European Mistletoe (Viscum album) Extract Is Cytotoxic to Canine High-Grade Astrocytoma Cells In Vitro and Has Additive Effects with Mebendazole

Malignant gliomas are associated with extremely poor clinical outcomes in both humans and dogs, and novel therapies are needed. Glioma-bearing canine patients may serve as promising preclinical models for human therapies, including complementary medicine. The objective of this study was to evaluate the effects of mistletoe extract (Viscum album) alone and in combination with mebendazole in an in vitro model of canine high-grade astrocytoma using the cell line SDT-3G. SDT-3G cells were exposed to a range of concentrations of mistletoe extract alone to obtain an IC50. In separate experiments, cells were exposed to mebendazole at a previously determined IC50 (0.03 µM) alone or in conjunction with varying concentrations of mistletoe extract to determine the additive effects. The IC50 for mistletoe alone was 5.644 ± 0.09 SD μg/mL. The addition of mistletoe at 5 μg/mL to mebendazole at 0.03 µM led to increased cell death compared to what would be expected for each drug separately. The cytotoxicity of mistletoe in vitro and its additive effect with mebendazole support future expanded in vitro and in vivo studies in dogs and supply early evidence that this may be a useful adjunct therapeutic agent for use in glioma-bearing dogs. To the authors’ knowledge, this is the first published report of Viscum album extract in canine glioma.

Simulating the Hydrodynamic Conditions of the Human Ascending Colon: A Digital Twin of the Dynamic Colon Model

The performance of solid oral dosage forms targeting the colon is typically evaluated using standardised pharmacopeial dissolution apparatuses. However, these fail to replicate colonic hydrodynamics. This study develops a digital twin of the Dynamic Colon Model; a physiologically representative in vitro model of the human proximal colon. Magnetic resonance imaging of the Dynamic Colon Model verified that the digital twin robustly replicated flow patterns under different physiological conditions (media viscosity, volume, and peristaltic wave speed). During local contractile activity, antegrade flows of 0.06–0.78 cm s−1 and backflows of −2.16–−0.21 cm s−1 were measured. Mean wall shear rates were strongly time and viscosity dependent although peaks were measured between 3.05–10.12 s−1 and 5.11–20.34 s−1 in the Dynamic Colon Model and its digital twin respectively, comparable to previous estimates of the USPII with paddle speeds of 25 and 50 rpm. It is recommended that viscosity and shear rates are considered when designing future dissolution test methodologies for colon-targeted formulations. In the USPII, paddle speeds >50 rpm may not recreate physiologically relevant shear rates. These findings demonstrate how the combination of biorelevant in vitro and in silico models can provide new insights for dissolution testing beyond established pharmacopeial methods.

Nuclear Fragility in Radiation-Induced Senescence: Blebs and Tubes Visualized by 3D Electron Microscopy

Irreparable DNA damage following ionizing radiation (IR) triggers prolonged DNA damage response and induces premature senescence. Cellular senescence is a permanent state of cell-cycle arrest characterized by chromatin restructuring, altered nuclear morphology and acquisition of secretory phenotype, which contributes to senescence-related inflammation. However, the mechanistic connections for radiation-induced DNA damage that trigger these senescence-associated hallmarks are poorly understood. In our in vitro model of radiation-induced senescence, mass spectrometry-based proteomics was combined with high-resolution imaging techniques to investigate the interrelations between altered chromatin compaction, nuclear envelope destabilization and nucleo-cytoplasmic chromatin blebbing. Our findings confirm the general pathophysiology of the senescence-response, with disruption of nuclear lamin organization leading to extensive chromatin restructuring and destabilization of the nuclear membrane with release of chromatin fragments into the cytosol, thereby activating cGAS-STING-dependent interferon signaling. By serial block-face scanning electron microscopy (SBF-SEM) whole-cell datasets were acquired to investigate the morphological organization of senescent fibroblasts. High-resolution 3-dimensional (3D) reconstruction of the complex nuclear shape allows us to precisely visualize the segregation of nuclear blebs from the main nucleus and their fusion with lysosomes. By multi-view 3D electron microscopy, we identified nanotubular channels formed in lamin-perturbed nuclei of senescent fibroblasts; the potential role of these nucleo-cytoplasmic nanotubes for expulsion of damaged chromatin has to be examined.

