Cell Monolayers Research Articles

Trans-nasal brain delivery of anti-TB drugs by methyl-β-cyclodextrin microparticles show efficient mycobacterial clearance from central nervous system.

Central nervous system tuberculosis (CNS-TB) is the most severe extra-pulmonary manifestation of tuberculosis (TB), facing significant challenges due to the limited penetration of anti-TB drugs (ATDs) across the blood-brain barrier (BBB) and their insufficient concentrations at the site of infection. This study aimed to enhance the efficacy of ATDs by encapsulating them in methyl-β-cyclodextrin (M-β-CD) microparticles (ATD-MP) using spray drying, intended for intranasal delivery to manage CNS-TB. M-β-CD microparticles loaded with isoniazid (INH) and rifampicin (RIF) exhibited spherical shapes with slightly deflated surfaces and particle sizes of 6.24 ± 0.77 μm and 5.97 ± 0.50 μm, respectively. M-β-CD improved the permeation of ATDs through RPMI-2650 cell monolayers while reducing drug cytotoxicity. Pharmacokinetic and biodistribution analysis demonstrated that intranasal administration of ATD-MP significantly enhanced the trans-nasal brain delivery of ATDs and their distribution in the brain, achieving the minimum inhibitory concentration. In a murine model of CNS-TB, intranasal insufflation of ATD-MP for four weeks led to a significant reduction (~0.78 Log10 CFU) in mycobacterial burden in the brain compared to the untreated group (~3.60 Log10 CFU). These preclinical results underscore the potential of intranasal administration of M-β-CD microparticles as an effective therapeutic strategy for combating brain inflammation in CNS-TB.

Assessment of in vitro dynamics of pathogenic environmental Acanthamoeba T4 and T9 genotypes isolated from three recreational lakes in Klang Valley, Malaysia over the HaCaT cell monolayer

ABSTRACT Free-living amoebae of the genus Acanthamoeba are causative agents of keratitis and amoebic encephalitis. They are widely found in various ecological environments. Therefore, the present study brings results that can help to better understand the genotypes of the environmental isolates and their pathogenicity. This study procured 26 Acanthamoeba isolates from three recreational lakes in 2022. Polymerase chain reaction amplification was performed on positive Acanthamoeba samples. The thermotolerance, osmotolerance, and cytopathogenicity in human keratinocyte (HaCaT) cells of the samples were also evaluated. The phylogenetic analysis demonstrated that 12 isolates were of genotype T4, two (T9), six (T17), four (T8), and one each from T5 and T11. The thermo- and osmotolerance assays indicated that eight Acanthamoeba samples were potentially pathogenic. Two T4 and one T9 genotype also recorded 33-, 42-, and 133-kDa serine-type proteases, respectively. The HaCaT cell monolayer revealed that three T4 and one T9 samples achieved cytopathic effects within the 50–100% range, hence significantly cytotoxic. The lactate dehydrogenase secretion results demonstrated that three (T4) and one (T9) sample exhibited exceptional toxicity (over 40%) compared to the other samples. The responses of Acanthamoeba members with similar genotypes to pathogenicity indicator assays varied considerably, rendering correlation of pathogenicity with specific genotypes challenging.

Toward “Green” Vessels: Characterization of Microstructure, Mechanics, and Endothelial Cell Interaction on Three Macro‐Tubular Plants for Vascular Tissue Engineering Applications

AbstractVascular tissue engineering aims to create vessel models for in vitro research and develop vascular grafts for in vivo applications using tubular scaffolds. Natural scaffolds outperform synthetic ones due to their biocompatibility and natural microenvironment supporting cell growth. Given the importance of producing biocompatible tubular scaffolds through cost‐effective and uncomplicated processes, this study introduces nature‐derived tubular structures from three decellularized tubular plants (Water Spinach, Green Onion, and Water Horsetail) as novel alternatives. Microstructural characterization on the luminal surfaces of the plants reveals unique surface topography for each. Water Spinach is the most promising graft candidate in suturability tests besides presenting the highest elongation before rupture in tensile test. Assessment of human endothelial cells on the luminal surfaces of decellularized scaffolds shows higher expression of Ki‐67 protein and a consistent increase in cell number on water spinach and green onion scaffolds compared to tissue culture plate as a control. Focal adhesion‐related molecule Vinculin is expressed more than twice on all scaffolds compared to control, and confluent cell monolayers are formed on water spinach and green onion scaffolds, as confirmed by VE‐cadherin. This study proposes an innovative approach to use the natural structure of macro‐tubular plants for the preparation of vascular scaffolds.

