cells. T3 has promoting effect on the cell proliferation of HKCSC, Caki-2, ASE, ACHN, SK-RC-42, SMKT-R2, Caki-1, 786-0, and SK-RC-45 cells. Tyrosine kinase inhibitor, sunitinib, directly inhibits proliferation of RCC cells, while thyroid hormone receptor antagonist 1-850 (CAS 251310‑57-3) has less significant inhibitory impact. T3 stimulation does not abrogate inhibitory effect of sunitinib. Renal cancer tumor cells hypostimulated with T3 may be more responsive to tyrosine kinase inhibition. Moreover, some tumors may be considered as T3-independent and present aggressive phenotype with thyroid hormone receptor activated independently from the ligand. On the contrary proliferation induced by deregulated VHL and or c-Met pathways may transgress normal T3 mediated regulation of the cell cycle.

Carbon-encapsulated iron nanoparticles (CEINs) qualified as metal-inorganic hybrid nanomaterials offer a potential scope for an increasing number of biomedical applications. In this study, we have focused on the investigation of cellular fate and resulting cytotoxic effects of CEINs synthesized using a carbon arc route and studied in murine endothelial (HECa-10) cells. The CEIN samples were characterized as pristine (the mean diameter between 47 and 56nm) and hydrodynamic (the mean diameter between 270 and 460nm) forms and tested using a battery of methods to determine the cell internalization extent and cytotoxicity effects upon to the exposures (0.0001-100μg/ml) in HECa-10 cells. Our studies evidenced that the incubation with CEINs for 24h is accompanied with substantial changes of Zeta potential in cells which can be considered as a key factor for affecting the membrane transport, cellular distribution and cytotoxicity of these nanoparticles. The results demonstrate that CEINs have entered the endothelial cell through the endocytic pathway rather than by passive diffusion and they were mainly loaded as agglomerates on the cell membrane and throughout the cytoplasm, mitochondria and nucleus. The studies show that CEINs induce the mitochondrial and cell membrane cytotoxicities in a dose-dependent manner resulting from the internal dosages due to CEIN agglomerates. Our results highlight the importance of the physicochemical characterization of CEINs in studying the magnetic nanoparticle-endothelial cell interactions because the CEIN mass agglomerates can sediment more or less rapidly in culture models., (Copyright © 2016 Elsevier B.V. All rights reserved.)

During the cell cycle, cyclin dependent kinase 1 (CDK1) and protein phosphatase 2A (PP2A) play major roles in the regulation of mitosis. CDK1 phosphorylates a series of substrates triggering M-phase entry. Most of these substrates are dephosphorylated by PP2A. To allow phosphorylation of CDK1 substrates, PP2A is progressively inactivated upon M-phase entry. We have shown previously that the interplay between these two activities determines the timing of M-phase entry. Slight diminution of CDK1 activity by the RO3306 inhibitor delays M-phase entry in a dose-dependent manner in Xenopus embryo cell-free extract, while reduction of PP2A activity by OA inhibitor accelerates this process also in a dose-dependent manner. However, when a mixture of RO3306 and OA is added to the extract, an intermediate timing of M-phase entry is observed. Here we use a mathematical model to describe and understand this interplay. Simulations showing acceleration and delay in M-phase entry match previously described experimental data. CDC25 phosphatase is a major activator of CDK1 and acts through CDK1 Tyr15 and Thr14 dephosphorylation. Addition of CDC25 activity to our mathematical model was also consistent with our experimental results. To verify whether our assumption that the dynamics of CDC25 activation used in this model are the same in all experimental variants, we analyzed the dynamics of CDC25 phosphorylation, which reflect its activation. We confirm that these dynamics are indeed very similar in control extracts and when RO3306 and OA are present separately. However, when RO3306 and OA are added simultaneously to the extract, activation of CDC25 is slightly delayed. Integration of this parameter allowed us to improve our model. Furthermore, the pattern of CDK1 dephosphorylation on Tyr15 showed that the real dynamics of CDK1 activation are very similar in all experimental variants. The model presented here accurately describes, in mathematical terms, how the interplay between CDK1, PP2A and CDC25 controls the flexible timing of M-phase entry.