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Analytical Biochemistry
Journal Prestige (SJR): 0.633
Citation Impact (citeScore): 2
Number of Followers: 198  
  Hybrid Journal Hybrid journal (It can contain Open Access articles)
ISSN (Print) 0003-2697 - ISSN (Online) 1096-0309
Published by Elsevier Homepage  [3161 journals]
  • Creation of thermostable L-tryptophan dehydrogenase by protein engineering
           and its application for L-tryptophan quantification
    • Abstract: Publication date: Available online 14 May 2019Source: Analytical BiochemistryAuthor(s): Daisuke Matsui, Yasuhisa Asano L-Tryptophan dehydrogenase is a new NAD+-dependent amino acid dehydrogenase discovered in Nostoc punctiforme. The enzyme is involved in scytonemin biosynthesis and is highly selective toward L-tryptophan. By a growth-dependent molecular evolution technique, a thermostable mutant enzyme was selected successfully. L-Tryptophan concentration in human plasma was successfully determined using the thermostable mutant of L-tryptophan dehydrogenase.Graphical abstractImage 1
  • A label-free fluorometric aptasensor for adenosine triphosphate (ATP)
           detection based on aggregation-induced emission probe
    • Abstract: Publication date: Available online 13 May 2019Source: Analytical BiochemistryAuthor(s): Hua Li, Zhijun Guo, Wancui Xie, Wenyi Sun, Shuang Ji, Jing Tian, Lei Lv Based on Aggregation-Induced Emission (AIE), the development of a label-free, simple and sensitive fluorometric aptasensor for adenosine triphosphate (ATP) detection is described. With ATP present, the aptamers will combine with ATP and the conformation of the aptamer will switch from a random coil to an antiparallel G-quadruplex, which impedes the digestion by exonuclease I (Exo I). Addition of 4,4 -(1E,1E)-2,2-(anthracene-9,10-diyl) bis (ethene-2,1-diyl) bis (N,N, N-trimethyl-benzenaminium iodide) (DSAI) into the solution will cause aggregation of DSAI on the surface of the aptamer/ATP complex and consequently give rise to strong emission. Additionally, a good linear relationship was observed under optimized conditions between the fluorescence intensities and the logarithm of ATP concentrations (R2 = 0.9908). The established aptamer sensor was highly sensitive and exhibited a low limit of detection of 32.8 nM, with superior specificity for ATP. It was also used in the quantification of ATP levels in human serum samples and demonstrated satisfactory recoveries in the scope of 93.2%–107.6%. The cellular ATP assay results indicated that the developed method can be used for monitoring ATP concentrations in cell extracts without the interference of other substances in the cells. This method offers several advantages such as simplicity, rapidity, low cost and excellent selectivity, which make it hold great potential for the detection of ATP in bioanalytical and biological studies.
  • Assessing the impact of product inhibition in a chromogenic assay
    • Abstract: Publication date: Available online 12 May 2019Source: Analytical BiochemistryAuthor(s): Michael T. Stobb, Dougald M. Monroe, Karin Leiderman, Suzanne S. Sindi Chromogenic substrates (CS) are synthetic substrates used to monitor the activity of a target enzyme. It has been reported that some CSs display competitive product inhibition with their target enzyme. Thus, in assays where enzyme activity is continuously monitored over long periods of time, the product inhibition may significantly interfere with the reactions being monitored. Despite this knowledge, it is rare for CSs to be directly incorporated into mathematical models that simulate these assays. This devalues the predictive power of the models. In this study, we examined the interactions between a single enzyme, coagulation factor Xa, and its chromogenic substrate. We developed, and experimentally validated, a mathematical model of a chromogenic assay for factor Xa that explicitly included product inhibition from the CS. We employed Bayesian inference, in the form of Markov-Chain Monte Carlo, to estimate the strength of the product inhibition and other sources of uncertainty such as pipetting error and kinetic rate constants. Our model, together with carefully calibrated biochemistry experiments, allowed for full characterization of the strength and impact of product inhibition in the assay. The effect of CS product inhibition in more complex reaction mixtures was further explored using mathematical models.Graphical abstractImage 1
  • Labeling microRNA precursors for Dicer assays
    • Abstract: Publication date: Available online 12 May 2019Source: Analytical BiochemistryAuthor(s): Verena Schlösser, Jonathan Hall Excision of the terminal loop of precursor microRNAs (pre-miRNA) by Dicer generates miRNA duplexes, comprising 5p and 3p strands. Dicer often cleaves the RNA at more than one site, producing mature miRNAs with heterogeneous 3′ and 5′ ends. As pre-miRNAs are most conveniently labeled at their 5′ ends, standard in vitro Dicer assays usually assay only 5p miRNA strands. In this work we present a straightforward protocol using the same [32P] isotope for both strands by placing an internal label into the 3p strand, thereby providing a tool to investigate miRNA processing, strand selection and isomiR formation for any pre-miRNA.
