Journal Cover Analytical Biochemistry
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   Hybrid Journal Hybrid journal (It can contain Open Access articles)
   ISSN (Print) 0003-2697 - ISSN (Online) 1096-0309
   Published by Elsevier Homepage  [3177 journals]
  • Prediction of lysine glutarylation sites by maximum relevance minimum
           redundancy feature selection
    • Authors: Zhe Ju; Jian-Jun He
      Pages: 1 - 7
      Abstract: Publication date: 1 June 2018
      Source:Analytical Biochemistry, Volume 550
      Author(s): Zhe Ju, Jian-Jun He
      Lysine glutarylation is new type of protein acylation modification in both prokaryotes and eukaryotes. To better understand the molecular mechanism of glutarylation, it is important to identify glutarylated substrates and their corresponding glutarylation sites accurately. In this study, a novel bioinformatics tool named GlutPred is developed to predict glutarylation sites by using multiple feature extraction and maximum relevance minimum redundancy feature selection. On the one hand, amino acid factors, binary encoding, and the composition of k-spaced amino acid pairs features are incorporated to encode glutarylation sites. And the maximum relevance minimum redundancy method and the incremental feature selection algorithm are adopted to remove the redundant features. On the other hand, a biased support vector machine algorithm is used to handle the imbalanced problem in glutarylation sites training dataset. As illustrated by 10-fold cross-validation, the performance of GlutPred achieves a satisfactory performance with a Sensitivity of 64.80%, a Specificity of 76.60%, an Accuracy of 74.90% and a Matthew's correlation coefficient of 0.3194. Feature analysis shows that some k-spaced amino acid pair features play the most important roles in the prediction of glutarylation sites. The conclusions derived from this study might provide some clues for understanding the molecular mechanisms of glutarylation.

      PubDate: 2018-04-15T10:34:19Z
      DOI: 10.1016/j.ab.2018.04.005
      Issue No: Vol. 550 (2018)
       
  • Principal coordinate analysis assisted chromatographic analysis of
           bacterial cell wall collection: A robust classification approach
    • Authors: Keshav Kumar; Felipe Cava
      Pages: 8 - 14
      Abstract: Publication date: 1 June 2018
      Source:Analytical Biochemistry, Volume 550
      Author(s): Keshav Kumar, Felipe Cava
      In the present work, Principal coordinate analysis (PCoA) is introduced to develop a robust model to classify the chromatographic data sets of peptidoglycan sample. PcoA captures the heterogeneity present in the data sets by using the dissimilarity matrix as input. Thus, in principle, it can even capture the subtle differences in the bacterial peptidoglycan composition and can provide a more robust and fast approach for classifying the bacterial collection and identifying the novel cell wall targets for further biological and clinical studies. The utility of the proposed approach is successfully demonstrated by analysing the two different kind of bacterial collections. The first set comprised of peptidoglycan sample belonging to different subclasses of Alphaproteobacteria. Whereas, the second set that is relatively more intricate for the chemometric analysis consist of different wild type Vibrio Cholerae and its mutants having subtle differences in their peptidoglycan composition. The present work clearly proposes a useful approach that can classify the chromatographic data sets of chromatographic peptidoglycan samples having subtle differences. Furthermore, present work clearly suggest that PCoA can be a method of choice in any data analysis workflow.

      PubDate: 2018-04-15T10:34:19Z
      DOI: 10.1016/j.ab.2018.04.008
      Issue No: Vol. 550 (2018)
       
  • Determination of enantiomeric excess of some amino acids by second-order
           calibration of kinetic-fluorescence data
    • Authors: Azadeh Naghashian-Haghighi; Bahram Hemmateenejad; Mojtaba Shamsipur
      Pages: 15 - 26
      Abstract: Publication date: 1 June 2018
      Source:Analytical Biochemistry, Volume 550
      Author(s): Azadeh Naghashian-Haghighi, Bahram Hemmateenejad, Mojtaba Shamsipur
      In this investigation a new non-separative kinetic-spectroflourimetric method is proposed for the determination of lysine (lys), leucine (leu) and phenylalanine (phe) enantiomers as their o-phthaldialdehyde (OPA) derivatives in the presence of an optically active chiral thiol compound, 1-mercapto-2-propanol (MP). At ambient temperature and in the borate buffer media of pH 9.6, MP, OPA, as highly selective fluorogenic reagents, and amino acid (AA) enantiomers reacts with each other to yield two fluorescent diasteriomers of D and L-AA with maximum difference in fluorescence intensity at about 450 nm. To achieve information from the small spectral changes, the data are analyzed by Multivariate Curve Resolution Alternating Least Squares (MCR-ALS) method. Linear calibration curves are achieved to distinct D and L-lys, leu and phe in different mole ratios by applying appropriate constraints in MCR-ALS procedures. This is the first application of MCR-ALS in determination of enantiomeric excess (ee) using OPA/MP adduct as chiral reagent, which benefits from direct time dependent-fluorescence spectral analysis and does not require prior separation of chiral analytes. Both the cross-validated correlation coefficient (Q 2) and root mean squares error of prediction (RMSEP) indicated satisfactory prediction ability of this method.

      PubDate: 2018-04-15T10:34:19Z
      DOI: 10.1016/j.ab.2018.04.004
      Issue No: Vol. 550 (2018)
       
  • Development of Quenching-qPCR (Q-Q) assay for measuring absolute
           intracellular cleavage efficiency of ribozyme
    • Authors: Min Woo Kim; Gwanggyu Sun; Jung Hyuk Lee; Byung-gee Kim
      Pages: 27 - 33
      Abstract: Publication date: 1 June 2018
      Source:Analytical Biochemistry, Volume 550
      Author(s): Min Woo Kim, Gwanggyu Sun, Jung Hyuk Lee, Byung-gee Kim
      Ribozyme (Rz) is a very attractive RNA molecule in metabolic engineering and synthetic biology fields where RNA processing is required as a control unit or ON/OFF signal for its cleavage reaction. In order to use Rz for such RNA processing, Rz must have highly active and specific catalytic activity. However, current methods for assessing the intracellular activity of Rz have limitations such as difficulty in handling and inaccuracies in the evaluation of correct cleavage activity. In this paper, we proposed a simple method to accurately measure the “intracellular cleavage efficiency” of Rz. This method deactivates unwanted activity of Rz which may consistently occur after cell lysis using DNA quenching method, and calculates the cleavage efficiency by analyzing the cleaved fraction of mRNA by Rz from the total amount of mRNA containing Rz via quantitative real-time PCR (qPCR). The proposed method was applied to measure “intracellular cleavage efficiency” of sTRSV, a representative Rz, and its mutant, and their intracellular cleavage efficiencies were calculated as 89% and 93%, respectively.

      PubDate: 2018-04-15T10:34:19Z
      DOI: 10.1016/j.ab.2018.04.007
      Issue No: Vol. 550 (2018)
       
  • Mucin and carbon nanotube-based biosensor for detection of glucose in
           human plasma
    • Authors: Fausto N. Comba; Marcelo R. Romero; Fernando S. Garay; Ana M. Baruzzi
      Pages: 34 - 40
      Abstract: Publication date: 1 June 2018
      Source:Analytical Biochemistry, Volume 550
      Author(s): Fausto N. Comba, Marcelo R. Romero, Fernando S. Garay, Ana M. Baruzzi
      This work reports an amperometric enzyme-electrode prepared with glucose oxidase, which have been immobilized by a cross-linking step with glutaraldehyde in a mixture containing albumin and a novel carbon nanotubes-mucin composite (CNT-muc). The obtained hydrogel matrix was trapped between two polycarbonate membranes and then fixed at the surface of a Pt working electrode. The developed biosensor was optimized by evaluating different compositions and the analytical properties of an enzymatic matrix with CNT-muc. Then, the performance of the resulting enzymatic matrix was evaluated for direct glucose quantification in human blood plasma. The novel CNT-muc composite provided a sensitivity of 0.44 ± 0.01 mA M−1 and a response time of 28 ± 2 s. These values were respectively 20% higher and 40% shorter than those obtained with a sandwich-type biosensor prepared without CNT. Additionally, CNT-muc based biosensor exhibited more than 3 orders of magnitude of linear dynamic calibration range and a detection limit of 3 μM. The short-term and long-term stabilities of the biosensors were also examined and excellent results were obtained through successive experiments performed within the first 60 days from their preparation. Finally, the storage stability was remarkable during the first 300 days.

      PubDate: 2018-04-15T10:34:19Z
      DOI: 10.1016/j.ab.2018.04.006
      Issue No: Vol. 550 (2018)
       
  • A recombinase polymerase amplification assay for the diagnosis of atypical
           pneumonia
    • Authors: Sebastian Kersting; Valentina Rausch; Frank F. Bier; Markus von Nickisch-Rosenegk
      Pages: 54 - 60
      Abstract: Publication date: 1 June 2018
      Source:Analytical Biochemistry, Volume 550
      Author(s): Sebastian Kersting, Valentina Rausch, Frank F. Bier, Markus von Nickisch-Rosenegk
      Pneumonia is one of the most common and potentially lethal infectious conditions worldwide. Streptococcus pneumoniae is the pathogen most frequently associated with bacterial community-acquired pneumonia, while Legionella pneumophila is the major cause for local outbreaks of legionellosis. Both pathogens can be difficult to diagnose since signs and symptoms are nonspecific and do not differ from other causes of pneumonia. Therefore, a rapid diagnosis within a clinically relevant time is essential for a fast onset of the proper treatment. Although methods based on polymerase chain reaction significantly improved the identification of pathogens, they are difficult to conduct and need specialized equipment. We describe a rapid and sensitive test using isothermal recombinase polymerase amplification and detection on a disposable test strip. This method does not require any special instrumentation and can be performed in less than 20 min. The analytical sensitivity in the multiplex assay amplifying specific regions of S. pneumoniae and L. pneumophila simultaneously was 10 CFUs of genomic DNA per reaction. In cross detection studies with closely related strains and other bacterial agents the specificity of the RPA was confirmed. The presented method is applicable for near patient and field testing with a rather simple routine and the possibility for a read out with the naked eye.

