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Analytical Biochemistry
Journal Prestige (SJR): 0.633
Citation Impact (citeScore): 2
Number of Followers: 184  
  Hybrid Journal Hybrid journal (It can contain Open Access articles)
ISSN (Print) 0003-2697 - ISSN (Online) 1096-0309
Published by Elsevier Homepage  [3161 journals]
  • Ultrasound-enhanced scintillation proximity assay for rapid diagnostics
    • Abstract: Publication date: Available online 13 February 2019Source: Analytical BiochemistryAuthor(s): So-Young Lee, Jae-Cheong Lim, Eun-Ha Cho, Seung-Kon Lee, Sung-Hee Jung Scintillation proximity assay (SPA) is a type of radioimmunoassay (RIA). We apply ultrasound enhancement to the general SPA. All assay procedures, including the antibody coating and radiolabeled antigen binding are achieved by simply mixing then standing for 5 min in an ultrasound chamber. No additional incubation time is required. To further demonstrate the capability of the UE-SPA, a quantitative measurement of CD55 in various grades of colon tumors was assessed on human tissue slides. The results showed a significant correlation between CD55 expression and tumorigenesis. In conclusion, we confirmed that UE-SPA is a reliable, rapid and alternative to RIA.
  • Development of a sandwich enzyme-linked immunosorbent assay for the
           quantification of ponatinib in serum
    • Abstract: Publication date: Available online 13 February 2019Source: Analytical BiochemistryAuthor(s): Yuta Yamamoto, Tetsuya Saita, Rintaro Sogawa, Kenji Ogata, Yutaro Yamamoto, Sakiko Kimura, Yutaka Narisawa, Shinya Kimura, Masashi Shin The tyrosine kinase inhibitor ponatinib is extensively metabolized in the body, and consequently the development of specific immunoassays for pharmacokinetic studies and therapeutic drug monitoring of ponatinib is challenging. If two antibodies simultaneously recognize the entire structure of ponatinib, they could be utilized to establish an ultra-specific sandwich immunoassay for ponatinib, free of any interference from ponatinib metabolites. In this study, we created two types of anti-ponatinib polyclonal antibodies that recognize two different ponatinib epitopes, and sandwiched almost all structural components of ponatinib in these two antibodies in order to develop an enzyme-linked immunosorbent assay (ELISA) technique not affected by any ponatinib metabolites. After optimization, this sandwich ELISA showed a linear detection range of 640 pg/mL to 2000 ng/mL and a limit of quantification of 640 pg/mL. This sandwich ELISA was specific to ponatinib and showed no cross-reactivity with the major metabolite M14. Comparison between the sandwich ELISA and HPLC, using serum samples from 15 rats orally administered a single dose of 15 mg/kg ponatinib, showed a linear regression (y = 0.9662x + 3.5354, r = 0.9683). Thus, in this study, we successfully developed the first ultra-specific sandwich ELISA for ponatinib in serum.
  • Reduced levels of modified nucleosides in the urine of autistic children.
           Preliminary studies
    • Abstract: Publication date: Available online 13 February 2019Source: Analytical BiochemistryAuthor(s): Barbara Bobrowska-Korczak, Paulina Gątarek, Angelina Rosiak, Joanna Giebultowicz, Joanna Kałużna-Czaplińska The aim of this study was to investigate and compare the levels of concentration of modified nucleosides in the urine of autistic and healthy children. The compounds have never been analyzed before. The levels of nucleosides in the urine of both groups were determined by validated high performance liquid chromatography coupled to mass spectrometry (LC-MS/MS) method using multiple reaction monitoring (MRM) mode. Chromatographic separation was achieved with HILIC column and tubercidin was used as the internal standard for the quantification of urinary nucleosides. The within run accuracy and precision ranged from 89 to 106% and from 0.8% to 4.9%, respectively. Lower levels of O-methylguanosine, 7-methylguanosine, 1-methyladenosine, 1-methylguanine, 7-methylguanine and 3-methyladenine in the urine of 22 children with autism, aged 3 to 16 were observed. The differences were not observed in 20 healthy volunteers, in a similar age group. These findings show that modified nucleosides there are metabolic disturbances and nutritional deficiencies in autistic children.
