Madin-Darby Canine Kidney Epithelial Cells Research Articles

Small RNA sequencing analysis reveals regulation of microRNA expression in Madin-Darby canine kidney epithelial cells infected with Canid alphaherpesvirus 1.

Canid alphaherpesvirus 1 (CHV-1) infection can cause spontaneous abortions in pregnant dams, and in young puppies, fatal systemic infections are common. MicroRNAs (miRNAs) affect viral infection by binding to messenger RNAs, and inhibiting expression of host and/or viral genes. We conducted deep sequencing of small RNAs in CHV-1-infected and mock-infected Madin-Darby Canine Kidney (MDCK) epithelial cells, and detected sequences corresponding to 282 cellular miRNAs. Of these, 18 were significantly upregulated at 12h post-infection, most of which were encoded on the X chromosome. We next quantified the mature forms of several of the miRNAs using stem loop RT-qPCR. Our results revealed a discordance between the levels of small RNAs corresponding to canine miRNAs, and levels of the corresponding mature miRNAs, which suggests a block in miRNA biogenesis in infected cells. Nevertheless, we identified several mature miRNAs that exhibited a statistically significant increase upon infection. These included cfa-miR-8908b, a miRNA of unknown function, and cfa-miR-146a, homologs of which target innate immune pathways and are known to play a role in other viral infections. Interestingly, ontology analysis predicted that cfa-miR-8908b targets factors involved in the ubiquitin-like protein conjugation pathway and peroxisome biogenesis among other cellular functions. This is the first study to evaluate changes in miRNA levels upon CHV-1 infection. Based on our findings, we developed a model whereby CHV-1 infection results in changes in levels of a limited number of cellular miRNAs that target elements of the host immune response, which may provide clues regarding novel therapeutic targets.

Impact of trypsin on cell cytoplasm during detachment of cells studied by terahertz sensing

Trypsin is a very common enzyme used in cell culture to harvest cells by cleaving the proteins responsible for cell adhesion. However, trypsin also induces undesirable effects on cells, such as altering membrane proteins and the cytoskeleton, changing the composition of the cytoplasm and the cell volume, and even leading to cell death when used improperly. Using attenuated total reflection in the terahertz domain, confocal microscopy, and the propidium iodide test, we quantified in real time the change in cytoplasmic content induced by trypsin proteolysis on Madin-Darby canine kidney epithelial cells. We have observed a cytoplasmic modification from the very first seconds of trypsinization, following the change of cell volume due to mechanical re-equilibrium of the membrane. We found that the cytoplasmic alteration is associated with a transfer of small solutes: electrolytes and metabolites. We also found a very good nonlinear correlation between the side effects monitored by terahertz sensing and the cell height, regardless of the dependence of the cell height on trypsin concentration and exposure time.

Calcium transients trigger switch-like discharge of prostaglandin E2 in an extracellular signal-regulated kinase-dependent manner.

Prostaglandin E2 (PGE2) is a key player in a plethora of physiological and pathological events. Nevertheless, little is known about the dynamics of PGE2 secretion from a single cell and its effect on the neighboring cells. Here, by observing confluent Madin-Darby canine kidney (MDCK) epithelial cells expressing fluorescent biosensors, we demonstrate that calcium transients in a single cell cause PGE2-mediated radial spread of PKA activation (RSPA) in neighboring cells. By in vivo imaging, RSPA was also observed in the basal layer of the mouse epidermis. Experiments with an optogenetic tool revealed a switch-like PGE2 discharge in response to the increasing cytoplasmic Ca2+ concentrations. The cell density of MDCK cells correlated with the frequencies of calcium transients and the following RSPA. The extracellular signal-regulated kinase (ERK) activation also enhanced the frequency of RSPA in MDCK and in vivo. Thus, the PGE2 discharge is regulated temporally by calcium transients and ERK activity.

Baicalin Exhibits a Protective Effect against Cisplatin-Induced Cytotoxic Damage in Canine Renal Tubular Epithelial Cells.