      PubDate: 2018-04-24T22:01:48Z
      DOI: 10.1016/j.ab.2018.04.014
      Issue No: Vol. 550 (2018)
       
  • Construction of Quenchbodies to detect and image amyloid β oligomers
    • Authors: Jinhua Dong; Richi Fujita; Tamotsu Zako; Hiroshi Ueda
      Pages: 61 - 67
      Abstract: Publication date: 1 June 2018
      Source:Analytical Biochemistry, Volume 550
      Author(s): Jinhua Dong, Richi Fujita, Tamotsu Zako, Hiroshi Ueda
      A quenchbody (Q-body) is an antibody-based biosensor that employs fluorescence quenching of the dye(s) attached to the antibody fragment, which are de-quenched upon antigen binding. In this study, we aimed to develop Fab type Q-bodies (UQ-bodies) to aid the diagnosis of Alzheimer's disease (AD). Characteristic senile plaques in AD consist of amyloid-β peptide (Aβ) generated from the amyloid precursor protein. Aβ42, one of the major peptide forms, aggregates fast and manifests higher neurotoxicity. Recent studies showed that Aβ oligomers, such as Aβ-derived diffusible ligand (ADDL), are more toxic than fibrils. Thus, detection of Aβ and its oligomers in body fluid might help detect deterioration caused by the disease. To this end, the Fab fragment of the anti-Aβ antibody h12A11, which binds preferentially to ADDL, was expressed in Escherichia coli, and labeled with a fluorescent dye at the N terminus of either the heavy chain, or the heavy and light chains, via Cys-containing tag(s) to prepare UQ-bodies. As a result, the double-labeled UQ-bodies detected ADDL with higher sensitivity than that for the Aβ peptide. In addition, the UQ-body could be used to image aggregated Aβ with a low background, which suggested the potential of UQ-bodies as a fast bioimaging tool.

      PubDate: 2018-04-24T22:01:48Z
      DOI: 10.1016/j.ab.2018.04.016
      Issue No: Vol. 550 (2018)
       
  • Modified beacon probe assisted dual signal amplification for visual
           detection of microRNA
    • Authors: Xiuwei Sun; Na Ying; Chuanjing Ju; Zhongyi Li; Na Xu; Guijuan Qu; Wensen Liu; Jiayu Wan
      Pages: 68 - 71
      Abstract: Publication date: 1 June 2018
      Source:Analytical Biochemistry, Volume 550
      Author(s): Xiuwei Sun, Na Ying, Chuanjing Ju, Zhongyi Li, Na Xu, Guijuan Qu, Wensen Liu, Jiayu Wan
      In a recent study, we reported a novel assay for the detection of microRNA-21 based on duplex-specific nuclease (DSN)-assisted isothermal cleavage and hybridization chain reaction (HCR) dual signal amplification. The Fam modified double-stranded DNA products were generated after the HCR, another biotin modified probe was digested by DSN and released from the magnetic beads after the addition of the target miRNA. The released sequence was then combined with HCR products to form a double-tagging dsDNA, which can be recognized by the lateral flow strips. In this study, we introduced a 2-OMethyl-RNA modified beacon probe (2-OMe-MB) to make some improvements based on the previous study. Firstly, the substitution of modified probe combined on magnetic beads avoids the fussy washing steps for the separation of un-reacted probes. Furthermore, the modification of 2-OMe on the stem of the probe avoided the unnecessary cleavage by DSN, which greatly reduce the background signal. Compared to the previous work, these improvements save us a lot of steps but possess the comparable sensitivity and selectivity.

      PubDate: 2018-05-01T22:25:05Z
      DOI: 10.1016/j.ab.2018.04.010
      Issue No: Vol. 550 (2018)
       
  • Characterization of therapeutic antibodies in the presence of human serum
           proteins by AU-FDS analytical ultracentrifugation
    • Authors: Robert T. Wright; David B. Hayes; Walter F. Stafford; Peter J. Sherwood; John J. Correia
      Pages: 72 - 83
      Abstract: Publication date: 1 June 2018
      Source:Analytical Biochemistry, Volume 550
      Author(s): Robert T. Wright, David B. Hayes, Walter F. Stafford, Peter J. Sherwood, John J. Correia
      The preclinical characterization of biopharmaceuticals seeks to determine the stability, state of aggregation, and interaction of the antibody/drug with other macromolecules in serum. Analytical ultracentrifugation is the best experimental method to understand these factors. Sedimentation velocity experiments using the AU-FDS system were performed in order to quantitatively characterize the nonideality of fluorescently labeled therapeutic antibodies in high concentrations of human serum proteins. The two most ubiquitous serum proteins are human serum albumin, HSA, and γ-globulins, predominantly IgG. Tracer experiments were done pairwise as a function of HSA, IgG, and therapeutic antibody concentration. The sedimentation coefficient for each fluorescently labeled component as a function of the concentration of the unlabeled component yields the hydrodynamic nonideality (ks). This generates a 3x3 matrix of ks values that describe the nonideality of each pairwise interaction. The ks matrix is validated by fitting both 2:1 mixtures of HSA (1–40 mg/ml) and IgG (0.5–20 mg/ml) as serum mimics, and human serum dilutions (10–100%). The data are well described by SEDANAL global fitting with the ks nonideality matrix. The ks values for antibodies are smaller than expected and appear to be masked by weak association. Global fitting to a ks and K2 model significantly improves the fits.

      PubDate: 2018-05-01T22:25:05Z
      DOI: 10.1016/j.ab.2018.04.002
      Issue No: Vol. 550 (2018)
       
  • A fluorescence polarization-based competition assay for measuring
           interactions between unlabeled ubiquitin chains and UCH37•RPN13
    • Authors: Jiale Du; Eric R. Strieter
      Pages: 84 - 89
      Abstract: Publication date: 1 June 2018
      Source:Analytical Biochemistry, Volume 550
      Author(s): Jiale Du, Eric R. Strieter
      Ubiquitin chains regulate distinct signaling events through cooperative interactions with effector proteins and deubiquitinases. Measuring the strength of these interactions is often challenging; either large amounts of material are required or one of the binding partners must be labeled for detection. We sought to develop a label-free method for measuring binding of ubiquitin chains to the proteasome-associated deubiquitinase UCH37 and its binding partner RPN13. The method we describe here is based on a fluorescence polarization competition (FPcomp) assay in which fluorescent monoubiquitin is competed off the UCH37•RPN13 complex by the addition of unlabeled ubiquitin chains. We show that the UCH37•RPN13 complex displays higher affinity toward chains with more than two ubiquitin subunits. Removing the ubiquitin-binding PRU domain of RPN13 does not change affinities. These results suggest UCH37•RPN13 acts to selectively recruit proteins modified with long chains (>2 subunits) to the proteasome for degradation. We also demonstrate that the FPcomp assay is suitable for high-throughput screening, which is important considering both UCH37 and RPN13 are potential targets for cancer therapy.

      PubDate: 2018-05-01T22:25:05Z
      DOI: 10.1016/j.ab.2018.04.018
      Issue No: Vol. 550 (2018)
       
  • A high-throughput screening assay for pyruvate carboxylase
    • Authors: Brittney N. Wyatt; Leggy A. Arnold; Martin St. Maurice
      Pages: 90 - 98
      Abstract: Publication date: 1 June 2018
      Source:Analytical Biochemistry, Volume 550
      Author(s): Brittney N. Wyatt, Leggy A. Arnold, Martin St. Maurice
      Pyruvate carboxylase (PC) catalyzes the conversion of pyruvate to oxaloacetate (OAA), an important metabolic reaction in a wide range of organisms. Small molecules directed against PC would enable detailed studies on the metabolic role of this enzyme and would have the potential to be developed into pharmacological agents. Currently, specific and potent small molecule regulators of PC are unavailable. To assist in efforts to find, develop, and characterize small molecule effectors of PC, a novel fixed-time assay has been developed based on the reaction of OAA with the diazonium salt, Fast Violet B (FVB), which produces a colored adduct with an absorbance maximum at 530 nm. This fixed time assay is reproducible, sensitive and responsive to known effectors of Rhizobium etli PC, Staphylococcus aureus PC, and Listeria monocytogenes PC, and is highly amenable to high-throughput screening. The assay was validated using a plate uniformity assessment test and a pilot screen of a library of 1280 compounds. The results indicate that the assay is suitable for screening small molecule libraries to find novel small molecule effectors of PC.

      PubDate: 2018-05-01T22:25:05Z
      DOI: 10.1016/j.ab.2018.04.012
      Issue No: Vol. 550 (2018)
       
  • Reactive oxygen species dynamics in roots of salt sensitive and salt
           tolerant cultivars of rice
    • Authors: Shivani Saini; Navdeep Kaur; Pratap Kumar Pati
      Pages: 99 - 108
      Abstract: Publication date: 1 June 2018
      Source:Analytical Biochemistry, Volume 550
      Author(s): Shivani Saini, Navdeep Kaur, Pratap Kumar Pati
      Salinity stress is one of the major constraints for growth and survival of plants that affects rice productivity worldwide. Hence, in the present study, roots of two contrasting salinity sensitive cultivars, IR64 (IR64, salt sensitive) and Luna Suvarna (LS, salt tolerant) were compared with regard to the levels of reactive oxygen species (ROS) to derive clues for their differential salt stress adaptation mechanisms. In our investigation, the tolerant cultivar exhibited longer primary roots, more lateral roots, higher root number leading to increased root biomass, with respect to IR64. It was observed that LS roots maintained higher level of H2O2 in comparison to IR64. The activities of various enzymes involved in enzymatic antioxidant defense mechanism (SOD, CAT, GPX, DHAR and MDHAR) were found to be greater in LS roots. Further, the higher transcript level accumulation of genes encoding ROS generating (RbohA, RbohD and RbohE) and scavenging enzymes (Fe-SOD, Chloroplastic Cu/Zn-SOD, CAT and DHAR) were noticed in the roots of tolerant cultivar, LS. Moreover, the content of other stress markers such as total protein and proline were also elevated in LS roots. While, the expression of proline biosynthesis gene (P5CS) and proline catabolism gene (PDH) was observed to be lower in LS.

