Comparison of primary and passaged tumor cell cultures and their application in personalized medicine.

Passaged cell lines represent currently an integral component in various studies of malignant neoplasms. These cell lines are utilized for drug screening both in monolayer cultures or as part of three-dimensional (3D) tumor models. They can also be used to model the tumor microenvironment in vitro and in vivo through xenotransplantation into immunocompromised animals. However, immortalized cell lines have some limitations of their own. The homogeneity of cell line populations and the extensive passaging in monolayer systems make these models distant from the original disease. Recently, there has been a growing interest among scientists in the use of primary cell lines, as these are passaged directly from human tumor tissues. In this case, cells retain the morphological and functional characteristics of the tissue from which they were derived, an advantage often not observed in passaged cultures. This review highlights the advantages and limitations of passaged and primary cell cultures, their similarities and differences, as well as existing test systems that are based on primary and passaged cell cultures for drug screening purposes.

Pro-inflammatory and genotoxic responses by metal oxide nanomaterials in alveolar epithelial cells and macrophages in submerged condition and air-liquid interface: An in vitro-in vivo correlation study.

Studies on in vitro-in vivo correlations of inflammatory and genotoxic responses are needed to advance new approach methodologies. Here, we assessed pro-inflammatory and genotoxic responses by 13 nanosized metal oxides (nMeOx) and quartz (DQ12) in alveolar epithelial cells (A549) and macrophages (THP-1a) exposed in submerged conditions, and in A549:THP-1a co-cultures in air-liquid interface (ALI) system. Soluble nMeOx produced the highest IL-8 expression in A549 and THP-1a cells in submerged conditions (≥2-fold, p < 0.05), whereas only CuO caused a strong response in co-cultures exposed in the ALI system (13-fold, p < 0.05). IL-8 expression in A549 cells with concentrations as nMeOx specific surface area (SSA) correlated with neutrophil influx in mice (r = 0.89-0.98, p < 0.05). Similarly, IL-8 expression in THP-1a with concentrations as mass and SSA (when excluding soluble nMeOx) correlated with neutrophil influx in mice (r = 0.81-0.84, p < 0.05). DNA strand breaks (SB) was measured by the comet assay. We used a scoring system that categorizes effects in standard deviation units for comparison of genotoxicity in different models. Concordant genotoxicity was observed between SB levels in vitro (A549 and co-culture) and in vivo (broncho-alveolar lavage fluid cells and lung tissue). In conclusion, this study shows in vitro-in vivo correlations of nMeOx-induced inflammatory and genotoxic responses.

Effect of Xanthohumol, a Bioactive Natural Compound from Hops, on Adenosine Pathway in Rat C6 Glioma and Human SH-SY5Y Neuroblastoma Cell Lines.

Xanthohumol (Xn) is an antioxidant flavonoid mainly extracted from hops (Humulus lupulus), one of the main ingredients of beer. As with other bioactive compounds, their therapeutic potential against different diseases has been tested, one of which is Alzheimer's disease (AD). Adenosine is a neuromodulatory nucleoside that acts through four different G protein-coupled receptors: A1 and A3, which inhibit the adenylyl cyclases (AC) pathway, and A2A and A2B, which stimulate this activity, causing either a decrease or an increase, respectively, in the release of excitatory neurotransmitters such as glutamate. This adenosinergic pathway, which is altered in AD, could be involved in the excitotoxicity process. Therefore, the aim of this work is to describe the effect of Xn on the adenosinergic pathway using cell lines. For this purpose, two different cellular models, rat glioma C6 and human neuroblastoma SH-SY5Y, were exposed to a non-cytotoxic 10 µM Xn concentration. Adenosine A1 and A2A, receptor levels, and activities related to the adenosine pathway, such as adenylate cyclase, protein kinase A, and 5'-nucleotidase, were analyzed. The adenosine A1 receptor was significantly increased after Xn exposure, while no changes in A2A receptor membrane levels or AC activity were reported. Regarding 5'-nucleotidases, modulation of their activity by Xn was noted since CD73, the extracellular membrane attached to 5'-nucleotidase, was significantly decreased in the C6 cell line. In conclusion, here we describe a novel pathway in which the bioactive flavonoid Xn could have potentially beneficial effects on AD as it increases membrane A1 receptors while modulating enzymes related to the adenosine pathway in cell cultures.

