Changes In Cell Morphology Research Articles

Surface plasmon resonance to trace and measure cancer cell apoptosis using morphological and refractive index changes

Morphological changes of cancer cells are often used as an important indicator within efficiency studies of anticancer drugs. Morphological cell analysis on cell size and shape distribution is typically performed using microscopic methods, which are time consuming and require skilled personnel. Recently, more advanced image processing and pattern recognition have enabled identification and quantitative analysis of the cell’s abnormality and classification in an automated way. However, these methods usually involve multiple staining steps. In addition to computational complexity, the processes greatly compromise real-time applications of the system. Therefore, a non-invasive, real-time method allowing for assessment of living cells’ reactions to a death inducer is very much needed. Here, we present an SPR biosensor that measures the changes in cancer cells’ size and detachment, relating the cell confluency with the changes of the refractive index on the cell-substrate interface. As a proof-of-concept, we chose HeLa cell and hydrogen peroxide (H2O2) induced apoptosis as the model system to study the morphological changes of the cell. The results show that the SPR response to cell apoptosis agreed with the cellular morphological changes observed via microscopy. Interestingly, we observed simultaneous apoptosis and necrosis at high H2O2 concentrations. This simultaneous occurrence was verified using a mathematical model which incorporated other important factors such as cell thickness and intercellular refractive index. This model helped resolve the disagreement between SPR signal and cell confluency at high H2O2 concentrations. Our results show the potential of SPR as a label free and real time monitoring method for morphological changes and surface detachment of cancer cells. This method can be fully expanded to other cell-based sensing applications.

Protective Action of Cannabidiol on Tiamulin Toxicity in Humans—In Vitro Study

The growing awareness and need to protect public health, including food safety, require a thorough study of the mechanism of action of veterinary drugs in consumers to reduce their negative impact on humans. Inappropriate use of veterinary drugs in animal husbandry, such as tiamulin, leads to the appearance of residues in edible animal tissues. The use of natural substances of plant origin, extracted from hemp (Cannabis sativa L.), such as cannabidiol (CBD), is one of the solutions to minimize the negative effects of tiamulin. This study aimed to determine the effect of CBD on the cytotoxicity of tiamulin in humans. The cytotoxic activity of tiamulin and the effect of its mixtures with CBD were tested after 72 h exposure to three human cell lines: SH-SY5Y, HepG2 and HEK-293. Cytotoxic concentrations (IC50) of the tested drug and in combination with CBD were assessed using five biochemical endpoints: mitochondrial and lysosomal activity, proliferation, cell membrane integrity and effects on DNA synthesis. Oxidative stress, cell death and cellular morphology were also assessed. The nature of the interaction between the veterinary drug and CBD was assessed using the combination index. The long-term effect of tiamulin inhibited lysosomal (SH-SY5SY) and mitochondrial (HepG2) activity and DNA synthesis (HEK-293). IC50 values for tiamulin ranged from 2.1 to >200 µg/mL (SH-SY5SY), 13.9 to 39.5 µg/mL (HepG2) and 8.5 to 76.9 µg/mL (HEK-293). IC50 values for the drug/CBD mixtures were higher. Reduced levels of oxidative stress, apoptosis and changes in cell morphology were demonstrated after exposure to the mixtures. Interactions between the veterinary drug and CBD showed a concentration-dependent nature of tiamulin in cell culture, ranging from antagonistic (low concentrations) to synergistic effects at high drug concentrations. The increased risk to human health associated with the presence of the veterinary drug in food products and the protective nature of CBD use underline the importance of these studies in food toxicology and require further investigation.

Small GTPase ARL4C Associated with Various Cancers Affects Microtubule Nucleation

Background/Objectives: The changes in the level of small GTPase ARL4C are associated with the initiation and progression of many different cancers. The content of ARL4C varies greatly between different tissues, and the induction of ARL4C expression leads to changes in cell morphology and proliferation. Although ARL4C can bind alpha-tubulin and affect intracellular transport, the role of ARL4C in the functioning of the tubulin cytoskeleton remained unclear. The aim of the present work is to study this role; Methods: The cells of the following lines were used for the experiments: HeLa (human cervical carcinoma), MCF7 (human breast cancer), U2OS (human osteosarcoma), Vero, BS-C-1, and COS7 (African green monkey kidney). The receptor activation by agonists followed by the preparation of cell lysates, electrophoresis, and immunoblotting, as well as cell fixation and immunofluorescent staining, were used to assess endogenous ARL4C/ABCA1 levels and the microtubule network morphology. The microtubule regrowth technique was performed to estimate the rate of microtubule nucleation, and the overexpression of different ARL4C constructs was used to affect ARL4C activity in the cells; Results: We showed that the changes in the endogenous ARL4C level or the ARL4C activity alter the microtubule nucleation process in the cells; Conclusions: small GTPase ARL4C may serve as one of the regulators of the microtubule nucleation process both in normal and cancer cells.

