Non-differentiated Cells Research Articles

8660 Bodipy Labeled Vitamin D3 Associates With Lipid Droplets In Adipocytes

Abstract Disclosure: N. Ucar: None. J.T. Deeney: None. R. Pickering, PhD: None. M.T. Kirber: None. T. Fan: None. R. Loo, PhD: Research Investigator; Self; Carbogen Amcis BV. M.F. Holick: Grant Recipient; Self; Carbogen Amcis BV. Research Investigator; Self; Carbogen Amcis BV. Obese patients are often found to be deficient in vitamin D, requiring higher levels of supplementation than normal weight subjects. While this has been attributed to vitamin D3‘s lipid solubility and accumulation in fat tissue, little is known about how vitamin D3 enters adipocytes and associates with the intracellular lipid droplet. The goal of this study was to investigate this association using a novel fluorescently labelled vitamin D3. Bodipy, 4,4-difluoro-1,3,5, 7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY 493/503), was covalently linked to vitamin D3 and its structure was confirmed by NMR and mass spectroscopy. Murine 3T3L1 and primary adipocytes were used for our study. Mouse 3T3L1 preadipocytes were cultured in multiwell plates or glass bottom dishes in high glucose DMEM media and differentiated for 7-14 days. Primary adipocytes were obtained by collagenase digestion after surgical extraction of adipose tissue from male/female C57BL/6J mice and attached to glass bottom dishes. The cultured adipocytes were exposed to 10 microM BODIPY labelled Vitamin D3 (vitD-B). BODIPY was added to cultured cells and served as the control. Cells were imaged at various times on a wide-field epifluorescence microscope and analysed with NIH ImageJ software. VitD-B adipocyte uptake and accumulation in lipid droplets was observed over 24 hrs. Isolated lipid droplets were also incubated with vitD-B and BODIPY. VitD-B in mature 3T3L1 cells was observed in lipid droplets after 1h incubation and increased fluorescent intensity was observed over the next 24 hrs. BODIPY was observed in the lipid droplets after 15-20 mins. As a negative control, non-differentiated 3T3L1 cells did not exhibit staining by BODIPY even after 24-hrs. When vitD-B was added to primary cultured adipocytes, it appeared in the adipocytes within 1-hr. Fluorescence intensity of primary adipocytes was increased 1.2-fold at 8 hrs and 5.7-fold at 24 hrs compared to 1 hr. For comparison the positive control treated with BODIPY exhibited rapid fluorescence uptake into primary adipocytes that was increased 29-fold higher than that of vitD-B at 1 hr. VitD-B and BODIPY both demonstrated rapid uptake into isolated lipid droplets (less than 2 min) based on fluorescence microscopy.These results hold promising implications for understanding of the interaction between vitamin D and intracellular lipid droplets. Keywords: Adipocyte, Vitamin D3, BODIPY, adipocytes, lipid droplet, fluorescent labelled vitamin D3 Presentation: 6/2/2024

Mass Spectrometry Analysis of Neurotransmitter Shifting during Neurogenesis and Neurodegeneration of PC12 Cells.

Parkinson's disease (PD) affects movement; however, most patients with PD also develop nonmotor symptoms, such as hyposmia, sleep disorder, and depression. Dopamine levels in the brain have a critical influence on movement control, but other neurotransmitters are also involved in the progression of PD. This study analyzed the fluctuation of neurotransmitters in PC12 cells during neurogenesis and neurodegeneration by performing mass spectrometry. We found that the dopaminergic metabolism pathway of PC12 cells developed vigorously during the neuron differentiation process and that the neurotransmitters were metabolized into 3-methoxytyramine, which was released from the cells. The regulation of the intracellular and extracellular concentrations of adenosine indicated that adenine nucleotides were actively utilized in neural differentiation. Moreover, we exposed the differentiated PC12 cells to rotenone, which is a suitable material for modeling PD. The cells exposed to rotenone in the early stage of differentiation exhibited stimulated serotoninergic metabolism, and the contents of the serotoninergic neurotransmitters returned to their normal levels in the late stage of differentiation. Interestingly, the nondifferentiated cells can resist the toxicant rotenone and produce normal dopaminergic metabolites. However, when differentiated neuron cells were exposed to rotenone, they were seriously damaged, leading to a failure to produce dopaminergic neurotransmitters. In the low-dosage damage process, the amino acids that functioned as dopaminergic pathway precursors could not be absorbed by the cells, and dopamine and L-dopa were secreted and unable to be reuptaken to trigger the cell damage.

