Modeling transthyretin (TTR) amyloid diseases, from monomer to amyloid fibrils.

ATTR amyloidosis is caused by deposition of large, insoluble aggregates (amyloid fibrils) of cross-ß-sheet TTR protein molecules on the intercellular surfaces of tissues. The process of amyloid formation from monomeric TTR protein molecules to amyloid deposits has not been fully characterized and is therefore modeled in this paper. Two models are considered: 1) TTR monomers in the blood spontaneously fold into a ß-sheet conformation, aggregate into short proto-fibrils that then circulate in the blood until they find a complementary tissue where the proto-fibrils accumulate to form the large, insoluble amyloid fibrils found in affected tissues. 2) TTR monomers in the native or ß-sheet conformation circulate in the blood until they find a tissue binding site and deposit in the tissue or tissues forming amyloid deposits in situ. These models only differ on where the selection for ß-sheet complementarity occurs, in the blood where wt-wt, wt-v, and v-v interactions determine selectivity, or on the tissue surface where tissue-wt and tissure-v interactions also determine selectivity. Statistical modeling in both cases thus involves selectivity in fibril aggregation and tissue binding. Because binding of protein molecules into fibrils and binding of fibrils to tissues occurs through multiple weak non-covalent bonds, strong complementarity between ß-sheet molecules and between fibrils and tissues is required to explain the insolubility and tissue selectivity of ATTR amyloidosis. Observation of differing tissue selectivity and thence disease phenotypes from either pure wildtype TTR protein or a mix of wildtype and variant molecules in amyloid fibrils evidences the requirement for fibril-tissue complementarity. Understanding the process that forms fibrils and binds fibrils to tissues may lead to new possibilities for interrupting the process and preventing or curing ATTR amyloidosis.

Reaction of KHP with excess NaOH or TRIS as standard reactions for calibration of titration calorimeters from 0 to 60 °C.

Calibration of titration calorimeters is an ongoing problem, particularly with calorimeters with reaction vessel volumes < 10 mL in which an electrical calibration heater is positioned outside the calorimetric vessel. Consequently, a chemical reaction with a known enthalpy change must be used to accurately calibrate these calorimeters. This work proposes the use of standard solutions of potassium acid phthalate (KHP) titrated into solutions of excess sodium hydroxide (NaOH) or excess tris(hydroxymethyl)aminomethane (TRIS) as standard reactions to determine the collective accuracy of the relevant variables in a determination of the molar enthalpy change for a reaction. KHP is readily available in high purity, weighable for easy preparation of solutions with accurately known concentrations, stable in solution, not compromised by side reactions with common contaminants such as atmospheric CO2, and non-corrosive to materials used in calorimeter construction. Molar enthalpy changes for these reactions were calculated from 0 to 60 °C from reliable literature data for the pKa of KHP, the molar enthalpy change for protonation of TRIS, and the molar enthalpy change for ionization of water. The feasibility of using these reactions as enthalpic standards was tested in several calorimeters; a 50 mL CSC 4300, a 185 µL NanoITC, a 1.4 mL VP-ITC, and a TAM III with 1 mL reaction vessels. The results from the 50 mL CSC 4300, which was accurately calibrated with an electric heater, verified the accuracy of the calculated standard values for the molar enthalpy changes of the proposed reactions.

Germination of Pisum sativum L. Seeds Is Associated with the Alternative Respiratory Pathway.

The alternative oxidase (AOX) is a ubiquinol oxidase with a crucial role in the mitochondrial alternative respiratory pathway, which is associated with various processes in plants. In this study, the activity of AOX in pea seed germination was determined in two pea cultivars, 'Maravilha d'América' (MA) and 'Torta de Quebrar' (TQ), during a germination trial using cytochrome oxidase (COX) and AOX inhibitors [rotenone (RT) and salicylic hydroxamic acid (SHAM), respectively]. Calorespirometry was used to assess respiratory changes during germination. In both cultivars, SHAM had a greater inhibitory effect on germination than RT, demonstrating the involvement of AOX in germination. Although calorespirometry did not provide direct information on the involvement of the alternative pathway in seed germination, this methodology was valuable for distinguishing cultivars. To gain deeper insights into the role of AOX in seed germination, the AOX gene family was characterized, and the gene expression pattern was evaluated. Three PsAOX members were identified-PsAOX1, PsAOX2a and PsAOX2b-and their expression revealed a marked genotype effect. This study emphasizes the importance of AOX in seed germination, contributing to the understanding of the role of the alternative respiratory pathway in plants.