Modeling development of breast cancer: from tumor microenvironment to preclinical applications.

Breast cancer is a complex disease and its progression is related not only to tumor cells but also to its microenvironment, which can not be sufficiently reflected by the traditional monolayer cell culture manner. The novel human cancer models comprising tumor microenvironment (TME), such as tumor organoids and organs-on-a-chip, has been established in recent years to help elucidate the underlying mechanisms of tumorigenesis and promote the development of cancer therapies. In this review, we first discuss the current state of breast cancer and their treatment strategies, and elucidates the complex properties of TME of breast cancer in vivo. The culture models used in breast cancer research are then summarized with insights into recent development. Finally, we also conclude by discussing the current limitations and future directions of culture models in breast cancer research for providing a preclinical reference for the precise treatment of cancer patients.

Dynamic three dimensional environment for efficient and large scale generation of smooth muscle cells from hiPSCs

BackgroundChronic ischemic limb disease often leads to amputation, which remains a significant clinical problem. Smooth-muscle cells (SMCs) are crucially involved in the development and progression of many cardiovascular diseases, but studies with primary human SMCs have been limited by a lack of availability. Here, we evaluated the efficiency of two novel protocols for differentiating human induced-pluripotent stem cells (hiPSCs) into SMCs and assessed their potency for the treatment of ischemic limb disease.MethodshiPSCs were differentiated into SMCs via a conventional two-dimensional (2D) protocol that was conducted entirely with cell monolayers, or via two protocols that consisted of an initial five-day three-dimensional (3D) spheroid phase followed by a six-day 2D monolayer phase (3D + 2D differentiation). The 3D phases were conducted in shaker flasks on an orbital shaker (the 3D + 2D shaker protocol) or in a PBS bioreactor (the 3D + 2D bioreactor protocol). Differentiation efficiency was evaluated via the expression of SMC markers (smooth-muscle actin [SMA], smooth muscle protein 22 [SM22], and Calponin-1), and the biological activity of the differentiated hiPSC-SMCs was evaluated via in-vitro assessments of migration (scratch assay), contraction in response to the treatment with a prostaglandin H2 analog (U46619), and tube formation on Geltrex, as well as in-vivo measurements of perfusion (fluorescence angiography) and vessel density in the limbs of mice that were treated with hiPSC-SMCs after experimentally induced hind-limb ischemia (HLI).ResultsBoth 3D + 2D protocols yielded > 5.6 × 107 hiPSC-SMCs/differentiation, which was ~ nine-fold more than that produced via 2D differentiation, and flow cytometry analyses confirmed that > 98% of the 3D + 2D-differentiated hiPSC-SMCs expressed SMA, > 81% expressed SM22, and > 89% expressed Calponin-1. hiPSC-SMCs obtained via the 3D + 2D shaker protocol also displayed typical SMC-like migratory, contraction, and tube-formation activity in-vitro and significantly improved measurements of perfusion, vessel density, and SMA-positive arterial density in the ischemic limb of mouse HLI model.ConclusionsOur dynamic 3D + 2D protocols produced an exceptionally high yield of hiPSC-SMCs. Transplantation of these hiPSC-SMCs results in significantly improved recovery of ischemic limb after ischemic injury in mice.

A Quantitative Real-Time PCR Assay for Measuring Poxvirus Replication and Cell Binding.

Quantitative real-time PCR (qPCR) is a fast and reliable method to quantify viral genomes as a surrogate to titering on monolayers of cells for measuring virus replication. Whether it be for determining the number of virions released, the total number of genomes produced during infection, or the number of virions bound to a cell, qPCR assays can be adapted to quickly enumerate total viral genomes in a broad range of experiments comparing virus replication under different conditions. In addition, qPCR offers several advantages compared to plaque assays including time, linearity over 9 logs, and scalability from tens-to-hundreds of samples, depending on the qPCR machine. Here we describe a qPCR assay for quantifying vaccinia virus' dsDNA genome that can be used to determine the total number of virions produced. Furthermore, we describe a straightforward protocol for a cell-binding assay that is sensitive enough to use with small concentrations of inoculating virions. This protocol is suitable for measuring the cell-binding ability of mutations that affect virus production and infectivity.

