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Open Access Publications from the University of California

Scholarly Works (17 results)

  • Article
  • Peer Reviewed
Issues in Applied Linguistics, Volume 21 (2020)

This paper builds on and extends Seedhouse (2004)’s study on conversational interaction and second language (L2) learning in formal pedagogical contexts through a longitudinal investigation of repair. Theoretically, the project engages with concepts of repair and language learning within the field of Conversation Analysis (CA) and attempts to re-examine the relationship between L2 repair and L2 classroom contexts proposed by Seedhouse. Methodologically, the research employs conversation analytic approach to L2 spoken data and as a departure from the traditional CA approach, it incooperates quantitative analysis as well as the researcher’s field notes and interviews to explore the complexities of L2 repair in terms of its sequential organization overtime.

The findings supported Seedhouse in that L2 repair is sequenced differently in accordance with the pedagogic goals set by the teacher. More importantly, this study adds to the previous research in that the learners orientated to achieving L2 accuracy in all pedagogic contexts regardless of the initial pedagogic focus.

    Cover page: A Longitudinal Study of Sequential Organization of L2 Repair in Classroom Contexts
    • Article
    • Peer Reviewed
    UCLA Previously Published Works (2018)
    Quiescence is a temporary, reversible state in which cells have ceased cell division, but retain the capacity to proliferate. Multiple studies, including ours, have demonstrated that quiescence is associated with widespread changes in gene expression. Some of these changes occur through changes in the level or activity of proliferation-associated transcription factors, such as E2F and MYC. We have demonstrated that mRNA decay can also contribute to changes in gene expression between proliferating and quiescent cells. In this protocol, we describe the procedure for establishing proliferating and quiescent cultures of human dermal foreskin fibroblasts. We then describe the procedures for inhibiting new transcription in proliferating and quiescent cells with Actinomycin D (ActD). ActD treatment represents a straightforward and reproducible approach to dissociating new transcription from transcript decay. A disadvantage of ActD treatment is that the time course must be limited to a short time frame because ActD affects cell viability. Transcript levels are monitored over time to determine transcript decay rates. This procedure allows for the identification of genes and isoforms that exhibit differential decay in proliferating versus quiescent fibroblasts.
      Cover page: Determining Genome-wide Transcript Decay Rates in Proliferating and Quiescent Human Fibroblasts.
      • Article
      • Peer Reviewed
      UCLA Previously Published Works (2020)
      Cell migration is essential for normal development, neural patterning, pathogen eradication, and cancer metastasis. Pre-mRNA processing events such as alternative splicing and alternative polyadenylation result in greater transcript and protein diversity as well as function and activity. A critical role for alternative pre-mRNA processing in cell migration has emerged in axon outgrowth during neuronal development, immune cell migration, and cancer metastasis. These findings suggest that migratory signals result in expression changes of post-translational modifications of splicing or polyadenylation factors, leading to splicing events that generate promigratory isoforms. We summarize this recent progress and suggest emerging technologies that may facilitate a deeper understanding of the role of alternative splicing and polyadenylation in cell migration.
        Cover page: Splicing Busts a Move: Isoform Switching Regulates Migration
        • Thesis
        • Peer Reviewed
        UCLA Electronic Theses and Dissertations (2019)

        For many types of animal cells, migration is essential for their physiological function. Long distance crawling is required for the development of a nervous system and for the ability of macrophages to accumulate at sites of infection. In response to a wound, resident fibroblasts migrate to the wound site and promote the wound healing process. Furthermore, cancer metastasis involves the ability of cancer cells to transform their morphology and acquire an ability to migrate. A better understanding of cell migration would have implications for normal development and could yield therapies for multiple different disease states, including fibrosis, atherosclerosis, and metastasis.

        Alternative polyadenylation (APA) is the process by which the same genetic locus can produce multiple transcripts ending at different polyadenylation sites. Coding sequence APA can change the length of the amino acid and can also switch the function of the protein. Proliferating cells, cancer cells, and stem cells in the early stages of mouse development and tumors tend to use proximal polyadenylation sites and commonly show rapid cell migration. Our laboratory has discovered that fibroblast migration can be regulated through polyadenylation site selection. To date, however, no studies have been performed to research how cell migration and invasion can be controlled by APA.

        Reversion-inducing-cysteine-rich protein with kazal-like motifs (RECK) suppresses cell migration. Previous studies have documented a role for RECK on the cell surface in inhibiting MMP activity via its Kazal-like motifs. RECK has also been implicated in tubulin dynamics and in anterior-posterior polarity. Our lab discovered through an RNA-Seq-based screen that there are short and long RECK isoforms that are generated by coding region alternative polyadenylation.

