Search

Madin–Darby Canine Kidney (MDCK) cells are a key cell line for influenza vaccine production, due to their high viral yield and low mutation resistance. In our laboratory, we established a tertiary cell bank (called M60) using a standard MDCK cell line imported from American Type Culture Collection (ATCC) in the USA. Due to their controversial tumourigenicity, we domesticated non-tumourigenic MDCK cells (named CL23) for influenza vaccine production via monoclonal screening in the early stage of this study, and the screened CL23 cells were characterised based on their low proliferative capacity, which had certain limitations in terms of expanding their production during cell resuscitation. It was thus our objective to enhance the proliferation efficiency of MDCK cells for influenza vaccine production following cell resuscitation, with a view to improving the production of non-tumourigenic MDCK cells for vaccines and enhancing the production of influenza virus lysate vaccines from MDCK cells through genetic intervention. We concentrated on the protein thrombosponin-1 (THBS1), which was markedly differentiated in the proteomics data of the two MDCK cells. By integrating this finding with related studies, we were able to ascertain that THBS1 exerts a significant influence on the level of cell proliferation and apoptosis. Consequently, our objective was to investigate the impact of THBS1 expression on MDCK cell apoptosis by verifying the differences in THBS1 expression between the two MDCK cells and by interfering with THBS1 expression in the MDCK cells. We found that the knockdown of THBS1 significantly increased the proliferation and apoptosis of CL23 cells without causing significant changes in cell migration and invasion, and its overexpression significantly decreased the proliferation of M60 cells and increased cell migration, invasion, and apoptosis. In addition, the TGF-β/Smad pathway target genes transforming growth factor-β1 (TGF-β1), mothers against decapentaplegic homolog 2 (Smad2), and mothers against decapentaplegic homolog 3 (Smad3), were significantly down-regulated in CL23 cells after THBS1 knockdown and up-regulated in M60 cells after overexpression, with consistent expression identified at both the mRNA and protein levels. The treatment of cells with TGF-β activators and inhibitors further demonstrated that THBS1 regulated MDCK cell proliferation and apoptosis through the TGF-β/Smad signalling pathway. Finally, we found that THBS1 also regulated H1N1 influenza virus replication. These findings enable a comprehensive understanding of the regulatory mechanisms of THBS1 regarding MDCK cell proliferation and apoptosis functions and the effects of influenza virus replication. [ABSTRACT FROM AUTHOR]

A single epithelial cell embedded in extracellular matrix (ECM) can proliferate to form an apical lumen‐harboring cyst, whose formation is a fundamental step in epithelial organ development. At an early two‐cell stage after cell division, the cell doublet typically displays "inverted" polarity, with apical and basolateral proteins being located to the ECM‐facing and cell–cell‐contacting plasma membranes, respectively. Correct cystogenesis requires polarity reorientation, a process containing apical protein endocytosis from the ECM‐abutting periphery and subsequent apical vesicle delivery to a cell–cell contact site for lumen formation. Here, we show that downstream of the ECM‐signal‐transducer β1‐integrin, Rac1, and its effector IQGAP1 promote apical protein endocytosis, contributing to polarity reorientation of mammalian epithelial Madin‐Darby canine kidney (MDCK) cells at a later two‐cell stage in three‐dimensional culture. Rac1–GTP facilitates IQGAP1 interaction with the Rac‐specific activator Tiam1, which also contributes to the endocytosis and enhances the effect of IQGAP1. These findings suggest that Tiam1 and IQGAP1 form a positive feedback loop to activate Rac1. With Rac1–GTP, IQGAP1 also binds to AP2α, an adaptor protein subunit for clathrin‐mediated endocytosis; depletion of the AP2 complex impairs apical protein endocytosis in MDCK doublets. Thus, Rac1 likely participates in polarity reorientation at the two‐cell stage via its interaction with IQGAP1. [ABSTRACT FROM AUTHOR]

Graphene oxide-mediated photothermal therapy using femtosecond lasers has recently shown promise in treating hepatocellular carcinoma. However, significant work remains to optimize irradiation parameters for specific nanoparticle types and cancer cells to improve nanomaterial-mediated photothermal anticancer therapy. This study investigated the photothermal potential of nGO and nGO-PEG nanoparticles (NPs) combined with femtosecond laser irradiation at 515 nm and 1030 nm wavelengths, with varying power (0.1 and 0.2 W/cm2) and duration (5 and 10 min), to optimize photothermal therapy for hepatocellular carcinoma. Conversion efficiency of NPs, morphology and viability of HepG2 and normal MDCK cells after treatments were evaluated using an electronic thermometer, phase-contrast microscopy, and WST-1 assay. The results revealed that nGO-PEG NPs exhibited better photothermal efficiency than nGO, with 515 nm of irradiation inducing a temperature increase up to 19.1 °C compared to 4.7 °C with 1030 nm of light. Laser exposure to 515 nm significantly reduced HepG2 cell viability, with the most intense conditions (10 min at 0.2 W/cm2) causing a decrease of up to 58.2% with nGO and 43.51% with nGO-PEG. Normal MDCK cells showed minimal impact or a slight viability increase, especially with nGO-PEG. Combined treatment with laser irradiation and NPs induced significant morphological changes in HepG2 cells, including cell detachment and apoptotic-like characteristics, particularly with 1030 nm of irradiation. MDCK cells exhibited minimal morphological changes, with some recovery observed under lower energy conditions. These findings suggest that low-energy lasers and engineered nanomaterials could provide a minimally invasive approach to photothermal cancer therapy with reduced side effects. [ABSTRACT FROM AUTHOR]

The development of serum-free media (SFM) is critical to advance cell culture techniques used in viral vaccine production and address the ethical concerns and contamination risks associated with fetal bovine serum (FBS). This study evaluated the effects of marine microalgal extracts and growth factor cocktails on the activity of Madin-Darby canine kidney (MDCK) and Vero cells. Five marine microalgal species were used: Spirulina platensis (SP), Dunaliella salina (DS), Haematococcus pluvialis (HP), Nannochloropsis salina (NS), and Tetraselmis sp. (TS). DS and SP extracts significantly increased the proliferation rate of both MDCK and Vero cells. DS had a proliferation rate of 149.56% and 195.50% in MDCK and Vero cells, respectively, compared with that in serum-free medium (SFM). Notably, DS and SP extracts significantly increased superoxide dismutase (SOD) activity, which was 118.61% in MDCK cells and 130.08% in Vero cells for DS, and 108.72% in MDCK cells and 125.63% in Vero cells for SP, indicating a reduction in intracellular oxidative stress. Marine microalgal extracts, especially DS and SP, are feasible alternatives to FBS in cell culture as they promote cell proliferation, ensure safety, and supply essential nutrients while reducing oxidative stress. [ABSTRACT FROM AUTHOR]

Madin–Darby canine kidney (MDCK) cells are commonly used to produce cell-based influenza vaccines. However, the role of the low-serum medium on the proliferation of MDCK cells and the propagation of the influenza virus has not been well studied. In the present study, we used 5 of 15 culture methods with different concentrations of a mixed medium and neonatal bovine serum (NBS) to determine the best culture medium. We found that a VP:M199 ratio of 1:2 (3% NBS) was suitable for culturing MDCK cells. Furthermore, the stable growth of MDCK cells and the production of the influenza virus were evaluated over long-term passaging. We found no significant difference in terms of cell growth and virus production between high and low passages of MDCK cells under low-serum culture conditions, regardless of influenza virus infection. Lastly, we performed a comparison of the transcriptomics and proteomics of MDCK cells cultured in VP:M199 = 1:2 (3% NBS) with those cultured in VP:M199 = 1:2 (5% NBS) before and after influenza virus infection. The transcriptome analysis showed that differentially expressed genes were predominantly enriched in the metabolic pathway and MAPK signaling pathway, indicating an activated state. This suggests that decreasing the concentration of serum in the medium from 5% to 3% may increase the metabolic activity of cells. Proteomics analysis showed that only a small number of differentially expressed proteins could not be enriched for analysis, indicating minimal difference in the protein levels of MDCK cells when the serum concentration in the medium was decreased from 5% to 3%. Altogether, our findings suggest that the screening and application of a low-serum medium provide a background for the development and optimization of cell-based influenza vaccines. [ABSTRACT FROM AUTHOR]