  • Methodological aspects of Universal immuno-PCR on standard tubes
    • Abstract: Publication date: Available online 12 February 2019Source: Analytical BiochemistryAuthor(s): J.E. Abud, C.G. Santamaría, M. Oggero, H.A. Rodriguez One of the most used formats in inmuno-polymerase chain reaction (IPCR) is known as “Universal” IPCR (signal-generating complexes is based on conjugates of biotinylated DNA, biotinylated IgG and avidin). In the present study, we evaluated the utility of using mono- and bi-biotinylated DNA probes, pre-self-assembled DNA-neutravidin complex, blocking step and glutaraldehyde pretreatment of standard PCR tubes to improve the analytical performance of the hTSH-IPCR assay. The use of pre-self-assembled mono-biotinylated DNA-neutravidin complex enhances both the sensitivity and the reproducibility of the hTSH-IPCR assay, even without blocking step: hTSH-IPCR assay showed an improved limit of detection (LOD: 0.01 μIU/ml), calibration sensitivity (SEN: 2.4) and analytic sensitivity (γ: 9 μIU/ml−1) in comparison with both a self-made ELISA and a commercial one.
  • Detection of phenol contamination in RNA samples and its impact on qRT-PCR
    • Abstract: Publication date: Available online 8 February 2019Source: Analytical BiochemistryAuthor(s): Conny Unger, Nicole Lokmer, Daniel Lehmann, Ilka M. Axmann Residual phenol, carried over from RNA purification, can alter RNA concentration measurements and is assumed to inhibit PCR. Here, we demonstrate that Impurities A260 values of spectral content profiling (SCP) UV/Vis measurements correlated with phenol concentration, whereas absorbance ratios of classical UV/Vis systems failed to reliably detect phenol in RNA samples. Phenol contamination led to over- or underestimation of RNA concentration on UV/Vis systems, whereas it had no influence on fluorometry quantification. Wrong RNA concentration results led to altered template input in qRT-PCR and consequently caused quantification cycle (Cq) shifts, although ≤ 0.01% phenol had no direct influence.
  • Development and characterization of novel 2′-F-RNA aptamers specific to
           human total and glycated hemoglobins
    • Abstract: Publication date: Available online 8 February 2019Source: Analytical BiochemistryAuthor(s): Anna Davydova, Mariya Vorobyeva, Eugenia Bashmakova, Pavel Vorobjev, Olga Krasheninina, Alexey Tupikin, Marsel Kabilov, Vasilisa Krasitskaya, Ludmila Frank, Alya Venyaminova Aptamers are short DNA and RNA fragments which bind their molecular targets with affinity and specificity comparable to those of antibodies. Here, we describe the selection of novel 2′-F-RNA aptamers against total human hemoglobin or its glycated form HbA1c. After SELEX and high-throughput sequencing of the enriched libraries, affinities and specificities of candidate aptamers and their truncated variants were examined by the solid-phase bioluminescent assay. As a result, we identified aptamers specific to both hemoglobins or only glycated HbA1c. The developed 2′-F-RNA aptamers have shown their applicability for detection of total and glycated hemoglobin in one sample.
  • Pyruvate kinase from Geobacillus stearothermophilus displays an unusual
           preference for Mn2+ in a cycling reaction
    • Abstract: Publication date: Available online 7 February 2019Source: Analytical BiochemistryAuthor(s): Shigeru Ueda, Shin-ichi Sakasegawa Previously, we developed a kinase cycling method using creatine kinase and pyruvate kinase (RMPK) both from rabbit muscle in the presence of an excess amount of ATP and IDP for the quantitative determination of substrate. To our surprise, the RMPK cycling reaction was 10-fold more efficient using Mn2+ rather than Mg2+. Here, we investigated PK from Geobacillus stearothermophilus (GSPK) as an alternative source of enzyme. Spectrophotometric real-time detection was accomplished by coupling the reaction to ADP-dependent glucokinase (ADP-GK) together with glucose-6-phosphate dehydrogenase (G6PD). The rate of increase in absorbance of NADH at 340 nm was monitored. GSPK displayed an even greater preference than RMPK for Mn2+ over Mg2+ in the cycling reaction with ATP and GDP or ATP and IDP. The much lower Km values for the substrate in the presence of Mn2+ rather than Mg2+ are consistent with the results of the cycling reaction observed in this study.