Morphometric analysis of epithelium in oral submucous fibrosis using open-source software

Background: Oral submucous fibrosis (OSMF) is a chronic oral mucosal disease characterized by progressive deposition of collagen in subepithelial connective tissue and epithelial atrophy. ImageJ is open-source software that aids researchers to visualize, inspect, quantify, and validate scientific image data. Objectives: To measure and compare the changes in epithelial thickness and cell morphology in OSMF in comparison with normal mucosa using computer-aided image analysis software. Materials and Methods: 15 OSMF and 10 cases of normal buccal mucosa were analyzed. Epithelial thickness and cell morphology of spinous cells of OSMF cases were quantified by ImageJ software and compared with that of normal tissue. Results: An increase in the cellular area of the spinous cells in OSMF compared to that of the normal tissue was observed. Conclusion: ImageJ represents a powerful tool that enhances the precision and efficiency of epithelial analysis in OSMF research compared to historical methods.

Attenuating Oxidative Damage with Macelignan in Glutamate-Induced HT22 Hippocampal Cells

Macelignan, from Myristica fragrans (nutmeg), is a bioactive compound with various pharmacological properties, including anti-inflammatory and neuroprotective activities. The purpose of this work was to investigate the antioxidant and anti-apoptotic effects of macelignan in glutamate-treated HT22 mouse hippocampal neurons. Macelignan was extracted and identified in a methanol extract of M. fragrans seeds. The DPPH was used to assess the antioxidative activity of macelignan. Glutamate (5 mM) was used to induce neurotoxicity in the HT22 cells. Neuroprotective effects were measured using relevant biochemical and imaging assays, including cell viability, ROS production, nuclear staining, apoptotic cell death, and protein expression. Macelignan markedly and concentration-dependently enhanced DPPH radical scavenging activity. In the HT22 cell model, glutamate induced cell damage by decreasing cell viability, promoting ROS generation, and increasing apoptotic cell death according to cell morphological changes. However, macelignan treatment restored cell viability, inhibited ROS generation concentration-dependently, and reduced apoptosis. Moreover, glutamate significantly up-regulated the phosphorylation of MAPK-pathway-related proteins, which was reversed by macelignan treatment. In conclusion, macelignan shows notable neuroprotective effects on oxidative stress and apoptotic cell death in glutamate-induced cells, and this study provides useful information on its potential therapeutic implications in neurological disorders.

To explore the protective mechanism of promethazine against hippocampal neuron injury based on network pharmacology and experimental verification.

This study aims to investigate the effect of promethazine (PMZ) on hippocampal neuronal injury through network pharmacology and in vivo experiments. Network pharmacology: The intersection genes of PMZ and Alzheimer Disease (AD) were obtained, and the core genes of PMZ in AD were screened. The intersection genes were enriched by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. In the in vitro experiment, mouse hippocampal neurons (HT22) were divided into control, glutamate (GLU) model, and GLU + PMZ treatment groups. The control group was given a complete culture medium, the model group was given GLU for 24 hours, the treatment group was given PMZ pretreatment for 3 hours, and then GLU was administered for 24 hours. Cell viability was determined, cell morphology was observed by microscopy, reactive oxygen species levels and glutathione content were detected, and protein expression of P53, PTGS2, SLC7A11, and GPX4 was detected by western blotting. Network pharmacology: A total of 317 PMZ targets, 1934 AD genes, 125 intersection genes, and 18 core genes, including P53 and PTGS2. Gene Ontology enrichment analysis showed that the effect of PMZ on AD was mainly related to cell proliferation, inflammation, hypoxia, synaptic structure, plasma membrane, and oxidoreductase activity. Kyoto Encyclopedia of Genes and Genomes results showed neuroactive ligand-receptor interaction, cell senescence, cancer pathway, PI3K-AKT signal pathway, neurodegeneration, and HIF-1 signal pathway. In vitro experiments: PMZ improved the GLU-induced decrease in cell viability and morphological changes in hippocampal neurons. PMZ inhibited reactive oxygen species levels and increased glutathione content in injured hippocampal neurons. Up-regulated of P53, SLC7A11 and GPX4 expression, and inhibited expression of PTGS2. PMZ regulates the SLC7A11-GPX4 antioxidant system to protect hippocampal neurons from oxidative stress injury.

