The bioelectric mechanisms of local calcium dynamics in cancer cell proliferation: an extension of the A549 in silico cell model.

Introduction: Advances in molecular targeting of ion channels may open up new avenues for therapeutic approaches in cancer based on the cells' bioelectric properties. In addition to in-vitro or in-vivo models, in silico models can provide deeper insight into the complex role of electrophysiology in cancer and reveal the impact of altered ion channel expression and the membrane potential on malignant processes. The A549 in silico model is the first computational cancer whole-cell ion current model that simulates the bioelectric mechanisms of the human non-small cell lung cancer cell line A549 during the different phases of the cell cycle. This work extends the existing model with a detailed mathematical description of the store-operated Ca2+ entry (SOCE) and the complex local intracellular calcium dynamics, which significantly affect the entire electrophysiological properties of the cell and regulate cell cycle progression. Methods: The initial model was extended by a multicompartmental approach, addressing the heterogenous calcium profile and dynamics in the ER-PM junction provoked by local calcium entry of store-operated calcium channels (SOCs) and uptake by SERCA pumps. Changes of cytosolic calcium levels due to diffusion from the ER-PM junction, release from the ER by RyR channels and IP3 receptors, as well as corresponding PM channels were simulated and the dynamics evaluated based on calcium imaging data. The model parameters were fitted to available data from two published experimental studies, showing the function of CRAC channels and indirectly of IP3R, RyR and PMCA via changes of the cytosolic calcium levels. Results: The proposed calcium description accurately reproduces the dynamics of calcium imaging data and simulates the SOCE mechanisms. In addition, simulations of the combined A549-SOCE model in distinct phases of the cell cycle demonstrate how Ca2+ - dynamics influence responding channels such as KCa, and consequently modulate the membrane potential accordingly. Discussion: Local calcium distribution and time evolution in microdomains of the cell significantly impact the overall electrophysiological properties and exert control over cell cycle progression. By providing a more profound description, the extended A549-SOCE model represents an important step on the route towards a valid model for oncological research and in silico supported development of novel therapeutic strategies.

Animals in Respiratory Research.

The respiratory barrier, a thin epithelial barrier that separates the interior of the human body from the environment, is easily damaged by toxicants, and chronic respiratory diseases are common. It also allows the permeation of drugs for topical treatment. Animal experimentation is used to train medical technicians, evaluate toxicants, and develop inhaled formulations. Species differences in the architecture of the respiratory tract explain why some species are better at predicting human toxicity than others. Some species are useful as disease models. This review describes the anatomical differences between the human and mammalian lungs and lists the characteristics of currently used mammalian models for the most relevant chronic respiratory diseases (asthma, chronic obstructive pulmonary disease, cystic fibrosis, pulmonary hypertension, pulmonary fibrosis, and tuberculosis). The generation of animal models is not easy because they do not develop these diseases spontaneously. Mouse models are common, but other species are more appropriate for some diseases. Zebrafish and fruit flies can help study immunological aspects. It is expected that combinations of in silico, in vitro, and in vivo (mammalian and invertebrate) models will be used in the future for drug development.

Liver X receptor activation mitigates oxysterol-induced dysfunction in fetoplacental endothelial cells.

Maintaining the homeostasis of the placental vasculature is of paramount importance for ensuring normal fetal growth and development. Any disruption in this balance can lead to perinatal morbidity. Several studies have uncovered an association between high levels of oxidized cholesterol (oxysterols), and complications during pregnancy, including gestational diabetes mellitus (GDM) and preeclampsia (PE). These complications often coincide with disturbances in placental vascular function. Here, we investigate the role of two oxysterols (7-ketocholesterol, 7ß-hydroxycholesterol) in (dys)function of primary fetoplacental endothelial cells (fpEC). Our findings reveal that oxysterols exert a disruptive influence on fpEC function by elevating the production of reactive oxygen species (ROS) and interfering with mitochondrial transmembrane potential, leading to its depolarization. Moreover, oxysterol-treated fpEC exhibited alterations in intracellular calcium (Ca2+) levels, resulting in the reorganization of cell junctions and a corresponding increase in membrane stiffness and vascular permeability. Additionally, we observed an enhanced adhesion of THP-1 monocytes to fpEC following oxysterol treatment. We explored the influence of activating the Liver X Receptor (LXR) with the synthetic agonist T0901317 (TO) on oxysterol-induced endothelial dysfunction in fpEC. Our results demonstrate that LXR activation effectively reversed oxysterol-induced ROS generation, monocyte adhesion, and cell junction permeability in fpEC. Although the effects on mitochondrial depolarization and calcium mobilization did not reach statistical significance, a strong trend towards stabilization of calcium mobilization was evident in LXR-activated cells. Taken together, our results suggest that high levels of systemic oxysterols link to placental vascular dysfunction and LXR agonists may alleviate their impact on fetoplacental vasculature.