Colon-Targeted Poly(ADP-ribose) Polymerase Inhibitors Synergize Therapeutic Effects of Mesalazine Against Rat Colitis Induced by 2,4-Dinitrobenzenesulfonic Acid

Background/Objectives: In addition to oncological applications, poly(ADP-ribose) polymerase (PARP) inhibitors have potential as anti-inflammatory agents. Colon-targeted delivery of PARP inhibitors has been evaluated as a pharmaceutical strategy to enhance their safety and therapeutic efficacy against gut inflammation. Methods: Colon-targeted PARP inhibitors 5-aminoisoquinoline (5-AIQ) and 3-aminobenzamide (3-AB) were designed and synthesized by azo coupling with salicylic acid (SA), yielding 5-AIQ azo-linked with SA (AQSA) and 3-AB azo-linked with SA (ABSA). Additional conjugation of AQSA with acidic amino acids yielded glutamic acid-conjugated AQSA (AQSA-Glu) and aspartic acid-conjugated AQSA, which further increased the hydrophilicity of AQSA. Results: The distribution coefficients of PARP inhibitors were lowered by chemical modifications, which correlated well with drug permeability via the Caco-2 cell monolayer. All derivatives were effectively converted to their corresponding PARP inhibitors in the cecal contents. Compared with observations in the oral administration of PARP inhibitors, AQSA-Glu and ABSA resulted in the accumulation of much greater amounts of each PARP inhibitor in the cecum. ABSA accumulated mesalazine (5-ASA) in the cecum to a similar extent as sulfasalazine (SSZ), a colon-targeted 5-ASA prodrug. In the DNBS-induced rat colitis model, AQSA-Glu enhanced the anticolitic potency of 5-AIQ. Furthermore, ABSA was more effective against rat colitis than SSZ or AQSA-Glu, and the anticolitic effects of AQSA-Glu were augmented by combined treatment with a colon-targeted 5-ASA prodrug. In addition, the colon-targeted delivery of PARP inhibitors substantially reduced their systemic absorption. Conclusions: Colon-targeted PARP inhibitors may improve the therapeutic and toxicological properties of inhibitors and synergize the anticolitic effects of 5-ASA.

Intestinal Cells-on-Chip for Permeability Studies

Background: To accurately measure permeability of compounds in the intestine, there is a need for preclinical in vitro models that accurately represent the specificity, integrity and complexity of the human small intestinal barrier. Intestine-on-chip systems hold considerable promise as testing platforms, but several characteristics still require optimization and further development. Methods: An established intestine-on-chip model for tissue explants was adopted for intestinal cell monolayer culture. A 3D-printed culture disc was designed to allow cell culture in static conditions and subsequent permeability studies in a dynamic environment. Membrane characteristics and standardized read-outs were investigated and compared to traditional permeability studies under static conditions. Results: By starting cultures outside the chip in conventional wells plates, the new cell disc design could support accurate cell monolayer formation for both Caco-2 and human enteroids. When transferred to the chip with laminar flow, there was accurate detection of barrier integrity (FD4 and Cascade Blue) and permeability (atenolol/antipyrine). Both flow and membrane characteristics had a significant impact on permeability outcomes. Conclusions: This novel intestinal cell-on-chip system offers large flexibility for intestinal permeability studies, although it still requires validation with more compounds to reveal its full potential.

Development and Characterization of 3-Dimensional Cell Culture Models of Adrenocortical Carcinoma.

Adrenocortical carcinoma (ACC) is a rare malignancy of the adrenal cortex that is associated with a poor prognosis. Developing effective treatment options for ACC is challenging owing to the current lack of representative preclinical models. This study addressed this limitation by developing and characterizing 3-dimensional (3D) cell cultures incorporating the ACC cell lines, MUC-1, HAC15, and H295R in a type I collagen matrix. ACC tissue samples were analyzed by immunohistochemistry to determine the presence of type I collagen in the tumor microenvironment. Cell viability and proliferation were assessed using flow cytometry and confocal microscopy. mRNA expression of steroidogenic enzymes and steroid secretion was analyzed by comparing the 3D and monolayer cell culture models. All cells were successfully cultured in a type I collagen matrix, which is highly expressed in the ACC tumor microenvironment and showed optimal viability until day 7. All 3 models showed increased metabolic and proliferative activity over time. Three-dimensional cell cultures were steroidogenic and demonstrated increased resistance to the gold standard chemotherapy, mitotane, compared with monolayer. The use of these models may lead to an improved understanding of disease pathology and provide a better representative platform for testing and screening of potential therapies.