Intricate ribosome composition and translational reprogramming in epithelial–mesenchymal transition

Epithelial-mesenchymal transition (EMT) involves profound changes in cell morphology, driven by transcriptional and epigenetic reprogramming. However, evidence suggests that translation and ribosome composition also play key roles in establishing pathophysiological phenotypes. Using genome-wide analyses, we reported significant rearrangement of the translational landscape and machinery during EMT. Specifically, a cell line overexpressing the EMT transcription factor ZEB1 displayed alterations in translational reprogramming and fidelity. Furthermore, using riboproteomics, we unveiled an increased level of the ribosomal protein RPL36A in mesenchymal ribosomes, indicating precise tuning of ribosome composition. Remarkably, RPL36A overexpression alone was sufficient to trigger the acquisition of mesenchymal features, including a switch in the molecular pattern, cell morphology, and behavior, demonstrating its pivotal role in EMT. These findings underline the importance of translational reprogramming and fine-tuning of ribosome composition in EMT.

Cell confinement by micropatterning induces phenotypic changes in cancer-associated fibroblasts

Recent advances in single-cell studies have revealed the vast transcriptomic heterogeneity of cancer-associated fibroblasts (CAFs), with each subset likely having unique roles in the tumor microenvironment. However, it is still unclear how different CAF subsets should be cultured in vitro to recapitulate their in vivo phenotype. The inherent plasticity of CAFs, or their ability to dynamically change their phenotype in response to different environmental stimuli, makes it highly challenging to induce and maintain a specific CAF state in vitro. In this study, we investigated how cell shape and confinement on two-dimensional culture substrates with different stiffnesses influence CAF transcriptomic profile and phenotype. Using micropatterning of polyacrylamide hydrogels to induce shape- and confinement-dependent changes in cell morphology, we observed that micropatterned CAFs exhibited phenotypic shifts towards more desmoplastic and inflammatory CAF subsets. Additionally, micropatterning enabled control over a range of CAF-specific markers and pathways. Lastly, we report how micropatterned and non-micropatterned CAFs respond differently to anti-cancer drugs, highlighting the importance of phenotype-oriented therapy that considers for CAF plasticity and regulatory networks. Control over CAF morphology offers a unique opportunity to establish highly robust CAF phenotypes in vitro, facilitating deeper understanding of CAF plasticity, heterogeneity, and development of novel therapeutic targets. Statement of SignificanceCancer-associated fibroblasts (CAFs) are the dominant stromal cell type in many cancers, and recent studies have revealed that they are highly heterogeneous and comprise several subpopulations. It is still unclear how different subsets of CAFs should be cultured in vitro to recapitulate their in vivo phenotype. In this study, we investigated how cell shape and confinement affect CAF transcriptomic profile and phenotype. We report that micropatterned CAFs resemble desmoplastic and inflammatory CAF subsets observed in vivo and respond differently to anti-cancer drugs as compared to non-patterned CAFs. Control over CAF morphology enables the generation of highly robust CAF phenotypes in vitro, facilitating deeper understanding of CAF plasticity and heterogeneity.

The physiological role of gamma-aminobutyric acid in relieving the effect of furfural inhibitor for improvement the production of lipid in D. intermedius Z8.

In order to improve the production and quality of lipid of D. intermedius Z8 under the furfural stress environment, different exogenous γ-aminobutyric acid (GABA) addition strategies and their reasons for improving the fermentation performance were investigated. For this purpose, the effect of different concentrations of furfural on the production of biomass and lipid in D. intermedius Z8 was researched. The result shows that the growth of D. intermedius Z8 nearly stopped when 1.0g/L furfural was adopted. However, when 1.5mmol/L GABA was used, the highest biomass and lipid production of 4.56g/L and 1.83g/L were obtained, respectively, which were 36.53% and 61.95% higher compared to the control group (the normal development BG11 medium). Additionally, the changes in microalgal cell morphology were analyzed using the scanning electron microscope (SEM) technology, and the results suggest that GABA addition could significantly mitigate the toxic effects of excessive furfural by maintaining the integrity of the cell. Simultaneously, the activities of key enzymes involved in fatty acid synthesis, such as nitrate reductase (NR), phosphatidic acid phosphatase (PAP), acetyl CoA carboxylase (ACC), and fatty acid synthase (FAS), were enhanced under the optimal condition, and thus improving lipid production. According to the results in this study, the exogenous GABA addition strategy is a simple and effective approach to improve microbial tolerance and enhance its fermentation performance.