  • Assessment of protein extraction and digestion efficiency of
           well-established shotgun protocols for heart proteomics
    • Abstract: Publication date: Available online 11 May 2019Source: Analytical BiochemistryAuthor(s): Lucas Rodrigues-Ribeiro, Marcella Nunes Melo-Braga, Frank Kjeldsen, Diana Paola Gómez-Mendoza, Thiago Verano-Braga Ischemic heart disease is the leading cause of deaths worldwide. Thus, understanding the molecular mechanisms underlying disease progression is needed. Due to heart importance and lack of studies evaluating different sample preparation methods for heart proteomics, we compared three well-established protocols in shotgun proteomics using dimethyl label quantitation to allow relative quantitation. The tested methods for the analysis of left ventricle (LV) tissue were: i) in-solution digestion (ISD); ii) on-pellet digestion (OPD); and iii) on-filter digestion (OFD). Protein extraction was done using SDS-containing buffer for OPD and OFD while this step was under urea-containing buffer for ISD. We used an optimized one-step reaction for reduction of disulfide bonds and alkylation of thiol groups in ISD and OPD. Using the same amount of tissue, we observed that OFD and ISD extracted significantly higher amount of protein than OPD. ISD outperformed OFD and OPD in the number of proteins identified. We did not observe significant bias related to physicochemical features of the identified proteins when comparing the three protocols. ISD was more efficient to identify low abundant proteins and yielded more proteins per protocol duration. Thus, we concluded that the optimized ISD suited better for heart proteomics than OFD and OPD.Graphical abstractImage 1
  • Profiling of VOCs released from different salivary bacteria treated with
           non-lethal concentrations of silver nitrate
    • Abstract: Publication date: Available online 11 May 2019Source: Analytical BiochemistryAuthor(s): Maciej Milanowski, Fernanda Monedeiro, Michał Złoch, Ileana-Andreea Ratiu, Paweł Pomastowski, Tomasz Ligor, Bruno S. De Martinis, Bogusław Buszewski Considering the shortcomings related to antibiotics usage, the introduction of other bacteriostatic and bactericidal agents that present synergetic effects or standalone properties is urgently needed. AgNO3 is an important bactericidal agent, which imparts various functions on bacteria dependent on its concentration. Therefore, an understanding of its mechanisms of action in infinitesimal concentrations plays an important role which can ultimately lead to AgNO3 involvement in the pharmaceutical industry. The monitoring of VOC (volatile organic compound) profiles emitted by bacteria is a simple method to assess changes occurring in bacterial metabolism. In this study, VOCs of Hafnia alvei, Pseudomonas luteola and Staphylococcus warneri cultures were analyzed both in the absence and in the presence of three concentrations of AgNO3. Headspace solid-phase microextraction gas chromatography/mass spectrometry (HS-SPME-GC/MS) was employed for extraction and analysis. After supplementation with AgNO3, changes in the emitted fingerprints were investigated. Odorants associated with mouth-related and systemic diseases, like dimethyl trisulfide, indole (halitosis) and 2-hexanone (celiac disease), were also affected by addition of AgNO3. Statistical tests proved discrimination between obtained profiles with more that 90% variability. Moreover, physiological states of bacteria after dosage with various concentration of stressing agent were investigated and explained by the mechanisms of action.Graphical abstractImage 1
  • Improved measurement of absolute mRNA quantity without reverse
    • Abstract: Publication date: Available online 9 May 2019Source: Analytical BiochemistryAuthor(s): Maasa Yokomori, Kenji Tsuge, Koh-ichiroh Shohda, Akira Suyama Gene expression studies using microarrays have provided important insights into understanding the mechanisms of transcriptional regulation in a variety of biological and disease phenomena. In a previous study, we developed Photo-DEAN, a universal-microarray-based RNA quantification method that enabled reverse transcription-free multiplex measurement of the absolute amount of RNA. Photo-DEAN promotes high-throughput and bias-less transcriptome analysis without the need for common controls or additional complicated normalization steps. In this study, we empirically identified two conditions (individual specificity and uniform duplex stability) necessary for in silico design of probe sequences, allowing the Photo-DEAN method to accurately measure the absolute amount of target RNA in total RNA. We then demonstrated that using the modified probe design conditions, the Photo-DEAN method successfully measured the absolute amount of pgi mRNA spiked into E. coli total RNA. The measurement was performed at five different sites in the coding region of pgi mRNA, exhibiting no significant site dependence. Theoretical considerations suggested that probe sequences longer than the previously used 30-bases better satisfy the necessary design conditions.
  • Adsorption of DNA by using polydopamine modified magnetic nanoparticles
           based on solid-phase extraction
    • Abstract: Publication date: Available online 9 May 2019Source: Analytical BiochemistryAuthor(s): Min Zhang, Lingxiao Li, Bucheng Li, Ning Tian, Meijuan Yang, Hui Zhang, Chongge You, Junping Zhang A polydopamine magnetic composite (PDA@Fe3O4) was prepared for the extraction of human genomic DNA and characterized by transmission electron microscopy, X-ray diffraction, FT-IR spectrometer, zeta potential and vibrating sample magnetometry. PDA@Fe3O4 based on magnetic solid phase extraction (MSPE) method have highly efficient capture of genomic deoxyribonucleic acid (DNA)and gene fragments ranging from about 100 bp to 200 bp. Compared with commercial beads (Shenggong, China) and spin column nucleic acid extraction kit (Tiangen, China), the PDA coated magnetic nanoparticles display superior genomic DNA extraction capacity (116 mg/g) and yield (90.2%). The isolation protocol used the solutions (composed of PEG and NaCl) with a specific pH for the binding and release of DNA. The procedure can be attributed to the charge switch of amino and hydroxyl groups on surface of the magnetic particle. The extracted DNA with high quality (A260/A280 = 1.82 ± 0.04) can be directly used as template for polymerase chain reaction (PCR) followed by agarose gel electrophoresis. The results showed the new composite to be an ideal adsorbent for separation of DNA which had the advantage of its low cost, high extraction capacity and yield.
  • Role of identified RNA N6-methyladenosine methylation in liver
    • Abstract: Publication date: Available online 7 May 2019Source: Analytical BiochemistryAuthor(s): Kechen Xu, Yuchao Sun, Baixiang Sheng, Yiwen Zheng, Xiaoli Wu, Keyang Xu N6-Methyladenosine (m6A) is the most abundant and important internal modification site of RNA methylation in viruses and eukaryotic. m6A RNA methylation plays key roles in the regulation of post-transcriptional gene expression, including messenger RNA (mRNA), microRNA (miRNA) and long noncoding RNA (lncRNA). And m6A methylation regulates the various aspects of RNA metabolism, including structure, maturation, stability, splicing, export, translation and decay. Liver is a vital metabolic and digestive organ in the pathophysiological processes. Recent studies suggested that m6A RNA modification highly regulates hepatic function and development of liver diseases. Here, we aim to summarize the biological and clinical significance of m6A modification in hepatic growth and hepatic disease including viral hepatitis, non-alcoholic fatty liver disease (NAFLD), and liver cancer.
