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All-RNA-mediated targeted gene integration methods, rendering reduced immunogenicity, effective deliverability with non-viral vehicles, and a low risk of random mutagenesis, are urgently needed for next-generation gene addition technologies. Naturally occurring R2 retrotransposons hold promise in this context due to their site-specific integration profile. Here, we systematically analyzed the biodiversity of R2 elements and screened several R2 orthologs capable of full-length gene insertion in mammalian cells. Robust R2 system gene integration efficiency was attained using combined donor RNA and protein engineering. Importantly, the all-RNA-delivered engineered R2 system showed effective integration activity, with efficiency over 60% in mouse embryos. Unbiased high-throughput sequencing demonstrated that the engineered R2 system exhibited high on-target integration specificity (99%). In conclusion, our study provides engineered R2 tools for applications based on hit-and-run targeted DNA integration and insights for further optimization of retrotransposon systems.
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ARN , Retroelementos , Animales , Retroelementos/genética , Ratones , Humanos , ARN/genética , ARN/metabolismo , Células HEK293 , Ingeniería Genética/métodos , Marcación de Gen/métodosRESUMEN
Insig-1 and Insig-2 are endoplasmic reticulum (ER) proteins that inhibit lipid synthesis by blocking transport of sterol regulatory element-binding proteins (SREBP-1 and SREBP-2) from ER to Golgi. In the Golgi, SREBPs are processed proteolytically to release their transcription-activating domains, which enhance the synthesis of fatty acids, triglycerides, and cholesterol. Heretofore, the two Insigs have redundant functions, and there is no rationale for two isoforms. The current data identify a specific function for Insig-2. We show that eicosapentaenoic acid (EPA), a polyunsaturated fatty acid, inhibits fatty acid synthesis in human fibroblasts and rat hepatocytes by activating adenylate cyclase, which induces protein kinase A (PKA) to phosphorylate serine-106 in Insig-2. Phosphorylated Insig-2 inhibits the proteolytic processing of SREBP-1, thereby blocking fatty acid synthesis. Phosphorylated Insig-2 does not block the processing of SREBP-2, which activates cholesterol synthesis. Insig-1 lacks serine-106 and is not phosphorylated at this site. EPA inhibition of SREBP-1 processing was reduced by the replacement of serine-106 in Insig-2 with alanine or by treatment with KT5720, a PKA inhibitor. Inhibition did not occur in mutant human fibroblasts that possess Insig-1 but lack Insig-2. These data provide an Insig-2-specific mechanism for the long-known inhibition of fatty acid synthesis by polyunsaturated fatty acids.
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Proteínas Quinasas Dependientes de AMP Cíclico , Fibroblastos , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana , Proteína 1 de Unión a los Elementos Reguladores de Esteroles , Humanos , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Animales , Fosforilación , Ratas , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Fibroblastos/metabolismo , Ácidos Grasos Insaturados/metabolismo , Ácidos Grasos/metabolismo , Ácidos Grasos/biosíntesis , Ácido Eicosapentaenoico/farmacología , Proteína 2 de Unión a Elementos Reguladores de Esteroles/metabolismo , Hepatocitos/metabolismoRESUMEN
Low-density lipoprotein (LDL) delivers cholesterol to mammalian cells through receptor-mediated endocytosis. The LDL cholesterol is liberated in lysosomes and transported to the plasma membrane (PM) and from there to the endoplasmic reticulum (ER). Excess ER cholesterol is esterified with a fatty acid for storage as cholesteryl esters. Recently, we showed that PM-to-ER transport of LDL cholesterol requires phosphatidylserine (PS). Others showed that PM-to-ER transport of cholesterol derived from other sources requires Asters (also called GRAMD1s), a family of three ER proteins that bridge between the ER and PM by binding to PS. Here, we use a cholesterol esterification assay and other measures of ER cholesterol delivery to demonstrate that Asters participate in PM-to-ER transport of LDL cholesterol in Chinese hamster ovary cells. Knockout of the gene encoding PTDSS1, the major PS-synthesizing enzyme, lowered LDL-stimulated cholesterol esterification by 85%, whereas knockout of all three Aster genes lowered esterification by 65%. The reduction was even greater (94%) when the genes encoding PTDSS1 and the three Asters were knocked out simultaneously. We conclude that Asters participate in LDL cholesterol delivery from PM to ER, and their action depends in large part, but not exclusively, on PS. The data also indicate that PS participates in another delivery pathway, so far undefined, that is independent of Asters.
