Prenatal Factors in the Development of Allergic Diseases.

Allergic diseases are showing increasing prevalence in Western societies. They are characterized by a heightened reactivity towards otherwise harmless environmental stimuli. Allergic diseases showing a wide range of severity of symptoms have a significant impact on the quality of life of affected individuals. This study aims to highlight the mechanisms that induce these reactions, how they progress, and which prenatal factors influence their development. Most frequently, the reaction is mediated by immunoglobulin E (IgE) produced by B cells, which binds to the surface of mast cells and basophils and triggers an inflammatory response. The antibody response is triggered by a shift in T-cell immune response. The symptoms often start in early childhood with eczema or atopic dermatitis and progress to allergic asthma in adolescence. An important determinant of allergic diseases seems to be parental, especially maternal history of allergy. Around 30% of children of allergic mothers develop allergic sensitization in childhood. Genes involved in the regulation of the epithelial barrier function and the T-cell response were found to affect the predisposition to developing allergic disorders. Cord blood IgE was found to be a promising predictor of allergic disease development. Fetal B cells produce IgE starting at the 20th gestation week. These fetal B cells could be sensitized together with mast cells by maternal IgE and IgE-allergen complexes crossing the placental barrier via the low-affinity IgE receptor. Various factors were found to facilitate these sensitizations, including pesticides, drugs, exposure to cigarette smoke and maternal uncontrolled asthma. Prenatal exposure to microbial infections and maternal IgG appeared to play a role in the regulation of T-cell response, indicating a protective effect against allergy development. Additional preventive factors were dietary intake of vitamin D and omega 3 fatty acids as well as decreased maternal IgE levels. The effect of exposure to food allergens during pregnancy was inconclusive, with studies having found both sensitizing and protective effects. In conclusion, prenatal factors including genetics, epigenetics and fetal environmental factors have an important role in the development of allergic disorders in later life. Children with a genetic predisposition are at risk when exposed to cigarette smoke as well as increased maternal IgE in the prenatal period. Maternal diet during pregnancy and immunization against certain allergens could help in the prevention of allergy in predisposed children.

Insect Cell-Expressed Major Ragweed Allergen Amb a 1.01 Exhibits Similar Allergenic Properties to Its Natural Counterpart from Common Ragweed Pollen.

Common ragweed pollen allergy has become a health burden worldwide. One of the major allergens in ragweed allergy is Amb a 1, which is responsible for over 90% of the IgE response in ragweed-allergic patients. The major allergen isoform Amb a 1.01 is the most allergenic isoform in ragweed pollen. So far, no recombinant Amb a 1.01 with similar allergenic properties to its natural counterpart (nAmb a 1.01) has been produced. Hence, this study aimed to produce a recombinant Amb a 1.01 with similar properties to the natural isoform for improved ragweed allergy management. Amb a 1.01 was expressed in insect cells using a codon-optimized DNA construct with a removable N-terminal His-Tag (rAmb a 1.01). The recombinant protein was purified by affinity chromatography and physicochemically characterized. The rAmb a 1.01 was compared to nAmb a 1.01 in terms of the IgE binding (enzyme-linked immunosorbent assay (ELISA), immunoblot) and allergenic activity (mediator release assay) in well-characterized ragweed-allergic patients. The rAmb a 1.01 exhibited similar IgE reactivity to nAmb a 1.01 in different IgE-binding assays (i.e., IgE immunoblot, ELISA, quantitative ImmunoCAP inhibition measurements). Furthermore, the rAmb a 1.01 showed comparable dose-dependent allergenic activity to nAmb a 1.01 regarding basophil activation. Overall, the results showed the successful expression of an rAmb a 1.01 with comparable characteristics to the corresponding natural isoform. Our findings provide the basis for an improvement in ragweed allergy research, diagnosis, and immunotherapy.

Non-Specific Lipid Transfer Protein Amb a 6 Is a Source-Specific Important Allergenic Molecule in Ragweed Pollen.

