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Analytical Biochemistry
Journal Prestige (SJR): 0.633
Citation Impact (citeScore): 2
Number of Followers: 209  
 
  Hybrid Journal Hybrid journal (It can contain Open Access articles)
ISSN (Print) 0003-2697 - ISSN (Online) 1096-0309
Published by Elsevier Homepage  [3185 journals]
  • Triethylamine improves MS signals stability of diluted oligonucleotides
           caused by sample containers
    • Abstract: Publication date: Available online 20 September 2019Source: Analytical BiochemistryAuthor(s): Rong Liu, Zhongqiu Liu, Lingzhi Gong The effect of sample containers made of different materials on the MS-based analysis of oligonucleotides remains unknown. Here, we evaluated five types of sample containers on the MS signal stability of oligonucleotide, and they were normal glass insert, silanized glass insert with three different silanization techniques, and polypropylene sample vial. Also, we attempted to tackle signal stability issue by varying modifiers in dissolution solvent. Our results showed that sample containers made of different materials can significantly influence the MS signal stability of oligonucleotide at low concentration. Triethylamine (TEA) evidently improved both the signal stability and intensity of oligonucleotide.
       
  • Detection of potential biomarkers associated with outrageous diseases and
           environmental pollutants by nanoparticle-based immuno-PCR assays
    • Abstract: Publication date: Available online 20 September 2019Source: Analytical BiochemistryAuthor(s): Bhawna Dahiya, Promod K. Mehta Immuno-polymerase chain reaction (I-PCR) assay with advantages of both enzyme-linked immunosorbent assay (ELISA) and PCR exhibits several-fold enhanced sensitivity in comparison to respective ELISA, which has wide applications for ultralow detection of several molecules, i.e. cytokines, protooncogenes and biomarkers associated with several diseases. Conjugation of reporter DNA to the detection antibodies is the most crucial step of I-PCR assay that usually employs streptavidin-protein A, streptavidin-biotin conjugate or succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) system by a covalent binding. However, coupling of antibodies and oligonucleotides to nanoparticles (NPs) is relatively easier in the NP- based I-PCR (NP–I-PCR) that also displays better accuracy. This article is mainly focused on the detection of important biomarkers associated with several outrageous infectious and non-infectious diseases by NP-I-PCR assays, which would expedite an early initiation of therapy thus human health would be improved. Similarly, ultralow detection of environmental pollutants by these assays and their elimination would certainly improve human health.
       
  • Explicit measurement of the endotoxin adsorption efficiency detects
           non-Langmuir behavior at low concentrations
    • Abstract: Publication date: Available online 19 September 2019Source: Analytical BiochemistryAuthor(s): Ekaterina G. Kholina, Alexey M. Nesterenko, Tatiana V. Galochkina, Danil A. Gvozdev, Irina V. Polyakova, Ilya B. Kovalenko, Marina G. Strakhovskaya, Oleg A. Pisarev Lipopolysaccharides (LPS) are the Gram-negative bacteria cell wall components capable to induce the system inflammatory response even at picomolar concentrations. LPS detection at these concentrations is necessary to develop new sorbents for the efficient purification of the biological fluids. LAL-test widely used for LPS concentration estimation is based on the LPS biological activity measurement and thus may depend on the LPS concentration in a non-linear way. Here we propose a new explicit method for the LPS concentration measurement based on fluorescently labeled LPS and direct photon counting and develop the new protocol for LPS adsorption efficiency measurement. Following the suggested protocol in the experiments on novel sorbents, we demonstrate that LPS adsorption at small biologically relevant concentrations is non-Langmuir.Graphical abstractImage 1
       
  • Comparison of seven methods for DNA extraction from prosomata of the acorn
           barnacle, Amphibalanus amphitrite
    • Abstract: Publication date: Available online 17 September 2019Source: Analytical BiochemistryAuthor(s): Janna N. Schultzhaus, Chris R. Taitt, Beatriz Orihuela, Madeline Smerchansky, Zachary S. Schultzhaus, Daniel Rittschof, Kathryn J. Wahl, Christopher M. Spillmann Next generation sequencing (NGS) technologies can provide an understanding of the molecular processes involved in marine fouling by Amphibalanus spp. barnacles. Here, seven methods for extracting DNA from A. amphitrite prosomata were assessed with respect to recovery, purity and size distribution. Methods incorporating organic extractions generally resulted in low recovery of fragmented DNA. The most promising method was the commercial E.Z.N.A. Blood DNA Mini kit, which provided tens of micrograms of DNA of sufficient molecular weight for use in long-read NGS library preparation. Other kits resulted in DNA preps suitable for short read length NGS platforms.
       