Multidrug-Resistant Profiles in Non-Small Cell Lung Carcinoma Patient-Derived Cells: Implications for Personalized Approaches with Tyrosine Kinase Inhibitors.

The impact of tyrosine kinase inhibitors (TKIs) on multidrug resistance (MDR) in non-small cell lung carcinoma (NSCLC) is a critical aspect of cancer therapy. While TKIs effectively target specific signaling pathways of cancer cells, they can also act as substrates for ABC transporters, potentially triggering MDR. The aim of our study was to evaluate the response of 17 patient-derived NSCLC cultures to 10 commonly prescribed TKIs and to correlate these responses with patient mutational profiles. Using an ex vivo immunofluorescence assay, we analyzed the expression of the MDR markers ABCB1, ABCC1, and ABCG2, and correlated these data with the genetic profiles of patients for a functional diagnostic approach. NSCLC cultures responded differently to TKIs, with erlotinib showing good efficacy regardless of mutation burden or EGFR status. However, the modulation of MDR mechanisms by erlotinib, such as increased ABCG2 expression, highlights the challenges associated with erlotinib treatment. Other TKIs showed limited efficacy, highlighting the variability of response in NSCLC. Genetic alterations in signaling pathways associated with drug resistance and sensitivity, including TP53 mutations, likely contributed to the variable responses to TKIs. The relationships between ABC transporter expression, gene alterations, and response to TKIs did not show consistent patterns. Our results suggest that in addition to mutational status, performing functional sensitivity screening is critical for identifying appropriate treatment strategies with TKIs. These results underscore the importance of considering drug sensitivity, off-target effects, MDR risks, and patient-specific genetic profiles when optimizing NSCLC treatment and highlight the potential for personalized approaches, especially in early stages.

From Ethnobotany to Biotechnology: Wound Healing and Anti-Inflammatory Properties of Sedum telephium L. In Vitro Cultures.

Sedum telephium is a succulent plant used in traditional medicine, particularly in Italy, for its efficacy in treating localized inflammation such as burns, warts, and wounds. Fresh leaves or freshly obtained derivatives are directly applied to the injuries for these purposes. However, challenges such as the lack of microbiologically controlled materials and product standardization prompted the exploration of more controlled biotechnological alternatives, utilizing in vitro plant cell cultures of S. telephium. In the present study, we used HPLC-DAD analysis to reveal a characteristic flavonol profile in juices from in vivo leaves and in vitro materials mainly characterized by several kaempferol and quercetin derivatives. The leaf juice exhibited the highest content in total flavonol and kaempferol derivatives, whereas juice from callus grown in medium with hormones and callus suspensions showed elevated levels of quercetin derivatives. The in vitro anti-inflammatory and wound-healing assays evidenced the great potential of callus and suspension cultures in dampening inflammation and fostering wound closure, suggesting quercetin may have a pivotal role in biological activities.

The Link Between Matrix Metalloproteinases and Alzheimer's Disease Pathophysiology.

Alzheimer's disease (AD) is a major contributor to dementia and the most common neurodegenerative disorder. In AD pathophysiology, matrix metalloproteinases (MMPs)-proteolytic enzymes, best known to be responsible for remodeling and degradation of the extracellular matrix-were suggested to play an important role. Due to the diverse nature of the published data and frequent inconsistent results presented in available papers, it was considered essential to analyze all aspects of MMP literature with respect to AD pathophysiology and attempt to outline a unifying concept for understanding their role in AD. Thus, the main contribution of this review article is to summarize the most recent research on the participation of MMP in AD pathophysiology obtained using the cell cultures to understand the molecular principles of their action. Furthermore, an updated comprehensive view regarding this topic based exclusively on papers from human studies is provided as well. It can be concluded that determining the exact role of any particular MMPs in the AD pathophysiology holds promise for establishing their role as potential biomarkers reflecting the severity or progression of this disease or for developing new therapeutic agents targeting the processes that lead to AD.

Characterization of ERα Signaling to Cell Proliferation Induced by Chronic and Pulsatile E2 Stimulation in 2D and 3D Cell Cultures.