Promyelocytic leukemia protein (PML) knockout increases mitochondrial Ca2+ uptake in HeLa cells

The multifunctional promyelocytic leukemia protein (PML) is involved in the regulation of various cellular processes in both physiological and pathological conditions. Specifically, PML is one of the inositol-1,4,5-trisphosphate receptors (IP3Rs) activity regulators and can influence Ca2+ transport from the endoplasmic reticulum (ER) to mitochondria. In this work, the effects of PML knockout on calcium homeostasis in the cytosol, ER, and mitochondria of HeLa cells were studied upon stimulation with histamine, which induces Ca2+ mobilization from the ER via IP3Rs. We utilized calcium indicators with different subcellular localizations, including synthetic dyes Fura-2 (cytosolic), Xrhod-5F (mitochondrial), and protein sensor R-CEPIAer (ER), as well as mitochondrial potential-sensitive probes Rh123 and TMRM. Our results show that PML knockout induced changes in HeLa cell and mitochondrial morphology, slightly decreased basal and integral Ca2+ levels, enhanced mitochondrial Ca2+ uptake from the cytoplasm, and maintained residual mitochondrial potential after depolarization. Additionally, it reduced the Ca2+ pool in ER membranes not associated with histamine receptor activation and, consequently, IP3Rs. These findings suggest that changes in calcium ion transport due to PML knockout in HeLa cells affect mitochondrial activity.

Effects of carbon black particles on human monocyte-derived macrophages: type-dependent pro-inflammatory activation in vitro.

Carbon black is a key component of air-borne particulate matter, linked to adverse health outcomes, such as increased susceptibility to respiratory infections and chronic pulmonary disease exacerbations. Fine and ultrafine particles can penetrate the lungs, enter the bloodstream, and induce pathogenetic events. Macrophages play a crucial role in responding to inhaled particles, including carbon black, by initiating an innate immune response and upregulating pro-inflammatory cytokines and anti-oxidative enzymes. This study investigates the effects of carbon black particles on human monocyte-derived macrophages in vitro at a concentration of 10µg/ml, offering insights into their potential role in disease pathogenesis. We have compared two commercially available carbon black particle types using various physicochemical techniques and assessed their biological effects on monocyte-derived macrophages. We have evaluated changes in cell viability, morphology, and particle uptake/phagocytosis. Western blot, ELISA, and RT-qPCR measured inflammatory and oxidative stress biomarkers. Both types of carbon black particles induced similar responses in macrophages, including particle uptake, cytokine production, and oxidative stress-related protein expression. The observed changes suggest activation of the Nrf2-mediated antioxidant response, impaired autophagy, and decreased cellular defense against oxidative stress, indicating potential pathways for chronic inflammatory lung disease development.

Tailoring glioblastoma treatment based on longitudinal analysis of post-surgical tumor microenvironment

Glioblastoma (GBM), an incurable primary brain tumor, typically requires surgical intervention followed by chemoradiation; however, recurrences remain fatal. Our previous work demonstrated that a nanomedicine hydrogel (GemC12-LNC) delays recurrence when administered post-surgery. However, tumor debulking also triggers time-dependent immune reactions that promote recurrence at the resection cavity borders. We hypothesized that combining the hydrogel with an immunomodulatory drug could enhance therapeutic outcomes. A thorough characterization of the post-surgical microenvironment (SMe) is crucial to guide combinatorial approaches.In this study, we performed cellular resolution imaging, flow cytometry and spatial hyperplexed immunofluorescence imaging to characterize the SMe in a syngeneic mouse model of tumor resection. Owing to our dynamic approach, we observed transient opening of the blood–brain barrier (BBB) during the first week after surgery. BBB permeability post-surgery was also confirmed in GBM patients. In our murine model, we also observed changes in immune cell morphology and spatial location post-surgery over time in resected animals as well as the accumulation of reactive microglia and anti-inflammatory macrophages in recurrences compared to unresected tumors since the first steps of recurrence growth. Therefore we investigated whether starting a systemic treatment with the SMAC mimetic small molecule (GDC-0152) directly after surgery would be beneficial for enhancing microglial anti-tumoral activity and decreasing the number of anti-inflammatory macrophages around the GemC12-LNC hydrogel-loaded tumor cavity. The immunomodulatory effects of this drug combination was firstly shown in patient-derived tumoroids. Its efficacy was confirmed in vivo by survival analysis and correlated with reversal of the immune profile as well as delayed tumor recurrence.This comprehensive study identified critical time frames and immune cellular targets within the SMe, aiding in the rational design of combination therapies to delay recurrence onset. Our findings suggest that post-surgical systemic injection of GDC-0152 in combination with GemC12-LNC local treatment is a promising and innovative approach for managing GBM recurrence, with potential for future translation to human patient.Graphical

