Cell Monolayers Research Articles

Tumor-derived extracellular vesicles disrupt the blood–brain barrier endothelium following high-frequency irreversible electroporation

AbstractHigh-frequency irreversible electroporation (H-FIRE), a nonthermal brain tumor ablation therapeutic, generates a central tumor ablation zone while transiently disrupting the peritumoral blood–brain barrier (BBB). We hypothesized that bystander effects of H-FIRE tumor cell ablation, mediated by small tumor-derived extracellular vesicles (sTDEV), disrupt the BBB endothelium. Monolayers of bEnd.3 cerebral endothelial cells were exposed to supernatants of H-FIRE or radiation (RT)-treated LL/2 and F98 cancer cells. Endothelial cell response was evaluated microscopically and via flow cytometry for apoptosis. sTDEV were isolated following H-FIRE and RT, characterized via nanoparticle tracking analysis (NTA) and transmission electron microscopy, and applied to a Transwell BBB endothelium model to quantify permeability changes. Supernatants of H-FIRE-treated tumor cells, but not supernatants of sham- or RT-treated cells, disrupted endothelial cell monolayer integrity while maintaining viability. sTDEV released by glioma cells treated with 3000 V/cm H-FIRE increased permeability of the BBB endothelium model compared to sTDEV released after lower H-FIRE doses and RT. NTA revealed significantly decreased sTDEV release after the 3000 V/cm H-FIRE dose. Our results demonstrate that sTDEV increase permeability of the BBB endothelium after H-FIRE ablation in vitro. sTDEV-mediated mechanisms of BBB disruption may be exploited for drug delivery to infiltrative margins following H-FIRE ablation.

Vinculin-Arp2/3 interaction inhibits branched actin assembly to control migration and proliferation.

Vinculin is a mechanotransducer that reinforces links between cell adhesions and linear arrays of actin filaments upon myosin-mediated contractility. Both adhesions to the substratum and neighboring cells, however, are initiated within membrane protrusions that originate from Arp2/3-nucleated branched actin networks. Vinculin has been reported to interact with the Arp2/3 complex, but the role of this interaction remains poorly understood. Here, we compared the phenotypes of vinculin knock-out (KO) cells with those of knock-in (KI-P878A) cells, where the point mutation P878A that impairs the Arp2/3 interaction is introduced in the two vinculin alleles of MCF10A mammary epithelial cells. The interaction of vinculin with Arp2/3 inhibits actin polymerization at membrane protrusions and decreases migration persistence of single cells. In cell monolayers, vinculin recruits Arp2/3 and the vinculin-Arp2/3 interaction participates in cell-cell junction plasticity. Through this interaction, vinculin controls the decision to enter a new cell cycle as a function of cell density.

Progesterone supplemented uterine epithelial cell co-culture improves in vitro quality embryo production in buffalo

Objectives: The present study was conducted to study the effect of progesterone and uterine luminal epithelial cells monolayer on blastocyst development and hatching rate. Material and Methods: The isolation, culture, and characterization of slaughterhouse-derived uterine epithelial cells were done using standard protocol. The steroid hormones estrogen and progesterone were supplemented in embryo developmental media (EDM), and day 04 embryos were cultured in different groups as progesterone supplementation (T1), co-cultured with epithelial cell monolayer (T2), co-cultured with progesterone supplemented epithelial cell monolayer (T3), or without any treatment (control). Finally, the effect of different treatments was analyzed in terms of blastocyst and hatching rate. Results: The isolated epithelial cells depicted compact cuboidal or columnar morphology at the confluence. Immunocytochemical localization and polymerase chain reaction study revealed positive expression of cytokeratin and absence of vimentin. Significantly higher blastocyst and hatching rates were noted in the T3 group, followed by T2, T1, and control groups. Conclusion: The present study revealed improved in vitro embryo production after co-culturing embryos with progesterone-supplemented uterine epithelial cells in buffalo.