Renal failure is a common chronic disease in dogs that substantially affects both their quality of life and longevity. The objective of this study was to assess the protective mechanisms of baicalin in cisplatin-induced Madin-Darby canine kidney (MDCK) epithelial cells' apoptosis model and explore the impacts of baicalin at varying doses on various indexes, such as cisplatin-induced MDCK cell apoptosis, oxidation and antioxidation, and inflammatory factors. (Methods) MDCK cells in the logarithmic growth phase were randomly divided into a control group, a model group (20 μmol/L cisplatin), and a baicalin-protection group (20 μmol/L cisplatin + 50, 25 μmol/L baicalin) and received the corresponding treatments for 24 h. The effects of cisplatin on MDCK cell apoptosis, oxidation and antioxidation, inflammatory factors, and other indicators were studied, and the relieving effect of baicalin on cisplatin-induced MDCK cell damage was explored. Calcein/PI staining and Annexin V-FITC/PI staining showed that cisplatin induced the apoptosis of MDCK cells, while baicalin effectively reduced the damage caused by cisplatin. The ELISA results demonstrated a significant elevation in the nitric oxide (NO) and malondialdehyde (MDA) levels within the MDCK cells following treatment with cisplatin (p < 0.01). In addition, superoxide dismutase (SOD), glutathione peroxidase (GSH), and catalase (CAT) activities remarkably declined (p < 0.01), while tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) expression within the MDCK cells were apparently elevated (p < 0.01). However, baicalin treatment resulted in opposite changes in these factors. The findings suggested that baicalin exhibits potential in mitigating cisplatin-induced oxidative stress and inflammation in MDCK cells. As revealed with the Western blot results, cisplatin promoted P62, P53, and BAX protein levels, increased mTOR phosphorylation, inhibited AMPK phosphorylation, and reduced Beclin1 and BCL-2 protein levels. However, a contrasting trend was observed following baicalin treatment. Cisplatin can inhibit the activity of MDCK cells, lead to abnormalities in oxidation and antioxidation functions and cell inflammatory factors, and accelerate cell apoptosis. Moreover, baicalin can significantly alleviate the damage of cisplatin to MDCK cells.

TRAF3 deficiency in MDCK cells improved sensitivity to the influenza A virus

Tumor necrosis factor receptor-associated factor 3 (TRAF3), an adaptor protein, has significant and varying effects on immunity depending on cell types. The role of TRAF3 in Madin-Darby Canine Kidney Epithelial (MDCK) cell resistance to influenza A virus (IVA) remains elusive. In the present study, CRISPR-Cas9 gene editing technology was used to construct the TRAF3 knockout MDCK cells (MDCK-TRAF3−/−). Hemagglutination assay, plaque assay, transcriptome, and quantitative real-time PCR were performed after IVA infection. The results showed that after IVA infection, HA titers and virus titers were promoted, interferon I-related pathways were significantly blocked, and transcription of several antiviral-related genes was significantly decreased in MDCK-TRAF3−/− cells. Thus, our study suggests that TRAF3 gene knockout reduced MDCK cell's resistance to IVA, thereby resulting in a promising way for IVA isolation and vaccine manufacturing.

Inhibitory Effects of Epithelial Cells on Fibrosis Mechanics of Microtissue and Their Spatiotemporal Dependence on the Epithelial-Fibroblast Interaction.

Cell-generated contraction force is the primary physical drive for fibrotic densification of biological tissues. Previous studies using two-dimensional culture models have shown that epithelial cells inhibit the myofibroblast-derived contraction force via the regulation of the fibroblast/myofibroblast transition (FMT). However, it remains unclear how epithelial cells interact with fibroblasts and myofibroblasts to determine the mechanical consequences and spatiotemporal regulation of fibrosis development. In this study, we established a three-dimensional microtissue model using an NIH/3T3 fibroblast-laden collagen hydrogel, incorporated with a microstring-based force sensor, to assess fibrosis mechanics. When Madin-Darby canine kidney epithelial cells were cocultured on the microtissue's surface, the densification, stiffness, and contraction force of the microtissue greatly decreased compared to the monocultured microtissue without epithelial cells. The key fibrotic features, such as enhanced protein expression of α-smooth muscle actin, fibronectin, and collagen indicating FMT and matrix deposition, respectively, were also significantly reduced. The antifibrotic effects of epithelial cells on the microtissue were dependent on the intercellular signaling molecule prostaglandin E2 (PGE2) with an effective concentration of 10 μM and their proximity to the fibroblasts, indicating paracrine cellular signaling between the two types of cells during tissue fibrosis. The effect of PGE2 on microtissue contraction was also dependent on the time point when PGE2 was delivered or blocked, suggesting that the presence of epithelial cells at an early stage is critical for preventing or treating advanced fibrosis. Taken together, this study provides insights into the spatiotemporal regulation of mechanical properties of fibrosis by epithelial cells, and the cocultured microtissue model incorporated with a real-time and sensitive force sensor will be a suitable system for evaluating fibrosis and drug screening.

