“Blind” mapping of genic DNA sequence polymorphisms in Lolium perenne L. by high resolution melting curve analysis

High resolution melting curve analysis (HRM) measures dissociation of double stranded DNA of a PCR product amplified in the presence of a saturating fluorescence dye. Recently, HRM proved successful to genotype DNA sequence polymorphisms such as SSRs and SNPs based on the shape of the melting curves. In this study, HRM was used for simultaneous screening and genotyping of genic DNA sequence polymorphisms identified in the Lolium perenne F2 mapping population VrnA. Melting profiles of PCR products amplified from previously published gene loci and from a novel gene putatively involved in vernalization response successfully discriminated genotypes in absence of allelic sequence information, and allowed to determine allele segregation in VrnA. Here we introduce the concept of “blind” mapping based on HRM as a powerful, fast and cheap method to map any DNA sequence polymorphisms without prior knowledge of allelic sequences in the key grassland species perennial ryegrass (Lolium perenne L.).     

A DNA marker assay based on high-resolution melting curve analysis for distinguishing species of the Festuca–Lolium complex

The grass breeding industry is interested in a fast and cheap method of identifying contamination in seeds of Italian and perennial ryegrass (Lolium perenne L. and L. multiflorum Lam., respectively). This study shows that high-resolution melting curve analysis in combination with an unlabelled probe assay is an effective method of detecting single nucleotide polymorphisms (SNPs) in diverse Italian and perennial ryegrass backgrounds. This method proved efficient in differentiating ryegrass species and reducing the effect of additional DNA sequence polymorphisms close to the target SNP on the melting curve profiles. For the identification of contamination in Italian and perennial ryegrass seed production, high-resolution melting curve analysis shows great potential, as it is a single closed-tube PCR reaction with an easy workflow, providing results in <2 h after DNA extraction.     

Sensitivity analysis of the Poisson Nernst–Planck equations: a finite element approximation for the sensitive analysis of an electrodiffusion model

Biological structures exhibiting electric potential fluctuations such as neuron and neural structures with complex geometries are modelled using an electrodiffusion or Poisson Nernst–Planck system of equations. These structures typically depend upon several parameters displaying a large degree of variation or that cannot be precisely inferred experimentally. It is crucial to understand how the mathematical model (and resulting simulations) depend on specific values of these parameters. Here we develop a rigorous approach based on the sensitivity equation for the electrodiffusion model. To illustrate the proposed methodology, we investigate the sensitivity of the electrical response of a node of Ranvier with respect to ionic diffusion coefficients and the membrane dielectric permittivity.

     

Systems for the detection and analysis of protein–protein interactions

The analysis of protein–protein interactions is important for developing a better understanding of the functional annotations of proteins that are involved in various biochemical reactions in vivo. The discovery that a protein with an unknown function binds to a protein with a known function could provide a significant clue to the cellular pathway concerning the unknown protein. Therefore, information on protein–protein interactions obtained by the comprehensive analysis of all gene products is available for the construction of interactive networks consisting of individual protein–protein interactions, which, in turn, permit elaborate biological phenomena to be understood. Systems for detecting protein–protein interactions in vitro and in vivo have been developed, and have been modified to compensate for limitations. Using these novel approaches, comprehensive and reliable information on protein–protein interactions can be determined. Systems that permit this to be achieved are described in this review.K. Kuroda, M. Kato and J. Mima contributed equally to this work.     