      PubDate: 2018-05-01T22:25:05Z
      DOI: 10.1016/j.ab.2018.04.019
      Issue No: Vol. 550 (2018)
       
  • iPhosT-PseAAC: Identify phosphothreonine sites by incorporating sequence
           statistical moments into PseAAC
    • Authors: Yaser Daanial Khan; Nouman Rasool; Waqar Hussain; Sher Afzal Khan; Kuo-Chen Chou
      Pages: 109 - 116
      Abstract: Publication date: 1 June 2018
      Source:Analytical Biochemistry, Volume 550
      Author(s): Yaser Daanial Khan, Nouman Rasool, Waqar Hussain, Sher Afzal Khan, Kuo-Chen Chou
      Among all the post-translational modifications (PTMs) of proteins, Phosphorylation is known to be the most important and highly occurring PTM in eukaryotes and prokaryotes. It has an important regulatory mechanism which is required in most of the pathological and physiological processes including neural activity and cell signalling transduction. The process of threonine phosphorylation modifies the threonine by the addition of a phosphoryl group to the polar side chain, and generates phosphothreonine sites. The investigation and prediction of phosphorylation sites is important and various methods have been developed based on high throughput mass-spectrometry but such experimentations are time consuming and laborious therefore, an efficient and accurate novel method is proposed in this study for the prediction of phosphothreonine sites. The proposed method uses context-based data to calculate statistical moments. Position relative statistical moments are combined together to train neural networks. Using 10-fold cross validation, 94.97% accurate result has been obtained whereas for Jackknife testing, 96% accurate results have been obtained. The overall accuracy of the system is 94.4% to sensitivity value 94% and specificity 94.6%. These results suggest that the proposed method may play an essential role to the other existing methods for phosphothreonine sites prediction.

      PubDate: 2018-05-01T22:25:05Z
      DOI: 10.1016/j.ab.2018.04.021
      Issue No: Vol. 550 (2018)
       
  • Enzyme-free isothermal target-recycled amplification combined with PAGE
           for direct detection of microRNA-21
    • Authors: Jiaqi Zhang; Wancun Zhang; Yueqing Gu
      Pages: 117 - 122
      Abstract: Publication date: 1 June 2018
      Source:Analytical Biochemistry, Volume 550
      Author(s): Jiaqi Zhang, Wancun Zhang, Yueqing Gu
      MicroRNA-21 (miR-21) has been regarded as a kind of potential biomarker in several types of cancers. Herein, in this study, a simple, sensitive and cost-effective miR-21 approach was developed utilizing the isothermal target-recycled enzyme-free amplification strategy and polyacrylamide gel electrophoresis (PAGE). In the target-recycled enzyme-free amplification strategy, two rational designed hairpin probes (HPs, HP1 and HP2) can form into HP1-HP2 duplex in the presence of miR-21 under the isothermal condition, producing target signal in PAGE. The sensitivity and the linear range of miR-21 approach were demonstrated by the in vitro detection of miR-21, with the detection limit of 10 pM and the linear range of 50 pM to 8 nM. In particular, the contents of miR-21 in cell extractions of different cell lines were successfully detected through miR-21 approach and the relative expression was highly coincident with the results of stem-loop RT-PCR approach. In summary, the developed approach can detect miR-21 sensitively and easily.
      Graphical abstract image

      PubDate: 2018-05-01T22:25:05Z
      DOI: 10.1016/j.ab.2018.04.024
      Issue No: Vol. 550 (2018)
       
  • A label free Ag+ sensing method via in situ formation of metal
           coordination polymer
    • Authors: Yan Lu; Lianjie Meng; Yan Gao; Dongli Liao; Yongxin Li; Yongxing Ai; Yuqin Ma; Cong Yu
      Pages: 21 - 25
      Abstract: Publication date: 15 May 2018
      Source:Analytical Biochemistry, Volume 549
      Author(s): Yan Lu, Lianjie Meng, Yan Gao, Dongli Liao, Yongxin Li, Yongxing Ai, Yuqin Ma, Cong Yu
      Metal ions sensing play critical roles in environmental monitoring and in biology. In this assay, we report the development of a facile fluorometric method for the sensing of Ag+ ions via the in situ formation of metal coordination polymer, based on the selective interactions of GSH with Ag+. The formation of coordination polymer with net multiple negative charges in an aqueous buffer solution (Tris-HAc, pH 9.0) resulted in aggregation and fluorescence quenching of a cationic perylene probe. The difference in emission intensity spurred us to develop a new strategy for sensing Ag+ ions. The proposed Ag+ detection method is simple, convenient, selective and sensitive, and can be used for Ag+ detection in lake water samples.

      PubDate: 2018-03-19T20:43:07Z
      DOI: 10.1016/j.ab.2018.03.005
      Issue No: Vol. 549 (2018)
       
  • Fluorescent microplate assay method for high-throughput detection of
           lipase transesterification activity
    • Authors: Jianyong Zheng; Wei Wei; Xing Lan; Yinjun Zhang; Zhao Wang
      Pages: 26 - 28
      Abstract: Publication date: 15 May 2018
      Source:Analytical Biochemistry, Volume 549
      Author(s): Jianyong Zheng, Wei Wei, Xing Lan, Yinjun Zhang, Zhao Wang
      This study describes a sensitive and fluorescent microplate assay method to detect lipase transesterification activity. Lipase-catalyzed transesterification between butyryl 4-methyl umbelliferone (Bu-4-Mu) and methanol in tert-butanol was selected as the model reaction. The release of 4-methylumbelliferone (4-Mu) in the reaction was determined by detecting the fluorescence intensity at λ ex 330 nm and λ em 390 nm. Several lipases were used to investigate the accuracy and efficiency of the proposed method. Apparent Michaelis constant (Km) was calculated for transesterification between Bu-4-Mu and methanol by the lipases. The main advantages of the assay method include high sensitivity, inexpensive reagents, and simple detection process.

      PubDate: 2018-03-19T20:43:07Z
      DOI: 10.1016/j.ab.2018.03.010
      Issue No: Vol. 549 (2018)
       
  • HPLC-UV assays for evaluation of inhibitors of mono and diamine oxidases
           using novel phenyltetrazolylalkanamine substrates
    • Authors: Kira Mergemeier; Matthias Lehr
      Pages: 29 - 38
      Abstract: Publication date: 15 May 2018
      Source:Analytical Biochemistry, Volume 549
      Author(s): Kira Mergemeier, Matthias Lehr
      Recently, we have described an HPLC-UV assay for the evaluation of inhibitors of plasma amine oxidase (PAO) using 6-(5-phenyl-2H-tetrazol-2-yl)hexan-1-amine (4) as a new type of substrate. Now we studied, whether this compound or homologues of it can also function as substrate for related amine oxidases, namely diamine oxidase (DAO), monoamine oxidase A (MAO A) and monoamine oxidase B (MAO B). Among these substances, 4 was converted by DAO with the highest rate. The best substrate for MAO A and B was 4-(5-phenyl-2H-tetrazol-2-yl)butan-1-amine (2). To validate the new assays, the inhibition values of known enzyme inhibitors were determined and the data were compared with those obtained with the substrate benzylamine, which is often used in amine oxidase assays. For the DAO inhibitor 2-(4-phenylphenyl)acetohydrazide an about 10fold lower IC50-value against DAO was obtained when benzylamine was applied instead of 4, indicating that 4 binds to the enzyme with higher affinity than benzylamine. The IC50-values of clorgiline and selegiline against MAO A and B, respectively, also decreased (two- and 30fold) replacing 2 by benzylamine. The discrepancies largely disappeared, when the enzymes were pre-incubated with the inhibitors for 15 min. This can be explained with the covalent inhibition mechanism of the inhibitors.

      PubDate: 2018-03-19T20:43:07Z
      DOI: 10.1016/j.ab.2018.03.007
      Issue No: Vol. 549 (2018)
       
  • Single step, direct fluorescence immunoassays based on metal enhanced
           fluorescence (MEF-FIA) applicable as micro plate-, array-, multiplexing-
           or point of care-format
    • Authors: G. Hawa; Linda Sonnleitner; A. Missbichler; A. Prinz; G. Bauer; C. Mauracher
      Pages: 39 - 44
      Abstract: Publication date: 15 May 2018
      Source:Analytical Biochemistry, Volume 549
      Author(s): G. Hawa, Linda Sonnleitner, A. Missbichler, A. Prinz, G. Bauer, C. Mauracher
      Although Enzyme Linked Immuno Sorbent Assay (ELISA) technology is approaching it's 45th year of existence since first described in 1971, it is still the main diagnostic tool in clinical research and routine diagnostics. However, despite its broad usage it suffers from some drawbacks, limiting its use especially in more advanced assay formats like multiplexing platforms, point of care devices or protein arrays. Those limitations result from the need for an enzyme label, a soluble enzyme substrate, washing steps (multiplexing, point care, arrays) and in some cases also insufficient sensitivity, because the majority of circulating proteins and thus potential biomarkers may be found in lower sub-picomolar concentrations. We hereby present a new assay platform based on metal enhanced fluorescence (MEF), that remedies these problems since it eliminates the need for washing steps, for using enzyme labels and allows detection of analytes down to sub-picomolar concentrations. In addition this technology is fully compatible to standard fluorescence reader equipment as it is found in many laboratories nowadays. Since our present work is focused on single biomarker evaluation, we chose a 96 well plate format for convenience, but any other formate like antibody arrays, strip-like point of care devices etc. is feasible too.

