Department of Biological Sciences, KAIST, Daejeon, 34141, Republic of Korea. Electronic address:
Published: November 2023
AI Article Synopsis
Article Abstract
Chinese hamster ovary (CHO) cells are the preferred mammalian host cells for therapeutic protein production that have been extensively engineered to possess the desired attributes for high-yield protein production. However, empirical approaches for identifying novel engineering targets are laborious and time-consuming. Here, we established a genome-wide CRISPR/Cas9 screening platform for CHO-K1 cells with 111,651 guide RNAs (gRNAs) targeting 21,585 genes using a virus-free recombinase-mediated cassette exchange-based gRNA integration method. Using this platform, we performed a positive selection screening under hyperosmotic stress conditions and identified 180 genes whose perturbations conferred resistance to hyperosmotic stress in CHO cells. Functional enrichment analysis identified hyperosmotic stress responsive gene clusters, such as tRNA wobble uridine modification and signaling pathways associated with cell cycle arrest. Furthermore, we validated 32 top-scoring candidates and observed a high rate of hit confirmation, demonstrating the potential of the screening platform. Knockout of the novel target genes, Zfr and Pnp, in monoclonal antibody (mAb)-producing recombinant CHO (rCHO) cells and bispecific antibody (bsAb)-producing rCHO cells enhanced their resistance to hyperosmotic stress, thereby improving mAb and bsAb production. Overall, the collective findings demonstrate the value of the screening platform as a powerful tool to investigate the functions of genes associated with hyperosmotic stress and to discover novel targets for rational cell engineering on a genome-wide scale in CHO cells.
Purpose: The purpose of this study was to investigate the potential roles of endoplasmic reticulum (ER) stress in the development of dry eye disease (DED).
Methods: Single-cell RNA sequencing (scRNA-seq) data from the Gene Expression Omnibus (GEO) database, derived from corneal tissues of a dry eye mouse model, was processed using the Seurat R program. The results were validated using a scopolamine-induced dry eye mouse model and a hyperosmotic-induced cell model involving primary human corneal epithelial cells (HCECs) and immortalized human corneal epithelial (HCE-2) cells.
Deciphering how natural selection emerges from demographic differences among genotypes, and reciprocally how evolution affects population dynamics, is key to understanding population responses to environmental stress. This is especially true in non-trivial ecological scenarios, such as programmed cell death (PCD) in unicellular organisms, which can lead to massive population decline in response to stress. To understand how selection may operate on this trait, we exposed monocultures and mixtures of two closely related strains of the microalga , one of which induces PCD, to multiple cycles of hyper-osmotic shocks, and tracked demography and selection throughout.
NmrA homologs have been reported as conserved regulators of the nitrogen metabolite repression (NMR) in various fungi. Here, we identified a NmrA homolog in and reported its functions in nitrogen utilization, growth and development, and pathogenesis. VdNmrA interacts with AreA protein and regulates the expression of a typical NCR target, the formamidase gene.
Institut de Neurociències, Departament de Bioquímica i Biologia Molecular, Unitat de Bioquímica, Facultad de Medicina, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Barcelona, Spain. Electronic address:
Hyperosmotic shock induces cytochrome c release and caspase-3 activation in Xenopus oocytes. Different signaling pathways engaged by osmostress converge on the mitochondria to trigger cell death. The mitogen activated protein kinases (MAPKs) JNK1-1 and JNK1-2 are early activated by hyperosmotic shock and sustained activation of both isoforms accelerates the apoptotic program.
Actin homeostasis is crucial for maintaining cell structure and is energy-intensive, with glycolytic enzymes playing a role in its interaction with cellular metabolism.
Hyperosmotic stress causes actin disorganization and promotes the condensation of tropomyosin 4 (TPM4), which then gathers important glycolytic enzymes and energy molecules like NADH and ATP.
This study shows that the condensation of TPM4 and glycolytic enzymes supports actin assembly under stress, indicating a link between energy production and cytoskeletal changes in cells.