Enhancing lung cancer growth inhibition with calcium ions: Role of mid- and high-frequency electric field pulses

Calcium electroporation (CaEP) involves the combination of calcium ions with electroporation, which is induced by pulsed electric fields (PEFs). This study explores the application of high-frequency unipolar nanosecond pulsed electric fields (nsPEFs: 8–14 kV/cm, 200 ns, 10 kHz, 100 kHz, 1 MHz repetition frequency pulse bursts, n = 100) and their potential in inhibiting lung cancer cell growth. As a reference, standard microsecond range parametric protocols were used (100 µs x 8 pulses). Methods included cell permeability quantification through Yo-Pro-1 uptake, cell viability assays, immunofluorescence studies for apoptosis and EMT markers, analysis of cell death types depending on repetition frequency pulse bursts. We determined the susceptibility of human lung cancer to electric pulses, characterized the efficacy of CaEP, and investigated cell death types depending on repetition frequency pulse bursts. We have shown that adding calcium ions to the applied nsPEF protocol increases cytotoxicity. Additionally, the use of these electroporation parameters can modulate key cellular processes, such as the epithelial-mesenchymal transition and apoptosis, as indicated by changes in the expression of markers such as E-cadherin, N-cadherin, BCL-2, and p53. Changes in cell morphology over time were observed using holotomographic microscopy. The study provides insights into the modulation of key cellular processes, indicating that nsPEF technology could improve the outcomes of conventional cancer treatments through enhanced efficacy and potentially mitigating drug resistance mechanisms. The promising results advocate for further research to optimize nsPEF protocols for clinical application, highlighting the potential of electrical fields in advancing cancer therapy.

Treatment of Melanoma Cells with Chloroquine and Everolimus Activates the Apoptosis Process and Alters Lipid Redistribution.

The balance between apoptosis and autophagy plays a key role in cancer biology and treatment strategies. The aim of this study was to assess the effect of the mTOR kinase inhibitor everolimus and chloroquine on the regulation of proliferation, caspase-3 activation, and apoptosis in melanoma cells. We studied the activity of caspase-3 and the levels of caspase-3 and -9 using the Western blot technique. Cellular apoptosis was examined using a DNA fragmentation assay, and changes in the cell nucleus and cytoskeleton were examined using fluorescence microscopy DAPI, OA/IP. We also studied the rearrangement of lipid structures using fluorescent dyes: Nile Red and Nile Blue. A low nanomolar concentration of the mTOR kinase inhibitor everolimus in combination with chloroquine activated the apoptosis process and decreased cell proliferation. These changes were accompanied by an obvious change in cell morphology and rearrangement of lipid structures. Alterations in lipid redistribution accompanying the process of apoptosis and autophagy are among the first to occur in the cell and can be easily monitored in in vitro studies. The combination of mTOR inhibitors and chloroquine represents a promising area of research in cancer therapy. It has the potential to enhance treatment efficacy through complementary mechanisms.

Müller Glia Co-Regulate Barrier Permeability with Endothelial Cells in an Vitro Model of Hyperglycemia.