Meniscus gene expression profiling of inner and outer zone meniscus tissue compared to cartilage and passaged monolayer meniscus cells

Meniscus injuries are common and while surgical strategies have improved, there is a need for alternative therapeutics to improve long-term outcomes and prevent post-traumatic osteoarthritis. Current research efforts in regenerative therapies and tissue engineering are hindered by a lack of understanding of meniscus cell biology and a poorly defined meniscus cell phenotype. This study utilized bulk RNA-sequencing to identify unique and overlapping transcriptomic profiles in cartilage, inner and outer zone meniscus tissue, and passaged inner and outer zone meniscus cells. The greatest transcriptomic differences were identified when comparing meniscus tissue to passaged monolayer cells (> 4,600 differentially expressed genes (DEGs)) and meniscus tissue to cartilage (> 3,100 DEGs). While zonal differences exist within the meniscus tissue (205 DEGs between inner and outer zone meniscus tissue), meniscus resident cells are more similar to each other than to either cartilage or passaged monolayer meniscus cells. Additionally, we identified and validated LUM, PRRX1, and SNTB1 as potential markers for meniscus tissue and ACTA2, TAGLN, SFRP2, and FSTL1 as novel markers for meniscus cell dedifferentiation. Our data contribute significantly to the current characterization of meniscus cells and provide an important foundation for future work in meniscus cell biology, regenerative medicine, and tissue engineering.

Unraveling Microviscosity Changes Induced in Cancer Cells by Photodynamic Therapy with Targeted Genetically Encoded Photosensitizer

Background: Despite the fundamental importance of cell membrane microviscosity, changes in this biophysical parameter of membranes during photodynamic therapy (PDT) have not been fully understood. Methods: In this work, changes in the microviscosity of membranes of live HeLa Kyoto tumor cells were studied during PDT with KillerRed, a genetically encoded photosensitizer, in different cellular localizations. Membrane microviscosity was visualized using fluorescence lifetime imaging microscopy (FLIM) with a viscosity-sensitive BODIPY2 rotor. Results: Depending on the localization of the phototoxic protein, different effects on membrane microviscosity were observed. With nuclear localization of KillerRed, a gradual decrease in microviscosity was detected throughout the entire observation period, while for membrane localization of KillerRed, a dramatic increase in microviscosity was observed in the first minutes after PDT, and then a significant decrease at later stages of monitoring. The obtained data on cell monolayers are in good agreement with the data obtained for 3D tumor spheroids. Conclusions: These results indicate the involvement of membrane microviscosity in the response of tumor cells to PDT, which strongly depends on the localization of reactive oxygen species attack via targeting of a genetically encoded photosensitizer.

A probiotic multi-strain mixture combined with hydroxyectoine improves intestinal barrier function by alleviating inflammation in lipopolysaccharide stimulated differentiated Caco-2 cells.

Many studies have highlighted the role of probiotics in re-establishing the gut microbiota balance and preventing intestinal barrier dysfunction. In fact, they can also contribute to the upregulation of anti-inflammatory genes and the downregulation of pro-inflammatory genes, which are known to contribute to the development of the inflammatory bowel disease (IBD) syndrome. The present study aims to investigate the effect of the compatible solute hydroxyectoine (HOE), to be used as a cryopreservant but also for its intrinsic biological properties, to obtain a new formula containing three probiotic strains (Limosilactobacillus fermentum (L. fermentum), Levilactobacillus brevis SP-48 (L. brevis), and Bifidobacterium lactis HN019 (B. lactis)), and evaluate the latter for its ability to prevent lipopolysaccharide (LPS)-induced inflammation in an in vitro bi-dimensional model of the intestinal barrier using a Caco-2 cell monolayer. The mRNA expression levels of the inflammatory cytokines (IL-6, IL-1β, and TNF-α) were analyzed by real-time PCR. Changes in the modulation of (TLR-4 and NF-κB) proteins were assessed by western blotting, and the effect of the HOE/PRO formula on the intestinal epithelial barrier function was also assessed using an immunofluorescence microscope for the tight junction protein zonula occludens-1 (ZO-1). This study found that this novel probiotic formulation containing HOE is capable of decreasing LPS-induced cytokines, as confirmed by the results of RT-PCR and ELISA and preserving the integrity of tight junctions as demonstrated by the relevant expression of ZO-1. HOE/PRO was shown to be effective in reducing the expression of TLR-4 and NF-κB. The latter plays a key role as an inflammation modulator as shown through experiments run with the THP-1/NF-κB reporter gene. Collectively, our data indicate that the HOE/PRO formula is a good candidate for potential preventive and/or therapeutic implementation in IBD.

