Soojin Yoon

Discovery of Hepatitis C Virus NS3 Helicase Inhibitors by a Multiplexed, High‐Throughput Helicase Activity Assay Based on Graphene Oxide

A GO‐to solution: A simple graphene oxide (GO)‐based assay to screen for selective inhibitors of a hepatitis C virus (HCV) helicase along with inhibitors of a severe acute respiratory syndrome coronavirus (SARS CoV) helicase was tested (see scheme). A single screen found five inhibitors highly selective for the HCV helicase orthologous to the SARS CoV helicase. Some of these hits were validated using the same GO‐based assay.WILEY-VCH

Exosomal proteins in the aqueous humor as novel biomarkers in patients with neovascular age-related macular degeneration.

Age-related macular degeneration (AMD) describes the progressive degeneration of the retinal pigment epithelium (RPE), retina, and choriocapillaris and is the leading cause of blindness in people over 50. The molecular mechanisms underlying this multifactorial disease remain largely unknown. To uncover novel secretory biomarkers related to the pathogenesis of AMD, we adopted an integrated approach to compare the proteins identified in the conditioned medium (CM) of cultured RPE cells and the exosomes derived from CM and from the aqueous humor (AH) of AMD patients by LC-ESI-MS/MS. Finally, LC-MRM was performed on the AH from patients and controls, which revealed that cathepsin D, cytokeratin 8, and four other proteins increased in the AH of AMD patients. The present study has identified potential biomarkers and therapeutic targets for AMD treatment, such as proteins related to the autophagy-lysosomal pathway and epithelial-mesenchymal transition, and demonstrated a novel and effective approach to identifying AMD-associated proteins that might be secreted by RPE in vivo in the form of exosomes. The proteomics-based characterization of this multifactorial disease could help to match a particular marker to particular target-based therapy in AMD patients with various phenotypes.

Autophagy and KRT8/keratin 8 protect degeneration of retinal pigment epithelium under oxidative stress

ABSTRACT Contribution of autophagy and regulation of related proteins to the degeneration of retinal pigment epithelium (RPE) in age-related macular degeneration (AMD) remain unknown. We report that upregulation of KRT8 (keratin 8) as well as its phosphorylation are accompanied with autophagy and attenuated with the inhibition of autophagy in RPE cells under oxidative stress. KRT8 appears to have a dual role in RPE pathophysiology. While increased expression of KRT8 following autophagy provides a cytoprotective role in RPE, phosphorylation of KRT8 induces pathologic epithelial-mesenchymal transition (EMT) of RPE cells under oxidative stress, which is mediated by MAPK1/ERK2 (mitogen-activated protein kinase 1) and MAPK3/ERK1. Inhibition of autophagy further promotes EMT, which can be reversed by inhibition of MAPK. Thus, regulated enhancement of autophagy with concurrent increased expression of KRT8 and the inhibition of KRT8 phosphorylation serve to inhibit oxidative stress-induced EMT of RPE cells as well as to prevent cell death, suggesting that pharmacological manipulation of KRT8 upregulation through autophagy with combined inhibition of the MAPK1/3 pathway may be attractive therapeutic strategies for the treatment of AMD.

Characterization of bacteriophage ϕPto-bp6g, a novel phage that lyses Pseudomonas tolaasii causing brown blotch disease in mushrooms

The bacteriophage, φPto-bp6g, exhibited strong bactericidal activity against Pseudomonas tolaasii, the bacterium that causes brown blotch disease in cultivated mushrooms. Analysis of phage morphology with an electron microscope revealed that φPto-bp6g contains an icosahedral head and a long tail, which is classified as the family of Siphoviridae. The phage was observed to lyse P. tolaasii in the broth about 4h after inoculation, indicating a putative lytic pathway exists during bacterial growth. The whole genome of φPto-bp6g was completely sequenced, with a length of 26,499 bp and a G+C content of 42.7%. A total of 77 open reading frames (ORFs) as putative coding sequences were identified and annotated, whereas 43 ORFs possessed no homologs. Proteins of several ORFs showed similarity with proteins of a diverse group of phages, including Siphoviridae (5 ORFs), Myoviridae (11 ORFs), and Podoviridae (4 ORFs). Phage proteins were grouped into three categories based on their predicted functions: (i) DNA replication and nucleotide metabolism, (ii) phage particle formation, and (iii) host interaction. Since there is no identified gene encoding integrase and toxins in phage genome, phage φPto-bp6g could be potentially applicable as a safe biological control reagent against brown blotch disease in mushroom cultivation.

Site‐specific cleavage of mutant ABL mRNA by DNAzyme is facilitated by peptide nucleic acid binding to RNA substrate

RNA‐cleaving DNAzymes were constructed to target the point mutation in the BCR–ABL transcript that causes imatinib resistance in leukemic cells. We examined the effect of 12mer peptide nucleic acids (PNAs) as facilitator oligonucleotides that bind to RNA substrate at the termini of the DNAzyme to improve DNAzyme‐mediated cleavage of full‐length RNA. When imatinib‐resistant cells were transfected with the facilitator PNA and DNAzyme, DNAzyme activity was enhanced and the cells were sensitized to imatinib treatment. Thus, facilitator PNA may be used to enhance activity of antisense oligonucleotide targeting the full‐length transcript.