      PubDate: 2018-04-11T10:25:23Z
      DOI: 10.1016/j.ab.2018.03.002
      Issue No: Vol. 549 (2018)
       
  • An improved immobilized enzyme reactor-mass spectrometry-based label free
           assay for butyrylcholinesterase ligand screening
    • Authors: Adriana Ferreira Lopes Vilela; Cláudia Seidl; Juliana Maria Lima; Carmen Lúcia Cardoso
      Pages: 53 - 57
      Abstract: Publication date: 15 May 2018
      Source:Analytical Biochemistry, Volume 549
      Author(s): Adriana Ferreira Lopes Vilela, Cláudia Seidl, Juliana Maria Lima, Carmen Lúcia Cardoso
      Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are key cholinesterase enzymes responsible for the hydrolysis of acetylcholine into choline and acetic acid, an essential process for the restoration of the cholinergic neuron. The loss of cholinergic function in the central nervous system contributes to the cognitive decline associated with advanced age and Alzheimer's disease (AD). Inhibitions assays represent a significant role in the drug discovery process. Herein, we describe an improved label free method to screen and characterize new BChE ligands. The liquid chromatography system uses an immobilized capillary enzyme reactor (ICER) as a low affinity and high selectivity column coupled to a mass spectrometer (MS). The enzyme activity was evaluated by monitoring the choline's precursor ion [M + H]+ m/z 104 for a brief period. The method was validated using two known cholinesterase inhibitors tacrine and galanthamine. The IC50 values were 0.03 ± 0.006 μM and 0.88 ± 0.2 for tacrine and galanthamine respectively, and Ki was 0.11 ± 0.2 for galanthamine. The efficient combination of the huBChE-ICER with sensitive enzymatic assay detection such as MS, improved the reliable, fast identification of new ligands. Moreover, specific direct quantitation of the product contributes to the reduction of false positive and negative results.
      Graphical abstract image

      PubDate: 2018-04-11T10:25:23Z
      DOI: 10.1016/j.ab.2018.03.012
      Issue No: Vol. 549 (2018)
       
  • Spherical-supported membranes as platforms for screening against membrane
           protein targets
    • Authors: V. Vasilca; A. Sadeghpour; S. Rawson; L.E. Hawke; S.A. Baldwin; T. Wilkinson; D. Bannister; V.L.G. Postis; M. Rappolt; S.P. Muench; L.J.C. Jeuken
      Pages: 58 - 65
      Abstract: Publication date: 15 May 2018
      Source:Analytical Biochemistry, Volume 549
      Author(s): V. Vasilca, A. Sadeghpour, S. Rawson, L.E. Hawke, S.A. Baldwin, T. Wilkinson, D. Bannister, V.L.G. Postis, M. Rappolt, S.P. Muench, L.J.C. Jeuken
      Screening assays performed against membrane protein targets (e.g. phage display) are hampered by issues arising from protein expression and purification, protein stability in detergent solutions and epitope concealment by detergent micelles. Here, we have studied a fast and simple method to improve screening against membrane proteins: spherical-supported bilayer lipid membranes (“SSBLM”). SSBLMs can be quickly isolated via low-speed centrifugation and redispersed in liquid solutions while presenting the target protein in a native-like lipid environment. To provide proof-of-concept, SSBLMs embedding the polytopic bacterial nucleoside transporter NupC were assembled on 100- and 200 nm silica particles. To test specific binding of antibodies, NupC was tagged with a poly-histidine epitope in one of its central loops between two transmembrane helices. Fluorescent labelling, small angle X-ray scattering (SAXS) and cryo-electron microscopy (cryo-EM) were used to monitor formation of the SSBLMs. Specific binding of an anti-his antibody and a gold-nitrilotriacetic acid (NTA) conjugate probe was confirmed with ELISAs and cryo-EM. SSBLMs for screening could be made with purified and lipid reconstituted NupC, as well as crude bacterial membrane extracts. We conclude that SSBLMs are a promising new means of presenting membrane protein targets for (biomimetic) antibody screening in a native-like lipid environment.

      PubDate: 2018-04-11T10:25:23Z
      DOI: 10.1016/j.ab.2018.03.006
      Issue No: Vol. 549 (2018)
       
  • In vitro selection and characterization of single stranded DNA aptamers
           for luteolin: A possible recognition tool
    • Authors: Jinan Tuma Sabah; Razauden Mohamed Zulkifli; Shafinaz Shahir; Farediah Ahmed; Mohammed Rafiq Abdul Kadir; Zarita Zakaria
      Pages: 72 - 79
      Abstract: Publication date: 15 May 2018
      Source:Analytical Biochemistry, Volume 549
      Author(s): Jinan Tuma Sabah, Razauden Mohamed Zulkifli, Shafinaz Shahir, Farediah Ahmed, Mohammed Rafiq Abdul Kadir, Zarita Zakaria
      Distinctive bioactivities possessed by luteolin (3′, 4′, 5, 7-tetrahydroxy-flavone) are advantageous for sundry practical applications. This paper reports the in vitro selection and characterization of single stranded-DNA (ssDNA) aptamers, specific for luteolin (LUT). 76-mer library containing 1015 randomized ssDNA were screened via systematic evolution of ligands by exponential enrichment (SELEX). The recovered ssDNA pool from the 8th round was amplified with unlabeled primers and cloned into PSTBlue-1 vector prior to sequencing. 22 of LUT-binding aptamer variants were further classified into one of the seven groups based on their N40 random sequence regions, wherein one representative from each group was characterized. The dissociation constant of aptamers designated as LUT#28, LUT#20 and LUT#3 was discerned to be 107, 214 and 109 nM, respectively with high binding affinity towards LUT. Prediction analysis of the secondary structure suggested discrete features with typical loop and stem motifs. Furthermore, LUT#3 displayed higher specificity with insignificant binding toward kaempferol and quercetin despite its structural and functional similarity compared to LUT#28 and LUT#20. Further LUT#3 can detect free luteolin within 0.2–1 mM in solution. It was suggested that LUT#3 aptamer were the most suitable for LUT recognition tool at laboratory scale based on the condition tested.

      PubDate: 2018-04-11T10:25:23Z
      DOI: 10.1016/j.ab.2018.03.004
      Issue No: Vol. 549 (2018)
       
  • A high-throughput pH-based colorimetric assay: application focus on
           alpha/beta hydrolases
    • Authors: Mariétou F. Paye; Harrison B. Rose; John M. Robbins; Diana A. Yunda; Seonggeon Cho; Andreas S. Bommarius
      Pages: 80 - 90
      Abstract: Publication date: 15 May 2018
      Source:Analytical Biochemistry, Volume 549
      Author(s): Mariétou F. Paye, Harrison B. Rose, John M. Robbins, Diana A. Yunda, Seonggeon Cho, Andreas S. Bommarius
      Research involving α/β hydrolases, including α-amino acid ester hydrolase and cocaine esterase, has been limited by the lack of an online high throughput screening assay. The development of a high throughput screening assay capable of detecting α/β hydrolase activity toward specific substrates and/or chemical reactions (e.g., hydrolysis in lieu of amidase activity and/or synthesis instead of thioesterase activity) is of interest in a broad set of scientific questions and applications. Here we present a general framework for pH-based colorimetric assays, as well as the mathematical considerations necessary to estimate de novo the experimental response required to assign a ‘hit’ or a ‘miss,’ in the absence of experimental standard curves. This combination is valuable for screening the hydrolysis and synthesis activity of α/β hydrolases on a variety of substrates, and produces data comparable to the current standard technique involving High Performance Liquid Chromatography (HPLC). In contrast to HPLC, this assay enables screening experiments to be performed with greater efficiency.

      PubDate: 2018-04-15T10:34:19Z
      DOI: 10.1016/j.ab.2018.03.009
      Issue No: Vol. 549 (2018)
       
  • The potential of aptamers for cancer research
    • Authors: Zhizhi Zhou; Mingying Liu; Jiahuan Jiang
      Pages: 91 - 95
      Abstract: Publication date: 15 May 2018
      Source:Analytical Biochemistry, Volume 549
      Author(s): Zhizhi Zhou, Mingying Liu, Jiahuan Jiang
      Aptamers are promising alternatives to antibodies and can be used as high affinity agents for the cancer detection and the targeted drug transportation. In this manuscript, we highlight the advantages of aptamers, such as high affinities, specificity and excellent chemical stabilities, which are likely to benefit for the diagnosis of cancer in its early stages and then achieve molecular-level treatment. Also, we discuss the challenges and problems in the current application of aptamers.

      PubDate: 2018-04-11T10:25:23Z
      DOI: 10.1016/j.ab.2018.03.008
      Issue No: Vol. 549 (2018)
       
  • Rapid quantification of tyrosine sulfation in therapeutic proteins
    • Authors: Iva Turyan; Ruth Frenkel; Zoran Sosic
      Pages: 96 - 98
      Abstract: Publication date: 15 May 2018
      Source:Analytical Biochemistry, Volume 549
      Author(s): Iva Turyan, Ruth Frenkel, Zoran Sosic
      Protein tyrosine sulfation (Tyr-O-SO3) is a common post-translational modification (PTM), which is important for protein function. Absolute quantitation of Tyr-O-SO3 in recombinant therapeutic proteins has been challenging. We report here an MRM method used for absolute quantitation of Tyr-O-SO3 in the hydrolysate of a recombinant Fc-fusion protein. Quantitation is achieved by monitoring the sum of two transitions: the loss of carboxylic acid from tyrosine sulfate (major transition) and sulfate group from tyrosine sulfate sodium salt. The method exhibits a good sensitivity with a limit of quantitation of 1.4 ng/mL, linearity over three orders of magnitude, good repeatability, precision and accuracy.

      PubDate: 2018-04-11T10:25:23Z
      DOI: 10.1016/j.ab.2018.03.001
      Issue No: Vol. 549 (2018)
       
  • Producing standard damaged DNA samples by heating: pitfalls and
           suggestions
    • Authors: Paolo Fattorini; Giorgio Marrubini; Serena Bonin; Barbara Bertoglio; Pierangela Grignani; Elisa Recchia; Paola Pitacco; Francesca Procopio; Carolina Cantoni; Irena Zupanič Pajnič; Solange Sorçaburu-Cigliero; Carlo Previderè
      Pages: 107 - 112
      Abstract: Publication date: 15 May 2018
      Source:Analytical Biochemistry, Volume 549
      Author(s): Paolo Fattorini, Giorgio Marrubini, Serena Bonin, Barbara Bertoglio, Pierangela Grignani, Elisa Recchia, Paola Pitacco, Francesca Procopio, Carolina Cantoni, Irena Zupanič Pajnič, Solange Sorçaburu-Cigliero, Carlo Previderè
      Heat-mediated hydrolysis of DNA is a simple and inexpensive method for producing damaged samples in vitro. Despite heat-mediated DNA hydrolysis is being widely used in forensic and clinical validation procedures, the lack of standardized procedures makes it impossible to compare the intra and inter-laboratory outcomes of the damaging treatments. In this work, a systematic approach to heat induced DNA hydrolysis was performed at 70 °C for 0–18 h to test the role both of the hydrolysis buffer and of the experimental conditions. Specifically, a trial DNA sample, resuspended in three different media (ultrapure water, 0.1% DEPC-water and, respectively, TE) was treated both in Eppendorf tubes (“Protocol P”) and in Eppendorf tubes provided with screwcaps (“Protocol S”). The results of these comparative tests were assessed by normalization of the qPCR results. DEPC-water increased the degradation of the samples up to about 100 times when compared to the ultrapure water. Conversely, the TE protected the DNA from degradation whose level was about 1700 times lower than in samples treated in ultrapure water. Even the employment of the “Protocol S” affected the level of degradation, by consistently increasing it (up to about 180 times in DEPC-water). Thus, this comparative approach showed that even seemingly apparently trivial and often underestimated parameters modify the degradation level up to 2–3 orders of magnitude. The chemical-physical reasons of these findings are discussed together with the role of potential factors such as enhanced reactivity of CO2, ROS, NOx and pressure, which are likely to be involved. Since the intra and inter-laboratory comparison of the outcomes of the hydrolytic procedure is the first step toward its standardization, the normalization of the qPCR data by the UV/qPCR ratio seems to be the simplest and most reliable way to allow this. Finally, the supplying (provided with the commercial qPCR kits) of a DNA sample whose degree of degradation is well documented could be helpful in ISO/IEC 17025 validation procedures and in proficiency testing.