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LDL-Colesterol/metabolismo , Proteínas de la Membrana/metabolismo , Fosfatidilserinas/metabolismo , Animales , Transporte Biológico , Células CHO , Membrana Celular/metabolismo , Colesterol/metabolismo , Ésteres del Colesterol/metabolismo , Cricetinae , Cricetulus , Endocitosis , Retículo Endoplásmico/metabolismo , Lisosomas/metabolismoRESUMEN
Phase separation of components of ER exit sites (ERES) into membraneless compartments, the Sec bodies, occurs in Drosophila cells upon exposure to specific cellular stressors, namely, salt stress and amino acid starvation, and their formation is linked to the early secretory pathway inhibition. Here, we show Sec bodies also form in secretory mammalian cells upon the same stress. These reversible and membraneless structures are positive for ERES components, including both Sec16A and Sec16B isoforms and COPII subunits. We find that Sec16A, but not Sec16B, is a driver for Sec body formation, and that the coalescence of ERES components into Sec bodies occurs by fusion. Finally, we show that the stress-induced coalescence of ERES components into Sec bodies precedes ER exit inhibition, leading to their progressive depletion from ERES that become non-functional. Stress relief causes an immediate dissolution of Sec bodies and the concomitant restoration of ER exit. We propose that the dynamic conversion between ERES and Sec body assembly, driven by Sec16A, regulates protein exit from the ER during stress and upon stress relief in mammalian cells, thus providing a conserved pro-survival mechanism in response to stress.
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Vesículas Cubiertas por Proteínas de Revestimiento , Retículo Endoplásmico , Animales , Retículo Endoplásmico/metabolismo , Vesículas Cubiertas por Proteínas de Revestimiento/metabolismo , Aparato de Golgi/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Vías Secretoras , Transporte de Proteínas , Mamíferos/metabolismoRESUMEN
The nanomechanical response of a cell depends on the frequency at which the cell is probed. The components of the cell that contribute to this property and their interplay are not well understood. Here, two force microscopy methods are integrated to characterize the frequency and/or the velocity-dependent properties of living cells. It is shown on HeLa and fibroblasts, that cells soften and fluidize upon increasing the frequency or the velocity of the deformation. This property was independent of the type and values (25 or 1000 nm) of the deformation. At low frequencies (2-10 Hz) or velocities (1-10 µm s-1 ), the response is dominated by the mechanical properties of the cell surface. At higher frequencies (>10 Hz) or velocities (>10 µm s-1 ), the response is dominated by the hydrodynamic drag of the cytosol. Softening and fluidization does not seem to involve any structural remodeling. It reflects a redistribution of the applied stress between the solid and liquid-like elements of the cell as the frequency or the velocity is changed. The data indicates that the quasistatic mechanical properties of a cell featuring a cytoskeleton pathology might be mimicked by the response of a non-pathological cell which is probed at a high frequency.
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Mamíferos , Fenómenos Mecánicos , Humanos , Animales , Módulo de Elasticidad , Microscopía de Fuerza Atómica , Células HeLa , Membrana CelularRESUMEN
In the biotechnology industry, ensuring the health and viability of mammalian cells, especially Chinese Hamster Ovary (CHO) cells, plays a significant role in the successful production of therapeutic agents. These cells are typically cultivated in aerated bioreactors, where they encounter fluid stressors from rapidly deforming bubbles. These stressors can disrupt essential biological processes and potentially lead to cell death. However, the impact of these transient, elevated stressors on cell viability remains elusive. In this study, we first employ /cgqamicrofluidics to expose CHO cells near to bubbles undergoing pinch-off, subsequently collecting and assaying the cells to quantify the reduction in viability. Observing a significant impact, we set out to understand this phenomenon. We leverage computational fluid dynamics and numerical particle tracking to map the stressor field history surrounding a rapidly deforming bubble. Separately, we expose CHO cells to a known stressor level in a flow constriction device, collecting and assaying the cells to quantify the reduction in viability. By integrating the numerical data and results from the flow constriction device experiments, we develop a predictive model for cell viability reduction. We validate this model by comparing its predictions to the earlier microfluidic results, observing good agreement. Our findings provide critical insights into the relationship between bubble-induced fluid stressors and mammalian cell viability, with implications for bioreactor design and cell culture protocol optimization in the biotechnology sector.
