Differential Mitochondrial, Oxidative Stress and Inflammatory Responses to SARS-CoV-2 Spike Protein Receptor Binding Domain in Human Lung Microvascular, Coronary Artery Endothelial and Bronchial Epithelial Cells.

Recent evidence indicates that the SARS-CoV-2 spike protein affects mitochondria with a cell type-dependent outcome. We elucidate the effect of the SARS-CoV-2 receptor binding domain (RBD) on the mitochondrial network and cristae morphology, oxygen consumption, mitoROS production, and inflammatory cytokine expression in cultured human lung microvascular (HLMVECs), coronary artery endothelial (HCAECs), and bronchial epithelial cells (HBECs). Live Mito Orange staining, STED microscopy, and Fiji MiNa analysis were used for mitochondrial cristae and network morphometry; an Agilent XFp analyser for mitochondrial/glycolytic activity; MitoSOX fluorescence for mitochondrial ROS; and qRT-PCR plus Luminex for cytokines. HLMVEC exposure to SARS-CoV-2 RBD resulted in the fragmentation of the mitochondrial network, mitochondrial swelling, increased cristae area, reduced cristae density, and suppressed mitochondrial oxygen consumption and glycolysis. No significant mitochondrial morphology or oxygen consumption changes were observed in HCAECs and HBECs. SARS-CoV-2 RBD induced mitoROS-mediated expression of cytokines GM-CSF and IL-1ß in all three investigated cell types, along with IL-8 expression in both endothelial cell types. The findings suggest mitochondrial ROS control SARS-CoV-2 RBD-induced inflammation in HLMVECs, HCAECs, and HBECs, with the mitochondria of HLMVECs being more sensitive to SARS-CoV-2 RBD.

Assessing the Therapeutic Impacts of HAMLET and FOLFOX on BRAF-Mutated Colorectal Cancer: A Study of Cancer Cell Survival and Mitochondrial Dynamics In Vitro and Ex Vivo.

Background and Objectives: Colorectal cancer (CRC) is a major global health challenge. The BRAF V600E mutation, found in 8-12% of CRC patients, exacerbates this by conferring poor prognosis and resistance to therapy. Our study focuses on the efficacy of the HAMLET complex, a molecular substance derived from human breast milk, on CRC cell lines and ex vivo biopsies harboring this mutation, given its previously observed selective toxicity to cancer cells. Materials and Methods: we explored the effects of combining HAMLET with the FOLFOX chemotherapy regimen on CRC cell lines and ex vivo models. Key assessments included cell viability, apoptosis/necrosis induction, and mitochondrial function, aiming to understand the mutation-specific resistance or other cellular response mechanisms. Results: HAMLET and FOLFOX alone decreased viability in CRC explants, irrespective of the BRAF mutation status. Notably, their combination yielded a marked decrease in viability, particularly in the BRAF wild-type samples, suggesting a synergistic effect. While HAMLET showed a modest inhibitory effect on mitochondrial respiration across both mutant and wild-type samples, the response varied depending on the mutation status. Significant differences emerged in the responses of the HT-29 and WiDr cell lines to HAMLET, with WiDr cells showing greater resistance, pointing to factors beyond genetic mutations influencing drug responses. A slight synergy between HAMLET and FOLFOX was observed in WiDr cells, independent of the BRAF mutation. The bioenergetic analysis highlighted differences in mitochondrial respiration between HT-29 and WiDr cells, suggesting that bioenergetic profiles could be key in determining cellular responses to HAMLET. Conclusions: We highlight the potential of HAMLET and FOLFOX as a combined therapeutic approach in BRAF wild-type CRC, significantly reducing cancer cell viability. The varied responses in CRC cell lines, especially regarding bioenergetic and mitochondrial factors, emphasize the need for a comprehensive approach considering both genetic and metabolic aspects in CRC treatment strategies.

The Effect of SARS-CoV-2 Spike Protein RBD-Epitope on Immunometabolic State and Functional Performance of Cultured Primary Cardiomyocytes Subjected to Hypoxia and Reoxygenation.

