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Canine coronavirus (CCoV) is widespread in dogs in several countries and causes mild enteric illness evolving to severe enteritis in young pups. In in vitro cultures canine coronaviruses generally induce extensive cell death, however nature of the events leading to cell death remains largely unknown. We analysed the induction of cytopathic effect by CCoV in a canine fibrosarcoma cell line (A-72) in order to characterize the apoptotic effect in homologous cell system. Following CCoV infection A-72 cell line, which is permissive to CCoV, showed reduced growth rate, as detected by MTT assay, a standard colorimetric assay for measuring cellular proliferation, and underwent to apoptotic death. Starting from 24h after CCoV infection, cells morphology appeared dramatically changed, with cells rounding and detachment from culture surface. Morphologic and biochemical features of apoptosis, such as blebbing of the plasma membrane, translocation of phosphatidilserine to cell surface and annexin V positive staining, nuclear fragmentation, apoptotic bodies formation and DNA laddering, were detected in CCoV-infected cells. Propidium iodide staining of infected culture indicated the appearance of hypodiploid DNA peak corresponding to apoptotic cell population. Commonly to other animal coronavirus infection caspase-3 is likely to contribute to the execution phase of apoptosis induced by CCoV in A-72 cells since we found activation of enzymatic activity as well as procaspase-3 activating cleavage. Apoptotic death of infected cells is detrimental as it causes cell and tissue destruction as well as inflammatory responses. Therefore in the case of CCoV associated gastroenteritis, apoptosis of epithelial mucosa cells may be responsible for pathology induced by CCoV infection.
We present an approach to fabricate biological matrix composites made entirely from cultured plant cells. We utilize the cell’s innate ability to synthesize nanofibrillar cell walls, which serve as the composite’s fundamental building blocks. Following a controlled compression/dehydration process, the cells arrange into lamellar structures with hierarchical features. We demonstrate that the native cell wall nanofibrils tether adjacent cells together through fibrillar interlocking and intermolecular hydrogen bonding. These interactions facilitate intercellular adhesion and eliminate the need for other binders. Our fabrication process utilizes the entire plant cell, grown in an in vitro culture; requires no harsh chemical treatments or waste-generating extraction or selection processes; and leads to bulk biocomposites that can be produced in situ and biodegrade in soil. The final mechanical properties are comparable to commodity plastics and can be further modulated by introducing filler particles.
Equine coronavirus (ECoV) was first isolated from a diarrheic foal and was found genetically similar to group II coronaviruses. However, its pathological characteristics were not adequately investigated. In our preliminary in vitro investigation, ECoV-induced cell death was observed in bovine kidney-derived MDBK cells. Based on this finding, we investigated whether the ECoV-induced CPE was apoptosis. Following ECoV infection, MDBK cells showed morphological changes such as cell rounding and detachment from the culture surface. Moreover, syncytium formation was observed as the other type of cytopathic effect in ECoV infection. Morphologic and biochemical features of apoptosis, such as nuclear fragmentation and DNA ladder formation, were also detected in ECoV-infected cells. Moreover, as is commonly observed in coronavirus infection in other animals, the activities of effecter caspases - caspase-3/7 - and initiator caspases - caspase-8 and caspase-9 - that are representative factors in the death receptor-mediated apoptotic pathway and mitochondrial apoptotic pathway, respectively, were increased in ECoV-infected MDBK cells. Therefore, it was suggested that ECoV can induce apoptosis in MDBK cells via a caspase-dependent pathway. Apoptotic death of infected cells is detrimental because it causes cell and tissue destruction and inflammatory responses. Although the pathological characteristics of ECoV are largely unknown, apoptosis may be the pathological basis of lesions of the digestive system in ECoV infection.
We previously isolated two human L1 elements (L1.2 and LRE2) as the progenitors of disease-producing insertions. Here, we show these elements can actively retrotranspose in cultured mammalian cells. When stably expressed from an episome in HeLa cells, both elements retrotransposed into a variety of chromosomal locations at a high frequency. The retrotransposed products resembled endogenous L1 insertions, since they were variably 5' truncated, ended in poly(A) tracts, and were flanked by target-site duplications or short deletions. Point mutations in conserved domains of the L1.2-encoded proteins reduced retrotransposition by 100- to 1000-fold. Remarkably, L1.2 also retrotransposed in a mouse cell line, suggesting a potential role for L1-based vectors in random insertional mutagenesis.