Development of RT-qPCR assay for assessing the expression of ACTB and SDHA housekeeping genes in the cell cultures of mammalian hosts of zoonotic infections

Background. The molecular mechanisms behind the maintenance of zoonotic pathogens in nature can be better understood by examining the gene expression in host cells in response to the infection. Reverse transcription and quantitative polymerase chain reaction (RT-qPCR) is a powerful method to study gene expression, especially with the use of endogenous reference genes (RG) to normalize the data. Therefore, it is critical to develop the reliable qRT-PCR assay and validate that selected RGs are stably expressed in the studied organism or cell culture.The aim. In this work, we aimed to develop the real-time qRT-PCR method for detecting mRNA of two candidate RGs, succinate dehydrogenase subunit A (SDHA) and beta-actin (ACTB), in the cell lines of mammalian hosts of zoonotic infections.Materials and methods. The SPEV (porcine embryonic kidney cell line) and ApnK (Korean field mouse kidney cell line) cells were grown in 24-well culture plates. Total RNA/DNA was isolated from trypsin-detached cell monolayers. Genomic DNA in the samples was removed with RNase-free DNase I, and one-step RT-qPCR was performed using primers for SDHA and ACTB gene fragments and the corresponding TaqMan hydrolysis probes. The experiment was performed in 4 independent replicates.Results. In the Korean field mouse cells, the linearity, efficiency, repeatability, and reproducibility of the RT-qPCR for ACTB gene mRNA corresponded to the modern requirements. However, RT-qPCR for SDHA exhibited good linearity and efficiency of the reaction, but CV values for repeatability and reproducibility slightly exceeded the recommended standards. In porcine cells, both assays had acceptable parameters. Thus, to use the SDHA as RG for ApnK cells, a detailed study of the stability of its expression in this particular model is required.Conclusions. New qRT-PCR assay was developed to assess the expression of housekeeping genes ACTB and SDHA in the cells of the Korean field mouse and domestic pig. Further research is necessary to validate these genes as references for quantitative assessment of gene expression in the cells of mammalian hosts of zoonotic infections.

Evaluation of the Biological Properties of the Bacteriodaceae Family Using the Caco-2 Cell Line.

Disorders in the composition of the microbiota are often associated with pathological processes in the body. The use of new biotherapeutic drugs containing bacteria from normal microflora to correct these disorders may contribute to more effective treatment. The Bacteroidaceae family dominating in microflora is a candidate for inclusion in bacterial preparations. The adhesion properties of five bacterial strains from the Bacteroidaceae family were analyzed using the Caco-2 cell line and their effect on the transepithelial electrical resistance parameters (TEER) was assessed to determine the extent of their impact on the cell monolayer integrity. This approach allowed the selection of strain B. intestinalis 181, which was characterized by a moderate adhesion activity combined with a relatively low effect on the TEER parameter.

Galectin-3 disrupts tight junctions of airway epithelial cell monolayers by inducing expression and release of matrix metalloproteinases upon influenza a infection.

