Journal of biochemical and biophysical methods最新文献

{"title":"Benchmarking protein classification algorithms via supervised cross-validation","authors":"Attila Kertész-Farkas ,&nbsp;Somdutta Dhir ,&nbsp;Paolo Sonego ,&nbsp;Mircea Pacurar ,&nbsp;Sergiu Netoteia ,&nbsp;Harm Nijveen ,&nbsp;Arnold Kuzniar ,&nbsp;Jack A.M. Leunissen ,&nbsp;András Kocsor ,&nbsp;Sándor Pongor","doi":"10.1016/j.jbbm.2007.05.011","DOIUrl":"10.1016/j.jbbm.2007.05.011","url":null,"abstract":"<div><p>Development and testing of protein classification algorithms are hampered by the fact that the protein universe is characterized by groups vastly different in the number of members, in average protein size, similarity within group, etc. Datasets based on traditional cross-validation (<em>k</em>-fold, leave-one-out, etc.) may not give reliable estimates on how an algorithm will generalize to novel, distantly related subtypes of the known protein classes. Supervised cross-validation, i.e., selection of test and train sets according to the known subtypes within a database has been successfully used earlier in conjunction with the SCOP database. Our goal was to extend this principle to other databases and to design standardized benchmark datasets for protein classification. Hierarchical classification trees of protein categories provide a simple and general framework for designing supervised cross-validation strategies for protein classification. Benchmark datasets can be designed at various levels of the concept hierarchy using a simple graph-theoretic distance. A combination of supervised and random sampling was selected to construct reduced size model datasets, suitable for algorithm comparison. Over 3000 new classification tasks were added to our recently established protein classification benchmark collection that currently includes protein sequence (including protein domains and entire proteins), protein structure and reading frame DNA sequence data. We carried out an extensive evaluation based on various machine-learning algorithms such as nearest neighbor, support vector machines, artificial neural networks, random forests and logistic regression, used in conjunction with comparison algorithms, BLAST, Smith-Waterman, Needleman-Wunsch, as well as 3D comparison methods DALI and PRIDE. The resulting datasets provide lower, and in our opinion more realistic estimates of the classifier performance than do random cross-validation schemes. A combination of supervised and random sampling was used to construct model datasets, suitable for algorithm comparison.</p><p>The datasets are available at <span>http://hydra.icgeb.trieste.it/benchmark</span><svg><path></path></svg>.</p></div>","PeriodicalId":15257,"journal":{"name":"Journal of biochemical and biophysical methods","volume":"70 6","pages":"Pages 1215-1223"},"PeriodicalIF":0.0,"publicationDate":"2008-04-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.jbbm.2007.05.011","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"26807453","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Analytical approach for selecting normalizing genes from a cDNA microarray platform to be used in q-RT-PCR assays: A cnidarian case study","authors":"Mauricio Rodriguez-Lanetty ,&nbsp;Wendy S. Phillips ,&nbsp;Sophie Dove ,&nbsp;Ove Hoegh-Guldberg ,&nbsp;Virginia M. Weis","doi":"10.1016/j.jbbm.2007.08.005","DOIUrl":"10.1016/j.jbbm.2007.08.005","url":null,"abstract":"<div><p>Research in gene function using Quantitative Reverse Transcription PCR (q-RT-PCR) and microarray approaches are emerging and just about to explode in the field of coral and cnidarian biology. These approaches are showing the great potential to significantly advance our understanding of how corals respond to abiotic and biotic stresses, and how host cnidarians/dinoflagellates symbioses are maintained and regulated. With these genomic advances, however, new analytical challenges are also emerging, such as the normalization of gene expression data derived from q-RT-PCR. In this study, an effective analytical method is introduced to identify candidate housekeeping genes (HKG) from a sea anemone (<em>Anthopleura elegantissima</em>) cDNA microarray platform that can be used as internal control genes to normalize q-RT-PCR gene expression data. It is shown that the identified HKGs were stable among the experimental conditions tested in this study. The three most stables genes identified, in term of gene expression, were beta-actin, ribosomal protein L12, and a Poly(a) binding protein. The applications of these HKGs in other cnidarian systems are further discussed.