In Vitro Study of the Bioavailability and Bioaccessibility of the Main Compounds Present in Ayahuasca Beverages

Ayahuasca is a psychoactive beverage that contains the psychoactive compound N,N-dimethyltryptamine and β-carboline alkaloids. This study aims at determining in vitro the bioavailability and bioaccessibility of the main compounds present in decoctions of four individual plants, in a commercial mixture and in four mixtures of two individual plants used in the preparation of Ayahuasca. The samples were subjected to an in vitro digestion process, and the Caco-2 cell line was used as an absorption model. The integrity and permeability of the cell monolayer were evaluated, as well as the cytotoxicity of the extracts. After digestion and cell incubation, the compounds absorbed by the cell monolayer were quantified by high-performance liquid chromatography coupled to a diode array detector. The results showed that compounds such as N,N-dimethyltryptamine, Harmine, Harmaline, Harmol, Harmalol and Tetrahydroharmine were released from the matrix during the in vitro digestion process, becoming bioaccessible. Similarly, some of these compounds, after being incubated with the cell monolayer, were absorbed, becoming bioavailable. The extracts did not show cytotoxicity after cell incubation, and the integrity and permeability of the cell monolayer were not compromised.

Short Assay Design for Micronucleus Detection in Human Lymphocytes

There has been a constant need to develop new and faster cytogenetic assays to measure the instability induced by genotoxic agents in the field of cytogenetic research, an example of which is the micronucleus assay. Micronuclei are fragments or complete chromosomes that remain in the cytoplasm during mitosis. With their high sensitivity and specificity detection, their presence can indicate environmental and occupational genotoxic effects. However, the prolonged periods of cell incubation this assay necessitates are costly and extensive. Hence, it is essential to develop an improved assay that can achieve standardization by being reproducible in practice. The standard protocol for the detection of micronuclei in lymphocytes uses a total assay time of 72 hours. Theoretically, it is possible to reduce the incubation period, and consequently, the total assay time, considering a lymphocyte, completes its mitosis in 24 hours. This study, after careful review of literature, proposes an experimental design to reduce the incubation period and demonstrates its usefulness in practice through the design of a collaborative trial.

Enhanced Biomechanically Mediated “Phagocytosis” in Detached Tumor Cells

Uptake of particles by cells involves various natural mechanisms that are essential for their biological functions. The same mechanisms are used in the engulfment of synthetic colloidal drug carriers, while the extent of the uptake affects the biological performance and selectivity. Thus far, little is known regarding the effect of external biomechanical stimuli on the capacity of the cells to uptake nano and micro carriers. This is relevant for anchorage-dependent cells that have detached from surfaces or for cells that travel in the body such as tumor cells, immune cells and various circulating stem cells. In this study, we hypothesize that cellular deformability is a crucial physical effector for the successful execution of the phagocytosis-like uptake in cancer cells. To test this assumption, we develop a well-controlled tunable method to compare the uptake of inert particles by cancer cells in adherent and non-adherent conditions. We introduce a self-designed 3D-printed apparatus, which enables constant stirring while facilitating a floating environment for cell incubation. We reveal a mechanically mediated phagocytosis-like behavior in various cancer cells, that was dramatically enhance in the detached cell state. Our findings emphasize the importance of including proper biomechanical cues to reliably mimic certain physiological scenarios. Beyond that, we offer a cost-effective accessible research tool to study mixed cultures for both adherent and non-adherent cells.