Development and Characterization of an In Vitro Intestinal Model Including Extracellular Matrix and Macrovascular Endothelium.

In vitro intestinal models are used to study biological processes, drug and food absorption, or cytotoxicity, minimizing the use of animals in the laboratory. They usually consist of enterocytes and mucus-producing cells cultured for 3 weeks, e.g., on Transwells, to obtain a fully differentiated cell layer simulating the human epithelium. Other important components are the extracellular matrix (ECM) and strong vascularization. The former serves as structural support for cells and promotes cellular processes such as differentiation, migration, and growth. The latter includes endothelial cells, which coordinate vascularization and immune cell migration and facilitate the transport of ingested substances or drugs to the liver. In most cases, animal-derived hydrogels such as Matrigel or collagen are used as ECM in in vitro intestinal models, and endothelial cells are only partially considered, if at all. However, it is well-known that animal-derived products can lead to altered cell behavior and incorrect results. To circumvent these limitations, synthetic and modifiable hydrogels (Peptigel and Vitrogel) were studied here to mimic xenofree ECM, and the data were compared with Matrigel. Careful rheological characterization was performed, and the effect on cell proliferation was investigated. The results showed that Vitrogel exhibited shear-thinning behavior with an internal structure recovery of 78.9 ± 11.2%, providing the best properties among the gels investigated. Therefore, a coculture of Caco-2 and HT29-MTX cells (ratio 7:3) was grown on Vitrogel, while simultaneously endothelial cells were cultured on the basolateral side by inverse cultivation. The model was characterized in terms of cell proliferation, differentiation, and drug permeability. It was found that the cells cultured on Vitrogel induced a 1.7-fold increase in cell proliferation and facilitated the formation of microvilli and tight junctions after 2 weeks of cultivation. At the same time, the coculture showed full differentiation indicated by high alkaline phosphatase release of Caco-2 cells (95.0 ± 15.9%) and a mucus layer produced by HT29-MTX cells. Drug tests led to ex vivo comparable permeability coefficients (Papp) (i.e., Papp; antipyrine = (33.64 ± 5.13) × 10-6 cm/s, Papp; atenolol = (0.59 ± 0.16) × 10-6 cm/s). These results indicate that the newly developed intestinal model can be used for rapid and efficient assessment of drug permeability, excluding unexpected results due to animal-derived materials.

Lipid-based particle engineering via spray-drying for targeted delivery of antibiotics to the lung.

Pulmonary delivery of antibiotics for the treatment of tuberculosis provides several benefits compared to conventional oral and parenteral administration. API-loaded particles delivered directly to alveolar macrophages, where Mycobacterium tuberculosis resides, can reduce the required dose and decrease the severe side effects of conventional treatment. In this work, lipid-microparticles loaded with rifampicin were engineered via spray-drying to be administered as a carrier-free dry powder for inhalation. Although, it is well-known that spray-drying of lipid-based excipients is strongly limited, a completely lipid-based formulation using diglycerol full ester of behenic acid was produced. The solid state of the lipid, providing high melting temperature, absence of polymorphism and monophasic crystallization, led to high yield of spray-dried particles (83%). Inhalable particles of mass median aerodynamic diameter of 2.36 µm, median geometric size of 2.05 µm, and negative surface (-50.03 mV) were engineered. Such attributes were defined for deep lung deposition and targeted delivery of antibiotics to alveolar macrophages. Superior aerodynamic performance as carrier-free DPI was associated to a high fine particle fraction of 79.5 %. No in vitro cytotoxic effects were found after exposing epithelial cell lines and alveolar macrophages. In vitro uptake of particles into alveolar macrophages indicated the efficiency of their targeted delivery. The use of highly processable and safe lipid-based excipients for particle engineering via spray-drying can extend the availability of materials for functionalized applications for pulmonary delivery.

Pars Distalis and Pars Tuberalis Thyroid-Stimulating Hormones and Their Roles in Macro-Thyroid-Stimulating Hormone Formation.