17ß-estradiol is a hormone that plays a vital role in human physiology. It acts through estrogen receptors, specifically estrogen receptor α and estrogen receptor ß, and its action is determined by the pulsatile secretion in the bloodstream. 17ß-estradiol affects cell proliferation, and dysregulation of 17ß-estradiol:estrogen receptor α signaling contribute to the development of breast cancer. Previous research on 17ß-estradiol:estrogen receptor α signaling has primarily used two-dimensional cell cultures, which do not fully recapitulate the complexity of tumors that exist in a three-dimensional environment and do not consider the pulsatile nature of this hormone. To address these limitations, we studied 17ß-estradiol:estrogen receptor α signaling in cell proliferation using both two-dimensional and three-dimensional breast cancer cell culture models under continuous and pulsatile stimulation conditions. Results revealed that breast cancer cells grown in an alginate-based three-dimensional matrix exhibited similar responsiveness to 17ß-estradiol compared with cells grown in conventional two-dimensional culture plates. 17ß-estradiol induced the expression of proteins containing estrogen response element in the three-dimensional model. The efficacy of the antiestrogen drugs fulvestrant (ICI182,280) and 4OH-tamoxifen was also demonstrated in the three-dimensional model. These results support the use of the three-dimensional culture model for studying tumor response to drugs and provide a more realistic microenvironment for such studies. Furthermore, the study revealed that a brief 5-min exposure to 17ß-estradiol triggered a physiological response comparable with continuous hormone exposure, suggesting that the cellular response to 17ß-estradiol is more important than the continuous presence of the hormone. In conclusion, the study demonstrates that the alginate-based three-dimensional culture model is suitable for studying the effects of 17ß-estradiol and antiestrogen drugs on breast cancer cells, offering a more realistic representation of tumor-microenvironment interactions. The results also highlight the importance of considering the physiological importance of the temporal dynamics in studying 17ß-estradiol signaling and cellular responses.

Growing Role of 3D In Vitro Cell Cultures in the Study of Cellular and Molecular Mechanisms: Short Focus on Breast Cancer, Endometriosis, Liver and Infectious Diseases.

Over the past decade, the development of three-dimensional (3D) models has increased exponentially, facilitating the unravelling of fundamental and essential cellular mechanisms by which cells communicate with each other, assemble into tissues and organs and respond to biochemical and biophysical stimuli under both physiological and pathological conditions. This section presents a concise overview of the most recent updates on the significant contribution of different types of 3D cell cultures including spheroids, organoids and organ-on-chip and bio-printed tissues in advancing our understanding of cellular and molecular mechanisms. The case studies presented include the 3D cultures of breast cancer (BC), endometriosis, the liver microenvironment and infections. In BC, the establishment of 3D culture models has permitted the visualization of the role of cancer-associated fibroblasts in the delivery of exosomes, as well as the significance of the physical properties of the extracellular matrix in promoting cell proliferation and invasion. This approach has also become a valuable tool in gaining insight into general and specific mechanisms of drug resistance. Given the considerable heterogeneity of endometriosis, 3D models offer a more accurate representation of the in vivo microenvironment, thereby facilitating the identification and translation of novel targeted therapeutic strategies. The advantages provided by 3D models of the hepatic environment, in conjunction with the high throughput characterizing various platforms, have enabled the elucidation of complex molecular mechanisms underlying various threatening hepatic diseases. A limited number of 3D models for gut and skin infections have been developed. However, a more profound comprehension of the spatial and temporal interactions between microbes, the host and their environment may facilitate the advancement of in vitro, ex vivo and in vivo disease models. Additionally, it may pave the way for the development of novel therapeutic approaches in diverse research fields. The interested reader will also find concluding remarks on the challenges and prospects of using 3D cell cultures for discovering cellular and molecular mechanisms in the research areas covered in this review.

Antineoplastic Effect of ALK Inhibitor Crizotinib in Primary Human Anaplastic Thyroid Cancer Cells with STRN-ALK Fusion In Vitro.

