• Bulletin of the Korean Chemical Society
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• 제35권5호
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• pp.1279-1284
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• 2014

The binding of TATA-binding protein (TBP) to the TATA-box containing promoter region is aided by many other transcriptional factors including TFIIA and TFIIB. The mechanistic insight into the assembly of RNA polymerase II preinitation complex (PIC) has been gained by either directly altering a function of target protein or perturbing molecular interactions using drugs, RNAi, or aptamers. Aptamers have been found particularly useful for studying a role of a subset of PIC on transcription for their ability to inhibit specific molecular interactions. One major hurdle to the wide use of aptamers as specific inhibitors arises from the difficulty with traditional assays to validate and determine specificity, affinity, and binding epitopes for aptamers against targets. Here, using a technique called the bio-layer interferometry (BLI) designed for a label-free, real-time, and multiplexed detection of molecular interactions, we studied the assembly of a subset of PIC, TBP binding to TATA DNA, and two distinct classes of aptamers against TPB in regard to their ability to inhibit TBP binding to TFIIA or TATA DNA. Using BLI, we measured not only equilibrium binding constants ($K_D$), which were overall in close agreement with those obtained by electrophoretic mobility shift assay, but also kinetic constants of binding ($k_{on}$ and $k_{off}$), differentiating aptamers of comparable KDs by their difference in binding kinetics. The assay developed in this study can readily be adopted for high throughput validation of candidate aptamers for specificity, affinity, and epitopes, providing both equilibrium and kinetic information for aptamer interaction with targets.

• Korean Chemical Engineering Research
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• 제49권3호
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• pp.348-351
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• 2011

본 연구에서는 폴리다이아세틸렌(polydiacetylene, PDA) 베시클을 이용하여 여러 가지 킬레이트제(chelating agent)를 쉽게 검출할 수 있는 센서 시스템을 개발하였다. 다른 센서들과 비교하여 PDA기반 센서는 많은 장점이 있다. 첫째로, 형광물질의 부착이 필요 없는 무표지 검출(label-free detection)이 가능하여 실험 절차가 간단하고 빠르다. 둘째로, PDA는 청색에서 외부 자극에 의해 적색으로 변화하는 색전이를 일으키므로 육안으로 쉽게 검출을 확인할 수 있었다. 끝으로, 특정 파장에서의 colorimetric response를 측정하여 각각의 킬레이트제의 농도에 따른 정량검출도 가능하다. 본 연구에서는 5가지 종류의 킬레이트제, 즉 EDTA, EGTA, NTA, DCTA, DTPA를 PDA 베시클과 반응시켰으며, 이중에서 EDTA, DCTA는 특히 강한 반응으로 PDA의 색전이를 유도함을 알 수 있었다. 본 연구를 통하여 PDA 베시클을 사용하여 어떠한 기계나 동력을 사용하지 않고 색전이를 이용하여 킬레이트를 성공적으로 검출할 수 있음을 보여주었다.

• 한국식품위생안전성학회지
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• 제34권6호
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• pp.509-518
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• 2019

사람이 섭취하는 식품 내의 항생제, 알레르기 유발 물질, 병원균 및 기타 오염물질의 수준을 모니터링하기 위해서는, 빠르고 정확하며 저렴한 비용으로 테스트 해야 한다. 이러한 문제 중 일부를 해결하기 위해 지난 10-15년 동안 진보된 기술(label-free biosensor assays)이 개발되어 왔다. 이 면역감지키트들은 실시간 측정이 가능하고, 높은 수준의 자동화를 제공하며, 향상된 처리율과 민감도를 가지고 있다. 또한, 기존의 방법과 비교하여 가격이 저렴하고, 덜 복잡하며, 분석 시간을 단축시켜주는 사용자 친화적 키트이다. 이 리뷰에서는 면역감지키트의 장단점, 그리고 미래의 식품안전검사에서의 사용성에 관한 것에 대해 논의해 볼 것이다.