α-Satellite DNA methylation in normal individuals and in ICF patients: heterogeneous methylation of constitutive heterochromatin in adult and fetal tissues

The methylation profile of ten α-satellites was investigated in normal individuals and in ICF (Immunodeficiency, Centromeric instability, Facial abnormalities) patients. Two out of three ICF patients showed modified methylation of these sequences, reproducing a placental profile. CENP-B boxes, the binding sites of centromeric protein B, were always skewed toward nonmethylation. Unexpected results were observed in normal individuals: in somatic adult tissues the methylation pattern of α-satellite DNA varied between chromosomes, and in fetal tissues these satellites were homogeneously undermethylated. Detailed methylation analysis of CENP-B boxes revealed that unmethylated α-satellite units coexist with thoroughly methylated regions. These observations showed that the two major components of constitutive heterochromatin are differently methylated in normal somatic and fetal tissues, since classical satellites are consistently methylated. The definite changes in the methylation profile of heterochromatin in somatic chromosomes and the asynchronous timing of methylation of classical and α-satellites during development may reflect specific roles of highly repeated sequences in genomic organization. Received: 29 October 1996 / Revised: 17 December 1996     

Towards a pathogenic Escherichia coli detection platform using multiplex SYBR®Green Real-time PCR methods and high resolution melting analysis

Escherichia coli is a group of bacteria which has raised a lot of safety concerns in recent years. Five major intestinal pathogenic groups have been recognized amongst which the verocytotoxin or shiga-toxin (stx1 and/or stx2) producing E. coli (VTEC or STEC respectively) have received a lot of attention recently. Indeed, due to the high number of outbreaks related to VTEC strains, the European Food Safety Authority (EFSA) has requested the monitoring of the "top-five" serogroups (O26, O103, O111, O145 and O157) most often encountered in food borne diseases and addressed the need for validated VTEC detection methods. Here we report the development of a set of intercalating dye Real-time PCR methods capable of rapidly detecting the presence of the toxin genes together with intimin (eae) in the case of VTEC, or aggregative protein (aggR), in the case of the O104:H4 strain responsible for the outbreak in Germany in 2011. All reactions were optimized to perform at the same annealing temperature permitting the multiplex application in order to minimize the need of material and to allow for high-throughput analysis. In addition, High Resolution Melting (HRM) analysis allowing the discrimination among strains possessing similar virulence traits was established. The development, application to food samples and the flexibility in use of the methods are thoroughly discussed. Together, these Real-time PCR methods facilitate the detection of VTEC in a new highly efficient way and could represent the basis for developing a simple pathogenic E. coli platform.     

Highly sensitive and selective photoelectrochemical DNA sensor for the detection of Hg²⁺ in aqueous solutions

A "turn-on" photoelectrochemical sensor for Hg(2+) detection based on thymine-Hg(2+)-thymine interaction is presented by using a thymine-rich oligonucleotide film and a double-strand DNA intercalator, Ru(bpy)(2)(dppz)(2+) (bpy=2,2'-bipyridine, dppz=dipyrido[3,2-a:2',3'-c]phenazine) as the photocurrent signal reporter. The presence of Hg(2+) induces the formation of a double helical DNA structure which provides binding sites for Ru(bpy)(2)(dppz)(2+). The double helical structure was confirmed by circular dichroism and fluorescence measurements. Under the optimized conditions, a linear relationship between photocurrent and Hg(2+) concentration was obtained over the range of 0.1 nM to 10 nM Hg(2+), with a detection limit of 20 pM. Interference by 10 other metal ions was negligible. Analytical results of Hg(2+) spiked into tap water and lake water by the sensor were in good agreement with mass spectrometry data. With the advantages of high sensitivity and selectivity, simple sensor construction, low instrument cost and low sample volume, this method is potentially suitable for the on-site monitoring of Hg(2+) contamination.     

Drosophila pupal macrophages – A versatile tool for combined ex vivo and in vivo imaging of actin dynamics at high resolution

Molecular understanding of actin dynamics requires a genetically traceable model system that allows live cell imaging together with high-resolution microscopy techniques. Here, we used Drosophila pupal macrophages that combine many advantages of cultured cells with a genetic in vivo model system. Using structured illumination microscopy together with advanced spinning disk confocal microscopy we show that these cells provide a powerful system for single gene analysis. It allows forward genetic screens to characterize the regulatory network controlling cell shape and directed cell migration in a physiological context. We knocked down components regulating lamellipodia formation, including WAVE, single subunits of Arp2/3 complex and CPA, one of the two capping protein subunits and demonstrate the advantages of this model system by imaging mutant macrophages ex vivo as well as in vivo upon laser-induced wounding.     

