• Journal of Microbiology and Biotechnology
• /
• 제29권2호
• /
• pp.274-282
• /
• 2019

Mercury-resistant ($Hg^R$) bacteria were isolated from heavy metal polluted wastewater and soil collected near to tanneries of district Kasur, Pakistan. Bacterial isolates AZ-1, AZ-2 and AZ-3 showed resistance up to $40{\mu}g/ml$ against mercuric chloride ($HgCl_2$). 16S rDNA ribotyping and phylogenetic analysis were performed for the characterization of selected isolates as Bacillus sp. AZ-1 (KT270477), Bacillus cereus AZ-2 (KT270478) and Bacillus cereus AZ-3 (KT270479). Phylogenetic relationship on the basis of merA nucleotide sequence confirmed 51-100% homology with the corresponding region of the merA gene of already reported mercury-resistant Gram-positive bacteria. The merE gene involved in the transportation of elemental mercury ($Hg^0$) via cell membrane was cloned for the first time into pHLV vector and transformed in overexpressed C43(DE3) E. coli cells. The recombinant plasmid (pHLMerE) was expressed and the native MerE protein was obtained after thrombin cleavage by size exclusion chromatography (SEC). The purification of fusion/recombinant and native protein MerE by Ni-NTA column, dialysis and fast protein liquid chromatography (FPLC/SEC) involved unfolding/refolding techniques. A small-scale reservoir of wastewater containing $30{\mu}g/ml$ of $HgCl_2$ was designed to check the detoxification ability of selected strains. It resulted in 83% detoxification of mercury by B. cereus AZ-2 and B. cereus AZ-3, and 76% detoxification by Bacillus sp. AZ-1 respectively (p < 0.05).

• 한국자원식물학회지
• /
• 제31권6호
• /
• pp.652-659
• /
• 2018

In this study, baicalin, as a marker substance of Scutellariae Radix, was quantitatively analyzed by a high performance liquid chromatography-photodiode array detector (HPLC-DAD). We identified wogonoside, baicalein, and wogonin in the Scutellariae Radix by a high performance liquid chromatography-electrospray ionization-mass spectrometer (HPLC-ESI-MS). The baicalin was separated on a Xterra C18 column ($5{\mu}m$, $4.6{\times}250mm$) using mobile phase consisting of 38% acetonitrile in 0.68% phosphoric acid. The baicalin spectrum in the Scutellariae Radix extracts was coincided by comparing with UV-visible spectrum (200-550 nm) of baicalin standard in the library. The amount of baicalin in Scutellariae Radix was 10.46%, which is higher than KFDA's guideline. The marker substances of Scutellariae Radix showed a strong base peak $[M]^+$ in the positive detection mode following as; baicalin (m/z; $271[MH^+-sugar]^+$, $447[M+H]^+$), wogonoside (m/z; $285[MH^+-sugar]^+$, $461[M+H]^+$), baicalein (m/z; $271\;[M+H]^+$), wogonin (m/z; $285[M+H]^+$). These results are consistent with the fragment pattern and molecular weight of standard components from literature.

• Environmental Engineering Research
• /
• 제24권3호
• /
• pp.389-396
• /
• 2019

The electrical energy consumption (EEC) in removal of NO by a $UV/H_2O_2$ oxidation process was introduced and related to removal efficiency of this gas. The absorption-reaction of NO was conducted in a bubble column reactor in the presence of $SO_2$. The variation in NO removal efficiency was investigated for various process parameters including NO and $SO_2$ inlet concentrations, initial concentration of $H_2O_2$ solution and gas flow rate. EEC values were obtained in these different conditions. The removal efficiency was increased from about 22% to 54.7% when $H_2O_2$ concentration increased from 0.1 to 1.5 M, while EEC decreased by about 70%. However, further increase in $H_2O_2$ concentration, from 1.5 to 2, had no significant effect on NO absorption and EEC. An increase in NO inlet concentration, from 200 to 500 ppm, decreased its removal efficiency by about 10%. However, EEC increased from $2.9{\times}10^{-2}$ to $3.9{\times}10^{-2}kWh/m^3$. Results also revealed that the presence of $SO_2$ had negative effect on NO removal percentage and EEC values. Some experiments were conducted to investigate the effect of $H_2O_2$ solution pH. The changing of pH of oxidation-absorption medium in the ranges between 3 to 10, had positive and negative effects on removal efficiency depending on pH value.