Characterization of RACK1-depleted mammalian cells by a palette of microscopy approaches reveals defects in cell cycle progression and polarity establishment

The Receptor for Activated C Kinase 1 (RACK1) is an evolutionarily conserved scaffold protein involved in the regulation of numerous cellular processes. Here, we used CRISPR/Cas9 and siRNA to reduce the expression of RACK1 in Madin-Darby Canine Kidney (MDCK) epithelial cells and Rat2 fibroblasts, respectively. RACK1-depleted cells were examined using coherence-controlled holographic microscopy, immunofluorescence, and electron microscopy. RACK1 depletion resulted in decreased cell proliferation, increased cell area and perimeter, and in the appearance of large binucleated cells suggesting a defect in the cell cycle progression. Our results show that the depletion of RACK1 has a pleiotropic effect on both epithelial and mesenchymal cell lines and support its essential role in mammalian cells.

Development of a simple cultured cell–anaerobic microbial co-culture system using liquid paraffin

Conventional co-culture systems are complicated, lack versatility, and do not adequately replicate the intestinal lumen environment. This study aimed to devise a system that allows for (i) arbitrary sampling of the culture medium, (ii) monitoring the growth of co-cultured cells, (iii) aerobic-anaerobic co-culture, (iv) simple operation, and (v) evaluation of multiple samples. We built a simple cell-anaerobic microorganism co-culture system using liquid paraffin to separate growth spaces for aerobic cells and anaerobic bacteria. Mineral oil was added to the top of the anaerobic bacterial cultivation space to seal the space and reduce gas exchange. Co-culture of anaerobic, Bifidobacterium bifidum and aerobic, epithelial Madin-Darby canine kidney (MDCK) cells demonstrated that the barrier function and viability of co-cultured MDCK cells were comparable to those of a pure MDCK culture after 24h, and the growth curve of co-cultured B.bifidum was similar to that of pure B.bifidum. Furthermore, the growth of B.bifidum pure culture under sealed conditions was approximately 1.5 times greater than that under non-sealed conditions at 24h. Glucose consumption at 24h of co-culture under sealed conditions was 10%-15% higher than that under non-sealed conditions. This highly versatile culture method enabled the quantitative characterisation of B.bifidum and MDCK cells upon co-culture. The newly established co-culture system could be applied to various aerobic cell-anaerobic bacteria co-culture which will provide a strategy for basic and applied research on host-microbe interactions.

The genetic diversity, replication, and transmission of 2009 pandemic H1N1 viruses in China.

The 2009 pandemic H1N1 influenza A virus (pdm09) continue to evolve, and few studies have systemically analyzed the evolution, replication, and transmission of pmd09 viruses in China. To better understand the evolution and pathogenicity of pdm09 viruses, we systematically analyzed viruses that were confirmed in 2009-2020 in China and characterized their replication and transmission ability. We extensively analyzed the evolution characteristics of pdm/09 in China over the past decades. The replication ability of 6B.1 and 6B.2 lineages on Madin-Darby canine kidney (MDCK) and human lung adenocarcinoma epithelial (A549) cells and their pathogenicity and transmission in guinea pigs were also compared. In total, 3,038 pdm09 viruses belonged to clade 6B.1 (62% of all pdm09 viruses) and clade 6B.2 (4%). Clade 6B.1 pdm09 viruses are the predominant clade, with proportions of 54.1%, 78.9%, 57.2%, 58.6%, 61.7%, 76.3%, and 66.6% in the North, Northeast, East, Central, South, Southwest, and Northeast regions in China, respectively. The isolation proportion of clade 6B.1 pdm/09 viruses was 57.1%, 74.3%, 96.1%, 98.2%, 86.7%, and 78.5% in 2015-2020, respectively. A clear differentiation time point appeared in 2015 before which the evolution trend of pdm09 viruses in China was similar to that in North America but then showed a different trend after that point. To characterize pdm09 viruses in China after 2015, we further analyzed 33 pdm09 viruses isolated in Guangdong in 2016-2017, among which A/ Guangdong/33/2016 and A/Guangdong/184/2016 (184/2016) belonged to clade 6B.2, and the other 31 strains belonged to clade 6B.1. A/Guangdong/887/2017 (887/2017) and A/Guangdong/752/2017 (752/2017) (clade 6B.1), 184/2016 (clade 6B.2) and A/California/04/2009 (CA04) replicated efficiently in MDCK cells and A549 cells, as well as the turbinates of guinea pigs. 184/2016 and CA04 could transmit among guinea pigs through physical contact. Our findings provide novel insights into the evolution, pathogenicity, and transmission of pdm09 virus. The results show that enhancing surveillance of pdm09 viruses and timely evaluation of their virulence are essential.