      PubDate: 2018-04-11T10:25:23Z
      DOI: 10.1016/j.ab.2018.03.011
      Issue No: Vol. 549 (2018)
       
  • FIA-MS/MS determination of creatinine in urine samples undergoing
           butylation
    • Authors: Helena Jurdáková; Renáta Górová; Gabriela Addová; Anna Šalingová; Ivan Ostrovský
      Pages: 113 - 118
      Abstract: Publication date: 15 May 2018
      Source:Analytical Biochemistry, Volume 549
      Author(s): Helena Jurdáková, Renáta Górová, Gabriela Addová, Anna Šalingová, Ivan Ostrovský
      Flow injection analysis-tandem mass spectrometry has become widely used for analysis of many biomarkers in various biological matrices. To improve the sensitivity, the compounds are often determined as their butylesters. Since the concentration of urinary excreted compounds are generally reported after normalization to creatinine, the aim of this study was to investigate the possibility of creatinine determination in urine samples which underwent butylation. The impact of derivatization on urinary creatinine determination was investigated by measuring of underivatized and derivatized samples. The 10% creatine to creatinine conversion was observed during butylation, what above 700 μmol creatine/mmol creatinine caused significant creatinine overestimation. In that case, correction for creatine conversion rate was done. QC samples at six concentration levels were examined and precision and accuracy values fulfill the European Medicine Agency validation requirements. The elaborated method was applied for determination of creatinine in 41 real human urine samples. Determined creatinine concentrations were in the range of 0.27–22.3 mmol/L, linearity was confirmed within the concentration range of 0.27–31.7 mmol/L. Obtained results highly correlated with routinely used enzymatic assay for all tested samples and proposed method provide reliable determination of creatinine in butylated urine in a single run with butylesters of other analytes of interest.

      PubDate: 2018-04-11T10:25:23Z
      DOI: 10.1016/j.ab.2018.03.018
      Issue No: Vol. 549 (2018)
       
  • A simple and reproducible protocol of glass surface silanization for TIRF
           microscopy imaging
    • Authors: Michał Szkop; Beata Kliszcz; Andrzej A. Kasprzak
      Pages: 119 - 123
      Abstract: Publication date: 15 May 2018
      Source:Analytical Biochemistry, Volume 549
      Author(s): Michał Szkop, Beata Kliszcz, Andrzej A. Kasprzak
      We describe a simple and reproducible protocol for the preparation of microscope glass slides for in vitro motility assays that use total internal reflection fluorescence microscopy. The developed method utilizes trimethylchlorosilane (TMCS) as a silanizing reagent, which in the presence of imidazole as a catalyst and under optimized conditions enables reproducible preparation of high-quality hydrophobic glass surfaces. This method presents a simplification and improvement in reproducibility over the commonly applied protocol utilizing dichlorodimethylsilane (DDS) as a silanizing agent. We demonstrate the applicability of the new method by performing the analysis of the interactions of a molecular motor, kinesin-1 with microtubules.

      PubDate: 2018-04-11T10:25:23Z
      DOI: 10.1016/j.ab.2018.03.020
      Issue No: Vol. 549 (2018)
       
  • Selection of specific aptamer against enrofloxacin and fabrication of
           graphene oxide based label-free fluorescent assay
    • Authors: Somayeh Dolati; Mohammad Ramezani; Maryam Sadat Nabavinia; Vahid Soheili; Khalil Abnous; Seyed Mohammad Taghdisi
      Pages: 124 - 129
      Abstract: Publication date: 15 May 2018
      Source:Analytical Biochemistry, Volume 549
      Author(s): Somayeh Dolati, Mohammad Ramezani, Maryam Sadat Nabavinia, Vahid Soheili, Khalil Abnous, Seyed Mohammad Taghdisi
      Specific ssDNA aptamers for the antibiotic enrofloxacin (ENR) were isolated from an enriched nucleotide library by SELEX (Systematic Evolution of Ligands by EXponential enrichment) method with high binding affinity. After seven rounds, five aptamers were selected and identified. Apt58 with highest affinity and sensitivity (Kd = 14.19 nM) was employed to develop a label-free fluorescent biosensing approach based on aptamer, graphene oxide (GO) and native fluorescence of ENR for determination of ENR residue in raw milk samples. Under optimized experimental conditions, the linear range was from 5 nM to 250 nM and LOD was calculated to be 3.7 nM, and the recovery rate was between 94.1% and 108.5%. The integration of aptamer and GO in this bioassay provides a promising way for rapid, sensitive and cost-effective detection of ENR in real samples like raw milk.

      PubDate: 2018-04-11T10:25:23Z
      DOI: 10.1016/j.ab.2018.03.021
      Issue No: Vol. 549 (2018)
       
  • An aptasensor for staphylococcus aureus based on nicking enzyme
           amplification reaction and rolling circle amplification
    • Authors: Jingguo Xu; Jia Guo; Sarah Wanjiku Maina; Yumeng Yang; Yimin Hu; Xuanxuan Li; Jiarong Qiu; Zhihong Xin
      Pages: 136 - 142
      Abstract: Publication date: 15 May 2018
      Source:Analytical Biochemistry, Volume 549
      Author(s): Jingguo Xu, Jia Guo, Sarah Wanjiku Maina, Yumeng Yang, Yimin Hu, Xuanxuan Li, Jiarong Qiu, Zhihong Xin
      An ultra-sensitive aptamer-based biosensor for the detection of staphylococcus aureus was established by adopting the nicking enzyme amplification reaction (NEAR) and the rolling circle amplification (RCA) technologies. Aptamer-probe (AP), containing an aptamer and a probe sequence, was developed to act as the recognition unit of the biosensor, which was specifically bound to S. aureus. The probe was released from AP and initiated into the subsequent DNA amplification reactions where S. aureus was present, converting the detection of S. aureus to the investigation of probe oligonucleotide. The RCA amplification products contained a G-quadruplex motif and formed a three dimensional structure in presence of hemin. The G4/hemin complex showed horseradish peroxidase (HRP)-mimic activity and catalyzed the chemiluminescence reaction of luminol mediated by H2O2. The results showed that the established biosensor could detect S. aureus specifically with a good linear correlation at 5–104 CFU/mL. The signal values based on NEAR-RCA two-step cycle were boosted acutely, much higher than that relied on one-cycle magnification. The limit of detection (LoD) was determined to be as low as 5 CFU/mL. The established aptasensor exhibited a good discrimination of living against dead S. aureus, and can be applied to detect S. aureus in the food industry.

      PubDate: 2018-04-11T10:25:23Z
      DOI: 10.1016/j.ab.2018.03.013
      Issue No: Vol. 549 (2018)
       
  • Characterization of antibody-C1q interactions by Biolayer Interferometry
    • Authors: Wei Zhou; Shanshan Lin; Rongying Chen; Jun Liu; Yali Li
      Pages: 143 - 148
      Abstract: Publication date: 15 May 2018
      Source:Analytical Biochemistry, Volume 549
      Author(s): Wei Zhou, Shanshan Lin, Rongying Chen, Jun Liu, Yali Li
      IgG molecules exert important effector functions including complement-dependent cytotoxicity (CDC). Different IgG isotypes induce CDC effect with variation, largely due to their differential binding to C1q, the initiating molecule of the classical CDC pathway. Here we report a method to characterize the binding of IgG to C1q using label-free technique. With this method, we determined the binding affinities of multiple IgG1, IgG2 and IgG4 antibodies to C1q. To explore whether antigen binding to antibodies affects C1q binding, we assessed the binding of Trastuzumab and Adalimumab with bound antigen proteins to C1q. The results showed that although the two tested IgG1 mAbs alone bind C1q similarly, their FC binding to C1q was significantly impacted by antigen binding to the Fab. The data suggested that the first step of complement pathway, whether C1q binds target cell bound antibody molecules, may significantly affect the CDC activities of antibody drugs.

      PubDate: 2018-04-11T10:25:23Z
      DOI: 10.1016/j.ab.2018.03.022
      Issue No: Vol. 549 (2018)
       
  • Prediction of DNase I hypersensitive sites in plant genome using multiple
           modes of pseudo components
    • Authors: Shanxin Zhang; Weichao Zhuang; Zhenghong Xu
      Pages: 149 - 156
      Abstract: Publication date: 15 May 2018
      Source:Analytical Biochemistry, Volume 549
      Author(s): Shanxin Zhang, Weichao Zhuang, Zhenghong Xu
      DNase I hypersensitive sites (DHSs) are accessible chromatin zones hypersensitive to DNase I endonucleases in plant genome. DHSs have been used as markers for the presence of transcriptional regulatory elements. It is an important complement to develop computational methods to identify DHSs for discovering potential regulatory elements. To the best of our knowledge, several machine learning approaches have been proposed for the DHSs prediction, but there is still room for improvements. In this work, a new predictor called pDHS-WE was proposed for prediction of DHSs in plant genome by using weighted ensemble learning framework. Here, five classes of heterogeneous features were used to represent the sequences. Five random forest (RF) operators were constructed based on these five classes of features. The proposed pDHS-WE was formed by fusing the five individual RF classifiers into an ensemble predictor. Genetic algorithm was employed to obtain the weights of different classes of features. In the experiments, pDHS-WE obtained accuracy of 88.5%, sensitivity of 89.1%, specificity of 88.0%, and AUC of 0.958, which was more than 2.7%, 2%, 3.5% and 2.6% higher than state-of-the-art methods, respectively. The results suggested that pDHS-WE may become a useful tool for transcriptional regulatory elements analysis in plant genome.