Neo-Glycolipid Oximes as Intestinal Permeation Enhancers for Peptide Hormone PYY3-36.

Herein, we describe the design and synthesis of 16 neo-glycolipids that are potential permeation enhancers for oral drug delivery of peptide therapeutics. These amphiphilic neo-glycolipids are composed of fatty acids and various carbohydrates (D-glucose, lactose, cellobiose, maltose) via an oxime linker. The ability of the synthesized neo-glycolipids to enhance permeation of fluorescein-labelled dextran (4 kDa) or 3H-mannitol across intestinal epithelium was investigated in vitro using monolayers of human epithelial Caco-2 cells. Their effects were compared with (pre-)clinically known enhancers as reference compounds; sodium salts of octanoic, decanoic, and dodecanoic acid, and sodium salcaprozate (SNAC). Most neo-glycolipids increased the permeation of the model compounds, proving that neo-glycolipids, which possess vastly different properties from the reference compounds, e.g., in terms of clogD and polar surface area, are effective permeation enhancers. The neo-glycolipid based on decanoic acid and glucose was more potent than related compounds based on disaccharides. Significant differences in solubility and cellular compatibility were found for neo-glyolipids based on different carbohydrates. Finally, neo-glycolipids were evaluated as permeation enhancers for the peptide hormone PYY3-36. Glucose- and maltose-derived neo-glycolipids based on decanoic and dodecanoic acid showed promising enhancements in PYY3-36 permeation while maintaining good cellular compatibility, relevant for oral delivery of obesity treatments.

Giardia antagonizes beneficial functions of indigenous and therapeutic intestinal bacteria during protein deficiency

ABSTRACT Undernutrition in children commonly disrupts the structure and function of the small intestinal microbial community, leading to enteropathies, compromised metabolic health, and impaired growth and development. The mechanisms by which diet and microbes mediate the balance between commensal and pathogenic intestinal flora remain elusive. In a murine model of undernutrition, we investigated the direct interactions Giardia lamblia, a prevalent small intestinal pathogen, on indigenous microbiota and specifically on Lactobacillus strains known for their mucosal and growth homeostatic properties. Our research reveals that Giardia colonization shifts the balance of lactic acid bacteria, causing a relative decrease in Lactobacillus spp. and an increase in Bifidobacterium spp. This alteration corresponds with a decrease in multiple indicators of mucosal and nutritional homeostasis. Additionally, protein-deficient conditions coupled with Giardia infection exacerbate the rise of primary bile acids and susceptibility to bile acid-induced intestinal barrier damage. In epithelial cell monolayers, Lactobacillus spp. mitigated bile acid-induced permeability, showing strain-dependent protective effects. In vivo, L. plantarum, either alone or within a Lactobacillus spp consortium, facilitated growth in protein-deficient mice, an effect attenuated by Giardia, despite not inhibiting Lactobacillus colonization. These results highlight Giardia’s potential role as a disruptor of probiotic functional activity, underscoring the imperative for further research into the complex interactions between parasites and bacteria under conditions of nutritional deficiency.

Calcium Transport Activity of UV/H2O2-Degraded Fucoidans and Their Structural Characterization

Calcium-chelated polysaccharides have been increasingly considered as promising calcium supplements. In this study, degraded fucoidans (DFs) with different molecular weights (Mws) were prepared after UV/H2O2 treatment; their calcium-chelating capacities and intestinal absorption properties were also investigated. The results showed that the calcium-chelating capacities of DFs were improved with a decrease in Mw. This was mainly ascribed to the increased carboxyl content, which was caused by free-radical-mediated degradation. Meanwhile, the conformation of DF changed from a rod-like chain to a shorter and softer chain. The thermodynamic analysis demonstrated that DF binding to calcium was spontaneously driven by electrostatic interactions. Additionally, DF-Ca chelates with lower Mw showed favorable transport properties across a Caco-2 cell monolayer and could effectively accelerate the calcium influx through intestinal enterocytes. Furthermore, these chelates also exhibited a protective effect on the epithelial barrier by alleviating damage to tight junction proteins. These findings provide an effective free-radical-related approach for the development of polysaccharide-based calcium supplements with improved intestinal calcium transport ability.

