Korea Research Institute of Bioscience and Biotechnology
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Quantitative proteomic analysis of primitive neural stem cells from LRRK2 G2019S-associated Parkinson's disease patient-derived iPSCs
No full textParkinson’s disease (PD) is a common neurodegenerative disease, causing movement defects. The incidence of PD is constantly increasing and this disease is still incurable. Thus, understanding PD pathophysiology would be pivotal for the development of PD therapy, and various PD models have thus been already developed. Through recent advances in reprogramming techniques, a primitive neural stem cell (pNSC) derived from PD patient induced pluripotent stem cells (iPSCs) could be potentially used as a reproducible and reliable experimental system to analyze the effect of the leucine-rich repeat kinase 2 G2019S mutation (LK2GS) in neural cells. Here, we investigated the advantages of such a model system through quantitative proteomic analysis of pNSCs from normal control iPSCs and familial PD patient iPSCs harboring LK2GS. We confirmed that the expression of molecules known to be involved in PD pathogenesis, such as oxidative stress-, cell adhesion-, and cytoskeleton-related proteins, were altered in the LK2GS pNSC. In addition, we showed that down-regulation of Ku80, which was found in the proteomic analysis with LK2GS pNSCs, resulted in apoptosis induced by DNA damage response. Taken together, we suggest that pNSCs from PD iPSCs could provide a reliable and useful model system to study PD. Moreover, the highly expandable pNSC is suitable for multi-omics approaches to understand PD pathologies and discover therapeutic targets for PD.
Efficient genome editing using CRISPR-Cas9 RNP delivery into cabbage protoplasts via electro-transfection
No full textNowadays, genome editing in plants has become much easier thanks to the recently developed clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (CRISPR？Cas9) nuclease system. However, to combine protoplast technology with the CRISPR？Cas9 system in plants, a stable and an efficient foreign DNA delivery system is essential for gene editing. In the present study, we developed an electro-transfection system for CRISPR？Cas9 ribonucleoprotein (RNP) delivery to cabbage protoplasts. Under 1000 V treatment, the frequency of initial cell division and total number of cell colonies formed were 47.7？±？2.5% and 52？±？7.5%, respectively. The total number of cell colonies formed following 1000 V treatment was 1.4 times higher than that following polyethylene glycol (PEG) treatment. However, the frequency of initial cell division and total number of cell colonies formed from protoplasts decreased with increasing voltage. Cy3？Cas9 protein delivery into the nucleus was confirmed through both electro-transfection and PEG-mediated transfection using confocal laser scanning microscopy. The frequency of insertions and deletions in the synthesized guide RNA of phytoene desaturase 1 was the highest at 3.4% following electro-transfection at 1000 V with a pulse width of 20 ms and only 1.8% following PEG-mediated transfection. These results indicate that electro-transfection is more efficient in RNP delivery to protoplast than PEG-mediated transfection in cabbage for PDS1 sgRNA delivery. Therefore, the electro-transfection system developed in the present study presents the possibility it could be used for DNA-free genome editing of other crops.
Acetylcholinesterase and butyrylcholinesterase inhibitory activities of khellactone coumarin derivatives isolated from Peucedanum japonicum Thurnberg
No full textCholinesterase (ChE) and monoamine oxidase (MAO) inhibitors have been attracted as candidate treatments for Alzheimer's disease (AD). Fifteen khellactone-type coumarins from the roots of Peucedanum japonicum Thunberg were tested for acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and MAO inhibitory activities. Compound 3′-angeloyl-4′-(2-methylbutyryl)khellactone (PJ13) most potently inhibited AChE (IC50？=？9.28 μM), followed by 3′-isovaleryl-4′-(2-methylbutyroyl)khellactone (PJ15) (IC50？=？10.0 μM). Compound senecioyl-4′-angeloyl-khellactone (PJ5) most potently inhibited BChE (IC50？=？7.22 μM) and had the highest selectivity index (>？5.54), followed by 3′-senecioyl-4′-(2-methylbutyryl)khellactone (PJ10) and 3′,4′-disenecioylkhellactone (PJ4) (IC50？=？10.2 and 10.7 μM, respectively). Compounds PJ13, PJ15, and PJ5 showed reversible and mixed-types of inhibition with Ki values of 5.98, 10.4 (for AChE), and 4.16 μM (for BChE), respectively. However, all 15 compounds weakly inhibited MAO-A and MAO-B. Molecular docking simulation revealed that PJ13 had a higher binding affinity (？？9.3 kcal/mol) with AChE than PJ15 (？？7.8 kcal/mol) or PJ5 (？ 5.4 kcal/mol), due to the formation of a hydrogen bond with Tyr121 (distance: 2.52 A). On the other hand, the binding affinity of PJ5 (？？10.0 kcal/mol) with BChE was higher than for PJ13 (？？7.7 kcal/mol) or PJ15 (？？8.1 kcal/mol), due to the formation of a hydrogen bond with Ser198 (distance: 2.05 A). These results suggest that PJ13 and PJ5 are potential reversible selective inhibitors of AChE and BChE, respectively, for the treatment of AD.