  • Real-time monitoring of D-Ala-D-Ala dipeptidase activity of VanX in living
           bacteria by isothermal titration calorimetry
    • Abstract: Publication date: Available online 6 May 2019Source: Analytical BiochemistryAuthor(s): Miao Lv, Yue-Juan Zhang, Fan Zhou, Ying Ge, Mu-Han Zhao, Ya Liu, Ke-Wu Yang The d,d-dipeptidase enzyme VanX is the main cause of vancomycin resistance in gram-positive bacteria because of hydrolysis of the D-Ala-D-Ala dipeptide used in cell-wall biosynthesis. Continuous assay of VanX has proven challenging due to lack of a chromophoric substrate. Here, we report a direct approach for continuous assay of VanX in vitro and in vivo from hydrolysis of D-Ala-D-Ala, based on the heat-rate changes measured with isothermal titration calorimetry (ITC). With the ITC approach, determination of kinetic parameters of VanX hydrolyzing D-Ala-D-Ala and the inhibition constant of d-cysteine inhibitor yielded KM of 0.10 mM, kcat of 11.5 s−1, and Ki of 18.8 μM, which are consistent with the data from ninhydrin/Cd(II) assays. Cell-based ITC studies demonstrated that the VanX expressed in E. coli and in clinical strain VRE was inhibited by d-cysteine with IC50 values of 29.8 and 28.6 μM, respectively. Also, the total heat from D-Ala-D-Ala (4 mM) hydrolysis decreases strongly (in absolute value) from 1.26 mJ for VRE to 0.031 mJ for E. faecalis, which is consistent with the large MIC value of vancomycin of 512 μg/mL for VRE and the much smaller value of 4 μg/mL for E. faecalis. The ITC approach proposed here could be applied to screen and evaluate small molecule inhibitors of VanX or to identify drug resistant bacteria.Graphical abstractA novel ITC approach for continuous assay of the activity and inhibition of VanX enzyme in vitro and in living bacterial by using D-Ala-D-Ala as the substrate.Image 1
  • Quantitative analysis of γ-glutamylpeptides by liquid chromatography-mass
           spectrometry and application for γ-glutamyltransferase assays
    • Abstract: Publication date: Available online 3 May 2019Source: Analytical BiochemistryAuthor(s): Sho Kobayashi, Yoshinori Tokairin, Takeru Miyakoshi, Takuya Saito, Keita Nagaoka, Yoshitaka Ikeda, Junichi Fujii, Hiroyuki Konno γ-Glutamylpeptides are largely produced via the action of γ-glutamylcysteine synthetase or γ-glutamyltransferase (GGT). GGT transfers the γ-glutamyl moiety from glutathione (GSH) and other γ-glutamyl compounds to amino acids, peptides, or water. A conventional GGT assay employs a synthetic donor substrate, which facilitates monitoring cleavage activity by means of colorimetric analyses but provides no information on the resulting γ-glutamylpeptides. In this study, we report on the use of liquid chromatography-mass spectrometry (LC-MS) to quantitatively measure the levels of 21 γ-glutamylpeptides including GSH and 45 amino acids, including Cys. Authentic compounds consisting of 17 chemically synthesized and commercially available 4 γ-glutamylpeptides were adopted as references. We applied this method to the characterization of γ-glutamylpeptides in blood plasma and livers of mice that had been treated with an overdose of acetaminophen. The established LC-MS-based assay was found to be useful for characterizing the γ-glutamylation reaction under in vivo and in vitro conditions and was clearly helpful for understanding the physiological significance of the production of γ-glutamylpeptides.Graphical abstractImage 1
  • Lab-scale preparation and QC of phytase assay substrate from rice bran
    • Abstract: Publication date: Available online 2 May 2019Source: Analytical BiochemistryAuthor(s): Claus Krogh Madsen, Charles Alistair Brearley, Henrik Brinch-Pedersen Phytases are involved in the phosphate acquisition and remobilization in plants, microbes and animals. They have become important technical enzymes in the feed industry and are used to make phosphate, present in animal feed as phytate, available for monogastric animal nutrition. Phytases may also be beneficial to human nutrition because phytate is known to interfere with the uptake of important micronutrients. Accordingly, phytases attract considerable research attention and phytate substrate lacking contaminants that interfere with commonly used phosphate-release assays is essential for this field of science. A procedure to prepare suitable sodium phytate from rice bran is presented. Extracted phytate is precipitated with barium hydroxide and re-dissolved in methanol after washing steps and sulphuric acid treatment. Remaining impurities are precipitated before the dissolved phytate is recovered as the sodium salt by addition of sodium hydroxide. In order to make the substrate widely available for research communities, the procedure relies solely on basic laboratory equipment and materials. Methods for quality control and monitoring of the purified sodium phytate or commercial alternatives are also presented.