Thyroid-stimulating hormone (TSH) and thyroid hormone levels are standard parameters in blood analysis. However, the immunoassays employed may lead to false-positive or false-negative results when the sample contains certain materials that interfere with the assay. Macro-TSH, a complex of TSH with immunoglobulin or albumin, may cause apparently increased TSH concentrations. TSH is produced in the pars tuberalis (PT) of the pituitary gland and by thyrotrophs of the pars distalis (PD). It was found that variable glycosylation can render the molecule more strongly bound to antibodies or albumin in the blood, leading to the hypothesis that macro-TSH consists mainly of PT-TSH. Although less known than PD-TSH, PT-TSH plays an important role in the central regulation of thyroid metabolism. The present review summarizes the physiological function of human PT-TSH and its role in macro-TSH formation. The prevalence of macro-hyperthyrotropinemia, the structure of PT-TSH and macro-TSH, problems in the measurement of TSH, and the action of PT-TSH in animals with seasonal breeding are discussed. Despite the absence of a specific function of macro-TSH in the organism, the identification of macro-TSH is important for avoiding unnecessary treatment based on a falsified readout of increased TSH concentrations as numerous individual case reports describe.

Lipid Nanoparticles as a Shuttle for Anti-Adipogenic miRNAs to Human Adipocytes.

Obesity and type 2 diabetes are major health burdens for which no effective therapy is available today. One treatment strategy could be to balance the metabolic functions of adipose tissue by regulating gene expressions using miRNAs. Here, we have loaded two anti-adipogenic miRNAs (miR26a and miR27a) into a pegylated lipid nanoparticle (PEG-LNP) formulation by a single-step microfluidic-assisted synthesis step. For the miRNA-loaded LNPs, the following system properties were determined: particle size, zeta potential, miRNA complexation efficiency, and cytotoxicity. We have used a human preadipocyte cell line to address the transfection efficiency and biological effects of the miRNA candidates at the gene and protein level. Our findings revealed that the upregulation of miR27a in preadipocytes inhibits adipogenesis by the downregulation of PPARγ and the reduction of lipid droplet formation. In contrast, miR26a transfection in adipocytes induced white adipocyte browning detected as the upregulation of uncoupling protein 1 (UCP1) as a marker of non-shivering thermogenesis. We conclude that the selective delivery of miRNAs by PEG-LNPs to adipocytes could offer new perspectives for the treatment of obesity and related metabolic diseases.

The Variety of 3D Breast Cancer Models for the Study of Tumor Physiology and Drug Screening.

Breast cancer is the most common cancer in women and responsible for multiple deaths worldwide. 3D cancer models enable a better representation of tumor physiology than the conventional 2D cultures. This review summarizes the important components of physiologically relevant 3D models and describes the spectrum of 3D breast cancer models, e.g., spheroids, organoids, breast cancer on a chip and bioprinted tissues. The generation of spheroids is relatively standardized and easy to perform. Microfluidic systems allow control over the environment and the inclusion of sensors and can be combined with spheroids or bioprinted models. The strength of bioprinting relies on the spatial control of the cells and the modulation of the extracellular matrix. Except for the predominant use of breast cancer cell lines, the models differ in stromal cell composition, matrices and fluid flow. Organoids are most appropriate for personalized treatment, but all technologies can mimic most aspects of breast cancer physiology. Fetal bovine serum as a culture supplement and Matrigel as a scaffold limit the reproducibility and standardization of the listed 3D models. The integration of adipocytes is needed because they possess an important role in breast cancer.

Assessment of Carbon Nanotubes on Barrier Function, Ciliary Beating Frequency and Cytokine Release in In Vitro Models of the Respiratory Tract.

The exposure to inhaled carbon nanotubes (CNT) may have adverse effects on workers upon chronic exposure. In order to assess the toxicity of inhaled nanoparticles in a physiologically relevant manner, an air-liquid interface culture of mono and cocultures of respiratory cells and assessment in reconstructed bronchial and alveolar tissues was used. The effect of CNT4003 reference particles applied in simulated lung fluid was studied in bronchial (Calu-3 cells, EpiAirway™ and MucilAir™ tissues) and alveolar (A549 +/-THP-1 and EpiAlveolar™ +/-THP-1) models. Cytotoxicity, transepithelial electrical resistance, interleukin 6 and 8 secretion, mucociliary clearance and ciliary beating frequency were used as readout parameters. With the exception of increased secretion of interleukin 6 in the EpiAlveolar™ tissues, no adverse effects of CNT4003 particles, applied at doses corresponding to the maximum estimated lifetime exposure of workers, in the bronchial and alveolar models were noted, suggesting no marked differences between the models. Since the doses for whole-life exposure were applied over a shorter time, it is not clear if the interleukin 6 increase in the EpiAlveolar™ tissues has physiological relevance.