The biosynthesis of selenoproteins was studied in relation to milk formation and mammary cell biology by incubating the bovine mammary cell line MAC-T with ((75)Se)selenite. Intracellular proteins and proteins secreted into the cell culture medium were separated by 2D electrophoresis, the selenoproteins were detected by autoradiography, and the proteins were identified by MALDI-TOF. Approximately 35 (75)Se-containing spots were found in the cell proteins from MAC-T cells. Among them, one-third showed high intensity. The strongest spot was identified as glutathione peroxidase 1. About 20 spots were observed in protein precipitated from cell culture medium, one-third of them being distinctly visible. In an attempt to study a perturbation of the system, the effect of retinoic acid (RA) on the formation of selenoproteins was investigated. The concentration of (75)Se in total cell protein was reduced by about 35% in cells cultured with RA compared with control cells, while the opposite effect was observed in protein precipitated from cell culture medium, which contained 60% more (75)Se in RA-treated samples than in controls. There were also indications that RA might affect different selenoproteins in different ways. The methods described provide a promising approach for further studies of the regulation of selenoprotein formation in the mammary gland.
The aim of this research is to determine whether oxidative stress induces cellular senescence in human retinal pigment epithelial cells. Cultured ARPE19 cells were subjected to different concentrations of hydrogen peroxide to induce oxidative stress. Cells were seeded into 24-well plates with hydrogen peroxide added to cell medium and incubated at 37°C + 5% CO2 for a 90-minute period [at 0, 300, 400 and 800 micromolar (MCM) hydrogen peroxide]. The number of viable ARPE19 cells were recorded using the Trypan Blue Dye Exclusion Method and cell senescence was measured by positive staining for senescence-associated beta-galactosidase (SA-beta-Gal) protein. Without hydrogen peroxide treatment, the number of viable ARPE19 cells increased significantly from 50,000 cells/well to 197,000 within 72 hours. Treatment with hydrogen peroxide reduced this level of cell proliferation significantly (to 52,167 cells at 400 MCM; to 49,263 cells at 800 MCM). Meanwhile, cells with a high level of positive senescence-indicator SA-Beta-Gal-positive staining was induced by hydrogen peroxide treatment (from a baseline level of 12% to 80% at 400 MCM and at 800 MCM). Our data suggests that oxidative stress from hydrogen peroxide treatment inhibited ARPE19 cell proliferation and induced cellular senescence.
Stochastic noise at the cellular level has been shown to play a fundamental role in circadian oscillations, influencing how groups of cells entrain to external cues and likely serving as the mechanism by which cell-autonomous rhythms are generated. Despite this importance, few studies have investigated how clock perturbations affect stochastic noise-even as increasing numbers of high-throughput screens categorize how gene knockdowns or small molecules can change clock period and amplitude. This absence is likely due to the difficulty associated with measuring cell-autonomous stochastic noise directly, which currently requires the careful collection and processing of single-cell data. In this study, we show that the damping rate of population-level bioluminescence recordings can serve as an accurate measure of overall stochastic noise, and one that can be applied to future and existing high-throughput circadian screens. Using cell-autonomous fibroblast data, we first show directly that higher noise at the single-cell results in faster damping at the population level. Next, we show that the damping rate of cultured cells can be changed in a dose-dependent fashion by small molecule modulators, and confirm that such a change can be explained by single-cell noise using a mathematical model. We further demonstrate the insights that can be gained by applying our method to a genome-wide siRNA screen, revealing that stochastic noise is altered independently from period, amplitude, and phase. Finally, we hypothesize that the unperturbed clock is highly optimized for robust rhythms, as very few gene perturbations are capable of simultaneously increasing amplitude and lowering stochastic noise. Ultimately, this study demonstrates the importance of considering the effect of circadian perturbations on stochastic noise, particularly with regard to the development of small-molecule circadian therapeutics.