• Bulletin of the Korean Chemical Society
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• 제34권3호
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• pp.815-819
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• 2013

Micropatterns of streptavidin and human epidermal carcinoma A431 cells were successfully imaged, as received and without any labeling, using cluster $Au_3{^+}$ ion beam-based time-of-flight secondary ion mass spectrometry (TOF-SIMS) together with a principal component analysis (PCA). Three different analysis ion beams ($Ga^+$, $Au^+$ and $Au_3{^+}$) were compared to obtain label-free TOF-SIMS chemical images of micropatterns of streptavidin, which were subsequently used for generating cell patterns. The image of the total positive ions obtained by the $Au_3{^+}$ primary ion beam corresponded to the actual image of micropatterns of streptavidin, whereas the total positive-ion images by $Ga^+$ or $Au^+$ primary ion beams did not. A PCA of the TOF-SIMS spectra was initially performed to identify characteristic secondary ions of streptavidin. Chemical images of each characteristic ion were reconstructed from the raw data and used in the second PCA run, which resulted in a contrasted - and corrected - image of the micropatterns of streptavidin by the $Ga^+$ and $Au^+$ ion beams. The findings herein suggest that using cluster-ion analysis beams and multivariate data analysis for TOF-SIMS chemical imaging would be an effectual method for producing label-free chemical images of micropatterns of biomolecules, including proteins and cells.

• 전자공학회논문지SC
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• 제48권2호
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• pp.86-90
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• 2011

전계효과 트랜지스터(FETs)를 이용한 전하 검출형 DNA센서는 DNA가 가지고 있는 음전하를 중성화 시키는 양이온의 영향은 매우 중요하다. 본 논문에서는 양이온 농도에 의존하는 Debye length에 관한 연구를 통해 DNA 검출감도를 평가하였다. Debye length는 낮은 농도의 NaCl 용액에서 긴 거리를 유지하며, Debye length가 높은 용액에서 DNA가 가지고 있은 음전하는 게이트 채널에 보다 많은 영향을 미친다. 용액내 NaCl농도가 1 mM인 버퍼 용액에서 상보적 DNA의 hybridization에 의한 전계효과 트랜지스터의 게이전압은 21 mV 시프트 했으며, NaCl 농도가 10 mM인 버퍼 용액에서는 7.2 mV, NaCl농도가 100 mM인 버퍼 용액에서는 전계효과 트랜지스터의 게이트 전압이 5.1 mV 각각 시프트 하였다. 이러한 결과를 바탕으로 전계효과 트랜지스터를 이용한 전하 검출형 DNA센서의 검출 감도는 Debye length에 의존하는 것을 규명하였다.

• Journal of Microbiology and Biotechnology
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• 제27권7호
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• pp.1345-1358
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• 2017

The impact of overproduction of a heterologous protein on the metabolic system of host Lactococcus lactis was investigated. The protein expression profiles of L. lactis IL1403 containing two near-identical plasmids that expressed high- and low-level of the green fluorescent protein (GFP) were examined via shotgun proteomics. Analysis of the two strains via high-throughput LC-MS/MS proteomics identified the expression of 294 proteins. The relative amount of each protein in the proteome of both strains was determined by label-free quantification using the spectral counting method. Although expression level of most proteins were similar, several significant alterations in metabolic network were identified in the high GFP-producing strain. These changes include alterations in the pyruvate fermentation pathway, oxidative pentose phosphate pathway, and de novo synthesis pathway for pyrimidine RNA. Expression of enzymes for the synthesis of dTDP-rhamnose and N-acetylglucosamine from glucose was suppressed in the high GFP strain. In addition, enzymes involved in the amino acid synthesis or interconversion pathway were downregulated. The most noticeable changes in the high GFP-producing strain were a 3.4-fold increase in the expression of stress response and chaperone proteins and increase of caseinolytic peptidase family proteins. Characterization of these host expression changes witnessed during overexpression of GFP was might suggested the metabolic requirements and networks that may limit protein expression, and will aid in the future development of lactococcal hosts to produce more heterologous protein.

• JSTS:Journal of Semiconductor Technology and Science
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• 제7권3호
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• pp.140-150
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• 2007

A simple, label-free microfluidic cell purification method is presented for separation of cancer cells by exploiting difference in cell adhesion. To maximize the adhesion difference, three types of polymeric nanostructures (50nm pillars, 50nm perpendicular and 50nm parallel lines with respect to the direction of flow) were fabricated using UV-assisted capillary moulding and included inside a polydimethylsiloxane (PDMS) microfluidic channel bonded onto glass substrate. The adhesion force of human breast epithelial cells (MCF10A) and human breast carcinoma (MCF7) was measured independently by injecting each cell line into the microfluidic device followed by culture for a period of time (e.g., one, two, and three hours). Then, the cells bound to the floor of a microfluidic channel were detached by increasing the flow rate of medium in a stepwise fashion. It was found that the adhesion force of MCF10A was always higher than that of MCF cells regardless of culture time and surface nanotopography at all flow rates, resulting in a label-free detection and separation of cancer cells. For the cell types used in our study, the optimum separation was found for 2 hours culture on 50nm parallel line pattern followed by flow-induced detachment at a flow rate of $300{\mu}l/min$.

