Browsing by Author "Wei, Fang"

Now showing 1 - 4 of 4
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    I. Charged pair hydrogen bonding interactions in collagen heterotrimers. II. Surface enhanced Raman spectroscopy of aromatic peptides
    (2010) Wei, Fang; Hartgerink, Jeffrey D.
    Eight ABC heterotrimers whose self-assembly are directed through electrostatic interactions were studied here. Oppositely charged pairs of amino acids, with varying side chain length, were assessed for their ability to stabilize a triple helix. Aspartate-lysine was found to result in the most thermally stable helix followed by lysine-glutamate, ornithine-aspartate and finally ornithine-glutamate. When the sequence position of these charged amino acids was reversed from what is normally observed in nature, triple helix stability and compositional purity was significantly reduced. The effect of salt on triple helix stability was explored and it was observed that increased salt concentration reduces the thermal stability of heterotrimers by an average of 5°C, but does not disrupt helix assembly. It was also found that positively charged homotrimers can be stabilized in the presence of phosphate anions. Raman and Surface-enhanced Raman spectroscopies (SERS) are potentially important tools in the characterization of biomolecules such as proteins and DNA. In this work, SERS spectra of three cysteine containing aromatic peptides: tryptophan-cysteine, tyrosine-cysteine, and phenylalanine-cysteine, bound to Au nanoshell substrates, were obtained and compared to their respective normal Raman spectra. While the full widths at half maximum of the SERS peaks are significantly broadened (up to 70%), no significant spectral shifts (<6 cm-1) of the major Stokes modes were observed between the two modalities. It is shown that the Raman and SERS spectra of penetratin, a cell-penetrating peptide, can be evaluated quite reliably from the spectra of its constituent aromatic amino acids except in the -CH2- bending and amide I and III regions where the spectral intensities are critically dependent on the chain length and/or protein conformations. From this study we conclude that with aromatic amino acid residues provide the overwhelmingly dominant features in the Raman and SERS spectra of peptides and proteins, and that the Raman modes of these three small constructed peptides can apply directly to the assignment of Raman and SERS features in the spectra of larger peptides and proteins.
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    Random Insertion of mCherry Into VP3 Domain of Adeno- associated Virus Yields Fluorescent Capsids With no Loss of Infectivity
    (American Society of Gene & Cell Therapy, 2012) Judd, Justin; Wei, Fang; Nguyen, Peter Q.; Tartaglia, Lawrence J.; Agbandje-McKenna, Mavis; Silberg, Jonathan J.; Suh, Junghae
    Adeno-associated virus (AAV)-derived vectors are promising gene delivery systems, and a number of design strategies have been pursued to improve their performance. For example, genetic insertion of proteins into the capsid may be used to achieve vector retargeting, reduced immunogenicity, or to track vector transport. Unfortunately, rational approaches to genetic insertion have experienced limited success due to the unpredictable context-dependent nature of protein folding and the complexity of the capsid's macroassembly. We report the construction and use of a frame-enriched DNase-based random insertion library based on AAV2 cap, called pAAV2_RaPID (Random Peptide Insertion by DNase). The fluorescent mCherry protein was inserted randomly throughout the AAV2 capsid and the library was selected for fluorescent and infectious variants. A capsid site was identified in VP3 that can tolerate the large protein insertion. In contrast to previous efforts to incorporate fluorescent proteins into the AAV2 capsid, the isolated mCherry mutant maintains native infectivity while displaying robust fluorescence. Collectively, these results demonstrate that the pAAV2_RaPID platform library can be used to create fully infectious AAV vectors carrying large functional protein domains on the capsid.
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    Structure-Dependent Thermal Defunctionalization of Single-Walled Carbon Nanotubes
    (American Chemical Society, 2015) Ghosh, Saunab; Wei, Fang; Bachilo, Sergei M.; Hauge, Robert H.; Billups, W.E.; Weisman, R. Bruce; Smalley Institute for Nanoscale Science and Technology
    Covalent sidewall functionalization of single-walled carbon nanotubes (SWCNTs) is an important tool for tailoring their properties for research purposes and applications. In this study, SWCNT samples were first functionalized by reductive alkylation using metallic lithium and 1-iodododecane in liquid ammonia. Samples of the alkyl-functionalized SWCNTs were then pyrolyzed under an inert atmosphere at selected temperatures between 100 and 500 °C to remove the addends. The extent of defunctionalization was assessed using a combination of thermogravimetric analysis, Raman measurements of the D, G, and radial breathing bands, absorption spectroscopy of the first- and second-order van Hove peaks, and near-IR fluorescence spectroscopy of (n,m)-specific emission bands. These measurements all indicate a substantial dependence of defunctionalization rate on nanotube diameter, with larger diameter nanotubes showing more facile loss of addends. The effective activation energy for defunctionalization is estimated to be a factor of ∼1.44 greater for 0.76 nm diameter nanotubes as compared to those with 1.24 nm diameter. The experimental findings also reveal the quantitative variation with functionalization density of the Raman D/G intensity ratio and the relative near-IR fluorescence intensity. Pyrolyzed samples show spectroscopic properties that are equivalent to those of SWCNTs prior to functionalization. The strong structure dependence of the defunctionalization rate suggests an approach for scalable diameter sorting of mixed SWCNT samples.
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    The Liquid Biopsy Consortium: Challenges and opportunities for early cancer detection and monitoring
    (Elsevier, 2023) Batool, Syeda Maheen; Yekula, Anudeep; Khanna, Prerna; Hsia, Tiffaney; Gamblin, Austin S.; Ekanayake, Emil; Escobedo, Ana K.; You, Dong Gil; Castro, Cesar M.; Im, Hyungsoon; Kilic, Tugba; Garlin, Michelle Andrea; Skog, Johan; Dinulescu, Daniela M.; Dudley, Jonathan; Agrawal, Nishant; Cheng, Jordan; Abtin, Fereidoun; Aberle, Denise R.; Chia, David; Elashoff, David; Grognan, Tristan; Krysan, Kostyantyn; Oh, Scott S.; Strom, Charles; Tu, Michael; Wei, Fang; Xian, Rena R.; Skates, Steven J.; Zhang, David Y.; Trinh, Thi; Watson, Mark; Aft, Rebecca; Rawal, Siddarth; Agarwal, Ashutosh; Kesmodel, Susan B.; Yang, Changhuei; Shen, Cheng; Hochberg, Fred H.; Wong, David T. W.; Patel, Abhijit A.; Papadopoulos, Nickolas; Bettegowda, Chetan; Cote, Richard J.; Srivastava, Sudhir; Lee, Hakho; Carter, Bob S.; Balaj, Leonora
    The emerging field of liquid biopsy stands at the forefront of novel diagnostic strategies for cancer and other diseases. Liquid biopsy allows minimally invasive molecular characterization of cancers for diagnosis, patient stratification to therapy, and longitudinal monitoring. Liquid biopsy strategies include detection and monitoring of circulating tumor cells, cell-free DNA, and extracellular vesicles. In this review, we address the current understanding and the role of existing liquid-biopsy-based modalities in cancer diagnostics and monitoring. We specifically focus on the technical and clinical challenges associated with liquid biopsy and biomarker development being addressed by the Liquid Biopsy Consortium, established through the National Cancer Institute. The Liquid Biopsy Consortium has developed new methods/assays and validated existing methods/technologies to capture and characterize tumor-derived circulating cargo, as well as addressed existing challenges and provided recommendations for advancing biomarker assays.