Biomedical subjects

Eun Jeong Cho

Publications and source records attributed to Eun Jeong Cho.

9 recordsLinked to original sources

PubMed2026

Derivation and characterization of ubiquitin-specific protease 18 inhibitors.

Ubiquitin-Specific Protease 18 (USP18) is a deISGylation enzyme and antineoplastic target. To develop USP18 inhibitors, an enzymatically active human recombinant USP18 protein was engineered suitable for high-throughput screening of ~80,000 chemical compounds. Three of them substantially inhibited USP18 enzymatic activity, with β-lapachone having prominent antineoplastic activity. Independent β-lapachone treatments of murine and human lung cancer cell lines statistically significantly reduced proliferation and increased apoptosis. Gain of USP18 expression antagonized these effects. β-Lapachone treatments statistically significantly repressed lung cancer xenograft growth. β-Lapachone increased reactive oxygen species (ROS), but antineoplastic effects occurred at dosages with negligible ROS production. ROS scavenger treatments did not rescue β-lapachone effects at these concentrations, consistent with an ROS-independent mechanism. IFN-Stimulated Response Element (ISRE) reporter assays following β-lapachone treatment activated this reporter. USP18 cotransfection antagonized this activity. β-Lapachone treatments increased global ISGylation. RNA-seq of lung cancer cells engineered with or without enhanced USP18 expression showed specific pathways affected by β-lapachone treatment. Proteomic analysis of these treated cells revealed known and new ISGylated proteins. In silico modeling identified a unique USP18 pocket where these USP18 inhibitors bind. Engineered mutation of this pocket disrupted β-lapachone activity. Taken together, β-lapachone is an antineoplastic tool compound useful for USP18 inhibitor development.

Humans↗

PubMed2006

Optimization of the biological component of a bioelectrochemical cell.

The efficiency of electron transduction by Shewanella oneidensis MR-1 was investigated both in batch culture and in a dual-chambered electrochemical cell. Aerobically grown bacteria were inoculated into an insoluble FeOOH suspension in an anaerobic environment. As the bacteria reduced Fe(III) to Fe(II) there was a visible color change from red to bluish black; this simple color change assay proved to be a robust method for determining the electrochemical activity of S. oneidensis MR-1. In an effort to improve electricity production by S. oneidensis MR-1, the performance of the electrochemical cell with lactate as a fuel was first optimized with respect to both poised potential and fuel concentration. Ultimately, electricity production proved to be proportional to both the poised potential and to fuel concentration. In particular, higher poised potentials increased charge production. Finally, we attempted to optimize the bacteria themselves for the efficient transduction of reduced carbon sources into electricity. The batch culture underwent a series of serial dilutions; after the 4th dilution the microbe population exhibited a 30% increase in charge production. We are further exploring whether this increase was due to metabolic adaptations or to genetic mutations, and examining additional ways to evolve electrogenic organisms.

Anaerobiosis↗

PubMed2005

Production and processing of aptamer microarrays.

Aptamers are nucleic acid species that are selected in vitro for their specific binding properties. We describe methods for the production and processing of aptamer microarrays, including detailed procedures for the high-throughput, enzymatic synthesis of 5' RNA biotinylated aptamers and for arraying them onto streptavidin-coated glass slides. Also presented are methods for processing the aptamer microarrays, including blocking, washing, drying, and scanning. Examples are shown for the specific capture of fluorescently labeled target proteins either alone in binding buffer or in competition with labeled intracellular proteins from cell lysates. Consideration is given to the challenges involved in producing multiplex aptamer chips composed of aptamers taken from disparate literature sources, and to the development of standardized methods for characterizing the performance of capture reagents used in biosensors.

Aptamers, Nucleotide↗

PubMed2005

Functional RNA microarrays for high-throughput screening of antiprotein aptamers.

High-throughput methods for generating aptamer microarrays are described. As a proof-of-principle, the microarrays were used to screen the affinity and specificity of a pool of robotically selected antilysozyme RNA aptamers. Aptamers were transcribed in vitro in reactions supplemented with biotinyl-guanosine 5'-monophosphate, which led to the specific addition of a 5' biotin moiety, and then spotted on streptavidin-coated microarray slides. The aptamers captured target protein in a dose-dependent manner, with linear signal response ranges that covered seven orders of magnitude and a lower limit of detection of 1 pg/mL (70 fM). Aptamers on the microarray retained their specificity for target protein in the presence of a 10,000-fold (w/w) excess of T-4 cell lysate protein. The RNA aptamer microarrays performed comparably to current antibody microarrays and within the clinically relevant ranges of many disease biomarkers. These methods should also prove useful for generating other functional RNA microarrays, including arrays for genomic noncoding RNAs that bind proteins. Integrating RNA aptamer microarray production with the maturing technology for automated in vitro selection of antiprotein aptamers should result in the high-throughput production of proteome chips.

Base Sequence↗

PubMed2005

Using a deoxyribozyme ligase and rolling circle amplification to detect a non-nucleic acid analyte, ATP.