Evidence for the expression of TRPM6 and TRPM7 in cardiomyocytes from all four chamber walls of the human heart

The expression of the channels-enzymes TRPM6 and TRPM7 in the human heart remains poorly defined, and TRPM6 is generally considered not to be expressed in cardiomyocytes. We examined their expression at protein and mRNA levels using right atrial samples resected from patients (n = 72) with or without ischemic heart disease (IHD) and samples from all chamber walls of explanted human hearts (n = 9). TRPM6 and TRPM7 proteins were detected using immunofluorescence on isolated cardiomyocytes, ELISA on tissue homogenates, and immunostaining of cardiac tissue, whereas their mRNAs were detected by RT-qPCR. Both TRPM6 and TRPM7 were present in all chamber walls, with TRPM7 being more abundant. TRPM6 was co-expressed with TRPM7. The expression levels were dependent on cell incubation conditions (presence or absence of divalent cations, pH of the extracellular milieu, presence of TRP channel inhibitors 2-aminoethoxydiphenyl-borate and carvacrol). These drugs reduced TRPM7 immunofluorescence but increased that of TRPM6. TRPM6 and TRPM7 expression was increased in tissues from IHD patients. This is the first demonstration of the presence and co-expression of TRPM6 and TRPM7 in cardiomyocytes from all chamber walls of the human heart. The increased TRPM6 and TRPM7 expression in IHD suggests that the chanzymes are involved in the pathophysiology of the disease.

Scaffolds Designing from Protein-loadable Coaxial Electrospun Fibermats of poly(acrylamide)-co-poly(diacetone acrylamide) and Gelatin

Background: A core-shell fiber mat is one of the attractive platforms for this purpose. However, very few details the importance of choosing the suitable material for the shell units that can endow efficient release properties. The tailored design of cytokine-releasing scaffolds aiming at in situ regenerative therapy is still one of the crucial issues to be studied. Objective: In this study, we characterized the effectiveness of core-shell fiber mats that possess cross-linked gelatin (CLG) as the shell layer of constituent nanofibers, as a protein-releasing cell-incubation scaffold. Methods: We utilized a crosslinked copolymer of poly (acrylamide)-co-poly (diacetone acrylamide) (poly (AM/DAAM)) and adipic acid dihydrazide (ADH), poly (AM/DAAM)/ADH for the core nanofibers in the core-shell fiber mats. By coaxial electrospinning and the subsequent crosslinking of the gelatin layer, we successfully constructed core-shell fiber mats consisting of double-layered nanofibers of poly (AM/DAAM)/ADH and CLG. Using fluorescein isothiocyanate-labeled lysozyme (FITC-Lys) as a dummy guest protein, we characterized the release behavior of the core-shell fiber mats containing a CLG layer. Upon loading essential fibroblast growth factor (bFGF) as cargo in our fiber mats, we also characterized the impacts of the released bFGF on the proliferation of the incubated cells. Results: Although the single-layered poly (AM/DAAM)/ADH nanofiber fiber mats did not adhere to the mammalian cells, the core-shell fiber mat with the CLG shell layer exhibited good adherence and subsequent proliferation. A sustained release of the preloaded FITC-Lys over 24 days without any burst release was observed, and the cumulative amount of released protein reached over 65% after 24 days. Upon loading bFGF in our fiber mats, we succeeded in promoting cell proliferation and highlighting its potential for therapeutic applications. Conclusion: We successfully confirmed that core-shell fiber mats with a CLG shell layer around the constituent nanofibers were practical as protein-releasing cell-incubation scaffolds.

Open-source personal pipetting robots with live-cell incubation and microscopy compatibility

Liquid handling robots have the potential to automate many procedures in life sciences. However, they are not in widespread use in academic settings, where funding, space and maintenance specialists are usually limiting. In addition, current robots require lengthy programming by specialists and are incompatible with most academic laboratories with constantly changing small-scale projects. Here, we present the Pipetting Helper Imaging Lid (PHIL), an inexpensive, small, open-source personal liquid handling robot. It is designed for inexperienced users, with self-production from cheap commercial and 3D-printable components and custom control software. PHIL successfully automated pipetting for e.g. tissue immunostainings and stimulations of live stem and progenitor cells during time-lapse microscopy. PHIL is cheap enough for any laboratory member to have their own personal pipetting robot(s), and enables users without programming skills to easily automate a large range of experiments.