Difference in Endocytosis Pathways Used by Differentiated Versus Nondifferentiated Epithelial Caco-2 Cells to Internalize Nanosized Particles.

Understanding the internalization of nanosized particles by mucosal epithelial cells is essential in a number of areas including viral entry at mucosal surfaces, nanoplastic pollution, as well as design and development of nanotechnology-type medicines. Here, we report our comparative study on pathways of cellular internalization in epithelial Caco-2 cells cultured in vitro as either a polarized, differentiated cell layer or as nonpolarized, nondifferentiated cells. The study reveals a number of differences in the extent that endocytic processes are used by cells, depending on their differentiation status and the nature of applied nanoparticles. In polarized cells, actin-driven and dynamin-independent macropinocytosis plays a prominent role in the internalization of both positively and negatively charged nanoparticles, contrary to its modest contribution in nonpolarized cells. Clathrin-mediated cellular entry plays a prominent role in the endocytosis of positive nanoparticles and cholesterol inhibition in negative nanoparticles. However, in nonpolarized cells, dynamin-dependent endocytosis is a major pathway in the internalization of both positive and negative nanoparticles. Cholesterol depletion affects both nonpolarized and polarized cells' internalization of positive and negative nanoparticles, which, in addition to the effect of cholesterol-binding inhibitors on the internalization of negative nanoparticles, indicates the importance of membrane cholesterol in endocytosis. The data collectively provide a new contribution to understanding endocytic pathways in epithelial cells, particularly pointing to the importance of the cell differentiation stage and the nature of the cargo.

Tailoring bisphosphonate-doped titanium films to optimally couple cellular responses and antibacterial activity for biomedical applications.

Titanium (Ti) is widely utilized as an implant material; nonetheless, its integration with bone tissue faces limitations due to a patient's comorbidities. To address this challenge, we employed a strategic approach involving the growth of thin films by spin-coating and surface functionalization with etidronate (ETI), alendronate (ALE), and risedronate (RIS). Our methodology involved coating of Ti cp IV disks with thin films of TiO2, hydroxyapatite (HA), and their combinations (1:1 and 1:2 v/v), followed by surface functionalization with ETI, ALE, and RIS. Bisphosphonate-doped films were evaluated in terms of surface morphology and physical-chemical properties by techniques such as electron microscopy, confocal microscopy, and x-ray photoelectron spectroscopy. The antibacterial potential of bisphosphonates alone or functionalized onto the Ti surface was tested against Staphylococcus aureus biofilms. Primary human bone mesenchymal stem cells were used to determine in vitro cell metabolism and mineralization. Although RIS alone did not demonstrate any antibacterial effect as verified by minimum inhibitory concentration assay, when Ti surfaces were functionalized with RIS, partial inhibition of Staphylococcus aureus growth was noted, probably because of the physical-chemical surface properties. Furthermore, samples comprising TiO2/HA (1:1 and 1:2 v/v) showcased an enhancement in the metabolism of nondifferentiated cells and can potentially enhance the differentiation of osteoblastic precursors. All samples demonstrated cell viability higher than 80%. Addition of hydroxyapatite and presence of bisphosphonates increase the metabolic activity and the mineralization of human bone mesenchymal cells. While these findings hold promise, it is necessary to conduct further studies to evaluate the system's performance in vivo and ensure its long-term safety. This research marks a significant stride toward optimizing the efficacy of titanium implants through tailored surface modifications.