  • Perfused murine heart optical transmission spectroscopy using optical
           catheter and integrating sphere: Effects of ischemia/reperfusion
    • Abstract: Publication date: Available online 17 September 2019Source: Analytical BiochemistryAuthor(s): Tyler M. Bauer, Abigail V. Giles, Junhui Sun, Armel Femnou, Raul Covian, Elizabeth Murphy, Robert S. Balaban Tissue transmission optical absorption spectroscopy provides dynamic information on metabolism and function. Murine genetic malleability makes it a major model for heart research. The diminutive size of the mouse heart makes optical transmission studies challenging. Using a perfused murine heart center mounted in an integrating sphere for light collection with a ventricular cavity optical catheter as an internal light source provided an effective method of optical data collection in this model. This approach provided high signal to noise optical spectra which when fit with model spectra provided information on tissue oxygenation and redox state. This technique was applied to the study of cardiac ischemia and ischemia reperfusion which generates extreme heart motion, especially during the ischemic contracture. The integrating sphere reduced motion artifacts associated with a fixed optical pickup and methods were developed to compensate for changes in tissue thickness. During ischemia, rapid decreases in myoglobin oxygenation occurred along with increases in cytochrome reduction levels. Surprisingly, when ischemic contracture occurred, myoglobin remained fully deoxygenated, while the cytochromes became more reduced consistent with a further, and critical, reduction of mitochondrial oxygen tension during ischemic contraction. This optical arrangement is an effective method of monitoring murine heart metabolism.
       
  • Verification and applicability of endogenous reference genes for
           quantifying GM rice by digital PCR
    • Abstract: Publication date: Available online 17 September 2019Source: Analytical BiochemistryAuthor(s): Tingting Deng, Wensheng Huang, Junan Ren, Xiuli Ma, Yiqiang Ge, Ying Chen To standardize the rice-specific quantification methods, the criteria of six genes of rice (gos9, PLD, SPS, RBE4, ppi-PPF and oriazain) were compared and evaluated by ddPCR. The results revealed that SPS, RBE4 and ppi-PPF were single copy genes per haploid genome and species specificity and stable among different rice cultivars, by employing Lectin gene of soybean as internal reference gene. The established ddPCR systems were precise and reliable with an absolute LOQ of 10–20 copies/reaction. Furthermore, the robustness of these three assays was verified by performing an intra-laboratory repeatability validation and the results showed that the three endogenous genes of rice could be quantitated repeatedly and precisely above the LOQ. These ddPCR methods can reliably quantified the GM content even if the content was low to 0.1%, which were much more reliable than the results from real-time PCR using the same primers and probes.Graphical abstractImage 1
       
  • Apparent degradation forms of rhG-CSF under forced conditions: Insights
           for better quality-control of bioproducts
    • Abstract: Publication date: Available online 15 September 2019Source: Analytical BiochemistryAuthor(s): Jamila Behi, Rym Hassiki, Nadia Ben Said, Ayoub Ksouri, Mohamed Lamine Benkhoud, Balkiss Bouhaouala-Zahar Stability and quality control of therapeutic protein formulations is a substantial part of drug development process. The objective of this study is to obtain information about stability of a recombinant human granulocyte colony stimulating factor (rhG-CSF) against various stress factors. This will play a crucial role in the finished product formulation development. In this study, rhG-CSF was exposed to various chemical and physical stress conditions at different levels in order to identify degradation pathways and the nature of impurities generated. Experiments were performed by a combination of orthogonal analytical techniques (reversed phase chromatography (RP-HPLC), size exclusion chromatography (SEC-HPLC), polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF)) to set and characterize the different degraded samples. The SEC-HPLC results suggest that the major degradation factors generating aggregated forms of the protein are basically thermal stress, freeze-thaw cycles and vortexing. Meanwhile, deamidated rhG-CSF was induced by basic pH as shown by IEF electrophoregram. As well, oxidized forms were generated increasingly with the time of exposure to hydrogen peroxide as outlined by RP-HPLC analysis. Based on these results, it was possible to define the storage and handling conditions of rhG-CSF finished product during its shelf life.Graphical abstractImage 1
       
  • Association of FSHR missense mutations with female infertility, in silico
           investigation of their molecular significance and exploration of possible
           treatments using virtual screening and molecular dynamics
    • Abstract: Publication date: Available online 12 September 2019Source: Analytical BiochemistryAuthor(s): Haniye Haqiqi, Marziye Farsimadan, Ardavan Abiri, Alireza Sharafshah, Hamidreza Vaziri, Ziba Zahiri This study investigated the association of A419T (rs121909661) and T449I (rs28928870) with infertility among Iranian women and possible treatments by agonizing the mutated receptor. 151 women were genotyped at A419T and T449I sites. Homology modeling, pharmacophore modeling, virtual screening, docking and molecular dynamics (MD) were performed. A419T and T449I indicated a significant and a weak association with infertility among Iranian women (P = 0.005 and P = 0.03, respectively). Significant differences found among three genotypes of A419T with FSH (P = 0.01) and LH (P 
       
  • Visual detection of kanamycin with DNA-functionalized gold nanoparticles
           probe in aptamer-based strip biosensor
    • Abstract: Publication date: Available online 12 September 2019Source: Analytical BiochemistryAuthor(s): Ying Ou, Xin Jin, Jing Liu, Yaping Tian, Nandi Zhou Kanamycin has been widely used to treat human and animal diseases. The excessive use of kanamycin causes its accumulation in animal-derived foods, and eventually threats human health. In the present study, we develop a lateral flow strip biosensor for fast and sensitive detection of kanamycin. The strip biosensor combines the easy separation of magnetic microspheres (MMS) with target-mediated chain displacement of single-stranded DNA and the capture of the visible DNA-functionalized gold nanoparticles (AuNPs) probe. The presence of kanamycin can competitively bind to the aptamer and release cDNA to the supernatant. The concentration of free cDNA, which is the direct target of the strip, is proportional to the concentration of kanamycin. The capture of DNA-functionalized AuNPs on the test zone of the strip through cDNA-induced hybridization provides a visual detection signal. The assay can be completed within 20 min. The visual detection limit by naked eyes of the strip is 50 nM. A linear detection range of 5–500 nM is derived for quantitative determination, with the detection limit of 4.96 nM (S/N = 3). This lateral flow strip biosensor can quickly and sensitively detect kanamycin in different food samples, which holds great application potential in medicine and daily life.Graphical abstractImage 1
       