</p></div>","PeriodicalId":15257,"journal":{"name":"Journal of biochemical and biophysical methods","volume":"70 6","pages":"Pages 985-991"},"PeriodicalIF":0.0,"publicationDate":"2008-04-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.jbbm.2007.08.005","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"27026287","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Investigation of coenzyme Q biosynthesis in human fibroblast and HepG2 cells","authors":"Michael Tekle ,&nbsp;Mikael Turunen ,&nbsp;Gustav Dallner ,&nbsp;Tadeusz Chojnacki ,&nbsp;Ewa Swiezewska","doi":"10.1016/j.jbbm.2007.08.003","DOIUrl":"10.1016/j.jbbm.2007.08.003","url":null,"abstract":"<div><p>Coenzyme Q (CoQ) deficiency occurs in genetic disorders, during aging and various diseases. Diagnosis requires skin fibroblasts in tissue culture. [³H]Mevalonate incorporation was appropriate to measure the rate of CoQ synthesis in fibroblasts and hepatoblastoma cells. [¹⁴C]<em>p</em>-Hydroxybenzoate had limited permeability, but it could be increased with Fugene and cyclodextrin. Inhibition of decaprenyl-4-hydroxybenzoate transferase results in the accumulation of decaprenyl diphosphate, an indicator of enzyme deficiency. Also, analysis of the corresponding mRNAs in this case is useful. <em>In vitro</em> assays to measure <em>trans</em>-prenyltransferase and decaprenyl-4-hydroxybenzoate transferase activities are not available. Neither measurement of methyltransferases is reliable in human cells. <em>In vitro</em> reconstruction of CoQ synthesis, in opposite to cholesterol synthesis, proved to be unsuccessful. Thus, the biochemical characterization of the CoQ biosynthetic system in human cells is restricted to a few reliable analytical procedures.</p></div>","PeriodicalId":15257,"journal":{"name":"Journal of biochemical and biophysical methods","volume":"70 6","pages":"Pages 909-917"},"PeriodicalIF":0.0,"publicationDate":"2008-04-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.jbbm.2007.08.003","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"27032216","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Reactivity of histidine and lysine side-chains with diethylpyrocarbonate — A method to identify surface exposed residues in proteins","authors":"Aleš Hnízda ,&nbsp;Jiří Šantrůček ,&nbsp;Miloslav Šanda ,&nbsp;Martin Strohalm ,&nbsp;Milan Kodíček","doi":"10.1016/j.jbbm.2007.07.004","DOIUrl":"10.1016/j.jbbm.2007.07.004","url":null,"abstract":"<div><p>The chemical modification of amino acid side-chains followed by mass spectrometric detection can reveal at least partial information about the 3-D structure of proteins. In this work we tested diethylpyrocarbonate, as a common histidyl modification agent, for this purpose. Appropriate conditions for the reaction and detection of modified amino acids were developed using angiotensin II as a model peptide. We studied the modification of several model proteins with a known spatial arrangement (insulin, cytochrome c, lysozyme and human serum albumin). Our results revealed that the surface accessibility of residues is a necessary, although in itself insufficient, condition for their reactivity; the microenvironment of side-chains and the dynamics of protein structure also affect the ability of residues to react. However the detection of modified residues can be taken as proof of their surface accessibility, and of direct contact with solvent molecules.</p></div>","PeriodicalId":15257,"journal":{"name":"Journal of biochemical and biophysical methods","volume":"70 6","pages":"Pages 1091-1097"},"PeriodicalIF":0.0,"publicationDate":"2008-04-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.jbbm.2007.07.004","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"26951432","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Cyanine dye–protein interactions: Looking for fluorescent probes for amyloid structures","authors":"K.D. Volkova ,&nbsp;V.B. Kovalska ,&nbsp;A.O. Balanda ,&nbsp;R.J. Vermeij ,&nbsp;V. Subramaniam ,&nbsp;Yu. L. Slominskii ,&nbsp;S.M. Yarmoluk","doi":"10.1016/j.jbbm.2007.03.008","DOIUrl":"10.1016/j.jbbm.2007.03.008","url":null,"abstract":"<div><p>We ascertained the ability to detect fibrillar β-lactoglobulin (BLG) of a series of mono-, tri-, penta-, and heptamethinecyanines based on benzothiazole and benzimidazole heterocycles, and of benzothiazole squaraine. Fluorescence properties of these cyanine dyes were measured in the unbound state and in the presence of monomeric and fibrillar BLG and compared with those for the commercially available benzothiazole dye Thioflavin T. The correlation between the chemical nature of the dye molecules and the ability of dyes to bind aggregated proteins was established. We found that meso-substituted cyanines with amino substituents in heterocycle in contrast to the corresponding unsubstituted dyes have a binding preference to fibrillar BLG and a noticeable fluorescence response in the presence of the aggregated protein. For the squaraines and benzimidazole penthamethinecyanines studied, fluorescence emission increased both in the presence of native and fibrillar protein. The trimethinecyanines T-49 and SH-516 exhibit specifically increased fluorescence in the presence of fibrillar BLG. These dyes demonstrated the same or higher emission intensity and selectivity to aggregated BLG as Thioflavin T, and are proposed for application in selective fluorescent detection of aggregated proteins.