Human immunocompetent choroid-on-chip: a novel tool for studying ocular effects of biological drugs

Disorders of the eye leading to visual impairment are a major issue that affects millions of people. On the other side ocular toxicities were described for e.g. molecularly targeted therapies in oncology and may hamper their development. Current ocular model systems feature a number of limitations affecting human-relevance and availability. To find new options for pharmacological treatment and assess mechanisms of toxicity, hence, novel complex model systems that are human-relevant and readily available are urgently required. Here, we report the development of a human immunocompetent Choroid-on-Chip (CoC), a human cell-based in vitro model of the choroid layer of the eye integrating melanocytes and microvascular endothelial cells, covered by a layer of retinal pigmented epithelial cells. Immunocompetence is achieved by perfusion of peripheral immune cells. We demonstrate controlled immune cell recruitment into the stromal compartments through a vascular monolayer and in vivo-like cytokine release profiles. To investigate applicability for both efficacy testing of immunosuppressive compounds as well as safety profiling of immunoactivating antibodies, we exposed the CoCs to cyclosporine and tested CD3 bispecific antibodies.

Untargeted Metabolomics Combined with Solid Phase Fractionation for Systematic Characterization of Bioactive Compounds in Hemp with Methane Mitigation Potential

This study systematically evaluates the presence of methane mitigating metabolites in two hemp (Cannabis sativa L.) varieties, Futura 75 and Finola. Hemp metabolites were extracted with methanol and fractionated using Solid Phase Extraction (SPE). Extracts, fractions, and the remaining pulp were screened for their methane mitigating potential using an in vitro model of rumen fermentation. The bioactive metabolites were identified with Liquid Chromatography-Mass Spectrometry (LC-MS). When incubated with a standard feed (maize silage), the extract of Futura 75 significantly reduced methane production compared to that of control (without added extract) and without negative effects on feed degradability and volatile fatty acid patterns. The compounds responsible for the methane mitigating effect were assigned to flavonoid glycosides. However, none of the fractions of Futura 75 or the pulp exhibited similar effect on methane emission. Butyric acid concentration in the fermentation inoculum was significantly increased, which could indicate why methane production was higher, when incubated with the fractions and the pulp. The extract of Finola did not show a similar significant effect, however, there was a numerical tendency towards lower methane production. The difference in methane mitigating properties between Cannabis sativa L. Futura 75 and Finola, could be related to the content of bioactive flavonoids.

Genotoxicity Response of Fibroblast Cells and Human Epithelial Adenocarcinoma In Vitro Model Exposed to Bare and Ozone-Treated Silica Microparticles

Indoor air pollutants (IAP), which can pose a serious risk to human health, include biological pollutants, nitric oxide (NO), nitrogen dioxide (NO2), volatile organic compounds (VOC), sulfur dioxide (SO2), carbon monoxide (CO), carbon dioxide (CO2), silica, metals, radon, and particulate matter (PM). The aim of our work is to conduct a multidisciplinary study of fine silica particles (<2.5 μm) in the presence or absence of ozone (O3), and evaluate their potential cytotoxicity using MTS, micronucleus, and the comet test in two cell lines. We analyzed A549 (human basal alveolar epithelial cell adenocarcinoma) and Hs27 (human normal fibroblasts) exposed to dynamic conditions by an IRC simulator under ozone flow (120 ppb) and in the presence of silica particles (40 μg/h). The viability of A549 and Hs27 cells at 48 and 72 h of exposure to silica or silica/ozone decreases, except at 72 h in Hs27 treated with silica/ozone. The micronucleus and comet tests showed a significant increase in the number of micronuclei and the % of DNA in the queue, compared to the control, in both lines in all treatments, even if in different cell times/types. We found that silica alone or with more O3 causes more pronounced genotoxic effects in A549 tumor cells than in normal Hs27 fibroblasts.