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Biotecnología , Microburbujas , Cricetinae , Animales , Cricetulus , Supervivencia Celular , Células CHO , Reactores BiológicosRESUMEN
Flavivirus virus-like particles (VLPs) exhibit a striking structural resemblance to viral particles, making them highly adaptable for various applications, including vaccines and diagnostics. Consequently, increasing VLPs production is important and can be achieved by optimizing expression plasmids and cell culture conditions. While attempting to express genotype III (GIII) Japanese encephalitis virus (JEV) VLPs containing the G104H mutation in the envelope (E) protein, we failed to generate VLPs in COS-1 cells. However, VLPs production was restored by cultivating plasmid-transfected cells at a lower temperature, specifically 28 °C. Furthermore, we observed that the enhancement in JEV VLPs production was independent of amino acid mutations in the E protein. The optimal condition for JEV VLPs production in plasmid-transfected COS-1 cells consisted of an initial culture at 37 °C for 6 h, followed by a shift to 28 °C (37/28 °C) for cultivation. Under 37/28 °C cultivation conditions, flavivirus VLPs production significantly increased in various mammalian cell lines regardless of whether its expression was transiently transfected or clonally selected cells. Remarkably, clonally selected cell lines expressing flavivirus VLPs consistently achieved yields exceeding 1 µg/ml. Binding affinity analyses using monoclonal antibodies revealed similar binding patterns for VLPs of genotype I (GI) JEV, GIII JEV, West Nile virus (WNV), and dengue virus serotype 2 (DENV-2) produced under both 37 °C or 37/28 °C cultivation conditions. In summary, our study demonstrated that the production of flavivirus VLPs can be significantly improved under 37/28 °C cultivation conditions without affecting the conformational structure of the E protein. KEYPOINTS: ⢠Low-temperature culture (37/28 °C) enhances production of flavivirus VLPs. ⢠Flavivirus VLPs consistently achieved yields exceeding 1 µg/ml. ⢠37/28 °C cultivation did not alter the structure of flavivirus VLPs.
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Virus de la Encefalitis Japonesa (Especie) , Encefalitis Japonesa , Flavivirus , Chlorocebus aethiops , Animales , Flavivirus/genética , Temperatura , Virus de la Encefalitis Japonesa (Especie)/genética , Frío , Células COS , MamíferosRESUMEN
Clustered, regularly interspaced, short palindromic repeat (CRISPR)/CRISPR-associated protein (CRISPR/Cas) system has revolutionized genetic research in the life sciences. Four classes of CRISPR/Cas-derived genome editing agents, such as nuclease, base editor, recombinase, and prime editor have been introduced for engineering the genomes of diverse organisms. The recently introduced prime editing system offers precise editing without many off-target effects than traditional CRISPR-based systems. Many researchers have successfully applied this gene-editing toolbox in diverse systems for various genome-editing applications. This review presents the mechanism of prime editing and summarizes the details of the prime editing system applied in plants and mammalian cells for precise genome editing. We also discuss the advantages, limitations, and potential future applications of prime editing in these systems. This review enables the researcher to gain knowledge on prime editing tools and their potential applications in plants and mammalian cells.
