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The Na+/K+ ATPase is a protein involved in the active transport of ions across the cellular membrane. Ouabain is a cardiotonic glycoside that, by inhibiting the Na+/K+ pump, interferes with cell processes mediated directly by the pump, but also indirectly influences other cellular processes, such as cellular cycle and proliferation, growth, cell differentiation, angiogenesis, migration, adhesion and invasion. We used the SK-BR-3 breast cancer cell line, mesenchymal stem cells (MSCs), and tumor-associated fibroblasts (TAFs) in vitro for determining the changes induced by Ouabain in concentration of 10-5 M, 10-6 M, 10-8 M, 10-9 M on these cellular types, employing specific methods: SEM for morphological evaluation; immunocytochemistry, flowcytometry to assess immunophenotypical markers expression, cell cycle and Annexin V/PI assay for cellular proliferation and viability testing; flowchamber assay to evaluate functional adhesion behavior; RT-PCR for Na+/K+ ATPase subunits molecular expression. Being indicative of the pro-apoptotic and inhibitory effect of Ouabain on tumor invasion and metastasis, the results support the addition of Ouabain to the oncological therapeutic arsenal, trailing the “repurposing drugs” approach.

Purpose The aim of this study was to evaluate the antioxidant potential, antimicrobial activity, the in vitro anticancer effect (tested on MCF-7 breast cancer cell line), as well as the antiangiogenic and immunomodulatory potential of Populus nigra L. bud (Pg) extract collected from the western part of Romania. Results Populus nigra L. bud extract presents an important antioxidant activity, due to the rich phytochemical composition. Regarding the biological activity, results have shown that poplar bud extract presents a significant inhibitory activity against Gram-positive bacteria and a dose-dependent decrease of MCF-7 tumor cell viability with an IC50 of 66.26 μg/mL, while not affecting healthy cells. Phenomena of early apoptotic events at the maximum concentration tested (150 μg/mL) were detected by Annexin V-PI double staining. The extract induced G0/G1 phase cell cycle arrest. In addition, Pg extract showed antiangiogenic potential on the chorioallantoic membrane. Also, at the highest concentration (150 μg/mL), good tolerability and no signs of toxicity upon vascular plexus were observed. Moreover, in low concentrations, the Pg extract had immunomodulatory activity on primary human dendritic cells by upregulating IL-12 and IL-23 subunits. Conclusion The study concludes that poplar bud extract elicited antioxidant activity, antitumor properties on the breast cancer cell line, followed by an antiangiogenic effect and an immunomodulatory potential on human primary dendritic cells. The biological activity of Populus nigra L. buds extract may open new directions of research on the topic addressed.

Heart transplantation remains the only curative treatment for end-stage heart failure. This life-saving option continues to be limited by the low number of organ donors, graft rejection and adverse effects of immunosuppressants. Engineering bioartificial hearts from acellular native-derived scaffolds and stem cells has gained attention because of its potential to overcome these limitations. In this study, rat hearts (n = 20) were decellularized by means of coronary perfusion with 1% sodium dodecyl sulfate (SDS) in a modified Langendorff device. The electrical field behavior of the SDS molecule was studied and it was assumed that when applying an alternating current, the exposure time of the tissue to the detergent might decrease. To repopulate the decellularized extracellular matrix (ECM), human mesenchymal stem cells (hMSCs) were used, induced to differentiate into cardiomyocytes (CMs) with 5-azacytidine (5-aza). The results showed no cellular debris and an intact ECM following decellularization. Decellularization in the presence of an electric field proved to be faster, decreasing the potential risk of ECM damage due to the detergent. After cell seeding and culturing of eight scaffolds with hMSCs, the recellularization process was analyzed using optic microscopy (OM), which showed cells suggestive for CMs. This study presents a novel and efficient decellularization protocol using an electric field and suggests that hMSCs can be useful in the generation of a bioartificial heart.

Previous experimental models showed that activation of the immune system, particularly T cells, is required for optimal healing following wounds or surgery in the oral cavity. Therefore, studies to explore the interactions between the immune system and the collagen matrix are mandated. The specific aim of the present study was to analyze the interactions between T lymphocytes and a resorbable three-dimensional (3D) collagen matrix routinely used for soft tissue regeneration during periodontal surgery. Peripheral venous blood samples were collected from five patients. Following Ficoll-Paque separation, mononuclear cells were grown on fully resorbable 3D collagen matrices for 5 days. Lymphocytes were analyzed by flow cytometry for different surface markers, including CD4, CD8, CD38 and CD69. Cell viability and late apoptosis/necrosis were assessed in each group using an apoptosis assay based on Annexin V/propidium iodide staining. After 5 days in contact with the collagen matrix, the T cells expressed different surface markers. The overall T cell population increased significantly in the collagen matrix group compared to the respective controls (31.9±6.5 vs. 38.7±3.8%). CD8 and CD69 also increased significantly compared to their controls (CD69: 19.7±3.0 vs. 27.1±4.5% for collagen vs. control groups). At the same time, CD4 and CD38 expression was similar in both groups. Viability and apoptosis/necrosis were also identical in the samples and controls. These results show that the interaction between the collagen matrix and the immune cells stimulated activation of T cells and did not impair the healing process.