  • CYP26A1 gene promoter is a useful tool for reporting RAR-mediated retinoid
    • Abstract: Publication date: Available online 27 April 2019Source: Analytical BiochemistryAuthor(s): Reza Zolfaghari, Floyd J. Mattie, Cheng-Hsin Wei, David R. Chisholm, Andrew Whiting, A. Catharine Ross Of numerous genes regulated by retinoic acid (RA), CYP26A1 is the most inducible gene by RA. In this study, we have used a shortened construct form, E4, of the CYP26A1 gene promoter, in a promoter-less vector with either luciferase or red fluorescent protein (RFP) as the reporter gene and have tested its responses to retinoids in transfected HepG2 and HEK293T cells. The promoter responded linearly to a wide concentration range of RA in cells cotransfected with retinoic acid receptors. It also responded quantitatively to retinol and other retinoids. An isolated clonal line of HEK293T cells permanently transfected with the promoter driving the expression of RFP responded to both RA and retinol, and the responses could be measured by fluorescence microscopy and flow cytometry. The promoter was used to assess the retinoid activity of 3 novel synthetic retinoid analogues, as well as of the intact serum samples of rats. Among the synthetic retinoid analogues tested, EC23 is more potent than RA at lower concentrations and was more stable than RA. The retinoid activities could be measured in control rat serum samples and were increased in the serum of RA-treated rats. This system offers a biologically-based alternative to mass-based retinoid analysis.
  • Portable detection of colorectal cancer SW620 cells by using a
           personal glucose meter
    • Abstract: Publication date: Available online 26 April 2019Source: Analytical BiochemistryAuthor(s): Jun Zhou, Lunxi Duan, Jiangsheng Huang, Zhongkun Zuo, Tenglong Tang, Ding Cao, Fei Ye, Shiwei Zhou, Yueying Zhou, Leiyi Zhang It is of great value to develop general, low-cost and even household methods for colorectal cancer detection. Here, a portable detection strategy based on a personal glucose meter (PGM) was designed for meeting this purpose. In this strategy, the anti-EpCAM coated magnet beads (MBs) were used as capture probes for enriching cancer cells and the aptamer modified and invertase loaded graphene oxides (GO) were used as report probes for producing glucose signal. This method is sensitive with detection limit as low as 560 cells, and demonstrates excellent detection specificity. Meanwhile, we succeeded in the specific detection of target cells in 20% human serum samples, indicating this method has great prospect in clinical diagnosis. Moreover, this method presents favorable universality for detecting different colorectal cancer cells by just using different recognition aptamers. Importantly, this method can be implemented for the target cell detection at room temperature without any expensive and large-scale instruments but a portable PGM. Therefore, this portable detection method possesses great potential in point-of-care detection of colorectal cancer cells.Graphical abstractA portable detection method for colorectal cancer SW620 cells was successfully constructed by using a personal glucose meter.Image 1
  • Metal organic framework MIL-53(Fe) as an efficient artificial oxidase for
           colorimetric detection of cellular biothiols
    • Abstract: Publication date: Available online 26 April 2019Source: Analytical BiochemistryAuthor(s): Ying Jiang, Qi-Meng Yang, Qiu-Ju Xu, Shi-Yu Lu, Lin-Yu Hu, Mao-Wen Xu, Ying-Shuai Liu Biothiols play critical roles in many biological processes and their aberrant is related to a variety of syndromes. A simple and reliable colorimetric method is developed in this work for biothiols detection based on an oxidase mimic, a metal organic framework (MOF) MIL-53(Fe), and a peroxidase substrate 3,3′,5,5′-tetramethylbenzidine (TMB). In this design, MIL-53(Fe) is utilized to catalyze the conversion of TMB to a blue colored 3,3′,5,5′-tetramethylbenzidine diimine, which can be read on a spectrophotometer at 652 nm. The oxidation-induced blue color generation can be efficiently inhibited by biothiols, thus a colorimetric analytical method is proposed for biothiols detection based on the above system. Under optimal conditions, a linear relationship in a range from 1 to 100 μM with a limit of detection (LOD) at 120 nM is achieved with Cys as a model target. The developed platform is further applied to evaluate cellular biothiols in normal (RWPE-1) and cancer (LNCap) cell lines, revealing that the overall biothiols level in LNCap is much higher than that in RWPE-1. This work renders a powerful tool for identifying cancer cells in a simple manner for biomedical diagnosis associated with biothiols.Graphical abstractImage 1
  • A novel immuno-PCR method using cDNA display
    • Abstract: Publication date: Available online 25 April 2019Source: Analytical BiochemistryAuthor(s): Hiroki Anzai, Takuya Terai, Chathuni Jayathilake, Takeru Suzuki, Naoto Nemoto Immuno-PCR (IPCR) provides sensitive and versatile detection of a variety of antigens by conjugating a PCR-amplifiable DNA reporter to a specific antibody or an aptamer. Several methodologies have been developed to prepare appropriate DNA-antibody conjugates, but in most cases, it remains difficult to label polypeptides with high site-specificity and fixed stoichiometry. To address this issue, we first demonstrated the feasibility of IPCR based on cDNA display, a 1:1 covalent complex of a polypeptide and its encoding cDNA via puromycin at the single molecule level. Several other in vitro display technologies (e.g., ribosome display, mRNA display) have similar simple nucleic acid-peptide linkage. However, they should be unsuitable for diagnostic applications because of their lability against heat and RNase. The newly developed system here, termed cDNA display mediated immuno-PCR (cD-IPCR), proved to work in direct- and sandwich-type detection of target proteins. Detection of a target in serum was also possible, using a VHH (variable domain of the heavy chain of a heavy chain antibody) antibody as a binding molecule. Although further improvement on sensitivity and quantitativity is necessary before the method becomes useful, we believe this work demonstrated a potential of cD-IPCR as an alternative novel format of IPCR.