Inhibition of Jurkat T cell proliferation 1 by active components of Rumex japonicus roots via induced mitochondrial damage and apoptosis promotion
No full textRumex japonicus Houtt (RJH) is a valuable plant used in traditional medicine to treat several diseases, such as scabies and jaundice. In this study, Jurkat cell growth inhibitory extracts of R. japonicus roots were subjected to bioassay-guided fractionation, resulting in the isolation of three naphthalene derivatives (3-5) along with one anthraquinone (6) and two phenolic compounds (1 and 2). Among these compounds, 2-methoxystypandrone (5) exhibited potent anti-proliferative effects on Jurkat cells. Analysis by flow cytometry confirmed that 2-methoxystypandrone (5) could significantly reduce mitochondrial membrane potential and promote increased levels of mitochondrial reactive oxygen species (ROS), suggesting a strong mitochondrial depolarization effect. Real-time quantitative polymerase chain reaction (qPCR) analysis was also performed, and the results revealed that the accumulation of ROS was caused by reduced mRNA expression levels of heme oxygenase (HO-1), catalase (CAT), glutathione peroxidase (GPx), and superoxide dismutase (SOD). In addition, 2-methoxystypandrone (5) triggered strong apoptosis that was mediated by the arrest of the G0/G1 phase of the cell cycle. Furthermore, 2-methoxystypandrone (5) downregulated p-IκB-α, p-NF-κB p65, Bcl2, and Bcl-xl and upregulated BAX proteins. Taken together, these findings revealed that 2-methoxystypandrone (5) isolated from RJH could potentially serve as an early lead compound for leukemia treatment involving intracellular signaling by increasing mitochondrial ROS and exerting anti-proliferative effects.
BLOTTING MEMBRANE APPLYING HYDROPHOBIC MATERIAL, METHOD OF MANUFACTURING THE SAME AND BLOTTING DEVICE INCLUDING THE SAME
No full text본 발명에 따른 소수성 물질을 적용한 블롯팅 멤브레인은 블롯팅(Blotting) 기기에 사용되는 블롯팅 멤브레인으로서, 소수성 물질로 형성된 베리어(barrier) 구조를 포함하고, 항체, 올리고 DNA 등과 같은 표지자를 포함한 용액의 주입 시, 상기 용액은 상기 베리어 구조에 의해 확산이 제한되는 것을 특징으로 한다. 이러한 소수성 물질을 적용한 블롯팅 멤브레인은 소수성 물질을 프린팅하여 표지자 용액과의 반응을 특정 부분으로 한정함으로써, 멤브레인 상의 표지자 용액의 확산을 억제하여 표지자 사용량을 감소시키고, 다수의 표지자를 이용한 블롯팅 을 동시에 진행할 수 있다. 또한, 본 발명에 따른 소수성 물질을 적용한 블롯팅 멤브레인은 블롯팅 분석 이미지 획득 시 관련 정보를 결과 이미지에 함께 전사되게 하여 유용한 여러 정보 들을 이미지 획득 과정 중에 쉽게 확보할 수 있다.국
TXNIP regulates natural killer cell-mediated innate immunity by inhibiting IFN-γ production during bacterial infection
No full textThe function of natural killer (NK) cell-derived interferon-γ (IFN-γ) expands to remove pathogens by increasing the ability of innate immune cells. Here, we identified the critical role of thioredoxin-interacting protein (TXNIP) in the production of IFN-γ in NK cells during bacterial infection. TXNIP inhibited the production of IFN-γ and the activation of transforming growth factor β-activated kinase 1 (TAK1) activity in primary mouse and human NK cells. TXNIP directly interacted with TAK1 and inhibited TAK1 activity by interfering with the complex formation between TAK1 and TAK1 binding protein 1 (TAB1). Txnip？/？ (KO) NK cells enhanced the activation of macrophages by inducing IFN-γ production during Pam3CSK4 stimulation or Staphylococcus aureus (S. aureus) infection and contributed to expedite the bacterial clearance. Our findings suggest that NK cell-derived IFN-γ is critical for host defense and that TXNIP plays an important role as an inhibitor of NK cell-mediated macrophage activation by inhibiting the production of IFN-γ during bacterial infection.