RKIP localizes to the nucleus through a bipartite nuclear localization signal and interaction with importin α to regulate mitotic progression

Raf kinase inhibitor protein (RKIP) is a multifunctional modulator of intracellular signal transduction. Although most of its functions have been considered cytosolic, we show here that the localization of RKIP is primarily nuclear in both growing and quiescent Madin-Darby canine kidney epithelial cells and in Cal-51 and BT-20 human breast cancer cells. We have identified a putative bipartite nuclear localization signal (NLS) in RKIP that maps to the surface of the protein surrounding a known regulatory region. Like classical NLS sequences, the putative NLS of RKIP is rich in arginine and lysine residues. Deletion of and point mutations in the putative NLS lead to decreased nuclear localization. Point mutation of all the basic residues in the putative NLS of RKIP particularly strongly reduces nuclear localization. We found consistent results in reexpression experiments with wildtype or mutant RKIP in RKIP-silenced cells. A fusion construct of the putative NLS of RKIP alone to a heterologous reporter protein leads to nuclear localization of the fusion protein, demonstrating that this sequence alone is sufficient for import into the nucleus. We found that RKIP interacts with the nuclear transport factor importin α in BT-20 and MDA-MB-231 human breast cancer cells, suggesting importin-mediated active nuclear translocation. Evaluating the biological function of nuclear localization of RKIP, we found that the presence of the putative NLS is important for the role of RKIP in mitotic checkpoint regulation in MCF-7 human breast cancer cells. Taken together, these findings suggest that a bipartite NLS in RKIP interacts with importin α for active transport of RKIP into the nucleus and that this process may be involved in the regulation of mitotic progression.

VAMP5 and distinct sets of cognate Q-SNAREs mediate exosome release.

Small extracellular vesicles (sEVs) are largely classified into two types, plasma-membrane derived sEVs and endomembrane-derived sEVs. The latter type (referred to as exosomes herein) is originated from late endosomes or multivesicular bodies (MVBs). In order to release exosomes extracellularly, MVBs must fuse with the plasma membrane, not with lysosomes. In contrast to the mechanism responsible for MVB-lysosome fusion, the mechanism underlying the MVB-plasma membrane fusion is poorly understood. Here, we systematically analyze soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family proteins and identify VAMP5 as an MVB-localized SNARE protein required for exosome release. Depletion of VAMP5 in HeLa cells impairs exosome release. Mechanistically, VAMP5 mediates exosome release by interacting with SNAP47 and plasma membrane SNARE Syntaxin 1 (STX1) or STX4 to release exosomes. VAMP5 is also found to mediate asymmetric exosome release from polarized Madin-Darby canine kidney (MDCK) epithelial cells through interaction with the distinct sets of Q-SNAREs, suggesting that VAMP5 is a general exosome regulator in both polarized cells and non-polarized cells.Key words: Exosome, small extracellular vesicle (sEV), multivesicular body, SNARE, VAMP5.

In Vitro and In Vivo Cell-Interactions with Electrospun Poly (Lactic-Co-Glycolic Acid) (PLGA): Morphological and Immune Response Analysis.

Random electrospun three-dimensional fiber membranes mimic the extracellular matrix and the interfibrillar spaces promotes the flow of nutrients for cells. Electrospun PLGA membranes were analyzed in vitro and in vivo after being sterilized with gamma radiation and bioactivated with fibronectin or collagen. Madin-Darby Canine Kidney (MDCK) epithelial cells and primary fibroblast-like cells from hamster’s cheek paunch proliferated over time on these membranes, evidencing their good biocompatibility. Cell-free irradiated PLGA membranes implanted on the back of hamsters resulted in a chronic granulomatous inflammatory response, observed after 7, 15, 30 and 90 days. Morphological analysis of implanted PLGA using light microscopy revealed epithelioid cells, Langhans type of multinucleate giant cells (LCs) and multinucleated giant cells (MNGCs) with internalized biomaterial. Lymphocytes increased along time due to undegraded polymer fragments, inducing the accumulation of cells of the phagocytic lineage, and decreased after 90 days post implantation. Myeloperoxidase+ cells increased after 15 days and decreased after 90 days. LCs, MNGCs and capillaries decreased after 90 days. Analysis of implanted PLGA after 7, 15, 30 and 90 days using transmission electron microscope (TEM) showed cells exhibiting internalized PLGA fragments and filopodia surrounding PLGA fragments. Over time, TEM analysis showed less PLGA fragments surrounded by cells without fibrous tissue formation. Accordingly, MNGC constituted a granulomatous reaction around the polymer, which resolves with time, probably preventing a fibrous capsule formation. Finally, this study confirms the biocompatibility of electrospun PLGA membranes and their potential to accelerate the healing process of oral ulcerations in hamsters’ model in association with autologous cells.