Actomyosin contractility represents an ancient feature of eukaryotic cells participating in many developmental and homeostasis events, including tissue morphogenesis, muscle contraction and cell migration, with dysregulation implicated in various pathological conditions, such as cancer. At the molecular level, actomyosin comprises actin bundles and myosin motor proteins that are sensitive to posttranslational modifications like phosphorylation. While the molecular components of actomyosin are well understood, the coordination of contractility by extracellular and intracellular signals, particularly from cellular signalling pathways, remains incompletely elucidated. This study focuses on WNT/planar cell polarity (PCP) signalling, previously associated with actomyosin contractility during vertebrate neurulation. Our investigation reveals that the main cytoplasmic PCP proteins, Prickle and Dishevelled, interact with key actomyosin components such as myosin light chain 9 (MLC9), leading to its phosphorylation and localized activation. Using proteomics and microscopy approaches, we demonstrate that both PCP proteins actively control actomyosin contractility through Rap1 small GTPases in relevant in vitro and in vivo models. These findings unveil a novel mechanism of how PCP signalling regulates actomyosin contractility through MLC9 and Rap1 that is relevant to vertebrate neurulation.

Novel swine Influenza A viruses (IAVs) have been described in South America. The objective of this study was to evaluate the replication kinetics of novel swine IAVs as a first step in vaccine production. Different swine IAV lineages (H1N1, H1N2, and H3N2), infection doses (MOI: 1, 0.1, 0.01, 0.001, 0.0001, and 0.00001), harvest times (every 12 h), and substrates (MDCK and Vero cells) were used. For all IAV strains, MDCK cells were the most efficient substrate, generating titers of =128 HAU/50 µL with an MOI of 0.00001 at 60 h post-infection. These data may be useful in vaccineproducing laboratories. [ABSTRACT FROM AUTHOR]

This study delves into the protectivemechanisms of Icariin (ICA) against cisplatin-induced damage in Madin-Darby canine kidney (MDCK) cells. Comprising two distinct phases, the investigation initially employed a single-factor randomized design to ascertain the minimal cisplatin concentration eliciting MDCK cell damage, spanning concentrations from 0 to 16 mmol/L. Concurrently, various concentrations of ICA (ranging from 5 to 50 mmol/L) were combined with 1 mmol/L cisplatin to determine the most efficacious treatment concentration. Subsequent investigations utilized four treatment groups: control, 1 mmol/L cisplatin, 1 mmol/L cisplatin + 20 mmol/L ICA, and 1 mmol/L cisplatin + 25 mmol/L ICA, aimed at elucidating ICA's protective mechanisms. Findings from the initial phase underscored a significant reduction in MDCK cell viability with 1 mmol/L cisplatin in comparison to the control (P < 0.01). Notably, the inclusion of 20 and 25 mmol/L ICA substantively ameliorated MDCK cell viability under 1 mmol/L cisplatin (P < 0.01). Moreover, cisplatin administration induced an elevation in inflammatory factors, malondialdehyde (MDA), reactive oxygen species (ROS), and Bax protein levels, while concurrently suppressing superoxide dismutase (SOD), catalase (CAT), and Bcl-2 expression (P < 0.01). Conversely, supplementation of 20 and 25 mmol/L ICA demonstrated a marked increase in mitochondrial membrane potential and levels of SOD, CAT, and Bcl-2 (P < 0.01). These interventions effectively attenuated inflammatory responses and suppressed Bax protein expression (P < 0.05), consequently mitigating cisplatin-induced apoptosis in MDCK cells (P < 0.01). In summary, these findings elucidate the role of ICA in impeding apoptosis in cisplatin-induced MDCK cells by regulating inflammatory responses, oxidative stress, and autophagic protein expression. [ABSTRACT FROM AUTHOR]

Influenza is a common respiratory disease caused by influenza viruses. MDCK（Madin-Darby Canine Kidney）cells have the characteristics of easy culture and high production, which can support the replication and proliferation of influenza viruses, and are widely used in the research and production of influenza viruses. With the deepening of research on influenza virus, and in order to further optimize the ability of MDCK cells to be used for influenza virus research and industrial application, researchers began to develop the domestication technology of MDCK cells. Through long-term cultivation and selection, the domesticated MDCK cells strain can better adapt to the growth environment of influenza virus, and improve the production and infectivity of influenza virus. In conclusion, the domestication technology of MDCK cells plays an important role in influenza virus research and industrial application, which may improve the production and infectivity of influenza viruses and provide better tools for influenza vaccine production and anti influenza drug development. Concurrently we have to also pay attention to the safety of MDCK cells and take appropriate measures to ensure the safety and reliability of its application. [ABSTRACT FROM AUTHOR]

H5N1 highly pathogenic avian influenza virus (HPAIV) infections pose a significant threat to human health, with a mortality rate of around 50%. Limited global approval of H5N1 HPAIV vaccines, excluding China, prompted the need to address safety concerns related to MDCK cell tumorigenicity. Our objective was to improve vaccine safety by minimizing residual DNA and host cell protein (HCP). We developed a downstream processing method for the cell-based H5N1 HPAIV vaccine, employing CaptoTM Core 700, a multimodal resin, for polishing. Hydrophobic-interaction chromatography (HIC) with polypropylene glycol as a functional group facilitated the reversible binding of virus particles for capture. Following the two-step chromatographic process, virus recovery reached 68.16%. Additionally, HCP and DNA levels were reduced to 2112.60 ng/mL and 6.4 ng/mL, respectively. Western blot, high–performance liquid chromatography (HPLC), and transmission electron microscopy (TEM) confirmed the presence of the required antigen with a spherical shape and appropriate particle size. Overall, our presented two-step downstream process demonstrates potential as an efficient and cost-effective platform technology for cell-based influenza (H5N1 HPAIV) vaccines. [ABSTRACT FROM AUTHOR]

Madin–Darby canine kidney (MDCK) cells are commonly used to produce cell-based influenza vaccines. However, the role of the low-serum medium on the proliferation of MDCK cells and the propagation of the influenza virus has not been well studied. In the present study, we used 5 of 15 culture methods with different concentrations of a mixed medium and neonatal bovine serum (NBS) to determine the best culture medium. We found that a VP:M199 ratio of 1:2 (3% NBS) was suitable for culturing MDCK cells. Furthermore, the stable growth of MDCK cells and the production of the influenza virus were evaluated over long-term passaging. We found no significant difference in terms of cell growth and virus production between high and low passages of MDCK cells under low-serum culture conditions, regardless of influenza virus infection. Lastly, we performed a comparison of the transcriptomics and proteomics of MDCK cells cultured in VP:M199 = 1:2 (3% NBS) with those cultured in VP:M199 = 1:2 (5% NBS) before and after influenza virus infection. The transcriptome analysis showed that differentially expressed genes were predominantly enriched in the metabolic pathway and MAPK signaling pathway, indicating an activated state. This suggests that decreasing the concentration of serum in the medium from 5% to 3% may increase the metabolic activity of cells. Proteomics analysis showed that only a small number of differentially expressed proteins could not be enriched for analysis, indicating minimal difference in the protein levels of MDCK cells when the serum concentration in the medium was decreased from 5% to 3%. Altogether, our findings suggest that the screening and application of a low-serum medium provide a background for the development and optimization of cell-based influenza vaccines.

