Korea Research Institute of Bioscience and Biotechnology
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A rapid quantitative on-site coronavirus disease 19 serological test
No full textOn-site severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) serological assays allow for timely in-field decisions to be made regarding patient status, also enabling population-wide screening to assist in controlling the coronavirus disease 2019 (COVID-19) pandemic. Here we propose a rapid microfluidic serological assay with two unique functions of nanointerstice filling and digitized flow control, which enable the fast/robust filling of the sample fluid as well as precise regulation of duration and volume of immune reaction. Developed microfluidic assay showed enhanced limit of detection, and 91.67% sensitivity and 100% specificity (n = 152) for clinical samples of SARS CoV-2 patients. The assay enables daily monitoring of IgM/IgG titers and patterns, which could be crucial parameters for convalescence from COVID-19 and provide important insight into how the immune system responds to SARS CoV-2. The developed on-site microfluidic assay presented the mean time for IgM and IgG seroconversions, indicating that these titers plateaued days after seroconversion. The mean duration from day 0 to PCR negativity was 19.4 days (median 20 d, IQR 16？21 d), with higher IgM/IgG titres being observed when PCR positive turns into negative. Simple monitoring of these titres promotes rapid on-site detection and comprehensive understanding of the immune response of COVID-19 patients.
Sodium phenylbutyrate reduces repetitive self-grooming behavior and rescues social and cognitive deficits in mouse models of autism
No full textAutism spectrum disorder (ASD) is a neurodevelopment disorder characterized by deficits in social interaction and restrictive, repetitive, and stereotypical patterns of behavior. However, there is no pharmacological drug that is currently used to target these core ASD symptoms. Sodium phenylbutyrate (NaPB) is a well-known long-term treatment of urea cycle disorders in children. In this study, we assessed the therapeutic effects of NaPB, which is a chemical chaperone as well as histone deacetylase inhibitor on a BTBR T + Itpr3tf/J (BTBR) mice model of ASD. We found that acute and chronic treatment of NaPB remarkably improved, not only core ASD symptoms, including repetitive behaviors and sociability deficit, but also cognitive impairment in the BTBR mice. NaPB substantially induced histone acetylation in the brain of the BTBR mice. Intriguingly, the therapeutic effects of NaPB on autistic-like behaviors, such as repetitive behaviors, impaired sociability, and cognitive deficit also showed in the valproic acid (VPA)-induced mouse model of autism. In addition, pentylenetetrazole (PTZ)-induced seizure was significantly attenuated by NaPB treatment in C57BL/6J and BTBR mice. These findings suggest that NaPB may provide a novel therapeutic approach for the treatment of patients with ASD.
Salmonella vaccine vector system for foot-and-mouth disease virus and evaluation of its efficacy with virus-like particles
No full textFoot-and-mouth disease virus (FMDV) causes a highly contagious and devastating disease in livestock animals and has a great potential to cause severe economic loss worldwide. The major antigen of FMDV capsid protein, VP1, contains the major B-cell epitope responsible for effectively eliciting protective humoral immunity. In this study, irradiated Salmonella Typhimurium (KST0666) were used as transgenic vectors containing stress-inducible plasmid pRECN-VP1 to deliver the VP1 protein from FMDV-type A/WH/CHA/09. Mice were orally inoculated with ATOMASal-L3 harboring pRECN-VP1, and FMDV virus-like particles, where (VLPFMDV)-specific humoral, mucosal, and cellular immune responses were evaluated. Mice vaccinated with attenuated Salmonella (KST0666) expressing VP1 (named KST0669) showed high levels of VLP-specific IgA in feces and IgG in serum, with high FMDV neutralization titer. Moreover, KST0669-vaccinated mice showed increased population of IFN-γ (type 1 T helper cells; Th1 cells)-, IL-5 (Th2 cells)-, and IL-17A (Th17 cells)-expressing CD4+ as well as activated CD8+ T cells (IFN-γ+CD8+ cells), detected by stimulating VLPFMDV. All data indicate that our Salmonella vector system successfully delivered FMDV VP1 to immune cells and that the humoral and cellular efficacy of the vaccine can be easily evaluated using VLPFMDV in a Biosafety Level I (BSL1) laboratory.
Compositional differences in hybrids between protoporphyrinogen IX oxidase (PPO)-inhibiting herbicide-resistant transgenic rice and weedy rice accessions
No full textWe characterized the metabolites in grains of transgenic protoporphyrinogen IX oxidase-inhibiting herbicide-resistant rice and weedy accessions using GC？MS and examined whether the chemical composition of their hybrids differed from that of the parents. We found that the metabolite profiles of transgenic rice and weedy rice were clearly separated. Although the metabolite profiles of F2 progeny were partially separated from their parents, zygosity did not affect the profiles. The F2 progeny had similar or intermediate levels of most major nutritional components compared with their parents. However, levels of galactopyranose, trehalose, xylofuranose, mannitol, and benzoic acid were higher in the F2 progeny. Some fatty acids and organic acids also showed prominent quantitative differences between the F2 progeny and the parents. Changes in the metabolite levels of transgenic crop-weed hybrids compared to their parents might influence not only the ecological consequences of the hybrids, but also the nutritional quality and food safety.