• Journal of Ginseng Research
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• 제44권1호
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• pp.50-57
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• 2020

Background: The cellular senescence of primary cultured cells is an irreversible process characterized by growth arrest. Restoration of senescence by ginsenosides has not been explored so far. Rg3(S) treatment markedly decreased senescence-associated β-galactosidase activity and intracellular reactive oxygen species levels in senescent human dermal fibroblasts (HDFs). However, the underlying mechanism of this effect of Rg3(S) on the senescent HDFs remains unknown. Methods: We performed a label-free quantitative proteomics to identify the altered proteins in Rg3(S)-treated senescent HDFs. Upregulated proteins induced by Rg3(S) were validated by real-time polymerase chain reaction and immunoblot analyses. Results: Finally, 157 human proteins were identified, and variable peroxiredoxin (PRDX) isotypes were highly implicated by network analyses. Among them, the mitochondrial PRDX3 was transcriptionally and translationally increased in response to Rg3(S) treatment in senescent HDFs in a time-dependent manner. Conclusion: Our proteomic approach provides insights into the partial reversing effect of Rg3 on senescent HDFs through induction of antioxidant enzymes, particularly PRDX3.

• Journal of Ginseng Research
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• 제43권1호
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• pp.143-153
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• 2019

Background: Ginseng is one of the well-known medicinal plants, exhibiting diverse medicinal effects. Its roots possess anticancer and antiaging properties and are being used in the medical systems of East Asian countries. It is grown in low-light and low-temperature conditions, and its growth is strongly inhibited at temperatures above $25^{\circ}C$. However, the molecular responses of ginseng to heat stress are currently poorly understood, especially at the protein level. Methods: We used a shotgun proteomics approach to investigate the effect of heat stress on ginseng leaves. We monitored their photosynthetic efficiency to confirm physiological responses to a high-temperature stress. Results: The results showed a reduction in photosynthetic efficiency on heat treatment ($35^{\circ}C$) starting at 48 h. Label-free quantitative proteome analysis led to the identification of 3,332 proteins, of which 847 were differentially modulated in response to heat stress. The MapMan analysis showed that the proteins with increased abundance were mainly associated with antioxidant and translation-regulating activities, whereas the proteins related to the receptor and structural-binding activities exhibited decreased abundance. Several other proteins including chaperones, G-proteins, calcium-signaling proteins, transcription factors, and transfer/carrier proteins were specifically downregulated. Conclusion: These results increase our understanding of heat stress responses in the leaves of ginseng at the protein level, for the first time providing a resource for the scientific community.

• 융합신호처리학회논문지
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• 제24권1호
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• pp.76-81
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• 2023

암세포와 정상세포를 구분하기 위해서는 H&E(Hematoxylin&Eosin) 염색이 필요하다. 병리 염색은 많은 비용과 시간이 필요하다. 최근 이러한 비용과 시간을 줄이고자 디지털 염색 방법이 소개되고 있다. 본 연구에서는 병리 H&E 염색의 디지털 변환 방법에 대한 새로운 알고리즘을 제안한다. 첫 번째 알고리즘은 Pair방법이다. 본 방법은 FPM(Fourier Ptychographic Microscopy)으로 촬영된 염색된 Phase 영상과 염색되지 않은 Amplitude 영상을 학습하여 염색된 Amplitude 영상으로 변환한다. 두 번째 알고리즘은 Unpair방법이다. 본 방법은 염색된 형광현미경 영상과 염색되지 않은 형광현미경 영상을 학습하여 모델링하여 디지털 염색을 수행한다. 본 연구에서는 GAN(generative Adversarial Network)를 활용하여 디지털 염색을 진행하였다. 연구 결과 Pair방법과 Unpair방법 모두 우수한 성능의 디지털 염색 결과를 확보하였다.