NDec is a novel combination of oral decitabine and tetrahydrouridine that is currently under clinical development for the treatment of sickle cell disease (SCD). Here, we investigate the potential for the tetrahydrouridine component of NDec to act as an inhibitor or substrate of key concentrative nucleoside transporters (CNT1–3) and equilibrative nucleoside transporters (ENT1–2). Nucleoside transporter inhibition and tetrahydrouridine accumulation assays were performed using Madin-Darby canine kidney strain II (MDCKII) cells overexpressing human CNT1, CNT2, CNT3, ENT1, and ENT2 transporters. Results showed that tetrahydrouridine did not influence CNT- or ENT-mediated uridine/adenosine accumulation in MDCKII cells at the concentrations tested (25 and 250 µM). Accumulation of tetrahydrouridine in MDCKII cells was initially shown to be mediated by CNT3 and ENT2. However, while time- and concentration-dependence experiments showed active accumulation of tetrahydrouridine in CNT3-expressing cells, allowing for estimation of K m (3,140 µM) and V max (1,600 pmol/mg protein/min), accumulation of tetrahydrouridine was not observed in ENT2-expressing cells. Potent CNT3 inhibitors are a class of drugs not generally prescribed to patients with SCD, except in certain specific circumstances. These data suggest that NDec can be administered safely with drugs that act as substrates and inhibitors of the nucleoside transporters included in this study. [ABSTRACT FROM AUTHOR]

IntroductionBioengineering an implantable artificial kidney (IAK) will require renal epithelial cells capable of reabsorption of salt and water. We used genome engineering to modify cells for improved Na+/H+ exchange and H2O reabsorption. The non-viral piggyBac transposon system enables genome engineering cells to stably overexpress one or more transgenes simultaneously.MethodsWe generated epitope-tagged human sodium hydrogen exchanger 3 (NHE3) and aquaporin-1 (AQP1) cDNA expressing piggyBac transposon vectors. Transgene expression was evaluated via western blot and immunofluorescence. Flow cytometry analysis was used to quantitate transporter expression in a library of genome engineered clones. Cell surface biotinylation was used evaluate surface protein localization. Blister formation assays were used to monitor cellular volumetric transport.ResultspiggyBac enabled stable transposon integration and overexpression of cumate-inducible NHE3 and/or constitutively expressing AQP1 in cultured renal (MDCK) epithelial cells. Cell surface delivery of NHE3 and AQP1 was confirmed using cell surface biotinylation assays. Flow cytometry of a library of MDCK clones revealed varying expression of AQP1 and NHE3. MDCK cells expressing AQP1 and cumate-inducible NHE3 demonstrated increased volumetric transport.ConclusionsOur results demonstrate that renal epithelial cells an be genome engineered for enhanced volumetric transport that will be needed for an IAK device. Our results lay the foundation for future studies of genome engineering human kidney cells for renal tubule cell therapy.

Background Madin-Darby canine kidney (MDCK) cells are a cellular matrix in the production of influenza vaccines. The proliferation rate of MDCK cells is one of the critical factors that determine the vaccine production cycle. It is yet to be determined if there is a correlation between cell proliferation and alterations in metabolic levels. This study aimed to explore the metabolic differences between MDCK cells with varying proliferative capabilities through the use of both untargeted and targeted metabolomics. Methods To investigate the metabolic discrepancies between adherent cell groups (MDCK-M60 and MDCK-CL23) and suspension cell groups (MDCK-XF04 and MDCK-XF06), untargeted and targeted metabolomics were used. Utilizing RT-qPCR analysis, the mRNA expressions of key metabolites enzymes were identified. Results An untargeted metabolomics study demonstrated the presence of 81 metabolites between MDCK-M60 and MDCK-CL23 cells, which were mainly affected by six pathways. An analysis of MDCK-XF04 and MDCK-XF06 cells revealed a total of 113 potential metabolites, the majority of which were impacted by ten pathways. Targeted metabolomics revealed a decrease in the levels of choline, tryptophan, and tyrosine in MDCK-CL23 cells, which was in accordance with the results of untargeted metabolomics. Additionally, MDCK-XF06 cells experienced a decrease in 5’-methylthioadenosine and tryptophan, while S-adenosylhomocysteine, kynurenine, 11Z-eicosenoic acid, 3-phosphoglycerate, glucose 6-phosphate, and phosphoenolpyruvic acid concentrations were increased. The mRNA levels of MAT1A, MAT2B, IDO1, and IDO2 in the two cell groups were all increased, suggesting that S-adenosylmethionine and tryptophan may have a significant role in cell metabolism. Conclusions This research examines the effect of metabolite fluctuations on cell proliferation, thus offering a potential way to improve the rate of MDCK cell growth.

In influenza vaccine development, Madin–Darby canine kidney (MDCK) cells provide multiple advantages, including large-scale production and egg independence. Several cell-based influenza vaccines have been approved worldwide. We cultured H5N1 virus in a serum-free MDCK cell suspension. The harvested virus was manufactured into vaccines after inactivation and purification. The vaccine effectiveness was assessed in the Wuhan Institute of Biological Products BSL2 facility. The pre- and postvaccination mouse serum titers were determined using the microneutralization and hemagglutination inhibition tests. The immunological responses induced by vaccine were investigated using immunological cell classification, cytokine expression quantification, and immunoglobulin G (IgG) subtype classification. The protective effect of the vaccine in mice was evaluated using challenge test. Antibodies against H5N1 in rats lasted up to 8 months after the first dose. Compared with those of the placebo group, the serum titer of vaccinated mice increased significantly, Th1 and Th2 cells were activated, and CD8+ T cells were activated in two dose groups. Furthermore, the challenge test showed that vaccination reduced the clinical symptoms and virus titer in the lungs of mice after challenge, indicating a superior immunological response. Notably, early after vaccination, considerably increased interferon-inducible protein-10 (IP-10) levels were found, indicating improved vaccine-induced innate immunity. However, IP-10 is an adverse event marker, which is a cause for concern. Overall, in the case of an outbreak, the whole-virion H5N1 vaccine should provide protection.

H5N1 highly pathogenic avian influenza virus (HPAIV) infections pose a significant threat to human health, with a mortality rate of around 50%. Limited global approval of H5N1 HPAIV vaccines, excluding China, prompted the need to address safety concerns related to MDCK cell tumorigenicity. Our objective was to improve vaccine safety by minimizing residual DNA and host cell protein (HCP). We developed a downstream processing method for the cell-based H5N1 HPAIV vaccine, employing CaptoTM Core 700, a multimodal resin, for polishing. Hydrophobic-interaction chromatography (HIC) with polypropylene glycol as a functional group facilitated the reversible binding of virus particles for capture. Following the two-step chromatographic process, virus recovery reached 68.16%. Additionally, HCP and DNA levels were reduced to 2112.60 ng/mL and 6.4 ng/mL, respectively. Western blot, high–performance liquid chromatography (HPLC), and transmission electron microscopy (TEM) confirmed the presence of the required antigen with a spherical shape and appropriate particle size. Overall, our presented two-step downstream process demonstrates potential as an efficient and cost-effective platform technology for cell-based influenza (H5N1 HPAIV) vaccines.

Local substrate stiffness is one of the major mechanical inputs for tissue organization during its development and remodeling. It is widely recognized that adherent cells use transmembrane proteins (integrins) at focal adhesions to translate ECM mechanical cues into intracellular bioprocess. Here we show that epithelial cells respond to substrate stiffening primarily via actin cytoskeleton organization, that requires activation of mechanosensitive Piezo1 channels. Piezo1 Knockdown cells eliminated the actin stress fibers that formed on stiff substrates, while it had minimal effect on cell morphology and spreading area. Inhibition of Piezo1 channels with GsMTx4 also significantly reduced stiffness-induced F-actin reorganization, suggesting Piezo1 mediated cation current plays a role. Activation of Piezo1 channels with specific agonist (Yoda1) resulted in thickening of F-actin fibers and enlargement of FAs on stiffer substrates, whereas it did not affect the formation of nascent FAs that facilitate spreading on the soft substrates. These results demonstrate that Piezo1 functions as a force sensor that couples with actin cytoskeleton to distinguish the substrate stiffness and facilitate epithelial adaptive remodeling.