Populus nigra L. (Salicaceae family) is one of the most popular trees that can be found in deciduous forests. Some particularities that characterize the Populus genus refer to the fact that it includes more than 40 species, being widespread especially in Europe and Asia. Many residues, parts of this tree can be used as a bioresource for different extracts as active ingredients in pharmaceuticals next to multiple benefits in many areas of medicine. The present review discusses the latest findings regarding the phytochemical composition and the therapeutic properties of Populus nigra L. buds. The vegetal product has been described mainly to contain phenolic compounds (phenols, phenolic acids and phenylpropanoids), terpenoids (mono and sesquiterpenoids), flavones (e.g., apigenol and crysin), flavanones (e.g., pinocembrin and pinostrombin), caffeic/ferulic acids and their derivates, and more than 48 phytocompounds in the essential oils. The resinous exudates present on the buds have been the major plant source used by bees to form propolis. Several studies depicted its antioxidant, anti-inflammatory, antibacterial, antifungal, antidiabetic, antitumor, hepatoprotective, hypouricemic properties and its effects on melanin production. All these lead to the conclusion that black poplar buds are a valuable and important source of bioactive compounds responsible for a wide range of therapeutic uses, being a promising candidate as a complementary and/or alternative source for a large number of health problems. The aim of the review is to gather the existing information and to bring an up to date regarding the phytochemical and therapeutic uses of Populus nigra L. buds.

The ability of cancer to adapt renders it one of the most challenging pathologies of all time. It is the most dreaded pathological entity because of its capacity to metastasize to distant sites in the body, and 90% of all cancer-related deaths recorded to date are attributed to metastasis. Currently, three main theories have been proposed to explain the metastatic pathway of cancer: the epithelial–mesenchymal transition (EMT) and mesenchymal–epithelial transition (MET) hypothesis (1), the cancer stem cell hypothesis (2), and the macrophage–cancer cell fusion hybrid hypothesis (3). We propose a new hypothesis, i.e., under the effect of particular biochemical and/or physical stressors, cancer cells can undergo nuclear expulsion with subsequent macrophage engulfment and fusion, with the formation of cancer fusion cells (CFCs). The existence of CFCs, if confirmed, would represent a novel metastatic pathway and a shift in the extant dogma of cancer; consequently, new treatment targets would be available for this adaptive pathology.

Given the present high incidence of melanoma and skin cancer, interest in potential drugs of plant origin has increased significantly. Pentacyclic lupane-type triterpenes are widely distributed in plants, offering numerous pharmacological benefits. Betulin is an important compound in the bark of Betula pendula Roth and has important therapeutic properties, including antitumor activities. Its biological effect is limited by its poor water solubility, which can be improved by cyclodextrin complexation. The best results have been obtained by using a novel cyclodextrin derivative, octakis-[6-deoxy-6-(2-sulfanyl ethanesulfonate)]-γ-CD. The complexes between betulin and the previously mentioned cyclodextrin were analyzed by scanning electron microscopy (SEM)and differential scanning calorimetry (DSC) and pharmacologically evaluated in vitro (MTT and immunocytochemistry tests) and in vivo in C57BL/6J mice. The solubility of betulin is improved by cyclodextrin complexation, which creates a stable complex that improves the in vitro and in vivo properties of the active compound.

The tumor microenvironment (TME) influences cancer progression. Therefore, engineered TME models are being developed for fundamental research and anti-cancer drug screening. This paper reports the biofabrication of 3D-printed avascular structures that recapitulate several features of the TME. The tumor is represented by a hydrogel droplet uniformly loaded with breast cancer cells (106 cells/mL), it is embedded in the same type of hydrogel containing primary cells—tumor-associated fibroblasts isolated from the peritumoral environment and peripheral blood mononuclear cells. Hoechst staining of cryosectioned tissue constructs demonstrated that cells remodeled the hydrogel and remained viable for weeks. Histological sections revealed heterotypic aggregates of malignant and peritumoral cells, moreover, the constituent cells proliferated in vitro. To investigate the interactions responsible for the experimentally observed cellular rearrangements, we built lattice models of the bioprinted constructs and simulated their evolution using Metropolis Monte Carlo methods. Although unable to replicate the complexity of the TME, the approach presented here enables the self-assembly and co-culture of several cell types of the TME. Further studies will evaluate whether the bioprinted constructs can evolve in vivo in animal models. If they become connected to the host vasculature, they may turn into a fully organized TME.