  • A toolkit for expression of Strep-tagged enhanced green fluorescent
           protein concatemers in mammalian cells
    • Abstract: Publication date: Available online 12 September 2019Source: Analytical BiochemistryAuthor(s): Aline Zweifel, Susanne Giehler, Marcus M. Nalaskowski Green fluorescent protein (GFP) and its variants are widely used tools in life sciences. Recently, we and others have used enhanced green fluorescent protein (EGFP) concatemers for determination of nuclear localization signal (NLS) strength, as natural fluorescence standards and for mapping mobility in living cell nuclei. In this study, we present a molecular toolbox of Strep-tagged EGFP concatemers ranging from 1 to 12 subunits (Addgene plasmids #122488–122499). EGFP concatemers can be easily fused to targeting motifs of any origin by oligonucleotide ligation. Subsequently, we used liposomal transfection for transient expression of EGFP concatemers in eukaryotic cells. In this study, we have tested multiple protocols for further processing of the cells and recommend use of formalin or paraformaldehyde/methanol fixation. After usage of these protocols, we were able to detect concatemers by both GFP fluorescence microscopy and αStrep immunomicroscopy. In addition, we observed a more reliable detection of the StrepTag polypeptide (SA-WSHPQFEK) when using αStrepTag antibody instead of StrepTag binding protein. Summing up, we present a toolbox for expression of a wide range of Strep-tagged EGFP concatemers for multiple applications. By use of EGFP fluorescence and/or StrepTag polypeptide, the expressed concatemers can be easily detected in the cell.Graphical abstractImage 1
       
  • A practical approach to steady-state kinetic analysis of cellulases acting
           on their natural insoluble substrate
    • Abstract: Publication date: Available online 11 September 2019Source: Analytical BiochemistryAuthor(s): Jeppe Kari, Stefan Jarl Christensen, Morten Andersen, Selene Sellés Baiget, Kim Borch, Peter Westh Measurement of steady-state rates (vSS) is straightforward in standard enzymology with soluble substrate, and it has been instrumental for comparative biochemical analyses within this area. For insoluble substrate, however, experimental values of vss remain controversial, and this has strongly limited the amount and quality of comparative analyses for cellulases and other enzymes that act on the surface of an insoluble substrate. In the current work, we have measured progress curves over a wide range of conditions for two cellulases, TrCel6A and TrCel7A from Trichoderma reesei, acting on their natural, insoluble substrate, cellulose. Based on this, we consider practical compromises for the determination of experimental vSS values, and propose a basic protocol that provides representative reaction rates and is experimentally simple so that larger groups of enzymes and conditions can be readily assayed with standard laboratory equipment. We surmise that the suggested experimental approach can be useful in comparative biochemical studies of cellulases; an area that remains poorly developed.Graphical abstractImage 1
       
  • The FlpTRAP system for purification of specific, endogenous chromatin
           regions
    • Abstract: Publication date: Available online 11 September 2019Source: Analytical BiochemistryAuthor(s): Ida S. Jensen, Juan Yuan, Jin He, Lin Lin, Bjoern Sander, Monika M. Golas The repressor element 1-silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) binds to repressor element 1/neuron-restrictive silencer element (RE1/NRSE) sites in the genome and recruits effector proteins to repress its target genes. Here, we developed the FlpTRAP system to isolate endogenously assembled DNA-protein complexes such as the REST/NRSF complex. In the FlpTRAP system, we take advantage of the step-arrest variant of the Flp recombinase, FlpH305L, which, in the presence of Flp recognition target (FRT) DNA, accumulates as FRT DNA-protein adduct. The FlpTRAP system consists of three elements: (i) FlpH305L-containing cell extracts or isolates, (ii) a cell line engineered to harbor the DNA motif of interest flanked by FRT sites, and (iii) affinity selection steps to isolate the target chromatin. Specifically, 3×FLAG-tagged FlpH305L was expressed in insect cell cultures infected with baculovirus, and cell lysates were prepared. The lysate was used to capture the FRT-SNAP25 RE1/NRSE-FRT chromatin from a human medulloblastoma cell line, and the target RE1/NRSE chromatin was isolated by anti-FLAG immunoaffinity chromatography. Using electrophoretic mobility shift assays (EMSAs) and chromatin immunopurification (ChIP), we show that FlpH305L recognized and bound to the FRT sites. Overall, we suggest the FlpTRAP system as a tool to purify endogenous, specific chromatin loci from eukaryotic cells.Graphical abstractImage 1
       