Galectins are β-galactosyl-binding lectins with key roles in early development, immune regulation, and infectious disease. Influenza A virus (IAV) infects the airway epithelia, and in severe cases may lead to bacterial superinfections and hypercytokinemia, and eventually, to acute respiratory distress syndrome (ARDS) through the breakdown of airway barriers. The detailed mechanisms involved, however, remain poorly understood. Our prior in vivo studies in a murine model system revealed that upon experimental IAV and pneumococcal primary and secondary challenges, respectively, galectin-1 and galectin-3 (Gal-3) are released into the airway and bind to the epithelium that has been desialylated by the viral neuraminidase, contributing to secondary bacterial infection and hypercytokinemia leading to the clinical decline and death of the animals. Here we report the results of in vitro studies that reveal the role of the extracellular Gal-3 in additional detrimental effects on the host by disrupting the integrity of the airway epithelial barrier. IAV infection of the human airway epithelia cell line A549 increased release of Gal-3 and its binding to the A549 desialylated cell surface, notably to the transmembrane signaling receptors CD147 and integrin-β1. Addition of recombinant Gal-3 to A549 monolayers resulted in enhanced expression and release of matrix metalloproteinases, leading to disruption of cell-cell tight junctions, and a significant increase in paracellular permeability. This study reveals a critical mechanism involving Gal-3 that may significantly contribute to the severity of IAV infections by promoting disruption of tight junctions and enhanced permeability of the airway epithelia, potentially leading to lung edema and ARDS.

Deficiency in glutathione peroxidase 4 (GPX4) results in abnormal lens development and newborn cataract

The human lens is composed of a monolayer of lens epithelial cells (LECs) and elongated fibers that align tightly but are separated by the plasma membrane. The integrity of the lens plasma membrane is crucial for maintaining lens cellular structure, homeostasis, and transparency. Glutathione peroxidase 4 (GPX4), a selenoenzyme, plays a critical role in protecting against lipid peroxidation. This study aims to elucidate the role of GPX4 in lens plasma membrane stability during lens development using in vitro, ex vivo, and in vivo systems. Our findings reveal that GPX4 deficiency triggers lens epithelial apoptosis-independent but ferroptosis-mediated cell death. Blocking lens GPX4 activity during ex vivo culture induces lens opacification, LEC death, and disruption of lens fiber cell arrangement. Deletion of lens-specific Gpx4 results in significant unsaturated phospholipid loss and an increase in oxidized phospholipids. Consequently, lenses with Gpx4 deficiency exhibit massive disruption of lens fiber cell structure, significant loss of LECs via ferroptosis, and formation of newborn cataracts. Remarkably, administering the lipid peroxidation inhibitor, liproxstatin-1, to pregnant mothers at embryonic days 9.5 significantly prevents lipid peroxidation, LEC death, and lens developmental defects. Our study unveils the crucial role of GPX4 in lens development and transparency, and also provides a successful intervention approach to prevent lens developmental defects through lipid peroxidation inhibition.

Endothelial cell-derived exosomes trigger a positive feedback loop in osteogenesis-angiogenesis coupling via up-regulating zinc finger and BTB domain containing 16 in bone marrow mesenchymal stem cell

The close spatial and temporal connection between osteogenesis and angiogenesis around type H vasculature is referred as “osteogenesis-angiogenesis coupling”, which is one of the basic mechanisms of osteogenesis. Endothelial cells (ECs), bone marrow mesenchymal stem cells (BMSCs), and their specific lineage constitute important cluster that participate in the regulation of osteogenesis and angiogenesis in bone microenvironment. However, the regulatory mechanism of osteogenesis-angiogenesis coupling under the condition of bone healing has not been unveiled. In this study, we demonstrated that the exosome derived from ECs (EC-exo) is an initiator of type H blood vessels formation, and EC-exo acts as a mediator in orchestrating osteogenesis-angiogenesis coupling by enhancing BMSC osteogenic differentiation and EC angiogenesis both in monolayer and stereoscopic co-culture system of primary human cells. The transcriptome array indicated that zinc finger and BTB domain containing 16 (ZBTB16) is a key gene in EC-exo-mediated osteogenesis, and ZBTB16 is indispensable in EC-exo-initiated osteogenesis-angiogenesis coupling. Mechanistically, EC-exo up-regulated the expression of ZBTB16 in BMSCs, thereby promoting osteoprogenitor phenotype transformation; the osteoprogenitors further promote ECs which constitute type H vessel (H-ECs) generation by activating HIF-1α pathway; and the H-ECs conversely promotes osteogenic differentiation of BMSCs. The crosstalk between BMSCs and ECs triggered by EC-exo constitutes a positive feedback loop that enhances osteogenesis-angiogenesis coupling. This study demonstrates that EC-exo can become an effective therapeutic tool to promote bone regeneration and repair.Graphical

Identification and molecular mechanism of novel antioxidant peptides from squid skin protein hydrolysates: In silico and in vitro analysis