Mesenchymal stem cells (MSCs) are increasingly being used in regenerative medicine to repair various tissue types. This study aimed to assess changes in MSCs properties as a result of their interaction with two biomaterials in order to evaluate their in vitro compatibility. We have used two types of biomaterials, as follows: hydroxyapatite anodized titanium (HA-Ti) is used in bone reconstruction; it is hard and remains in the body, while patches of collagen combined with poly-e-caprolactone (C-PCL) are utilized in the reconstruction of soft tissue (particularly the skin); these patches resorb in the body, being only a temporary matrix that fosters and accelerates tissue formation. We have chosen to assess changes in morphology, viability and proliferation of MSCs obtained by lipoaspirate, through direct contact with the biomaterial or in eluate. MSCs properties were assessed by optical microscopy, fluorescence microscopy after acridine orange/ethidium bromide (AO/EB) staining, and the MTT assay. After result interpretation, we gained an image of the cytotoxicity phenomenon seen during the experiment and were able to assess viability of cell in contact with the biomaterials used. HA-Ti shows better interaction with cells, which preserve their viability, and does not influence MSCs growth, proliferation or morphology. In contrast, cells on C-PCL matrix become small, spherical, were detached from the plate and had lower viability percentage.

A severe, chronic and irreversible kidney disease affecting discrete rural populations in the Balkan Peninsula countries, Balkan endemic nephropathy (BEN) has been a scientific puzzle for more than half a century. Many environmental and other factors have been suggested as the primary cause and recent significant findings have linked BEN to aristolochic acids, phytotoxins derived from the plant Aristolochia clematitis, found in high density in the endemic areas. However, given that the incidence of BEN is less than 10% in affected villages, and it tends to have a family aggregation, as yet unidentified genetic factors may also play a role. To further explore this possibility, a pilot study was initiated to investigate the DNA methylation of CYP1A1, CYP1A2, NAT1, NQO1 and GSTT1 in blood samples from a group of Romanian BEN patients, compared to healthy controls and non-BEN chronic kidney disease (CKD) subjects. Our study revealed a more pronounced hypomethylation pattern in BEN and non-BEN CKD groups, compared to the healthy control group at specific CpGs across all five genes interrogated. Average methylation across the five regions investigated indicated significant differences only at GSTT1, in both BEN patients (p = 0.028) and non-BEN disease subjects (p = 0.015), relative to healthy individuals. Since GSTT1 active genotype appears to be a common feature of Serbian and Romanian BEN patients, GSTT1 epigenetic variation and increased gene activity could act as a predisposing (co)factor in BEN populations from the affected countries. BEN and non-BEN CKD groups show similar methylation patterns with exception of GSTT1 CpG8 (p = 0.046).

Apigenin (4&prime, 5,7-trihydroxyflavone) (Api) is an important component of the human diet, being distributed in a wide number of fruits, vegetables and herbs with the most important sources being represented by chamomile, celery, celeriac and parsley. This study was designed for a comprehensive evaluation of Api as an antiproliferative, proapoptotic, antiangiogenic and immunomodulatory phytocompound. In the set experimental conditions, Api presents antiproliferative activity against the A375 human melanoma cell line, a G2/M arrest of the cell cycle and cytotoxic events as revealed by the lactate dehydrogenase release. Caspase 3 activity was inversely proportional to the Api tested doses, namely 30 &mu, M and 60 &mu, M. Phenomena of early apoptosis, late apoptosis and necrosis following incubation with Api were detected by Annexin V-PI double staining. The flavone interfered with the mitochondrial respiration by modulating both glycolytic and mitochondrial pathways for ATP production. The metabolic activity of human dendritic cells (DCs) under LPS-activation was clearly attenuated by stimulation with high concentrations of Api. Il-6 and IL-10 secretion was almost completely blocked while TNF alpha secretion was reduced by about 60%. Api elicited antiangiogenic properties in a dose-dependent manner. Both concentrations of Api influenced tumour cell growth and migration, inducing a limited tumour area inside the application ring, associated with a low number of capillaries.

Chamomile, parsley, and celery represent major botanical sources of apigenin, a well-known flavone with chemopreventive properties. The aim of this study was to assess the phytochemical composition, antioxidant, and anti-inflammatory potential of methanol extracts obtained from chamomile, parsley, and celery collected from Romania, as well as the biological activity against A375 human melanoma and human dendritic cells. Results have shown that all three extracts are rich in polyphenolic compounds and flavonoids, and they generate a radical scavenger capacity, iron chelation potential, as well as lipoxygenase inhibition capacity. Chamomile and celery extracts present weak antiproliferative and pro-apoptotic properties in the set experimental conditions, while parsley extract draws out significant pro-apoptotic potential against A375 human melanoma cells. Parsley and chamomile extracts affected the fibroblast-like morphology of the screened tumor cell line. On the other hand, chamomile and celery extracts abrogated the expansion of LPS-activated dendritic cells, while the metabolic activity was attenuated by stimulation with celery extract, chamomile and parsley extracts had no effect upon this parameter. Chamomile and parsley extracts incubation with naive dendritic cells did not trigger cytokine secretion (TNF-alpha, IL-6, IL-10), but celery extract stimulation significantly reduced the anti-inflammatory, cytokine IL-10.