Cardio complications such as arrhythmias and myocardial damage are common in COVID-19 patients. SARS-CoV-2 interacts with the cardiovascular system primarily via the ACE2 receptor. Cardiomyocyte damage in SARS-CoV-2 infection may stem from inflammation, hypoxia-reoxygenation injury, and direct toxicity; however, the precise mechanisms are unclear. In this study, we simulated hypoxia-reoxygenation conditions commonly seen in SARS-CoV-2-infected patients and studied the impact of the SARS-CoV-2 spike protein RBD-epitope on primary rat cardiomyocytes to gain insight into the potential mechanisms underlying COVID-19-related cardiac complications. Cell metabolic activity was evaluated with PrestoBlueTM. Gene expression of proinflammatory markers was measured by qRT-PCR and their secretion was quantified by Luminex assay. Cardiomyocyte contractility was analysed using the Myocyter plugin of ImageJ. Mitochondrial respiration was determined through Seahorse Mito Stress Test. In hypoxia-reoxygenation conditions, treatment of the SARS-CoV-2 spike RBD-epitope reduced the metabolic activity of primary cardiomyocytes, upregulated Il1ß and Cxcl1 expression, and elevated GM-CSF and CCL2 cytokines secretion. Contraction time increased, while amplitude and beating frequency decreased. Acute treatment with a virus RBD-epitope inhibited mitochondrial respiration and lowered ATP production. Under ischaemia-reperfusion, the SARS-CoV-2 RBD-epitope induces cardiomyocyte injury linked to impaired mitochondrial activity.

Species-Specific Plant-Derived Nanoparticle Characteristics.

Medicinal and agricultural plants contain numerous phytochemical compounds with pronounced biological effects on human health. They are known to encapsulate most of their characteristic bioactive compounds within membranous elements of intercellular communication known as exosomes. These nanovesicles serve as capsules protecting their biological activity and improving their penetration into the tissue. Therefore, the application of plant exosome preparations holds considerable potential for cosmetics and pharmacy, but the quality and consistency of plant material for exosome isolation is of critical importance. Therefore, in this study, we aimed to evaluate yield, size distribution patterns, and antioxidant properties between nanovesicle preparations of the following portfolio of medicinal plants: Kalanchoe daigremontiana, Artemisia absinthium, Hypericum perforatum, Silybum marianum, Chelidonium majus, and Scutellaria baicalensis. Results showed that nanoparticle yield, size distribution, and antioxidant activities were specific to plant species. Compared to other plants, nanoparticle preparations from Artemisia absinthium were distinguished by remarkably higher yield and concentration, while the highest antioxidant activity of plant-derived nanoparticle preparations per weight and per particle was determined to occur in Chelidonium majus and Hypericum perforatum samples. Results showed no significant correlation in DPPH (2-diphenyl-1-picrylhydrazyl) free radical scavenging activity and FRAP (ferric reducing antioxidant power) between plant material and nanoparticle preparations. More detailed biochemical analysis of exosome preparations is necessary to validate their biological activity and its relation to source plant cells.

Comparison of Microglial Morphology and Function in Primary Cerebellar Cell Cultures on Collagen and Collagen-Mimetic Hydrogels.

Neuronal-glial cell cultures are usually grown attached to or encapsulated in an adhesive environment as evenly distributed networks lacking tissue-like cell density, organization and morphology. In such cultures, microglia have activated amoeboid morphology and do not display extended and intensively branched processes characteristic of the ramified tissue microglia. We have recently described self-assembling functional cerebellar organoids promoted by hydrogels containing collagen-like peptides (CLPs) conjugated to a polyethylene glycol (PEG) core. Spontaneous neuronal activity was accompanied by changes in the microglial morphology and behavior, suggesting the cells might play an essential role in forming the functional neuronal networks in response to the peptide signalling. The present study examines microglial cell morphology and function in cerebellar cell organoid cultures on CLP-PEG hydrogels and compares them to the cultures on crosslinked collagen hydrogels of similar elastomechanical properties. Material characterization suggested more expressed fibril orientation and denser packaging in crosslinked collagen than CLP-PEG. However, CLP-PEG promoted a significantly higher microglial motility (determined by time-lapse imaging) accompanied by highly diverse morphology including the ramified (brightfield and confocal microscopy), more active Ca2+ signalling (intracellular Ca2+ fluorescence recordings), and moderate inflammatory cytokine level (ELISA). On the contrary, on the collagen hydrogels, microglial cells were significantly less active and mostly round-shaped. In addition, the latter hydrogels did not support the neuron synaptic activity. Our findings indicate that the synthetic CLP-PEG hydrogels ensure more tissue-like microglial morphology, motility, and function than the crosslinked collagen substrates.