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Sistemas CRISPR-Cas , Edición Génica , Animales , Sistemas CRISPR-Cas/genética , Endonucleasas , Genoma , Mamíferos/genética , Plantas/genéticaRESUMEN
Kemerovo virus (KEMV) is a tick-borne orbivirus transmitted by ticks of the genus Ixodes. Previous animal experimentation studies with orbiviruses, in particular the interferon receptor double knock-out (IFNAR(-/-)) mouse model, did not indicate bias that is related to age or sex. We endeavoured to assess the effect of serial and alternated passages of KEMV in mammalian or Ixodes cells on virus replication and potential virulence in male or female IFNAR(-/-) mice, with important age differences: younger males (4-5 months old), older males (14-15 months old), and old females (14-15 months old). After 30 serial passages in mammalian or tick cells, or alternated passages in the two cell types, older female mice which were inoculated with the resulting virus strains were the first to show clinical signs and die. Younger males behaved differently from older males whether they were inoculated with the parental strain of KEMV or with any of the cell culture-passaged strains. The groups of male and female mice inoculated with the mammalian cell culture-adapted KEMV showed the lowest viraemia. While older female and younger male mice died by day 6 post-inoculation, surprisingly, the older males survived until the end of the experiment, which lasted 10 days. RNA extracted from blood and organs of the various mice was tested by probe-based KEMV real-time RT-PCR. Ct values of the RNA extracts were comparable between older females and younger males, while the values for older males were >5 Ct units higher for the various organs, indicating lower levels of replication. It is noteworthy that the hearts of the old males were the only organs that were negative for KEMV RNA. These results suggest, for the first time, an intriguing age- and sex-related bias for an orbivirus in this animal model. Changes in the amino acid sequence of the RNA-dependent RNA polymerase of Kemerovo virus, derived from the first serial passage in Ixodes cells (KEMV Ps.IRE1), were identified in the vicinity of the active polymerase site. This finding suggests that selection of a subpopulation of KEMV with better replication fitness in tick cells occurred.
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Ixodes , Orbivirus , Animales , Femenino , Masculino , Ratones , Secuencia de Aminoácidos , Técnicas de Cultivo de Célula , Ixodes/genética , Mamíferos/genética , Orbivirus/genética , ARN Viral/genéticaRESUMEN
A mathematical model for the mammalian cell cycle is developed as a system of 13 coupled nonlinear ordinary differential equations. The variables and interactions included in the model are based on detailed consideration of available experimental data. A novel feature of the model is inclusion of cycle tasks such as origin licensing and initiation, nuclear envelope breakdown and kinetochore attachment, and their interactions with controllers (molecular complexes involved in cycle control). Other key features are that the model is autonomous, except for a dependence on external growth factors; the variables are continuous in time, without instantaneous resets at phase boundaries; mechanisms to prevent rereplication are included; and cycle progression is independent of cell size. Eight variables represent cell cycle controllers: the Cyclin D1-Cdk4/6 complex, APCCdh1, SCFßTrCP, Cdc25A, MPF, NuMA, the securin-separase complex, and separase. Five variables represent task completion, with four for the status of origins and one for kinetochore attachment. The model predicts distinct behaviors corresponding to the main phases of the cell cycle, showing that the principal features of the mammalian cell cycle, including restriction point behavior, can be accounted for in a quantitative mechanistic way based on known interactions among cycle controllers and their coupling to tasks. The model is robust to parameter changes, in that cycling is maintained over at least a five-fold range of each parameter when varied individually. The model is suitable for exploring how extracellular factors affect cell cycle progression, including responses to metabolic conditions and to anti-cancer therapies.
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Proteínas de Ciclo Celular , Mamíferos , Animales , Separasa , Ciclo Celular/fisiología , Proteínas de Ciclo Celular/metabolismo , División Celular , Modelos TeóricosRESUMEN
As the potential applications of DNA diagnostics continue to expand, there is a need for improved methods and standards for DNA analysis. This report describes several methods that could be considered for the production of reference materials for the quantitative measurement of DNA damage in mammalian cells. With the focus on DNA strand breaks, potentially useful methods for assessing DNA damage in mammalian cells are reviewed. The advantages and limitations of each method, as well as additional concerns with respect to reference material development, are also discussed. In conclusion, we outline strategies for developing candidate DNA damage reference materials that could be adopted by research laboratories in a wide variety of applications.
