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Open Access Publications from the University of California

Scholarly Works (7 results)

  • Thesis
  • Peer Reviewed
UC Davis Electronic Theses and Dissertations (2024)

Demyelinating disorders, including multiple sclerosis and Canavan disease, afflict millions worldwide, yet their underlying mechanisms remain largely elusive. Our study on Canavan disease utilized single-cell spatial-temporal transcriptomics and mass spectrometry imaging. This approach enabled us to profile 37,083 genes at 500 nm resolution and 85 lipids at 10 μm resolution, offering detailed insights into the dysregulated genes and metabolites affecting signal transduction, osmotic homeostasis, vision, auditory function, and circadian rhythm. The Canavan brain lacks aspartoacylase; therefore, N-acetylaspartate (NAA) cannot be utilized to generate acetyl-CoA, an essential precursor for lipid synthesis. Our data demonstrate that the alternate route for acetyl-CoA production, mediated by ATP:citrate lyase in citrate cleavage, is also impaired, thus contributing to the deficit in myelin lipid synthesis. Additionally, this impairment results in a broad attenuation of gene expression due to histone hypoacetylation and disturbances in the tricarboxylic acid (TCA) cycle. Such disruptions could manifest as increased oxidative stress, widespread reduction in protein synthesis, and decreased ATP production. Notably, our research also revealed that oligodendrocytes are more abundant but less communicative with neurons in Canavan disease. To restore their function at an early stage, we delivered adeno-associated viruses (AAVs) intracerebroventricularly to fetal mice. These AAVs were engineered to express the ASPA gene in oligodendrocytes, significantly alleviating disease symptoms. Additionally, we introduced two new methods, BrainSegmenter and TransMetaSegmentation (TMS), to address challenges in brain cell segmentation and metabolite allocation, respectively. We anticipate that this study will advance the understanding and treatment of neurological and genetic disorders.