The use of Quantitative Agarose Gel Electrophoresis for rapid analysis of the integrity of protein–DNA complexes

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.     

Construction and analysis of the protein–protein interaction network for the olfactory system of the silkworm Bombyx mori

Olfaction plays an essential role in feeding and information exchange in insects. Previous studies on the olfaction of silkworms have provided a wealth of information about genes and proteins, yet, most studies have only focused on a single gene or protein related to the insect's olfaction. The aim of the current study is to determine key proteins in the olfactory system of the silkworm, and further understand protein–protein interactions (PPIs) in the olfactory system of Lepidoptera. To achieve this goal, we integrated Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and network analyses. Furthermore, we selected 585 olfactory-related proteins and constructed a (PPI) network for the olfactory system of the silkworm. Network analysis led to the identification of several key proteins, including GSTz1, LOC733095, BGIBMGA002169-TA, BGIBMGA010939-TA, GSTs2, GSTd2, Or-2, and BGIBMGA013255-TA. A comprehensive evaluation of the proteins showed that glutathione S-transferases (GSTs) had the highest ranking. GSTs also had the highest enrichment levels in GO and KEGG. In conclusion, our analysis showed that key nodes in the biological network had a significant impact on the network, and the key proteins identified via network analysis could serve as new research targets to determine their functions in olfaction.     

Molecular dynamics simulation of the melting processes of core–shell and pure nanoparticles

Core–shell nanoparticles are nanosized particles that consist of a core and a shell, constructed from different metallic elements. Core–shell nanoparticles have received extensive attention, owing to their various potential applications such as paints, optical films and catalysts. Herein, we investigate the melting behaviours of different core–shell nanoparticles under continuous heating using molecular dynamics simulation. Different metallic elements were examined as core–shell and pure nanoparticles. Five different processes were observed during the melting of core–shell nanoparticles. In contrast, only one process was identified during the melting of pure nanoparticles. These processes were influenced by the nanoparticle size, shell thickness and differences between the lattice constants and melting point temperatures of the metallic elements. Our simulation provides microscopic insights into the melting behaviours of existing and proposed core–shell nanoparticles that would be highly beneficial towards the fabrication of materials with different chemical coatings.     

Use of the DBD–FISH technique for detecting DNA breakage in response to high doses of X-rays

The aim of this study was to generate a dose–response curve using the DNA breakage detection–fluorescent in situ hybridization (DBD–FISH) test as a biomarker of initial genetic effects induced by high doses of X-rays. A dose–response curve was obtained by measuring the ex vivo responses to increasing doses (0–50 Gy) of X-rays in the peripheral blood lymphocytes of ten healthy donors. The overall dose–response curve was constructed using integrated density (ID; area × fluorescence intensity) as a measure of genetic damage induced by irradiation. The correlation coefficient was high (r = 0.934, b ₀ = 10.408, and b ₁ = 0.094). One-way ANOVA with the Student–Newman–Keuls test for multiple comparisons showed significant differences among the average ln ID values according to dose. Our results suggest the usefulness of the DBD–FISH technique for measuring intrinsic individual cellular radio sensitivity ex vivo.     