• Mass Spectrometry Letters
• /
• 제10권1호
• /
• pp.18-26
• /
• 2019

We developed a bioanalytical method for simultaneous determination of nine NBOMe derivatives (25H-NBOMe, 25B-NBOMe, 25E-NBOMe, 25N-NBOMe, 25C-NBOH, 25I-NBOH, 25B-NBF, 25C-NBF, and 25I-NBF) in human plasma using liquid chromatography tandem mass spectrometry (LC-MS/MS). Human plasma samples were pre-treated using solid-phase extraction. Separation was achieved on a C18 column under gradient elution using a mobile phase containing 0.1% formic acid in acetonitrile and 0.1% formic acid in water at a flow rate of 0.3 mL/min. Mass detection was performed in the positive ion mode using multiple reaction monitoring. The calibration range was 1-100 ng/mL for all quantitative analytes, with a correlation coefficient greater than 0.99. The intra- and inter-day precision and accuracy varied from 0.85 to 6.92% and from 90.19 to 108.69%, respectively. The recovery ranged from 86.36 to 118.52%, and the matrix effects ranged from 27.09 to 99.72%. The stability was acceptable in various conditions. The LC-MS/MS method was validated for linearity, accuracy, precision, matrix effects, recovery and stability in accordance with the FDA guidance. The proposed method is suitable for reliable and robust routine screening and analysis of nine NBOMe derivatives in forensic field.

• Biomolecules & Therapeutics
• /
• 제27권2호
• /
• pp.193-200
• /
• 2019

Ceramide metabolism is known to be an essential etiology for various diseases, such as atopic dermatitis and Gaucher disease. Glucosylceramide synthase (GCS) is a key enzyme for the synthesis of glucosylceramide (GlcCer), which is a main ceramide metabolism pathway in mammalian cells. In this article, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to determine GCS activity using synthetic non-natural sphingolipid C8-ceramide as a substrate. The reaction products, C8-GlcCer for GCS, could be separated on a C18 column by reverse-phase high-performance liquid chromatography (HPLC). Quantification was conducted using the multiple reaction monitoring (MRM) mode to monitor the precursor-to-product ion transitions of m/z $588.6{\rightarrow}264.4$ for C8-GlcCer at positive ionization mode. The calibration curve was established over the range of 0.625-160 ng/mL, and the correlation coefficient was larger than 0.999. This method was successfully applied to detect GCS in the human hepatocellular carcinoma cell line (HepG2 cells) and mouse peripheral blood mononuclear cells. We also evaluated the inhibition degree of a known GCS inhibitor 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) on GCS enzymatic activity and proved that this method could be successfully applied to GCS inhibitor screening of preventive and therapeutic drugs for ceramide metabolism diseases, such as atopic dermatitis and Gaucher disease.