The impact of ATP-sensitive potassium channel modulation on mitochondria in a Parkinson’s disease model using SH-SY5Y cells depends on their differentiation state

Inward rectifying potassium channels sensitive to ATP levels (KATP) have been the subject of investigation for several decades. Modulators of KATP channels are well-established treatments for metabolic as well as cardiovascular diseases. Experimental studies have also shown the potential of KATP modulation in neurodegenerative disorders. However, to date, data regarding the effects of KATP antagonists/agonists in experiments related to neurodegeneration remain inconsistent. The main source of confusion in evaluating available data seems to be the choice of experimental models. The present study aims to provide a comprehensive understanding of the effects of both opening and blocking KATP channels in two forms of SH-SY5Y cells. Our results offer valuable insights into the significance of metabolic differences between differentiated and non-differentiated SH-SY5Y cells, particularly in the context of glibenclamide and diazoxide effects under normal conditions and during the initiation of pathological events simulating Parkinson’s disease in vitro. We emphasize the analysis of mitochondrial functions and changes in mitochondrial network morphology. The heightened protein expression of KATP channels identified in non-differentiated SH-SY5Y cells seems to be a platform for a more significant impact of KATP modulators in this cell type. The efficiency of rotenone treatment in inducing morphological changes in the mitochondrial network depends on the differentiation status of SH-SY5Y cells.

Computational Studies Using COMSOL Multiphysics on Dielectrophoretic Characterization and Separation of Mesenchymal Stem Cells Undergoing Tenogenic Differentiation

Stem cells have unique self-renewal and differentiation capacities, which are advantageous for regenerative medicine and tissue engineering applications. Stem cells can recreate cells needed to repair injured tissues and organs in the body, for example, by regenerating connective tissues, which provide support, protection, and a structural framework for various organs and other tissues. However, a significant limitation is the susceptibility of tendons to injury with long-term loss of function. Mesenchymal Stem Cell (MSC) therapies are promising for healing tendon injuries and tears but controlling stem cell differentiation and generating homogeneous tenogenic stem cell populations remains challenging. Therefore, separating differentiating and non-differentiating MSCs to collect homogenous cells is critical for further tissue repair after transplantation. To substantiate our hypothesis that differentiating and non-differentiating stem cells have unique dielectric properties, we focused on characterizing-obtaining dielectric signatures for differentiating tenogenic and non-tenogenic MSCs on the third day of differentiation using dielectrophoresis (DEP) focused on crossover frequencies [1, 2]. The estimated values for membrane capacitance, conductivity, and permittivity after 3-day treatment of undifferentiated cells differentiating into tenogenic MSCs are 2.46±0.1 pF, 0.82±0.01 S/m, and 1.97±0.05, respectively, and cytoplasm conductivity 0.82±0.02 S/m. The treated stem cells undergo differentiation, but the untreated cells don’t. The results showed a significant difference between the 3-day treatment and no-treatment group. The estimated properties from the dielectrophoretic characterization studies are crucial in designing a DEP-based enrichment microdevice to collect homogeneous differentiated stem cell populations for tendon repair. Using particle tracing, creeping flow (transport of diluted species model), and electric current physics in COMSOL Multiphysics simulation software, we designed a microdevice that achieves 100% separation of untreated and treated mesenchymal stem cells undergoing differentiation towards tendon. Applying dielectrophoresis to the microdevice architecture demonstrated high selectivity and yield, processing one million treated and untreated stem cells in 6.4 and 27.8 hours, respectively.

