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A distinct boundary exists between the progenitor cells in the basal limbal epithelium and the more differentiated corneal epithelial basal cells. We have shown that reciprocal expression patterns of EphA2 and Ephrin-A1 are likely to contribute to normal limbal-corneal epithelial compartmentalization as well as play a role in response to injury. How this signaling axis is regulated remains unclear. We have demonstrated that microRNAs (miRNAs) play critical roles in corneal epithelial wound healing and several miRNAs (e.g. miR-210) have been predicted to target Ephrins. Previous expression profiling experiments demonstrated that miR-210 is prominently expressed in corneal epithelial cells. RNA-seq data acquired from miR-210-depleted HCECs showed up-regulation of genes involved in cellular migration. In addition, miR-210 is decreased after corneal injury while EphA2 is increased. Moreover, antago-210-treated HCECs markedly enhanced wound closure in a scratch wound assay. Antago-210 treatment resulted in increased EphA2 protein levels as well as pS897-EphA2, the pro-migratory form of EphA2. As expected, Ephrin-A1 levels were reduced, while levels of a well-known target of miR-210, Ephrin-A3, were increased by antago-210 treatment. The increase in migration with antago-210 could be inhibited by Ephrin-A1 overexpression, Ephrin-A1-Fc treatment or siRNA depletion of EphA2. However, depletion of Ephrin-A3 did not have effects on the antago-210-induced increase in migration. In addition, Ephrin-A1 overexpression and siEphA2 dampened EGFR signaling, which is increased by antago-210. Our data clearly demonstrate a link between miR-210 and EphA2/Ephrin-A1 signaling that regulates, in part, corneal epithelial migration. This interaction might potentially control the limbal-corneal epithelial boundary.

Mycobacteria and related organisms in the Corynebacterineae suborder are characterized by a distinctive outer membrane referred to as the mycomembrane. Biosynthesis of the mycomembrane occurs through an essential process called mycoloylation, which involves antigen 85 (Ag85)-catalyzed transfer of mycolic acids from the mycoloyl donor trehalose monomycolate (TMM) to acceptor carbohydrates and, in some organisms, proteins. We recently described an alkyne-modified TMM analogue (O-AlkTMM-C7) which, in conjunction with click chemistry, acted as a chemical reporter for mycoloylation in intact cells and allowed metabolic labeling of mycoloylated components of the mycomembrane. Here, we describe the synthesis and evaluation of a toolbox of TMM-based reporters bearing alkyne, azide, trans-cyclooctene, and fluorescent tags. These compounds gave further insight into the substrate tolerance of mycoloyltransferases (e.g., Ag85s) in a cellular context and they provide significantly expanded experimental versatility by allowing one- or two-step cell labeling, live cell labeling, and rapid cell labeling via tetrazine ligation. Such capabilities will facilitate research on mycomembrane composition, biosynthesis, and dynamics. Moreover, because TMM is exclusively metabolized by Corynebacterineae, the described probes may be valuable for the specific detection and cell-surface engineering of Mycobacterium tuberculosis and related pathogens. We also performed experiments to establish the dependence of probe incorporation on mycoloyltransferase activity, results from which suggested that cellular labeling is a function not only of metabolic incorporation (and likely removal) pathway(s), but also accessibility across the envelope. Thus, whole-cell labeling experiments with TMM reporters should be carefully designed and interpreted when envelope permeability may be compromised. On the other hand, this property of TMM reporters can potentially be exploited as a convenient way to probe changes in envelope integrity and permeability, facilitating drug development studies.

Aggregates or oligomeric forms of many intrinsically disordered proteins (IDPs), including α-synuclein, are hallmarks of neurodegenerative diseases, like Parkinson's and Alzheimer's disease, and key contributors to their pathogenesis. Due to their disordered nature and therefore lack of defined drug-binding pockets, IDPs are difficult targets for traditional small molecule drug design and are often referred to as "undruggable". The 20S proteasome is the main protease that targets IDPs for degradation and therefore small molecule 20S proteasome enhancement presents a novel therapeutic strategy by which these undruggable IDPs could be targeted. The concept of 20S activation is still relatively new, with few potent activators having been identified thus far. Herein, we synthesized and evaluated a library of dihydroquinazoline analogues and discovered several promising new 20S proteasome activators. Further testing of top hits revealed that they can enhance 20S mediated degradation of α-synuclein, the IDP associated with Parkinson's disease.

