Korea Research Institute of Bioscience and Biotechnology
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Protein dosage of the lldPRD operon is correlated with RNase E-dependent mRNA processing
No full textThe ability of Escherichia coli to grow on L-lactate as a sole carbon source depends on the expression of the lldPRD operon. A striking feature of this operon is that the gene encoding the transcriptional regulator (LldR) is located between the genes encoding the permease (LldP) and the dehydrogenase (LldD). In this study, we report that the dosages of the LldP, LldR, and LldD proteins are not modulated on the transcriptional level. Instead, modulation of the protein dosage is correlated primarily with RNase E-dependent mRNA-processing events that take place within the lldR mRNA, leading to the immediate inactivation of lldR, to differential segmental stabilities of the resulting cleavage products, and to differences in the translation efficiencies of the three cistrons. A model for the processing events controlling the molar quantities of the proteins in the lldPRD operon is presented and discussed. IMPORTANCE Adjustment of gene expression is critical for proper cell function. In the case of polycistronic transcripts, posttranscriptional regulatory mechanisms can be used to fine-tune the expression of individual cistrons. Here, we elucidate how the protein dosage of the Escherichia coli lldPRD operon, which presents the paradox of having the gene encoding a regulator protein located between genes that code for a permease and an enzyme, is regulated. Our results demonstrate that the key event in this regulatory mechanism involves the RNase E-dependent cleavage of the primary lldPRD transcript at an internal site(s) located within the lldR cistron, resulting in a drastic decrease in the amount of intact lldR mRNA, in differential segmental stabilities of the resulting cleavage products, and in differences in the translation efficiencies of the three cistrons.
Development of generation and production technologies for therapeutic enzymes with the improved lysosome targeting capability, based on synthetic biology
No full text합성생물학 기반 라이소좀 타겟팅 효율이 향상된 효소 치료제 제작 및 생산기술 개발ABM427202
Lacticaseibacillus absianus sp. nov., isolated from the cecum of a mini-pig
No full textA rod-shaped, facultative anaerobic, Gram-stain-positive bacteria, isolated from the cecum of a mini-pig, was designated as strain YH-lac23T. Analysis of 16S rRNA gene sequences revealed that the strain was closely related to Lacticaseibacillus daqingensis JCM 33273T (97.9？%), Lacticaseibacillus porcinae KCTC 21027T (96.2？%) and Lacticaseibacillus manihotivorans KCTC 21010T (95.7？%). Analysis of housekeeping gene sequences (pheS and recA) revealed that the strain formed a sub-cluster with L. daqingensis . The average nucleotide identity value for YH-lac23T and its most closely related strain ( L. daqingensis ) is 80.7？%. The main fatty acids are C18？:？1ω9c and C16？:？0. The cell wall contains the peptidoglycan of meso-diaminopimelic acid. The G+C content of the genomic DNA is 59.8？mol%. In view of the chemotaxonomic, phenotypic and phylogenetic properties, YH-lac23T (=KCTC 25006=JCM 33998) represents a novel taxon. The name Lacticaseibacillus absianus sp. nov. is proposed.
Development of functional lung organoids to mimin human lung and disease & discovery of NGS-based therapeutic target
No full text폐질환 인체 모방을 위한 기능성 폐오가노이드 개발 및 NGS기반 치료표적 탐색OGM508201
Transcriptomic analysis of cellular senescence: one step closer to senescence atlas
No full textSenescent cells that gradually accumulate during aging are one of the leading causes of aging. While senolytics can improve aging in humans as well as mice by specifically eliminating senescent cells, the effect of the senolytics varies in different cell types, suggesting variations in senescence. Various factors can induce cellular senescence, and the rate of accumulation of senescent cells differ depending on the organ. In addition, since the heterogeneity is due to the spatiotemporal context of senescent cells, in vivo studies are needed to increase the understanding of senescent cells. Since current methods are often unable to distinguish senescent cells from other cells, efforts are being made to find markers commonly expressed in senescent cells using bulk RNA-sequencing. Moreover, single-cell RNA (scRNA) sequencing, which analyzes the transcripts of each cell, has been utilized to understand the in vivo characteristics of the rare senescent cells. Recently, transcriptomic cell atlases for each organ using this technology have been published in various species. Novel senescent cells that do not express previously established marker genes have been discovered in some organs. However, there is still insufficient information on senescent cells due to the limited throughput of the scRNA sequencing technology. Therefore, it is necessary to improve the throughput of the scRNA sequencing technology or develop a way to enrich the rare senescent cells. The in vivo senescent cell atlas that is established using rapidly developing single-cell technologies will contribute to the precise rejuvenation by specifically removing senescent cells in each tissue and individual.
Pharmacokinetic characterization of LW6, a novel hypoxia-inducible factor-1α (HIF-1α) inhibitor in mice
No full textLW6, an (aryloxyacetylamino)benzoic acid derivative, was recently identified to be an inhibitor of hypoxia-inducible factor-1α (HIF-1α), which is an attractive target for cancer therapeutics. Although LW6 is known to act by inhibiting the accumulation of HIF-1α, pharmacokinetics needs to be evaluated to assess its potential as an anti-tumor agent. Here, we investigated the plasma pharmacokinetics and metabolism of LW6 in mice. LW6 exhibited a small volume of distribution (0.5 ± 0.1 L/kg), and a short terminal half-life (0.6 ± 0.1 h). Following intravenous or oral administration, LW6 was rapidly converted to its active metabolite, (4-adamantan-1-yl-phenoxy)acetic acid (APA). Although LW6 was rapidly absorbed, its oral bioavailability, estimated using AUClast values, was low (1.7 ± 1.8%). It was slowly degraded in mouse liver microsomes (t1/2 > 1 h) and serum (t1/2 > 6 h). About 54% or 44.8% of LW6 was available systemically as APA in the mouse after a single intravenous or oral administration, respectively. Thus, our results indicated the need to simultaneously consider the active metabolite as well as the parent compound for successful evaluation during lead optimization.