Involvement of pro-inflammatory mediators and cell cycle disruption in neuronal cells induced by gliotoxin and ochratoxin A after individual and combined exposure

Mycotoxins such as gliotoxin (GTX) and ochratoxin A (OTA) are secondary metabolites of Aspergillus and Penicillum found in food and feed. Both mycotoxins have shown to exert a detrimental effect on neuronal activity. The following study was carried out to elucidate the mechanisms by which GTX and OTA exert their toxicity. Non-differentiated SH-SY5Y neuronal-like cells were treated with GTX, OTA and their combinations to assess their cytotoxic effect using the MTT assay during 24, 48 and 72 h of exposure. Based on the results of the cytotoxic assays, cell cycle proliferation and immunological mediators were measured by determining the production of IL-6 and TNF-α using flow cytometry and ELISA, respectively. The IC50 values obtained were 1.24 and 1.35 µM when SH-SY5Y cells were treated with GTX at 48 h and 72 h, respectively. IC50 values of 8.25, 5.49 and 4.5 µM were obtained for OTA treatment at 24 h, 48 h and 72 h, respectively. The SubG0 phase increased in both treatments at 24 and 48 h. On the other hand, IL-6 and TNF-α production was increased in all mycotoxin treatments studied and was more pronounced for [GTX + OTA] after 48 h exposure. The additive and synergistic effect observed by the isobologram analysis between GTX and OTA resulted to a higher cytotoxicity which can be explained by the increased production of IL-6 and TNF-α inflammatory mediators that play an important role in the toxicity mechanism of these mycotoxins.

Differentiated and non-differentiated HepaRG™ cells: A possible in-vitro model system for early hepatocarcinogenesis and non-genotoxic carcinogens

Many xenobiotics are non-genotoxic carcinogens (NGC) in rodent liver. Their mode of action (MoA) and health risks for humans are unclear and no in-vitro tests are available to predict NGC. Human HepaRG™ cells in the differentiated (d-HepaRG) and non-differentiated state (nd-HepaRG) were studied as new approach methodology (NAM) for NGC. Cell-biological assays were performed with d-/nd-HepaRG and human hepatoma/hepatocarcinoma cell lines to characterize the benign/malignant phenotype. Reaction of d-/nd-HepaRG to several liver growth factors and NGC (phenobarbital, PB; cyproterone acetate, CPA; WY-14643) was compared to unaltered and premalignant rat hepatocytes in ex-vivo culture. Enzyme induction by NGC was checked by RT-qPCR/oligo-arrays. Growth, anchorage-independency, migration, clonogenicity, and in-vivo tumorigenicity of nd-HepaRG ranged between benign d-HepaRG and malignant hepatoma/hepatocarcinoma cells. All growth factors elevated DNA replication of d-/nd-HepaRG cells, similarly to unaltered/premalignant rat hepatocytes. NGC induced their prototypical enzymes in the rat and human cells, but elicited a growth response only in the unaltered/premalignant rat hepatocytes and not in human d-/nd-HepaRG cells. To conclude, a benign/premalignant phenotype of d-/nd-HepaRG cells and a reactivity towards several hepatic growth factors and NGC, as known from human hepatocytes, are essential components for an in-vitro model for early stage human hepatocarcinogenesis.The potential value as new approach methodology (NAM) for NGC is discussed.

Lupenone-Rich Fraction Derived from Cissus quadrangularis L. Suppresses Lipid Accumulation in 3T3-L1 Adipocytes.

Cissus quadrangularis L. (CQ) has potential as a therapeutic for managing obesity and balancing metabolic activity, but the main bioactive compound and regulatory mechanism remain unknown. Herein, the CQ hexane extract was fractionated into 30 fractions (CQ-H) using flash column chromatography and analyzed using thin-layer chromatography. The direct antiadipogenesis effect of CQ-H fractions was tested on 3T3-L1 preadipocytes. Lupenone-rich fractions 2H and 3H were identified as containing potent antiadipogenesis agents that reduced differentiated cell numbers and intracellular lipid droplet size. Although the overall mitochondrial density remained unchanged, differentiated cells exhibited a higher mitochondrial density than that in non-differentiated cells. Additionally, 2H increased mitochondrial activity in both cell types as shown by their differentiation and lipid formation stages. Lupenone was isolated from 2H (Lu-CQ) and shown to dose-dependently inhibit adipogenesis, with 2H being more potent than Lu-CQ. Lu-CQ and 2H downregulated the expression of Pparg2 mRNA and upregulated that of glucose transporter genes, Slc2a1 and Slc2a4. Lu-CQ and 2H induced increased glucose uptake by 3T3-L1 cells. These findings suggest that lupenone-rich fractions in CQ contribute to balancing metabolic activity and reducing adipose tissue formation. Further exploration of CQ and its components may prompt innovative strategies for managing obesity and metabolic disorders.