Endothelial Dysfunctions in Blood-Brain Barrier Breakdown in Alzheimer's Disease: From Mechanisms to Potential Therapies.

Recent research has shown the presence of blood-brain barrier (BBB) breakdown in Alzheimer's disease (AD). BBB is a dynamic interface consisting of a continuous monolayer of brain endothelial cells (BECs) enveloped by pericytes and astrocytes. The restricted permeability of BBB strictly controls the exchange of substances between blood and brain parenchyma, which is crucial for brain homeostasis by excluding blood-derived detrimental factors and pumping out brain-derived toxic molecules. BBB breakdown in AD is featured as a series of BEC pathologies such as increased paracellular permeability, abnormal levels and functions of transporters, and inflammatory or oxidative profile, which may disturb the substance transportation across BBB, thereafter induce CNS disorders such as hypometabolism, Aβ accumulation, and neuroinflammation, eventually aggravate cognitive decline. Therefore, it seems important to protect BEC properties for BBB maintenance and neuroprotection. In this review, we thoroughly summarized the pathological alterations of BEC properties reported in AD patients and numerous AD models, including paracellular permeability, influx and efflux transporters, and inflammatory and oxidative profiles, and probably associated underlying mechanisms. Then we reviewed current therapeutic agents that are effective in ameliorating a series of BEC pathologies, and ultimately protecting BBB integrity and cognitive functions. Regarding the current drug development for AD proceeds extremely hard, this review aims to discuss the therapeutic potentials of targeting BEC pathologies and BBB maintenance for AD treatment, therefore expecting to shed a light on the future AD drug development by targeting BEC pathologies and BBB protection.

Tailoring metabolic activity assays for tumour-engineered 3D models

Monitoring cell behaviour in hydrogel-based 3D models is critical for assessing their growth and response to cytotoxic treatment. Resazurin-based PrestoBlue and AlamarBlue reagents are frequently used metabolic activity assays when determining cell responses. However, both assays are largely applied to cell monolayer cultures but yet to have a defined protocol for use in hydrogel-based 3D models. The assays' performance depends on the cell type, culture condition and measurement sensitivity. To better understand how both assays perform, we grew pancreatic cancer cells in gelatin methacryloyl and collagen hydrogels and evaluated their metabolic activity using different concentrations and incubation times of the PrestoBlue and AlamarBlue reagents. We tested reagent concentrations of 4 % and 10 % and incubation times of 45 min, 2 h and 4 h. In addition, we co-cultured cancer cells together with cancer-associated fibroblasts and peripheral blood mononuclear cells in gelatin methacryloyl hydrogels and subjected them to gemcitabine and nab-paclitaxel to evaluate how both assays perform when characterising cell responses upon drug treatment. CyQuant assays were conducted on the same samples and compared to data from the metabolic activity assays. In cancer monocultures, higher reagent concentration and incubation time increased fluorescent intensity. We found a reagent concentration of 10 % and an incubation time of 2 h suitable for all cell lines and both hydrogels. In multicellular 3D cultures, PrestoBlue and AlamarBlue assays detected similar cell responses upon drug treatment but overestimated cell growth. We recommend to assess cell viability and growth in conjunction with CyQuant assays that directly measure cell functions.

A Distinct Genotype, D/Ep6, Detected in Korean Female Patients: Chlamydia trachomatis Characterization from 2017 - 2018.

The objective of this study lies in identifying the dominant genotype of C. trachomatis isolated in Republic of Korea (ROK) between 2017 and 2018. A total of 504 clinical cervicovaginal swabs from patients were collected and inoculated on McCoy cell monolayers to isolate Chlamydial agents. C. trachomatis isolates were analyzed by sequencing of its ompA gene. A total of 54 C. trachomatis isolates were obtained. The constructed phylogenetic tree revealed the genotypes of isolates are D/Ep6 (48, 88.89%), D/Ep6-like (5, 9.26%), and Ja (1, 1.85%). The C. trachomatis D/Ep6-like have one single nucleotide polymorphism (SNP) compared to C. trachomatis D/Ep6, leading to E870K amino acid change. Phylogenetic analysis demonstrated globally rare C. trachomatis D/Ep6 was dominant in the ROK from 2017 to 2018. Findings in this study act as a keystone, bridging past and present molecular epidemiology of C. trachomatis in context of ROK.