Whereas much is known about the genes regulated by ΔNp63α in keratinocytes, how ΔNp63α is regulated is less clear. During studies with the hydroxylase, factor inhibiting hypoxia-inducible factor 1 (FIH-1), we observed increases in epidermal ΔNp63α expression along with proliferative capacity in a conditional FIH-1 transgenic mouse. Conversely, loss of FIH-1 in vivo and in vitro attenuated ΔNp63α expression. To elucidate the FIH-1/p63 relationship, BioID proteomics assays identified FIH-1 binding partners that had the potential to regulate p63 expression. FIH-1 interacts with two previously unknown partners, Plectin1 and signal transducer and activator of transcription 1 (STAT1) leading to the regulation of ΔNp63α expression. Two known interactors of FIH-1, apoptosis-stimulating of P53 protein 2 (ASPP2) and histone deacetylase 1 (HDAC1), were also identified. Knockdown of ASPP2 upregulated ΔNp63α and reversed the decrease in ΔNp63α by FIH-1 depletion. Additionally, FIH-1 regulates growth arrest and DNA damage-45 alpha (GADD45α), a negative regulator of ΔNp63α by interacting with HDAC1. GADD45α knockdown rescued reduction in ΔNp63α by FIH-1 depletion. Collectively, our data reveal that FIH-1 positively regulates ΔNp63α in keratinocytes via variety of signaling partners: (a) Plectin1/STAT1, (b) ASPP2, and (c) HDAC1/GADD45α signaling pathways.

Rod-shaped mycobacteria expand from their poles, yet d-amino acid probes label cell wall peptidoglycan in this genus at both the poles and sidewall. We sought to clarify the metabolic fates of these probes. Monopeptide incorporation was decreased by antibiotics that block peptidoglycan synthesis or l,d-transpeptidation and in an l,d-transpeptidase mutant. Dipeptides complemented defects in d-alanine synthesis or ligation and were present in lipid-linked peptidoglycan precursors. Characterizing probe uptake pathways allowed us to localize peptidoglycan metabolism with precision: monopeptide-marked l,d-transpeptidase remodeling and dipeptide-marked synthesis were coincident with mycomembrane metabolism at the poles, septum and sidewall. Fluorescent pencillin-marked d,d-transpeptidation around the cell perimeter further suggested that the mycobacterial sidewall is a site of cell wall assembly. While polar peptidoglycan synthesis was associated with cell elongation, sidewall synthesis responded to cell wall damage. Peptidoglycan editing along the sidewall may support cell wall robustness in pole-growing mycobacteria.

Trehalosamine (2-amino-2-deoxy-α,α-d-trehalose) is an aminoglycoside with antimicrobial activity against Mycobacterium tuberculosis, and it is also a versatile synthetic intermediate used to access imaging probes for mycobacteria. To overcome inefficient chemical synthesis approaches, we report a two-step chemoenzymatic synthesis of trehalosamine that features trehalose synthase (TreT)-catalyzed glycosylation as the key transformation. Soluble and recyclable immobilized forms of TreT were successfully employed. We demonstrate that chemoenzymatically synthesized trehalosamine can be elaborated to two complementary imaging probes, which label mycobacteria via distinct pathways.

Since late 2002, the National Oceanic and Atmospheric Administration's (NOAA) Office of Ocean Exploration and Research (OER) has been collaborating with the National Oceanographic Data Center (NODC) and its three divisions, the NOAA Central Library (NCL), the Marine Data Stewardship Division (MDSD), and the National Coastal Data Development Center (NCDDC) to address the requirements for archiving, preserving, managing, and providing online access to digital videos and still images from OER oceanographic expeditions. The Video Data Management System (VDMS), which was developed to facilitate managing online information and access to video and images obtained during NOAA-sponsored oceanographic expeditions, now enables access to hundreds of digital video clips, highlight movies, still images and related documents and products from OER expeditions. Using discovery tools available via NOAALINC, the NCL online catalog (http://www.lib.noaa.gov/), NOAA scientists and other researchers can discover and download online video and still images to deep sea coral ecosystems areas. Upon special arrangement, the NCL can provide equipment and an appropriate environment for users to view, copy and/or download requested off-line scientific video data or view original expedition tapes from the NOAA Library Archives. Additional online information includes related cruise reports, educational lesson plans, original video and image annotation documents, digital maps and Web sites, and links to other oceanographic observation data. During the development of the VDMS project plan, which is a part of a larger comprehensive OER Data Management Project, the VDMS team defined and established several 'best practices' to support OER video data management requirements. Metadata guidelines for digital video (DV12) and digital still images (DI12) help scientists and data managers in the field to create complete descriptive metadata about their image data. Scientists, librarians and archivists then use this information to create MARC21, FGDC, or Dublin Core metadata records. The VDMS team also developed a work-flow for managing digital video by defining the process for moving video data from ship to library to archive, including steps for creating archival backup copies and web-accessible video clips and highlights. The VDMS presently manages off-line access to more than 1500 MiniDV and 500 DVCAM tapes, over 1500 DVDs, and online access to more than 300 digital video clips and highlights collected during NOAA ocean exploration cruises. Over 80% of all digital video and image data are from OER expeditions to various deep-sea coral areas. A growing collection of the digital data, including in situ physical and chemical ocean observations are archived in NCL and NODC. In situ data are accessible through the search and retrieval functions of the NODC Ocean Archive System (OAS) at http://www.nodc.noaa.gov/Archive/Search/.