  • Rolling circle amplification and graphene-based sensor-on-a-chip for
           sensitive detection of serum circulating miRNAs
    • Abstract: Publication date: Available online 25 April 2019Source: Analytical BiochemistryAuthor(s): Kiatnida Treerattrakoon, Thanakorn Jiemsakul, Chookiat Tansarawiput, Preedee Pinpradup, Tawin Iempridee, Patraporn Luksirikul, Krissana Khoothaim, Tararaj Dharakul, Deanpen Japrung In this study, we developed a simple multiplex miRNA detection platform based on rolling circle amplification and the fluorescence quenching property of reduced graphene oxide. The detection platform could be applied on a microfluidics chip with a mobile system controller to eliminate contamination and to facilitate potential use in remote areas. As a proof of concept, two fluorescence-labeled ssDNA tags were used for detection of miR-29a and miR-144*, two miRNAs that are highly expressed in the blood circulation of some patients with cancer or tuberculosis. The circular ssDNA probes in this study were designed to have an advantage over padlock probes as they can be prepared in advance. Our multiplex miRNA detection platform exhibited high sensitivity and selectivity, with a limit of detection of 0.05 pmol. In addition, our platform could detect target miRNAs from the total miRNA population extracted from human serum or a cancer cell line. These results indicated that our miRNA sensor has the potential to provide simple and high throughput miRNA analysis for disease diagnosis and prognosis.
  • A miniature electrochemical detection system based on GOQDs/MWCNTs /SPCE*
           for determination the purine in cells
    • Abstract: Publication date: Available online 25 April 2019Source: Analytical BiochemistryAuthor(s): Shi Zhou, Xiaoling Guo, Lingren Meng, Jiwen Cui, Jinlian Li, Xing Yuan, Dongmei Wu The development of a simple, portable and accurate instrument used in the evaluation of in vitro cytotoxicity is particularly important and urgent in the field of toxicology research at present. In the current study, a miniature electrochemical detection system was constructed that integrated graphene oxide quantum dots and multiwall carbon nanotubes modified anodized screen printed carbon electrode (GOQDs/MWCNTs/SPCE*) with a mini reaction vessel, which reduced the consumption of sample from 500 to 80 μL. Four electrochemical signals could be detected in cells distinctly for the first time which were attributed to the oxidation of uric acid, guanine/xanthine, adenine and hypoxanthine, respectively. This miniature electrochemical detection system has better selectivity, sensitivity and its detection limits were lower than those of most electrochemical sensors. Furthermore it has been found that the level of purine nucleotide metabolism in BALB/3T3 cells and MCF-7 cells was different, which could be related to the different purine nucleotide metabolisms of cancer cells and non cancer cells. The simple, portable and miniature electrochemical detection system could be used as a convenient instrument for toxicology detection.Graphical abstractImage 1
  • Evidence by GC-MS that lysine is an arginase-catalyzed metabolite of
           homoarginine in vitro and in vivo in humans
    • Abstract: Publication date: Available online 24 April 2019Source: Analytical BiochemistryAuthor(s): Alexander Bollenbach, Kathrin Cordts, Erik Hanff, Dorothee Atzler, Chi-un Choe, Edzard Schwedhelm, Dimitrios Tsikas lL-Homoarginine (hArg) is biosynthesized from lL-arginine (Arg) and lL-lysine (Lys) by arginine:glycine amidinotransferase (AGAT). AGAT also catalyzes the formation of guanidinoacetate (GAA) from Arg and glycine (Gly). GAA is converted to creatine (N-methyl guanidinoacetate) by guanidinoacetate N-methyl-transferase (GAMT). Low circulating and excretory concentrations of hArg are associated with worse cardiovascular outcome and mortality. hArg is a poor substrate of nitric oxide synthase (NOS) and a weak inhibitor of arginase. The metabolism of hArg in humans is little investigated. Previously, we found that orally administered hArg (125 mg/day) increased the plasma concentration of hArg, but not of Arg, the substrate of NOS, in healthy subjects. We newly analyzed the plasma samples collected in that study for Lys and other amino acids. Repeated measures ANOVA revealed statistically significant differences between the groups (P = 0.008) with respect to plasma Lys concentration which increased by about 8% after a 4-week hArg supplementation. In vitro, recombinant human arginase and bovine liver arginase I were demonstrated by a specific and sensitive stable-isotope GC-MS assay to hydrolyze hArg to Lys. Our results suggest that Lys is a metabolite of hArg produced by the hydrolytic activity of arginase. Arginase may play a key role in hArg homeostasis in humans.
  • Using word embedding technique to efficiently represent protein sequences
           for identifying substrate specificities of transporters
    • Abstract: Publication date: Available online 22 April 2019Source: Analytical BiochemistryAuthor(s): Trinh-Trung-Duong Nguyen, Nguyen-Quoc-Khanh Le, Quang-Thai Ho, Dinh-Van Phan, Yu-Yen Ou Membrane transport proteins and their substrate specificities play crucial roles in various cellular functions. Identifying the substrate specificities of membrane transport proteins is closely related to protein-target interaction prediction, drug design, membrane recruitment, and dysregulation analysis, thus being an important problem for bioinformatics researchers. In this study, we applied word embedding approach, the main cause for natural language processing breakout in recent years, to protein sequences of transporters. We defined each protein sequence based on the word embeddings and frequencies of its biological words. The protein features were then fed into machine learning models for prediction. We also varied the lengths of protein sequence's constituent biological words to find the optimal length which generated the most discriminative feature set. Compared to four other feature types created from protein sequences, our proposed features can help prediction models yield superior performance. Our best models reach an average area under the curve of 0.96 and 0.99, respectively on the 5-fold cross validation and the independent test. With this result, our study can help biologists identify transporters based on substrate specificities as well as provides a basis for further research that enriches a field of applying natural language processing techniques in bioinformatics.Graphical abstractIn this study, we aimed at identifying the substrate specificities of transport proteins, which is closely related to protein-target interaction prediction, drug design, and dysregulation analysis. We defined each protein sequence based on the word embeddings and frequencies of its biological words. The protein features were then fed into machine learning models for prediction. We also varied the lengths of protein sequence's constituent biological words to find the optimal length which generated the most discriminative feature set. Our final model built based on the best choice of biological word lengths and machine learning algorithms achieves a high average accuracy of 95% and 97%, average area under the curve of 0.96 and 0.99, respectively on the 5-fold cross validation and independent test.Image 1
  • An improved method for isolation of mitochondria from cell lines that
           enables reconstitution of calcium-dependent processes
    • Abstract: Publication date: Available online 22 April 2019Source: Analytical BiochemistryAuthor(s): Masaaki Ishii, Gyda Beeson, Craig Beeson, Bärbel Rohrer Optimum cytosolic calcium concentrations support balanced mitochondrial respiration. However, cytosolic Ca2+ concentrations vary among cell types and excess Ca2+ can cause mitochondrial dysfunction. We optimized an isolation protocol to eliminate excess Ca2+ and thereby minimizing structural damage. Ca2+ uptake was monitored by measuring mitochondrial Ca2+-dependent PKA activity using cAMP ELISAs, and O2 consumption levels during mitochondrial respiration using high-resolution respirometry. 3 nM Ca2+ was found to increase cAMP levels and produce optimal state III respiration. Hence, optimized isolation of mitochondria from cell lines using calcium denudation provides the best platform for the study of Ca2+-dependent regulation of mitochondrial signaling.