  • High-throughput method for in process monitoring of 3-O-sulfotransferase
           catalyzed sulfonation in bioengineered heparin synthesis
    • Abstract: Publication date: Available online 10 September 2019Source: Analytical BiochemistryAuthor(s): Lei Lin, Yanlei Yu, Fuming Zhang, Xing Zhang, Robert J. Linhardt Bioengineered heparin (BEH) offers a potential alternative for the preparation of a safer pharmacological heparin. Construction of in-process control assays for tracking each enzymatic step during bioengineered heparin synthesis remains a challenge. Here, we report a high-throughput sensing platform based on enzyme-linked immunosorbent assay (ELISA) and enzymatic signal amplification that allows the rapid and accurate monitoring of the 3-OST sulfonation in BEH synthesis process. The anticoagulant activity of target BEH was measured to reflect the degree of sulfonation by testing its competitive antithrombin (AT) binding ability. BEH samples with different sulfonation degrees show different AT protein binding capacity and thus changes the UV response to a different extent. This BEH-induced signal can be conveniently and sensitively monitored by the plate sensing system, which benefits from its high sensitivity brought in by the enzymatic signal amplification. Furthermore, modification convenience and mechanical robustness also ensure the stability of the test platform. This proposed strategy exhibits excellent analytical performance in both BEH activity analysis and 3-OST sulfonation evaluation. The simple and sensitive plate system shows great potential in developing on-chip, high-throughput methods for fundamental biochemical process research, drug discovery, and clinic diagnostics.Graphical abstractImage 1
       
  • Differential effects of hydrogen peroxide (H2O2) treatment on epitope
           recognition in western blotting
    • Abstract: Publication date: Available online 7 September 2019Source: Analytical BiochemistryAuthor(s): Shuting Han, Yan Cui, Dario-Lucas Helbing Inactivation of horseradish peroxidase by hydrogen peroxide (H2O2) treatment is a convenient alternative to stripping for sequential chemiluminescent western blotting (WB). However, little evidence exists on whether H2O2 treatment affects epitope recognition. Here we show that H2O2 treatment had a negligible effect for most of tested antibodies, whereas it could also eliminate or enhance antibody binding. Thus, H2O2 treatment has unpredictable effects on epitope recognition. Moreover, we demonstrate potential steric hindrance from previously bound antibodies. Hence, it would be advantageous to pre-test the suitability of antibodies for H2O2-treated WB, and to optimize conditions to mitigate steric interference for re-probing.
       
  • A microtubule crosslinking protocol for integrative structural modeling
           activities
    • Abstract: Publication date: Available online 6 September 2019Source: Analytical BiochemistryAuthor(s): Atefeh Rafiei, David C. Schriemer Microtubules (MTs) are key components in the cytoskeleton of the eukaryotic cell, and play roles in processes such as intracellular transport and cell division. An improved understanding MT regulation requires structural analysis of the extensive interactions between the MT lattice and its regulatory proteins, but MT interactions are challenging for even the most advanced structural methods to characterize. Integrative methods involving crosslinking mass spectrometry (XL-MS) can extend structural analysis to many interaction classes, but the representation of MTs in crosslinking data-sets has been surprisingly low. Here, we explore the basis for the underrepresentation of the MT lattice and present an enhanced method for mapping MT structural features using an optimized set of reagents, together with fluorescence detection to ensure MT structural integrity. Through the application of stringent identification criteria, 91 unique crosslinks were identified, 78 of which were uniquely matched to 7 distinct structural features of the MT lattice. Of note, 4 crosslinks were detected for the lattice-A protofilament organization. The lattice-A structure defines a “seam” or discontinuity in MTs and is an emerging site of interest for MT regulation. Our methodology should be broadly applicable to integrative structural studies involving any MT-protein interaction.Graphical abstractImage 1
       
  • Efficient data acquisition with three-channel centerpieces in
           sedimentation velocity
    • Abstract: Publication date: Available online 4 September 2019Source: Analytical BiochemistryAuthor(s): Kristian Juul-Madsen, Huaying Zhao, Thomas Vorup-Jensen, Peter Schuck Three-channel 3D printed centerpieces with two sample sectors next to a joint solvent reference sector were recently described as a strategy to double the throughput of sedimentation velocity analytical ultracentrifugation experiments [Anal. Chem. 91 (2019) 5866–5873]. They are compatible with Rayleigh interference optical detection in commercial analytical ultracentrifuges, but require the rotor angles of data acquisition to be repeatedly adjusted during the experiment to record from the two sample sectors. Here we present an approach to automate this data acquisition mode through the use of a secondary, general-purpose automation software, and an accompanying data pre-processing software for scan sorting.
       