Pollen from common ragweed is an important allergen source worldwide and especially in western and southern Romania. More than 100 million patients suffer from symptoms of respiratory allergy (e.g., rhinitis, asthma) to ragweed pollen. Among the eleven characterized allergens, Amb a 6 is a non-specific lipid transfer protein (nsLTP). nsLTPs are structurally stable proteins in pollen and food from different unrelated plants capable of inducing severe reactions. The goal of this study was to produce Amb a 6 as a recombinant and structurally folded protein (rAmb a 6) and to characterize its physicochemical and immunological features. rAmb a 6 was expressed in Spodoptera frugiperda Sf9 cells as a secreted protein and characterized by mass spectrometry and circular dichroism (CD) spectroscopy regarding molecular mass and fold, respectively. The IgE-binding frequency towards the purified protein was evaluated using sera from 150 clinically well-characterized ragweed-allergic patients. The allergenic activities of rAmb a 6 and the nsLTP from the weed Parietaria judaica (Par j 2) were evaluated in basophil activation assays. rAmb a 6-specific IgE reactivity was associated with clinical features. Pure rAmb a 6 was obtained by insect cell expression. Its deduced molecular weight corresponded to that determined by mass spectrometry (i.e., 10,963 Da). rAmb a 6 formed oligomers as determined by SDS-PAGE under non-reducing conditions. According to multiple sequence comparisons, Amb a 6 was a distinct nsLTP with less than 40% sequence identity to currently known plant nsLTP allergens, except for nsLTP from Helianthus (i.e., 52%). rAmb a 6 is an important ragweed allergen recognized by 30% of ragweed pollen allergic patients. For certain patients, rAmb a 6-specific IgE levels were higher than those specific for the major ragweed allergen Amb a 1 and analysis also showed a higher allergenic activity in the basophil activation test. rAmb a 6-positive patients suffered mainly from respiratory symptoms. The assumption that Amb a 6 is a source-specific ragweed allergen is supported by the finding that none of the patients showing rAmb a 6-induced basophil activation reacted with Par j 2 and only one rAmb a 6-sensitized patient had a history of plant food allergy. Immunization of rabbits with rAmb a 6 induced IgG antibodies which strongly inhibited IgE binding to rAmb a 6. Our results demonstrate that Amb a 6 is an important source-specific ragweed pollen allergen that should be considered for diagnosis and allergen-specific immunotherapy of ragweed pollen allergy.

Stem Cells Derived from Human Exfoliated Deciduous Teeth Functional Assessment: Exploring the Changes of Free Fatty Acids Composition during Cultivation.

The metabolic regulation of stemness is widely recognized as a crucial factor in determining the fate of stem cells. When transferred to a stimulating and nutrient-rich environment, mesenchymal stem cells (MSCs) undergo rapid proliferation, accompanied by a change in protein expression and a significant reconfiguration of central energy metabolism. This metabolic shift, from quiescence to metabolically active cells, can lead to an increase in the proportion of senescent cells and limit their regenerative potential. In this study, MSCs from human exfoliated deciduous teeth (SHEDs) were isolated and expanded in vitro for up to 10 passages. Immunophenotypic analysis, growth kinetics, in vitro plasticity, fatty acid content, and autophagic capacity were assessed throughout cultivation to evaluate the functional characteristics of SHEDs. Our findings revealed that SHEDs exhibit distinctive patterns of cell surface marker expression, possess high self-renewal capacity, and have a unique potential for neurogenic differentiation. Aged SHEDs exhibited lower proliferation rates, reduced potential for chondrogenic and osteogenic differentiation, an increasing capacity for adipogenic differentiation, and decreased autophagic potential. Prolonged cultivation of SHEDs resulted in changes in fatty acid composition, signaling a transition from anti-inflammatory to proinflammatory pathways. This underscores the intricate connection between metabolic regulation, stemness, and aging, crucial for optimizing therapeutic applications.

Relationship between IgE Levels Specific for Ragweed Pollen Extract, Amb a 1 and Cross-Reactive Allergen Molecules.

Ragweed (Ambrosia artemisiifolia) pollen is a major endemic allergen source responsible for severe allergic manifestations in IgE-sensitized allergic patients. It contains the major allergen Amb a 1 and cross-reactive allergen molecules, such as the cytoskeletal protein profilin, Amb a 8 and calcium-binding allergens Amb a 9 and Amb a 10. To assess the importance of Amb a 1, profilin and calcium-binding allergen, the IgE reactivity profiles of clinically well-characterized 150 ragweed pollen-allergic patients were analysed regarding specific IgE levels for Amb a 1 and cross-reactive allergen molecules by quantitative ImmunoCAP measurements, IgE ELISA and by basophil activation experiments. By quantifying allergen-specific IgE levels we found that Amb a 1-specific IgE levels accounted for more than 50% of ragweed pollen-specific IgE in the majority of ragweed pollen-allergic patients. However, approximately 20% of patients were sensitized to profilin and the calcium-binding allergens, Amb a 9 and Amb a 10, respectively. As shown by IgE inhibition experiments, Amb a 8 showed extensive cross-reactivity with profilins from birch (Bet v 2), timothy grass (Phl p 12) and mugwort pollen (Art v 4) and was identified as a highly allergenic molecule by basophil activation testing. Our study indicates that molecular diagnosis performed by the quantification of specific IgE to Amb a 1, Amb a 8, Amb a 9 and Amb a 10 is useful to diagnose genuine sensitization to ragweed pollen and to identify patients who are sensitized to highly cross-reactive allergen molecules present in pollen from unrelated plants, in order to enable precision medicine-based approaches for the treatment and prevention of pollen allergy in areas with complex pollen sensitization.