  • Rapid time-resolved luminescence based screening of bacteria in urine with
           luminescence modulating biosensing phages
    • Abstract: Publication date: Available online 5 February 2019Source: Analytical BiochemistryAuthor(s): Janne Kulpakko, Kaisu Rantakokko-Jalava, Erkki Eerola, Pekka E. Hänninen Urinary tract infections (UTIs) are a common problem worldwide. The most prevalent causative pathogen of UTI is Escherichia coli, focus of this study. The current golden standard for detecting UTI is bacterial culture, creating a major workload for hospital laboratories - cost-effective and rapid mass screening of patient samples is needed. Here we present an alternative approach to screen patient samples with a single-step assay utilising time-resolved luminescence and luminescence modulating biosensing phages. Filamentous phage M13 was biopanned for binding luminescence quenching metal (copper) and further E. coli. The screening assay luminescence modulation was further enhanced by selecting right chemical environment for the functioning phage clones. Semi-specific interaction between phage, target bacteria and metal was detected by modulation in the signal of a weakly chelating, easily quenchable lanthanide complex. In the presence of the target pathogen, the phages collected quenching metal from solution to the bacterial surface changing the quenching effect on the lanthanide label and thus modulating the signal. Our method was compared with the bacterial culture data obtained from 70 patient samples. The developed proof-of-principle screening assay showed sensitivity and a specificity at the 90% mark when compared to culture method although some samples had high turbidity and even blood. The detection limit of E. coli was in the range of 1000–10 000 colony forming units/mL. Untreated urine sample was screened and time-resolved luminescence signal result was achieved within 10 min in a single incubation step.
  • Single-site phosphorylation within the His-tag sequence attached to a
           recombinant protein
    • Abstract: Publication date: Available online 5 February 2019Source: Analytical BiochemistryAuthor(s): Himanshu Singh, Deepshikha Verma, Benjamin Bardiaux We report the observation of single-site phosphorylation in a His-tag sequence N-terminally attached to a recombinant protein (UVI31+) in vitro. This modification was detected at position 23 at a serine residue of the His-tag sequence encoded by the vector pET28a. Furthermore, the phosphorylated tag sequence was found to be dephosphorylated by the action of alkaline phosphatases. The functional activity and dynamics of the protein carrying the His-tag sequence were unchanged after phosphorylation. The possibility of phosphorylation within the N-terminal His-tag demonstrates that care has to be taken upon analysis of post-translational modifications via mass spectrometry for recombinant protein expression strategies.
  • Surface plasmon resonance and cytotoxicity assays of drug efficacies
           predicted computationally to inhibit p53/MDM2 interaction
    • Abstract: Publication date: Available online 2 February 2019Source: Analytical BiochemistryAuthor(s): Xiaoying Wang, Patrycja Magdziarz, Ernest Enriquez, Wang Zhao, Chris Quan, Narek Darabedian, Jamil Momand, Feimeng Zhou Docking on the p53-binding site of murine double minute 2 (MDM2) by small molecules restores p53's tumor-suppressor function. We previously assessed 3244 FDA-approved drugs via “computational conformer selection” for inhibiting MDM2 and p53 interaction. Here, we developed a surface plasmon resonance method to experimentally confirm the inhibitory effects of the known MDM2 inhibitor, nutlin-3a, and two drug candidates predicted by our computational method. This p53/MDM2 interaction displayed a dosage-dependent weakening when MDM2 is pre-mixed with drug candidates. The inhibition efficiency order is nutlin-3a (IC50 = 97 nM) > bepridil (206 nM) > azelastine (307 nM). Furthermore, we verified their anti-proliferation effects on SJSA-1 (wild-type p53 and overexpressed MDM2), SW480 (mutated p53), and SaOs-2 (deleted p53) cancer cell lines. The inhibitory order towards SJSA-1 cell line is nutlin-3a (IC50 = 0.8 μM) > bepridil (23 μM) > azelastine (25 μM). Our experimental results are in line with the computational prediction, and the higher IC50 values from the cell-based assays are due to the requirement of higher drug concentrations to penetrate cell membranes. The anti-proliferation effects of bepridil and azelastine on the cell lines with mutated and deleted p53 implied some p53-independent anti-proliferation effects.