Optogenetic control of apical constriction induces synthetic morphogenesis in mammalian tissues

The emerging field of synthetic developmental biology proposes bottom-up approaches to examine the contribution of each cellular process to complex morphogenesis. However, the shortage of tools to manipulate three-dimensional (3D) shapes of mammalian tissues hinders the progress of the field. Here we report the development of OptoShroom3, an optogenetic tool that achieves fast spatiotemporal control of apical constriction in mammalian epithelia. Activation of OptoShroom3 through illumination in an epithelial Madin-Darby Canine Kidney (MDCK) cell sheet reduces the apical surface of the stimulated cells and causes displacements in the adjacent regions. Light-induced apical constriction provokes the folding of epithelial cell colonies on soft gels. Its application to murine and human neural organoids leads to thickening of neuroepithelia, apical lumen reduction in optic vesicles, and flattening in neuroectodermal tissues. These results show that spatiotemporal control of apical constriction can trigger several types of 3D deformation depending on the initial tissue context.

3D printed devices with integrated collagen scaffolds for cell culture studies including transepithelial/transendothelial electrical resistance (TEER) measurements

In this paper, we describe the use of 3D printed devices for both static and flow studies that contain electrospun collagen scaffolds and can accommodate transepithelial/transendothelial electrical resistance (TEER) measurements. Electrospinning was used to create the collagen scaffold, followed by an optimized 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-Hydroxysuccinimide (EDC/NHS) cross-linking procedure to produce stable collagen fibers that are similar in size to fibers in vivo. LC/MS was used to study the leaching of solvent and NHS from the scaffold, with several rinsing steps being shown to eliminate the leaching and promote the culture of Madin-Darby Canine Kidney (MDCK) epithelial cells on the scaffold. Both static and flow 2-part devices were successfully fabricated by 3D printing using either VeroClear or MED610 material (PolyJet printing) and assembling the scaffold between laser cut Teflon gaskets. The devices were designed to easily accommodate commonly used STX2 chopstick electrodes for TEER measurements. A detailed comparison was made between the use of collagen scaffolds vs other electrospun materials for cell culture. The collagen extracellular matrix model displayed a high barrier functionality for up to 7 days. In addition, a different 3D printed device with a collagen scaffold is described to incorporate continuous flow and replenishment of media under the cell layer in a manner that also enables periodic recording of TEER measurements. Overall, this work shows that the combination of biological ECM materials such as collagen into microfluidic devices that incorporate flow have great potential to form more realistic cell culture models in areas such as blood brain barrier research.

Rab39 and its effector UACA regulate basolateral exosome release from polarized epithelial cells.

Exosomes are small extracellular vesicles that originate from the intraluminal vesicles of multivesicular bodies (MVBs). We previously reported that polarized Madin-Darby canine kidney (MDCK) epithelial cells secrete two types of exosomes, apical and basolateral exosomes, from different MVBs. However, how these MVBs are selectively targeted to the apical or basolateral membrane remained unknown. Here, we analyze members of the Rab family small GTPases and show that different sets of Rabs mediate asymmetrical exosome release. Rab27, the best-known regulator of MVB transport for exosome release, is specifically but partially involved in apical exosome release, and Rab37, a close homolog of Rab27, is an additional apical exosome regulator. By contrast, Rab39 functions as a specific regulator of basolateral exosome release. Mechanistically, Rab39 interacts with its effector UACA, and UACA then recruits Lyspersin, a component of BLOC-1-related complex (BORC). Our findings suggest that the Rab39-UACA-BORC complex specifically mediates basolateral exosome release.

Protective effect of morin by targeting mitochondrial reactive oxygen species induced by hydrogen peroxide demonstrated at a molecular level in MDCK epithelial cells.

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. 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. 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. These findings suggest that morin reduces H2O2-induced oxidative stress, reduces DNA oxidative damage, and prevents the depletion of antioxidant genes in MDCK cells.