Role of Gpcpd1 in intestinal alpha-glycerophosphocholine metabolism and trimethylamine N-oxide production

Glycerophosphocholine (GPC) is an intracellular metabolite in phosphatidylcholine metabolism and has been studied for endogenous choline supply in cells. GPC, as a water-soluble supplement, has been expected to play a role in preventing brain disorders; however, recent studies have shown that intake of high levels of choline-containing compounds is related to trimethylamine N-oxide (TMAO) production in the liver, which is reportedly associated with the progression of atherosclerosis. In this study, we aimed to explore the mechanisms underlying the intestinal absorption and metabolism of GPC. Caco-2 cell monolayer experiments showed that exogenously added GPC was hydrolyzed to choline in the apical medium, and the resulting choline was transported into the Caco-2 cells and further to the basolateral medium. Subsequently, we focused on glycerophosphodiesterase 1 (Gpcpd1/GDE5), which hydrolyzes GPC to choline in vitro and is widely expressed in the gastrointestinal epithelium. Our results revealed that the Gpcpd1 protein was located not only in cells but also in the medium in which Caco-2 cells were cultured. Gpcpd1 siRNA decreased the GPC-hydrolyzing activity both inside Caco-2 cells and in conditioned medium, suggesting the involvement of Gpcpd1 in luminal GPC metabolism. Finally, we generated intestinal epithelial–specific Gpcpd1-deficient mice and found that Gpcpd1 deletion in intestinal epithelial cells affected GPC metabolism in intestinal tissues and partially abolished the increase in blood TMAO levels induced by GPC administration. These observations demonstrate that Gpcpd1 triggers choline production from GPC in the intestinal lumen and is a key endogenous enzyme that regulates TMAO levels following GPC supplementation.

Ultrasonication coupled to enzymatic hydrolysis of soybean okara proteins for producing bioactive and bioavailable peptides

This work was aimed to explore the antioxidative properties, bioavailability and the safety of bioactive peptides obtained by the enzymatic hydrolysis of ultrasound-treated (UO) and untreated (nUO) soybean okara proteins. Particularly, the peptidomic profiles of both hydrolysates were examined using an untargeted metabolomics technique for suspect screening that was specifically designed for the profiling of short-chain peptides and relied on ultra-high-performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS) and bioinformatics.Next, both UO and nUO hydrolysates reduce Dipeptidyl peptidase-IV (DPP-IV) enzyme activity until 39.54 ± 0.26 % and 43.29 ± 0.36 % respectively and inhibit angiotensin converting enzyme (ACE) activities by 30.54 ± 0.42 % and 30.76 ± 0.02 %, respectively. Moreover, they demonstrate to exerted antioxidant properties. Particularly, they show a comparable in vitro antioxidant activity but when the oxidative stress is induced by H2O2 in Caco-2 cells, UO hydrolysate is more active in lowering the levels of reactive oxygen species (ROS) and of lipid peroxidation induced of 48% and 20% respectively.In addition, UO- and nUO-derived peptides trans-epithelial transported by human differentiated intestinal cell monolayer, were identified. Lastly, the possible hepatotoxicity of UO and nUO hydrolysates in HepG2 cells has not been observed by measuring alanine transferase (ALT) and aspartate transferase (AST) levels and cytotoxic effects.

Gastrointestinal metabolism of Astragalus membranaceus polysaccharides and its related hypoglycemic mechanism based on gut microbial transformation

Astragalus membranaceus polysaccharides (AMP) was reported to exhibit hypoglycemic potential in diabetic host. However, the metabolic fate of AMP in gastrointestinal tract and its underlying hypoglycemic mechanisms remained unclear. Our current study aimed to reveal the structure alteration of AMP in gastrointestinal tract and its hypoglycemic mechanism from the perspective of microbial transformation. Caco-2 monolayer cell model revealed that AMP exhibited poor intestinal absorption. The in-vitro digestion and fermentation study revealed that AMP remained intact after gastrointestinal digestion while it could be degraded and utilized by gut microbiota with increased SCFA formation and decreased levels of all the monosaccharides in AMP except for mannose. Additionally, Diversity of gut microbiota was improved with the increased abundance of Dubosiella and Monoglobus and decreased abundance of Escherichia-Shigella and Acinetobacter after fermentation of AMP. Further hypoglycemic mechanism study for the first time revealed that both AMP and its potential microbial metabolites, SCFA salt mixture, could enhance intestinal integrity significantly on LPS induced Caco-2 cell model, while only SCFA salt mixture rather than AMP could significantly stimulate GLP-1 secretion in NCI-H716 cell model possibly via promoting GPCR43 expression. Such findings provided insights into the hypoglycemic mechanism of AMP from the perspective of microbial transformation.