Anaplastic thyroid cancer (ATC) is one of the deadliest human cancers and represents <2% of thyroid carcinomas. A therapeutic target for ATC is represented by anaplastic lymphoma kinase (ALK) rearrangements, involved in tumor growth. Crizotinib is an oral small-molecule tyrosine kinase inhibitor of the ALK, MET, and ROS1 kinases, approved in ALK-positive non-small cell lung cancer. Until now, the effect of crizotinib in "primary human ATC cells" (pATCs) with transforming striatin (STRN)-ALK fusion has not been reported in the literature. In this study, we aimed to obtain pATCs with STRN-ALK in vitro and evaluate the in vitro antineoplastic action of crizotinib. Thyroid surgical samples were obtained from 12 ATC patients and 6 controls (who had undergone parathyroidectomy). A total of 10/12 pATC cultures were obtained, 2 of which with transforming STRN-ALK fusion (17%). Crizotinib inhibited proliferation, migration, and invasion and increased apoptosis in 3/10 pATC cultures (2 of which with/1 without STRN-ALK), particularly in those with STRN-ALK. Moreover, crizotinib significantly inhibited the proliferation of AF cells (a continuous cell line obtained from primary ATC cells). In conclusion, the antineoplastic activity of crizotinib has been shown in human pATCs (with STRN-ALK) in preclinical studies in vitro, opening the way to future clinical evaluation in these patients.

[Information support for the bioresource collection: a biological information system «NeuroOnc¼]. / Informatsionnaya podderzhka bioresursnoi kollektsii: biologicheskaya informatsionnaya sistema «NeiroOnk¼.

The manuscript is devoted to development of information support system for a bioresource collection - biological information system «NeuroOnc¼. Architecture and main functions of system are presented. This system was formed in the project «Development of bioresource collection of tumors of the human nervous system with molecular genetic certification for personalized treatment of patients with neuro-oncological diseases¼. The purpose of this project was not only formation of bioresource collection, but also development of various molecular genetic methods for analysis of biospecimens in context of clinical researches. Biological information systems created to support the work of bioresource collections in hospitals should become a natural part of information infrastructure. Information support of bioresource collections cannot imply only «warehouse¼ functions. This system should have tools to support various scientific and clinical researches. Biological information systems can sometimes expand medical information systems but remain sufficiently autonomous. It is advisable to develop biological information systems in large specialized companies that can support their products for many years.

Oxytocin Exhibits Neuroprotective Effects on Hippocampal Cultures under Severe Oxygen-Glucose Deprivation Conditions.

Perinatal asphyxia (PA) and hypoxic-ischemic encephalopathy can result in severe, long-lasting neurological deficits. In vitro models, such as oxygen-glucose deprivation (OGD), are used experimentally to investigate neuronal response to metabolic stress. However, multiple variables can affect the severity level of OGD/PA and may confound any measured treatment effect. Oxytocin (OXT) has emerged as a potential neuroprotective agent against the deleterious effects of PA. Previous studies have demonstrated OXT's potential to enhance neuronal survival in immature hippocampal cultures exposed to OGD, possibly by modulating gamma-aminobutyric acid-A receptor activity. Moreover, OXT's precise impact on developing hippocampal neurons under different severities of OGD/PA remains uncertain. In this study, we investigated the effects of OXT (0.1 µM and 1 µM) on 7-day-old primary rat hippocampal cultures subjected to 2 h OGD/sham normoxic conditions. Cell culture viability was determined using the resazurin assay. Our results indicate that the efficacy of 1 µM OXT treatment varied according to the severity of the OGD-induced lesion, exhibiting a protective effect (p = 0.022) only when cellular viability dropped below 49.41% in non-treated OGD cultures compared to normoxic ones. Furthermore, administration of 0.1 µM OXT did not yield significant effects, irrespective of lesion severity (p > 0.05). These findings suggest that 1 µM OXT treatment during OGD confers neuroprotection exclusively in severe lesions in hippocampal neurons after 7 days in vitro. Further research is warranted to elucidate the mechanisms involved in OXT-mediated neuroprotection.

Cytotoxic and Immunomodulatory Effects of Hypericin as a Photosensitizer in Photodynamic Therapy Used on Skin Cell Cultures.

