Korea Research Institute of Bioscience and Biotechnology
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Complete mitochondrial genome sequence of Afla-Guard®, commercially available non-toxigenic Aspergillus flavus
No full textAfla-Guard® is a commercial non-toxigenic Aspergillus flavus strain used to decrease aflatoxin contamination level in field. Its mitochondrial genome was sequenced, showing that its length is 29,208？bp with typical configuration of Aspergillus mitochondrial genome. 17 SNPs and 27 INDELs were identified by comparing with previous A. flavus mitochondrial genome. Phylogenetic trees present that A. flavus of Afla-Guard® was clustered with the previous A. flavus mitochondrial genome.
Genetically encoded biosensor-based screening for directed bacteriophage T4 lysozyme evolution
No full textLysozyme is widely used as a model protein in studies of structure？function relationships. Recently, lysozyme has gained attention for use in accelerating the degradation of secondary sludge, which mainly consists of bacteria. However, a high-throughput screening system for lysozyme engineering has not been reported. Here, we present a lysozyme screening system using a genetically encoded biosensor. We first cloned bacteriophage T4 lysozyme (T4L) into a plasmid under control of the araBAD promoter. The plasmid was expressed in Escherichia coli with no toxic effects on growth. Next, we observed that increased soluble T4L expression decreased the fluorescence produced by the genetic enzyme screening system. To investigate T4L evolution based on this finding, we generated a T4L random mutation library, which was screened using the genetic enzyme screening system. Finally, we identified two T4L variants showing 1.4-fold enhanced lytic activity compared to native T4L. To our knowledge, this is the first report describing the use of a genetically encoded biosensor to investigate bacteriophage T4L evolution. Our approach can be used to investigate the evolution of other lysozymes, which will expand the applications of lysozyme.
Phase separation of the Cep63.Cep152 complex underlies the formation of dynamic supramolecular self-assemblies at human centrosomes
No full textThe centrosome is a unique membraneless organelle that plays a pivotal role in the orderly progression of the cell cycle in animal cells. It has been shown that two pericentriolar scaffold proteins, Cep63 and Cep152, generate a heterotetrameric complex to self-assemble into a higher-order cylindrical architecture around a centriole. However, the mechanisms underlying how they reach their threshold concentrations in the vast intracellular space and generate a self-assembled architecture remain mysterious. Here we demonstrate that, like liquid-like assemblies, Cep63 and Cep152 cooperatively generate amorphous aggregates capable of undergoing dynamic turnover and inter-aggregate fusion in vivo and a significant level of internal rearrangemefnt within a condensate in vitro. Consistently, 1,6-hexanediol, a liquid-liquid phase separation disruptor, greatly diminished the ability of endogenous Cep63 and Cep152 to localize to centrosomes. Interestingly, a purified Cep63？Cep152 complex generated either a cylindrical structure or a vesicle-like hollow sphere in a spatially controlled manner. It also formed condensate-like solid spheres in the presence of a macromolecular crowder. At the molecular level, two hydrophobic motifs, one each from Cep63 and Cep152, were required for generating phase-separating condensates and a high molecular-weight assembly. Thus, we propose that the self-assembly of the Cep63？Cep152 complex is triggered by an intrinsic property of the complex undergoing density transition through the hydrophobic-motif-mediated phase separation.