Virus Mimetic Poly (I:C)-Primed Airway Exosome-like Particles Enter Brain and Induce Inflammatory Cytokines and Mitochondrial Reactive Oxygen Species in Microglia.

Viral infections induce extracellular vesicles (EVs) containing viral material and inflammatory factors. Exosomes can easily cross the blood-brain barrier during respiratory tract infection and transmit the inflammatory signal to the brain; however, such a hypothesis has no experimental evidence. The study investigated whether exosome-like vesicles (ELVs) from virus mimetic poly (I:C)-primed airway cells enter the brain and interact with brain immune cells microglia. Airway cells were isolated from Wistar rats and BALB/c mice; microglial cell cultures-from Wistar rats. ELVs from poly (I:C)-stimulated airway cell culture medium were isolated by precipitation, visualised by transmission electron microscopy, and evaluated by nanoparticle analyser; exosomal markers CD81 and CD9 were determined by ELISA. For in vitro and in vivo tracking, particles were loaded with Alexa Fluor 555-labelled RNA. Intracellular reactive oxygen species (ROS) were evaluated by DCFDA fluorescence and mitochondrial superoxide-by MitoSOX. ELVs from poly (I:C)-primed airway cells entered the brain within an hour after intranasal introduction, were internalised by microglia and induced intracellular and intramitochondrial ROS production. There was no ROS increase in microglial cells was after treatment with ELVs from airway cells untreated with poly (I:C). In addition, poly (I:C)-primed airway cells induced inflammatory cytokine expression in the brain. The data indicate that ELVs secreted by virus-primed airway cells might enter the brain, cause the activation of microglial cells and neuroinflammation.

The Role of Mitochondria in Brain Cell Protection from Ischaemia by Differently Prepared Propolis Extracts.

Mitochondria are both the primary targets and mediators of ischaemic damage in brain cells. Insufficient oxygen causes reactive oxygen species that damage the mitochondria, leading to the loss of functionality and viability of highly energy-demanding neurons. We have recently found that aqueous (AqEP), polyethylene glycol-aqueous (Pg-AqEP) and ethanolic propolis extracts (EEP) can modulate mitochondria and ROS production in C6 cells of astrocytic origin. The aim of this study was to investigate the effect of the extracts on viability, mitochondrial efficiency and superoxide generation, and inflammatory cytokine release in primary rat cerebellar neuronal-glial cell cultures affected by ischaemia (mimicked by hypoxia +/- deoxyglucose). AqEP and Pg-AqEP (15-60 µg/mL of phenolic compounds, or PC) significantly increased neuronal viability in ischaemia-treated cultures, and this was accompanied by a reduction in mitochondrial superoxide levels. Less extended protection against ischaemia-induced superoxide production and death was exhibited by 2 to 4 µg/mL of PC EEP. Both Pg-AqEP and Ag-EP (but not EEP) significantly protected the cultures from hypoxia-induced elevation of TNF-α, IL-1ß and IL-6. Only Pg-AqEP (but not AqEP or EEP) prevented hypoxia-induced loss of the mitochondrial basal and ATP-coupled respiration rate, and significantly increased the mitochondrial respiratory capacity. Summarising, the study revealed that hydrophilic propolis extracts might protect brain cells against ischaemic injury by decreasing the level of mitochondrial superoxide and preventing inflammatory cytokines, and, in the case of Pg-AqEP, by protecting mitochondrial function.

Investigation of Phenolic Composition and Anticancer Properties of Ethanolic Extracts of Japanese Quince Leaves.