Determination of the hypericin-photodynamic (HY-PDT) effect on the secretion of cytokines secreted by the skin cells, may be the basis for using the immunomodulatory effect of photodynamic action in the treatment of inflammatory skin diseases. The study aimed to evaluate the cytotoxic and immunomodulatory effects of hypericin (HY) in photodynamic therapy (PDT) performed in vitro on cultures of selected skin cell lines. The study used two human cell lines, primary dermal fibroblast (HDFa) and primary epidermal keratinocytes (HEKa). The MTT test was used to define the metabolic activity of treated cells. Cell supernatants subjected to sublethal PDT were assessed to determine the interleukins: IL-2, IL-8, IL-10, IL-11, IL-19, IL-22, and metalloproteinase 1 (MMP-1). The results confirm the destructive effect of HY-PDT and the immunomodulatory effects of sublethal doses on the selected skin cells, depending on the concentration of HY and the light doses. No statistically significant differences were noted in IL-2 and IL-10 concentration after HY-PDT for HEKa and HDFa lines. After using HY-PDT, the concentration of IL-8, MMP-1, IL-22, and IL-11 significantly decreased in the HEKa line. Moreover, the concentration of IL-19 and MMP-1 significantly decreased in the HDFa line. The concentration of IL-11 in the HDFa line after using only the HY, without the light, increased but decreased after HY-PDT. Our experiment confirmed that HY-PDT has not only a cytotoxic effect but, used in sublethal doses, also presents immunomodulatory properties. These may be an advantage of HY-PDT when used in the treatment of persistent skin inflammation, connected with the release of pro-inflammatory cytokines resistant to conventional treatment methods.

In vitro analysis of Porcine Endogenous Retroviruses in different pig cell types.

The shortage of organs for human transplantation is a topic of extreme interest, and xenotransplantation with porcine organs has been recognized as a promising solution. However, the potential spillover linked to infectious agents present in pigs remains a concern. Among these, Pig Endogenous Retroviruses (PERVs), whose proviral DNAs are integrated in the genome of all pig breeds, represent an extremely important biological risk. This study aims to evaluate PERVs distribution in several swine cell lines and samples of domestic and feral pigs. Moreover, the capacity of PERVs to infect human and non-human primate cells and to integrate in the cellular genome was tested by Real-Time PCR and by Reverse Transcriptase assay. Results indicated a widespread diffusion of PERVs both in cell lines and samples analysed: the viral genome was found in all the established cell lines, in 40% of the primary cell lines and in 60% of the tissue samples tested. The assays indicated that the virus can be transmitted from porcine to human cells: in the specific case, infected NSK and NPTr cells allow passage to human 293 and MRC-5 cells with active production of the virus demonstrable via PCR and RT assay. In light of these aspects and also the lack of studies on PERVs, it appears clear that there are still many questions to be clarified, also by means of future studies, before xenotransplantation can be considered microbiologically safe.

EcoHIV Infection of Primary Murine Brain Cell Cultures to Model HIV Replication and Neuropathogenesis.

BACKGROUND: EcoHIV is a chimeric HIV that replicates in mice in CD4+ T cells, macrophages, and microglia (but not in neurons), causing lasting neurocognitive impairment resembling neurocognitive disease in people living with HIV. The present study was designed to develop EcoHIV-susceptible primary mouse brain cultures to investigate the indirect effects of HIV infection on neuronal integrity. RESULTS: We used two EcoHIV clones encoding EGFP and mouse bone marrow-derived macrophages (BMM), mixed mouse brain cells, or enriched mouse glial cells from two wild-type mouse strains to test EcoHIV replication efficiency, the identity of productively infected cells, and neuronal apoptosis and integrity. EcoHIV replicated efficiently in BMM. In mixed brain cell cultures, EcoHIV targeted microglia but did not cause neuronal apoptosis. Instead, the productive infection of the microglia activated them and impaired synaptophysin expression, dendritic density, and axonal structure in the neurons. EcoHIV replication in the microglia and neuronal structural changes during infection were prevented by culture with an antiretroviral. CONCLUSIONS: In murine brain cell cultures, EcoHIV replication in the microglia is largely responsible for the aspects of neuronal dysfunction relevant to cognitive disease in infected mice and people living with HIV. These cultures provide a tool for further study of HIV neuropathogenesis and its control.