  • Fluorescence polarization assay for the identification and evaluation of
           inhibitors at YAP–TEAD protein–protein interface 3
    • Abstract: Publication date: Available online 31 August 2019Source: Analytical BiochemistryAuthor(s): Wei Zhou, Yiping Li, Jinhua Song, Chenglong Li The Hippo signaling pathway controls cell–cell contact, cell proliferation, as well as organ size by integrating changes in the cellular microenvironment. In recent years, the pivotal role of Hippo signaling in cancers has been well recognized. Inhibition of the pathway promotes the translocation of the major Hippo pathway effectors, the yes-associated protein (YAP) and its paralog TAZ, to the nucleus, where they interact with the transcription factor family transcriptional enhancer associate domain (TEAD), thus coactivating the expression of downstream genes, leading to cell transformation, tissue overgrowth, and tumor development. Therefore, the interruption of the YAP–TEAD transcriptional complex represents a novel opportunity for the treatment of cancer. Here, we established a fluorescence polarization (FP)-based assay for the identification and evaluation of YAP–TEAD protein–protein interface (PPI) inhibitors at the YAP Ω-loop binding region of TEAD, which is also called interface 3 at the YAP–TEAD binding surface. Furthermore, a patented small molecule (Patent-22) was evaluated by the FP assay, which confirmed that it was a YAP–TEAD PPI inhibitor at interface 3. Possessing great application value, this FP method is reliable, robust, and economical for inhibitor assessment and drug discovery.Graphical abstractImage 1
       
  • Review on nanomaterials-enabled electrochemical sensors for ascorbic acid
           detection
    • Abstract: Publication date: Available online 31 August 2019Source: Analytical BiochemistryAuthor(s): Keerthy Dhara, Debiprosad Roy Mahapatra This review (with 307 refs.) addresses the recent advances in electrochemical nonenzymatic ascorbic acid (AA) sensors using various nanomaterials as sensing elements. In general, nanomaterials have paved the way for a novel and advanced sensing device due to their unique physical and chemical properties. AA sensors based on noble metals, their nanoparticles, transition metals/metal nanoparticles, alloys/bimetallic nanoparticles, conducting polymers and carbon nanomaterials have been reviewed. Also, there has been a focus on describing the details of many significant articles explaining the design of sensors and utilities of the prepared sensors, so that readers might get the principles behind such devices and relevant detection strategies. Finally, the challenges and prospects for the application of nanomaterials-enabled electrochemical sensors for AA analysis have also been incorporated.Graphical abstractSchematic representation of electrochemical oxidation of ascorbic acid on nanomaterials modified electrode.Image 1
       
  • Sensitivity and reproducibility improvements in a human plasma immunoassay
           with removal of clotting factors
    • Abstract: Publication date: 15 November 2019Source: Analytical Biochemistry, Volume 585Author(s): Jiwon Kwak, Soo Suk Lee Interferences in human plasma immunoassay are severe challenge that affects the sensitivity and reproducibility of the assay. The clotting factor fibrinogen is a negatively charged protein and is one of the most common sources of interference in immunoassays, and its removal increases the sensitivity and reproducibility. Here, we present a highly sensitive and reproducible method for the detection of prostate specific antigen (PSA) in human plasma immunoassays. Protamine sulfate, a highly positively charged protein, was used to precipitate fibrinogen via ionic interaction to improve the sensitivity and reproducibility of human plasma immunoassay. In a sandwich ELISA for PSA using plasma and protamine-treated plasma samples, the limit of detection was improved from 413 pg/mL in plasma to 235 pg/mL in protamine-treated plasma samples, and the coefficient of variation known as a measure of reproducibility was significantly lowered by protamine treatment. The use of protamine sulfate in human plasma immunoassays for detection of PSA using quartz crystal microbalance (QCM) biosensors resulted in increased sensitivity and reproducibility by about 2-fold and 3-fold, respectively, relative to when not using protamine sulfate. Based on these results, protamine sulfate was the best choice to increase the sensitivity and reproducibility in immunoassays using plasma samples.Graphical abstractWe have demonstrated that the sensitivity and reproducibility of PSA detection in protamine-treated plasma samples were significantly improved compared with untreated plasma samples in the sandwich ELISA and the QCM sensor assays.Image 1
       
  • Improvisation of a spectrophotometric method to quantify hydroxycitric
           acid
    • Abstract: Publication date: Available online 29 August 2019Source: Analytical BiochemistryAuthor(s): Disha Patel, Aditi Buch Existing spectrophotometric method to quantify hydroxycitric acid (HCA), although is specific and sensitive; finds limited use owing to poor stability of HCA-metavanadate complex. Present study describes improvisation of this method with respect to source of HCA standard and assay parameters. Assay system consisting of HCA and metavanadate reagent was modified to include 1 M NaOH to neutralize excess acidity. Resulting complex showed λmax at 485 nm, obeying Beer-Lambert's law within concentration range of 33–677 μg/ml, with linear calibration curve showing a good coefficient of determination (R2 = 0.998). Moreover, HCA-metavanadate complex showed enhanced stability retaining up to 70% absorbance even after 60 min of its formation. Similar consistency in scaled-down assay system renders the method suitable for high-throughput screening of HCA-producing microbes. Of the tested metabolites and media components, only tartrate interfered with the spectrophotometric estimation of HCA; a correction factor to eliminate which was also established. Accordingly measured HCA level in the culture supernatant of a bacterial isolate IT6 was comparable to that determined using the standardized HPLC method. The proposed procedure therefore is a convenient, sensitive, accurate and high-throughput method suitable for primary screening of HCA producing microbes; the only ecofriendly alternative source of optically pure HCA.Graphical abstractImage 1
       
  • An effective and rapid method for RNA preparation from non-conventional
           yeast species
    • Abstract: Publication date: Available online 27 August 2019Source: Analytical BiochemistryAuthor(s): Dong Wook Lee, Chang Pyo Hong, Hyun Ah Kang The increased use of high-throughput RNA-based analysis has spurred the demand for rapid and simple preparation of high quality RNA. RNA preparation from non-conventional yeasts having diverse cell wall and morphological characteristics is often inefficient using current methods adapted for the model yeast, Saccharomyces cerevisiae. We report a simple RNA preparation method based on glass bead-mediated breakage in a formamide/EDTA solution. High quality RNA is generated within 15 min from various non-conventional yeasts species. The obtained RNA can be directly used for experimentation without further RNA purification and buffer exchange.
       