Developmental Brain Asymmetry. The Good and the Bad Sides

Brain asymmetry is a hallmark of the human brain. Recent studies report a certain degree of abnormal asymmetry of brain lateralization between left and right brain hemispheres can be associated with many neuropsychiatric conditions. In this regard, some questions need answers. First, the accelerated brain asymmetry is programmed during the pre-natal period that can be called “accelerated brain decline clock”. Second, can we find the right biomarkers to predict these changes? Moreover, can we establish the dynamics of these changes in order to identify the right time window for proper interventions that can reverse or limit the neurological decline? To find answers to these questions, we performed a systematic online search for the last 10 years in databases using keywords. Conclusion: we need to establish the right in vitro model that meets human conditions as much as possible. New biomarkers are necessary to establish the “good” or the “bad” borders of brain asymmetry at the epigenetic and functional level as early as possible.

Human Brain Organoids as an In Vitro Model System of Viral Infectious Diseases

Brain organoids, or brainoids, have shown great promise in the study of central nervous system (CNS) infection. Modeling Zika virus (ZIKV) infection in brain organoids may help elucidate the relationship between ZIKV infection and microcephaly. Brain organoids have been used to study the pathogenesis of SARS-CoV-2, human immunodeficiency virus (HIV), HSV-1, and other viral infections of the CNS. In this review, we summarize the advances in the development of viral infection models in brain organoids and their potential application for exploring mechanisms of viral infections of the CNS and in new drug development. The existing limitations are further discussed and the prospects for the development and application of brain organs are prospected.

Neuroprotective Potential of Bacopa monnieri: Modulation of Inflammatory Signals

Background: To date, much evidence has shown theincreased interest in natural molecules and traditional herbal medicine as alternative bioactive compounds to fight many inflammatory conditions, both in relation to immunomodulation and in terms of their wound healing potential. Bacopa monnieri is a herb that is used in the Ayurvedic medicine tradition for its anti-inflammatory activity. Objective: In this study, we evaluate the anti-inflammatory and regenerative properties of the Bacopa monnieri extract (BME) in vitro model of neuroinflammation. Methods: Neuronal SH-SY5Y cells were stimulated with TNF and IFN and used to evaluate the effect of BME on cell viability, cytotoxicity, cytokine gene expression, and healing rate. Results: Our results showed that BME protects against the Okadaic acid-induced cytotoxicity in SH-SY5Y cells. Moreover, in TNF and IFN primed cells, BME reduces IL-1, IL-6, COX-2, and iNOS, mitigates the mechanical trauma injury-induced damage, and accelerates the healing of wounds. Conclusion: This study indicates that BME might become a promising candidate for the treatment of neuroinflammation.

A Novel in vitro Model Delineating Hair Cell Regeneration and Neural Reinnervation in Adult Mouse Cochlea

The study of an adult mammalian auditory system, such as regeneration, has been hampered by the lack of an in vitro system in which hypotheses can be tested efficiently. This is primarily due to the fact that the adult inner ear is encased in the toughest bone of the body, whereas its removal leads to the death of the sensory epithelium in culture. We hypothesized that we could take advantage of the integral cochlear structure to maintain the overall inner ear architecture and improve sensory epithelium survival in culture. We showed that by culturing adult mouse cochlea with the (surrounding) bone intact, the supporting cells (SCs) survived and almost all hair cells (HCs) degenerated. To evaluate the utility of the explant culture system, we demonstrated that the overexpression of Atoh1, an HC fate-determining factor, is sufficient to induce transdifferentiation of adult SCs to HC-like cells (HCLCs). Transdifferentiation-derived HCLCs resemble developmentally young HCs and are able to attract adult ganglion neurites. Furthermore, using a damage model, we showed that degenerated adult ganglions respond to regenerated HCLCs by directional neurite outgrowth that leads to HCLC-neuron contacts, strongly supporting the intrinsic properties of the HCLCs in establishing HCLC-neuron connections. The adult whole cochlear explant culture is suitable for diverse studies of the adult inner ear including regeneration, HC-neuron pathways, and inner ear drug screening.