Transcriptional response in the whiteleg shrimp (Penaeus vannamei) to short-term microplastic exposure
No full textThe ingestion of microplastics by diverse marine organisms induces behavioral disorders, physiological changes, and immune and stress responses. The negative effects of microplastic exposure in penaeid shrimps are still unclear despite the annual increase in the consumption of these food sources by humans and in marine aquaculture. Therefore, this study aimed to investigate the potential negative effects of microplastic exposure and gain a deeper understanding of its impacts on culturing penaeid shrimp. We generated RNA sequencing data from the whiteleg shrimp (Penaeus vannamei) artificially exposed to microplastics and analyzed the differentially expressed genes. Based on transcriptional comparisons, exposure to microplastics induces cardiac muscle dysfunction and promotes stress and immune responses in whiteleg shrimp. Thus, we confirmed the negative impact of microplastic exposure in whiteleg shrimp. These results provide a foundation for studies on the biological effects of microplastic ingestion on aquaculture species and their potential impact on humans through their consumption of these important marine resources.
Implications of access and benefit-sharing of genetic resources for researchers
No full textIn the era of the Nagoya Protocol, the way researchers have perceived genetic resources needs to change-genetic resources are no longer a common heritage of mankind. Many countries have recently amended their legal systems and are ready to adopt new legal procedures for access and benefit-sharing (ABS) of genetic resources to implement the Nagoya Protocol, claiming their sovereign rights over these resources for research use. Although both Korean government and academics have been striving to respond to the Nagoya Protocol, the understanding of ABS among researchers and the governmental guidelines for the use of genetic resources are not satisfactory yet. Researchers may perceive the implementation of the Nagoya Protocol as another burden for their research. However, it is evident that resource-rich countries are more likely to protect their genetic resources than other countries, and this tendency is expected to last long. Therefore, Korean researchers need to be prepared to minimize any damage that might be caused by ABS. This paper aims to raise awareness of ABS among Korean researchers by reviewing the key contents and overall structure of the Nagoya Protocol. It also identifies ABS procedures and presents specific measures for researchers to respond to the resulting changes in their research environments. Accordingly, this paper will serve as a guide for researchers to conduct research and development in accordance with the Nagoya Protocol.
Production of recombinant miraculin protein in carrot callus via Agrobacterium-mediated transformation
No full textMiraculin is a taste-modifying protein that interacts with human sweet-taste receptors and transforms a sour taste into sweet taste. Miraculin is extracted from the miracle fruit (Synsepalum dulcificum). Since mass production of miraculin is difficult because of regional and seasonal limitations, several attempts have been made to express miraculin in various cell systems. In this study, a binary vector containing the miraculin gene under the control of the SWPA2 promotor was introduced into carrot (Daucus carota) callus via Agrobacterium-mediated transformation to synthesize miraculin in carrot cell cultures. After 4 weeks of co-cultivation with Agrobacterium tumefaciens, 20 tentative transgenic callus (TC) lines were obtained on kanamycin selection medium. PCR analysis confirmed that 18 of these 20 lines (TC1？TC18) carried the miraculin gene, and 4 TC lines with high cell growth and gene expression (determined by RT-qPCR) were selected for further analysis. Protein analysis of these four TC lines by SDS-PAGE and Western blot showed that the miraculin protein was stably produced in TC lines. The cell growth showed no correlation with gene expression levels. The DNA content and G1 phase ratio were negatively correlated, whereas the S and G2/M phase ratios were positively correlated with gene expression. The ratio of cell cycle was determined by counting the number of cells in each step through flow cytometric analysis. These results indicate that gene expression was higher in TC lines with active cell division. Overall, our results demonstrate the feasibility of mass production of recombinant miraculin protein in transgenic cell culture systems.
Cβ-selective aldol addition of D-threonine aldolase by spatial constraint of aldehyde binding
No full textd-Threonine aldolase (DTA) is a useful biocatalyst that reversibly converts glycine and aldehyde to β-hydroxy-α-d-amino acid. However, low activity and poor diastereoselectivity limit its applications. Here we report DTA from Filomicrobium marinum (FmDTA) that shows much higher activity and Cβ-stereoselectivity in d-threonine production compared with those of other known DTAs. We determine the FmDTA structure at a 2.2 A resolution and propose a DTA catalytic mechanism with a kernel of the Lys49 inner proton sink and metal ion in the aldol reaction cycle. The enzyme is rationally engineered to have high Cβ-stereoselectivity based on spatial constraint at the anti-specific aldehyde position in the mechanism, and the rational strategy is further applied to other DTAs for syn-production. The final FmDTAG179A/S312A variant exhibits a near-perfect 99.5% de value for d-threonine and maintains the de value above 93% even under kinetically unfavorable conditions. This study demonstrates how a detailed understanding of the reaction mechanism can be used for rational protein engineering.