  • Direct and highly sensitive measurement of fluorescent molecules in bulk
           solutions using flow cytometry
    • Abstract: Publication date: Available online 30 January 2019Source: Analytical BiochemistryAuthor(s): Matthias Wurm, Sibel Ilhan, Uwe Jandt, An-Ping Zeng Utilizing flow cytometry to monitor progress of bulk biochemical reactions and concentration of chemical species normally relies on the utilization of cells carrying intrinsic fluorescence or modified beads. We present a method for a simple measurement of the fluorescent marker molecule fluorescein and GFPuv in bulk solutions with high sensitivity using a CytoFLEX flow cytometer and without the need for modified beads. Polystyrene beads were used to trigger measurements based on their high scatter signal, to detect the fluorescence signal from two different fluorophores present in the sample solution. We report sensitivities of 33 pg/mL for fluorescein and 50 ng/mL for GFPuv. This method is comparable in sensitivity to a typical spectrometric fluorescence assay tested with fluorescein, and approximately ten times more sensitive for the measurement of GFPuv. PEG was added to the sample at a low concentration of 0.001% (w/v) to block unspecific GFPuv binding to the beads. The method was further applied to measure the GFPuv concentration in crude cell lysate samples used for cell free protein expression. An advantage of this method over spectrometric assays is the ability to differentiate signal subpopulations in the sample based on their individual fluorescence intensities.
  • A simple fluorescent assay for the discovery of protein-protein
           interaction inhibitors
    • Abstract: Publication date: Available online 30 January 2019Source: Analytical BiochemistryAuthor(s): Mona Al-Mugotir, Carol Kolar, Krysten Vance, David L. Kelly, Amarnath Natarajan, Gloria E.O. Borgstahl Due to the therapeutic potential of targeting protein-protein interactions (PPIs) there is a need for easily executed assays to perform high throughput screening (HTS) of inhibitors. We have developed and optimized an innovative and robust fluorescence-based assay for detecting PPI inhibitors, called FluorIA (Fluorescence-based protein-protein Interaction Assay). Targeting the PPI of RAD52 with replication protein A (RPA) was used as an example, and the FluorIA protocol design, optimization and successful application to HTS of large chemical libraries are described. Here enhanced green fluorescent protein (EGFP)-tagged RAD52 detected the PPI using full-length RPA heterotrimer coated, black microtiter plates and loss in fluorescence intensity identified small molecule inhibitors (SMIs) that displaced the EGFP-tagged RAD52. The FluorIA design and protocol can be adapted and applied to detect PPIs for other protein systems. This should push forward efforts to develop targeted therapeutics against protein complexes in pathological processes.Graphical abstractImage 1
  • Effects of incubation temperature and acetonitrile amount on
           microwave-assisted tryptic digestion of proteins
    • Abstract: Publication date: Available online 29 January 2019Source: Analytical BiochemistryAuthor(s): Yeoseon Kim, Dabin Lee, Jeongkwon Kim The effects of incubation temperature and acetonitrile (ACN) amount on microwave-assisted tryptic digestion of horse skeletal muscle myoglobin (MYG) and bovine serum albumin (BSA) were investigated. Microwave-assisted tryptic digestion was performed on BSA or MYG solutions containing different amounts (0, 10, and 20%) of ACN for different times (10, 20, 30, 40, and 50 min) at different temperatures (25, 37, and 55 °C). Conventional overnight tryptic digestion was also conducted with gentle mixing at 37 °C for 16 h. Digested samples were analyzed using matrix-assisted laser desorption/ionization mass spectrometry. Similar sequence coverage (SC) values were obtained under most conditions except when the protein sample solutions were digested with 20% ACN at 55 °C, which provided the lowest SC for both MYG and BSA for all investigated digestion times. Considering the missed cleavage (MC) ratios for 50-min microwave-assisted digestion, the highest MC ratio, (i.e., lowest trypsin activity) was observed for the digestion condition of 20% ACN at 55 °C for both proteins, while the lowest MC ratio was observed with 0% ACN at 25 °C for MYG and with 0% ACN at 55 °C for BSA. Conventional overnight tryptic digestion at 37 °C provided more completely cleaved peptides than 50-min microwave-assisted tryptic digestion at the same temperature.Graphical abstractImage 1
  • Polydopamine-functionalized magnetic iron oxide for the determination of
           trace levels of lead in bovine milk