Summary: Neratinib, an irreversible pan-HER tyrosine kinase inhibitor, has been approved for the treatment of HER2-positive (HER2+) early-stage and brain metastatic breast cancer. Thus far, the pharmacology effects and pharmacodynamics of neratinib have been well studied. However, the disposition of neratinib and its influencing factors in vivo remain unclear. P-glycoprotein (P-gp), one of the most extensively studied transporters, substantially restricts penetration of drugs into the body or deeper compartments (i.e., blood-brain barrier, BBB), regarding drug resistance and drug-drug interactions. Thereby, the aim of this study was to investigate the influence of verapamil (a P-gp inhibitor) on the pharmacokinetics of neratinib in rats. Here, we have established a high specific, selective and sensitive ultra-performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method to quantify plasma concentrations of neratinib in rats. Pharmacokinetic results showed that verapamil significantly increased the system exposure of neratinib, as Cmax increased by 2.09-fold and AUC0-t increased by 1.64-fold, respectively. Additionally, the in vitro transport of neratinib was evaluated using Madin-Darby canine kidney II (MDCK II) and human MDR1 gene overexpressed MDCK (MDCK-MDR1) cell line models. As a result, the net flux ratio was over than 2 and decreased over 50% by verapamil, suggesting that neratinib was a substrate of P-gp. Hence, our findings have highlighted the important role of P-gp in the system exposure of neratinib in vivo, and drug-drug interaction should be considered when coadministration of P-gp inhibitors with neratinib. These findings may support the further clinical development and application of neratinib. [ABSTRACT FROM AUTHOR]

Sideritis scardica (S. scardica) is a mountain plant, endemic to the central part of the Balkan Peninsula, including the southern regions of Bulgaria. It is widely used in traditional medicine to treat some respiratory, gastrointestinal tract, or neurodegenerative diseases, but research data for its cytotoxic and anticancer properties are limited. In the present study, a hydroethanolic extract of S. scardica from region Trigrad, Bulgaria was prepared and its total polyphenolic and flavonoid content was determined. Further, its antioxidant, cytotoxic, and anticancer activities toward a mouse adenocarcinoma cell line, Colon 26 was evaluated in vitro. Higher antioxidant activity and higher total phenolic and flavonoid content were detected in the S. scardica hydroethanolic extract from region Trigrad as compared to the publish data for other ecotypes of this species. Also, the extract of S. scardica with a concentration higher than 200 µg mL-1 was found to inhibit the viability and growth of colon cancer cells but not those of the normal kidney MDCK cells pointing to a cell-specific and concentration-dependent cytotoxic effect. Thus, the current study demonstrates a high oxidation potential of S. scardica hydroethanolic extract from the region Trigrad and its cytotoxic effects on colon cancer cells when the extract was applied with concentrations of 200 µg mL-1 and higher. [ABSTRACT FROM AUTHOR]

Aminoglycoside antibiotics such as gentamicin are used frequently to treat bacterial infections in humans. Excessive consumption of these antibiotics lead to renal dysfunction. One of the factors contributing to renal dysfunction is oxidative damage, which causes apoptosis. Hence, this study investigates the effect of the antioxidant compound deacetyl epoxyazadiradione (DEA) in reducing cell death induced by gentamicin treatment in kidney cells (Madin–Darby canine kidney cells). The antioxidant experiments showed that reactive oxygen species level is decreased up to 27.06 ± 0.18% in 150 µM of DEA treatment. At this concentration, the activity of antioxidant enzymes such as superoxide dismutase increased from 0.4 ± 0.04 to 1.46 ± 0.05 µmol/min/L and catalase increased from 7.48 ± 0.39 to 17.6 ± 0.74 U/mg. The relative folds of gene expression of mitochondrial enzymes such as GST, GPx and GR restored from 0.596 ± 0.019, 0.521 ± 0.013 and 0.775 ± 0.014 to 0.866 ± 0.013, 0.669 ± 0.015 and 0.8615 ± 0.028, respectively. Consequently, the percentage of cell viability increases upto 91.8 ± 2.01 from 61.93 ± 1.63 with much less fragmentation in genomic DNA. Additionally, molecular docking results showed that DEA could bind to Bax, Bcl‐ 2, Caspase‐ 3 and Caspase‐ 9 proteins. These results indicate that DEA could reduce cell apoptosis by reducing oxidative stress due to antibiotics and interrupting the apoptotic signal pathway in kidney cells. [ABSTRACT FROM AUTHOR]

Conditions that cause the loss of epithelial barrier integrity are often accompanied by dysregulation of tight junction protein expression and/or localization. Recently, we have reported that patients with mutations in SLC12A2, the gene encoding the basolateral Na+-K+-2Cl- cotransporter (NKCC1), suffer from severe gastrointestinal deficits, including chronic gastrointestinal inflammation, gastrointestinal hemorrhage, intestinal obstruction, and constipation. Although the intestinal inflammation observed in patients with loss of NKCC1 function may or may not be due to tight junction dysfunction, we investigated whether the loss of NKCC1 function affects paracellular ion transport and epithelial barrier function. Wild-type HT29-MTX-E12 and CRISPR/Cas9-mediated NKCC1 knockout (KO) HT29 clones were tested for tight junction protein expression and localization. Tightness of epithelial cell monolayer was assessed by measurement of transepithelial electrical resistance and permeability of molecular tracers in transwell filters. Tight junction protein localization was assessed by immunofluorescence. Loss of NKCC1 expression strongly increases the expression of claudin-2 and occludin in epithelial cell monolayers. Loss of NKCC1 significantly reduces the transepithelial electrical resistance (TER) indicating an increase in paracellular ions flux, consistent with upregulation of the cation-selective and channel-forming claudin-2. In addition, NKCC1-KO monolayers showed a significant increase in the paracellular flux of small molecules like fluorescein (0.33 kDa), whereas the permeability of higher molecular weight TRITC-Dextran (4 kDa and 70 kDa) remained unchanged. Thus, NKCC1 regulates tight junction protein expression and loss of NKCC1 function affects epithelial barrier integrity. [ABSTRACT FROM AUTHOR]

Influenza is a global public health issue leading to widespread morbidity and mortality with devastating economic loss annually. Madin-Darby Canine Kidney (MDCK) cell line has been a major cell line for influenza vaccine applications. Though many details of the host metabolic responses upon influenza A virus (IAV) infection have been documented, little is known about the metabolic reprogramming features of a hyper-productive host for IAV vaccine production. In this study, a MDCK cell clone H1 was shown to have a particular high productivity of 30 × 103 virions/cell. The glucose and amino acid metabolism of H1 were evaluated, indicating that the high producer had a particular metabolic reprogramming phenotype compared to its parental cell line (P): elevated glucose uptake, superior tricarboxylic acid cycle flux, moderate amino acid consumption, and better regulation of reactive oxygen species. Combined with the stronger mitochondrial function and mild antiviral and inflammatory responses characterized previously, our results indicated that the high producer had a sufficient intracellular energy supply, and balanced substrate distribution for IAV and host protein synthesis as well as the intracellular redox status. Understanding of these metabolic alterations paves the way for the rational cell line development and reasonable process optimization for high-yield influenza vaccine production. [ABSTRACT FROM AUTHOR]

The uptake and trafficking of cell surface receptors can be monitored by a technique called 'antibody-feeding' which uses an externally applied antibody to label the receptor on the surface of cultured, live cells. Here, we adapt the traditional antibody-feeding experiment to polarized epithelial cells (Madin-Darby Canine Kidney) grown on permeable Transwell supports. By adding two tandem extracellular Myc epitope tags to the C-terminus of the SNARE protein syntaxin 3 (Stx3), we provided a site where an antibody could bind, allowing us to perform antibody-feeding experiments on cells with distinct apical and basolateral membranes. With this procedure, we observed the endocytosis and intracellular trafficking of Stx3. Specifically, we assessed the internalization rate of Stx3 from the basolateral membrane and observed the ensuing endocytic route in both time and space using immunofluorescence microscopy on cells fixed at different time points. For cell lines that form a polarized monolayer containing distinct apical and basolateral membranes when cultured on permeable supports, e.g., MDCK or Caco-2, this protocol can measure the rate of endocytosis and follow the subsequent trafficking of a target protein from either limiting membrane.