Premexotac: Machine learning bitterants predictor for advancing pharmaceutical development.

Bitter taste receptors were recently found to be involved in numerous physiological and pathological conditions other than taste and are suggested as potential drug targets. In vivo and in vitro techniques for screening bitterants as ligands come with economical, time and ethic challenges. Therefore, in silico tools can represent a valuable alternative due to their practicality. Yet, the main challenge of already established ligand-based (LB) classifiers is the low number of experimentally confirmed bitterants and non-bitterants. Premexotac models were constructed as a LB bitterants screener, exploring novel combinations of feature extraction, feature selection and learning algorithms as a contrast with the already available screeners. Premexotac came among the top performers, exhibiting a F-1 score up to 81% on external validation. Premexotac identified as well insights on physicochemical and topological descriptors important for bitter prediction. Among the key insights, important molecular substructures from Extended Connectivity Fingerprints for bitterness classification were identified. Also, the importance of a selection of physicochemical/topological descriptors was ranked using mutual information and it was found that descriptors related to the ramification of the molecular structure and molecular weight came at the top of the ranking. The remaining challenges for improving performance were discussed and stated, widening the LB bitterness prediction outlook.

Biomechanical properties of native and cultured red blood cells-Interplay of shape, structure and biomechanics.

Modern medicine increases the demand for safe blood products. Ex vivo cultured red blood cells (cRBC) are eagerly awaited as a standardized, safe source of RBC. Established culture models still lack the terminal cytoskeletal remodeling from reticulocyte to erythrocyte with changes in the biomechanical properties and interacts with membrane stiffness, viscosity of the cytoplasm and the cytoskeletal network. Comprehensive data on the biomechanical properties of cRBC are needed to take the last step towards translation into clinical use in transfusion medicine. Aim of the study was the comparative analysis of topographical and biomechanical properties of cRBC, generated from human CD34+ adult hematopoietic stem/progenitor cells, with native reticulocytes (nRET) and erythrocytes (nRBC) using cell biological and biomechanical technologies. To gain the desired all-encompassing information, a single method was unsatisfactory and only the combination of different methods could lead to the goal. Topographical information was matched with biomechanical data from optical tweezers (OT), atomic force microscopy (AFM) and digital holographic microscopy (DHM). Underlying structures were investigated in detail. Imaging, deformability and recovery time showed a high similarity between cRBC and nRBC. Young's modulus and plasticity index also confirmed this similarity. No significant differences in membrane and cytoskeletal proteins were found, while lipid deficiency resulted in spherical, vesiculated cells with impaired biomechanical functionality. The combination of techniques has proven successful and experiments underscore a close relationship between lipid content, shape and biomechanical functionality of RBC.

Insight into Potential Interactions of Thyroid Hormones, Sex Hormones and Their Stimulating Hormones in the Development of Non-Alcoholic Fatty Liver Disease.

Non-Alcoholic Fatty Liver Disease (NAFLD) is a common manifestation of metabolic syndrome. In addition to lifestyle, endocrine hormones play a role in the dysregulation of hepatic metabolism. The most common endocrine hormones contributing to metabolic syndrome are alterations in the levels of thyroid hormones (THs, predominantly in subclinical hypothyroidism) and of sex hormones (in menopause). These hormonal changes influence hepatic lipid and glucose metabolism and may increase hepatic fat accumulation. This review compares the effects of sex hormones, THs and the respective stimulating hormones, Thyroid-Stimulating Hormone (TSH) and Follicle-Stimulating Hormone (FSH), on the development of hepatosteatosis. TSH and FSH may be more relevant to the dysregulation of hepatic metabolism than the peripheral hormones because metabolic changes were identified when only levels of the stimulating hormones were abnormal and the peripheral hormones were still in the reference range. Increased TSH and FSH levels appear to have additive effects on the development of NAFLD and to act independently from each other.

Non-Cellular Layers of the Respiratory Tract: Protection against Pathogens and Target for Drug Delivery.