Ursolic and oleanolic acids have been brought into the spotlight of research due to their chemopreventive, anti-inflammatory and immunomodulatory properties. The most important disadvantage of ursolic and oleanolic acids is their weak water solubility which limits their bioavailability. Pentacyclic triterpenes can form inclusion complexes with different types of cyclodextrins which provide the hydrophilic matrix requested for the molecular dispersion of drugs in order to become more water soluble. The aim of the current study is the complexation of ursolic and oleanolic acids with hydrophilic cyclodextrins in order to achieve an improvement of their pharmacological effect. After the virtual screening of the binding affinities between ursolic and oleanolic acids and various cyclodextrins, 2-hydroxypropyl-β-cyclodextrin and 2-hydroxypropil-γ-cyclodextrin were selected as host-molecules for the inclusion complexation. Using the scanning electron microscopy, differential scanning calorimetry and X-ray diffraction the formation of real inclusion complexes between ursolic and oleanolic acids and the two cyclodextrins was confirmed. The anti-proliferative potential of the complexes was tested in vitro on several melanoma cell lines, using the pure compounds as reference. The complexes exhibited higher in vitro anti-proliferative activity as compared to the pure compounds; this improvement was significant for ursolic acid complexes, the highest activity being reported for the 2-hydroxypropil-γ-cyclodextrin complex. Weaker results were recorded for the oleanolic acid complexes where 2-hydroxypropyl-β-cyclodextrin proved to be the most fitted inclusion partner. The entrapment of the two active compounds inside ramified hydrophilic cyclodextrins proved to be a suitable option to increase their anti-proliferative activity.

This study deals with the comparative analysis of thermal degradation (including kinetic study) in dynamic air atmosphere, for two similar-structured triterpenoid acids—ursolic (URS) and oleanolic (OLE) acids. As kinetic methods, two integral methods (Kissinger–Akahira–Sunose and Flynn–Wall–Ozawa) and one differential method (Friedman) were used. The “in-depth” kinetic analysis was completed by applying the nonparametric kinetics (NPK) method, which confirmed the multiprocess degradation suggested by isoconversional methods (the observed variation in E a vs. conversion degree). The NPK method revealed that ursolic acid is degraded solely by two parallel chemical processes, while in the case of oleanolic acid, the processes are accompanied by physical ones. The physicochemical investigation was completed with a bioactivity evaluation regarding the apoptotic process caused by the two compounds in A2058 human melanoma cell line. It was found that the similar-structured triterpenoids possess different biological effects, OLE being antiapoptotic, while URS being strong apoptotic.

Products of natural origin have become important agents in the treatment of cancer, and the active principles of natural sources could be used in combination with chemotherapeutic agents to increase their effects and to minimize their toxicity. Oleanolic (OA) and ursolic (UA) acids are intensely studied for their promising anticancer potential. The aim of this study was focused on the in vitro toxicological effects induced by UA and OA human mesenchymal stem cells and on melanoma, one of the most frequent cancers whose incidence is increasing every year. The two compounds were tested for their cytotoxic, cell cycle arrest and pro-apoptotic effects on melanoma cells (A375 and B164A5) and mesenchymal stem cells. UA exerted a cytotoxic effect in a dose-dependent manner on melanoma cells, while OA's activity has been shown to be low or moderate. Both compounds produced alterations of the cell cycle, arresting cells in the G0/G1 phase. Furthermore, UA induced significant apoptosis through the bcl-2 genes family pathway, with the decrease of the bcl-2 gene expression. The two compounds exerted selective effects on melanoma cells with no effects on human mesenchymal stem cells. The presented results reveal the anticancer potential of UA on melanoma cells, with no detectable toxicity on the mesenchymal stem cells.

There are few major morphologies of cell death that have been described so far: apoptosis (type I), cell death associated with autophagy (type II), necrosis (type III) and anchorage-dependent mechanisms-anoikis. Here, we show for the first time a possibly novel mechanism inducing tumour cell death under in vitro conditions-enucleation. We pursued the influence of colloidal suspensions of Fe3 O4 nanoparticles on tumour cell lines (SK-BR-3 and MCF-7 breast cancer cell lines) grown according to standard cell culture protocols. Magnetite nanoparticles were prepared by combustion synthesis and double layer coated with oleic acid. Scanning and transmission electron microscopy revealed that tumour cells developed a network of intracytoplasmic stress fibres, which induce extrusion of nuclei, and enucleated cells die. Normal adult mesenchymal stem cells, used as control, did not exhibit the same behaviour. Intact nuclei were found in culture supernatant of tumour cells, and were visualized by immunofluorescence. Enucleation as a potential mechanism of tumour cell death might open new horizons in cancer biology research and development of therapeutic agents capable of exploiting this behaviour.