An allosteric ribozyme (aptazyme) has been used to transduce the binding of a small organic analyte (ATP) into the ligation of a circular template for rolling circle amplification (RCA). An ATP-activated deoxyribozyme ligase was immobilized on a glass slide and, upon addition of ATP, catalyzed the ligation of a circular padlock probe. The ligated products could be directly amplified and visualized via RCA. The coupled reaction exhibited could detect as little as 1 muM of ATP and could discriminate against structurally similar nucleotides such as GTP, CTP, and UTP. Cooperative ATP activation of the deoxyribozyme was faithfully mimicked by RCA, yielding an amplified "switch" that was responsive to ATP concentration.

Adenosine Triphosphate↗

PubMed2004

Aptamer-based sensor arrays for the detection and quantitation of proteins.

Aptamer biosensors have been immobilized on beads, introduced into micromachined chips on the electronic tongue sensor array, and used for the detection and quantitation of proteins. Aptamer chips could detect proteins in both capture and sandwich assay formats. Unlike most protein-based arrays, the aptamer chips could be stripped and reused multiple times. The aptamer chips proved to be useful for screening aptamers from in vitro selection experiments and for sensitively quantitating the biothreat agent ricin.

Base Sequence↗

PubMed2003

Tailored delivery of active keratinocyte growth factor from biodegradable polymer formulations.

We report the results of a high throughput screening campaign that is aimed to develop a biodegradable polymer-based formulation to deliver active keratinocyte growth factor (KGF) and provide a means to tune the KGF delivery rate. A statistical design strategy was used to prepare and screen a series of polymer blends that were composed of poly(lactic acid) (PLA), poly(glycolic acid) (PGA), and the surfactant sodium bis(ethylhexyl)sulfosuccinate (Aerosol-OT, AOT). Chloroform was the solvent. Our high throughput screening method used a two-tiered assessment strategy. At Level 1, we identified "lead" KFG-loaded formulations that exhibited KGF emission spectra that were the most similar to the native KGF spectrum recorded in buffer. At Level 2, we used steady-state emission and a homogeneous polarization immunoassay strategy to determine the concentration of total and active KGF, respectively, liberated from the lead formulations during biodegradation. After preparing and screening 2500 formulations, we identified several viable, lead formulations. An analysis of the data showed that the combination of PLA, PGA, and AOT were important to yield a high fraction of active KGF upon release from the formulation; no combination of any two together produced an effect as good as the ternary formulation. The optimum formulations that yielded the highest fraction of active KGF upon release had the following general features: PLA/PGA (w/w) near unity, AOT loading of 100-200 mM, water/AOT mole ratio of 10-20, and a pH between 6 and 8. PLA alone cast from chloroform delivered KGF, but that KGF did not bind to anti-KGF antibodies (i.e., it was inactive). We can tune the KGF release kinetics by more than two orders of magnitude while maintaining the KGF activity upon liberation from the formulation by adjusting the PLA molecular weight.

Biocompatible Materials↗

PubMed2002

Multianalyte pin-printed biosensor arrays based on protein-doped xerogels.

We report the first biosensor arrays based on pin printing protein-doped xerogels. The individual biosensor elements are on the order of 100 microm in diameter. Arrays are formed (1) onto a planar substrate that is excited by an external source (laser) or (2) directly on the face of a light-emitting diode. We illustrate the potential of our approach by fabricating, testing, and characterizing four types of pin-printed biosensor arrays (PPBSA) for the simultaneous detection of glucose and O2. The analytically reliable operating ranges for the PPBSAs are 0.1-10 mM for glucose and 0.1-100% for O2. The PPBSAs exhibit short- and long-term reproducibilities of no worse than 4 and 8%, respectively. The overall array-to-array response reproducibilities are < or = 12%. These results demonstrate for the first time the combination sol-gel processing and pin printing methods as a way to rapidly form ensembles of integrated, reusable, and stable biosensor arrays for simultaneous multianalyte detection.

Biosensing Techniques↗

PubMed2002

Pin-printed chemical sensor arrays for simultaneous multianalyte quantification.

A new approach to rapidly produce micrometer-scale sensor elements into reusable multianalyte chemical sensor arrays is demonstrated. By using pin printing technology in concert with sol-gel processing methods, we form discrete xerogel-based microsensors on a planar substrate. We illustrate the new approach by forming discrete 02- and pH-responsive sensing elements into arrays that allow one to simultaneously determine O2 and pH in aqueous samples. The pin printing method allows one to prepare sensor elements that are on the order of 100 microm in diameter, 1-2 microm thick, at a rate of approximately one sensor element per second with a single pin. Within a given calibrated array, the sensor element-to-sensor element response is reproducible to within 5%, the sensor element short- and long-term reproducibilities are 3 and 6%, respectively, and the array-to-array response reproducibility is 11%. These results demonstrate the potential of this methodology for rapidly forming ensembles of reusable sensor arrays for simultaneous multianalyte detection.

Journal Article↗