Incorporation of Elastin to Improve Polycaprolactone-Based Scaffolds for Skeletal Muscle via Electrospinning

Skeletal muscle regeneration is increasingly necessary, which is reflected in the increasing number of studies that are focused on improving the scaffolds used for such regeneration, as well as the incubation protocol. The main objective of this work was to improve the characteristics of polycaprolactone (PCL) scaffolds by incorporating elastin to achieve better cell proliferation and biocompatibility. In addition, two cell incubation protocols (with and without dynamic mechanical stimulation) were evaluated to improve the activity and functionality yields of the regenerated cells. The results indicate that the incorporation of elastin generates aligned and more hydrophilic scaffolds with smaller fiber size. In addition, the mechanical properties of the resulting scaffolds make them adequate for use in both bioreactors and patients. All these characteristics increase the biocompatibility of these systems, generating a better interconnection with the tissue. However, due to the low maturation achieved in biological tests, no differences could be found between the incubation with and without dynamic mechanical stimulation.

A Membrane Filter-Assisted Mammalian Cell-Based Biosensor Enabling 3D Culture and Pathogen Detection

We have developed a membrane filter-assisted cell-based biosensing platform by using a polyester membrane as a three-dimensional (3D) cell culture scaffold in which cells can be grown by physical attachment. The membrane was simply treated with ethanol to increase surficial hydrophobicity, inducing the stable settlement of cells via gravity. The 3D membrane scaffold was able to provide a relatively longer cell incubation time (up to 16 days) as compared to a common two-dimensional (2D) cell culture environment. For a practical application, we fabricated a cylindrical cartridge to support the scaffold membranes stacked inside the cartridge, enabling not only the maintenance of a certain volume of culture media but also the simple exchange of media in a flow-through manner. The cartridge-type cell-based analytical system was exemplified for pathogen detection by measuring the quantities of toll-like receptor 1 (TLR1) induced by applying a lysate of P. aeruginosa and live E. coli, respectively, providing a fast, convenient colorimetric TLR1 immunoassay. The color images of membranes were digitized to obtain the response signals. We expect the method to further be applied as an alternative tool to animal testing in various research areas such as cosmetic toxicity and drug efficiency.

Secretion and gene expression of proatherosclerotic cytokines IL6 and IL8 by endothelial cells exposed to mutagen