    • Article
    • Peer Reviewed
    UC Davis Previously Published Works (2023)
    Technologies assessing the lipidomics, genomics, epigenomics, transcriptomics, and proteomics of tissue samples at single-cell resolution have deepened our understanding of physiology and pathophysiology at an unprecedented level of detail. However, the study of single-cell spatial metabolomics in undecalcified bones faces several significant challenges, such as the fragility of bone, which often requires decalcification or fixation leading to the degradation or removal of lipids and other molecules. As such, we describe a method for performing mass spectrometry imaging on undecalcified spine that is compatible with other spatial omics measurements. In brief, we use fresh-frozen rat spines and a system of carboxyl methylcellulose embedding, cryofilm, and polytetrafluoroethylene rollers to maintain tissue integrity while avoiding signal loss from variations in laser focus and artifacts from traditional tissue processing. This reveals various tissue types and lipidomic profiles of spinal regions at 10 μm spatial resolutions using matrix-assisted laser desorption/ionization mass spectrometry imaging. We expect this method to be adapted and applied to the analysis of the spinal cord, shedding light on the mechanistic aspects of cellular heterogeneity, development, and disease pathogenesis underlying different bone-related conditions and diseases. This study furthers the methodology for high spatial metabolomics of spines and adds to the collective efforts to achieve a holistic understanding of diseases via single-cell spatial multiomics.
      Cover page: Sample Preparation Method for MALDI Mass Spectrometry Imaging of Fresh-Frozen Spines
      • Article
      • Peer Reviewed
      UC Davis Previously Published Works (2022)
      Human serum albumin (HSA) is the most abundant protein in human blood plasma. It plays a critical role in the native transportation of numerous drugs, metabolites, nutrients, and small molecules. HSA has been successfully used clinically as a noncovalent carrier for insulin (e.g., Levemir), GLP-1 (e.g., Liraglutide), and paclitaxel (e.g., Abraxane). Site-specific bioconjugation strategies for HSA only would greatly expand its role as the biocompatible, non-toxic platform for theranostics purposes. Using the enabling one-bead one-compound (OBOC) technology, we generated combinatorial peptide libraries containing myristic acid, a well-known binder to HSA at Sudlow I and II binding pockets, and an acrylamide. We then used HSA as a probe to screen the OBOC myristylated peptide libraries for reactive affinity elements (RAEs) that can specifically and covalently ligate to the lysine residue at the proximity of these pockets. Several RAEs have been identified and confirmed to be able to conjugate to HSA covalently. The conjugation can occur at physiological pH and proceed with a high yield within 1 h at room temperature. Tryptic peptide profiling of derivatized HSA has revealed two lysine residues (K225 and K414) as the conjugation sites, which is much more specific than the conventional lysine labeling strategy with N-hydroxysuccinimide ester. The RAE-driven site-specific ligation to HSA was found to occur even in the presence of other prevalent blood proteins such as immunoglobulin or whole serum. Furthermore, these RAEs are orthogonal to the maleimide-based conjugation strategy for Cys34 of HSA. Together, these attributes make the RAEs the promising leads to further develop in vitro and in vivo HSA bioconjugation strategies for numerous biomedical applications.
        Cover page: Site-Specific Albumin-Selective Ligation to Human Serum Albumin under Physiological Conditions
        • Article
        • Peer Reviewed
        UC Davis Previously Published Works (2023)
        Nanoparticle-based drug delivery systems have the potential to revolutionize medicine, but their low vascular permeability and rapid clearance by phagocytic cells have limited their medical impact. Nanoparticles delivered at the in utero stage can overcome these key limitations due to the high rate of angiogenesis and cell division in fetal tissue and the under-developed immune system. However, very little is known about nanoparticle drug delivery at the fetal stage of development. In this report, using Ai9 CRE reporter mice, we demonstrate that lipid nanoparticle (LNP) mRNA complexes can deliver mRNA in utero, and can access and transfect major organs, such as the heart, the liver, kidneys, lungs and the gastrointestinal tract with remarkable efficiency and low toxicity. In addition, at 4 weeks after birth, we demonstrate that 50.99 ± 5.05%, 36.62 ± 3.42% and 23.7 ± 3.21% of myofiber in the diaphragm, heart and skeletal muscle, respectively, were transfected. Finally, we show here that Cas9 mRNA and sgRNA complexed to LNPs were able to edit the fetal organs in utero. These experiments demonstrate the possibility of non-viral delivery of mRNA to organs outside of the liver in utero, which provides a promising strategy for treating a wide variety of devastating diseases before birth.
          Cover page: In utero delivery of mRNA to the heart, diaphragm and muscle with lipid nanoparticles
          • Article
          • Peer Reviewed
          UC Davis Previously Published Works (2023)
          Conventional approaches for screening anticancer drugs rely on chemical reactions, which are time consuming, labor intensive, and costly. Here, we present a protocol for label-free and high-throughput assessment of drug efficacy using a vision transformer and a Conv2D. We describe the steps for cell culture, drug treatment, data collection, and preprocessing. We then detail the building of deep learning models and their use to predict drug potency. This protocol can be adapted for screening chemicals that affect the density or morphological features of cells. For complete details on the use and execution of this protocol, please refer to Wang et al.1.
            Cover page: Protocol for vision transformer-based evaluation of drug potency using images processed by an optimized Sobel operator
            • Article
            • Peer Reviewed
            UC Davis Previously Published Works (2023)
            As a measure of cytotoxic potency, half-maximal inhibitory concentration (IC50) is the concentration at which a drug exerts half of its maximal inhibitory effect against target cells. It can be determined by various methods that require applying additional reagents or lysing the cells. Here, we describe a label-free Sobel-edge-based method, which we name SIC50, for the evaluation of IC50. SIC50 classifies preprocessed phase-contrast images with a state-of-the-art vision transformer and allows for the continuous assessment of IC50 in a faster and more cost-efficient manner. We have validated this method using four drugs and 1,536-well plates and also built a web application. We anticipate that this method will assist in the high-throughput screening of chemical libraries (e.g., small-molecule drugs, small interfering RNA [siRNA], and microRNA and drug discovery).
              Cover page: SIC50: Determining drug inhibitory concentrations using a vision transformer and an optimized Sobel operator
              • Article
              • Peer Reviewed
              UC Davis Previously Published Works (2022)
              Immune checkpoint blockade (ICB) therapy has revolutionized clinical oncology. However, the efficacy of ICB therapy is limited by the ineffective infiltration of T effector (Teff) cells to tumors and the immunosuppressive tumor microenvironment (TME). Here, we report a programmable tumor cells/Teff cells bispecific nano-immunoengager (NIE) that can circumvent these limitations to improve ICB therapy. The peptidic nanoparticles (NIE-NPs) bind tumor cell surface α3β1 integrin and undergo in situ transformation into nanofibrillar network nanofibers (NIE-NFs). The prolonged retained nanofibrillar network at the TME captures Teff cells via the activatable α4β1 integrin ligand and allows sustained release of resiquimod for immunomodulation. This bispecific NIE eliminates syngeneic 4T1 breast cancer and Lewis lung cancer models in mice, when given together with anti-PD-1 antibody. The in vivo structural transformation-based supramolecular bispecific NIE represents an innovative class of programmable receptor-mediated targeted immunotherapeutics to greatly enhance ICB therapy against cancers.
                Cover page: Programmable Bispecific Nano-immunoengager That Captures T Cells and Reprograms Tumor Microenvironment
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