Out-of-plane curvature is an important, but poorly explored geometric parameter that influences cell behavior. We address the impact of curvature on epithelial proliferation through monitoring how MDCK cells proliferate on planar and curved toroidal hydrogel substrates with a broad range of Gaussian curvatures. We illustrate in detail the imaging processing methodology to characterize curved surfaces and quantify proliferation of cells. We find that MDCK cells grow readily on both curved and flat surfaces and can cover the entire surface of the toroidal structure as long as the initial seeding is uniform. Our analysis shows that proliferation does not depend on Gaussian curvature within the range probed in our experiment, but rather on cell density. Despite epithelial proliferation is insensitive to the curvature range presented in this study, the toroidal-construct fabrication technique and image processing methodology may find utility for probing cell processes like collective migration, as it involves long-range force transmission.

The practical use of cell‐based seasonal influenza vaccines is currently being considered in Japan. From the perspective of adventitious virus contamination, we assessed the suitability of NIID‐MDCK cells (NIID‐MDCK‐Cs) as a safe substrate for the isolation of influenza viruses from clinical specimens. We first established a sensitive multiplex real‐time PCR system to screen for 27 respiratory viruses and used it on 34 virus samples that were isolated by passaging influenza‐positive clinical specimens in NIID‐MDCK‐Cs. Incidentally, the limit of detection (LOD) of the system was 100 or fewer genome copies per reaction. In addition to influenza viruses, human enterovirus 68 (HEV‐D68) genomes were detected in two samples after two or three passages in NIID‐MDCK‐Cs. To further investigate the susceptibility of NIID‐MDCK‐Cs to adventitious viruses, eight common respiratory viruses were subjected to passages in NIID‐MDCK‐Cs. The genome copy numbers of seven viruses other than parainfluenza 3 decreased below the LOD by passage 4. By passaging in NIID‐MDCK‐Cs, the genome numbers of the input HEV‐D68, 1 × 108 copies, declined to 102 at passage 3 and to under the LOD at passage 4, whereas those of the other six viruses were under the LOD by passage 3. These results implied that during the process of isolating influenza viruses with NIID‐MDCK‐Cs, contaminating viruses other than parainfluenza 3 can be efficiently removed by passages in NIID‐MDCK‐Cs. NIID‐MDCK‐Cs could be a safe substrate for isolating influenza viruses that can be used to develop cell‐based influenza vaccine candidate viruses. [ABSTRACT FROM AUTHOR]

Background: The development of diabetic nephropathy is aided by the presence of oxidative stress. Morin, a natural flavonoid molecule, has been shown to have antioxidant and anti-diabetic properties. However, little is known about the mechanism of its protective effect in diabetic nephropathy pathogenesis caused by oxidative stress. Methods: Using Madin-Darby canine kidney (MDCK) cells as a working model, the current study investigates the detailed mechanism of morin's beneficial action. In hydrogen peroxide-induced oxidative stressed MDCK cells, there was a considerable rise in intracellular ROS and decreased antioxidant enzyme levels. Results: Morin has a higher binding affinity for the antioxidant receptor; according to in silico study using molecular docking and ADMET, it is predicted to be an orally active molecule. While morin administration increased SOD and CAT activity in oxidative stress-induced MDCK cells, it also reduced mitochondrial oxidative stress and apoptosis. Furthermore, the present study discovered the molecular mechanism through which morin reduced oxidative stress in MDCK cells by upregulating antioxidant enzyme molecules including GST, GPx, and GCS. Conclusion: These findings suggest that morin reduces H2O2-induced oxidative stress, reduces DNA oxidative damage, and prevents the depletion of antioxidant genes in MDCK cells. [ABSTRACT FROM AUTHOR]

βCDPEG5 and βCDPEG2 are two derivatives comprising seven PEG linear chains of 5 and 2 kDa, respectively, conjugated to βCD. As βCDPEGs display different physicochemical properties than their precursors, they could also trigger distinct cellular responses. To investigate the biological behavior of βCDPEGs in comparison to their parent compounds, we performed broad toxicological assays on RAW 264.7 macrophages, MC3T3-E1 osteoblasts, and MDCK cells. By analyzing ROS and NO2− overproduction in macrophages, we found that βCDPEGs induced a moderate stress response without affecting cell viability. Although MC3T3-E1 osteoblasts were more sensitive than MDCK cells to βCDPEGs and the parent compounds, a similar pattern was observed: the effect of βCDPEG5 on cell viability and cell cycle progression was larger than that of βCDPEG2; PEG2 affected cell viability and cell cycle more than βCDPEG2; cell post-treatment recovery was favorable in all cases, and the compounds had similar behaviors regarding ROS generation. The effect on MDCK cell migration followed a similar pattern. In contrast, for osteoblasts, the interference of βCDPEG5 with cell migration was smaller than that of βCDPEG2; likewise, the effect of PEG2 was shorter than its conjugate. Overall, the covalent conjugation of βCD and PEGs, particularly to yield βCDPEG2, improved the biocompatibility profile, evidencing that a favorable biological response can be tuned through a thoughtful combination of materials. Moreover, this is the first time that an in vitro evaluation of βCD and PEG has been presented for MC3T3-E1 and MDCK cells, thus providing valuable knowledge for designing biocompatible nanomaterials constructed from βCD and PEGs. [ABSTRACT FROM AUTHOR]

Human health may benefit from the study of natural compounds and phytoconstituents that can protect from inflammation. We investigated Nimbin (N1), a member of the ring C Seco‐tetranortriterpenoids family, and its semi‐natural analog deacetyl Nimbin namely N2 and N3 for their anti‐inflammatory properties. As key findings, N1, N2, and N3 were able to improve wound healing by cell proliferation in a period of 24 h and were able to reduce the reactive oxygen species (ROS) production in Madin–Darby Canine Kidney cells which were screened using dichloro‐dihydro fluorescein diacetate (DCF‐DA) staining. When the zebrafish larvae were subjected to DCF‐DA assay N1, N2, and N3 were able to substantially reduce the ROS levels in a dose‐dependent manner. In zebrafish larvae, the cell death indicates the fluorescent intensity due to acridine orange staining that was found to be dramatically decreasing upon the treatment of N1, N2, and N3. The cell membrane lipid peroxidation levels were also reduced in a dose‐dependent manner upon the treatment of Nimbin and its analogs indicating lesser blue fluorescent levels. Among the Nimbin and its analogs, N2 was subjected to have better activity. To confirm the activity of N1, N2, and N3, in silico characterization was performed using Density functional theory and molecular docking. As a result, N2 exhibited the lowest electronegative value and highest binding energy when docked with anti‐inflammatory and antioxidant proteins CAT, COX, GP, IL‐1, and MPO. Furthermore, the therapeutic potential of N2 must be explored at the molecular level as well as in clinical studies for the treatment of inflammation‐associated diseases. [ABSTRACT FROM AUTHOR]