Wnt signaling preserves progenitor cell multipotency during adipose tissue development

Mesenchymal stem/progenitor cells are essential for tissue development and repair throughout life, but how they are maintained under chronic differentiation pressure is not known. Using single-cell transcriptomics of human progenitor cells we find that adipose differentiation stimuli elicit two cellular trajectories: one toward mature adipocytes and another toward a pool of non-differentiated cells that maintain progenitor characteristics. These cells are induced by transient Wnt pathway activation and express numerous extracellular matrix genes and are therefore named structural Wnt-regulated adipose tissue cells. We find that the genetic signature of structural Wnt-regulated adipose tissue cells is present in adult human adipose tissue and adipose tissue developed from human progenitor cells in mice. Our results suggest a mechanism whereby adipose differentiation occurs concurrently with the maintenance of a mesenchymal progenitor cell pool, ensuring tissue development, repair and appropriate metabolic control over the lifetime.

Analysis of Nipah Virus Replication and Host Proteome Response Patterns in Differentiated Porcine Airway Epithelial Cells Cultured at the Air-Liquid Interface.

Respiratory tract epithelium infection plays a primary role in Nipah virus (NiV) pathogenesis and transmission. Knowledge about infection dynamics and host responses to NiV infection in respiratory tract epithelia is scarce. Studies in non-differentiated primary respiratory tract cells or cell lines indicate insufficient interferon (IFN) responses. However, studies are lacking in the determination of complex host response patterns in differentiated respiratory tract epithelia for the understanding of NiV replication and spread in swine. Here we characterized infection and spread of NiV in differentiated primary porcine bronchial epithelial cells (PBEC) cultivated at the air-liquid interface (ALI). After the initial infection of only a few apical cells, lateral spread for 12 days with epithelium disruption was observed without releasing substantial amounts of infectious virus from the apical or basal sides. Deep time course proteomics revealed pronounced upregulation of genes related to type I/II IFN, immunoproteasomal subunits, transporter associated with antigen processing (TAP)-mediated peptide transport, and major histocompatibility complex (MHC) I antigen presentation. Spliceosomal factors were downregulated. We propose a model in which NiV replication in PBEC is slowed by a potent and broad type I/II IFN host response with conversion from 26S proteasomes to immunoproteasomal antigen processing and improved MHC I presentation for adaptive immunity priming. NiV induced cytopathic effects could reflect the focal release of cell-associated NiV, which may contribute to efficient airborne viral spread between pigs.

Toxic effects of the cylindrospermopsin and chlorpyrifos combination on the differentiated SH-SY5Y human neuroblastoma cell line

Due to climate change and anthropogenic activities, the levels of pollution of aquatic and terrestrial environments have increased in the last decades. In this sense, the rise of cyanobacterial blooms, which release secondary metabolites with toxic properties, and the global use of pesticides for agricultural purposes have a negative impact on ecosystems. Thus, it would be interesting to study the concomitance of both types of toxicants in the same sample, since it is possible that they appear together. The aim of the present work was to state the effects of the interaction between the cyanotoxin cylindrospermopsin and the pesticide chlorpyrifos in differentiated SH-SY5Y neuronal cells to assess how they could affect the nervous system. To this end, cytotoxicity, morphological, and acetylcholinesterase activity studies were performed during 24 and 48 h. The results revealed a concentration-dependent decrease in viability and interaction between both toxicants, together with clear signs of apoptosis and necrosis induction. In this sense, different stages on the differentiation process would lead to differences in the toxicity exerted by the compounds both isolated as in combination, which it is not observed in non-differentiated cells. Additionally, the acetylcholinesterase activity appeared not to be affected, which is a clear difference compared to non-differentiated cells. These results show the importance of studying not only the toxicants themselves, but also in combination, to assess their possible effects in a more realistic scenario.