Hepatotoxicity assessment of innovative nutritional supplements based on olive-oil formulations enriched with natural antioxidants.

Introduction: This study focuses on the assessment of extra virgin olive-oil and olive fruit-based formulations enriched with natural antioxidants as potential nutritional supplements for alleviating symptoms and long-term consequences of illnesses whose molecular pathophysiology is affected by oxidative stress and inflammation, such as Alzheimer's disease (AD). Methods: Besides evaluating cell viability and proliferation capacity of human hepatocellular carcinoma HepG2 cells exposed to formulations in culture, hepatotoxicity was also considered as an additional safety measure using quantitative real-time PCR on RNA samples isolated from the cell cultures and applying approaches of targeted molecular analysis to uncover potential pathway effects through gene expression profiling. Furthermore, the formulations investigated in this work contrast the addition of natural extract with chemical forms and evaluate the antioxidant delivery mode on cell toxicity. Results: The results indicate minimal cellular toxicity and a significant beneficial impact on metabolic molecular pathways in HepG2 cell cultures, thus paving the way for innovative therapeutic strategies using olive-oil and antioxidants in dietary supplements to minimize the long-term effects of oxidative stress and inflammatory signals in individuals being suffered by disorders like AD. Discussion: Overall, the experimental design and the data obtained support the notion of applying innovative molecular methodologies and research techniques to evidently advance the delivery, as well as the scientific impact and validation of nutritional supplements and dietary products to improve public health and healthcare outcomes.

Classifying contaminated cell cultures using time series features.

We examine the use of time series data, derived from Electric Cell-substrate Impedance Sensing (ECIS), to differentiate between standard mammalian cell cultures and those infected with a mycoplasma organism. With the goal of easy visualization and interpretation, we perform low-dimensional feature-based classification, extracting application-relevant features from the ECIS time courses. We can achieve very high classification accuracy using only two features, which depend on the cell line under examination. Initial results also show the existence of experimental variation between plates and suggest types of features that may prove more robust to such variation. Our paper is the first to perform a broad examination of ECIS time course features in the context of detecting contamination; to combine different types of features to achieve classification accuracy while preserving interpretability; and to describe and suggest possibilities for ameliorating plate-to-plate variation.

Metabolome and Transcriptome Analysis Revealed the Pivotal Role of Exogenous Melatonin in Enhancing Salt Tolerance in Vitis vinifera L.

Vitis vinifera L. possesses high economic value, but its growth and yield are seriously affected by salt stress. Though melatonin (MT) has been widely reported to enhance tolerance towards abiotic stresses in plants, the regulatory role melatonin plays in resisting salt tolerance in grapevines has scarcely been studied. Here, we observed the phenotypes under the treatment of different melatonin concentrations, and then transcriptome and metabolome analyses were performed. A total of 457 metabolites were detected in CK- and MT-treated cell cultures at 1 WAT (week after treatment) and 4 WATs. Exogenous melatonin treatment significantly increased the endogenous melatonin content while down-regulating the flavonoid content. To be specific, the melatonin content was obviously up-regulated, while the contents of more than a dozen flavonoids were down-regulated. Auxin response genes and melatonin synthesis-related genes were regulated by the exogenous melatonin treatment. WGCNA (weighted gene coexpression network analysis) identified key salt-responsive genes; they were directly or indirectly involved in melatonin synthesis and auxin response. The synergistic effect of salt and melatonin treatment was investigated by transcriptome analysis, providing additional evidence for the stress-alleviating properties of melatonin through auxin-related pathways. The present study explored the impact of exogenous melatonin on grapevines' ability to adapt to salt stress and provided novel insights into enhancing their tolerance to salt stress.

Reversal of Postnatal Brain Astrocytes and Ependymal Cells towards a Progenitor Phenotype in Culture.