Атеросклероз занимает лидирующую позицию в структуре заболеваемости и смертности среди всей сердечно-сосудистой патологии как в России, так и во всем мире. Имеются свидетельства того, что в развитии эндотелиальной дисфункции, являющейся первым этапом атерогенеза, помимо классических факторов риска также играют роль соматические мутации, а в атеросклеротических бляшках и пораженных сосудах отмечается повышенный уровень различных ДНК аддуктов. Однако остается открытым вопрос о фундаментальных основах роли соматических мутаций в патогенезе атеросклероза, что наиболее важно в условиях увеличения генотоксической нагрузки на организм человека, особенно в регионах с развитой промышленностью. Целью данного исследования явилось изучение особенностей секреции проатеросклеротических цитокинов IL-6 и IL-8 и экспрессии их генов в культурах первичных эндотелиальных клеток коронарной и внутренней грудной артерий, различающихся по степени подверженности атеросклерозу и экспонированных мутагеном алкилирующего механизма действия митомицином С. Концентрация изученных цитокинов в культуральной среде и уровень мРНК соответствующих генов измерялись методами количественной полимеразной цепной реакции и иммуноферментного анализа в двух временных точках - непосредственно после 6 часов экспозиции клеток митомицином С (первая временная точка) и после 6 часов экспозиции клеток мутагеном с последующими сутками культивирования в культуральной среде без митомицина С (вторая временная точка). Установлено, что в первой временной точке в экспонированных митомицином С культурах наблюдается статистически значимое снижение секреции и экспрессии гена IL8, а также снижение уровня мРНК гена IL6. Во второй временной точке, наоборот, концентрация и уровень мРНК гена IL8, а также генная экспрессия IL6 резко возрастали в культурах, экспонированных митомицином С по сравнению с неэкспонированным контролем (p<0,01), причем эндотелиальные клетки коронарной артерии, наиболее часто поражаемой атеросклерозом, были более чувствительны к мутагенному воздействию, чем эндотелиальные клетки внутренней грудной артерии, в наименьшей степени подверженной атерогенезу. Таким образом, впервые в эксперименте in vitro показаны мутаген-индуцированные изменения характера секреции и экспрессии генов проатеросклеротических цитокинов IL-6 и IL-8 в культурах первичных эндотелиальных клеток различных артерий человека. Atherosclerosis is a leader in morbidity and mortality among all cardiovascular pathologies both in Russia and around the world. Nowadays there are evidences that somatic mutations in addition to classical risk factors can play a role in the development of endothelial dysfunction - the initial stage of atherogenesis, and atherosclerotic plaques are characterized by increased level of various DNA adducts. At the same time, a fundamental base of the contribution of mutagenesis to atherogenesis is still unstudied thought it is important for industrial regions with high genotoxic risk The aim of this research was to study the secretion and gene expression of proatherosclerotic cytokines IL-6 and IL-8 by primary human coronary- and internal thoracic artery endothelial cells characterized by different sensitivity to atherogenesis and exposed to alkylating mutagen mitomycin C. Concentration of the studied cytokines in culture medium and mRNA level of the corresponding genes were measured using quantitative polymerase chain reaction and enzyme-linked immunosorbent assay at two time points - immediately after 6 hours of cell incubation with mitomycin C and after 6 hours of cell incubation with mutagen followed by 24 hours incubation in the complete media without mitomycin C. It was found that at the first time point, a significant decrease in the secretion and gene expression of IL-8, as well as a decrease in the mRNA level of the IL-6 gene in the exposed culture was discovered. At the second time point, on the contrary, the concentration and mRNA level of IL-8, as well as expression of the IL6 gene sharply increased in cultures exposed to mitomycin C in comparison with control (p <0.01), and the coronary artery cells were more sensitive to mutagenic effects than the cells of the internal thoracic artery. Thus, for the first time in in vitro experiment, mutagen-induced changes in the secretion and gene expression of proatherosclerotic cytokines IL-6 and IL-8 in primary human endothelial cells derived from various arteries have been shown.

Sex dependent action of Aroclor 1254 on basal and sGnRHa-stimulated secretion of LH from the pituitary cells of common carp, Cyprinus carpio L.

Polychlorinated biphenyls (PCBs) affect the hypothalamic-pituitary-gonadal axis in many vertebrates, changing the hormonal regulation of reproduction. To identify one of the possible sites of action of PCBs on gonadotropin release in common carp, the direct effects of Aroclor 1254 on luteinizing hormone (LH) secretion from dispersed pituitary cells were investigated. Pituitary cells were obtained from sexually mature male and female common carp (Cyprinus carpio L.) at the time of natural spawning. The cells were incubated with different concentrations of Aroclor 1254 (5, 10, 50 and 100 ng mL−1 medium) and/or salmon gonadotropin-releasing hormone analogue (sGnRHa) at a concentration of 10−8 M. LH levels were measured in the cultured medium by the ELISA method after 10 hours of cell incubation. Incubation of male pituitary cells in the presence of tested concentrations of Aroclor did not change the basal LH secretion to the media. In the female pituitary cell incubations Aroclor (5, 10 and 100 ng mL−1 medium) caused a significant increase in LH concentrations in comparison to control incubations. In the case of sGnRHa-stimulated LH secretion in incubations of cells of both sexes, all the concentrations of Aroclor significantly stimulated LH release and potentiated stimulatory effects of sGnRH analogue. These results indicate that endocrine disrupters, such as Aroclor 1254, may affect reproduction in fish, acting also directly on gonadotrophs at the level of the pituitary gland, changing LH secretion.