Epsilon toxin (Etx) from Clostridium perfringens is the third most potent toxin after the botulinum and tetanus toxins. Etx is the main agent of enterotoxemia in ruminants and is produced by Clostridium perfringens toxinotypes B and D, causing great economic losses. Etx selectively binds to target cells, oligomerizes and inserts into the plasma membrane, and forms pores. A series of mutants have been previously generated to understand the cellular and molecular mechanisms of the toxin and to obtain valid molecular tools for effective vaccination protocols. Here, two new non-toxic Etx mutants were generated by selective deletions in the binding (Etx-ΔS188-F196) or insertion (Etx-ΔV108-F135) domains of the toxin. As expected, our results showed that Etx-ΔS188-F196 did not exhibit the usual Etx binding pattern but surprisingly recognized specifically an O-glycoprotein present in the proximal tubules of the kidneys in a wide range of animals, including ruminants. Although diminished, Etx-ΔV108-F135 maintained the capacity for binding and even oligomerization, indicating that the mutation particularly affected the pore-forming ability of the toxin. [ABSTRACT FROM AUTHOR]

The voltage-gated sodium (Na V ) channel is critical for cardiomyocyte function since it is responsible for action potential initiation and its propagation throughout the cell. It consists of a protein complex made of a pore forming α subunit and associated β subunits, which regulate α subunit function and subcellular localization. We previously showed the implication of N-linked glycosylation and S-acylation of β2 in its polarized trafficking. Here, we present evidence of β2 dimerization. Moreover, we demonstrate the implication of the cytoplasmic tail, extracellular loop, and transmembrane domain on proper β2 folding and export to the cell surface of polarized Madin-Darby canine kidney cells. Substantial alteration, or lack of any of these domains, leads to accumulation of β2 in the endoplasmic reticulum, along with impaired complex N-glycosylation, which is needed for its efficient surface delivery. We also show that these alterations to β2 affected to a certain extent Na V 1.5 surface localization. Conversely, however, Na V 1.5 had little or no influence on β2 trafficking, its localization to the surface, or homodimer formation. Altogether, our data link the architecture of the β2 domains to the establishment of its proper subcellular localization. These findings could provide valuable insights to gain a deeper comprehension of the elusive biology of β subunits in excitable cells, such as neurons and cardiomyocytes., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Cell cultured-based influenza virus production is a viable option for vaccine manufacturing. In order to achieve a high concentration of viable cells, is requirement to have not only optimal process conditions, but also an active metabolism capable of intracellular synthesis of viral components. Experimental metabolic data collected in such processes are complex and difficult to interpret, for which mathematical models are an appropriate way to simulate and analyze the complex and dynamic interaction between the virus and its host cell. A dynamic model with 35 states was developed in this study to describe growth, metabolism, and influenza A virus production in shake flask cultivations of suspension Madin-Darby Canine Kidney (MDCK) cells. It considers cell growth (concentration of viable cells, mean cell diameters, volume of viable cells), concentrations of key metabolites both at the intracellular and extracellular level and virus titers. Using one set of parameters, the model accurately simulates the dynamics of mock-infected cells and correctly predicts the overall dynamics of virus-infected cells for up to 60 h post infection (hpi). The model clearly suggests that most changes observed after infection are related to cessation of cell growth and the subsequent transition to apoptosis and cell death. However, predictions do not cover late phases of infection, particularly for the extracellular concentrations of glutamate and ammonium after about 12 hpi. Results obtained from additional in silico studies performed indicated that amino acid degradation by extracellular enzymes resulting from cell lysis during late infection stages may contribute to this observed discrepancy. [ABSTRACT FROM AUTHOR]

Background: Madin–Darby canine kidney (MDCK) cells are widely used for vaccine production, however, the safety of MDCK cells needs to be considered seriously because of high tumorigenicity. Micro RNAs (miRNAs) that are involved in the tumorigenicity of MDCK cells have been never been reported. Objective: To reveal the role of miRNA in the tumorigenic phenotype of MDCK cell line. Methods: The miRNA expression profiles of two monoclonal MDCK cells (M09CL and M35CL) with low tumorigenicity and one MDCK cell line (M73P) with high tumorigenicity were characterized and investigated by using small RNA-seq technology. Results: A total of 5 known miRNAs and 5 novel miRNAs were highly expressed in M73P. In addition, 4 known miRNAs and 4 novel miRNAs were highly expressed in M09CL and M35CL. The target genes of the differentially expressed miRNAs were significantly enriched in several biological processes, and the majority of these genes were involved in pathways in cancer and the MAPK signaling pathway. Through interaction analysis, 4 up-regulated miRNAs (cfa-miR-452, cfa-miR-8826, cfa-miR-224, and cfa-miR-2387) and their crucial target genes related to the tumor regulation network were identified. Results indicated these 4 miRNAs might play crucial roles in the tumorigenesis of MDCK cells. Conclusion: Our findings, which were based on the functional prediction of miRNAs and target genes, suggested that miRNAs might influence the tumorigenicity of MDCK cells by regulating target genes. Moreover, the results provided important data for understanding the miRNA-mediated regulatory networks that control the tumorigenicities of MDCK cells. [ABSTRACT FROM AUTHOR]

Permeation across Caco-2 cells in lipolysis-permeation setups can predict the rank order of in vivo drug exposure obtained with lipid-based formulations (LBFs). However, Caco-2 cells require a long differentiation period and do not capture all characteristics of the human small intestine. We therefore evaluated two in vitro assays with artificial lecithin-in-dodecane (LiDo) membranes and MDCK cells as absorptive membranes in the lipolysis-permeation setup. Fenofibrate-loaded LBFs were used and the results from the two assays compared to literature plasma concentrations in landrace pigs administered orally with the same formulations. Aqueous drug concentrations, supersaturation, and precipitation were determined in the digestion chamber and drug permeation in the receiver chamber. Auxiliary in vitro parameters were assessed, such as permeation of the taurocholate, present in the simulated intestinal fluid used in the assay, and size of colloidal structures in the digestion medium over time. The LiDo membrane gave a similar drug distribution as the Caco-2 cells and accurately reproduced the equivalent rank-order of fenofibrate exposure in plasma. Permeation of fenofibrate across MDCK monolayers did not, however, reflect the in vivo exposure rankings. Taurocholate flux was negligible through either membrane. This process was therefore not considered to significantly affect the in vitro distribution of fenofibrate. We conclude that the artificial LiDo membrane is a promising tool for lipolysis–permeation assays to evaluate LBF performance. [ABSTRACT FROM AUTHOR]

In influenza vaccine development, Madin–Darby canine kidney (MDCK) cells provide multiple advantages, including large-scale production and egg independence. Several cell-based influenza vaccines have been approved worldwide. We cultured H5N1 virus in a serum-free MDCK cell suspension. The harvested virus was manufactured into vaccines after inactivation and purification. The vaccine effectiveness was assessed in the Wuhan Institute of Biological Products BSL2 facility. The pre- and postvaccination mouse serum titers were determined using the microneutralization and hemagglutination inhibition tests. The immunological responses induced by vaccine were investigated using immunological cell classification, cytokine expression quantification, and immunoglobulin G (IgG) subtype classification. The protective effect of the vaccine in mice was evaluated using challenge test. Antibodies against H5N1 in rats lasted up to 8 months after the first dose. Compared with those of the placebo group, the serum titer of vaccinated mice increased significantly, Th1 and Th2 cells were activated, and CD8+ T cells were activated in two dose groups. Furthermore, the challenge test showed that vaccination reduced the clinical symptoms and virus titer in the lungs of mice after challenge, indicating a superior immunological response. Notably, early after vaccination, considerably increased interferon-inducible protein-10 (IP-10) levels were found, indicating improved vaccine-induced innate immunity. However, IP-10 is an adverse event marker, which is a cause for concern. Overall, in the case of an outbreak, the whole-virion H5N1 vaccine should provide protection. [ABSTRACT FROM AUTHOR]