Precise Tuning and Characterization of Viscoelastic Interfaces for the Study of Early Epithelial-Mesenchymal Transition Behaviors.

There is growing evidence that cellular functions are regulated by the viscoelastic nature of surrounding matrices. This study aimed to investigate the impact of interfacial viscoelasticity on adhesion and epithelial-mesenchymal transition (EMT) behaviors of epithelial cells. The interfacial viscoelasticity was manipulated using spin-coated thin films composed of copolymers of ε-caprolactone and d,l-lactide photo-cross-linked with benzophenone, whose mechanical properties were characterized using atomic force microscopy and a rheometer. The critical range for the morphological transition of epithelial Madin-Darby canine kidney (MDCK) cells was of the order of 102 ms relaxation time, which was 1-2 orders of magnitude smaller than the relaxation times reported (10-102 s). An analysis of strain rate-dependent viscoelastic properties revealed that the difference was caused by the different strain rate/frequency used for the mechanical characterization of the interface and bulk. Furthermore, decoupling of the interfacial viscous and elastic terms demonstrated that E/N-cadherin expression levels were regulated differently by interfacial relaxation and elasticity. These results confirm the significance of precise manipulation and characterization of interfacial viscoelasticity in mechanobiology studies on EMT progression.

A novel role for PRL in regulating epithelial cell density by inducing apoptosis at confluence.

Maintaining proper epithelial cell density is essential for the survival of multicellular organisms. Although regulation of cell density through apoptosis is well known, its mechanistic details remain elusive. Here, we report the involvement of membrane-anchored phosphatase of regenerating liver (PRL), originally known for its role in cancer malignancy, in this process. In epithelial Madin-Darby canine kidney cells, upon confluence, doxycycline-induced expression of PRL upregulated apoptosis, reducing cell density. This could be circumvented by artificially reducing cell density via stretching the cell-seeded silicon chamber. Moreover, small interfering RNA-mediated knockdown of endogenous PRL blocked apoptosis, leading to greater cell density. Mechanistically, PRL promoted apoptosis by upregulating the translation of E-cadherin and activating the TGF-β pathway. Morpholino-mediated inhibition of PRL expression in zebrafish embryos caused developmental defects, with reduced apoptosis and increased epithelial cell density during convergent extension. Overall, this study revealed a novel role for PRL in regulating density-dependent apoptosis in vertebrate epithelia. This article has an associated First Person interview with the first author of the paper.

Effect of Treponema Denticola Infection on Epithelial Cells.

Chronic periodontitis is an infectious disease caused by periodontopathic bacteria in subgingival plaque. One major pathogen of this disease, Treponema denticola, has several virulence factors, including a major surface protein (Msp) and the surface protease dentilisin. The cytopathic effects of periodontopathic bacteria on epithelial cells disrupt the integrity of the barrier junction, resulting in the inflammation of periodontal tissue. The aim of this study was to investigate the effect of T. denticola virulence factors dentilisin and Msp on epithelial cells. The effects of T. denticola wild-type, Msp-mutant, and dentilisin-mutant strains on the contact junction in Madin-Darby canine kidney epithelial cells was evaluated based on ohmic values. Cultured oral carcinoma epithelial cells were scratched and exposed to the selected T. denticola strains and cell migration determined. Subsequent degradation of adherence proteins and proteins in the contact junctions was evaluated. Dissociation of cell contact junctions was detected in cells infected with wild-type T. denticola approximately 30 min after infection, but not in those exposed to the mutants. Inhibition of migration was observed in the wild-type and Msp-deficient mutants. The adherent proteins focal adhesion kinase, ZO-1, and paxillin were hydrolyzed by infection with the wild-type and Msp mutants. These results indicate that T. denticola disrupts the function of epithelial cells by hydrolyzing proteins at the intercellular junction and inhibiting healing of epithelial cells via hydrolyzed proteins associated with focal adhesion; Msp was also associated with these effects.

Cover Image: Entry of the Varicellovirus Canid herpesvirus 1 into Madin–Darby canine kidney epithelial cells is pH‐independent and occurs via a macropinocytosis‐like mechanism but without increase in fluid uptake (Cellular Microbiology 12/2021)

Confocal micrograph showing Canid herpesvirus 1 (red) bound to MDCK cells, and DAPI-stained nuclei (blue). Primary amines of viral glycoproteins were labelled with Alexa Fluor succinimidyl esters 568 dye. For further details, readers are referred to the article by Eisa et al. on p. e13398 of this issue.