Effects of the mobile phase on the chromatographic separation of L-lysine and 5-aminovaleric acid
No full text5-Aminovaleric acid (5AVA), an attractive bio-based carbon-5 building block for polymer synthesis, is produced from l-lysine. However, their structural similarity makes separating 5AVA from l-lysine challenging. The simulated moving bed (SMB), continuous chromatographic separation process provides a solution with high productivity and low eluent consumption. Here, the effects of the mobile phase on the separation of 5AVA and l-lysine on a C18 column are reported. Pre-column derivatization using diethylethoxymethylene malonate enhanced the detection sensitivity and separation performance, which was strongly affected by the phosphate buffer pH. A 70/30 (v/v) mobile phase of 20？mM phosphate buffer at pH 6.8 and acetonitrile showed the best separation performance in isocratic mode. The selectivity at adsorption equilibrium was determined from pulse experiments, and the optimal operating conditions of the SMB process were determined from the equilibrium constants of both components. The feasibility of separating 5AVA and l-lysine by the SMB process was confirmed with purity and recovery of greater than 99%. The SMB process using pre-column derivatization can provide the competiveness of the bio-based chemical production because separation processes typically contribute to more than 70% of the total production cost.
Differential proteomic analyses of green microalga Ettlia sp. at various dehydration levels
No full textWater deprivation could be a lethal stress for aquatic and aero-terrestrial organisms. Ettlia sp. is a unicellular photosynthetic freshwater microalga. In the present study, proteomic alterations and physiological characteristics of Ettlia sp. were analyzed to comprehend the molecular changes in dehydrated conditions. Varying levels of dehydration were achieved by incubating drained Ettlia sp. in different relative humidity environments for 24？ hours. Using a comparative proteomic analysis, 52 differentially expressed protein spots were identified that could be divided into eight functional groups. The PCA analysis of normalized protein expression values demonstrated a clear segregation of protein expression profiles among different dehydration levels. Identified proteins could be grouped into four clusters based on their expression profiles. Proteins relating to photosynthesis comprised the largest group with 25% of the identified proteins that were decreased in dehydrated samples and belonged to cluster I. The photosynthetic activities were measured with rehydrated Ettlia sp. These results revealed that photosynthesis remained inhibited over extended time in response to dehydration. The expressions of reactive oxygen species (ROS) scavenger proteins increased in strong dehydration and were assigned to cluster III. Carbon metabolism proteins were suppressed, which might limit energy consumption, whereas glycolysis was activated at mild dehydration. The accumulation of desiccation-associated late embryogenesis proteins might inhibit the aggregation of housekeeping proteins. DNA protective proteins were expressed higher in the dehydrated state, which might reduce DNA damage, and membrane-stabilizing proteins increased in abundance in desiccation. These findings provide an understanding of Ettlia's adaptation and survival capabilities in a dehydrated state.
Idiopathic chronic diarrhea associated with dysbiosis in a captive cynomolgus macaque (Macaca fascicularis)
No full textChronic inflammatory enteric diseases occur commonly in humans and animals, especially in captive bred macaques. However, information about the etiology of idiopathic chronic inflammatory diarrhea in cynomolgus monkeys is limited. In this paper, we reported the unusual case of idiopathic chronic diarrhea in a captive cynomolgus monkey based on microbial, imaging, and microbiome examinations.
Increased O-GlcNAcylation of c-Myc promotes Pre-B cell proliferation
No full textO-linked β-N-acetylglucosamine (O-GlcNAc) modification regulates the activity of hundreds of nucleocytoplasmic proteins involved in a wide variety of cellular processes, such as gene expression, signaling, and cell growth; however, the mechanism underlying the regulation of B cell development and function by O-GlcNAcylation remains largely unknown. Here, we demonstrate that changes in cellular O-GlcNAc levels significantly affected the growth of pre-B cells, which rapidly proliferate to allow expansion of functional clones that express successfully rearranged heavy chains at the pro-B stage during early B cell development. In our study, the overall O-GlcNAc levels in these proliferative pre-B cells, which are linked to the glucose uptake rate, were highly induced when compared with those in pro-B cells. Thus, pharmacologically, genetically, or nutritionally, inhibition of O-GlcNAcylation in pre-B cells markedly downregulated c-Myc expression, resulting in cell cycle arrest via blockade of cyclin expression. Importantly, the population of B cells after the pro-B cell stage in mouse bone marrow was severely impaired by the administration of an O-GlcNAc inhibitor. These results strongly suggest that O-GlcNAcylation-dependent expression of c-Myc represents a new regulatory component of pre-B cell proliferation, as well as a potential therapeutic target for the treatment of pre-B cell-derived leukemia.