CHO microRNA engineering is growing up : recent successes and future challenges

microRNAs with their ability to regulate complex pathways that control cellular behavior and phenotype have been proposed as potential targets for cell engineering in the context of optimization of biopharmaceutical production cell lines, specifically of Chinese Hamster Ovary cells. However, until recently, research was limited by a lack of genomic sequence information on this industrially important cell line. With the publication of the genomic sequence and other relevant data sets for CHO cells since 2011, the doors have been opened for an improved understanding of CHO cell physiology and for the development of the necessary tools for novel engineering strategies. In the present review we discuss both knowledge on the regulatory mechanisms of microRNAs obtained from other biological models and proof of concepts already performed on CHO cells, thus providing an outlook of potential applications of microRNA engineering in production cell lines

The effect of collagen organization on tensile strength loss in anterior cruciate ligament grafts post-reconstruction surgery

Gemstone Team LEGSGrafts used for anterior cruciate ligament (ACL) reconstructions fall short of restoring native mechanics. This study investigated a morphological cause for tension loss by comparing native ACL and two common grafts, bone-patellar tendonbone (BPTB) and semitendinosus/gracilis hamstring tendon (ST/G), in a cadaveric system. Tension loss during continuous passive motion was quantified via force transducer. Microstructural changes were assessed by measuring collagen crimp angles. No significant differences were found for rates of percent tension loss relative to total tension loss among grafts. However, all groups displayed exponential decay, implying rapid tension loss. The crimp angles for the unstressed grafts were significantly different from each other, suggesting innate differences. The percent change experienced by stressed grafts, normalized to their unstressed baselines, showed that ST’s crimp behavior was significantly different from that of ACL and BPTB, implying the BPTB graft is superior for ACL reconstruction because it better mimics the ACL’s morphological behavior

Evidence-based Kernels: Fundamental Units of Behavioral Influence

This paper describes evidence-based kernels, fundamental units of behavioral influence that appear to underlie effective prevention and treatment for children, adults, and families. A kernel is a behavior–influence procedure shown through experimental analysis to affect a specific behavior and that is indivisible in the sense that removing any of its components would render it inert. Existing evidence shows that a variety of kernels can influence behavior in context, and some evidence suggests that frequent use or sufficient use of some kernels may produce longer lasting behavioral shifts. The analysis of kernels could contribute to an empirically based theory of behavioral influence, augment existing prevention or treatment efforts, facilitate the dissemination of effective prevention and treatment practices, clarify the active ingredients in existing interventions, and contribute to efficiently developing interventions that are more effective. Kernels involve one or more of the following mechanisms of behavior influence: reinforcement, altering antecedents, changing verbal relational responding, or changing physiological states directly. The paper describes 52 of these kernels, and details practical, theoretical, and research implications, including calling for a national database of kernels that influence human behavior

Elucidation of the CHO Super-Ome (CHO-SO) by Proteoinformatics

Chinese hamster ovary (CHO) cells are the preferred host cell line for manufacturing a variety of complex biotherapeutic drugs including monoclonal antibodies. We performed a proteomics and bioinformatics analysis on the spent medium from adherent CHO cells. Supernatant from CHO-K1 culture was collected and subjected to in-solution digestion followed by LC/LC–MS/MS analysis, which allowed the identification of 3281 different host cell proteins (HCPs). To functionally categorize them, we applied multiple bioinformatics tools to the proteins identified in our study including SignalP, TargetP, SecretomeP, TMHMM, WoLF PSORT, and Phobius. This analysis provided information on the presence of signal peptides, transmembrane domains, and cellular localization and showed that both secreted and intracellular proteins were constituents of the supernatant. Identified proteins were shown to be localized to the secretory pathway including ones playing roles in cell growth, proliferation, and folding as well as those involved in protein degradation and removal. After combining proteins predicted to be secreted or having a signal peptide, we identified 1015 proteins, which we termed as CHO supernatant-ome (CHO-SO), or superome. As a part of this effort, we created a publically accessible web-based tool called GO–CHO to functionally categorize proteins found in CHO-SO and identify enriched molecular functions, biological processes, and cellular components. We also used a tool to evaluate the immunogenicity potential of high-abundance HCPs. Among enriched functions were catalytic activity and structural constituents of the cytoskeleton. Various transport related biological processes, such as vesicle mediated transport, were found to be highly enriched. Extracellular space and vesicular exosome associated proteins were found to be the most enriched cellular components. The superome also contained proteins secreted from both classical and nonclassical secretory pathways. The work and database described in our study will enable the CHO community to rapidly identify high-abundance HCPs in their cultures and therefore help assess process and purification methods used in the production of biologic drugs

Lessons from the Hamster: <i>Cricetulus griseus</i> Tissue and CHO Cell Line Proteome Comparison

Chinese hamster ovary cells represent the dominant host for therapeutic recombinant protein production. However, few large-scale data sets have been generated to characterize this host organism and derived CHO cell lines at the proteomics level. Consequently, an extensive label-free quantitative proteomics analysis of two cell lines (CHO-S and CHO DG44) and two Chinese hamster tissues (liver and ovary) was used to identify a total of 11 801 unique proteins containing at least two unique peptides. 9359 unique proteins were identified specifically in the cell lines, representing a 56% increase over previous work. Additionally, 6663 unique proteins were identified across liver and ovary tissues, providing the first Chinese hamster tissue proteome. Protein expression was more conserved within cell lines during both growth phases than across cell lines, suggesting large genetic differences across cell lines. Overall, both gene ontology and KEGG pathway analysis revealed enrichment of cell-cycle activity in cells. In contrast, upregulated molecular functions in tissue include glycosylation and lipid transporter activity. Furthermore, cellular components including Golgi apparatus are upregulated in both tissues. In conclusion, this large-scale proteomics analysis enables us to delineate specific changes between tissues and cells derived from these tissues, which can help explain specific tissue function and the adaptations cells incur for applications in biopharmaceutical productions

In Sickness and in Health: The Co-Regulation of Inflammation and Social Behavior

Although it has commonly been assumed that the immune system and the processes that govern social behavior are separate, non-communicating entities, research over the past several decades suggests otherwise. Considerable evidence now shows that inflammatory processes and social behavior are actually powerful regulators of one another. This review first summarizes evidence that inflammatory processes regulate social behavior, leading to characteristic changes that may help an individual navigate the social environment during times of sickness. Specifically, this review shows that inflammation: (1) increases threat-related neural sensitivity to negative social experiences (eg, rejection, negative social feedback), presumably to enhance sensitivity to threats to well-being or safety in order to avoid them and (2) enhances reward-related neural sensitivity to positive social experiences (eg, viewing close others and receiving positive social feedback), presumably to increase approach-related motivation towards others who might provide support and care during sickness. Next, this review summarizes evidence showing that social behavior also regulates aspects of inflammatory activity, preparing the body for situations in which wounding and infection may be more likely (social isolation). Here, we review research showing: (1) that exposure to social stressors increases proinflammatory activity, (2) that individuals who are more socially isolated (ie, lonely) show increased proinflammatory activity, and (3) that individuals who are more socially isolated show increased proinflammatory activity in response to an inflammatory challenge or social stressor. The implications of the co-regulation of inflammation and social behavior are discussed