        In this dissertation, I explain how cell migration and invasion can be controlled by APA via alternative isoform expression of the RECK protein. I show that the short RECK isoform promotes cell migration and that this result is mediated by short RECK and long (canonical) RECK interaction. Furthermore, I propose that RECK APA can regulate cell migration not only through its established role of modifying matrix metalloproteinases but also via its roles in affecting tubulin post-translational modifications (PTMs). These studies provide a much more detailed and mechanistic model for the biological functions of RECK alternative isoform use.

          Cover page: To Define the Mechanistic Basis for the Effect of RECK Alternative Isoform Expression on Cell Migration
          • Article
          • Peer Reviewed
          UCLA Previously Published Works (2019)
          Cell migration is essential for proper development and the defense against pathogens. Our previous work detailed a pathway of REversion-inducing-Cysteine-rich protein with Kazal motifs (RECK) isoform-mediated invasion in which a shorter RECK protein competes with MMP9 for interaction with the canonical RECK protein on the cell surface. Here we demonstrate that the mechanism through which RECK isoforms affect cell migration is mediated through changes in the levels of post-translational modifications (PTM) of α-tubulin. We show that both the canonical and short RECK isoforms modulate levels of tubulin acetylation and detyrosination. We demonstrate that these changes are sufficient to modulate the rate of fibroblast migration. If these tubulin PTMs are not altered, the effects of the canonical RECK isoform on cell migration are reversed. In defining the molecular pathway linking RECK and tubulin PTMs, we found that MMP9 and integrin activity both act as upstream regulators of tubulin acetylation and detyrosination. Overall, we propose a mechanism in which RECK isoforms on the cell surface have opposing effects on cell migration through MMP9-modulated changes to integrin-extracellular matrix (ECM) interactions that, in turn, affect microtubule PTMs.
            Cover page: RECK isoforms differentially regulate fibroblast migration by modulating tubulin post-translational modifications
            • Article
            • Peer Reviewed
            UC San Diego Previously Published Works (2020)
            Cells rely on a complex network of spatiotemporally regulated signaling activities to effectively transduce information from extracellular cues to intracellular machinery. To probe this activity architecture, researchers have developed an extensive molecular tool kit of fluorescent biosensors and optogenetic actuators capable of monitoring and manipulating various signaling activities with high spatiotemporal precision. The goal of this review is to provide readers with an overview of basic concepts and recent advances in the development and application of genetically encodable biosensors and optogenetic tools for understanding signaling activity.
              Cover page: Recent advances in the use of genetically encodable optical tools to elicit and monitor signaling events
              • Article
              • Peer Reviewed
              UC Davis Previously Published Works (2018)
              Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that causes serious infections in immunocompromised individuals and cystic fibrosis patients. ExoS and ExoT are two homologous bifunctional Type III Secretion System (T3SS) virulence factors that induce apoptosis in target host cells. They possess a GTPase Activating Protein (GAP) domain at their N-termini, which share ~76% homology, and an ADP-ribosyltransferase (ADPRT) domain at their C-termini, which target non-overlapping substrates. Both the GAP and the ADPRT domains contribute to ExoTs cytotoxicity in target epithelial cells, whereas, ExoS-induced apoptosis is reported to be primarily due to its ADPRT domain. In this report, we demonstrate that ExoS/GAP domain is both necessary and sufficient to induce mitochondrial apoptosis. Our data demonstrate that intoxication with ExoS/GAP domain leads to enrichment of Bax and Bim into the mitochondrial outer-membrane, disruption of mitochondrial membrane and release of and cytochrome c into the cytosol, which activates initiator caspase-9 and effector caspase-3, that executes cellular death. We posit that the contribution of the GAP domain in ExoS-induced apoptosis was overlooked in prior studies due to its slower kinetics of cytotoxicity as compared to ADPRT. Our data clarify the field and reveal a novel virulence function for ExoS/GAP as an inducer of apoptosis.
                Cover page: Pseudomonas aeruginosa ExoS Induces Intrinsic Apoptosis in Target Host Cells in a Manner That is Dependent on its GAP Domain Activity.
                • Article
                • Peer Reviewed
                UC San Diego Previously Published Works (2018)