Glioblastoma multiforme is an aggressive and invasive disease with no efficient therapy available, and there is a great need for finding alternative treatment strategies. This study aimed to investigate anticancer activity of the extracts of the Japanese quince (JQ) cultivars 'Darius', 'Rondo', and 'Rasa' leaf extracts on glioblastoma C6 and HROG36 cells. As identified by ultra high performance liquid chromatography electrospray ionization tandem mass spectrometry, the extracts contained three prevailing groups of phenols: hydroxycinnamic acid derivatives; flavan-3-ols; and flavonols. Sixteen phenols were detected; the predominant compound was chlorogenic acid. The sum of detected phenols varied significantly between the cultivars ranging from 9322 µg/g ('Rondo') to 17,048 µg/g DW ('Darius'). Incubation with the extracts decreased the viability of glioblastoma HROG36 cells with an efficiency similar to temozolomide, a drug used for glioblastoma treatment. In the case of C6 glioblastoma cells, the extracts were even more efficient than temozolomide. Interestingly, primary cerebellar neuronal-glial cells were significantly less sensitive to the extracts compared to the cancer cell lines. The results showed that JQ leaf ethanol extracts are rich in phenolic compounds, can efficiently reduce glioblastoma cell viability while preserving non-cancerous cells, and are worth further investigations as potential anticancer drugs.

Investigation of Cancer Cell Migration and Proliferation on Synthetic Extracellular Matrix Peptide Hydrogels.

Chemical and mechanical properties of a tumor microenvironment are essential players in cancer progression, and it is important to precisely control the extracellular conditions while designing cancer in vitro models. The study investigates synthetic hydrogel matrices from multi-arm polyethylene glycol (PEG) functionalized with collagen-like peptide (CLP) CG(PKG)4(POG)4(DOG)4 alone and conjugated with either cell adhesion peptide RGD (mimicking fibronectin) or IKVAV (mimicking laminin). Human glioblastoma HROG36, rat C6 glioma cells, and A375 human melanoma cells were grown on the hydrogels and monitored for migration, proliferation, projected cell area, cell shape index, size and number, distribution of focal contacts in individual cells, and focal adhesion number. PEG-CLP-RGD induced migration of both glioma cell lines and also stimulated proliferation (assessed as metabolic activity) of HROG36 cells. Migration of C6 cells were also stimulated by PEG-CLP-IKVAV. These responses strongly correlated with the changes in adhesion and morphology parameters of individual cells - projected cell area, cell shape index, and focal contact number. Melanoma A375 cell proliferation was increased by PEG-CLP-RGD, and this was accompanied by a decrease in cell shape index. However, neither RGD nor IKVAV conjugated to PEG-CLP stimulated migratory capacity of A375 cells. Taken together, the study presents synthetic scaffolds with extracellular matrix (ECM)-mimicking peptides that allow for the exploration of the effect of ECM signaling to cancer cells.

Cerebellar Cells Self-Assemble into Functional Organoids on Synthetic, Chemically Crosslinked ECM-Mimicking Peptide Hydrogels.

Hydrogel-supported neural cell cultures are more in vivo-relevant compared to monolayers formed on glass or plastic substrates. However, there is a lack of synthetic microenvironment available for obtaining standardized and easily reproducible cultures characterized by tissue-mimicking cell composition, cell-cell interactions, and functional networks. Synthetic peptides representing the biological properties of the extracellular matrix (ECM) proteins have been reported to promote the adhesion-driven differentiation and functional maturation of neural cells. Thus, such peptides can serve as building blocks for engineering a standardized, all-synthetic environment. In this study, we have compared the effect of two chemically crosslinked hydrogel compositions on primary cerebellar cells: collagen-like peptide (CLP), and CLP with an integrin-binding motif arginine-glycine-aspartate (CLP-RGD), both conjugated to polyethylene glycol molecular templates (PEG-CLP and PEG-CLP-RGD, respectively) and fabricated as self-supporting membranes. Both compositions promoted a spontaneous organization of primary cerebellar cells into tissue-like clusters with fast-rising Ca2+ signals in soma, reflecting action potential generation. Notably, neurons on PEG-CLP-RGD had more neurites and better synaptic efficiency compared to PEG-CLP. For comparison, poly-L-lysine-coated glass and plastic surfaces did not induce formation of such spontaneously active networks. Additionally, contrary to the hydrogel membranes, glass substrates functionalized with PEG-CLP and PEG-CLP-RGD did not sufficiently support cell attachment and, subsequently, did not promote functional cluster formation. These results indicate that not only chemical composition but also the hydrogel structure and viscoelasticity are essential for bioactive signaling. The synthetic strategy based on ECM-mimicking, multifunctional blocks in registry with chemical crosslinking for obtaining tissue-like mechanical properties is promising for the development of fast and well standardized functional in vitro neural models and new regenerative therapies.