  • Integrated immunochromatographic assay for qualitative and quantitative
           detection of clenbuterol
    • Abstract: Publication date: Available online 22 April 2019Source: Analytical BiochemistryAuthor(s): Yuan Chen, Zhen Huang, Song Hu, Ganggang Zhang, Juan Peng, Jun Xia, Weihua Lai In this study, colloidal gold (CG) and time-resolved fluorescent nanobead (TRFN) probes were used to establish an integrated immunochromatographic assay (ICA) to qualitatively and quantitatively detect clenbuterol (CLE). The best experimental conditions for the two probes in separate ICAs were obtained by optimizing the antibody labeling concentration, the amount of antigen, and the concentration of probe. When the CG and TRFN probes co-existed in the ICA, the latter had no effect on the sensitivity of qualitative detection of the CG probe-based ICA. However, the CG probe optimized the linear range of quantitative detection in the TRFN probe-based ICA. The integrated test strip can be used for qualitative and quantitative detection of CLE in one step. When the amount of antigen reached 0.4 mg/mL, the CG probe concentration reached 1.2 μg/mL, and the TRFN probe concentration reached 0.68 μg/mL. The qualitative sensitivity of the integrated ICA was 0.5 ng/mL and its quantitative limit of detection was 0.04 ng/mL with a detection range of 0.1–2.7 ng/mL. This developed method is of great significance for large-scale samples screening and positive monitoring in the field of food safety testing.Graphical abstractImage 1
  • A reliable fluorimetric method to screen the nitric oxide synthase
           inhibitors in 96 well plate
    • Abstract: Publication date: Available online 20 April 2019Source: Analytical BiochemistryAuthor(s): Saurabh Agrawal, Rita Kumari, Pratibha Mehta Luthra In general, 4 amino-5-methylamino-2′,7′-difluorescein diacetate (DAF-FM-DA) dye is used to detect nitric oxide in biological systems through cell imaging. In this study, we have used 96 well plate format to quantify nitric oxide using DAF-FM-DA through a multimode reader (or independently using fluorospectrometer) and could be visualized in fluorescence microscope. Similar study otherwise will require a high-end instrument. The method has been validated to screen NOS inhibitors in the HEK 293T cell lines over-expressing the NOS isoforms. We observed that the method is very simple to use, adaptive, sensitive and most importantly it saves time.Reagents/toolsEthanol (70% [v/v] in distilled water), Nω-Nitro-l-arginine (l-NAME), 7-Nitro-Indazole (7-NI) (Sigma, St. Louis, MO), HEK 293T cell lines (National Centre for Cell Science (NCCS), Pune, India), DMEM (Himedia laboratories pvt), Fetal Bovine Serum (FBS) (Invitrogen, Carlsbad, CA), 100 U/mL penicillin, and 0.1 mg/mL streptomycin in a 5% CO2 atmosphere.Hank's Balanced Salt Solution (HBSS) without Phenol Red of pH 7.4 was prepared with the following composition: NaCl, 8.0g, KCl, 0.4g, CaCl2, 0.14g, MgSO4•7H2O, 0.1g, MgCl2·6H2O, 0.1g, Na2HPO4·2H2O, 0.06g, KH2PO4, 0.06g, glucose, 1.0g, NaHCO3, 0.35g, H2O, to 1000 ml, Sterilized and refrigerated, Calcium Ionophore A23187 (sigma Aldrich 52665-69-7) DAF-FM Di Aceatate (Molecular Probes Life Technologies), and DAF-FM Di Aceatate was prepared as a stock solution (5 mM) in DMSO, divided into aliquots and stored at −20 °C, followed by dilution to the required concentration in HBSS buffer before use.EquipmentNeubauer chamber, Microtube centrifuges (1.5 mL), Micropipettors,10,100, and 1000 mL with corresponding tips, multimode reader (Teacan, Synergy-HT), inverted fluorescence microscope (Nikon, eclipse Ti-S), black flat bottom Microplates (96-well) (Corning 3603).Graphical abstractImage 1
  • Template-ready PCR method for detection of human telomerase reverse
           transcriptase mRNA in sputum
    • Abstract: Publication date: Available online 13 April 2019Source: Analytical BiochemistryAuthor(s): Enguo Chen, Zhang Bao, Hui Zhen, Yu Chen, Chunyan Wu, Jun Zhang, Hangdi Xu, Yi Ding, Yanzhong Wang, Fei Yu, Guohua Lu, Junjun Chen, Pei Zhang, Yake Yao, Songlin Liu, Jincong Xuan, Fang Xia, Ran Chen, Bo Su, Jianying Zhou Human telomerase reverse transcriptase (hTERT) mRNA in tissue is a biomarker of lung cancer, but hTERT mRNA in sputum had not been successfully detected with conventional reverse transcription PCR methods. Here, we developed a novel PCR protocol: Template-Ready PCR (TRPCR), to detect sputum hTERT mRNA, in which probes serve as templates of amplification. While free probes and dsDNA were removed in template preparation through aspiration and restriction digestion, probes that formed into heterocomplex with target RNA remained intact for PCR amplification. By fishing out the heterocomplex and amplifying the probes, TRPCR achieved sensitivity higher than reverse transcription-quantitative PCR (RT-qPCR). ROC curve of sputum hTERT mRNA by TRPCR assay showed the discrimination in high sensitivity and specificity between patients with lung cancer and lung cancer-free donors at the PCR Ct cutoff of 33. We further validated this approach through TRPCR assay of sputum from 858 lung cancer patients and 480 non-malignant pulmonary disease patients. 722 (84.2%) cases from 858 with lung cancer patients were detected as positive, whereas 461 (96.0%) cases from 480 non-malignant pulmonary disease patients were detected as negative, suggesting that TRPCR assay of sputum hTERT mRNA can serve as a non-invasive molecular diagnosis of lung cancer.