Introduction The purpose of the ongoing research is to improve our current skills and knowledge in stem cell isolation, cultivation and differentiation from the vestibular epithelia of young mice. Matherials and Methods We harvested utricles and sacculi from 7 days old mice. Obtained cells were cultivated at 37ºC and 5% CO2 in DMEM with F12 Nutrient mixture, B27, N2 supplement, IGF-1 and EGF. Sphere pluripotency was established with Nanog and Oct-4 stem cell markers. We mechanically dissociated primary spheres and cultivated them. Cells were characterized by immunofluorescence and immunohistochemistry for myosinVIIA (hair cell marker), nestin (intermediate filament VI marker) and beta III tubulin. Results We proved that vestibular epithelia contains pluripotent stem cells which formed spheres. Sphere-derived cells’ pluripotency was demonstrated by the expression of nanog, oct 4 and nestin markers. We obtained through differentiation different cell types including neuronal cells which were positive for myosin VIIA, nestin, beta III tubulin. We wanted to know the type of neurons and performed Brn3a staining for immature neurons, and TrKB staining for mature neurons. We cultivated vestibular stem cells on different substrates to observe their behavior. Conclusions Cells obtained from utricular epithelia are pluripotent because they express nanog, oct 4, nestin, characteristic for cell progenitors and they generate spheres.

In recent years, identification of new sources of adult stem cells developed rapidly, pursuing to find easily available tissues, which will give rise to homogenous stem cells populations. Up to present, bone marrow-derived mesenchymal stem cells (BM-MSCs) are unanimously considered to fulfill the criteria for being used in clinical settings, but adipose stem cells, placental and umbilical cord stem cells, and other tissue-derived stem cells are making their way to being used at least in autologous transplantation. We isolated cellular populations from placental tissue and umbilical cord using the explants method. The placental (PL) and umbilical cord (UC)-derived cells were cultured and expanded in appropriate conditions for generation of stem cells. We assessed the stemness characteristics of the tissue-isolated cells and compared them to an established MSCs line. For this purpose, we determined the immunophenotype, morphological and ultrastructural characteristics, as well as functional abilities of PL- and UC-derived cells. Flow cytometric evaluation of cells revealed presence of CD90, CD73, and CD105 stem cells markers, while the cells were negative for CD34, CD45 and HLA-DR. Immunocytochemical staining showed that 100% of PL- and UC-derived cells are positive for vimentin and CD105 expression, while cytokeratin was revealed in less than 10% in both tissue-isolated cells. Morphological and ultrastructural characteristics of cells exposed analogous cellular size and intracellular organization, similar to MSCs, but detailed view of UC-derived cells by transmission electron microscopy (TEM) demonstrated presence of intercellular junctions-desmosomes, similar to epithelial cells. Both PL- and UC-derived cells confirmed their trilineage potential, being able to differentiate into adipocytes, osteoblasts, and chondrocytes in different proportions. Flow chamber in vitro assay was used to determine to what extent PL- and UC-derived cells are able to adhere to substrates (VCAM and ICAM) and we showed progressively decreased adhesion of both cellular types, inversely proportional to the generated shear stress. We may conclude that explants-isolated placental and umbilical cord cells are endowed with characteristics of both epithelial and mesenchymal stem cells, and purification procedures are additionally required for safe use of these cells in diverse clinical applications.

Background The aim of the present study is to analyze the behavior of selected populations of oral keratinocytes and T-lymphocytes, responsible for re-constructing and maintaining the oral epithelial tissue architecture, following augmentation of the keratinized oral mucosa using a 3D-collagen matrix. Methods Different groups of oral keratinocytes were isolated from biopsies harvested from 3 patients before the surgical procedure, as well as 7 and 14 days after the augmentation procedure. T-lymphocytes were isolated from peripheral blood at same timepoints. Keratinocytes were characterized for stem and differentiation markers, such as p63, cytokeratin 10 and 14, and in vitro parameters, such as cell viability, cell size and colony-forming efficiency. T-lymphocytes were analyzed for viability and the expression of various cluster of differentiation markers. The methods included magnetic separation of cell populations, immunofluorescence, flow cytometry, and histology of oral biopsies. Results Both at 7 and 14 days, the majority of cells that repopulate the matrix were actively proliferating/progenitor oral keratinocytes with the phenotype integrin alfa6beta4 + CD71+. These cells display in vitro characteristics similar to the progenitor cells analyzed before the matrix placement. T-lymphocytes expressed CD8 and CD69 markers, while CD25 was absent. Conclusion The study shows that two weeks after the collagen membrane placement, the healing process appeared to be histologically complete, with no abnormal immune response induced by the matrix, however, with a higher than usual content of active proliferating cells, the majority of keratinocytes being characterized as transit amplifying cells.

Oleanolic and ursolic acids are antitumor and antibacterial agents which are extensively studied. Their major disadvantage is the poor water solubility which limits their applications.Oleanolic and ursolic acid were encapsulated into polyurethane nanostructures that act as drug carriers. In order to evaluate the effectiveness of the particles, anti-microbial and anti-proliferative activity compared to un-encapsulated active compounds was tested.Using an interfacial polycondensation technique, combined with spontaneous emulsification, structures with nanoscale dimensions were obtained. Scanning electron microscopy, differential scanning calorimetry and X-ray assays confirmed the encapsulation process. Concentrations of 10 and 30 μM particles and un-encapsulated compounds were tested by MTT viability assay for several breast cancer lines, with an exposure time of 72 h. For the antibacterial studies, the dilution method with MIC determination was used.Ursolic acid had an excellent inhibitory effect with ICAlthough the active compounds did not fully exert their anti-proliferative activity following encapsulation inside polymeric nanoparticles, in vivo evaluation is needed in order to obtain an exhaustive conclusion, as the active compounds could be released as a result of metabolic activity.