아프리카돼지열병 바이러스 생산 동물세포주의 무혈청 부유형 배양 공정 개발 연구
No full text아프리카돼지열병 바이러스 생산 동물세포주의 무혈청 부유형 배양 공정 개발 연구IGM071201
Identification and enumeration of cyanobacteria species using a deep neural network
No full textCell classification and cell counting are essential for the detection, monitoring, forecasting, and management of harmful algae populations. Conventional methods of algae classification and cell counting are known to be time-consuming, labor-intensive, and subjective, depending on the expertise of the observers. The objectives of this study were to classify and quantify five cyanobacteria using the deep learning techniques of a fast regional convolutional neural network (R-CNN) and convolutional neural network (CNN). Water samples taken from the Haman weir of Nakdong River and Baekje weir of the Geum River were observed under the optical microscope. The images captured by the microscope were used to classify cyanobacteria species using the fast R-CNN model. Post-processing of the classified images generated by the model reduced the noises of the cell features, thereby improving the accuracy of the CNN model in quantifying cyanobacteria cells. The distinctive morphological features of the five species were extracted by the fast R-CNN model. This model was able to achieve a reasonable agreement with the manual classification results, yielding average precision (AP) values of 0.929, 0.973, 0.829, 0.890, and 0.890 for Microcystis aeruginosa, Microcystis wesenbergii, Dolichospermum, Oscillatoria, and Aphanizomenon, respectively. The CNN model for the Microcystis species obtained an R2 value of 0.775 and RMSE value of 26 cells for training, and an R2 of 0.854 and RMSE of 23 cells for validation. A minor underestimation and overestimation for a population with <50 cells and >250 cells were observed, respectively, which are due to the overlapping of cells and the presence of blurry regions in the input images. In conclusion, this study was able to demonstrate the reliable performance of cyanobacteria classification and cell counting using deep learning approaches.
Comprehensive analysis of translationally controlled tumor protein (TCTP) provides insights for lineage-specific evolution and functional divergence
No full textBackground Translationally controlled tumor protein (TCTP) is a conserved, multifunctional protein involved in numerous cellular processes in eukaryotes. Although the functions of TCTP have been investigated sporadically in animals, invertebrates, and plants, few lineage-specific activities of this molecule, have been reported. An exception is in Arabidopsis thaliana, in which TCTP (AtTCTP1) functions in stomatal closuer by regulating microtubule stability. Further, although the development of next-generation sequencing technologies has facilitated the analysis of many eukaryotic genomes in public databases, inter-kingdom comparative analyses using available genome information are comparatively scarce. Methodology To carry out inter-kingdom comparative analysis of TCTP, TCTP genes were identified from 377 species. Then phylogenetic analysis, prediction of protein structure, molecular docking simulation and molecular dynamics analysis were performed to investigate the evolution of TCTP genes and their binding proteins. Results A total of 533 TCTP genes were identified from 377 eukaryotic species, including protozoa, fungi, invertebrates, vertebrates, and plants. Phylogenetic and secondary structure analyses reveal lineage-specific evolution of TCTP, and inter-kingdom comparisons highlight the lineage-specific emergence of, or changes in, secondary structure elements in TCTP proteins from different kingdoms. Furthermore, secondary structure comparisons between TCTP proteins within each kingdom, combined with measurements of the degree of sequence conservation, suggest that TCTP genes have evolved to conserve protein secondary structures in a lineage-specific manner. Additional tertiary structure analysis of TCTP-binding proteins and their interacting partners and docking simulations between these proteins further imply that TCTP gene variation may influence the tertiary structures of TCTP-binding proteins in a lineage-specific manner. Conclusions Our analysis suggests that TCTP has undergone lineage-specific evolution and that structural changes in TCTP proteins may correlate with the tertiary structure of TCTP-binding proteins and their binding partners in a lineage-specific manner. ⓒ 2020 Koo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Overexpression of swpa4 peroxidase enhances tolerance to hydrogen peroxide and high salinity-mediated oxidative stress in transgenic sweetpotato plants
No full textSecretory class III peroxidases are involved in plant responses to a range of biological stress conditions, including oxidative stress. To investigate the stress-related functions of the swpa4 peroxidase gene in sweetpotato (Ipomoea batatas L.), transgenic plants overexpressing the swpa4 gene under the control of the CaMV 35S promoter were generated using Agrobacterium-mediated transformation. Peroxidase activity was 3- to 13-fold higher in transgenic lines than in control plants. Transgenic plants were tested for tolerance to stress. Following treatment with 400？mM hydrogen peroxide (H2O2), leaf discs from transgenic plants showed approximately 13？26% less damage than control plants. Transgenic plants also showed enhanced tolerance to high salinity conditions. Following treatment with NaCl, photosynthetic capacity and total chlorophyll contents were less severely impacted in swpa4 transgenic plants than in control plants. Aerial plant parts and storage root yields were not significantly different between transgenic and control plants following cultivation in field conditions. These results indicate that transgenic sweetpotato lines were able to respond efficiently to oxidative and saline stress via overexpression of swpa4.