  • An enzymatic assay for quantification of inositol in human term placental
           tissue
    • Abstract: Publication date: Available online 27 August 2019Source: Analytical BiochemistryAuthor(s): Mohammed Omedul Islam, Preben Selvam, Reshma Appukuttan Pillai, Oliver Claire Watkins, Shiao-Yng Chan A modified sensitive, cheap and simple enzymatic assay method is described for the quantitation of inositol (6-carbon polyol) in human placental tissue. Water-soluble and total (water-soluble and lipid-bound) inositol isomers were extracted and quantified using a 96-well adaption of the Megazyme® assay. This assay specifically recognized myo-inositol (predominant isomer), d-chiro-, epi-, and allo-inositols, but not scyllo-inositol, glucose or fucose. In term placenta, water-soluble and total inositol contents were high [489(±58) and 635(±69) μg/g respectively], and reliably quantified with good reproducibility. This modified assay could facilitate placental inositol biology research, particularly pertinent now with interest in myo-inositol supplementation for GDM prevention.
       
  • New switch on fluorescent probe with AIE characteristics for selective and
           reversible detection of mercury ion in aqueous solution
    • Abstract: Publication date: Available online 26 August 2019Source: Analytical BiochemistryAuthor(s): Yanglei Yuan, Xin Chen, Qing Chen, Guoyu Jiang, Hongmei Wang, Jianguo Wang A new tetraphenylethylene derivative based fluorescenct probe (probe 2) was synthesized in a simple two-step process for selectively switch on and reversible detection of Hg2+ in aqueous solution based on aggregation-induced emission phenomenon. Probe 2 exhibited weak emission in aqueous solution due to the fast non-radiative decay of the excited singlet state facilitated by the free rotation of four phenyl rotors. While after coordination with Hg2+, the Hg2+-promoted aggregation formation will occur and restrict the intramolecular rotation, which blocks the non-radiative pathways and opens up the emission channel, resulting in the switch on response of probe 2 toward Hg2+. Probe 2 exhibited high sensitivity and good selectivity toward Hg2+ with a detection limit of 45.4 nM. Moreover, the detection can be reversible by subsequent addition of S2- into the detection system, which may be applied in the removal of toxic Hg2+ from water. Elsevier B.V. All rights reserved.Graphical abstractA new tetraphenylethylene derivative based fluorescenct probe (probe 2) was synthesized for selectively switch on and reversible detection of Hg2+ in aqueous solution using aggregation-induced emission features.Image 1
       
  • Capillary electrophoretic reactor for estimation of spontaneous
           dissociation rate of Trypsin–Aprotinin complex
    • Abstract: Publication date: 15 November 2019Source: Analytical Biochemistry, Volume 585Author(s): Yumiko Sasaki, Yosuke Sato, Toru Takahashi, Mitsuo Umetsu, Nobuhiko Iki A capillary electrophoretic reactor was used to analyze the dissociation kinetics of an enzyme–inhibitor complex in a homogeneous solution without immobilization. The complex consisting of trypsin (Try) and aprotinin (Apr) was used as the model. Capillary electrophoresis provided a reaction field for Try–Apr complex to dissociate through the steady removal of free Try and Apr from the Try–Apr zone. By analyzing the dependence of peak height of Try–Apr on separation time, the dissociation rate kdH was obtained as 2.73 × 10−4 s−1 (298 K) at pH 2.46. The dependence of kdH on the proton concentration (pH = 2.09–3.12) revealed a first-order dependence of kdH on [H+]; kdH = kd + k1[H+], where kd is the spontaneous dissociation rate and was 5.65 × 10−5 s−1, and k1 is the second-order rate constant and was 5.07 × 10−2 M−1 s−1. From the kd value, the half-life of the Try–Apr complex at physiological pH was determined as 3.4 h. The presence of the proton-assisted dissociation can be explained by the protonation of -COO– of the Asp residue in Try, which breaks the salt bridge with the –NH3+ group of Lys in Apr.Graphical abstractImage 1
       