Epithelial barriers separate the human body from the environment to maintain homeostasis. Compared to the skin and gastrointestinal tract, the respiratory barrier is the thinnest and least protective. The properties of the epithelial cells (height, number of layers, intercellular junctions) and non-cellular layers, mucus in the conducting airways and surfactant in the respiratory parts determine the permeability of the barrier. The review focuses on the non-cellular layers and describes the architecture of the mucus and surfactant followed by interaction with gases and pathogens. While the penetration of gases into the respiratory tract is mainly determined by their hydrophobicity, pathogens use different mechanisms to invade the respiratory tract. Often, the combination of mucus adhesion and subsequent permeation of the mucus mesh is used. Similar mechanisms are also employed to improve drug delivery across the respiratory barrier. Depending on the payload and target region, various mucus-targeting delivery systems have been developed. It appears that the mucus-targeting strategy has to be selected according to the planned application.

Thiolated Chitosan Conjugated Liposomes for Oral Delivery of Selenium Nanoparticles.

This study aimed to design a hybrid oral liposomal delivery system for selenium nanoparticles (Lip-SeNPs) to improve the bioavailability of selenium. Thiolated chitosan, a multifunctional polymer with mucoadhesive properties, was used for surface functionalization of Lip-SeNPs. Selenium nanoparticle (SeNP)-loaded liposomes were manufactured by a single step microfluidics-assisted chemical reduction and assembling process. Subsequently, chitosan-N-acetylcysteine was covalently conjugated to the preformed Lip-SeNPs. The Lip-SeNPs were characterized in terms of composition, morphology, size, zeta potential, lipid organization, loading efficiency and radical scavenging activity. A co-culture system (Caco-2:HT29-MTX) that integrates mucus secreting and enterocyte-like cell types was used as a model of the human intestinal epithelium to determine adsorption, mucus penetration, release and transport properties of Lip-SeNPs in vitro. Thiolated Lip-SeNPs were positively charged with an average size of about 250 nm. Thiolated Lip-SeNPs tightly adhered to the mucus layer without penetrating the enterocytes. This finding was consistent with ex vivo adsorption studies using freshly excised porcine small intestinal tissues. Due to the improved mucoadhesion and retention in a simulated microenvironment of the small intestine, thiolated Lip-SeNPs might be a promising tool for oral selenium delivery.

Acute Respiratory Distress Syndrome: Focus on Viral Origin and Role of Pulmonary Lymphatics.

Acute respiratory distress syndrome (ARDS) is a serious affection of the lung caused by a variety of pathologies. Great interest is currently focused on ARDS induced by viruses (pandemic influenza and corona viruses). The review describes pulmonary changes in ARDS and specific effects of the pandemic viruses in ARDS, and summarizes treatment options. Because the known pathogenic mechanisms cannot explain all aspects of the syndrome, the contribution of pulmonary lymphatics to the pathology is discussed. Organization and function of lymphatics in a healthy lung and in resorption of pulmonary edema are described. A future clinical trial may provide more insight into the role of hyaluronan in ARDS but the development of promising pharmacological treatments is unlikely because drugs play no important role in lymphedema therapy.

Initial Biological Assessment of Upconversion Nanohybrids.

Nanoparticles for medical use should be non-cytotoxic and free of bacterial contamination. Upconversion nanoparticles (UCNPs) coated with cucurbit[7]uril (CB[7]) made by combining UCNPs free of oleic acid, here termed bare UCNPs (UCn), and CB[7], i.e., UC@CB[7] nanohybrids, could be used as photoactive inorganic-organic hybrid scaffolds for biological applications. UCNPs, in general, are not considered to be highly toxic materials, but the release of fluorides and lanthanides upon their dissolution may cause cytotoxicity. To identify potential adverse effects of the nanoparticles, dehydrogenase activity of endothelial cells, exposed to various concentrations of the UCNPs, was determined. Data were verified by measuring lactate dehydrogenase release as the indicator of loss of plasma membrane integrity, which indicates necrotic cell death. This assay, in combination with calcein AM/Ethidium homodimer-1 staining, identified induction of apoptosis as main mode of cell death for both particles. The data showed that the UCNPs are not cytotoxic to endothelial cells, and the samples did not contain endotoxin contamination. Higher cytotoxicity, however, was seen in HeLa and RAW 264.7 cells. This may be explained by differences in lysosome content and particle uptake rate. Internalization of UCn and UC@CB[7] nanohybrids by cells was demonstrated by NIR laser scanning microscopy.

"Biology and Medicine": A Section of Nanomaterials Addressing Interactions of Nanomaterials with All Forms of Life.

"Biology and Medicine" is a Section of Nanomaterials publishing original articles, reviews, and commentaries on any aspect related to the interaction of nanomaterials with biology [...].