      PubDate: 2018-04-11T10:25:23Z
      DOI: 10.1016/j.ab.2018.03.025
      Issue No: Vol. 549 (2018)
       
  • Application of nanomaterials for the electrical and optical detection of
           the hepatitis B virus
    • Authors: Danielle A. Oliveira; Jussara V. Silva; José M.R. Flauzino; Ana C.H. Castro; Anna C.R. Moço; Márcia M.C.N. Soares; João M. Madurro; Ana G. Brito-Madurro
      Pages: 157 - 163
      Abstract: Publication date: 15 May 2018
      Source:Analytical Biochemistry, Volume 549
      Author(s): Danielle A. Oliveira, Jussara V. Silva, José M.R. Flauzino, Ana C.H. Castro, Anna C.R. Moço, Márcia M.C.N. Soares, João M. Madurro, Ana G. Brito-Madurro
      This work describes different approaches for the detection of hepatitis B virus (HBV) genomic DNA, using electrochemical and optical techniques. The platforms consisted of a single-stranded DNA probe (HEPB1S), specific to HBV, grafted on a gold electrode modified with reduced graphene oxide or gold nanoparticles. Differential pulse voltammetry analysis indicates that the addition of HBV genomic DNA caused an increase of about 1.4 times in the current peak value, when compared to the negative control. It was observed a linear dependence with the log HBV-genomic DNA concentration and the electrochemical biosensor detected until 7.65 pg μL−1 of the target. Electrochemical impedance spectroscopy measurements showed an increase of about 2 times in the charge transfer resistance, after the addition of HBV genomic DNA. Assays using colloidal suspension of gold nanoparticles showed a shift of the peak wavelength, linearly proportional to the HBV-genomic DNA concentration, with a detection limit of 0.15 ng μL−1. The applicability of the gold nanoparticles for clinical samples was tested with success in the blood plasma. All the approaches used in this work were effective in detecting genomic DNA or blood plasma in positive samples for HBV.

      PubDate: 2018-04-11T10:25:23Z
      DOI: 10.1016/j.ab.2018.03.023
      Issue No: Vol. 549 (2018)
       
  • A fluorometric assay for lysosomal phospholipase A2 activity using
           fluorescence-labeled truncated oxidized phospholipid
    • Authors: Akira Abe; Miki Hiraoka; James A. Shayman; Hiroshi Ohguro
      Pages: 164 - 170
      Abstract: Publication date: 15 May 2018
      Source:Analytical Biochemistry, Volume 549
      Author(s): Akira Abe, Miki Hiraoka, James A. Shayman, Hiroshi Ohguro
      Lysosomal phospholipase A2 (LPLA2) is a key enzyme involved in the homeostasis of cellular phospholipids. Recently, LPLA2 was reported to preferentially degrade some truncated oxidized phospholipids at the sn-1 position. A commercially available, truncated oxidized phospholipid conjugated with a fluorescent dye, 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphoethanolamine-N-[4-(dipyrrometheneboron difluoride) butanoyl] (PGPE-BODIPY), was used to develop a specific assay for this enzyme. When recombinant mouse LPLA2 was incubated with liposomes consisting of 1,2-O-octadecyl-sn-glycero-3-phosphocholine/PGPE-BODIPY under acidic conditions, PGPE-BODIPY was converted to palmitic acid and a polar BODIPY-product. After phase partitioning by chloroform/methanol, the polar BODIPY-product was recovered in the aqueous phase and identified as 1-lyso-PGPE-BODIPY. The formation of 1-lyso-PGPE-BODIPY was quantitatively determined by fluorescent measurements. The Km and Vmax values of the recombinant LPLA2 for PGPE-BODIPY were 5.64 μM and 20.7 μmol/min/mg protein, respectively. Detectable activity against PGPE-BODIPY was present in LPLA2 deficient mouse sera, but the deacylase activity was completely suppressed by treatment with 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF). AEBSF had no effect on LPLA2 activity. The LPLA2 activity of mouse serum pre-treated with AEBSF was specifically and quantitatively determined by this assay method. The PGPE-BODIPY and AEBSF based LPLA2 assay is convenient and can be used to measure LPLA2 activity in a variety of biological specimens.

      PubDate: 2018-04-11T10:25:23Z
      DOI: 10.1016/j.ab.2018.03.024
      Issue No: Vol. 549 (2018)
       
  • The detection of a mismatched DNA by using hairpin DNA-templated silver
           nanoclusters
    • Authors: Seyeon Kim; Jongback Gang
      Pages: 171 - 173
      Abstract: Publication date: 15 May 2018
      Source:Analytical Biochemistry, Volume 549
      Author(s): Seyeon Kim, Jongback Gang
      Fluorescence assays have been developed to detect biomolecules using DNA-templated silver nanoclusters (DNA-AgNCs) utilizing their unique physical and optical properties. This study was designed to detect single-mismatched DNA by the hybridization of target DNA to template DNA either before or after DNA-AgNCs synthesis. The results showed that the detection specificity of a single-mismatched DNA was clearly enhanced when the target DNA (cDNA) was hybridized to template DNA prior to DNA-AgNCs synthesis compared with cDNA hybridization subsequent to DNA-AgNCs synthesis.

      PubDate: 2018-04-11T10:25:23Z
      DOI: 10.1016/j.ab.2018.03.026
      Issue No: Vol. 549 (2018)
       
  • Quantification of receptor activation by oxytocin and vasopressin in
           endocytosis-coupled bioluminescence reduction assay using nanoKAZ
    • Authors: Isao Kii; Shino Hirahara-Owada; Masataka Yamaguchi; Takashi Niwa; Yuka Koike; Rie Sonamoto; Harumi Ito; Kayo Takahashi; Chihiro Yokoyama; Takuya Hayashi; Takamitsu Hosoya; Yasuyoshi Watanabe
      Pages: 174 - 183
      Abstract: Publication date: 15 May 2018
      Source:Analytical Biochemistry, Volume 549
      Author(s): Isao Kii, Shino Hirahara-Owada, Masataka Yamaguchi, Takashi Niwa, Yuka Koike, Rie Sonamoto, Harumi Ito, Kayo Takahashi, Chihiro Yokoyama, Takuya Hayashi, Takamitsu Hosoya, Yasuyoshi Watanabe
      Oxytocin (OXT) and arginine vasopressin (AVP) are structurally similar neuropeptide hormones that function as neurotransmitters in the brain, and have opposite key roles in social behaviors. These peptides bind to their G protein-coupled receptors (OXTR and AVPRs), inducing calcium ion-dependent signaling pathways and endocytosis of these receptors. Because selective agonists and antagonists for these receptors have been developed as therapeutic and diagnostic agents for diseases such as psychiatric disorders, facile methods are in demand for the evaluation of selectivity between these receptors. In this study, we developed a quantitative assay for OXT- and AVP-induced endocytosis of their receptors. The mutated Oplophorus luciferase, nanoKAZ, was fused to OXTR and AVPRs to enable rapid quantification of agonist-induced endocytosis by bioluminescence reduction. Agonist stimulation significantly decreases bioluminescence of nanoKAZ-fused receptors in living cells. Using this system, we evaluated clinically used OXTR antagonist atosiban and a reported pyrazinyltriazole derivative, hereby designated as PF13. Atosiban acted as an antagonist of AVPR1a, as well as an agonist for AVPR1b, whereas PF13 antagonized OXTR more selectively than atosiban, as reported previously. This paper shows a strategy for quantification of agonist-induced endocytosis of OXTR and AVPRs, and confirms its potent utility in the evaluation of agonists and antagonists.
      Graphical abstract image

      PubDate: 2018-04-24T22:01:48Z
      DOI: 10.1016/j.ab.2018.04.001
      Issue No: Vol. 549 (2018)
       
  • Quantification of proteins by data independent acquisition: Performance
           assessment of the Hi3 methodology
    • Authors: Guillaume Chevreux; Nolwenn Tilly; Nicolas Bihoreau
      Pages: 184 - 187
      Abstract: Publication date: 15 May 2018
      Source:Analytical Biochemistry, Volume 549
      Author(s): Guillaume Chevreux, Nolwenn Tilly, Nicolas Bihoreau
      Proteomics greatly benefited from the development of mass spectrometry. Over the last years, data-independent acquisitions increased in popularity in an effort to provide routine label free quantitative information. In this report, the performance of the Hi3 label free method was assessed based on the analysis of a plasma-derived protein mixture. The following parameters of the method (CVs) were determined: repeatability 13.8%, intermediate precision 27.6%, bias 32.3% and linearity observed over 3 orders of magnitude. Finally an accuracy of 42.5% corresponding to a confidence interval within 2 fold the expected protein abundance should be a good approximation of the method performance.

      PubDate: 2018-04-15T10:34:19Z
      DOI: 10.1016/j.ab.2018.03.019
      Issue No: Vol. 549 (2018)
       
  • A method for the extraction of the endogenous tryptic peptides (peptidome)
           from human EDTA plasma
    • Authors: Jaimie Dufresne; Angelique Florentinus-Mefailoski; Pete Bowden; John G. Marshall
      Pages: 188 - 196
      Abstract: Publication date: 15 May 2018
      Source:Analytical Biochemistry, Volume 549
      Author(s): Jaimie Dufresne, Angelique Florentinus-Mefailoski, Pete Bowden, John G. Marshall
      The proteins identified from endogenous peptides agreed between serum versus plasma, and tryptic versus non-tryptic peptides, when collected by C18 alone and analyzed by liquid chromatography electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS) including amyloids, apolipoproteins, haptoglobin, complements, fibrinogens, hemopexin, antitrypsin and alpha 2 macroglobulin. Precipitation of polypeptides from plasma in 9 vol of 100% organic solvent followed by stepwise extraction of the insoluble pellet with an increasing fraction of water identified thousands of proteins. A Coomassie-blue protein binding assay, and tricine SDS-PAGE, showed that Acetonitrile-Water (AH) resulted in a greater relative enrichment of low molecular weight plasma polypeptides than Acetonitrile-Methanol Water (AMH). A total of 905,386 MS/MS spectra greater than ~10,000 (E4) counts were correlated by X!TANDEM to a federated human protein library of 153,124 different protein sequences that resulted in 58,223 fully tryptic peptides from 3463 Gene Symbols of which 1880 had ≥ 5 independent peptides (p ≤ 0.00001). The results were filtered and organized in an SQL database for analysis using the generic R statistical analysis system. Cellular proteins including secreted and exosome proteins, signaling factors, nucleic acid binding proteins, metabolic enzymes and uncharacterized factors were observed with a significant enrichment of expected protein-protein interactions by STRING analysis.