Oleanolic and ursolic acids (OA and UA) are two pentacyclic triterpenes, ubiquitously spread in plants, previously known for their chemopreventive capacity on different types of cancer. The major pharmacological disadvantage of these phytocompounds is their poor water solubility, which often limits their applicability.Using the interfacial polycondensation combined with spontaneous emulsification technique, polyurethane nanostructures (PU) were synthetized in order to improve this problem. In order to test the in vivo chemopreventive potential of the two pure compounds, as well as the encapsulated compounds in PU used as drug carriers, a chemically-induced skin carcinogenesis model was constructed.UA and OA have a moderate chemopreventive activity against tumors induced by 7,12-dimethylbenzantracene (DMBA) and 12-O-tetradecanoilphorbol-13-acetate (TPA) application. Incorporation of active agents in PU did not lead to increased chemopreventive effect.PU is not a suitable formulation of UA and OA.

Background: Fisetin,quercetin and kaempferol are among the important representatives of flavonols, biological active phytocomounds, with low water solubility. Objective: To evaluate the antimicrobial effect, respectively the antiproliferative and pro apoptotic activity on the B164A5 murine melanoma cell line of pure flavonols and their beta cyclodextrins complexes. Method: Incorporation of fisetin, quercetin and kaempferol in beta cyclodextrins was proved by scanning electron microscopy (SEM), differencial scanning calorimetry (DSC) and X-ray powder diffraction (XRPD). Pure compounds and their complexes were tested for antiproliferative (MTT) and pro-apoptotic activity (Annexin V-PI) on the B164A5 murine melanoma cell line and for the antimicrobial properties (Disk Diffusion Method) on the selected strains. Results: The phytocompounds presented in a different manner in vitro chemopreventive activity against B164A5 murine melanoma cell line and weak antimicrobial effect. Conclusion: The three flavonols: fisetin, quercetin and kaempferol were successfully incorporated in beta-cyclodextrin (BCD) and hydroxylpropyl-beta-cyclodextrin (HPBCD). Incorporation in beta cyclodextrins had a mix effect on the biological activity conducing to decrease, increase or consistent effect compared to pure phytocompound, depending on the screened process and on the chosen combination.

Adult bone marrow mesenchymal stem cells (BMSCs) can be differentiated in vitro to become adipocyte-like cells with lipid vacuoles, similar to adipocytes derived from adult adipose tissue. Little is known regarding the composition of free fatty acids (FFAs) of the in vitro-differentiated adipocytes, or whether it resembles that of native adult adipocytes. We used gas chromatography-mass spectrometry to identify FFA species in BMSC-derived adipocytes and compared them with FFAs found in adipocytes derived from adult adipose tissue. We found that adult adipocytes contained significant percentages of saturated and monounsaturated FFAs, including palmitic acid (C16:0), stearic acid (C18:0), and oleic acid (C18:1); some polyunsaturated FFAs, such as linoleic acid (C18:2), a small percentage of arachidonic acid (C20:4), and very little linolenic acid (C18:3). In comparison, 80%–90% confluent BMSCs contained comparable percentages of palmitic and oleic acids, significantly more arachidonic and stearic acids, very little linoleic acid, and no linolenic acid. After differentiation, compared with adult adipocytes, BMSC-derived adipocytes contained a comparable percentage of palmitic acid, more stearic and arachidonic acids, less oleic acid, almost no linoleic acid, and no detectable linolenic acid. This composition was quite similar to that of undifferentiated BMSCs. The differentiation medium contained only palmitic and stearic acids, with traces of oleic acid; it did not contain the essential polyunsaturated fatty acids. Thus, the composition of FFAs in BMSC-derived adipocytes was altered compared with adult adipocytes. BMSC-derived adipocytes had an altered composition of saturated and monounsaturated FFAs and lacked essential FFAs that may directly affect signaling related to their lipolysis/lipogenesis functions.

Wound healing involves complex interactions between the motile epithelial cells and the extracellular matrix in response to activation by a high number of growth factors and cytokines involved both...