    • Abstract: Publication date: Available online 25 January 2019Source: Analytical BiochemistryAuthor(s): Mohtaram Ramezanpour, Shahram Naghizadeh Raeisi, Seyed-Ahmad Shahidi, Sorour Ramezanpour, Shahram Seidi In this work, a novel sorbent based on polydopamine-functionalized magnetic ferric oxide (Fe3O4) was synthesized and applied for dispersive solid phase extraction of Pb(II) in bovine milk samples. The extracts were analyzed by flame atomic absorption spectrometry (FAAS). The sorbent was characterized with different analytical techniques (XRD, FT-IR, SEM, TEM, VSM and EDX). To reach the maximum extraction efficiency of Pb(II), some effective parameters on both adsorption and desorption steps were optimized with the aid of central composite design and response surface methodology. Under the optimal conditions, the method provided an enhancement factor of 40.5 corresponding to the absolute recovery of 81%. LOD and LOQ were found as 0.13 and 0.43 ng mL−1, respectively. The calibration curve was linear over the concentration range of 3.0–300 ng mL−1 (R2 = 0.9957). The intra and inter-day precisions (RSD%) of the method were calculated as 3.2% and 5.6%, respectively. Finally, the method was successfully applied for the determination of Pb(II) in different bovine milk samples. The Pb(II) content in one of the investigated milk samples was found to exceed the maximum permissible limit (20 μg L−1) set by Codex Alimentarius Commission. The relative recoveries were obtained within the range of 86.5–93.6.Graphical abstractImage 1
  • An easily-performed high-throughput method for plant genomic DNA
    • Abstract: Publication date: Available online 25 January 2019Source: Analytical BiochemistryAuthor(s): Deshui Yu, Ju Zhang, Guangxuan Tan, Ningshu Yu, Qiuyue Wang, Qiqi Duan, Xin Qi, Mingjiao Cheng, Chunxue Yan, Zhangkun Wei, Zhenmiao Yu, Wenchao Huang, Chengwei Li Genomic DNA isolation is a crucial technique for researchers studying plant molecular biology. A current widely-used protocol for DNA extraction needs a pestle and mortal for each sample and consumes a large amount of liquid nitrogen in grinding the samples. Most high-throughput methods depend on expensive machines for tissue homogenization. Here we developed a CTAB-based DNA extraction method using 2.0 ml microcentrifuge tubes for sample processing. This protocol has the advantages that it is suitable for a variety of plants, easily-performed without special equipment, and high-throughput; it effectively avoids sample cross-contamination, and is inexpensive, rapid and safe.
  • EPAI-NC: Enhanced prediction of adenosine to inosine RNA editing sites
           using nucleotide compositions
    • Abstract: Publication date: Available online 18 January 2019Source: Analytical BiochemistryAuthor(s): Ahsan Ahmad, Swakkhar Shatabda RNA editing process like Adenosine to Intosine (A-to-I) often influences basic functions like splicing stability and most importantly the translation. Thus knowledge about editing sites is of great importance in molecular biology. With the growth of known editing sites, machine learning or data centric approaches are now being applied to solve this problem of prediction of RNA editing sites. In this paper, we propose EPAI-NC, a novel method for prediction of RNA editing sites. We have used l-mer composition and n-gapped l-mer composition as features and used Pearson Correlation Coefficient to select features according to Pareto Principle. Locally deep support vector machines were used to train the classification model of EPAI-NC. EPAI-NC significantly enhances the prediction accuracy compared to the previous state-of-the-art methods when tested on standard benchmark and independent dataset.
  • Acetonitrile-assisted enzymatic digestion can facilitate the bottom-up
           identification of proteins of cancer origin
    • Abstract: Publication date: Available online 17 January 2019Source: Analytical BiochemistryAuthor(s): M. Laštovičková, P. Bobál, D. Strouhalová, J. Bobálová The main objective of this study was to develop an effective in-gel trypsin digestion protocol using aqueous-acetonitrile solvent system to facilitate MS analysis and maximize the number of identified proteins from biological samples. The procedure, where 80% acetonitrile was present in the trypsin reaction mixture, increased the number of matched peptides, and allowed the identification of more proteins with higher coverage than the common digestion protocol. Vimentin, annexins, tubulin, actin, peptidyl-prolyl cis-trans isomerase or alpha-enolase are examples of important proteins that change during the progress cancer. These were isolated from human breast cancer cells and were used for this study.