Decellularized Extracellular Matrix Scaffolds for Cardiovascular Tissue Engineering: Current Techniques and Challenges.

Cardiovascular diseases are the leading cause of global mortality. Over the past two decades, researchers have tried to provide novel solutions for end-stage heart failure to address cardiac transplantation hurdles such as donor organ shortage, chronic rejection, and life-long immunosuppression. Cardiac decellularized extracellular matrix (dECM) has been widely explored as a promising approach in tissue-regenerative medicine because of its remarkable similarity to the original tissue. Optimized decellularization protocols combining physical, chemical, and enzymatic agents have been developed to obtain the perfect balance between cell removal, ECM composition, and function maintenance. However, proper assessment of decellularized tissue composition is still needed before clinical translation. Recellularizing the acellular scaffold with organ-specific cells and evaluating the extent of cardiomyocyte repopulation is also challenging. This review aims to discuss the existing literature on decellularized cardiac scaffolds, especially on the advantages and methods of preparation, pointing out areas for improvement. Finally, an overview of the state of research regarding the application of cardiac dECM and future challenges in bioengineering a human heart suitable for transplantation is provided.

Aristolochic acid I: an investigation into the role of food crops contamination, as a potential natural exposure pathway.

Aristolochic acid I (AAI) is a potent nephrotoxic and carcinogenic compound produced by plants of the Aristolochiaceae family and thoroughly investigated as a main culprit in the etiology of Balkan endemic nephropathy (BEN). So far, the AAI exposure was demonstrated to occur through the consumption of Aristolochia clematitis plants as traditional remedies, and through the contamination of the surrounding environment in endemic areas: soil, food and water contamination. Our study investigated for the first time the level of AAI contamination in 141 soil and vegetable samples from two cultivated gardens in non-endemic areas, A. clematitis being present in only one of the gardens. We developed and validated a simple and sensitive ultra-high-performance liquid chromatography-ion trap mass spectrometry method for qualitative and quantitative AAI analysis. The results confirmed the presence of AAI at nanogram levels in soil and vegetable samples collected from the non-endemic garden, where A. clematitis grows. These findings provide additional evidence that the presence of A. clematitis can cause food crops and soil contamination and unveil the pathway through which AAI could move from A. clematitis to other plant species via a common matrix: the soil. Another issue regarding the presence of AAI, in a non-endemic BEN area from Romania, could underlie a more widespread environmental exposure to AAI and explain certain BEN-like cases in areas where BEN has not been initially described.

A Theoretical Study of the Molecular Coupled Structures of Aristolochic Acids and Humic Acid, Potential Environmental Contaminants.

An understanding of the fate of organic compounds originating from plants in soil is crucial for determining their persistence and concentrations in the environment. Aristolochic acids are believed to be the causal agents that induce Balkan endemic nephropathy by food contamination through soil adsorption of humic acids, major components of soil. Aristolochic acids are active chemicals in Aristolochia plant species found in endemic villages. In this article, molecular structure interactions between 18 structures of aristolochic acids with an inserted humic acid structure were studied. These structures were optimized in vacuo and by periodic box simulation with water solvate using the computational molecular mechanics MM+ method with HyperChem software. The QSPR models were used for correlation of the relationship between the hydrophobicity values of 18 AA structures coupled with a HA structure by MM+ and QSAR+ properties. Computational hydrophobicity values were considered dependent variables and were related to the structural features obtained by molecular and quantum mechanics calculations by multiple linear regression approaches. The obtained model was validated, and the results indicated differing hydrophobicity between the MM+ and QSAR+ properties.

Cannabidiol-from Plant to Human Body: A Promising Bioactive Molecule with Multi-Target Effects in Cancer.

