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Open Access Publications from the University of California

Scholarly Works (8 results)

  • Thesis
  • Peer Reviewed
UC Berkeley Electronic Theses and Dissertations (2012)

Synthetic biologists engineer genetic circuits for applications ranging from biosynthesis to biotherapeutics. Although the application of engineering strategies such as standardization, abstraction, and modularity has long been highlighted as the path to designing complex biological systems, early work generally relied on an ad hoc strategy, limiting applications to relatively simple systems. More recently, several groups have explicitly applied modular design to the development of biosynthetic pathways, biological computation, and increasingly sophisticated logic functions. However, connection of distinct functions to create a useful system-level behavior remains a key challenge. We assessed a possibility and limitation of a modular design when engineering a complex biological system.

By applying a modular design, we engineered E. coli to deliver macromolecules to the cytoplasm of cancer cells in vitro. Fabrication, testing, composition, and troubleshooting of five functional modules produced an efficient system capable of delivering proteins to over 80 % of targeted cancer cells. The modular design strategy enabled facile system modification for both troubleshooting and optimization. These devices were then mixed and matched to build a new type of delivery device that enabled E. coli to escape from the vacuole and secrete payloads. The delivery system was then further modified to deliver payloads to other eukaryotic organisms.

Successful application of modular design to the delivery system demonstrates that abstraction is a simple yet powerful tool for making the design of complex biological systems tractable. We expect that continued refinement of modular design, including incorporation of relevant quantitative information, will enable construction of increasingly complex systems. We envision that the bacterial delivery system developed here may itself become a high level module that can be incorporated into the design of more complex systems, such as a therapeutic bacterium delivering cancer-cell specific microRNA.

