Calorimetric Analysis Using DNA Thermal Stability to determine protein concentration

It was recently reported for two globular proteins and a short DNA hairpin in NaCl buffer that values of the transition heat capacities, Cp,DNA and Cp,PRO for equal concentrations (mg/mL) of DNA and proteins, are essentially equivalent (differ by less than 1%). Additional evidence for this equivalence is presented that reveals this phenomenon does not depend on DNA sequence, buffer salt, or Tm. Sequences of two DNA hairpins were designed to confer a near 20°C difference in their Tm’s. For the molecules, in NaCl and CsCl buffer the evaluated Cp,PRO and Cp,DNA were equivalent. Based on the equivalence of transition heat capacities, a calorimetric method was devised to determine protein concentrations in pure and complex solutions. The scheme uses direct comparisons between the thermodynamic stability of a short DNA hairpin standard of known concentration, and thermodynamic stability of protein solutions of unknown concentrations. In all cases, evaluated protein concentrations determined from the DNA standard curve agreed with the UV-Vis concentration for monomeric proteins. For samples of multimeric proteins, streptavidin (tetramer), Herpes Simplex Virus glycoprotein D (trimer/dimer), and a 16 base pair DNA duplex (dimer), evaluated concentrations were greater than determined by UV-Vis by factors of 3.94, 2.65, and 2.15, respectively

DNA-based Assay for Calorimetric determination of protein concentrations in pure or mixed solutions

It was recently reported that values of the transition heat capacities, as measured by differential scanning calorimetry, for two globular proteins and a short DNA hairpin in NaCl buffer are essentially equivalent, at equal concentrations (mg/mL). To validate the broad applicability of this phenomenon, additional evidence for this equivalence is presented that reveals it does not depend on DNA sequence, buffer salt, or transition temperature (Tm). Based on the equivalence of transition heat capacities, a calorimetric method was devised to determine protein concentrations in pure and complex solutions. The scheme uses direct comparisons between the thermodynamic stability of a short DNA hairpin standard of known concentration, and thermodynamic stability of protein solutions of unknown concentrations. Sequences of two DNA hairpins were designed to confer a near 20˚C difference in their Tm values. In all cases, evaluated protein concentrations determined from the DNA standard curves agreed with the UV-Vis concentration for monomeric proteins. For multimeric proteins evaluated concentrations were greater than determined by UV-Vis suggesting the calorimetric approach can also be an indicator of molecular stoichiometry

Multiplex SNP Discrimination

Multiplex hybridization reactions of perfectly matched duplexes and duplexes containing a single basepair mismatch (SNPs) were investigated on DNA microarrays. Effects of duplex length, G-C percentage, and relative position of the SNP on duplex hybridization and SNP resolution were determined. Our theoretical model of multiplex hybridization accurately predicts observed results and implicates target concentration as a critical variable in multiplex SNP detection

Microscopic formulation of the Zimm-Bragg model for the helix-coil transition

A microscopic spin model is proposed for the phenomenological Zimm-Bragg model for the helix-coil transition in biopolymers. This model is shown to provide the same thermophysical properties of the original Zimm-Bragg model and it allows a very convenient framework to compute statistical quantities. Physical origins of this spin model are made transparent by an exact mapping into a one-dimensional Ising model with an external field. However, the dependence on temperature of the reduced external field turns out to differ from the standard one-dimensional Ising model and hence it gives rise to different thermophysical properties, despite the exact mapping connecting them. We discuss how this point has been frequently overlooked in the recent literature.Comment: 11 pages, 2 figure

DNA multiplex hybridization on microarrays and thermodynamic stability in solution: a direct comparison

Hybridization intensities of 30 distinct short duplex DNAs measured on spotted microarrays, were directly compared with thermodynamic stabilities measured in solution. DNA sequences were designed to promote formation of perfect match, or hybrid duplexes containing tandem mismatches. Thermodynamic parameters ΔH°, ΔS° and ΔG° of melting transitions in solution were evaluated directly using differential scanning calorimetry. Quantitative comparison with results from 63 multiplex microarray hybridization experiments provided a linear relationship for perfect match and most mismatch duplexes. Examination of outliers suggests that both duplex length and relative position of tandem mismatches could be important factors contributing to observed deviations from linearity. A detailed comparison of measured thermodynamic parameters with those calculated using the nearest-neighbor model was performed. Analysis revealed the nearest-neighbor model generally predicts mismatch duplexes to be less stable than experimentally observed. Results also show the relative stability of a tandem mismatch is highly dependent on the identity of the flanking Watson–Crick (w/c) base pairs. Thus, specifying the stability contribution of a tandem mismatch requires consideration of the sequence identity of at least four base pair units (tandem mismatch and flanking w/c base pairs). These observations underscore the need for rigorous evaluation of thermodynamic parameters describing tandem mismatch stability

Competition for hydrogen bond formation in the helix-coil transition and protein folding

The problem of the helix-coil transition of biopolymers in explicit solvents, like water, with the ability for hydrogen bonding with solvent is addressed analytically using a suitably modified version of the Generalized Model of Polypeptide Chains. Besides the regular helix-coil transition, an additional coil-helix or reentrant transition is also found at lower temperatures. The reentrant transition arises due to competition between polymer-polymer and polymer-water hydrogen bonds. The balance between the two types of hydrogen bonding can be shifted to either direction through changes not only in temperature, but also by pressure, mechanical force, osmotic stress or other external influences. Both polypeptides and polynucleotides are considered within a unified formalism. Our approach provides an explanation of the experimental difficulty of observing the reentrant transition with pressure; and underscores the advantage of pulling experiments for studies of DNA. Results are discussed and compared with those reported in a number of recent publications with which a significant level of agreement is obtained.Comment: 21 pages, 3 figures, submitted to Phys Rev