Expression of osteogenic factors in FasL-deficient calvarial cells.

During bone development, FasL acts not only through the traditional apoptotic mechanism regulating the amount of bone-resorbing osteoclasts, but there is also growing evidence about its effect on cell differentiation. Expression of osteoblastic factors was followed in non differentiated and differentiating primary calvarial cells obtained from FasL-deficient (gld) mice. The gld cells showed decreased expression of the key osteoblastic molecules osteocalcin (Ocn), osteopontin (Opn), and alkaline phosphatase (Alpl) in both groups. Notably, receptor activator of nuclear factor kappa-B ligand (Rankl) was unchanged in non-differentiated gld vs. wild type (wt) cells but decreased in differentiating gld cells. Osteoprotegerin (Opg) in the gld samples was increased in both groups. Opg vs. Rankl expression levels favored Opg in the case of non-differentiated cells but Rankl in differentiating ones. These results expand information on the involvement of FasL in non-apoptotic cell pathways related to osteoblastogenesis and consequently also osteoclastogenesis and pathologies such as osteoporosis.

Hydrogen Peroxide Produced from Selective Phenolic Acids in Cell Culture Underlies Caco-2 Changes in Cell Proliferation Parameters

The physicochemical property of phenolic acids to generate hydrogen peroxide (H2O2) in cell culture media has been underreported when describing multiple biological effects in vitro. Our aim was to focus on examining the relative capacity of four common phenolic acids widely consumed in the Western diet for autoxidation potential to generate H2O2 during in vitro culture. Furthermore, quantifying H2O2 derived from different phenolic acids cultured in Dulbecco's modified Eagle's medium (DMEM) was associated with changes in cell proliferation in non-differentiated human intestinal carcinoma cells. Results showed that the different percentage losses of phenolic acids, namely, caffeic (84.78 ± 1.51), chlorogenic (37.3 ± 0.38), ferulic (1.26 ± 0.78), and gallic (100%), paralleled a relative capacity to generate H2O2 when present in DMEM media for 24 h. The rate and total H2O2 generated was dependent on both phenolic acid type and concentration (p < 0.05). Gallic acid had the greatest capacity to generate H2O2 in culture without the presence of cells (p < 0.05). When cultured with non-differentiated Caco-2 cells, gallic acid evoked the greatest bioactivity that included cytotoxicity, anti-proliferation, apoptosis, and nuclear condensation, respectively (p < 0.05). Corresponding treatments with cells with phenolic acids in the presence of catalase confirmed that H2O2 generated from phenolic acid autoxidation was involved in cell proliferation and apoptosis.

Sphingosine-1-phosphate receptor 2 plays a dual role depending on the stage of cell differentiation in renal epithelial cells

Epithelial renal cells have the ability to adopt different cellular phenotypes through epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET). These processes are increasingly recognized as important repair factors following acute renal tubular injury. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid with impact on proliferation, growth, migration, and differentiation which has significant implication in various diseases including cancer and kidney fibrosis. Here we demonstrated that S1P can exert by activating S1P receptor 2 (S1PR2) different functions depending on the stage of cell differentiation. We observed that the differences in the migratory profile of Madin-Darby canine kidney (MDCK) cells depend both on their stage of cell differentiation and the activity of S1PR2, a receptor that can either promote or inhibit the migratory process. Meanwhile in non-differentiated cells S1PR2 activation avoids migration, it is essential on fully differentiated cells. This is the first time that an antagonist effect of S1PR2 was reported for the same cell type. Moreover, in fully differentiated cells, S1PR2 activation is crucial for the progression of EMT - characterized by adherent junctions disassembly, β-catenin and SNAI2 nuclear translocation and vimentin expression- and depends on ERK 1/2 activation and nuclear translocation. These findings provide a new perspective about the different S1PR2 functions depending on the stage of cell differentiation that can be critical to the modulation of renal epithelial cell plasticity, potentially paving the way for innovative research with pathophysiologic relevance.