</p></div>","PeriodicalId":15257,"journal":{"name":"Journal of biochemical and biophysical methods","volume":"70 5","pages":"Pages 727-733"},"PeriodicalIF":0.0,"publicationDate":"2007-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.jbbm.2007.03.008","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"26693967","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The use of Quantitative Agarose Gel Electrophoresis for rapid analysis of the integrity of protein–DNA complexes","authors":"Nicholas L. Adkins ,&nbsp;J. Adam. Hall ,&nbsp;Philippe T. Georgel","doi":"10.1016/j.jbbm.2007.03.006","DOIUrl":"10.1016/j.jbbm.2007.03.006","url":null,"abstract":"<div><p>Recent biochemical studies evaluated the affinity of histones to DNA in the context of nucleosome core particle (NCP). These have indicated a concentration-dependence for nucleosome stability. However, when studying chromatin the preferred templates are nucleosome arrays (NA) and not the NCP. Biochemical methods are poorly suited for structural analysis of chromatin. To overcome that technical hindrance, and investigate the effect of concentration on stability of the histone–DNA interactions, we have applied the multigel Quantitative Agarose Gel Electrophoresis (QAGE) method to in vitro-assembled nucleosomal arrays. The results demonstrated the method to be extremely valuable for the evaluation of the effect of low concentration on NA. However, QAGE is a fairly time-demanding and complex method. To maximize the efficiency of use of this technology, we devised a protocol that allowed for multiple sets of templates to be analyzed simultaneously. Briefly, samples can be loaded at regular intervals and analyzed individually for their molecular composition. The technique presented in this study describes the calibration steps and proof of concept necessary to validate the use of multiple loading of multigel to evaluate the composition of nucleosomal arrays as a function of concentration.</p></div>","PeriodicalId":15257,"journal":{"name":"Journal of biochemical and biophysical methods","volume":"70 5","pages":"Pages 721-726"},"PeriodicalIF":0.0,"publicationDate":"2007-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.jbbm.2007.03.006","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"26807004","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A convenient and efficient protocol for isolating high-quality RNA from latex of Hevea brasiliensis (para rubber tree)","authors":"Chaorong Tang,&nbsp;Jiyan Qi,&nbsp;Heping Li,&nbsp;Cunliang Zhang,&nbsp;Yuekun Wang","doi":"10.1016/j.jbbm.2007.04.002","DOIUrl":"10.1016/j.jbbm.2007.04.002","url":null,"abstract":"<div><p>Isolating high-quality RNA from latex of <em>H. brasiliensis</em> is a prerequisite to elucidating the molecular mechanisms of rubber biosynthesis and its regulation. Here, an improved protocol was developed for latex collection, transportation, storage, and RNA isolation. Compared with existing ones, our protocol eliminated liquid nitrogen for latex collection and subsequent low-temperature (−<!--> <!-->70 °C) condition for latex storage, making it more convenient and feasible when latex was collected in remote sampling sites, and latex storage and RNA isolation were conducted in poorly-equipped laboratories. Different methods (UV absorbance scans, denaturing gel electrophoresis, autoradiograph monitoring of cDNA synthesis) were used to confirm the high quality of the RNA prepared with this protocol, whose usefulness was further verified by several practical applications, including construction of one high-quality cDNA library, cloning of the full-length cDNAs of 3 novel <em>Hevea</em> sucrose transporter genes, and semi-quantitative RT-PCR analysis of two rubber-biosynthesis essential genes and one sucrose transporter gene.</p></div>","PeriodicalId":15257,"journal":{"name":"Journal of biochemical and biophysical methods","volume":"70 5","pages":"Pages 749-754"},"PeriodicalIF":0.0,"publicationDate":"2007-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.jbbm.2007.04.002","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"26713034","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Application of Fourier linear spectral analyses to the characterization of smooth muscle contractile signals","authors":"Mario Díaz","doi":"10.1016/j.jbbm.2007.03.007","DOIUrl":"10.1016/j.jbbm.2007.03.007","url":null,"abstract":"<div><p>In intestinal smooth muscle, peristaltic activity emerges from a depolarization wave generated by Cajal's interstitial cells that travels longitudinally and transversally towards adjacent smooth muscle. The electrophysiological diversity of cell populations involved