Astrocytes and ependymal cells have been reported to be able to switch from a mature cell identity towards that of a neural stem/progenitor cell. Astrocytes are widely scattered in the brain where they exert multiple functions and are routinely targeted for in vitro and in vivo reprogramming. Ependymal cells serve more specialized functions, lining the ventricles and the central canal, and are multiciliated, epithelial-like cells that, in the spinal cord, act as bi-potent progenitors in response to injury. Here, we isolate or generate ependymal cells and post-mitotic astrocytes, respectively, from the lateral ventricles of the mouse brain and we investigate their capacity to reverse towards a progenitor-like identity in culture. Inhibition of the GSK3 and TGFß pathways facilitates the switch of mature astrocytes to Sox2-expressing, mitotic cells that generate oligodendrocytes. Although this medium allows for the expansion of quiescent NSCs, isolated from live rats by "milking of the brain", it does not fully reverse astrocytes towards the bona fide NSC identity; this is a failure correlated with a concomitant lack of neurogenic activity. Ependymal cells could be induced to enter mitosis either via exposure to neuraminidase-dependent stress or by culturing them in the presence of FGF2 and EGF. Overall, our data confirm that astrocytes and ependymal cells retain a high capacity to reverse to a progenitor identity and set up a simple and highly controlled platform for the elucidation of the molecular mechanisms that regulate this reversal.

Relationship of Cytotoxic and Antimicrobial Effects of Triphenylphosphonium Conjugates with Various Quinone Derivatives.

Quinone derivatives of triphenylphosphonium have proven themselves to be effective geroprotectors and antioxidants that prevent oxidation of cell components with participation of active free radicals - peroxide (RO2·), alkoxy (RO·), and alkyl (R·) radicals, as well as reactive oxygen species (superoxide anion, singlet oxygen). Their most studied representatives are derivatives of plastoquinone (SkQ1) and ubiquinone (MitoQ), which in addition to antioxidant properties also have a strong antibacterial effect. In this study, we investigated antibacterial properties of other quinone derivatives based on decyltriphenylphosphonium (SkQ3, SkQT, and SkQThy). We have shown that they, just like SkQ1, inhibit growth of various Gram-positive bacteria at micromolar concentrations, while being less effective against Gram-negative bacteria, which is associated with recognition of the triphenylphosphonium derivatives by the main multidrug resistance (MDR) pump of Gram-negative bacteria, AcrAB-TolC. Antibacterial action of SkQ1 itself was found to be dependent on the number of bacterial cells. It is important to note that the cytotoxic effect of SkQ1 on mammalian cells was observed at higher concentrations than the antibacterial action, which can be explained by (i) the presence of a large number of membrane organelles, (ii) lower membrane potential, (iii) spatial separation of the processes of energy generation and transport, and (iv) differences in the composition of MDR pumps. Differences in the cytotoxic effects on different types of eukaryotic cells may be associated with the degree of membrane organelle development, energy status of the cell, and level of the MDR pump expression.

Intestinal tissue-resident memory T cells maintain distinct identity from circulating memory T cells after in vitro restimulation.

Resident memory T (TRM) cells have been recently established as an important subset of memory T cells that provide early and essential protection against reinfection in the absence of circulating memory T cells. Recent findings showing that TRM expand in vivo after repeated antigenic stimulation indicate that these memory T cells are not terminally differentiated. This suggests an opportunity for in vitro TRM expansion to apply in an immunotherapy setting. However, it has also been shown that TRM may not maintain their identity and form circulating memory T cells after in vivo restimulation. Therefore, we set out to determine how TRM respond to antigenic activation in culture. Using Listeria monocytogenes and LCMV infection models, we found that TRM from the intraepithelial compartment of the small intestine expand in vitro after antigenic stimulation and subsequent resting in homeostatic cytokines. A large fraction of the expanded TRM retained their phenotype, including the expression of key TRM markers CD69 and CD103 (ITGAE). The optimal culture of TRM required low O2 pressure to maintain the expression of these and other TRM-associated molecules. Expanded TRM retained their effector capacity to produce cytokines after restimulation, but did not acquire a highly glycolytic profile indicative of effector T cells. The proteomic analysis confirmed TRM profile retention, including expression of TRM-related transcription factors, tissue retention factors, adhesion molecules, and enzymes involved in fatty acid metabolism. Collectively, our data indicate that limiting oxygen conditions supports in vitro expansion of TRM cells that maintain their TRM phenotype, at least in part, suggesting an opportunity for therapeutic strategies that require in vitro expansion of TRM.