  • Temporal influence of different antibiotics onto the inhibition of
           Escherichia coli bacterium grown in different media
    • Abstract: Publication date: 15 November 2019Source: Analytical Biochemistry, Volume 585Author(s): Ileana Andreea Ratiu, Viorica Railean Plugaru, Pawel Pomastowski, Maciej Milanowski, Radik Mametov, Victor Bocos-Bintintan, Boguslaw Buszewski Escherichia coli (E. coli) is a Gram-negative bacterium commonly found in the lower intestine of warm-blooded organisms, including humans. Although the majority of the strains are considerably harmless, some serotypes are pathogenic, frequently causing diarrhea and other illnesses outside the intestinal tract. The standard antidote against bacteria is the use of antibiotics. Depending on their type, the antibiotics have various mechanisms of action on bacteria. Moreover, in case of in-vitro cultivation of bacteria, the used growth media plays a crucial role, since it influences bacterial inhibition as well.In the present study, we emphasize the importance of cultivability in bacterial inhibition under the treatment with five different antibiotics belonging to different classes. Consequently, E. coli was cultivated in three different growth media: trypcase soy broth (TSB), Mueller Hinton (MH), and minimal salts (M9) enriched with glucose, respectively. MALDI-TOF MS (matrix-assisted laser desorption ionization time-of-flight mass spectrometry) analyses, that were used for fast characterization of changes that occur in ribosomal protein profiles, revealed differentiation and similarities between investigated cases, while flow cytometry (FCM) tests better explained the given changes that occurred in the analyzed samples after 3, 24 and 48 h of experimental campaign.Graphical abstractImage 1
       
  • A comparison of methods for DNA preparation prior to microarray analysis
    • Abstract: Publication date: Available online 22 August 2019Source: Analytical BiochemistryAuthor(s): Chris R. Taitt, Tomasz A. Leski, Sophie Colston, Manuela Bernal, Enrique Canal, James Regeimbal, Paul Rios, Gary J. Vora Microarrays are a valuable tool for analysis of both bacterial and eukaryotic nucleic acids. As many of these applications use non-specific amplification to increase sample concentration prior to analysis, the methods used to fragment and label large amplicons are important to achieve the desired analytical selectivity and specificity. Here, we used eight sequenced ESKAPE pathogens to determine the effect of two methods of whole genome amplicon fragmentation and three methods of subsequent labeling on microarray performance; nick translation was also assessed. End labeling of both initial DNase-treated and sonication-fragmented amplicons failed to provide detectable material for a significant number of sequence-confirmed genes. However, processing of amplicons by nick translation, or by sequential fragmentation and labeling by Universal Labeling System or Klenow fragment/random primer provided good sensitivity and selectivity, with marginally better results obtained by Klenow fragment labeling. Nick-translation provided 91–100% sensitivity and 100% specificity in the tested strains, requiring half as many hands-on manipulations and less than 4h to process samples for hybridization; full sample processing from whole genome amplification to final data analysis could be performed in less than 10h. The method of template denaturation before amplification did affect detection sensitivity/selectivity of nick-labeled amplicons, however.Graphical abstractImage 1
       
  • Development of an effective sample transfer device for biomarker detection
           in nasal secretions
    • Abstract: Publication date: Available online 21 August 2019Source: Analytical BiochemistryAuthor(s): Young Ju Lee, Jae-Chul Lee, Young Gyu Eun, Gi-Ja Lee Nasal secretions (NS) reflect inflammatory activity of the nasal mucosa and thus can be utilized for disease diagnosis and determining treatment effects in Allergic rhinitis (AR). However, non-standardized collection of samples can affect the measured concentration of inflammatory biomarker in NS. In this study, we aimed to develop and evaluate new devices capable of standardizing the collection, storage, and preprocessing methods of NS samples. First, we chose the best swab as polyester (PE) and selected a stimulation method, twirling for 10 s at 1 Hz, to efficiently release AR biomarkers from a PE swab. Storage of sample solutions at −20 °C was optimal for the stability of biomarkers for the detection of AR. The new swab sample transfer device showed excellent concentration recovery efficiency (90–100%) for tryptase (Trp) and eosinophil cationic protein (ECP) without crosstalk between the two biomarkers. Finally, we compared the concentration of Trp in human NS samples of AR patients (n = 6) pre-processed by the new device with that by centrifuge as a standard method. As a result, the concentrations of Trp in NS were very similar in both groups. Therefore, this device can be utilized as an effective sample transfer and pre-processing device for point-of-care testing of AR.Graphical abstractImage 1
       
  • SEAP activity measurement in reporter cell-based assays using BCIP / NBT
           as substrate
    • Abstract: Publication date: Available online 20 August 2019Source: Analytical BiochemistryAuthor(s): Valérie Jérôme, Ruth Freitag, Dirk Schüler, Frank Mickoleit SEAP (secreted embryonic alkaline phosphatase) has been suggested as versatile reporter protein inter alia for cell ligand interaction. Generic photometric assay formats for this enzyme are currently lacking. Using the interaction of recombinant hCD40 ligand with HEK-Blue sensor cells expressing the CD40 receptor as example, we show that such an assay can be developed based on BCIP/NBT (5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium chloride) as substrate. Supplementation of the reaction buffer with a micelle-forming detergent (TWEEN 20) stabilizes the water-insoluble reactions products thereby allowing reproducible photometric quantification of the colloidal dispersion. After optimizing the assay in terms of incubation time, cell number and environmental conditions, a cellular response to stimulation was already visible for 0.25 ng mL−1 of rhCD40L. Moreover, the sensitivity of the assay was significantly better than reported previously for alternative assays used in combination with the commercially available reporter cells. The use of BCIP/NBT as substrate therefore provides a robust and sensitive method to monitor SEAP activity in solution, which could conceivably be extended to other cell-based and biological assays using SEAP as reporter protein.
       