Diabetic retinopathy is a complex, microvascular disease that impacts millions of working adults each year. High blood glucose levels from Diabetes Mellitus lead to the accumulation of advanced glycation end-products (AGEs), which promote inflammation and the breakdown of the inner blood retinal barrier (iBRB), resulting in vision loss. This study used an in vitro model of hyperglycemia to examine how endothelial cells (ECs) and Müller glia (MG) collectively regulate molecular transport. Changes in cell morphology, the expression of junctional proteins, and the reactive oxygen species (ROS) of ECs and MG were examined when exposed to a hyperglycemic medium containing AGEs. Trans-endothelial resistance (TEER) assays were used to measure the changes in cell barrier resistance in response to hyperglycemic and inflammatory conditions, with and without an anti-VEGF compound. Both of the cell types responded to hyperglycemic conditions with significant changes in the cell area and morphology, the ROS, and the expression of the junctional proteins ZO-1, CX-43, and CD40, as well as the receptor for AGEs. The resistivities of the individual and dual ECs and MG barriers decreased within the hyperglycemia model but were restored to that of basal, normoglycemic levels when treated with anti-VEGF. This study illustrated significant phenotypic responses to an in vitro model of hyperglycemia, as well as significant changes in the expression of the key proteins used for cell-cell communication. The results highlight important, synergistic relationships between the ECs and MG and how they contribute to changes in barrier function in combination with conventional treatments.

Development and Validation of a Flow-Dependent Endothelialized 3D Model of Intracranial Atherosclerotic Disease.

Intracranial atherosclerotic disease (ICAD) is a major cause of stroke globally, with mechanisms presumed to be shared with atherosclerosis in other vascular regions. Due to the scarcity of relevant animal models, testing biological hypotheses specific to ICAD is challenging. We developed a workflow to create patient-specific models of the middle cerebral artery (MCA) from neuroimaging studies, such as CT angiography. These models, which can be endothelialized with human endothelial cells and subjected to flow forces, provide a reproducible ICAD model. Using imaging from the SAMMPRIS clinical trial, we validated this novel model. Computational fluid dynamics flow velocities correlated strongly with particle-derived flow, regardless of stenosis degree. Post-stenotic flow disruption varied with stenosis severity. Single-cell RNA-seq identified flow-dependent endothelial gene expression and specific endothelial subclusters in diseased MCA segments, including upregulated genes linked to atherosclerosis. Confocal microscopy revealed flow-dependent changes in endothelial cell proliferation and morphology in vessel segments related to stenosis. This platform, rooted in the specific anatomy of cerebral circulation, enables detailed modeling of ICAD lesions and pathways. Given the high stroke risk associated with ICAD and the lack of effective treatments, these experimental models are crucial for developing new ICAD-related stroke therapies.

Translational implications of a novel combination of iPRF and collagen scaffold for proliferation of gingival mesenchymal stem cells

“Tissue engineering,” is a concept that involves the use of scaffolds, cells, and growth factors/signaling molecules in the field of regenerative medicine. The present study was carried out to evaluate the attachment and depth of penetration of the Gingival Mesenchymal Stem Cells (GMSCs) onto the collagen scaffold preconditioned with Fetal Bovine Serum (FBS), Injectable Platelet Rich Fibrin (i-PRF) and a combination of FBS with i-PRF using histological analysis and environmental scanning electron microscopy (ESEM) respectively. In the present study, commercially available collagen membranes were used as scaffolds and divided into 3 groups where Group I was preconditioned with FBS, Group II was preconditioned with i-PRF, and Group III with a combination of FBS and i-PRF. Scaffolds were then seeded with GMSCs and incubated in a CO2 incubator at 37 0C for 7 days. Both histological and ESEM analysis showed proliferation, attachment, and depth of penetration along with the combination of cell morphological changes in all the three groups. In group I cells showed mild depth of penetration along with a round morphological appearance. In group II the cells showed moderate depth of penetration and rounded morphology. In group III maximum depth of penetration was seen along with the round and spheroidal morphology of cells. The combination of the growth media showed the best effect on cell attachment, depth of penetration, and cell morphology. This is the first report demonstrating the effect of the combination of collagen and i-PRF supports the proliferation of GMSCs. Since FBS alone and i-PRF alone exhibited similar cell proliferation it is recommended that i-PRF could be used in clinical scenarios making it xenofree.

Mechanical Properties of Inflamed Appendix Tissues.