Ochratoxin A (OTA) is universally known to induce nephrotoxicity via inducing oxidative stress and apoptosis, inhibiting protein synthesis and activating autophagy. Our previous studies have proved that OTA induces nephrotoxicity in vitro and in vivo by adjusting the NOD‐like receptor protein 3 (NLRP3) inflammasome activation and caspase‐1‐dependent pyroptosis. Based on these findings, we further investigated the protective role of selenomethionine (SeMet) on OTA‐caused nephrotoxicity using the Madin‐Darby canine kidney (MDCK) epithelial cells as an in vitro model, proposing to offer a new way for remedying OTA‐induced nephrotoxicity by nutritional manipulation. We measured the cell vitality, lactate dehydrogenase (LDH) activity and the expression of renal fibrotic genes, NLRP3 inflammasome and pyroptosis related genes. MTT and LDH results indicated that SeMet supplementation significantly mitigated 2.0 μg/ml OTA‐induced cytotoxicity in MDCK cells (p < 0.05). Meanwhile, SeMet alleviated OTA induced increase of reactive oxygen species in MDCK cells. Then, the expressions of α‐SMA, Vimentin, and TGF‐β were detected both in mRNA and protein levels. The results indicated 8 μM SeMet supplementation could significantly downregulate the expression of OTA‐induced renal fibrosis‐related genes (p < 0.05). In addition, the upregulation of OTA‐induced NLRP3 inflammasome and pyroptosis downstream genes was also significantly inhibited by 8 μM of SeMet (p < 0.05). In summary, SeMet could alleviate OTA‐induced renal fibrotic genes expression and reduce NLRP3‐caspase‐1‐dependent pyroptosis. Therefore, SeMet supplementation may become an effective approach for preserving animals from renal injury exposed to OTA. [ABSTRACT FROM AUTHOR]

Glycosylation is considered as a critical quality attribute for the production of recombinant biopharmaceuticals such as hormones, blood clotting factors, or monoclonal antibodies. In contrast, glycan patterns of immunogenic viral proteins, which differ significantly between the various expression systems, are hardly analyzed yet. The influenza A virus (IAV) proteins hemagglutinin (HA) and neuraminidase (NA) have multiple N‐glycosylation sites, and alteration of N‐glycan micro‐ and macroheterogeneity can have strong effects on virulence and immunogenicity. Here, we present a versatile and powerful glycoanalytical workflow that enables a comprehensive N‐glycosylation analysis of IAV glycoproteins. We challenged our workflow with IAV (A/PR/8/34 H1N1) propagated in two closely related Madin–Darby canine kidney (MDCK) cell lines, namely an adherent MDCK cell line and its corresponding suspension cell line. As expected, N‐glycan patterns of HA and NA from virus particles produced in both MDCK cell lines were similar. Detailed analysis of the HA N‐glycan microheterogeneity showed an increasing variability and a higher complexity for N‐glycosylation sites located closer to the head region of the molecule. In contrast, NA was found to be exclusively N‐glycosylated at site N73. Almost all N‐glycan structures were fucosylated. Furthermore, HA and NA N‐glycan structures were exclusively hybrid‐ and complex‐type structures, to some extent terminated with alpha‐linked galactose(s) but also with blood group H type 2 and blood group A epitopes. In contrast to the similarity of the overall glycan pattern, differences in the relative abundance of individual structures were identified. This concerned, in particular, oligomannose‐type, alpha‐linked galactose, and multiantennary complex‐type N‐glycans. [ABSTRACT FROM AUTHOR]

Background: The classification of natural antimicrobials as potential antibiotic replacements is still hampered by the absence of clear biological mechanisms behind their mode of action. This study investigated the mechanisms underlying the anti-bacterial effect of a mixture of natural antimicrobials (maltodextrin, citric acid, sodium citrate, malic acid, citrus extract and olive extract) against Campylobacter jejuni RC039, Salmonella enterica SE 10/72 and Clostridium perfringens ATCC® 13124 invasion of Madin–Darby Canine Kidney cells (MDCK). Results: Minimum sub-inhibitory concentrations were determined for Campylobacter jejuni (0.25%), Salmonella enterica (0.50%) and Clostridium perfringens (0.50%) required for the in vitro infection assays with MDCK cells. The antimicrobial mixture significantly reduced the virulence of all three pathogens towards MDCK cells and restored the integrity of cellular tight junctions through increased transepithelial resistance (TEER) and higher expression levels of ZO-1 (zonula occludens 1) and occludin. This study also identified the ERK (external regulated kinase) signalling pathway as a key mechanism in blocking the pro-inflammatory cytokine production (IL-1β, IL-6, IL-8, TNF-α) in infected cells. The reduction in hydrogen peroxide (H2O2) production and release by infected MDCK cells, in the presence of the antimicrobial mixture, was also associated with less tetrathionate formed by oxidation of thiosulphate (p < 0.0001). Conclusion: The present study describes for the first time that mixtures of natural antimicrobials can prevent the formation of substrates used by bacterial pathogens to grow and survive in anaerobic environments (e.g. tetrathionate). Moreover, we provide further insights into pathogen invasion mechanisms through restoration of cellular structures and describe their ability to block the ERK–MAPK kinase pathway responsible for inflammatory cytokine release [ABSTRACT FROM AUTHOR]

TRPV4 is a nonselective cationic channel responsive to several physical and chemical stimuli. Defects in TRPV4 channel function result in human diseases, such as skeletal dysplasias, arthropathies, and peripheral neuropathies. Nonetheless, little is known about the role of TRPV4 in other cellular functions, such as nuclear Ca2+ homeostasis or Ca2+‐regulated transcription. Here, we confirmed the presence of the full‐length TRPV4 channel in the nuclei of nonpolarized Madin‐Darby canine kidney cells. Confocal Ca2+ imaging showed that activation of the channel increases cytoplasmic and nuclear Ca2+ leading to translocation of TRPV4 out of the nucleus together with β‐catenin, a transcriptional regulator in the Wnt signaling pathway fundamental in embryogenesis, organogenesis, and cellular homeostasis. TRPV4 inhibits β‐catenin transcriptional activity through a direct interaction dependent upon channel activity. This interaction also occurs in undifferentiated osteoblastoma and neuroblastoma cell models. Our results suggest a mechanism in which TRPV4 may regulate differentiation in several cellular contexts. [ABSTRACT FROM AUTHOR]

Ochratoxin A (OTA), a prevalent nephrotoxic mycotoxin contaminant in food and feedstuff, has been reported to induce renal injury. To disclose the nephrotoxicity of continuous administration of OTA and to investigate potential mechanisms related to pyroptosis, male C57BL/6 mice were intraperitoneally injected with 1.0 and 2.0 mg/kg B.W. OTA every other day for 14 days. At 2.0 mg/kg B.W. OTA administration significantly increased histological injury and renal fibrosis molecules (α-SMA, Vimentin, TGF-β) and activated the NOD-like receptor protein 3 (NLRP3) inflammasome and induced pyroptosis compared with control. In the in vitro tests, Madin–Darby canine kidney (MDCK) epithelial cells were exposed to 0–4.0 μg/ml OTA for 24 h in serum-free medium. Data showed that OTA dose-dependently affected cell viability and significantly up-regulated renal fibrosis genes (α-SMA, Vimentin, TGF-β). 2.0 μg/ml OTA significantly induced NLRP3 inflammasome activation and caspase-1-dependent pyroptosis, increasing the expression and secretion of pro-inflammatory cytokines (IL-6, TNF-α) and pyroptosis-related genes (GSDMD, IL-1β, IL-18) in MDCK cells. These outcomes were significantly abrogated after inhibiting NLRP3 activation with inhibitor MCC950 and silencing NLRP3 with small interfering RNA (siRNA). Furthermore, knockdown of caspase-1 also ameliorated OTA-induced renal fibrosis via the inhibition of pyroptosis. Collectively, the chosen doses of OTA-triggered nephrotoxicity through NLRP3 inflammasome activation and caspase-1-dependent pyroptosis both in vitro and in vivo. [ABSTRACT FROM AUTHOR]

Abstract Morphogenesis is a tightly‐regulated developmental process by which tissues acquire the morphology that is critical to their function. For example, epithelial cells exhibit different 2D and 3D morphologies, induced by distinct biochemical and biophysical cues from their environment. In this work, novel hybrid matrices composed of a Matrigel and synthetic oligo(ethylene glycol)‐grafted polyisocyanides (PICs) hydrogels are used to form a highly tailorable environment. Through precise control of the stiffness and cell‐matrix interactions, while keeping other properties constant, a broad range of morphologies induced in Madin‐Darby Canine Kidney (MDCK) cells is observed. At relatively low matrix stiffness, a large morphological shift from round hollow cysts to 2D monolayers is observed, without concomitant translocation of the mechanotransduction protein Yes‐associated protein (YAP). At higher stiffness levels and enhanced cell‐matrix interactions, tuned by controlling the adhesive peptide density on PIC, the hybrid hydrogels induce a flattened cell morphology with simultaneous YAP translocation, suggesting activation. In 3D cultures, the latter matrices lead to the formation of tubular structures. Thus, mixed synthetic and natural gels, such as the hybrids presented here, are ideal platforms to dissect how external physical factors can be used to regulate morphogenesis in MDCK model system, and in the future, in more complex environments.