Modification of the structural stability of human serum albumin in rheumatoid arthritis.

Differential scanning calorimetry (DSC) can indicate changes in structure and/or concentration of the most abundant proteins in a biological sample via heat denaturation curves (HDCs). In blood serum for example, HDC changes result from either concentration changes or altered thermal stabilities for 7-10 proteins and has previously been shown capable of differentiating between sick and healthy human subjects. Here, we compare HDCs and proteomic profiles of 50 patients experiencing joint-inflammatory symptoms, 27 of which were clinically diagnosed with rheumatoid arthritis (RA). The HDC of all 50 subjects appeared significantly different from expected healthy curves, but comparison of additional differences between the RA and the non-RA subjects allowed more specific understanding of RA samples. We used mass spectrometry (MS) to investigate the reasons behind the additional HDC changes observed in RA patients. The HDC differences do not appear to be directly related to differences in the concentrations of abundant serum proteins. Rather, the differences can be attributed to modified thermal stability of some fraction of the human serum albumin (HSA) proteins in the sample. By quantifying differences in the frequency of artificially induced post translational modifications (PTMs), we found that HSA in RA subjects had a much lower surface accessibility, indicating potential ligand or protein binding partners in certain regions that could explain the shift in HSA melting temperature in the RA HDCs. Several low abundance proteins were found to have significant changes in concentration in RA subjects and could be involved in or related to binding of HSA. Certain amino acid sites clusters were found to be less accessible in RA subjects, suggesting changes in HSA structure that may be related to changes in protein-protein interactions. These results all support a change in behavior of HSA which may give insight into mechanisms of RA pathology.

Temperature Response of Metabolic Activity of an Antarctic Nematode.

Because of climate change, the McMurdo Dry Valleys of Antarctica (MCM) have experienced an increase in the frequency and magnitude of summer pulse warming and surface ice and snow melting events. In response to these environmental changes, some nematode species in the MCM have experienced steady population declines over the last three decades, but Plectus murrayi, a mesophilic nematode species, has responded with a steady increase in range and abundance. To determine how P. murrayi responds to increasing temperatures, we measured metabolic heat and CO2 production rates and calculated O2 consumption rates as a function of temperature at 5 °C intervals from 5 to 50 °C. Heat, CO2 production, and O2 consumption rates increase approximately exponentially up to 40 °C, a temperature never experienced in their polar habitat. Metabolic rates decline rapidly above 40 °C and are irreversibly lost at 50 °C due to thermal stress and mortality. Caenorhabditis elegans, a much more widespread nematode that is found in more temperate environments reaches peak metabolic heat rate at just 27 °C, above which it experiences high mortality due to thermal stress. At temperatures from 10 to 40 °C, P. murrayi produces about 6 times more CO2 than the O2 it consumes, a respiratory quotient indicative of either acetogenesis or de novo lipogenesis. No potential acetogenic microbes were identified in the P. murrayi microbiome, suggesting that P. murrayi is producing increased CO2 as a byproduct of de novo lipogenesis. This phenomenon, in conjunction with increased summer temperatures in their polar habitat, will likely lead to increased demand for carbon and subsequent increases in CO2 production, population abundance, and range expansion. If such changes are not concomitant with increased carbon inputs, we predict the MCM soil ecosystems will experience dramatic declines in functional and taxonomic diversity.

Exploring the Applicability of Calorespirometry to Assess Seed Metabolic Stability Upon Temperature Stress Conditions-Pisum sativum L. Used as a Case Study.