Background/Objectives: Histopathological examination enables visualization of morphological changes in cells and tissues. In recent years, there has been increasing interest in assessing the mechanical properties of tissues that cannot be determined by standard histopathological examinations. Mechanobiology is crucial in human physiology and holds promise for uncovering new diagnostic markers for disease processes such as carcinogenesis and inflammation. In this study, we concentrated on measuring the mechanical properties of appendix biopsy specimens to identify potential mechanomarkers of inflammation. Appendix tissues provided the opportunity to measure mechanical properties both with an atomic force microscope and a shear rheometer. Methods: The atomic force microscope AFM-NanoWizard 4 BioScience JPK/Bruker was used for the evaluation of the elastic modulus (i.e., Young's modulus) of appendix tissues. Young's modulus was derived from the Hertz-Sneddon model applied to force-indentation curves. The rheological properties of macroscopic samples were measured on a parallel-plate, strain-controlled shear rheometer Anton Paar MCR302. Results: The data collected suggest that elasticity, expressed as Young's modulus and the storage modulus, could be considered a marker indicating appendix tissue inflammation. Young's modulus of inflamed appendix tissues was found to be significantly lower than that of healthy ones, with an average reduction of 67%. Furthermore, it was observed that inflamed appendix tissues, in comparison to healthy ones, respond differently under varying axial and shear stresses, enabling their identification. Conclusions: Our findings suggest that the specific mechanical properties of inflamed vermiform appendices could serve as novel mechanomarkers for the early detection and monitoring of appendicitis.

High-Throughput Measurement of Single-Fission Yeast Cell Volume Using Fluorescence Exclusion.

Cell volume is a critical parameter for the biology of living species and has an impact on virtually all cellular functions. In Schizosaccharomyces pombe, the regulation of cell size has been the focus of intense investigation, and mechanisms that couple size control with cell cycle progression have been identified. In fission yeast, cell length at division is generally used as a proxy for determining cell size. However, it only allows for an inaccurate evaluation of this critical parameter and neglects potential changes in cell morphology and diameter, which can strongly impact cell volume. Until recently, one of the major obstacles for studying the complexity of cell size regulation in fission yeast has been the lack of a robust method for high-throughput, direct measurement of single-cell volume. Here, we provide a comprehensive protocol for S. pombe cell volume determination based on the Fluorescence eXclusion method and microfluidics technologies. This approach makes it possible to reliably describe cell volume and its distribution in populations of fission yeast cells.

Antioxidant, neuroprotective, and neuroblastoma cells (SH-SY5Y) differentiation effects of melanins and arginine-modified melanins from Daedaleopsis tricolor and Fomes fomentarius

BackgroundMicrobial melanins possess a broad spectrum of biological activities. However, there is little understanding of their neuroprotective and neuronal cell differentiation properties. This study aimed to extract, purify, and modify melanins from two medicinal fungi (Daedaleopsis tricolor and Fomes fomentarius), and to evaluate their antioxidant activity, as well as their cell protective ability against neurotoxins. In addition, the study also investigated the feasibility of combining melanins or modified melanins with retinoic acid (RA) to induce neuronal differentiation.MethodsMelanin was extracted and purified using alkaline acid-based methods. Antioxidant activities and neuroprotective effects were evaluated using the DPPH (1,1-diphenyl-2-picrylhydrazyl) and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assays, respectively. In addition, morphological changes of SH-SY5Y cells were recorded by using a Pannoramic MIDI scanner.ResultsAll melanins and arginine-modified melanins displayed mild DPPH scavenging activities, which were statistically lower than that of ascorbic acid (p < 0.05). In terms of neuroprotection, both melanins and arginine-modified melanins exhibited significant cell protection against H2O2 after 24 h exposure (p < 0.05). Notably, there is no significant difference between F. fomentarius melanin and its modified form as they both increased cell viability by about 20%. Contrarily, while D. tricolor melanin enhanced the cell viability with 16%, its modified form increased the cell viability with 21%. These activities, however, are significantly lower than the positive control (N-acetylcysteine, p < 0.05). Regarding MPTP, only the arginine-modified melanins of the two fungi significantly protected the cells after 24 h exposure to the toxin (p < 0.05). Specifically, F. fomentarius and D. tricolor modified melanins enhanced the cell viability with 10.2% and 11.1%, respectively, whereas that of the positive control was 13.2%. Interestingly, combining RA (10 µM) with 20 µg/mL of either F. fomentarius, or especially D. tricolor arginine-modified melanin, significantly promoted neuroblastoma cell differentiation into mature neuronal cells compared to using RA alone (p < 0.05).ConclusionsThe arginine-modified melanins of D. tricolor and F. fomentarius have potential for neuroprotection against Parkinsonian neurotoxins. In addition, the arginine-modified melanin of D. tricolor may serve as an excellent material for research in neuroblastoma treatment.