  • Rapid detection of insulin by immune-enrichment with
           silicon-nanoparticle-assisted MALDI-TOF MS
    • Abstract: Publication date: Available online 13 April 2019Source: Analytical BiochemistryAuthor(s): Yichao Wang, Xinwei Han, Zhaoyun Li, Jiaogui Xie BackgroundInsulin is central to regulating fat and carbohydrate metabolism in the body. However, it is difficult to detect insulin using mass spectrometry (MS). The integration of nanotechnology with mass spectrometry for selective and sensitive detection is an important research area. Our aim was to establish a method to detect insulin using silicon nanoparticle-assisted high-throughput MS.MethodsDifferent nanomaterials with the potential for use as MALDI components to enhance the MS signal by increasing peptide ionization were investigated in the present study. Insulin in samples was enriched with antibody-coated silicon nanoparticles and then analyzed by MALDI-TOF MS. Method validation was performed in the present study.ResultsA platform for insulin detection with small sample volumes (100 μL) and a simplified procedure was successfully developed. The silicon nanoparticle-MS assay exhibited high sensitivity (LOQ, 0.1 nM) and good linear correlation of MS intensity with insulin concentration (R2 = 0.99). Intra-assay precision (% coefficient of variation) ranged from 1.81 to 4.53%, and interassay precision ranged from 2.71 to 8.09%. In addition, a correlation between the MALDI assay and a chemiluminescence immunoassay (CIA) was completed in patient samples, and the resulting Deming regression revealed good agreement (R2 = 0.981).ConclusionsIn our study, we found that the insulin signal could be enhanced with silicon nanoparticles. A new insulin determination method, immunoaffinity-based mass spectrometry, that saves time and involves simple processes, has been successfully established. The present assay was validated to detect insulin with low limits of detection.
  • Quantitative in situ proximity ligation assays examining protein
           interactions and phosphorylation during smooth muscle contractions
    • Abstract: Publication date: Available online 11 April 2019Source: Analytical BiochemistryAuthor(s): Yeming Xie, Brian A. Perrino Antibody-based in situ proximity ligation assays (isPLA) have the potential to study protein phosphorylation and protein interactions with spatial resolution in intact tissues. However, the application of isPLA at the tissue level is limited by a lack of appropriate positive and negative controls and the difficulty in accounting for changes in tissue shape. Here we demonstrate a set of experimental and computational approaches using gastric fundus smooth muscles to improve the validity of quantitative isPLA. Appropriate positive and negative biological controls and PLA technical controls were selected to ensure experimental rigor. To account for changes in morphology between relaxed and contracted smooth muscles, target PLA spots were normalized to smooth muscle myosin light chain 20 PLA spots or the cellular cross-sectional areas. We describe the computational steps necessary to filter out false-positive improperly sized spots and set the thresholds for counting true positive PLA spots to quantify the PLA signals. We tested our approach by examining protein phosphorylation and protein interactions in smooth muscle myofilament Ca2+ sensitization pathways from resting and contracted gastric fundus smooth muscles. In conclusion, our tissue-level isPLA method enables unbiased quantitation of protein phosphorylation and protein-protein interactions in intact smooth muscle tissues, suggesting the potential for quantitative isPLA applications in other types of intact tissues.
  • Crossly charged microfluidic device for spontaneous filtration without an
           external power supply
    • Abstract: Publication date: Available online 11 April 2019Source: Analytical BiochemistryAuthor(s): Kiwoong Kim, Sang Joon Lee Spontaneous filtration without a power supply was investigated using highly charged precursor mixtures instead of recognized microfluidic desalination techniques. The proposed filtration method consisted of a main channel (depth: 1 mm) and shallow channels (depth: 400 μm) in which the precursor mixture was injected. The crossly charged channel surfaces had the same surface charge between diagonal-directional shallow channels. The filtration performance of the proposed device was evaluated by injecting a fluorescent dye that moved toward the centerline of the main channel. The respective rejection rates of CaCl2, NaCl, CaSO4, and Na2SO4 were 3.16%, 30.13%, 43.74%, and 36.32% as revealed by the mechanism of Donnan exclusion theory and focusing of charged species toward centerline of the main channel caused by highly charged surfaces. The proposed filtration method exhibited a migration of charged microparticles toward a certain direction. This particle migration behavior was consistent with the simulation data. These results suggested that the proposed filtration method showed strong potential for desalinating brackish water or salty surface water without an external power supply.