                Daily PrEP is highly effective at preventing HIV-1 acquisition, but risks of long-term tenofovir disoproxil fumarate plus emtricitabine (TDF-FTC) include renal decline and bone mineral density decrease in addition to initial gastrointestinal side effects. We investigated the impact of TDF-FTC on the enteric microbiome using rectal swabs collected from healthy MSM before PrEP initiation and after 48 to 72 weeks of adherent PrEP use. The V4 region of the 16S ribosomal RNA gene sequencing showed that Streptococcus was significantly reduced from 12.0% to 1.2% (p = 0.036) and Erysipelotrichaceae family was significantly increased from 0.79% to 3.3% (p = 0.028) after 48-72 weeks of daily PrEP. Catenibacterium mitsuokai, Holdemanella biformis and Turicibacter sanguinis were increased within the Erysipelotrichaceae family and Streptococcus agalactiae, Streptococcus oralis, Streptococcus mitis were reduced. These changes were not associated with host factors including PrEP duration, age, race, tenofovir diphosphate blood level, any drug use and drug abuse, suggesting that the observed microbiome shifts were likely induced by daily PrEP use. Long-term PrEP resulted in increases of Catenibacterium mitsuokai and Holdemanella biformis, which have been associated with gut microbiome dysbiosis. Our observations can aid in characterizing PrEP's side effects, which is likely to improve PrEP adherence, and thus HIV-1 prevention.
                  Cover page: Daily HIV pre-exposure prophylaxis (PrEP) with tenofovir disoproxil fumarate-emtricitabine reduced Streptococcus and increased Erysipelotrichaceae in rectal microbiota
                  • Article
                  • Peer Reviewed
                  UC Davis Previously Published Works (2020)
                  The atypical trichromatic cyanobacterial phytochrome NpTP1 from Nostoc punctiforme ATCC 29133 is a linear tetrapyrrole (bilin)-binding photoreceptor protein that possesses tandem-cysteine residues responsible for shifting its light-sensing maximum to the violet spectral region. Using bioinformatics and phylogenetic analyses, here we established that tandem-cysteine cyanobacterial phytochromes (TCCPs) compose a well-supported monophyletic phytochrome lineage distinct from prototypical red/far-red cyanobacterial phytochromes. To investigate the light-sensing diversity of this family, we compared the spectroscopic properties of NpTP1 (here renamed NpTCCP) with those of three phylogenetically diverged TCCPs identified in the draft genomes of Tolypothrix sp. PCC7910, Scytonema sp. PCC10023, and Gloeocapsa sp. PCC7513. Recombinant photosensory core modules of ToTCCP, ScTCCP, and GlTCCP exhibited violet-blue-absorbing dark-states consistent with dual thioether-linked phycocyanobilin (PCB) chromophores. Photoexcitation generated singly-linked photoproduct mixtures with variable ratios of yellow-orange and red-absorbing species. The photoproduct ratio was strongly influenced by pH and by mutagenesis of TCCP- and phytochrome-specific signature residues. Our experiments support the conclusion that both photoproduct species possess protonated 15E bilin chromophores, but differ in the ionization state of the noncanonical "second" cysteine sulfhydryl group. We found that the ionization state of this and other residues influences subsequent conformational change and downstream signal transmission. We also show that tandem-cysteine phytochromes present in eukaryotes possess similar amino acid substitutions within their chromophore-binding pocket, which tune their spectral properties in an analogous fashion. Taken together, our findings provide a roadmap for tailoring the wavelength specificity of plant phytochromes to optimize plant performance in diverse natural and artificial light environments.
                    Cover page: Spectral and photochemical diversity of tandem cysteine cyanobacterial phytochromes
                    • Article
                    • Peer Reviewed
                    UC San Diego Previously Published Works (2022)
                    G-protein-coupled receptors (GPCRs), the largest family of signalling receptors, as well as important drug targets, are known to activate extracellular-signal-regulated kinase (ERK)-a master regulator of cell proliferation and survival1. However, the precise mechanisms that underlie GPCR-mediated ERK activation are not clearly understood2-4. Here we investigated how spatially organized β2-adrenergic receptor (β2AR) signalling controls ERK. Using subcellularly targeted ERK activity biosensors5, we show that β2AR signalling induces ERK activity at endosomes, but not at the plasma membrane. This pool of ERK activity depends on active, endosome-localized Gαs and requires ligand-stimulated β2AR endocytosis. We further identify an endosomally localized non-canonical signalling axis comprising Gαs, RAF and mitogen-activated protein kinase kinase, resulting in endosomal ERK activity that propagates into the nucleus. Selective inhibition of endosomal β2AR and Gαs signalling blunted nuclear ERK activity, MYC gene expression and cell proliferation. These results reveal a non-canonical mechanism for the spatial regulation of ERK through GPCR signalling and identify a functionally important endosomal signalling axis.
                      Cover page: Non-canonical β-adrenergic activation of ERK at endosomes
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