Effective production of human growth factors in Escherichia coli by fusing with small protein 6HFh8
No full textBackground: Growth factors (GFs) are signaling proteins that affect cellular processes such as growth, proliferation, and differentiation. GFs are used as cosmeceuticals, exerting anti-wrinkle, anti-aging, and whitening effects, and also as pharmaceuticals to treat wounds, growth failure, and oral mucositis. However, in mammalian and bacterial cells, low productivity and expression in inclusion bodies, respectively, of GFs does not satisfy the consumer demand. Here, we aimed to develop a bacterial expression system that produces high yields of soluble GFs that can be purified in their native forms.
Results: We present Fh8, an 8-kDa peptide from Fasciola hepatica with an N-terminal hexa-histidine (6HFh8), as a fusion partner for enhanced human GF production in recombinant Escherichia coli. The fusion partner harboring a tobacco etch virus (TEV) protease cleavage site was fused to the N-terminus of 10 human GFs: acidic and basic fibroblast growth factors (aFGF and bFGF, respectively), epidermal growth factor (EGF), human growth hormone (hGH), insulin-like growth factor 1 (IGF-1), vascular endothelial growth factor 165 (VEGF165), keratinocyte growth factor 1 (KGF-1), placental growth factor (PGF), stem cell factor (SCF), and tissue inhibitor of metalloproteinase 1 (TIMP-1). The fusion proteins were expressed in E. coli under the control of T7 promoter at three temperatures (25 °C, 30 °C, and 37 °C). All individual fusion proteins, except for SCF and TIMP-1, were successfully overexpressed in cytoplasmic soluble form at more than one temperature. Further, the original aFGF, IGF-1, EGF, and VEGF165 proteins were cleaved from the fusion partner by TEV protease. Five-liter fed-batch fermentation approaches for the 6HFh8-aFGF (lacking disulfide bonds) and 6HFh8-VEGF165 (a cysteine-rich protein) were devised to obtain the target protein at concentrations of 9.7 g/l and 3.4 g/l, respectively. The two GFs were successfully highly purified (> 99% purity). Furthermore, they exerted similar cell proliferative effects as those of their commercial equivalents.
Conclusions: We demonstrated that 6HFh8-GF fusion proteins could be overexpressed on a g/l scale in the cytoplasm of E. coli, with the GFs subsequently highly purified and maintaining their biological activity. Hence, the small protein 6HFh8 can be used for efficient mass-production of various GFs.
Discovery of natural inhibitors of cholinesterases from Hydrangea: in vitro and in silico approaches
No full textAlzheimer's disease (AD) is a neurodegenerative disease conceptualized as a clinical-biological neurodegenerative construct where amyloid-beta pathophysiology is supposed to play a role. The loss of cognitive functions is mostly characterized by the rapid hydrolysis of acetylcholine by cholinesterases including acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Moreover, both enzymes are responsible for non-catalytic actions such as interacting with amyloid β peptide (Aβ) which further leads to promote senile plaque formation. In searching for a natural cholinesterase inhibitor, the present study focused on two isocoumarines from hydrangea, thunberginol C (TC) and hydrangenol 8-O-glucoside pentaacetate (HGP). Hydrangea-derived compounds were demonstrated to act as dual inhibitors of both AChE and BChE. Furthermore, the compounds exerted selective and non-competitive mode of inhibition via hydrophobic interaction with peripheral anionic site (PAS) of the enzymes. Overall results demonstrated that these natural hydrangea-derived compounds acted as selective dual inhibitors of AChE and BChE, which provides the possibility of potential source of new type of anti-cholinesterases with non-competitive binding property with PAS.
Polyacetylene (9Z,16S)-16-hydroxy-9,17-octadecadiene-12,14-diynoic acid in Dendropanax morbifera leaves
No full textDendropanax morbifera extracts were obtained from three different tissues: the heartwood, stem bark, and leaves. The 95% ethanol extract from leaf showed the strongest inhibitory activity (88% inhibition at 50 μg/mL with HepG2 cells) against triglyceride (TG) biosynthesis. Bioactivity-guided fractionation and metabolite investigation of the leaf extracts resulted in the separation and identification of three polyacetylene derivatives that inhibited newly synthesized TG in HepG2 cells. A method for the profile and contents analyses of the leaf extracts were measured using QTOF-MS with a charged aerosol detector (CAD), including (9Z,16S)-16-hydroxy-9,17-octadecadiene-12,14-diynoic acid (1), which was decomposed during the temperature and storage studies. All metabolites decomposed at different temperatures, especially the polyacetylene, which was rapidly oxidized when the isolated form was exposed to air. However, it was fairly stable in the extract at low temperature <？20 °C. The aim of this study was to provide a way to protect the active ingredients in the process of developing and distributing functional foods containing D. morbifera. The application of this quality control/quality assurance step may improve the chemistry, manufacturing, and control for raw materials.