      PubDate: 2018-04-24T22:01:48Z
      DOI: 10.1016/j.ab.2018.02.025
      Issue No: Vol. 549 (2018)
       
  • Simultaneous detection of Mycoplasma pneumoniae IgG and IgM using
           dual-label time resolved fluoroimmunoassay
    • Authors: Yi Zhang; Xue Yang; Jun Qian; Xiaohong Gu; Jue Zhang; Jie Liu; Zhigang Hu
      Pages: 1 - 6
      Abstract: Publication date: 1 May 2018
      Source:Analytical Biochemistry, Volume 548
      Author(s): Yi Zhang, Xue Yang, Jun Qian, Xiaohong Gu, Jue Zhang, Jie Liu, Zhigang Hu
      Anti-Mycoplasma pneumoniae (MP) IgM and IgG are useful serological markers for detection of MP infection. In this study, a simultaneous quantification of MP IgM and IgG was performed by time-resolved fluoroimmunoassay (TRFIA). The europium-labeled anti-human IgM and samarium-labeled anti-human IgG were used as tracers, and MP IgM and IgG were recognized in serum samples. After dissociating europium and samarium ions from the immune complex, their fluorescence intensity was recorded and used to calculate the concentrations. The linear range and sensitivity of detection were 2–5500 BU/mL and 0.5 BU/mL for IgM, and 1.5–1500 BU/mL and 0.2 BU/mL for IgG, respectively. The intra- and inter-assay coefficients of variation were 5.14% and 8.41% for IgM, and 5.44% and 8.76% for IgG, respectively. The recovery rate was 94.9–106.8% for IgM and 96.1–109.4% for IgG. The correlation rates of serum detection for 38 respiratory infected patients between dual-label TRFIA and ELISA were 0.9294 and 0.9366 for IgM and IgG, respectively. The coincidence rate between passive particle agglutination and TRFIA is 93.3%. Dual-label TRFIA is a sensitive and reliable technique for measuring MP IgM and IgG levels and could be useful for the early diagnosis of MP infection.

      PubDate: 2018-02-26T00:26:16Z
      DOI: 10.1016/j.ab.2018.02.015
      Issue No: Vol. 548 (2018)
       
  • An efficient screening method for purifying and crystallizing membrane
           proteins using modified clear-native PAGE
    • Authors: Nanao Suzuki; Yuuki Takamuku; Tomohiro Asakawa; Makoto Inai; Tomoya Hino; So Iwata; Toshiyuki Kan; Takeshi Murata
      Pages: 7 - 14
      Abstract: Publication date: 1 May 2018
      Source:Analytical Biochemistry, Volume 548
      Author(s): Nanao Suzuki, Yuuki Takamuku, Tomohiro Asakawa, Makoto Inai, Tomoya Hino, So Iwata, Toshiyuki Kan, Takeshi Murata
      Membrane proteins, such as G-protein coupled receptors, control communication between cells and their environments and are indispensable for many cellular functions. Nevertheless, structural studies on membrane proteins lag behind those on water-soluble proteins, due to their low structural stability, making it difficult to obtain crystals for X-ray crystallography. Optimizing conditions to improve the stability of membrane proteins is essential for successful crystallization. However, the optimization usually requires large amounts of purified samples, and it is a time-consuming and trial-and-error process. Here, we report a rapid method for precrystallization screening of membrane proteins using Clear Native polyacrylamide gel electrophoresis (CN-PAGE) with the modified Coomassie Brilliant Blue G-250 (mCBB) stain that was reduced in sodium formate. A2A adenosine receptor (A2AAR) was selected as a target membrane protein, for which we previously obtained the crystal structure using an antibody, and was expressed as a red fluorescent protein fusion for in-gel fluorescence detection. The mCBB CN-PAGE method enabled the optimization of the solubilization, purification, and crystallization conditions of A2AAR using the solubilized membrane fraction expressing the protein without purification procedures. These data suggest the applicability of mCBB CN-PAGE technique to a wide variety of integral membrane proteins.

      PubDate: 2018-02-26T00:26:16Z
      DOI: 10.1016/j.ab.2018.02.007
      Issue No: Vol. 548 (2018)
       
  • Functional electrospun nanofibers-based electrochemiluminescence
           immunosensor for detection of the TSP53 using RuAg/SiO2NPs as signal
           enhancers
    • Authors: Xiaoying Wang; Yijie Wang; Meng Jiang; Yanqun Shan; Xin Jin; Miao Gong; Xiaoning Wang
      Pages: 15 - 22
      Abstract: Publication date: 1 May 2018
      Source:Analytical Biochemistry, Volume 548
      Author(s): Xiaoying Wang, Yijie Wang, Meng Jiang, Yanqun Shan, Xin Jin, Miao Gong, Xiaoning Wang
      A functional electrospun nanofibers-based electrochemiluminescence (ECL) immunosensor for detection of the tumor suppressor protein p53 (TSP53) at trace level using core-shell nanocomposite as signal enhancers is fabricated. The gold nanoparticles (AuNPs) were decorated on the surface of the electrospun carbon-nanotubes (MWCNTs) doped chitosan (CTS) nanofibers (MWCNTs-CTS) by in-situ electrodeposition. The functional electrospun nanofibers (MWCNTs-CTS-AuNPs) was utilized as supporting scaffolds for TSP53 capture antibody (CAb) immobilization firstly. They can dramatically increase the amount and stability of CAb attachment, and efficiently enhance the sensitivity of detection. After a sandwich immunoreaction, TSP53 and Ru(bpy)3 2+/silver nanoparticles doped silica core-shell nanocomposite (RuAg/SiO2NPs)-labeled detection antibody (RuAg/SiO2NPs@DAb) captured onto the electrode surface. It was observed that the increase of response ECL signal was proportional to the TSP53 concentration in the range of 1 pg mL−1 to 1 ng mL−1. The detection limit was 0.5 pg mL−1, which is comparable or better than that in reported TSP53 assays. The immunosensor was successfully applied to determination of TSP53 in normal human cubital vein blood samples with good recovery, and the results are basically consistent with the TSP53 Kit (ELISA). Excellent sensitivity and selectivity make the developed ECL immunosensor a potential and simple tool for the detection of tumor biomarkers.
      Graphical abstract image

      PubDate: 2018-02-26T00:26:16Z
      DOI: 10.1016/j.ab.2018.02.006
      Issue No: Vol. 548 (2018)
       
  • A methodological approach for the thermal stability and stress exposure
           studies of a model antibody
    • Authors: Gaëlle Coussot; Clément Faye; Aurélie Le Postollec; Michel Dobrijevic
      Pages: 23 - 31
      Abstract: Publication date: 1 May 2018
      Source:Analytical Biochemistry, Volume 548
      Author(s): Gaëlle Coussot, Clément Faye, Aurélie Le Postollec, Michel Dobrijevic
      The anti-horseradish peroxidase (HRP) antibody is conventionally used in immunohistochemistry. More recently, it has been used as the key element in a gold standard method to evaluate the functionality of antibody-based materials. However, few information are available about its melting temperature and its stability after exposition to laboratory stress conditions including freeze-drying and freeze-thawing cycles. The aim of this study was to evaluate the effects of these environmental constraints on the anti-HRP antibody in order to further use it as a reference in quality control and in the development of new antibody-based materials. In the developed method, the anti-HRP antibody is covalently immobilized onto a solid surface. After the direct recognition of its antigen HRP, the signal is proportional to the number of antibody active binding sites. The method was successfully utilized to accurately evaluate the anti-HRP antibody melting temperature (Tm was 73.5 ± 0.2 °C). The method is a rapid and reliable tool with minimal cost for studying the anti-HRP antibody stability to solvent stress, freeze-thawing cycles, and freeze-drying process. The obtained information may be useful for routine analysis or in the development of antibody-based materials. This can be also proposed as an easy way to control antibody freeze-drying process.
      Graphical abstract image

      PubDate: 2018-03-08T03:19:02Z
      DOI: 10.1016/j.ab.2018.02.019
      Issue No: Vol. 548 (2018)
       
  • A highly sensitive electrochemical sensor for the determination of
           methanol based on PdNPs@SBA-15-PrEn modified electrode
    • Authors: Ziba Karimi; Mojtaba Shamsipur; Mahmoud Amouzadeh Tabrizi; Sadegh Rostamnia
      Pages: 32 - 37
      Abstract: Publication date: 1 May 2018
      Source:Analytical Biochemistry, Volume 548
      Author(s): Ziba Karimi, Mojtaba Shamsipur, Mahmoud Amouzadeh Tabrizi, Sadegh Rostamnia
      In this study, a novel electrochemical sensor for the determination of methanol based on palladium nanoparticles supported on Santa barbara amorphous-15- PrNHEtNH2 (PdNPs@SBA-15-PrEn) as nanocatalysis platform is presented. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD) and electrochemical methods are employed to characterize the PdNPs@SBA-15-PrEn nanocomposite. The Nafion-Pd@SBA-15-PrEn modified glassy carbon electrode (Nafion-PdNPs@SBA-15-PrEn/GCE) displayed the high electrochemical activity and excellent catalytic characteristic for electro-oxidation of methanol in an alkaline solution. The electro-oxidation performance of the proposed sensor was investigated using cyclic voltammetry (CV) and amperometry. The sensor exhibits a good sensitivity of 0.0905 Amol−1 Lcm−2, linear range of 20–1000 μM and the corresponding detection limit of 12 μM (3σ). The results demonstrate that the Nafion-PdNPs@SBA-15-PrEn/GCE has potential as an efficient and integrated sensor for methanol detection.