• Steel and Composite Structures
• /
• 제39권1호
• /
• pp.65-80
• /
• 2021

Irregularities of a building in plan and elevation, which results in the change in stiffness on different floors highly affect the seismic performance and resistance of a structure. This study motivated to investigate the seismic responses of high-rise steel-frame buildings of twelve stories with various stiffness irregularities. The building has five spans of 3200 mm distance in both X- and Z-directions in the plan. The design package SAP2000 was adopted for the design of beams and columns and resulted in the profile IPE500 for the beams of all floors and box sections for columns. The column cross-section dimensions vary concerning the number of the story; one to three: 0.50×0.50×0.05m, four to seven: 0.45×0.45×0.05 m, and eight to twelve: 0.40×0.40×0.05 m. Real recorded ground accelerations obtained from the Vrancea earthquake in Romania together with dead and live loads corresponding to each story were considered for the applied load. The model was validated by comparing the results of the current method and literature considering a three-bay steel moment-resisting frame of eight-story height subject to seismic load. To investigate the seismic performance of the buildings, the time-history analysis was performed using ABAQUS. Deformed shapes corresponding to negative and positive peaks were provided followed by the story drifts and fragility curves which were used to examine the probability of collapse of the building. From the results, it was concluded that regular buildings provided a seismic performance much better than irregular buildings. Furthermore, it was observed that building with torsional irregularity was more vulnerable to seismic failure.

• Mass Spectrometry Letters
• /
• 제12권1호
• /
• pp.1-10
• /
• 2021

A simple, specific, and economical LC-MS/MS method was investigated for the screening of 43 prescribed antihypertensive and related drugs in human urine. The urine samples were simply prepared by diluting and mixing with internal standard before directly introduced to the LC-MS/MS system, which is fast, straightforward, and cost-effective. Fractional factorial, Box-Behnken, and I-optimal design were applied to screen and optimize the mass spectrometric and chromatographic factors. The analysis was carried out on a triple quadrupole mass spectrometer system utilizing multiple reaction monitoring with positive and negative electrospray ionization method. Chromatographic separation was performed on a Thermo Scientific Accucore RP-MS column (50 × 3.0 mm ID., 2.6 ㎛) using two separate gradient elution programs established with the same mobile phases. Chromatographic separation was performed within 12 min. The optimal method was validated based on FDA guideline. The results indicated that the assay was specific, reproducible, and sensitive with the limit of detection from 0.1 to 50.0 ㎍/L. The method was linear for all analytes with coefficient of determination ranging from 0.9870 to 0.9981. The intra-assay precision was from 1.44 to 19.87% and the inter-assay precision was between 2.69 and 18.54% with the recovery rate ranges from 84.54 to 119.78% for all drugs measured. All analytes in urine samples were stable for 24 h at 25℃, and for 2 weeks at -60℃. The developed method improves on currently existing methods by including larger number of cardiovascular medications and better sensitivity of 12 analytes.

• 대한한의학방제학회지
• /
• 제32권3호
• /
• pp.223-233
• /
• 2024

Objectives : Natural products containing bioactive compounds with high antioxidant activity are potentially important sources that can contribute to the improvement of various diseases. Therefore, the aim of this study was to investigate phenolic compounds of Cinnamomum cassia (C. cassia) ethanolic extract (CCEE). And then we evaluated the antioxidant effect. Methods : We used liquid chromatography with tandem mass spectrometry (LC-MS/MS) to identify the compounds in CCEE. LC-MS/MS was performed in positive ion mode using Shimadzu, Nexera HPLC system and IDA TOF mass system. Solvent A was distilled water and solvent B was acetonitrile as mobile phase. The analysis was performed at a flow rate of 0.5 ml/min, column temperature of 35 ℃ and wavelength of 284 nm. The antioxidant effect of CCEE was analyzed using DPPH (2,2-diphenyl-2-picrylhydrazyl free radical) and ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)). In addition, total phenolics and total flavonoids contents were measured to determine antioxidant effects. Results : Analysis using LC-MS/MS identified four compounds: Coumarin, Trans-cinnamaldehyde, Trans-cinnamic acid, and 2-Methoxycinnamaldehyde. Free radicals decreased in a concentration-dependent manner starting from 10 ㎍/ml of CCEE, and decreased to a level similar to Ascorbic acid (AA) from a concentration of 60 ㎍/ml onwards. Conclusions : Based on the findings, CCEE exhibits strong antioxidant activity as evidenced by the presence of Coumarin, Trans-cinnamaldehyde, Trans-cinnamic acid, and 2-Methoxycinnamaldehyde. Consequently, this study suggests that CCEE can serve as an important source of natural antioxidants and can be efficiently used in the management of oxidative stress diseases.