  • Spectrophotometric method for simultaneous measurement of zinc and copper
           in metalloproteins using 4-(2-pyridylazo)resorcinol
    • Abstract: Publication date: Available online 20 March 2019Source: Analytical BiochemistryAuthor(s): Colleen Doyle, Dalia Naser, Heather Bauman, Jessica Rumfeldt, Elizabeth Meiering Bound metals are observed in a great many natural proteins, where they perform diverse roles in determining protein folding, stability and function. Due to the diverse impact of bound metals on biophysical and biochemical properties of proteins, it is valuable to have accurate and facile methods for determining the metal content of proteins. Here we describe an optimized methodology using 4-(2-pyridylazo)resorcinol (PAR) to simultaneously quantify two metal ions in solution. The assay is demonstrated for quantification of Cu2+ and Zn2+ ions in human Cu, Zn superoxide dismutases (SOD1s); however, the method is general and can be applied to various combinations of metal ions. Advantages of the assay are that it is rapid and inexpensive, requires little sample and preparation, and has simple data analysis. We show that spectral decomposition software can accurately resolve the absorption bands of Cu2+ and Zn2+ with high accuracy and precision. Using the PAR assay, we determined that metal binding is altered in disease-associated mutants of SOD1, with comparable results to those determined by ICP-AES. In addition, we highlight key issues for using spectrophotometric chelators such as PAR for metal analysis of proteins.
  • AFFINImeter: A software to analyze molecular recognition processes from
           experimental data
    • Abstract: Publication date: Available online 5 March 2019Source: Analytical BiochemistryAuthor(s): Ángel Piñeiro, Eva Muñoz, Juan Sabín, Miguel Costas, Margarida Bastos, Adrián Velázquez-Campoy, Pablo F. Garrido, Philippe Dumas, Eric Ennifar, Luis García-Río, Javier Rial, Daniel Pérez, Patricia Fraga, Aurelio Rodríguez, Carmen Cotelo The comprehension of molecular recognition phenomena demands the understanding of the energetic and kinetic processes involved. General equations valid for the thermodynamic analysis of any observable that is assessed as a function of the concentration of the involved compounds are described, together with their implementation in the AFFINImeter software. Here, a maximum of three different molecular species that can interact with each other to form an enormous variety of surpramolecular complexes are considered. The corrections currently employed to take into account the effects of dilution, volume displacement, concentration errors and those due to external factors, especially in the case of ITC measurements, are included. The methods used to fit the model parameters to the experimental data, and to generate the uncertainties are described in detail. A simulation tool and the so called kinITC analysis to get kinetic information from calorimetric experiments are also presented. An example of how to take advantage of the AFFINImeter software for the global multi-temperature analysis of a system exhibiting cooperative 1:2 interactions is presented and the results are compared with data previously published. Some useful recommendations for the analysis of experiments aimed at studying molecular interactions are provided.
  • Cloning of a novel trypsin inhibitor from the Traditional Chinese medicine
           decoction pieces, Radix Trichosanthis
    • Abstract: Publication date: Available online 1 March 2019Source: Analytical BiochemistryAuthor(s): Mengru Sang, Yuan Ying, Qinan Wu, Chengbang Ma, Xinping Xi, Mei Zhou, Lei Wang, Olaf R.P. Bininda-Emonds, Tianbao Chen Most herbs of traditional Chinese medicine (TCM) are used as air-dried decoction pieces that are manufactured and kept at ambient temperature for long periods. Given the ability of some desiccation-tolerant plants to conserve RNA, it could be worthwhile to isolate mRNA from TCM decoction pieces as part of a transcriptomic strategy to identify new substances with potential pharmaceutical application. Here, we report the molecular cloning of a novel trypsin inhibitor (as the probable alleleic variants TKTI-2 and TKTI-3) from the decoction piece of Radix Trichosanthis, representing the dried root of Trichosanthes kirilowii. From this material, the total RNA was extracted and a cDNA library was constructed from the isolated mRNA from which the cDNAs of two precursors were successfully cloned and sequenced. TKTI-3 showed an amino-acid substitution in the otherwise highly-conserved P1-P1′ reaction site of the mature peptide, which we confirmed to not be an artefact. Subsequent analysis using LC-MS/MS detected the presence of specific tryptic peptides expected from TKTI-3, confirming the presence and expression of this locus in Radix Trichosanthis. More generally, this study indicates that mRNA can persist in decoction pieces and so could present a viable option for the molecular cloning from other TCMs.
  • anti-EGFR capture mitigates EMT- and chemoresistance-associated
           heterogeneity in a resistance-profiling CTC platform
    • Abstract: Publication date: Available online 18 February 2019Source: Analytical BiochemistryAuthor(s): Fredrik I. Thege, Conor N. Gruber, Ian I. Cardle, Sophie H. Cong, Timothy B. Lannin, Brian J. Kirby Capture and analysis of circulating tumor cells (CTCs) holds promise for diagnosing and guiding treatment of pancreatic cancer. To accurately monitor disease progression, capture platforms must be robust to processes that increase the phenotypic heterogeneity of CTCs. Most CTC-analysis technologies rely on the recognition of epithelial-specific markers for capture and identification, in particular the epithelial cell-adhesion molecule (EpCAM) and cytokeratin. As the epithelial-to-mesenchymal transition (EMT) and the acquisition of chemoresistance are both associated with loss of epithelial markers and characteristics, the effect of these processes on the expression of commonly used CTC markers, specifically EpCAM, EGFR and cytokeratin, requires further exploration. To determine this effect, we developed an in vitro model of EMT and acquired gemcitabine resistance in human pancreatic cancer cell lines. Using this model, we show that EMT-induction and acquired chemoresistance decrease EpCAM expression and microfluidic anti-EpCAM capture performance. Furthermore, we find that EGFR capture is more robust to these processes. By measuring the expression of known mediators of chemoresistance in captured cells using automated imaging and image processing, we demonstrate the ability to resistance-profile cells on-chip. We expect that this approach will allow for the development of improved non-invasive biomarkers of pancreatic cancer progression.Graphical abstractImage 1
School of Mathematical and Computer Sciences
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