Human skeletal muscle tissue displays specific cellular architecture easily damaged during individual existence, requiring multiple resources for regeneration. Congruent with local prerequisites, heterogeneous muscle stem cells (MuSCs) are present in the muscle interstitium. In this study, we aimed to characterize the properties of human muscle interstitial cells that had the characteristic morphology of telocytes (TCs). Immunocytochemistry and immunofluorescence showed that cells with TC morphology stained positive for c-kit/CD117 and VEGF. C-kit positive TCs were separated with magnetic-activated cell sorting, cultured in vitro and expanded for study. These cells exhibited high proliferation capacity (60% expressed endoglin/CD105 and 80% expressed nuclear Ki67). They also exhibited pluripotent capacity limited to Oct4 nuclear staining. In addition, 90% of c-kit positive TCs expressed VEGF. C-kit negative cells in the MuSCs population exhibited fibroblast-like morphology, low trilineage differential potential and negative VEGF staining. These results suggested that c-kit/CD117 positive TCs represented a unique cell type within the MuSC niche.

Few are known about the molecular mechanism controlling the early embryo development. The reduce dimension of the embryos, only a few μm, the small quantities of proteins synthesized and the artificial environment influence makes difficult to decode the mechanisms controlling early embryonic stages of development. Although, in the last few years many genes have been showed to be active in the early embryonic stages of development, only a few have been characterized and found to be implicated in the molecular mechanism responsible of preimplantational embryos development. Ped gene (Preimplantational embryo development) is considered to be involved in regulation of embryonic cleavage division and subsequent embryo survival. This review presents, based on a rich documentation, the main mechanisms involved in early embryo development.

Tumour-associated fibroblasts (TAFs) are part of the tumour stroma, providing functional and structural support for tumour progression and development. The origin and biology of TAFs are poorly understood, but within the tumour environment, TAFs become activated and secrete different paracrine and autocrine factors involved in tumorigenesis. It has been shown that bone marrow mesenchymal stem cells (MSCs) can be recruited into the tumours, where they proliferate and acquire a TAF-like phenotype. We attempted to determine to what extent TAFs characteristics in vitro juxtapose to MSCs’ definition, and we showed that TAFs and MSCs share immunophenotypic similarities, including the presence of certain cell surface molecules [human leukocyte antigen-DR subregion (HLA-DR), CD29, CD44, CD73, CD90, CD106 and CD117]; the expression of cytoskeleton and extracellular matrix proteins, such as vimentin, α-smooth muscle actin, nestin and trilineage differentiation potential (to adipocytes, chondrocytes and osteoblasts). When compared to MSCs, production of cytokines, chemokines and growth factors showed a significant increase in TAFs for vascular endothelial growth factor, transforming growth factor-β1, interleukins (IL-4, IL-10) and tumour necrosis factor α. Proliferation rate was highly increased in TAFs and fibroblast cell lines used in our study, compared to MSCs, whereas ultrastructural details differentiated the two cell types by the presence of cytoplasmic elongations, lamellar content lysosomes and intermediate filaments. Our results provide supportive evidence to the fact that TAFs derive from MSCs and could be a subset of ‘specialized’ MSCs.

The purpose of the ongoing research is to improve our current skills and knowledge in stem cell isolation, cultivation and differentiation from the utricular and saccular epithelia of young mice. We harvested utricles and sacculi from 7 days old NMRI mice. Utricles were trypsinized in order to isolate single cells. Obtained cells were cultivated in DMEM with F12 Nutrient mixture, B27, N2 supplement, IGF-1 and EGF. Sphere pluripotency was established with stem cell markers Nanog and Oct-4. We mechanically dissociated primary spheres and cultivated. Secondary spheres were placed on fibronectin coated tissue culture slide chambers in the absence of IGF-1 and EGF. Cells were characterized by immunofluorescence and immunohistochemistry for myosinVIIA (hair cell marker) and nestin (intermediate filament VI marker). Vestibular epithelia contain pluripotent stem cells able to form cell clusters (spheres). Pluripotency was demonstrated by the expression of nanog, oct 4 and nestin markers (cell progenitors). Also sphere dissociation and separate cultivation of the isolated cells lead to the formation of larger spheres and in higher amounts compared to the original spheres, proving that these cells are not only pluripotent but also capable of self-renewal. Single spheres harvested and cultivated on fibronectin produced through differentiation different cell types including neuron like-cells, positive for myosin VIIA and nestin.