Biophysical Analysis of EGCG’s Protective Effects on Camptothecin-Induced Oxidative Stress in Bone-like Cancer Cells Using Electric Cell-Substrate Impedance Sensing (ECIS)

Various medical treatments aim to counteract the impact of oxidants on mammalian cells. One such antioxidant is Epigallocatechin-3-gallate (EGCG), an active ingredient in green tea, which has demonstrated protective effects against cellular oxidants like camptothecin (CAMPT). This study examines how EGCG mitigates CAMPT’s effects on UMR cells, focusing on cell proliferation and biophysical parameters. UMR cells were treated with different CAMPT concentrations and incubated for 72 h. Subsequently, cell proliferation and viability were assessed. In a separate experiment, UMR cells were co-treated with CAMPT and varying EGCG concentrations to evaluate EGCG’s ability to mitigate CAMPT’s oxidative effect. Electric Cell–Substrate Impedance Sensing (ECIS) technology was also used to assess the biophysical parameters of CAMPT-treated UMR cells, including cell monolayer resistance, cell spreading, and cell attachment. The results showed a concentration-dependent decrease in cell proliferation for CAMPT-treated UMR cells. However, co-treatment with EGCG reversed CAMPT’s oxidative effects in a concentration-dependent manner. ECIS technology revealed a decrease in biophysical parameters when UMR cells were treated with CAMPT alone. Statistical analysis indicated significant differences with p-values < 0.05. This study suggests that EGCG effectively protects UMR cells from oxidative stress and highlights its potential role in mitigating oxidative stress in mammalian cells. Additionally, the use of ECIS technology validates its application in corroborating the biological effects of CAMPT and EGCG on UMR cells.

Modulatory potentials of nitric oxide in obesity-induced endothelial dysfunction

Obesity is caused by a significant increase in adipose tissue due to excessive caloric intake that exceeds energy expenditure. Obesity has emerged as a significant public health issue in both poor resource and economic developed nations, owing to its increased incidence and links to chronic diseases, such osteoarthritis, hypertension, non-alcoholic fatty liver disease (NAFLD), and cardiovascular diseases (CVDs). It is believed that uncoupled eNOS, nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, xanthine oxidase, lipoxygenase, cyclooxygenase, microsomal P-450 enzymes, and pro-oxidant heme molecules are responsible for the drastic decrease in NO production and the relative increase in reactive oxygen species (ROS) secretion, which are considered as the molecular mechanisms associated with obesity-induced endothelial dysfunction. Vascular endothelium defines a monolayer of cells, which lies between the vascular smooth muscle cells (VSMCs) and the vessel lumen. It performs a number of protective actions facilitated by the release of nitric oxide (NO), a soluble gas that is generated in endothelial cells by the amino acid L-arginine through the action of endothelial nitric oxide synthase (eNOS). The maintenance of vascular homeostasis is facilitated by various biological actions of NO, such as modulation of vascular dilator tone, control of local cell growth, and protection of the endothelium from the harmful effects of cellular components in blood circulation, while maintaining normal endothelial functions. Decreased peripheral arterial endothelium-dependent dilation (EDD) in response to mechanical (intravascular shear) or chemical (acetylcholine) stimuli suggests that decreased NO bioavailability has been recognised as major molecular mechanisms associated with the progress and development of obesity-induced endothelial dysfunction. In this review, the modulatory effects of NO in obesity-induced endothelial dysfunction will be discussed.

Effect of Oral Administration of Collagen Peptide OG-5 on Advanced Atherosclerosis Development in ApoE-/- Mice.