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Daño del ADN , Reparación del ADN , Animales , ADN , Mamíferos/genéticaRESUMEN
For the production of recombinant protein therapeutics in mammalian cells, a high rate of gene expression is desired and hence strong viral-derived promoters are commonly used. However, they usually induce cellular stress and can be susceptible to epigenetic silencing. Endogenous promoters, which coordinates their activity with cellular and bioprocess dynamics while at the same time they maintain high expression levels, may help to avoid such drawbacks. In this work, new endogenous promoters were discovered based on high expression levels in RNA-seq data of CHO-K1 cells cultured in high density. The promoters of Actb, Ctsz, Hmox1, Hspa5, Vim and Rps18 genes were selected for generating new expression vectors for the production of recombinant proteins in mammalian cells. The in silico-derived promoter regions were experimentally verified and the majority showed transcriptional activity comparable or higher than CMV. Also, stable expression following a reduction of culture temperature was investigated. The characterized endogenous promoters (excluding Rps18) constitute a promising alternative to CMV promoter due to their high strength, long-term expression stability and integration into the regulatory network of the host cell. These promoters may also comprise an initial panel for designing cell engineering strategies and synthetic promoters, as well as for industrial cell line development.
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Técnicas de Cultivo de Célula , Infecciones por Citomegalovirus , Animales , Células CHO , Cricetinae , Mamíferos , Plásmidos , Regiones Promotoras Genéticas , Proteínas Recombinantes/genéticaRESUMEN
To generate stable cell lines that express high levels of recombinant genes often requires screening of a large number of transfected cells using ELISA. The most widely used alternative to ELISA screening is to use an intracellularly expressed GFP reporter construct which allows sorting of recombinant gene expression cells based on GFP fluorescence intensity. The disadvantage of cell sorting, however, is that the resulting population will be polyclonal with the danger of instability and overgrowth of low producers. In addition, GFP or its variants can be toxic to host cells at high concentrations, and thus may reduce growth and robustness of high producer cells or even cause them to become apoptotic. We have developed a new mammalian expression system in which a recombinant protein and a fluorescence protein, AcGFP1, are expressed on the same plasmid separated by an internal ribosome entry site (IRES). A signal peptide was incorporated upstream of AcGFP1 so that the fluorescent protein is secreted from cells, preventing cellular toxicity from intracellular accumulation and enabling convenient and accurate measurement of the protein. Expression tests of Ebola viral envelope GP1 and HIV gp120 proteins using this expression system in 293-H cells showed recombinant protein expression levels were closely correlated with AcGFP1 yield. Therefore, AcGFP1 can serve as an accurate reporter for recombinant protein expression and measuring AcGFP1 concentration provides a convenient, product independent and universal way for efficient clone screening.
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Expresión Génica , Proteínas Fluorescentes Verdes/genética , Sitios Internos de Entrada al Ribosoma , Proteínas Recombinantes/genética , Línea Celular , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Plásmidos/genética , Plásmidos/metabolismo , Proteínas Recombinantes/metabolismoRESUMEN
The technology of clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease Cas9 (CRISPR-Cas9) is a powerful system for protein depletion resulting from insertions and deletions following Cas9 cleavage of genome at specific site in vitro and in vivo. We herein present a relatively standard protocol for protein depletion in a step-by-step procedure, including guide RNA designation and vector construction, lentivirus production, cell selection, and experimentally validate the function of targeted protein. We exemplified this approach by editing PDGFRß in human epithelial cells, and expected that this simplified and detailed protocol will be more broadly applied on specific genes to aid understanding gene functions.