  • Elastin-like polypeptide fusions for high-level expression and
           purification of human IFN-γ in Escherichia coli
    • Abstract: Publication date: Available online 20 August 2019Source: Analytical BiochemistryAuthor(s): Reza Heidari-Japelaghi, Raheem Haddad, Mostafa Valizadeh, Ebrahim Dorani-Uliaie, Mokhtar Jalali-Javaran In this study, the ELP sequence was fused to human interferon-γ (hIFN-γ) and hIFN-γ-ELP fusion protein accumulated with high levels of yield and purity, compared with the corresponding unfused hIFN-γ protein. The hIFN-γ was exclusively produced in the form of insoluble inclusion bodies while the hIFN-γ was relatively soluble when expressed as an ELP fusion protein. The insoluble inclusion bodies were then solubilized under denaturing conditions, refolded in the presence of arginine and purified by single-step ion-exchange chromatography. The fusion to ELP signidficantly increased the accumulation of hIFN-γ by 10-fold with a stable expression on average of 46.85% of total soluble protein (TSP). Furthermore, three rounds of Inverse Transition Cycling (ITC) purification increased overall purity of the hIFN-γ-ELP to 98 ± 5%. The recovery amount of the fusion protein found to be dependent on the NaCl concentration, with increase of NaCl concentration, a greater fraction of the hIFN-γ-ELP was aggregated. However, due to the presence of an aliphatic guest residue in ELP sequence, the high concentration of salt was necessary to trigger the inverse phase transition of hIFN-γ-ELP fusion protein. Moreover, recombinant hIFN-γ and hIFN-γ-ELP proteins purified from E. coli possessed a relatively similar bioactivity based on viral cytopathic assay.
       
  • The determination of human peripheral blood mononuclear cell counts using
           a genomic DNA standard and application in tenofovir diphosphate
           quantitation
    • Abstract: Publication date: Available online 19 August 2019Source: Analytical BiochemistryAuthor(s): Deqing Xiao, Kah Hiing John Ling, Thomas Tarnowski, Sophia R. Majeed, Brian Kearney, Christos Kolaris, Susan Zondlo A fluorescent quantitation method to determine PBMC-derived DNA amounts using purified human genomic DNA (gDNA) as the reference standard was developed and validated. gDNA was measured in a fluorescence-based assay using a DNA intercalant, SYBR green. The fluorescence signal was proportional to the amount (mass) of DNA in the sample.The results confirmed a linear fit from 0.0665 to 1.17 μg/μL for gDNA, corresponding to 2.0 × 106 to 35.0 × 106 cells/PBMC sample. Intra-batch and inter-batch accuracy (%RE) was within ±15%, and precision (%CV) was
       
  • The conversion of azo-quenchers to fluorophores
    • Abstract: Publication date: Available online 19 August 2019Source: Analytical BiochemistryAuthor(s): O. Hofstetter, H. Hofstetter, T. Miron, M. Wilchek In this short note we describe the conversion of the widely used fluorescence quenching azo-dyes DABCYL and HABA to fluorophores. The dyes were conjugated to the proteins RNase and human serum albumin (HSA) and subsequently reduced using sodium dithionite (Na2S2O4), thus forming amine-containing fluorophores. Since this chemical reaction can be applied to any azo-containing quencher compound, a great variety of substances can be readily obtained synthetically. This approach provides a promising tool in the use of fluorescence-based investigations of biomolecular interactions.
       
  • Label-free detection of miRNA cancer markers based on terminal
           deoxynucleotidyl transferase-induced copper nanoclusters
    • Abstract: Publication date: Available online 8 August 2019Source: Analytical BiochemistryAuthor(s): Yiting Li, Dihong Tang, Li Zhu, Jingting Cai, Chaonan Chu, Jing Wang, Man Xia, Zhenzhen Cao, Hong Zhu The variations in microRNA (miRNA) expression levels can be useful biomarkers for the diagnosis of different cancers. In this work, a label-free and sensitive fluorescent method for detection of miRNA-21 is described based on duplex-specific nuclease (DSN) assist target recycling and terminal deoxynucleotidyl transferase (TdT) induced copper nanoclusters (CuNCs). In the absence of target, the 3′-phosphorylated probe DNA cannot be hydrolyzed by DSN and extended by TdT, and failed to synthesizing fluorescent CuNCs. However, the target miRNA-21 can caused the digestion of probe DNA with DSN, releasing primer DNA with 3′-OH. After that, the primer DNA can forms long poly T with the assistance of TdT, leading to synthesize high fluorescent CuNCs. The fluorescence change of CuNCs can be used to identify the concentration of target miRNA-21. Under optimal experimental conditions, this strategy could quantitatively detect miRNA-21 down to 18.7 pM. We have also demonstrated the practical application of our proposed method for monitoring miRNA-21 expression levels in cancer cells. Moreover, this method show good specificity for miRNA-21 detection due to the strong preference of DSN for cutting perfectly matched DNA/RNA duplex, which holds great potential for highly specific quantification of biomarkers in bioanalysis and clinical diagnosis.Graphical abstractA label-free and sensitive fluorescent method for detection of miRNA-21 is described based on duplex-specific nuclease (DSN) assist target recycling and terminal deoxynucleotidyl transferase (TdT) induced copper nanoclusters (CuNCs). This strategy was also applied to detecting miRNA-21 in cancer cell samples.Image 1
       
 
 
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