      PubDate: 2018-03-08T03:19:02Z
      DOI: 10.1016/j.ab.2018.01.033
      Issue No: Vol. 548 (2018)
       
  • Near infrared optical biosensor based on peptide functionalized
           single-walled carbon nanotubes hybrids for 2,4,6-trinitrotoluene (TNT)
           explosive detection
    • Authors: Jin Wang
      Abstract: Publication date: 1 June 2018
      Source:Analytical Biochemistry, Volume 550
      Author(s): Jin Wang
      A near infrared (NIR) optical biosensor based on peptide functionalized single-walled carbon nanotubes (SWCNTs) hybrids for 2,4,6-trinitrotoluene (TNT) explosive detection was developed. The TNT binding peptide was directly anchored on the sidewall of the SWCNTs using the π-π interaction between the aromatic amino acids and SWCNTs, forming the peptide-SWCNTs hybrids for near infrared absorption spectra measurement. The evidence of the morphology of peptide-SWCNTs hybrids was obtained using atomic force microscopy (AFM). The results demonstrated that peptide-SWCNTs hybrids based NIR optical biosensor exhibited sensitive and highly selective for TNT explosive determination, addressing a promising optical biosensor for security application.

      PubDate: 2018-04-24T22:01:48Z
       
  • A mass spectrometry based transport assay for studying EmrE transport of
           unlabeled substrates
    • Authors: Anne E. Robinson; Jeffrey P. Henderson; Katherine A. Henzler-Wildman
      Abstract: Publication date: Available online 17 March 2018
      Source:Analytical Biochemistry
      Author(s): Anne E. Robinson, Jeffrey P. Henderson, Katherine A. Henzler-Wildman
      Membrane transporters are an important class of proteins which remain challenging to study. Transport assays are crucial to developing our understanding of such proteins as they allow direct measurement of their transport activity. However, currently available methods for monitoring liposomal loading of organic substrates primarily rely on detection of radioactively or fluorescently labeled substrates. The requirement of a labeled substrate significantly restricts the systems and substrates that can be studied. Here we present a mass spectrometry based detection method for liposomal uptake assays that eliminates the need for labeled substrates. We demonstrate the efficacy of the assay with EmrE, a small multidrug resistance transporter found in E. coli that has become a model transport system for the study of secondary active transport. Furthermore, we develop a method for differentiation between bound and transported substrate, enhancing the information gained from the liposomal uptake assay. The transport assay presented here is readily applicable to other transport systems and substrates.
      Graphical abstract image

      PubDate: 2018-03-19T20:43:07Z
      DOI: 10.1016/j.ab.2018.03.017
       
  • Facile preparation of highly active casein kinase 1 using Escherichia coli
           constitutively expressing lambda phosphatase
    • Authors: Kazutoshi Akizuki; Taku Toyama; Masashi Yamashita; Yasunori Sugiyama; Atsuhiko Ishida; Isamu Kameshita; Noriyuki Sueyoshi
      Abstract: Publication date: Available online 17 March 2018
      Source:Analytical Biochemistry
      Author(s): Kazutoshi Akizuki, Taku Toyama, Masashi Yamashita, Yasunori Sugiyama, Atsuhiko Ishida, Isamu Kameshita, Noriyuki Sueyoshi
      Casein kinase 1 (CK1) is a widely expressed Ser/Thr kinase in eukaryotic organisms that is involved in various cellular processes (e.g., circadian rhythm and apoptosis). Therefore, preparing highly active CK1 and investigating its properties in vitro have important implications for understanding the biological roles of the kinase. However, recombinant CK1 undergoes autoinactivation via autophosphorylation in Escherichia coli cells and thus is undesirably prepared as a phosphorylated and inactivated kinase. To circumvent this problem, we established a protein expression system using E. coli strain BL21(DE3)pλPP in which λ protein phosphatase (λPPase) is constitutively expressed. Using this system, recombinant CK1 isoforms (α, δ and ε) were readily prepared as unphosphorylated forms. Furthermore, we found that CK1s prepared using BL21(DE3)pλPP showed markedly higher activity than those prepared by the conventional BL21(DE3). Finally, we demonstrated that the kinase activity of CK1δ from BL21(DE3)pλPP was higher than that prepared by a conventional method consisting of troublesome steps such as in vitro λPPase treatment. Thus, this simple method using BL21(DE3)pλPP is valuable for preparing highly active CK1s. It may also be applicable to other kinases that are difficult to prepare because of phosphorylation in E. coli cells.
      Graphical abstract image

      PubDate: 2018-03-19T20:43:07Z
      DOI: 10.1016/j.ab.2018.03.015
       
  • An ELISA for the study of calcineurin-NFAT unstructured region interaction
    • Authors: Nesly Dotan; Vera Gayder; Itai Bloch; Maayan Gal
      Abstract: Publication date: Available online 16 March 2018
      Source:Analytical Biochemistry
      Author(s): Nesly Dotan, Vera Gayder, Itai Bloch, Maayan Gal
      Calcineurin is a phosphatase that targets the transcription factor, nuclear factor of activated T-cells (NFAT) dephosphorylates multiple sites along NFAT's regulatory domain. The calcineurin-NFAT complex interaction is mediated through two conserved binding motifs known as the PxIxIT and LxVP, which are located at the N- and C- terminus to the phosphorylation sites. The vast range of cellular processes regulated by the calcineurin-NFAT interaction has aroused great interest in the investigation of the structural aspects that govern their complex formation and in the discovery of protein-protein interaction inhibitors; the latter interfere with calcineurin-NFAT complex formation while keeping calcineurin's catalytic site free. To assist additional biophysical study of the calcineurin-NFAT structure-function relation and to screen for new inhibitors, we present a robust and cost-effective Enzyme Linked Immuno Sorbent Assay (ELISA) that is based on the interaction of calcineurin with the NFAT homology region. The latter includes the two calcineurin's binding sites, in addition to the phosphorylation sites. The ELISA experiment shown here can thus be applied towards the study of important structural aspects of the complex and for the discovery of new inhibitors. This will allow for a better understanding of T-cell activation switch.

      PubDate: 2018-03-19T20:43:07Z
      DOI: 10.1016/j.ab.2018.03.014
       
  • Metabolic profiling of moulds with laser desorption/ionization mass
           spectrometry on gold nanoparticle enhanced target
    • Authors: Adrian Arendowski; Justyna Szulc; Joanna Nizioł; Beata Gutarowska; Tomasz Ruman
      Abstract: Publication date: Available online 16 March 2018
      Source:Analytical Biochemistry
      Author(s): Adrian Arendowski, Justyna Szulc, Joanna Nizioł, Beata Gutarowska, Tomasz Ruman
      Surface-assisted laser desorption/ionization mass spectrometry on gold nanoparticle enhanced target (AuNPET) technique was used for metabolomic analysis and secondary metabolites detection of two mould strains – Aspergillus versicolor and Penicillium chrysogenum in model conditions on microbiological malt extract agar medium. Results obtained with the use of AuNPET-based mass spectrometry technique were compared with traditional matrix-assisted laser desorption/ionization (MALDI) method based on α-cyano-4-hydroxycinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB) matrices. Gold nanoparticle enhanced target method enabled effective ionization of microbial cellular extract ingredients without interference from the matrix and also improved calibration of spectra resulting in the detection of much higher amount of characteristic metabolites for studied organisms than MALDI.
      Graphical abstract image

      PubDate: 2018-03-19T20:43:07Z
      DOI: 10.1016/j.ab.2018.03.016
       
  • 3-Amino-1-phenyl-2-pyrazoline-5-ketone as a heterobifunctional chromogenic
           reagent to derivatize reducing glycans for subsequent online LC/MS
           analysis
    • Authors: Yu Lu; Chengjian Wang; Rendan Liu; Wanjun Jin; Yanan Wen; Linjuan Huang; Zhongfu Wang
      Abstract: Publication date: Available online 7 March 2018
      Source:Analytical Biochemistry
      Author(s): Yu Lu, Chengjian Wang, Rendan Liu, Wanjun Jin, Yanan Wen, Linjuan Huang, Zhongfu Wang
      Sensitive analysis of glycans by liquid chromatography/mass spectrometry is significantly hampered by the lack of chromogenic or fluorescent groups on the glycan structures, as well as, their poor ionization properties. In the present, a heterobifunctional chromogenic reagent 3-amino-1-phenyl-2-pyrazoline-5-ketone (PAP) bearing amino and active methylene groups, which readily reacts with reducing glycans, was used for detection of the pre-column-labeled glycans via high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS). The PAP derivatives with active methylene and amino groups were obtained via reductive amination in acidic medium and condensation of an active PAP methylene group with the reducing end of glycans in alkaline medium, respectively, and the PAP derivatives could be further functionalized, e.g., via glycan microarray preparation. The conditions for the two reaction modes were optimized, the HPLC separation method of PAP derivatives was investigated, and the PAP derivatives of some glycans derived from biological samples were obtained and analyzed by ESI-MS and LC-MS. Using this new reagent, reducing glycans can be selectively derivatized by different reaction mechanisms, having great importance for functional glycomics studies.

      PubDate: 2018-03-08T03:19:02Z
      DOI: 10.1016/j.ab.2018.02.029
       
  • Analyzing mitochondrial function in human peripheral blood mononuclear
           cells
    • Authors: Chao-Pin Hsiao; Charles Hoppel
      Abstract: Publication date: Available online 2 March 2018
      Source:Analytical Biochemistry
      Author(s): Chao-Pin Hsiao, Charles Hoppel
      Mitochondrial oxidative phosphorylation (OXPHOS) is responsible for producing most of the adenosine triphosphate required by eukaryotic cells. Lymphocytes make up the majority of the peripheral blood mononuclear cells. Peripheral blood mononuclear cells are readily obtainable, providing an ideal sample to monitor systemic changes and understand molecular signaling mechanisms in disease processes. Mitochondrial energy metabolism of lymphocyte has been used to screen for OXPHOS disorders. While there are increasing studies of lymphocyte OXPHOS, few studies examined activity of electron transport chain of lymphocyte mitochondria. We present an optimal protocol to harvest fresh peripheral blood mononuclear cells from human whole blood, determine integrated mitochondrial function, and analyze electron transport chain complex activity. Analyzing integrated mitochondrial function using OXPHOS provides data to uncover defects in the transport of substrates into the mitochondria, generation of reducing equivalents, the electron transport chain, and coupling to the production of adenosine triphosphate. The optimal conditions to harvest peripheral blood mononuclear cells were using blood anticoagulated with ethylenediaminetetraacetic acid, processed utilizing Lymphoprep™, and washed in phosphate buffered saline, all at room temperature. Using isolated peripheral blood mononuclear cells, integrated mitochondrial function and the activities of electron transport chain were determined.

      PubDate: 2018-03-08T03:19:02Z
      DOI: 10.1016/j.ab.2018.03.003
       
 
 
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