Squid skin is rich in collagen and a high-quality source of bioactive peptides. This study aimed to identify novel antioxidant peptides derived from squid skin and to explore their potential molecular mechanism using in silico and in vitro approaches. Two novel peptides QDGFQGAH and SGDNLRFP were identified and demonstrated disparate of free radical scavenging and cytoprotective activities. Subsequent investigations exhibited that peptides were capable of reducing intracellular MDA level, and elevating the levels of antioxidant enzymes, including SOD, CAT, and GSH-Px. Furthermore, compared to the control, the expressions of Nrf2 treated with peptides were increased, while the levels of Keap1 were decreased, and the peptide QDGFQGAH showed the best activity. Molecular docking and dynamic simulations demonstrated that the peptides could form stable interactions with key residues of Keap1 by influencing the amino acid regions (Gly571-Asp589, Ala376-Cys395). Transport experiments on Caco-2 cell monolayers revealed that two novel peptides could traverse the intestinal epithelium. The findings suggest that the two peptides may be considered as potential antioxidant agents in functional foods, and provide a theoretical foundation for the extensive processing of squid skin.

Interplay of geometry and mechanics in epithelial wound healing.

Wound healing is a complex biological process critical for maintaining an organism's structural integrity and tissue repair following an infection or injury. Recent studies have unveiled the mechanisms involving the coordination of biochemical and mechanical responses in the tissue in wound healing. In this article, we focus on the healing property of an epithelial tissue as a material while the effects of biological mechanisms such as cell proliferation, tissue intercalation, cellular migration, cell crawling, and filopodia protrusion is minimal. We present a mathematical framework that predicts the fate of a wounded tissue based on the wound's geometrical features and the tissue's mechanical properties. Precisely, adapting the vertex model of tissue mechanics, we predict whether a wound of a specific size in an epithelial monolayer characterized by certain levels of actomyosin contractility and cell-cell adhesion will heal (i.e., close), shrink in size, or rupture the tissue further. Moreover, we show how tissue-mediated mechanisms such as purse-string tension at the wound boundary facilitate wound healing. Finally, we validate the predictions of our model by designing an experimental setup that enables us to create wounds of specific sizes in kidney epithelial cells (MDCK) monolayers. Altogether, this work sets up a basis for interpreting the interplay of mechanical and geometrical features of a tissue in the process of wound healing.

Assessment of Digestion and Absorption Properties of 1,3-Dipalmitoyl-2-Oleoyl Glycerol-Rich Lipids Using an In Vitro Gastrointestinal Digestion and Caco-2 Cell-Mediated Coupled Model.

The digestion and absorption properties of 1,3-dipalmitoyl-2-oleoyl glycerol (POP)-rich lipids was evaluated using in vitro gastrointestinal digestion and a Caco-2 cell-mediated coupled model. Caco-2 cell viability and monolayer integrity were assessed by an MTT assay and transepithelial electrical resistance. The IC50 for bile salts, pancreatin, and free fatty acid (FFA) were 0.22 mM, 0.22 mg/mL, and 1.47 mM, respectively, and no cytotoxicity was observed for bovine serum albumin (0.01-0.20 mM) or triacylglycerol (1.00-10.00 mM). The in vitro-digested POP-rich lipid containing FFA > 2.95 mM caused the disruption of monolayer tight junctions in Caco-2 cells. The major triacylglycerols (TAG) of POP-rich lipids were POP (50.8%), POO (17.8%), POL/OPL/PLO (7.6%), PPO (7.1%), and PLP (6.8%). Following digestion and uptake into Caco-2 cells, the resynthesized TAGs included PPO (20.6%), PPP (15.9%), POO (14.0%), POL/OPL/PLO (12.2%), POP (10.9%), OOO (7.5%), OPO (7.0%), OOL/OLO (6.7%), PLP (3.1%), and PPL (2.2%). The secreted major TAGs were POL/OPL/PLO (50.8%), PPP (11.1%), and OOL/OLO (8.4%), indicating a diverse TAG profile in newly synthesized lipids. This study provides a coupled model for lowering cytotoxicity and maintaining the monolayer in Caco-2 cells, and for evaluating the digestion and absorption properties of functional lipids containing specific fatty acids incorporated into TAG.

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

High-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.