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Edición Génica , Sistemas CRISPR-Cas/genética , Endonucleasas , Genoma , Humanos , ARN Guía de Kinetoplastida/genéticaRESUMEN
Optimizing appropriate signal peptides in mammalian cell-based protein production is crucial given that most recombinant proteins produced in mammalian cells are thought to be secreted proteins. Until now, most studies on signal peptide in mammalian cells have replaced native signal peptides with well-known heterologous signal peptides and bioinformatics-based signal peptides. In the present study, we successfully established an in vitro screening system for synthetic signal peptide in CHO cells by combining a degenerate codon-based oligonucleotides library, a site-specific integration system, and a FACS-based antibody detection assay. Three new signal peptides were screened using this new screening system, confirming to have structural properties as signal peptides by the SignalP web server, a neural network-based algorithm that quantifies the signal peptide-ness of amino acid sequences. The novel signal peptides selected in this study increased Fc-fusion protein production in CHO cells by increasing specific protein productivity, whereas they did not negatively affect cell growth. Particularly, the SP-#149 clone showed the highest qp, 0.73 ± 0.01 pg/cell/day from day 1 to day 4, representing a 1.47-fold increase over the native signal peptide in a serum-free suspension culture mode. In addition, replacing native signal peptide with the novel signal peptides did not significantly affect sialylated N-glycan formation, N-terminal cleavage pattern, and biological function of Fc-fusion protein produced in CHO cells. The overall results indicate the utility of a novel in vitro screening system for synthetic signal peptide for mammalian cell-based protein production. KEY POINTS: ⢠An in vitro screening system for synthetic signal peptide in mammalian cells was established ⢠This system combined a degenerate codon-based library, site-specific integration, and a FACS-based detection assay ⢠The novel signal peptides selected in this study could increase Fc-fusion protein production in mammalian cells.
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Péptidos , Señales de Clasificación de Proteína , Animales , Células CHO , Cricetinae , Cricetulus , Péptidos/química , Péptidos/genética , Señales de Clasificación de Proteína/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Serological tests detect antibodies generated by infection or vaccination, and are indispensable tools along different phases of a pandemic, from early monitoring of pathogen spread up to seroepidemiological studies supporting immunization policies. This work discusses the development of an accurate and affordable COVID-19 antibody test, from production of a recombinant protein antigen up to test validation and economic analysis. We first developed a cost-effective, scalable technology to produce SARS-COV-2 spike protein and then used this antigen to develop an enzyme-linked immunosorbent assay (ELISA). A receiver operator characteristic (ROC) analysis allowed optimizing the cut-off and confirmed the high accuracy of the test: 98.6% specificity and 95% sensitivity for 11+ days after symptoms onset. We further showed that dried blood spots collected by finger pricking on simple test strips could replace conventional plasma/serum samples. A cost estimate was performed and revealed a final retail price in the range of one US dollar, reflecting the low cost of the ELISA test platform and the elimination of the need for venous blood sampling and refrigerated sample handling in clinical laboratories. The presented workflow can be completed in 4 months from first antigen expression to final test validation. It can be applied to other pathogens and in future pandemics, facilitating reliable and affordable seroepidemiological surveillance also in remote areas and in low-income countries.
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BACKGROUND: Resistance and tolerance are two coexisting defense strategies for fighting infections. Resistance is mediated by signaling pathways that induce transcriptional activation of resistance factors that directly eliminate the pathogen. Tolerance refers to adaptations that limit the health impact of a given pathogen burden, without targeting the infectious agent. The key players governing immune tolerance are largely unknown. In Drosophila, the histone H3 lysine 9 (H3K9) methyltransferase G9a was shown to mediate tolerance to virus infection and oxidative stress (OS), suggesting that abiotic stresses like OS may also evoke tolerance mechanisms. In response to both virus and OS, stress resistance genes were overinduced in Drosophila G9a mutants, suggesting an intact but overactive stress response. We recently demonstrated that G9a promotes tolerance to OS by maintaining metabolic homeostasis and safeguarding energy availability, but it remained unclear if this mechanism also applies to viral infection, or is conserved in other species and stress responses. To address these questions, we analyzed publicly available datasets from Drosophila, mouse, and human in which global gene expression levels were measured in G9a-depleted conditions and controls at different time points upon stress exposure. RESULTS: In all investigated datasets, G9a attenuates the transcriptional stress responses that confer resistance against the encountered stressor. Comparative analysis of conserved G9a-dependent stress response genes suggests that G9a is an intimate part of the design principles of stress resistance, buffering the induction of promiscuous stress signaling pathways and stress-specific resistance factors. Importantly, we find stress-dependent downregulation of metabolic genes to also be dependent on G9a across all of the tested datasets. CONCLUSIONS: These results suggest that G9a sets the balance between activation of resistance genes and maintaining metabolic homeostasis, thereby ensuring optimal organismal performance during exposure to diverse types of stress across different species. We therefore propose G9a as a potentially conserved master regulator underlying the widely important, yet poorly understood, concept of stress tolerance.
