Biology subjects

Clapper, J. C.

Publications and source records attributed to Clapper, J. C..

5 recordsLinked to original sources

bioRxiv2022

Expression and functional evaluation of recombinant human ABO blood group antigen cleaving glycoside hydrolases α-N-acetylgalactosaminidase, α-galactosidase, and endo-β-galactosidase produced in Escherichia coli

Blood transfusions are an integral component of healthcare; however, availability of viable blood is limited by patient-donor blood type specificity, which contributes to seasonal shortages as well as shortages worldwide, especially in developing countries, and during pandemics or natural disasters. Attempts to increase blood supply with commercial incentives have raised ethical concerns, and current proposed artificial blood substitutes are unable to fully replicate the function of native red blood cells (RBCs). In this study, we explore the potential strategy of alleviating blood shortages through enzymatic conversion of A, B, and AB blood types to blood type O. In theory, this process eliminates ABO patient-donor incompatibility, which increases the supply of universal donor blood. Three glycoside hydrolases, -N-acetylgalactosaminidase, -galactosidase, and endo-{beta}-galactosidase, were selected to act as molecular scissors to cleave terminal residues on A and B RBC surface antigens and catalyze the conversion process. These enzymes were recombinantly expressed in BL21(DE3) Escherichia coli and purified through nickel ion affinity chromatography. A combination of colorimetric substrate assays, thin-layer chromatography, and mass spectroscopy were utilized to evaluate enzyme functionality. Enzyme efficiency was modeled using Michaelis-Menten kinetics. Partial enzymatic A-to-O blood type conversion on porcine red blood cells was observed with slide agglutination tests. Results confirm recombinant enzyme-mediated blood type conversion as a potential strategy for alleviating blood shortages.

biochemistry↗

bioRxiv2021

The Effects of Phenolic Compounds Isolated from Chinese Herbal Medicine Rhodiola Rosea on Preventing Photoaging of Keratinocytes

Among the many health benefits Chinese herbal medicine (CHM) presents, anti-aging is of special interest (Shen, Jiang, Yang, Wang, & Zhu, 2017; Zhao & Luo, 2017). Reported to possess anti-aging effects, the CHM Rhodiola rosea L., known colloquially as the "golden root", has been widely incorporated in various drinks, daily supplements, and even cosmetics. This study investigates the effects of commercial R. rosea extracts and natural R. rosea roots on preventing UV-induced photoaging of the skin and correlates such effects with the composition of active ingredients in the extracts. To simulate the photoaging process, drug treated HaCaT cells were exposed to UVA and UVB radiation. The pharmacological anti-aging effects of R. rosea extracts were evaluated qualitatively and quantitatively through confocal immunofluorescence images with {gamma}-H2A.X marker and telomerase activity assay (Telo TAGGG Telomerase PCR-ELISA). Preparatory thin layer chromatography and high-performance liquid chromatography were performed to isolate and quantify active ingredients. Cultured HaCaT cells showed morphological change after exposure to both UVA (>15.0 J) and UVB (>2000 mJ). The photoaging of keratinocytes was rescued by pretreating cells with R. rosea extracts as well as salidroside and rosavin active ingredients (P < 0.05). R. rosea-treated cells were characterized by increased telomerase activity and fewer {gamma}-H2A.X foci compared to that of the control. Extracts with better preventative effects contained higher salidroside and rosavin content. The findings in this study reaffirm R. roseas efficacy as an anti-aging remedy and provide a basis for CHMs integration into the mainstream of global healthcare.

biochemistry↗

bioRxiv2020

Room Temperature Isothermal Colorimetric Padlock Probe Rolling Circle Amplification for Viral RNA Detection

Seasonal flu and pandemics, which account for millions of infections and hundreds of thousands of deaths, require rapid and reliable detection mechanisms to implement preventive and therapeutic measures. Current detection methods of viral infections have limitations in speed, accuracy, accessibility, and usability. This project presents a novel, widely applicable viral diagnostic test that uses a modified version of rolling circle amplification (RCA) to be sensitive, specific, direct RNA targeted, colorimetric and operable at room temperature. We are specifically detecting the following high-impact viruses: SARS-CoV-2, Influenza A (H1N1pdm09), and Influenza B (Victoria Lineage), although our test can be adapted to any viral infection. Results using synthetic viral DNA and RNA sequences show that our diagnostic test takes approximately one hour, detects femtomolar concentrations of RNA strands, and differentiates between virus strains. We believe implementing our diagnostic test will provide faster responses to future viral-related outbreaks for quicker societal recovery.

synthetic biology↗

bioRxiv2020

Recombinant Expression of Metal-Binding Proteins (Bacterioferritin, MBP, Metallothionein, and OprF) and Pesticide-Binding Proteins (LC-Cutinase and OpdA) in Escherichia coli for Detection of Agricultural Contaminants

We consume fruits and vegetables every day without knowing whether or not agricultural residues (i.e. pesticides & heavy metals) are present on them. Current methods of agricultural residue detection are not easily accessible to the public and are inconvenient for everyday use. Thus, we want to develop a convenient visualization of agricultural residues by designing metal-binding and pesticide-binding proteins along with colored proteins that can directly interact with these residues. We worked with metal-binding proteins including Bacterioferritin, MBP, Metallothionein, and OprF. We also utilized pesticide-binding proteins LC Cutinase and OpdA. In this paper, we envision a system in which our residue-binding proteins are fused with chromoproteins to allow for the visible detection of agricultural residues on produce. Our final product can be used by consumers, distributors and farmers alike.Competing Interest StatementThe authors have declared no competing interest.View Full Text

synthetic biology↗

bioRxiv2018

Recombinant expression of Proteorhodopsin and biofilm regulators in Escherichia coli for nanoparticle binding and removal in a wastewater treatment model

The small size of nanoparticles is both an advantage and a problem. Their high surface-area-to-volume ratio enables novel medical, industrial, and commercial applications. However, their small size also allows them to evade conventional filtration during water treatment, posing health risks to humans, plants, and aquatic life. This project aims to remove nanoparticles during wastewater treatment using genetically modified Escherichia coli in two ways: 1) binding citrate-capped nanoparticles with the membrane protein Proteorhodopsin, and 2) trapping nanoparticles using Escherichia coli biofilm produced by overexpressing two regulators: OmpR234 and CsgD. We demonstrate experimentally that Escherichia coli expressing Proteorhodopsin binds to 60 nm citrate-capped silver nanoparticles. We also successfully upregulate biofilm production and show that Escherichia coli biofilms are able to trap 30 nm gold particles. Finally, both Proteorhodopsin and biofilm approaches are able to bind and remove nanoparticles in simulated wastewater treatment tanks. We envision integrating our trapping system in both rural and urban wastewater treatment plants to efficiently capture all nanoparticles before treated water is released into the environment.\n\nFinancial DisclosureThis work was funded by the Taipei American School. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\nCompeting InterestsThe authors have declared that no competing interests exist.\n\nEthics StatementN/A\n\nData AvailabilityYes - all data are fully available without restriction. Sequences for the plasmids used in this study are available through the Registry of Standard Biological Parts. Links to raw data are included in Supplementary Information.

synthetic biology↗