In vivo monitoring platform of transplanted human stem cells using magnetic resonance imaging
No full textAs stem cells show great promise in regenerative therapy, stem cell-mediated therapeutic efficacy must be demonstrated through the migration and transplantation of stem cells into target disease areas at the pre-clinical level. In this study, we developed manganese-based magnetic nanoparticles with hollow structures (MnOHo) and modified them with the anti-human integrin β1 antibody (MnOHo-Ab) to enable the minimal-invasive monitoring of transplanted human stem cells at the pre-clinical level. Compared to common magnetic resonance imaging (MRI)-based stem cell monitoring systems that use pre-labeled stem cells with magnetic particles before stem cell injection, the MnOHo-Ab is a new technology that does not require stem cell modification to monitor the therapeutic capability of stem cells. Additionally, MnOHo-Ab provides improved T1 MRI owing to the hollow structure of the MnOHo. Particularly, the anti-integrin β1 antibody (Ab) introduced in the MnOHo targets integrin β1 expressed in the entire stem cell lineage, enabling targeted monitoring regardless of the differentiation stage of the stem cells. Furthermore, we verified that intravenously injected MnOHo-Ab specifically targeted human induced pluripotent stem cells (hiPSCs) that were transferred to mice testes and differentiated into various lineages. The new stem cell monitoring method using MnOHo-Ab demonstrates whether the injected human stem cells have migrated and transplanted themselves in the target area during long-term stem cell regenerative therapy.
Morphology and phylogeny of Scrippsiella precaria Montresor & Zingone (Thoracosphaerales, Dinophyceae) from Korean coastal waters
No full textThe dinoflagellate genus Scrippsiella is a common member of phytoplankton and their cysts are also frequently reported in coastal sediments worldwide. However, the diversity of Scrippsiella in Korean waters has not been fully investigated. Here, several isolates of Scrippsiella precaria collected from Korean waters and germinated from resting cysts were examined using light and scanning electron microscopy. The resting cysts were characterized by pointed calcareous spines and one or two red accumulation bodies, and the archeopyle was mesoepicystal, representing the loss of 2？4′ and 1？3a paraplates. Rounded resting cysts were found in culture, and an increase in spine length was observed until 8 days of development. Korean isolates of S. precaria had the plate formula of Po, X, 4′, 3a, 7″, 6C, 4S, 5？, 2？. There were differences in the cell size and location of the red body between Korean isolates and previously described cells of S. precaria. In addition, the Korean isolates of S. precaria had two types of the 5″ plate that either contacted the 2a plate or not. Molecular phylogeny based on internal transcribed spacer (ITS) and large subunit (LSU) rDNA sequences revealed that the Korean isolates were nested within the subclade of PRE (S. precaria and related species) in the clade of Scrippsiella sensu lato, and that the PRE subclade had two ribotypes: ribotype 1 consisting of the isolates from Korea, China, and Australia, and ribotype 2 consisting of the isolates from Italy and Greece. Lineages between isolates of ribotype 1 were likely to be related to the dispersal by ocean currents and ballast waters from international shipping, and the two types of spine shapes and locations of the 5″ plates may be a distinct feature for ribotype 1.
Population pharmacokinetic method to predict within-subject variability using single-period clinical data
No full textSample sizes for single-period clinical trials, including pharmacokinetic studies, are statistically determined by within-subject variability (WSV). However, it is difficult to determine WSV without replicate-designed clinical trial data, and statisticians typically estimate optimal sample sizes using total variability, not WSV. We have developed an efficient population-based method to predict WSV accurately with single-period clinical trial data and demonstrate method performance with eperisone. We simulated 1000 virtual pharmacokinetic clinical trial datasets based on single-period and dense sampling studies, with various study sizes and levels of WSV and interindividual variabilities (IIVs). The estimated residual variability (RV) resulting from population pharmacokinetic methods were compared with WSV values. In addition, 3 × 3 bioequivalence results of eperisone were used to evaluate method performance with a real clinical dataset. With WSV of 40% or less, regardless of IIV magnitude, RV was well approximated by WSV for sample sizes greater than 18 subjects. RV was underestimated at WSV of 50% or greater, even with datasets having low IIV and numerous subjects. Using the eperisone dataset, RV was 44% to 48%, close to the true value of 50%. In conclusion, the estimated RV accurately predicted WSV in single-period studies, validating this method for sample size estimation in clinical trials.