Study of the potential toxicity of adrenaline to neurons, using the SH-SY5Y human cellular model

Prolonged overexposure to catecholamines causes toxicity, usually credited to continuous adrenoceptor stimulation, autoxidation, and the formation of reactive pro-oxidant species. Non-differentiated SH-SY5Y cells were used to study the possible contribution of oxidative stress in adrenaline (ADR)-induced neurotoxicity, as a model to predict the toxicity of this catecholamine to peripheral nerves. Cells were exposed to several concentrations of ADR (0.1, 0.25, 0.5 and 1mM) and two cytotoxicity assays [lactate dehydrogenase (LDH) release and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction] were performed at several time-points (24, 48, and 96h). The cytotoxicity of ADR was concentration- and time-dependent in both assays, since the lowest concentration tested (0.1mM) also caused significant cytotoxicity at 96h. N-acetyl-cysteine (1mM), a precursor of glutathione synthesis, prevented ADR-induced toxicity elicited by 0.5mM and 0.25mM ADR following a 96-h exposure, while the antioxidant Tiron (100µM) was non-protective. In conclusion, ADR led to mitochondrial distress and ultimately cell death in non-differentiated SH-SY5Y cells, possibly because of ADR oxidation products. The involvement of such processes in the catecholamine-induced peripheral neuropathy requires further analysis.

Rapid, label-free classification of glioblastoma differentiation status combining confocal Raman spectroscopy and machine learning.

Label-free identification of tumor cells using spectroscopic assays has emerged as a technological innovation with a proven ability for rapid implementation in clinical care. Machine learning facilitates the optimization of processing and interpretation of extensive data, such as various spectroscopy data obtained from surgical samples. The here-described preclinical work investigates the potential of machine learning algorithms combining confocal Raman spectroscopy to distinguish non-differentiated glioblastoma cells and their respective isogenic differentiated phenotype by means of confocal ultra-rapid measurements. For this purpose, we measured and correlated modalities of 1146 intracellular single-point measurements and sustainingly clustered cell components to predict tumor stem cell existence. By further narrowing a few selected peaks, we found indicative evidence that using our computational imaging technology is a powerful approach to detect tumor stem cells in vitro with an accuracy of 91.7% in distinct cell compartments, mainly because of greater lipid content and putative different protein structures. We also demonstrate that the presented technology can overcome intra- and intertumoral cellular heterogeneity of our disease models, verifying the elevated physiological relevance of our applied disease modeling technology despite intracellular noise limitations for future translational evaluation.

Evaluation of the Anticancer and Probiotic Potential of Autochthonous (Wild) Lacticaseibacillus paracasei Strains from New Ecological Niches as a Possible Additive for Functional Dairy Foods.

Probiotics such as Lactobacillus spp. could modulate the intestinal microbiota composition, supporting gastrointestinal tract barrier function and benefiting human health. To evaluate the anticancer and probiotic properties of potentially active autochthonous Lacticaseibacillus paracasei strains on proliferating and differentiated enterocytes, human colon adenocarcinoma cell line HT29 was used as a model. The lactic acid bacteria (LAB) were isolated from new ecological niches-mountain anthills populated by redwood ants (Formica rufa L.). Human colorectal adenocarcinoma cells (HT29, ATCC, HTB-38™) were treated for twenty-four hours with supernatants (SNs) derived from four strains of Lacticaseibacillus paracasei: P4, C8, C15 and M2.1. An MTT assay, alkaline phosphatase activity, IAP, Bax and Bcl-2 gene expression analysis (RT-qPCR) and the Bax/Bcl-2 ratio were evaluated. The MTT assay revealed that the observed effects varied among groups. However, 10% neutralized supernatants from P4, C8, C15 and M2.1 strains did not show cytotoxic effects. In contrast to non-differentiated cells, a significant (p &lt; 0.001) rise in ALP activity in all treatments, with an average of 18%, was established in differentiated cells. The IAP expression was remarkably downregulated in the differentiated M2.1 group (p &lt; 0.05) and upregulated in the non-differentiated P4 (p &lt; 0.05) and M2.1 (p &lt; 0.05) groups. The Bax/Bcl-2 quantity expression ratio in P4 was significantly (p &lt; 0.05) upregulated in proliferating cancer cells, but in P4- and M2.1-differentiated cells these values were downregulated (p &lt; 0.05). The obtained results indicate that the isolated L. paracasei strains possess anticancer and probiotic properties and could be used as additives for functional dairy foods and thus benefit human health.