Electrical detection of the temperature induced melting transition of a DNA hairpin covalently attached to gold interdigitated microelectrodes

The temperature induced melting transition of a self-complementary DNA strand covalently attached at the 5′ end to the surface of a gold interdigitated microelectrode (GIME) was monitored in a novel, label-free, manner. The structural state of the hairpin was assessed by measuring four different electronic properties of the GIME (capacitance, impedance, dissipation factor and phase angle) as a function of temperature from 25°C to 80°C. Consistent changes in all four electronic properties of the GIME were observed over this temperature range, and attributed to the transition of the attached single-stranded DNA (ssDNA) from an intramolecular, folded hairpin structure to a melted ssDNA. The melting curve of the self-complementary single strand was also measured in solution using differential scanning calorimetry (DSC) and UV absorbance spectroscopy. Temperature dependent electronic measurements on the surface and absorbance versus temperature values measured in solution experiments were analyzed assuming a two-state process. The model analysis provided estimates of the thermodynamic transition parameters of the hairpin on the surface. Two-state analyses of optical melting data and DSC measurements provided evaluations of the thermodynamic transition parameters of the hairpin in solution. Comparison of surface and solution measurements provided quantitative evaluation of the effect of the surface on the thermodynamics of the melting transition of the DNA hairpin

Bullying behaviors and victimization experiences among adolescent students: the role of resilience

The role of resilience in the relationship between bullying behaviours, victimisation experiences, and self-efficacy was examined. Three hundred and 93 (191 male, 202 female) adolescents (mean age = 15.88, SD = .64) from schools in Coimbatore, India completed scales to assess bullying behaviours and victimisation experiences, resilience, and self-efficacy. Multigroup SEM, with separate groups created according to participant sex, revealed that resilience mediated the relationship between bullying behaviours and self-efficacy in males. Males engaged in bullying behaviours and experienced victimisation more frequently than females. The findings of the study have implication for designing intervention programs to enhance resilience among adolescents and young adults to enable them to manage bullying behaviours

Reliability and validity of the Japanese version of the Resilience Scale and its short version

<p>Abstract</p> <p>Background</p> <p>The clinical relevance of resilience has received considerable attention in recent years. The aim of this study is to demonstrate the reliability and validity of the Japanese version of the Resilience Scale (RS) and short version of the RS (RS-14).</p> <p>Findings</p> <p>The original English version of RS was translated to Japanese and the Japanese version was confirmed by back-translation. Participants were 430 nursing and university psychology students. The RS, Center for Epidemiologic Studies Depression Scale (CES-D), Rosenberg Self-Esteem Scale (RSES), Social Support Questionnaire (SSQ), Perceived Stress Scale (PSS), and Sheehan Disability Scale (SDS) were administered. Internal consistency, convergent validity and factor loadings were assessed at initial assessment. Test-retest reliability was assessed using data collected from 107 students at 3 months after baseline. Mean score on the RS was 111.19. Cronbach's alpha coefficients for the RS and RS-14 were 0.90 and 0.88, respectively. The test-retest correlation coefficients for the RS and RS-14 were 0.83 and 0.84, respectively. Both the RS and RS-14 were negatively correlated with the CES-D and SDS, and positively correlated with the RSES, SSQ and PSS (all p < 0.05), although the correlation between the RS and CES-D was somewhat lower than that in previous studies. Factor analyses indicated a one-factor solution for RS-14, but as for RS, the result was not consistent with previous studies.</p> <p>Conclusions</p> <p>This study demonstrates that the Japanese version of RS has psychometric properties with high degrees of internal consistency, high test-retest reliability, and relatively low concurrent validity. RS-14 was equivalent to the RS in internal consistency, test-retest reliability, and concurrent validity. Low scores on the RS, a positive correlation between the RS and perceived stress, and a relatively low correlation between the RS and depressive symptoms in this study suggest that validity of the Japanese version of the RS might be relatively low compared with the original English version.</p

Methylthioadenosine reprograms macrophage activation through adenosine receptor stimulation

Regulation of inflammation is necessary to balance sufficient pathogen clearance with excessive tissue damage. Central to regulating inflammation is the switch from a pro-inflammatory pathway to an anti-inflammatory pathway. Macrophages are well-positioned to initiate this switch, and as such are the target of multiple therapeutics. One such potential therapeutic is methylthioadenosine (MTA), which inhibits TNFα production following LPS stimulation. We found that MTA could block TNFα production by multiple TLR ligands. Further, it prevented surface expression of CD69 and CD86 and reduced NF-KB signaling. We then determined that the mechanism of this action by MTA is signaling through adenosine A2 receptors. A2 receptors and TLR receptors synergized to promote an anti-inflammatory phenotype, as MTA enhanced LPS tolerance. In contrast, IL-1β production and processing was not affected by MTA exposure. Taken together, these data demonstrate that MTA reprograms TLR activation pathways via adenosine receptors to promote resolution of inflammation. © 2014 Keyel et al