  • A fluorescence-based activity assay for immobilized lipases in non-native
    • Abstract: Publication date: Available online 17 January 2019Source: Analytical BiochemistryAuthor(s): Kim N. Ingenbosch, Anna Rousek, Dennis Wunschik, Kerstin Hoffmann-Jacobsen A new method for the analysis of lipase activity in the immobilized state is developed. The fluorescence assay aims to quantify the potential of lipases for the application in organic solvents. As lipases are universally immobilized on polymeric carriers for the use in bioorganic synthesis, the assay includes an immobilization step on the walls of polymeric cuvettes. The activity of the immobilized lipase is probed by 4-methylumbelliferone hydrolysis. The activity retention as a function of solvent concentration is used as a measure for the solvent resistance of the enzyme variant. The method is applied to two different lipases, Candida antarctica lipase B (CalB) and Bacillus subtilis lipase A (BSLA) in the presence of the solvents acetonitrile and ethanol. By comparison of the assay results with a commercial biocatalyst consisting of CalB on polymeric carrier (Novozyme 435) it is demonstrated that the assay allows a good prediction of the activity of the respective lipase as immobilisate on polymeric carriers. The assay surpasses the respective analysis in solution in terms of accuracy and precision.Graphical abstractImage 1
  • High-throughput Carbonyl Content Method of Therapeutic mAb using
    • Abstract: Publication date: Available online 14 January 2019Source: Analytical BiochemistryAuthor(s): Yunyu Yi, Li Zang Monoclonal antibody (mAb), one of the major types of therapeutic proteins in the pharmaceutical industry, is predominantly manufactured using mammalian cell culture [1]. Oxidative stress, potentially present during cell culture process, may increase the protein carbonyl content in the mAb product, which was reported to positively correlate with aggregate burst rate during storage [2]. In order to monitor carbonyl content during mAb process development, we developed a high-throughput screening method for therapeutic mAbs using size-exclusion chromatography followed by ultraviolet and fluorescence detection (SEC-UV/FL), optimized from a fluorescein thiosemicarbazide (FTC) semi-microplate method. The method demonstrated a good correlation with conventional ELISA assay and FTC-based fluorometric semi-microplate method with improved throughput and precision. The method was successfully applied in three case studies to improve our understanding of mAb carbonylation, including the impact of metal-catalyzed oxidation on an IgG4 mAb, comparison of carbonyl content between several mAbs expressed by CHO cell culture with human serum antibody pool, as well as the surface charge property of carbonylated mAb assessed by ion-exchange chromatography.
  • Enhanced protoplast assay by transfecting PCR-assembled gene expression
           cassettes with telomeric repeats and thiophosphate modifications
    • Abstract: Publication date: Available online 12 January 2019Source: Analytical BiochemistryAuthor(s): Xiangjuan Huang, Jiao Xue, Feng-Zhu Wang, Jian-Feng Li Transient expression assays are invaluable complements to the stable transgenic assay for studying gene functions, because they possess desirable time, labor efficiencies and high-throughput potential or circumvent technical difficulties of stable transgenic expression. The protoplast transient expression system is one of the mainstream transient expression assays used in plant research. Here, we developed a PCR amplicon-mediated protoplast transient (PROMPT) assay by using overlapping PCR assembled gene expression cassettes for Arabidopsis protoplast transfection instead of plasmid DNA, thereby bypassing the need for time- and labor-consuming plasmid construction. When 200 μl of Arabidopsis protoplasts were transfected with 1 μg of PCR amplicons or plasmid DNA, we detected substantially higher gene expression in the former. Moreover, we found that adding telomeric repeats and thiophosphate modifications to the 5’ end of the nonsense strand through the reverse primer could further increase the PCR amplicon-mediated gene expression in protoplasts. Importantly, these improvements could also be applied to the protoplast assays in other dicot and monocot species including tobacco, rice and wheat. In addition, the subcellular localization of immune receptor FLS2 could be analyzed by PROMPT method. The PROMPT assay allows an accelerated and robust transient gene expression in protoplasts from diverse plant species.