Cannabis sativa L. is a plant long used for its textile fibers, seed oil, and oleoresin with medicinal and psychoactive properties. It is the main source of phytocannabinoids, with over 100 compounds detected so far. In recent years, a lot of attention has been given to the main phytochemicals present in Cannabis sativa L., namely, cannabidiol (CBD) and Δ9-tetrahydrocannabinol (THC). Compared to THC, CBD has non-psychoactive effects, an advantage for clinical applications of anti-tumor benefits. The review is designed to provide an update regarding the multi-target effects of CBD in different types of cancer. The main focus is on the latest in vitro and in vivo studies that present data regarding the anti-proliferative, pro-apoptotic, cytotoxic, anti-invasive, anti-antiangiogenic, anti-inflammatory, and immunomodulatory properties of CBD together with their mechanisms of action. The latest clinical evidence of the anticancer effects of CBD is also outlined. Moreover, the main aspects of the pharmacological and toxicological profiles are given.

The antiviral activity and cytotoxicity of 15 natural phenolic compounds with previously demonstrated antifungal activity.

The present study attempted to evaluate the carcinogenicity of natural phenolic compounds with previously demonstrated antifungal activity, using a computational structure-cytotoxicity approach, namely the quantum structure cytotoxicity relationship model. The cytotoxicity of 15 phenolic compounds with antiviral activity 96 h after treatment was studied using the AdmetSAR computational program. Per the EPA classification, four of the investigated compounds would be included in the second cytotoxicity category, four in the third category, and six showed no toxicity, rendering the studied natural phenolic compounds much less toxic to aquatic life than synthetic pesticides, the organophosphorus compounds, which mostly fall into the first and second categories of toxicity.

Botanical Therapeutics: Phytochemical Screening and Biological Assessment of Chamomile, Parsley and Celery Extracts against A375 Human Melanoma and Dendritic Cells.

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.

Balkan endemic nephropathy and aristolochic acid I: an investigation into the role of soil and soil organic matter contamination, as a potential natural exposure pathway.

Aristolochic acids (AAs) are carcinogenic and nephrotoxic plant alkaloids present in Aristolochia species, used in traditional medicine. Recent biomolecular and environmental studies have incriminated these toxins as an etiological agent in Balkan endemic nephropathy (BEN), a severe kidney disease occurring in the Balkan Peninsula. The questions on how the susceptible populations are exposed to these toxins have not yet been clearly answered. Exposure to AAs through the food chain, and environmental pollution (soil/dust), could provide an explanation for the presence of BEN in the countries where no folkloric use of the plant has been documented (Bulgaria, Croatia). Additional exposure pathways are likely to occur, and we have shown previously that AAs can contaminate crop plants through absorption from soil, under controlled laboratory environment. Here, we attempt to provide additional support to this potential exposure pathway, by revealing the presence of AAI in soil and soil organic matter samples collected from BEN and non-BEN areas. The samples were processed in order to be analyzed by high-pressure liquid chromatography, and ion trap mass spectrometry. Our results showed the presence of AAI in small concentrations, both in BEN and non-BEN soils, especially where Aristolochia plants and seeds were present.

Selective in vitro anti-melanoma activity of ursolic and oleanolic acids.

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.

Synthesis, Characterization, Antimicrobial and Antiproliferative Activity Evaluation of Cu(II), Co(II), Zn(II), Ni(II) and Pt(II) Complexes with Isoniazid-Derived Compound.

Hydrazone complexes of Cu(II), Co(II), Zn(II), Ni(II) and Pt(II) with N-isonicotinoyl-N'-(3-metoxy-2 hydroxybenzaldehyde)-hydrazone (HL) were synthesized and characterized by different physico-chemical techniques including elemental and thermal analysis, magnetic susceptibility measurements, molar electric conductivity, as well as IR (infrared), ¹H-NMR and 13C-NMR (hydrogen and carbon nuclear magnetic resonance, UV-Vis (ultraviolet-visible), FAB (fast atom bombardment), EPR (electron paramagnetic resonance), and mass spectroscopies. The crystal structure of ligand was determined by single crystal X-ray diffraction studies. Spectral data showed that hydrazone behaves as an ONO tridentate ligand through the azomethine nitrogen, phenolate and keto oxygen atoms. For the copper(II) complexes, metal-ligand bonding parameters were evaluated from the EPR spectra. These parameters indicate the presence of in-plane π bonding. In addition, the f values of complexes 1-4 indicate small distortion from planarity. The effect of these complexes on proliferation of human breast cancer (MCF-7 and SKBR-3), human melanoma (A375), lung adenocarcinoma cells (NCI-H1573) and their antibacterial activity against Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus and Candida albicans strains were studied and compared with those of free ligand. The ligand and complexes 1-3 showed significant antimicrobial activity against the Gram-positive bacteria Staphylococcus aureus and Candida albicans in comparison to the control drugs. The complexes 2-4 could be potential antitumor agents, leading to a significant improvement of the cytotoxic activity when compared with HL.