Background In the past years, ultraviolet-B (UVB)-induced morphological changes in cultured fibroblasts have been systematically studied in relation with photoprotective strategies. The present study was aimed at providing a comprehensive characterization of a human dermal fibroblast cell line, HDFa, in terms of: (i) morphological and phenotypical aspects, and (ii) mitochondrial function and bioenergetics. Methods Morphology of HDFa line was assessed by light microscopy and flow cytometry was used for the evaluation of the the following surface markers: CD34, CD45, CD90, CD73, CD44, CD29, CD117, CD105, CD109, CD26, IL-10R, HLA-DR and TGF-RIII. Cell monolayers were fixed and stained for CD105, CD117, Ki67, Vimentin, -smooth muscle actin, Caldesmon, Cytokeratin, and smooth muscle myosin by immunofluorescence. Mitochondrial bioenergetics was measured by using Seahorse Bioscience XF24 extracellular flux analyzer. Different number of cells were seeded in order to obtain the optimal seeding density needed to identify a measurable OCR (oxygen consumption rate) and ECAR (extracellular acidification rate). The following protocol for mitochondrial function assay was tested at 37C: Basal respiration followed by sequential injections of Oligomycin, FCCP, and Antimycin A (BOFA). Parameters of mitochondrial respiratory function (basal OCR, maximal respiratory capacity, ATP production, and proton leak) together with those of glycolysis (glycolytic flux, glycolytic capacity, and glycolytic reserve), respectively were measured. After each BOFA assay, cells were lysed to quantify total protein by Bradford assay. Results HDFa cells showed elongated flattened form, were adherent and rapidly expanded in culture. HDFa cells were positive for CD90, CD73, CD26, and CD29 at flowcytometry, whereas imunofluorescence microscopy revealed that the cell line was positive for CD105, Ki67, and Vimentin. The optimal mitochondrial and glycolytic flux parameters were reported at 20.000 cells/well, resulting that this seeding density will be used in our further experiments. Conclusions Characterization of HDFa cell line was performed in order to further report on their changes in response to different protocols of UVB exposure in the presence vs. the absence of protective phytochemicals. Research supported by the doctoral fellowship POSDRU/159 /1.5/S/ 136893.

In recent years, the design and synthesis of colloidal magnetic suspensions have attracted an increased interest especially in the fields of biotechnology and biomedicine because they have many applications including targeted drug delivery, cell labeling and magnetic cell separation, hyperthermia, tissue repairing, magnetic resonance imaging (MRI) contrast enhancement, enzyme immobilization, immunoassays, protein purification, etc.

Betulinic acid, a very promising anti-melanoma agent, has very low water solubility that causes low bioavailability. To overcome this inconvenience, a highly water-soluble cyclodextrin was used (octakis-[6-deoxy-6-(2-sulfanyl ethanesulfonic acid)]-γ-cyclodextrin). The complex was physico-chemically analyzed using differential scanning calorimetry (DSC), X-ray and scanning electron microscopy (SEM) methods and then in vitro tested for its antiproliferative activity by the MTT assay and by cell cycle analysis. Finally, the complex was tested in vivo using an animal model of murine melanoma developed in C57BL/6J mice, where it caused a reduction in tumor volume and weight. The study revealed the beneficial influence of betulinic acid inclusion into the cyclodextrin in terms of antiproliferative activity and in vivo tumor development.

BackgroundOne of the most popular and versatile model of murine melanoma is by inoculating B16 cells in the syngeneic C57BL6J mouse strain. A characterization of different B16 modified cell sub-lines will be of real practical interest. For this aim, modern analytical tools like surface enhanced Raman spectroscopy/scattering (SERS) and MTT were employed to characterize both chemical composition and proliferation behavior of the selected cells.MethodsHigh quality SERS signal was recorded from each of the four types of B16 cell sub-lines: B164A5, B16GMCSF, B16FLT3, B16F10, in order to observe the differences between a parent cell line (B164A5) and other derived B16 cell sub-lines. Cells were incubated with silver nanoparticles of 50–100 nm diameter and the nanoparticles uptake inside the cells cytoplasm was proved by transmission electron microscopy (TEM) investigations. In order to characterize proliferation, growth curves of the four B16 cell lines, using different cell numbers and FCS concentration were obtained employing the MTT proliferation assay. For correlations doubling time were calculated.ResultsSERS bands allowed the identification inside the cells of the main bio-molecular components such as: proteins, nucleic acids, and lipids. An "on and off" SERS effect was constantly present, which may be explained in terms of the employed laser power, as well as the possible different orientations of the adsorbed species in the cells in respect to the Ag nanoparticles. MTT results showed that among the four tested cell sub-lines B16 F10 is the most proliferative and B164A5 has the lower growth capacity. Regarding B16FLT3 cells and B16GMCSF cells, they present proliferation ability in between with slight slower potency for B16GMCSF cells.ConclusionMolecular fingerprint and proliferation behavior of four B16 melanoma cell sub-lines were elucidated by associating SERS investigations with MTT proliferation assay.

Aristolochia clematitis, member of the Aristolochiaceae family is a medicinal plant used from immemorial time in folkloric medicine to succesfully treats various ailments, from snake bites to cancer. Recently Aristolochia plant use as a natural or pharmacologic remedy was banned by the Food and Drug Administration (FDA), because it contains aristolochic acids, carcinogenic and nephrotoxic compounds. The purpose of this study is to assess the effects of different concentrations of Aristolochia clematitis aqueous extract on human breast adenocarcinoma cell lines (MDA-MB-231 and MDA-MB-468), tumor-associated fibroblasts (TAFs), and mesenchymal stem cells (MSCs). Viability and proliferation MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, and a flow chamber adhesion assay were used in order to test the adhesion capacity of TAFs and MSCs treated with A. clematitis extracts on MDA-MB-231 or MDA-MB-468 cellular coatings. Our studies demonstrated a significant inhibitory effect of A. clematitis extracts on the viability and function of normal cells (MSCs) compared to tumor cells