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Gutierrez, J. M.

Publications and source records attributed to Gutierrez, J. M..

24 records · Page 2Linked to original sources

Changes in subjective preference do not require dopamine signaling or the orbital frontal cortex

Sunk or unrecoverable costs impact proximal reward-based decisions across species. However, it is not known if these incurred costs elicit a long-lasting change in reward value. To address this, we identified the relative preference between different flavored food pellets in rats. Animals were then trained to experience the initially preferred reward after short delays and the initially less preferred reward after long delays. This training regimen enhanced the preference for the initially less desirable food reward. We probed whether this change in subjective preference involved dopamine signaling or the orbital frontal cortex (OFC) given that these neural systems contribute to reward valuation. Systemic dopamine receptor antagonism attenuated anticipatory responding during training sessions but did not prevent the change in reward preference elicited by incurred temporal costs. OFC lesions had no effect on anticipatory responding during training or on the change in reward preference. These findings collectively illustrate that the neural systems involved with economic assessments of reward value are not contributing to changes in subjective preference.

neuroscience↗

Multiplex secretome engineering enhances recombinant protein production and purity

Host cell proteins (HCPs) are process-related impurities generated during biotherapeutic protein production. HCPs can be problematic if they pose a significant metabolic demand, degrade product quality, or contaminate the final product. Here, we present an effort to create a \"clean\" Chinese hamster ovary (CHO) cell by disrupting multiple genes to eliminate HCPs. Using a model of CHO cell protein secretion, we predicted the elimination of unnecessary HCPs could have a non-negligible impact on protein production. We analyzed the total HCP content of 6-protein, 11-protein, and 14-protein knockout clones and characterized their growth in shake flasks and bioreactors. These cell lines exhibited a substantial reduction in total HCP content (40%-70%). We also observed higher productivity and improved growth characteristics, in specific clones. With the reduced HCP content, protein A and ion exchange chromatography more efficiently purified a monoclonal antibody (mAb) produced in these cells during a three-step purification process. Thus, substantial improvements can be made in protein titer and purity through large-scale HCP deletion, providing an avenue to increased quality and affordability of high-value biopharmaceuticals.

bioengineering↗

The human secretome - the proteins secreted from human cells

The proteins secreted by human tissues (the secretome) are important for the basic understanding of human biology, but also for identification of potential targets for future diagnosis and therapy. Here, we present an annotation of all predicted secreted proteins (n=2,623) with information about their spatial distribution in the human body. A high-throughput mammalian cell factory was established to create a resource of recombinant full-length proteins. This resource was used for phenotypic assays involving {beta}-cell dedifferentiation and for development of targeted proteomics assays. A comparison between host cells, including omics analysis, shows that many of the proteins that failed to be generated in CHO cells could be rescued in human HEK293 cells. In conclusion, the human secretome has been mapped and characterized to facilitate further exploration of the human secretome.

molecular biology↗

Deficient skeletal muscle regeneration after injury induced by a Clostridium perfringens strain associated with gas gangrene

Very little is known about the muscle regeneration process that follows myonecrosis induced by C. perfringens, the main agent of gas gangrene. This study revealed that, in a murine model of the infection with a sublethal inoculum of C. perfringens, muscle necrosis occurs concomitantly with significant vascular damage, which limits the migration of inflammatory cells. A significant increase in cytokines that promote inflammation explains the presence of inflammatory infiltrate; however, an impaired IFN{gamma} expression, a reduced number of Ml macrophages, a deficient phagocytic activity, and the prolongation of the permanence of inflammatory cells, lead to deficient muscle regeneration. The expression of TGF{beta}1 and the consequent accumulation of collagen in the muscle, likely contribute to the fibrosis observed 30 days after infection. These results provide new information on the pathogenesis of gas gangrene caused by C. perfringens, shed light on the basis of the poor muscle regenerative activity, and may open new perspectives for the development of novel therapies for patients suffering this disease.

microbiology↗

Combating viral contaminants in CHO cells by engineering STAT1 mediated innate immunity

Viral contamination in biopharmaceutical manufacturing can lead to shortages in the supply of critical therapeutics. To facilitate the protection of bioprocesses, we explored the basis for the susceptibility of CHO cells, the most commonly used cell line in biomanufacturing, to RNA virus infection. Upon infection with certain ssRNA and dsRNA viruses, CHO cells fail to generate a significant interferon (IFN) response. Nonetheless, the downstream machinery for generating IFN responses and its antiviral activity is intact in these cells: treatment of cells with exogenously-added type I IFN or poly I:C prior to infection limited the cytopathic effect from Vesicular stomatitis virus (VSV), Encephalomyocarditis virus (EMCV), and Reovirus-3 virus (Reo-3) in a STAT1-dependent manner. To harness the intrinsic antiviral mechanism, we used RNA-Seq to identify two upstream repressors of STAT1: Gfi1 and Trim24. By knocking out these genes, the engineered CHO cells exhibited increased resistance to the prototype RNA viruses tested. Thus, omics-guided engineering of mammalian cell culture can be deployed to increase safety in biotherapeutic protein production among many other biomedical applications.

bioengineering↗

Genome-scale reconstructions of the mammalian secretory pathway predict metabolic costs and limitations of protein secretion

In mammalian cells, >25% of synthesized proteins are exported through the secretory pathway. The pathway complexity, however, obfuscates its impact on the secretion of different proteins. Unraveling its impact on diverse proteins is particularly important for biopharmaceutical production. Here we delineate the core secretory pathway functions and integrate them with genome-scale metabolic reconstructions of human, mouse, and Chinese hamster cells. The resulting reconstructions enable the computation of energetic costs and machinery demands of each secreted protein. By integrating additional omics data, we find that highly secretory cells have adapted to reduce expression and secretion of other expensive host cell proteins. Furthermore, we predict metabolic costs and maximum productivities of biotherapeutic proteins and identify protein features that most significantly impact protein secretion. Finally, the model successfully predicts the increase in secretion of a monoclonal antibody after silencing a highly expressed selection marker. This work represents a knowledgebase of the mammalian secretory pathway that serves as a novel tool for systems biotechnology.

systems biology↗