Practice of solid-phase extraction and protein precipitation in the 96-well format combined with high-performance liquid chromatography–ultraviolet detection for the analysis of drugs in plasma and brain

C₁₈ Empore 96-well extraction disc plates have been employed for the analysis of three drugs with different polarities in plasma in conjunction with HPLC–UV, rufinamide, ICL670 and an anticonvulsant agent (AA1) in an early stage of development. With the most polar compound (AA1), ion-pair extraction at pH 12 was applied. The method developed for the assay of AA1 in plasma was applied to its determination in brain using an Oasis HLB plate following homogenisation in a pH 7.4 buffer and protein precipitation with NaOH–ZnSO₄, thereby saving time for method development. Protein precipitation in the 96-well format with filtration of the precipitate was applied to the determination of ICL670, a highly protein-bound compound (>99.5%), with a good recovery (78%). Reversed-phase chromatography was applied using a short 5 cm column packed with 3 μm particles for the determination of ICL670 and AA1 and two parallel columns (15 cm long) for the determination of rufinamide. The methods were used routinely, one plate per analysis day being processed, resulting in increase in sample throughput and saving in solvents.     

A method for genome-wide analysis of DNA helical tension by means of psoralen–DNA photobinding

The helical tension of chromosomal DNA is one of the epigenetic landmarks most difficult to examine experimentally. The occurrence of DNA crosslinks mediated by psoralen photobinding (PB) stands as the only suitable probe for assessing this problem. PB is affected by chromatin structure when is done to saturation; but it is mainly determined by DNA helical tension when it is done to very low hit conditions. Hence, we developed a method for genome-wide analysis of DNA helical tension based on PB. We adjusted in vitro PB conditions that discern DNA helical tension and applied them to Saccharomyces cerevisiae cells. We selected the in vivo cross-linked DNA sequences and identified them on DNA arrays. The entire procedure was robust. Comparison of PB obtained in vivo with that obtained in vitro with naked DNA revealed that numerous chromosomal regions had deviated PB values. Similar analyses in yeast topoisomerase mutants uncovered further PB alterations across specific chromosomal domains. These results suggest that distinct chromosome compartments might confine different levels of DNA helical tension in yeast. Genome-wide analysis of psoralen–DNA PB can be, therefore, a useful approach to uncover a trait of the chromosome architecture not amenable to other techniques.     

In vitro and in silico analysis of the Aspergillus nidulans DNA–CreA repressor interactions

The CreA protein mediates carbon catabolite repression in the fungus Aspergillus nidulans. Its DNA-binding domain belongs to the Cys₂-His₂ class, binding specifically to a 5′ SYGGRG 3′ nucleotide sequence. EMSA experiments showed that the CreA(G27D) mutation resulted in a 30-fold increase of the K_diss, and footprinting revealed a altered pattern of protein/DNA contacts. We modeled the CreA and the CreA(G27D) complexes in silico. A 15?ns molecular dynamics simulation of the solvated CreA(G27D) and CreA models was carried out using the MOE 2007.09 suite and the Amber99 force field. We have focused our analysis in residues Arg14, Glu16, His17, and Arg20 and Arg44, Asp46, and Arg50, previously, shown to be responsible for the specific contacts of the two Zn fingers. The electrostatic and the total potential energies showed the CreA(G27D) mutation to decrease the affinity of the complex, in agreement with the K_diss′s values. The in silico approach highlighted the role of the inter-finger linker. We identified several differential structural characteristics of the CreA and CreA(G27D)/DNA complexes and observed that the latter resulted in a lower dynamic flexibility of the complex.     

Epigenetic diagnostics of cancer — the application of DNA methylation markers

In recent years it has become apparent that epigenetic events are potentially equally responsible for cancer initiation and progression as genetic abnormalities. DNA methylation is the main epigenetic modification in humans. Two DNA methylation lesions coexist in human neoplasms: hypermethylation of promoter regions of specific genes within a context of genomic hypomethylation. Aberrant methylation is found at early stages of carcinogenesis and distinct types of cancer exhibit specific patterns of methylation changes. Tumor specific DNA is readily obtainable from different clinical samples and methylation status analysis often permits sensitive disease detection. Methylation markers may also serve for prognostic and predictive purposes as they often reflect the metastatic potential and sensitivity to therapy. As current findings show a great potential of recently characterised methylation markers, more studies in the field are needed in the future. Large clinical studies of newly developed markers are especially needed. The review describes the diagnostic potential of DNA methylation markers.