Perspectives of using of somatic cells fusion process in some aspects of car¬cino¬ge-nesis

Normal somatic cells sensitive to carcinogenic effects and capable of proliferation form firstly a binuclear cells (dikaryons) and then hybrid cells (mononucleated synkaryons) by means of fusion with another cells of the same organism, in particular with differentiated and non-differentiated cells of corresponding tissue or with cells capable to migrate. In all probability, during the perforation of the plasma membrane, i.e., after the formation of po­res, induced by different carcinogenic (and noncarcinogenic) agents and factors, the total negative charge of plasma membrane changes (decreases) and the cells develop the ability to come closer to each other, whi­ch will probably be the prerequisite to a fusion process. On the other hand, it is not excluded that perforation can assist the fusion of only neighboring cells. Initially, a set of chromosomes in a precancerous cell retains, even for a short time, the condition of tetraploidy. Because of cell fusion generates tetraploidy, it potentially might cause chromo­somal instability. The article emphasizes a possibility of using the fusion process in different aspects of carcino­genesis (therapy, prevention, etc.).

Equine Stomach Development in the Foetal Period of Prenatal Life-A Histological and Histometric Study.

Histological and morphometrical analysis of the stomach wall was performed during the foetal period divided into three age groups (4th-11th month of gestation). The material was taken from non-glandular (the blind ventricular sac) and glandular parts (the plicated edge margin/cardiac part, the body of stomach and the pyloric part) of the stomach. It was preserved and prepared according to the standard protocol. The histological slides were stained (H-E, Masson-Goldner and PAS). The analyses were performed using the light microscope. All measurements were statistically elaborated. The crown-rump length growth rate was estimated as isometric. The blind ventricular sac growth rate was lower than CRL (negative allometric) and the partition of stomach mucosa into non-glandular and glandular part occurred in the 1st age group. The plicated edge margin/cardiac part and the pyloric part shoved similar tendencies. Only the body of stomach demonstrated a higher growth rate than CRL (positive allometric), which can be explained due to the strongest development of fundic glands. Moreover, comparing the adult reference group to the three parts of the foetal period, all metric values were lower than those achieved prenatally. The blind ventricular sac was covered with the multiple plane epithelium. The glandular parts of stomach that formed the superficial concave areas were covered with the simple columnar epithelium in the 1st age group, which developed to the cardiac, fundic, and pyloric glands in the 2rd and 3rd age groups. The propria mucosae was built with the mesenchyme, which differentiated later to the loose connective tissue. The muscular layer of mucosa was not clearly distinguishable in the 1st age group. The muscular layer of the stomach wall was formed with myoblasts in the 1st age group and later in the 2nd and the 3rd age groups built with fusiform myocytes divided into internal and external layers. The non-differentiated cells of glandular epithelium transformed into the parietal and chief cells. The first were visible in the gastric glands of the 2nd age group. Both of them were present in the 3rd age group gastric mucosa. The PAS staining proved a moderate PAS-positive reaction in the 2rd age group, while it was estimated as intense Pas-positive in the gastric glands in the 3rd age group and was comparable to postnatal observation (the adult reference group).