• 한국미생물·생명공학회지
• /
• 제26권2호
• /
• pp.137-142
• /
• 1998

본 실험에서는 만성위염, 위궤양, 십이지장궤양, 위암발생의 원인균으로 알려진 H. pylori를 저해하는 특이항균물질을 생성하는 균주를 토양에서 분리한 방선균액과 곰팡이 추출액을 대상으로 탐색하여 곰팡이 균주번호 60686을 선별하였다. Jar fermentor로 배양하여 얻은 균체를 acetone 및 EtOAc로 추출하였고, 얻어진 추출액을 silica column chromatography와 LH-20 gel chromatography를 수행하여 활성분획을 농축하였고 HPLC를 사용하여 항균 활성을 나타내는 단일물질 IDA를 분리 정제하였다. 항균 활성물질 IDA의 구조를 MS, NMR 분석등을 통해 추정한 결과 곰팡이의 2차 대사산물인 cytochalasan의 전형적 구조를 갖는 분자식 $C_{32}H_{36}N_2O_5$의 chaetoglobosin A라는 물질로 판명되었다. 항균물질 IDA의 항균력을 paper disk법으로 실험한 결과 그람양성 균주중에서는 S. aureus SG 511, 285와 503 3주에서만 항균력을 보였고, 그람음성 균주중에서는 H. pylori 4주에서만 항균력을 보였으나 동일농도 처리시 H. pylori에 대한 항균력이 S. aureus에 비해 우수한 항균효과를 가진다고 판단되었다.

• 한국식품저장유통학회지
• /
• 제22권4호
• /
• pp.559-566
• /
• 2015

본 연구에서는 곡류 중 트리코테센류 곰팡이독소인 T-2 독소 및 HT-2 독소의 LC-MS/MS 분석방법을 검증하고 국내 유통 곡류 중 T-2 독소 및 HT-2 독소의 오염실태를 파악하였다. 곡류 중의 T-2 독소 및 HT-2 독소를 분석하기 위해, 염화나트륨을 포함한 90% 메탄올 용액으로 추출, 원심분리, 여과, 4% 염화나트륨용액으로 희석하고, 원심분리한 후, 여과한 후 면역친화성칼럼에 의해 정제한 시료를 LC-MS/MS 동시정량 분석하였다. T-2 독소 및 HT-2 독소의 검출한계 및 정량한계는 각각 $0.5{\mu}g/kg$ 및 $1.5{\mu}g/kg$ 얻었다. matrix-matched 표준 검량식에서 상관계수 0.99 이상의 직선성을 얻었으며, T-2독소와 HT-2 독소 2배에서 10배의 정량한계로 표준용액을 첨가한 시료에서 회수율은 T-2독소와 HT-2 독소 각각 $100.6{\pm}7.2%$, $96.8{\pm}9.4%$로 EU 가이드라인에서 제시하는 유효성 기준을 만족하였다. LC-MS/MS 정량법을 이용하여 국내 곡류 9품목 115건에 대해 T-2 독소와 HT-2 독소의 오염도를 조사하여 본 결과, 전체 곡류 115건 중에서 T-2 독소는 83건, HT-2 독소는 93건 검출되었으며 오염도는 T-2 독소는 N.D~37.1 ug/kg, HT-2 독소는 N.D~5.4 ug/kg 으로 낮은 수준이었으며, 오염도는 유럽기준치($100{\mu}g/kg$)이내 이었다. 본 연구에서 개발된 곡류 중 T-2 독소 및 HT-2 독소에 대한 분석법은 향후 우리나라 곡류 중 곰팡이독소 안전관리를 위한 시험법으로 활용가능하며, 오염도 자료는 안전성 평가의 기초자료로 활용이 가능할 것으로 사료된다.