in the generation and transmission of excitation between nerve and muscle cells, as well as the specialization in the excitation–contraction coupling of smooth muscle cells, makes it difficult to extrapolate individual cell responses to overall peristaltic activity. It is conceivable intestinal contractile activity as the macroscopic output from a multicellular system in time and frequency domains. Given that contractile signals are usually linear and stationary, application of frequency analyses using the discrete Fourier transform allows the accurate definition of amplitudes and phases of harmonic components in the frequency spectrum of contractile activity records, as well as the power spectrum of the signal. In addition, by using the short-time Fourier transform it is also possible to obtain the spectrogram of contractile signals, which allows the identification of non-stationary events. Often, the combined usage of these types of analyses together with specific pharmacological and molecular biology tools is sufficient to unveil the cellular and molecular locus of action of modulators of peristaltism, including hormones and different natural and synthetic compounds.</p></div>","PeriodicalId":15257,"journal":{"name":"Journal of biochemical and biophysical methods","volume":"70 5","pages":"Pages 803-808"},"PeriodicalIF":0.0,"publicationDate":"2007-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.jbbm.2007.03.007","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"26693965","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"DNA immobilization chemical interference due to aggregates study by Dip and Drop approach","authors":"Ram Ajore ,&nbsp;Rakesh Kumar ,&nbsp;Inderpreet Kaur ,&nbsp;R.C. Sobti ,&nbsp;Lalit M. Bharadwaj","doi":"10.1016/j.jbbm.2007.05.006","DOIUrl":"10.1016/j.jbbm.2007.05.006","url":null,"abstract":"<div><p>In the present manuscript, we report the studies and observations for chemical interference due to aggregates formation during covalent immobilization of thiolated λ-DNA between gold microelectrodes. Dip and Drop approaches were employed to study DNA immobilization using thiolated oligos (oligoA 5′ GGGCGGCGACCT 3′ and oligoB 5′ AGGTCGCCGCCC 3′). As a result of aggregation, less interference was observed in Dip approach as compared to Drop approach. Atomic Force Microscopy (AFM) analysis of piranha treated gold surface revealed 47.5% increase in height roughness, contributing in interference by creating active sites. Cyclic voltammetry (CV) studies ascertain the multitude of adsorption states existing in long strand of DNA on surface. Surface coverage was found to be ∼ 72% (1.35<!--> <!-->×<!--> <!-->10¹⁰ molecules/cm²), and ∼ 42% (7.89<!--> <!-->×<!--> <!-->10⁹ molecules/ cm²) in Dip and Drop approach, respectively. Dip approach can be used as a measure to minimize interference due to aggregation.</p></div>","PeriodicalId":15257,"journal":{"name":"Journal of biochemical and biophysical methods","volume":"70 5","pages":"Pages 779-785"},"PeriodicalIF":0.0,"publicationDate":"2007-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.jbbm.2007.05.006","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"26801805","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Novel fluorescence detection technique for non-contact temperature sensing in microchip PCR","authors":"Sudip Mondal,&nbsp;V. Venkataraman","doi":"10.1016/j.jbbm.2007.05.001","DOIUrl":"10.1016/j.jbbm.2007.05.001","url":null,"abstract":"<div><p>DNA amplification using Polymerase Chain Reaction (PCR) in a small volume is used in Lab-on-a-chip systems involving DNA manipulation. For few microliters of volume of liquid, it becomes difficult to measure and monitor the thermal profile accurately and reproducibly, which is an essential requirement for successful amplification. Conventional temperature sensors are either not biocompatible or too large and hence positioned away from the liquid leading to calibration errors. In this work we present a fluorescence based detection technique that is completely biocompatible and measures directly the liquid temperature. PCR is demonstrated in a 3 μL silicon-glass microfabricated device using non-contact induction heating whose temperature is controlled using fluorescence feedback from SYBR green I dye molecules intercalated within sensor DNA. The performance is compared with temperature feedback using a thermocouple sensor. Melting curve followed by gel electrophoresis is used to confirm product specificity after the PCR cycles.</p></div>","PeriodicalId":15257,"journal":{"name":"Journal of biochemical and biophysical methods","volume":"70 5","pages":"Pages 773-777"},"PeriodicalIF":0.0,"publicationDate":"2007-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.jbbm.2007.05.001","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"26778839","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}