The coordination of cell proliferation and migration in growing tissues is crucial in development and regeneration but remains poorly understood. Here, we find that, while expanding with an edge speed independent of initial conditions, millimeter-scale epithelial monolayers exhibit internal patterns of proliferation and migration that depend not on the current but on the initial tissue size, indicating memory effects. Specifically, the core of large tissues becomes very dense, almost quiescent, and ceases cell-cycle progression. In contrast, initially-smaller tissues develop a local minimum of cell density and a tissue-spanning vortex. To explain vortex formation, we propose an active polar fluid model with a feedback between cell polarization and tissue flow. Taken together, our findings suggest that expanding epithelia decouple their internal and edge regions, which enables robust expansion dynamics despite the presence of size- and history-dependent patterns in the tissue interior.

Similar to the recent COVID-19 pandemic, influenza A virus poses a constant threat to the global community. For the treatment of flu disease, both antivirals and vaccines are available with vaccines the most effective and safest approach. In order to overcome limitations in egg-based vaccine manufacturing, cell culture–based processes have been established. While this production method avoids egg-associated risks in face of pandemics, process intensification using animal suspension cells in high cell density perfusion cultures should allow to further increase manufacturing capacities worldwide. In this work, we demonstrate the development of a perfusion process using Madin-Darby canine kidney (MDCK) suspension cells for influenza A (H1N1) virus production from scale-down shake flask cultivations to laboratory scale stirred tank bioreactors. Shake flask cultivations using semi-perfusion mode enabled high-yield virus harvests (4.25 log10(HAU/100 μL)) from MDCK cells grown up to 41 × 106 cells/mL. Scale-up to bioreactors with an alternating tangential flow (ATF) perfusion system required optimization of pH control and implementation of a temperature shift during the infection phase. Use of a capacitance probe for on-line perfusion control allowed to minimize medium consumption. This contributed to a better process control and a more economical performance while maintaining a maximum virus titer of 4.37 log10(HAU/100 μL) and an infectious virus titer of 1.83 × 1010 virions/mL. Overall, this study clearly demonstrates recent advances in cell culture–based perfusion processes for next-generation high-yield influenza vaccine manufacturing for pandemic preparedness. Key points: • First MDCK suspension cell–based perfusion process for IAV produciton was established. • "Cell density effect" was overcome and process was intensified by reduction of medium use and automated process control. • The process achieved cell density over 40 × 106cells/mL and virus yield over 4.37 log10(HAU/100 μL). [ABSTRACT FROM AUTHOR]

Morphogenesis is a tightly‐regulated developmental process by which tissues acquire the morphology that is critical to their function. For example, epithelial cells exhibit different 2D and 3D morphologies, induced by distinct biochemical and biophysical cues from their environment. In this work, novel hybrid matrices composed of a Matrigel and synthetic oligo(ethylene glycol)‐grafted polyisocyanides (PICs) hydrogels are used to form a highly tailorable environment. Through precise control of the stiffness and cell‐matrix interactions, while keeping other properties constant, a broad range of morphologies induced in Madin‐Darby Canine Kidney (MDCK) cells is observed. At relatively low matrix stiffness, a large morphological shift from round hollow cysts to 2D monolayers is observed, without concomitant translocation of the mechanotransduction protein Yes‐associated protein (YAP). At higher stiffness levels and enhanced cell‐matrix interactions, tuned by controlling the adhesive peptide density on PIC, the hybrid hydrogels induce a flattened cell morphology with simultaneous YAP translocation, suggesting activation. In 3D cultures, the latter matrices lead to the formation of tubular structures. Thus, mixed synthetic and natural gels, such as the hybrids presented here, are ideal platforms to dissect how external physical factors can be used to regulate morphogenesis in MDCK model system, and in the future, in more complex environments. [ABSTRACT FROM AUTHOR]

The ionic gelation method was used to study the effect of the crosslinking agent, sodium tripolyphosphate on average particle size (Dp) and zeta potential (ζp) of chitosan microparticles (CSMP) unloaded and loaded with trans-cinnamaldehyde (TCIN). The obtained values of Dp and ζp trend as 117.6 ± 0.4 ≤ Dp ≤ 478.5 ± 3.5 nm and +27.8 ± 1.3 ≤ ζp ≤ +103.5 ± 4.2 mV, respectively. The entrapment efficiency of TCIN in CSMP was 9.1 ± 2.0% and 71.5 ± 2.9% was released after 360 min (pH = 6.5) which reveals a potential anti-cancer activity in acidic environment. Cytotoxicity of TCIN in DMSO (0–50 μM) was evaluated on MDCK and HeLa cell lines and exhibited low effect at either 24 or 48 h of exposure; whereas TCIN-loaded CSMP (0–50 μM) showed, after 24 h of exposure, 67.6 ± 7.0 and 64.5 ± 3.9% cytotoxicity for MDCK and HeLa cell lines, respectively. At 48 h of exposure, TCIN-loaded CSMP achieved 81.1 ± 0.26 and 77.9 ± 4.2% cytotoxicity for MDCK and HeLa cell lines, respectively. • TCIN-loaded CSMP significantly enhanced its activity on HeLa cancer cells. • Higher cumulative TCIN releasing was found at pH 6.5 than 7.4 pH. • CSMP shows cytotoxic effects of TCIN on HeLa and MDCK cell lines. • Dp and ζp of the obtained TCIN-loaded CSMP (<200 nm) could facilitate EPR effect. [ABSTRACT FROM AUTHOR]

P-glycoprotein (P-gp) efflux assay is an integral part of discovery screening, especially for drugs requiring brain penetration as P-gp efflux ratio (ER) inversely correlates with brain exposure. However, significant variability in P-gp ER generated across cell lines can lead to misclassification of a P-gp substrate and subsequently disconnect with brain exposure data. We hypothesized that the ER depends on P-gp protein expression level in the in vitro assay. Quantitative proteomics and immunofluorescence staining were utilized to characterize P-gp protein expression and localization in four recombinant cell lines, over-expressing human or mouse P-gp isoforms, followed by functional evaluation. Efflux data generated in each cell line was compared against available rodent brain distribution data. The results suggested that the cell line with highest P-gp expression (hMDCK-MDR1 sourced from NIH) led to greatest dynamic range for efflux; thus, proving to be the most sensitive model to predict brain penetration. Cell lines with lower P-gp expression exhibited the greatest tendency for compound-dependent in vitro efflux saturation leading to false negative results. Ultimately, P-gp kinetics were characterized using a compartmental model to generate system-independent parameters to resolve such discrepancy. This study highlights the need for careful choice of well characterized P-gp in vitro tools and utility of modeling techniques to enable appropriate interpretation of the data. [ABSTRACT FROM AUTHOR]