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Epigénesis Genética , Animales , Drosophila/genética , Drosophila/metabolismo , Epigenómica , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Ratones , Estrés Oxidativo/genética , Transcripción GenéticaRESUMEN
Cultured mammalian cells have been shown to respond to microgravity (µG), but the molecular mechanism is still unknown. The study we report here is focused on molecular and cellular events that occur within a short period of time, which may be related to gravity sensing by cells. Our assumption is that the gravity-sensing mechanism is activated as soon as cells are exposed to any new gravitational environment. To study the molecular events, we exposed cells to simulated µG (SµG) for 15 min, 30 min, 1 h, 2 h, 4 h, and 8 h using a three-dimensional clinostat and made cell lysates, which were then analyzed by reverse phase protein arrays (RPPAs) using a panel of 453 different antibodies. By comparing the RPPA data from cells cultured at 1G with those of cells under SµG, we identified a total of 35 proteomic changes in the SµG samples and found that 20 of these changes took place, mostly transiently, within 30 min. In the 4 h and 8 h samples, there were only two RPPA changes, suggesting that the physiology of these cells is practically indistinguishable from that of cells cultured at 1 G. Among the proteins involved in the early proteomic changes were those that regulate cell motility and cytoskeletal organization. To see whether changes in gravitational environment indeed activate cell motility, we flipped the culture dish upside down (directional change in gravity vector) and studied cell migration and actin cytoskeletal organization. We found that compared with cells grown right-side up, upside-down cells transiently lost stress fibers and rapidly developed lamellipodia, which was supported by increased activity of Ras-related C3 botulinum toxin substrate 1 (Rac1). The upside-down cells also increased their migratory activity. It is possible that these early molecular and cellular events play roles in gravity sensing by mammalian cells. Our study also indicated that these early responses are transient, suggesting that cells appear to adapt physiologically to a new gravitational environment.
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Actinas , Ingravidez , Actinas/metabolismo , Animales , Movimiento Celular , Células Cultivadas , Mamíferos/metabolismo , ProteómicaRESUMEN
Type V Cas12a nucleases are DNA editors working in a wide temperature range and using expanded protospacer-adjacent motifs (PAMs). Though they are widely used, there is still a demand for discovering new ones. Here, we demonstrate a novel ortholog from Ruminococcus bromii sp. entitled RbCas12a, which is able to efficiently cleave target DNA templates, using the particularly high accessibility of PAM 5'-YYN and a relatively wide temperature range from 20 °C to 42 °C. In comparison to Acidaminococcus sp. (AsCas12a) nuclease, RbCas12a is capable of processing DNA more efficiently, and can be active upon being charged by spacer-only RNA at lower concentrations in vitro. We show that the human-optimized RbCas12a nuclease is also active in mammalian cells, and can be applied for efficient deletion incorporation into the human genome. Given the advantageous properties of RbCas12a, this enzyme shows potential for clinical and biotechnological applications within the field of genome editing.
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Sistemas CRISPR-Cas , Endonucleasas , Acidaminococcus/genética , Acidaminococcus/metabolismo , Animales , ADN/metabolismo , Endonucleasas/metabolismo , Edición Génica , Humanos , Mamíferos/metabolismo , RuminococcusRESUMEN
Three-dimensional cell culture has become a reliable method for reproducing in vitro cellular growth in more realistic physiological conditions. The surface hydrophobicity strongly influences the promotion of cell aggregate formation. In particular, for spheroid formation, highly water-repellent coatings seem to be required for the significant effects of the process. In this work, surfaces at different wettability have been compared to observe their influence on the growth and promotion of aggregates of representative mammalian cell lines, both tumoral and non-tumoral (3T3, HaCat and MCF-7 cell lines). The effect of increased hydrophobicity from TCPS to agarose hydrogel to mixed organic-inorganic superhydrophobic (SH) coating has been investigated by optical and fluorescence microscopy, and by 3D confocal profilometry, in a time scale of 24 h. The results show the role of less wettable substrates in inducing the formation of spheroid-like cell aggregates at a higher degree of sphericity for the studied cell lines.