  • The study of the constituents and source of toxicants in poisonous honey
    • Abstract: Publication date: Available online 9 January 2019Source: Analytical BiochemistryAuthor(s): Huiqin Wu, Huitai Luo, Fang Huang, Xi Zhou, Xiaolan Huang, Jianghan Chen A novel method for non-target screening of toxicants in poisonous honey was established in this study. Poisonous honey and nontoxic honey were contrastive detected using liquid chromatography quadrupole-time-of-flight mass spectrometry and analyzed by Mass Profiler Professional Software. 4 poisonous alkaloids were screened out and confirmed by comparison with reference compounds. In order to investigate the source of these poisonous alkaloids, 6 poisonous alkaloids, ubiquitous in Gelsemium elegan, from honey, honeybees, pollen in honeycomb and different organs of Gelsemium elegan, were quantified by liquid chromatography triple-quadrupole tandem mass spectrometry. The results showed that alkaloids composition characteristics in honey, honeybees, and pollen were similar to those in the flower and bud of Gelsemium elegan and significant different from those in leave, stem and root. This result demonstrated that poisonous alkaloids in honey were come from the gathering honey process. This strategy provided an efficient and rapid method for non-target screening of toxicants in food.
  • Development of a novel L-histidine assay method using histamine
           dehydrogenase and a stable mutant of histidine decarboxylase
    • Abstract: Publication date: Available online 23 December 2018Source: Analytical BiochemistryAuthor(s): Hiroki Yamaguchi, Kunio Nakata, Moemi Tatsumi, Masayuki Sugiki, Hiroshi Miyano, Toshimi Mizukoshi L-Histidine analysis is essential in physiological research and clinical applications because L-histidine concentrations in biofluids are associated with various diseases. However, an enzymatic method for L-histidine quantitation has not yet been established. Here, we describe a novel L-histidine quantitation assay using a combination of histidine decarboxylase (HDC) and histamine dehydrogenase (HDH) enzymes. Wild-type HDC was unstable and completely lost its activity within 50 days of storage at 4 °C in solution. We rationally designed a HDC C57S mutant with markedly improved stability (storage at 4 °C for over 200 days) without altering the enzyme's substrate specificity. Together with HDH, the HDC C57S mutant was applied to quantify L-histidine concentrations in human plasma. The assay showed high precision (
  • Profiling of carboxyl-containing metabolites in smokers and non-smokers by
           stable isotope labeling combined with LC-MS/MS
    • Abstract: Publication date: Available online 10 December 2018Source: Analytical BiochemistryAuthor(s): Yunlu He, Yanbo Luo, Huan Chen, Jian Chen, Yaning Fu, Hongwei Hou, Qingyuan Hu Profiling of carboxyl-containing metabolites in smokers and non-smokers provides insight into the smoking-related biological events and causal relationships between exposure and adverse events. However, more comprehensive analysis of carboxyl-containing metabolites in bio-matrices with high sensitivity and accuracy is challenging. In this work, stable isotope labeling in combination with liquid chromatography-tandem mass spectrometry was used for untargeted profiling and relative quantification of carboxyl-containing metabolites in plasma of smokers and non-smokers. A pair of isotope labeling reagents, N, N-dimethylethylenediamine (DMED) and d4-DMED was used to label carboxyl-containing metabolites. Since the isotope labeled dimethylamino moieties of DMED and d4-DMED are easily fragmented and lost as characteristic neutral fragments of 45 and 49 Da, respectively, double neutral loss scans can be used to profiling of carboxyl-containing metabolites. Subsequently, based on the ion pair parameters obtained from double neutral loss scans, relative quantification method was developed. As a result, 269 carboxyl-containing metabolite candidates were discovered, and 88 metabolite candidates were found to have significantly alterations between smokers and non-smokers. 7Z, 10Z-hexadecadienoic acid, myristic acid and 3β-hydroxy-5-cholestenoic acid with significant difference confirmed by standard comparison are linked to smoking related inflammation, abnormal bile acid synthesis and cholesterol metabolism.
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Heriot-Watt University
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