Atherosclerosis is a chronic inflammatory disease of the arterial wall, which involves multiple cell types. Peptide OG-5 is identified from collagen hydrolysates derived from Salmo salar and exhibits an inhibitory effect on early atherosclerosis. The primary objective of this study was to investigate the impact of OG-5 on advanced atherosclerotic lesions as well as its stability during absorption. In this study, the ApoE-/- mice were employed to establish advanced atherosclerosis model to investigate the treatment effect of peptide OG-5. The results showed that oral administration of OG-5 at a dosage of 150 mg/kg bw resulted in a 30% reduction in the aortic plaque formation area in ApoE-/- mice with few bleeding risks. Specifically, intervention with a low dose of OG-5 (50 mg/kg bw), initiated in the early stage of atherosclerosis, continues to provide benefits into the middle and late stages without bleeding risks. Furthermore, treatment of OG-5 increased expression levels of contractile phenotype markers and reduced the accumulation of lipoprotein in VSMCs induced by ox-LDL. Peptide OG-5 could ensure transport across Caco-2 cell monolayers, exhibiting a Papp value of 1.80 × 10-5 cm/s, and exhibited a robust stability in plasma with remaining content >70% after 8 h incubation. In vivo studies revealed that OG-5 reached maximum concentration in blood after 120 min. The present results demonstrate the potential efficacy of peptide OG-5 as a promising agent for intervention in anti-atherogenesis strategies.

Mechanism of Marinobufagenin-Induced Hyperpermeability of Human Brain Microvascular Endothelial Cell Monolayer: A Potential Pathogenesis of Seizure in Preeclampsia.

Preeclampsia (preE) is a hypertensive disorder in pregnancies. It is the third leading cause of mortality among pregnant women and fetuses worldwide, and there is much we have yet to learn about its pathophysiology. One complication includes cerebral edema, which causes a breach of the blood-brain barrier (BBB). Urinary marinobufagenin (MBG) is elevated in a preE rat model prior to developing hypertension and proteinuria. We investigated what effect MBG has on the endothelial cell permeability of the BBB. Human brain microvascular endothelial cells (HBMECs) were utilized to examine the permeability caused by MBG. The phosphorylation of ERK1/2, Jnk, p38, and Src was evaluated after the treatment with MBG. Apoptosis was evaluated by examining caspase 3/7. MBG ≥ 1 nM inhibited the proliferation of HBMECs by 46-50%. MBG induced monolayer permeability, causing a decrease in the phosphorylation of ERK1/2 and the activated phosphorylation of Jnk, p38, and Src. MBG increased the caspase 3/7 expression, indicating the activation of apoptosis. Apoptotic signaling or the disruption of endothelia tight junction proteins was not observed when using the p38 inhibitor as a pretreatment in MBG-treated cells. The MBG-induced enhancement of the HBMEC monolayer permeability occurs by the downregulation of ERK1/2, the activation of Jnk, p38, Src, and apoptosis, resulting in the cleavage of tight junction proteins, and are attenuated by p38 inhibition.

A programmable platform for probing cell migration and proliferation.

The advent of advanced robotic platforms and workflow automation tools has revolutionized the landscape of biological research, offering unprecedented levels of precision, reproducibility, and versatility in experimental design. In this work, we present an automated and modular workflow for exploring cell behavior in two-dimensional culture systems. By integrating the BioAssemblyBot® (BAB) robotic platform and the BioApps™ workflow automater with live-cell fluorescence microscopy, our workflow facilitates execution and analysis of in vitro migration and proliferation assays. Robotic assistance and automation allow for the precise and reproducible creation of highly customizable cell-free zones (CFZs), or wounds, in cell monolayers and "hands-free," schedulable integration with real-time monitoring systems for cellular dynamics. CFZs are designed as computer-aided design models and recreated in confluent cell layers by the BAB 3D-Bioprinting tool. The dynamics of migration and proliferation are evaluated in individual cells using live-cell fluorescence microscopy and an in-house pipeline for image processing and single-cell tracking. Our robotics-assisted approach outperforms manual scratch assays with enhanced reproducibility, adaptability, and precision. The incorporation of automation further facilitates increased flexibility in wound geometry and allows for many experimental conditions to be analyzed in parallel. Unlike traditional cell migration assays, our workflow offers an adjustable platform that can be tailored to a wide range of applications with high-throughput capability. The key features of this system, including its scalability, versatility, and the ability to maintain a high degree of experimental control, position it as a valuable tool for researchers across various disciplines.