    Cover page: Protein Delivery to Eukaryotic Cells by Engineered Bacteria
    • Article
    • Peer Reviewed
    UC San Diego Previously Published Works (2021)
    Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease and is strongly associated with obesity-related ectopic fat accumulation in the liver. Hepatic lipid accumulation encompasses a histological spectrum ranging from simple steatosis to nonalcoholic steatohepatitis (NASH), which can progress to cirrhosis and hepatocellular carcinoma. Given that dysregulated hepatic lipid metabolism may be an onset factor in NAFLD, understanding how hepatic lipid metabolism is modulated in healthy subjects and which steps are dysregulated in NAFLD subjects is crucial to identify effective therapeutic targets. Additionally, hepatic inflammation is involved in chronic hepatocyte damage during NAFLD progression. As a key immune signaling hub that mediates NF-κB activation, the IκB kinase (IKK) complex, including IKKα, IKKβ, and IKKγ (NEMO), has been studied as a crucial regulator of the hepatic inflammatory response and hepatocyte survival. Notably, TANK-binding kinase 1 (TBK1), an IKK-related kinase, has recently been revealed as a potential link between hepatic inflammation and energy metabolism. Here, we review (1) the biochemical steps of hepatic lipid metabolism; (2) dysregulated lipid metabolism in obesity and NAFLD; and (3) the roles of IKKs and TBK1 in obesity and NAFLD.
      Cover page: Roles of IκB kinases and TANK-binding kinase 1 in hepatic lipid metabolism and nonalcoholic fatty liver disease
      • Article
      • Peer Reviewed
      UC San Diego Previously Published Works (2021)
      Macrophage proinflammatory activation is an important etiologic component of the development of insulin resistance and metabolic dysfunction in obesity. However, the underlying mechanisms are not clearly understood. Here, we demonstrate that a mitochondrial inner membrane protein, adenine nucleotide translocase 2 (ANT2), mediates proinflammatory activation of adipose tissue macrophages (ATMs) in obesity. Ant2 expression was increased in ATMs of obese mice compared with lean mice. Myeloid-specific ANT2-knockout (ANT2-MKO) mice showed decreased adipose tissue inflammation and improved insulin sensitivity and glucose tolerance in HFD/obesity. At the molecular level, we found that ANT2 mediates free fatty acid-induced mitochondrial permeability transition, leading to increased mitochondrial reactive oxygen species production and damage. In turn, this increased HIF-1α expression and NF-κB activation, leading to proinflammatory macrophage activation. Our results provide a previously unknown mechanism for how obesity induces proinflammatory activation of macrophages with propagation of low-grade chronic inflammation (metaflammation).
        Cover page: ANT2 drives proinflammatory macrophage activation in obesity
        • Article
        • Peer Reviewed
        UC Berkeley Previously Published Works (2019)
        The Arabidopsis DEMETER (DME) DNA glycosylase demethylates the maternal genome in the central cell prior to fertilization and is essential for seed viability. DME preferentially targets small transposons that flank coding genes, influencing their expression and initiating plant gene imprinting. DME also targets intergenic and heterochromatic regions, but how it is recruited to these differing chromatin landscapes is unknown. The C-terminal half of DME consists of 3 conserved regions required for catalysis in vitro. We show that this catalytic core guides active demethylation at endogenous targets, rescuing dme developmental and genomic hypermethylation phenotypes. However, without the N terminus, heterochromatin demethylation is significantly impeded, and abundant CG-methylated genic sequences are ectopically demethylated. Comparative analysis revealed that the conserved DME N-terminal domains are present only in flowering plants, whereas the domain architecture of DME-like proteins in nonvascular plants mainly resembles the catalytic core, suggesting that it might represent the ancestral form of the 5mC DNA glycosylase found in plant lineages. We propose a bipartite model for DME protein action and suggest that the DME N terminus was acquired late during land plant evolution to improve specificity and facilitate demethylation at heterochromatin targets.
          Cover page: The catalytic core of DEMETER guides active DNA demethylation in Arabidopsis
          • Article
          • Peer Reviewed
          UC San Diego Previously Published Works (2020)
          Hepatic TANK (TRAF family member associated NFκB activator)-binding kinase 1 (TBK1) activity is increased during obesity, and administration of a TBK1 inhibitor reduces fatty liver. Surprisingly, liver-specific TBK1 knockout in mice produces fatty liver by reducing fatty acid oxidation. TBK1 functions as a scaffolding protein to localize acyl-CoA synthetase long-chain family member 1 (ACSL1) to mitochondria, which generates acyl-CoAs that are channeled for β-oxidation. TBK1 is induced during fasting and maintained in the unphosphorylated, inactive state, enabling its high affinity binding to ACSL1 in mitochondria. In TBK1-deficient liver, ACSL1 is shifted to the endoplasmic reticulum to promote fatty acid re-esterification in lieu of oxidation in response to fasting, which accelerates hepatic lipid accumulation. The impaired fatty acid oxidation in TBK1-deficient hepatocytes is rescued by the expression of kinase-dead TBK1. Thus, TBK1 operates as a rheostat to direct the fate of fatty acids in hepatocytes, supporting oxidation when inactive during fasting and promoting re-esterification when activated during obesity.
            Cover page: TANK-Binding Kinase 1 Regulates the Localization of Acyl-CoA Synthetase ACSL1 to Control Hepatic Fatty Acid Oxidation
            • Article
            • Peer Reviewed
            UC San Diego Previously Published Works (2019)
            Decreased adipose tissue oxygen tension and increased HIF-1α expression can trigger adipose tissue inflammation and dysfunction in obesity. Our current understanding of obesity-associated decreased adipose tissue oxygen tension is mainly focused on changes in oxygen supply and angiogenesis. Here, we demonstrate that increased adipocyte O2 demand, mediated by ANT2 activity, is the dominant cause of adipocyte hypoxia. Deletion of adipocyte Ant2 improves obesity-induced intracellular adipocyte hypoxia by decreasing obesity-induced adipocyte oxygen demand, without effects on mitochondrial number or mass, or oligomycin-sensitive respiration. This led to decreased adipose tissue HIF-1α expression and inflammation with improved glucose tolerance and insulin resistance in both a preventative or therapeutic setting. Our results suggest that ANT2 may be a target for the development of insulin sensitizing drugs and that ANT2 inhibition might have clinical utility.
              Cover page: Knockdown of ANT2 reduces adipocyte hypoxia and improves insulin resistance in obesity
              • Article
              • Peer Reviewed
              UC San Francisco Previously Published Works (2024)
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                Cover page: Author Correction: Highly secreted tryptophanyl tRNA synthetase 1 as a potential theranostic target for hypercytokinemic severe sepsis.
                • Article
                • Peer Reviewed
                UC San Francisco Previously Published Works (2024)
                Despite intensive clinical and scientific efforts, the mortality rate of sepsis remains high due to the lack of precise biomarkers for patient stratification and therapeutic guidance. Secreted human tryptophanyl-tRNA synthetase 1 (WARS1), an endogenous ligand for Toll-like receptor (TLR) 2 and TLR4 against infection, activates the genes that signify the hyperinflammatory sepsis phenotype. High plasma WARS1 levels stratified the early death of critically ill patients with sepsis, along with elevated levels of cytokines, chemokines, and lactate, as well as increased numbers of absolute neutrophils and monocytes, and higher Sequential Organ Failure Assessment (SOFA) scores. These symptoms were recapitulated in severely ill septic mice with hypercytokinemia. Further, injection of WARS1 into mildly septic mice worsened morbidity and mortality. We created an anti-human WARS1-neutralizing antibody that suppresses proinflammatory cytokine expression in marmosets with endotoxemia. Administration of this antibody into severe septic mice attenuated cytokine storm, organ failure, and early mortality. With antibiotics, the antibody almost completely prevented fatalities. These data imply that blood-circulating WARS1-guided anti-WARS1 therapy may provide a novel theranostic strategy for life-threatening systemic hyperinflammatory sepsis.
                  Cover page: Highly secreted tryptophanyl tRNA synthetase 1 as a potential theranostic target for hypercytokinemic severe sepsis.
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