Synthesis and Characterization of Novel Cu(II), Pd(II) and Pt(II) Complexes with 8-Ethyl-2-hydroxytricyclo(7.3.1.0(2,7))tridecan-13-one-thiosemicarbazone: Antimicrobial and in Vitro Antiproliferative Activity.

New Cu(II), Pd(II) and Pt(II) complexes, (Cu(L)(H2O)2(OAc)) (1), (Cu(HL)(H2O)2(SO4)) (2), (Cu(L)(H2O)2(NO3)) (3), (Cu(L)(H2O)2(ClO4)) (4), (Cu(L)2(H2O)2) (5), (Pd(L)(OAc))H2O (6), and (Pt(L)2) (7) were synthesized from 8-ethyl-2-hydroxytricyclo(7.3.1.0(2,7))tridecan-13-one thiosemicarbazone (HL). The ligand and its metal complexes were characterized by IR, ¹H-NMR, (13)C-NMR, UV-Vis, FAB, EPR, mass spectroscopy, elemental and thermal analysis, magnetic susceptibility measurements and molar electric conductivity. The free ligand and the metal complexes have been tested for their antimicrobial activity against E. coli, S. enteritidis, S. aureus, E. faecalis, C. albicans and cytotoxicity against the NCI-H1573 lung adenocarcinoma, SKBR-3 human breast, MCF-7 human breast, A375 human melanoma and HL-60 human promyelocytic leukemia cell lines. Copper complex 2 exhibited the best antiproliferative activities against MCF-7 human breast cancer cells. A significant inhibition of malignant HL-60 cell growth was observed for copper complex 2, palladium complex 6 and platinum complex 7, with IC50 values of 1.6 µM, 6.5 µM and 6.4 µM, respectively.

Evaluation of oral keratinocyte progenitor and T-lymphocite cells response during early healing after augmentation of keratinized gingiva with a 3D collagen matrix - a pilot study.

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.

Anti-proliferative and antibacterial in vitro evaluation of the polyurethane nanostructures incorporating pentacyclic triterpenes.

CONTEXT: 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. OBJECTIVES: 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. MATERIALS AND METHODS: 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. RESULTS: Ursolic acid had an excellent inhibitory effect with IC50 value of 2.47, 1.20, 1.26 and 1.34 µM on MCF7, T47D, MDA-MB-231 and MDA-MB-361, respectively. Oleanolic acid did not show anti-proliferative activity. The pure compounds showed their antibacterial activity only against Bacillus species and Candida albicans, but MIC values were too high to be considered efficient antimicrobial agents (2280 and 4570 µg mL - 1, respectively). Polyurethane nanoparticles which incorporated the agents did not show any biological activity. DISCUSSION AND CONCLUSION: Although 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.

Enucleation: a possible mechanism of cancer cell death.

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.

Hydrogel Extrusion Speed Measurements for the Optimization of Bioprinting Parameters.

Three-dimensional (3D) bioprinting is the use of computer-controlled transfer processes for assembling bioinks (cell clusters or materials loaded with cells) into structures of prescribed 3D organization. The correct bioprinting parameters ensure a fast and accurate bioink deposition without exposing the cells to harsh conditions. This study seeks to optimize pneumatic extrusion-based bioprinting based on hydrogel flow rate and extrusion speed measurements. We measured the rate of the hydrogel flow through a cylindrical nozzle and used non-Newtonian hydrodynamics to fit the results. From the videos of free-hanging hydrogel strands delivered from a stationary print head, we inferred the extrusion speed, defined as the speed of advancement of newly formed strands. Then, we relied on volume conservation to evaluate the extrudate swell ratio. The theoretical analysis enabled us to compute the extrusion speed for pressures not tested experimentally as well as the printing speed needed to deposit hydrogel filaments of a given diameter. Finally, the proposed methodology was tested experimentally by analyzing the morphology of triple-layered square-grid hydrogel constructs printed at various applied pressures while the printing speeds matched the corresponding extrusion speeds. Taken together, the results of this study suggest that preliminary measurements and theoretical analyses can simplify the search for the optimal bioprinting parameters.