Hepatic stellate cells (HSCs) play a vital role in liver fibrosis, and a greater understanding of their regulation is required. Recent studies have focused on relationships between extracellular matrix (ECM) stiffness and gene expression or cellular metabolism, but none have provided a detailed metabolic analysis of HSC changes in spheroid cultures. Accordingly, in the present study, we created an HSC spheroid culture and analyzed changes in gene expression and metabolism. Expression of α-smooth muscle actin (α-SMA) decreased in the spheroids, suppressing proliferation. Gene expression analysis revealed the cell cycle, sirtuin signaling, mitochondrial dysfunction, and the Hippo pathway to be canonical pathways, believed to result from decreased proliferative ability or mitochondrial suppression. In the Hippo pathway, nuclear translocation of the yes-associated protein (YAP) was decreased in the spheroid, which was associated with the stiffness of the ECM. Metabolome analysis showed glucose metabolism changes in the spheroid, including glutathione pathway upregulation and increased lipid synthesis. Addition of the glycolytic product phosphoenolpyruvate (PEP) led to increased spheroid size, with increased expression of proteins such as α-SMA and S6 ribosomal protein (RPS6) phosphorylation, which was attributed to decreased suppression of translation. The results of our study contribute to the understanding of metabolic changes in HSCs and the progression of hepatic fibrosis.
Mammalian cells can die by apoptosis or by one of several non-apoptotic mechanisms, such as ferroptosis. Here, we present a protocol to distinguish ferroptosis from other cell death mechanisms in cultured cells. We describe steps for seeding cells, administering mechanism-specific cell death inducers and inhibitors, and measuring cell death and viability. We then detail the use of molecular markers to verify mechanisms of cell death. This protocol can be used to identify and distinguish ferroptosis in 2D and 3D cultures. For complete details on the use and execution of this protocol, please refer to Ko, et al. (2019),1 Magtanong, et al. (2022),2 and Armenta, et al. (2022).3.
The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. The potential throughput of this assay has, however, been limited by the need to individually clone and assay the activity of each sequence on interest using protein fluorescence or enzymatic activity as a proxy for regulatory activity. Advances in high-throughput DNA synthesis and sequencing technologies have recently made it possible to overcome these limitations by multiplexing the construction and interrogation of large libraries of reporter constructs. This protocol describes implementation of a Massively Parallel Reporter Assay (MPRA) that allows direct comparison of hundreds of thousands of putative regulatory sequences in a single cell culture dish.
Medium conditioned by cultured hippocampal glial contains an inhibitory factor that can hyperpolarize and suppress neuronal activity. Using biochemistry, electrophysiology, pharmacology, and mass spectrometry, we have identified the inhibitory factor as GABA (gamma-aminobutyric acid). Like GABA, the inhibitory factor increases chloride and potassium currents in neurons, which can be blocked by bicuculline. Mass spectrometry analysis of conditioned medium reveals peaks that are identical to that for GABA. Up to 500 micromolar GABA is found in conditioned medium from glial cultures. No GABA is found in conditioned medium from neuronal cultures. Hippocampal glia make much more GABA than cortical glia or glia from other brain regions. It is not clear how hippocampal glia synthesize GABA. Although they express GAD mRNA and adding glutamate to the culture medium increases the amount of GABA produced, other data suggest that glia do not use GAD to make GABA. Identifying the mechanism(s) by which GABA is produced by hippocampal glia would help clarify its role in modulating neuronal activity in the brain.
Primary retinal cell cultures and immunocytochemistry are important experimental platforms in ophthalmic research. Translation of retinal cells from their native environment to the in vitro milieu leads to cellular stress, jeopardizing their in vivo phenotype features. Moreover, the specificity and stability of many retinal immunochemical markers are poorly evaluated in retinal cell cultures. Hence, we here evaluated the expression profile of 17 retinal markers, that is, recoverin, rhodopsin, arrestin, Chx10, PKC, DCX, CRALBP, GS, vimentin, TPRV4, RBPMS, Brn3a, β-tubulin III, NeuN, MAP2, GFAP, and synaptophysin. At 7 and 18 days of culture, the marker expression profiles of mouse postnatal retinal cells were compared with their age-matched in vivo retinas. We demonstrate stable in vitro expression of all markers, except for arrestin and CRALBP. Differences in cellular expression and location of some markers were observed, both over time in culture and compared with the age-matched retina. We hypothesize that these differences are likely culture condition dependent. Taken together, we suggest a thorough evaluation of the antibodies in specific culture settings, before extrapolating the in vitro results to an in vivo setting. Moreover, the identification of specific cell types may require a combination of different genes expressed or markers with structural information.
Different classes of antidepressant drugs are used as a treatment for depression by activating the catecholinergic system. In addition, depression has been associated with decrease of growth factors, which causes insufficient axonal sprouting and reduced neuronal damage repair. In this study, antidepressant treatments are analyzed in a cell culture system, to study the modulation of growth factors.
The proton motive force (PMF) is bio-energetically important for various cellular reactions to occur. We developed PMF-photogenerating mitochondria in cultured mammalian cells. An archaebacterial rhodopsin, delta-rhodopsin, which is a light-driven proton pump derived from Haloterrigena turkmenica, was expressed in the mitochondria of CHO-K1 cells. The constructed stable CHO-K1 cell lines showed suppression of cell death induced by rotenone, a pesticide that inhibits mitochondrial complex I activity involved in PMF generation through the electron transport chain. Delta-rhodopsin was also introduced into the mitochondria of human neuroblastoma SH-SY5Y cells. The constructed stable SH-SY5Y cell lines showed suppression of dopaminergic neuronal cell death induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), an inducer of Parkinson's disease models, which acts through inhibition of complex I activity. These results suggest that the light-activated proton pump functioned as a PMF generator in the mitochondria of mammalian cells, and suppressed cell death induced by inhibition of respiratory PMF generation.
To comply with guiding principles for the ethical use of animals for experimental research, the field of mutation research has witnessed a shift of interest from large-scale in vivo animal experiments to small-sized in vitro studies. Mutation assays in cultured cells of transgenic rodents constitute, in many ways, viable alternatives to in vivo mutagenicity experiments in the corresponding animals. A variety of transgenic rodent cell culture models and mutation detection systems have been developed for mutagenicity testing of carcinogens. Of these, transgenic Big Blue® (Stratagene Corp., La Jolla, CA, USA, acquired by Agilent Technologies Inc., Santa Clara, CA, USA, BioReliance/Sigma-Aldrich Corp., Darmstadt, Germany) mouse embryonic fibroblasts and the λ Select cII Mutation Detection System have been used by many research groups to investigate the mutagenic effects of a wide range of chemical and/or physical carcinogens. Here, we review techniques and principles involved in preparation and culturing of Big Blue® mouse embryonic fibroblasts, treatment in vitro with chemical/physical agent(s) of interest, determination of the cII mutant frequency by the λ Select cII assay and establishment of the mutation spectrum by DNA sequencing. We describe various approaches for data analysis and interpretation of the results. Furthermore, we highlight representative studies in which the Big Blue® mouse cell culture model and the λ Select cII assay have been used for mutagenicity testing of diverse carcinogens. We delineate the advantages of this approach and discuss its limitations, while underscoring auxiliary methods, where applicable.
Human pluripotent stem cells would be invaluable for in vitro studies of aspects of human embryogenesis. With the goal of establishing pluripotent stem cell lines, gonadal ridges and mesenteries containing primordial germ cells (PGCs, 5-9 weeks postfertilization) were cultured on mouse STO fibroblast feeder layers in the presence of human recombinant leukemia inhibitory factor, human recombinant basic fibroblast growth factor, and forskolin. Initially, single PGCs in culture were visualized by alkaline phosphatase activity staining. Over a period of 7-21 days, PGCs gave rise to large multicellular colonies resembling those of mouse pluripotent stem cells termed embryonic stem and embryonic germ (EG) cells. Throughout the culture period most cells within the colonies continued to be alkaline phosphatase-positive and tested positive against a panel of five immunological markers (SSEA-1, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81) that have been used routinely to characterize embryonic stem and EG cells. The cultured cells have been continuously passaged and found to be karyotypically normal and stable. Both XX and XY cell cultures have been obtained. Immunohistochemical analysis of embryoid bodies collected from these cultures revealed a wide variety of differentiated cell types, including derivatives of all three embryonic germ layers. Based on their origin and demonstrated properties, these human PGC-derived cultures meet the criteria for pluripotent stem cells and most closely resemble EG cells.
Indoxyl sulfate (IS), an organic anion uremic toxin, promotes the progression of renal dysfunction. Some studies have suggested that IS inhibits osteoclast differentiation and suppresses parathyroid hormone (PTH)-stimulated intracellular cAMP production, decreases PTH receptor expression, and induces oxidative stress in primary mouse calvaria osteoblast cell culture. However, the direct effects of IS on osteoblast apoptosis have not been fully evaluated. Hence, we investigated whether IS acts as a bone toxin by studying whether IS induces apoptosis and inhibits differentiation in the cultured osteoblast cell line MC3T3-E1.
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