The availability of phenotyping tools to assist breeding programs in the selection of high-quality crop seeds is of obvious interest with consequences for both seed producers and consumers. Seed germination involves the activation of several metabolic pathways, such as cellular respiration to provide the required ATP and reducing power. This work tested the applicability of calorespirometry, the simultaneous measurement of heat and CO2 rates, as a phenotyping tool to assess seed respiratory properties as a function of temperature. The effect of temperature on seed germination was evaluated after 16 h of seed imbibition by calorespirometric experiments performed in isothermal mode at 15, 20, 25, and 28°C on the seeds of three cultivars of peas (Pisum sativum L.) commonly used in conventional agriculture (cvs. 'Rondo', 'Torta de Quebrar', and 'Maravilha d'América'). Significant differences in metabolic heat rate and CO2 production rate (R CO2 ) as well as in the temperature responses of these parameters were found among the three cultivars. A seed germination trial was conducted during the 6 days of imbibition to evaluate the predictive power of the parameters derived from the calorespirometric measurements. The germination trial showed that the optimal germination temperature was 20°C and low germination rates were observed at extreme temperatures (15 or 28°C). The cv. 'Torta de Quebrar' showed significantly higher germination in comparison with the other two cultivars at all three temperatures. In comparison with the other two cultivars, 'Torta de Quebrar' has the lowest metabolic heat and CO2 rates and the smallest temperature dependence of these measured parameters. Additionally, 'Torta de Quebrar' has the lowest values of growth rate and carbon use efficiency calculated from the measured variables. These data suggest that calorespirometry is a useful tool for phenotyping physiologic efficiency at different temperatures during early germination stages, and can determine the seeds with the highest resilience to temperature variation, in this case 'Torta de Quebrar'.

Transformation of matter in living organisms during growth and evolution.

Growth of an organism involves transformations of the state of matter from unstructured food or photosynthate into the highly organized matter in the living organism. Biological evolution involves random changes in the structure of DNA that lead to changes in the organization of the matter in an organism. Thermodynamic data show the organized biomass in living organisms has the same thermodynamic properties as a random mixture of the same elemental composition and is not in an energetically metastable, low entropy state. Therefore, the central thesis of this work is that building biological structures and organization from foodstuffs incurs no direct thermodynamic cost. The implication is that growth and evolution occur with little or no thermodynamic cost. In consequence, the fundamental difference between living biomass and lifeless organic sludge is in the information constraints that direct and govern the organization of the system. These constraints within a living organism override random processes to produce an organized distribution of biomass within the organism. Similarly, the information in DNA constrains the outcome of biological evolution across organisms within a population of a species in a predictable way that leads to convergent evolution. Although individuals and molecules act or are acted upon in a random manner, the outcome in a constrained system is predictable within an organism and across organisms. As a consequence evolution will produce similar outcomes at the macro level in similar environments. Stochastic determinism is proposed as a method that could be used to model convergent evolution.

Proposing a minimal set of metrics and methods to predict probabilities of amyloidosis disease and onset age in individuals.

Many amyloid-driven pathologies have both genetic and stochastic components where assessing risk of disease development requires a multifactorial assessment where many of the variables are poorly understood. Risk of transthyretin-mediated amyloidosis is enhanced by age and mutation of the transthyretin (TTR) gene, but amyloidosis is not directly initiated by mutated TTR proteins. Nearly all of the 150+ known mutations increase dissociation of the homotetrameric protein structure and increase the probability of an individual developing a TTR amyloid disease late in life. TTR amyloidosis is caused by dissociated monomers that are destabilized and refold into an amyloidogenic form. Therefore, monomer concentration, monomer proteolysis rate, and structural stability are key variables that may determine the rate of development of amyloidosis. Here we develop a unifying biophysical model that quantifies the relationships among these variables in plasma and suggest the probability of an individual developing a TTR amyloid disease can be estimated. This may allow quantification of risk for amyloidosis and provide the information necessary for development of methods for early diagnosis and prevention. Given the similar observation of genetic and sporadic amyloidoses for other diseases, this model and the measurements to assess risk may be applicable to more proteins than just TTR.

Response of Mycorrhizal 'Touriga Nacional' Variety Grapevines to High Temperatures Measured by Calorespirometry and Near-Infrared Spectroscopy.

Heat stress negatively affects several physiological and biochemical processes in grapevine plants. In this work, two new methods, calorespirometry, which has been used to determine temperature adaptation in plants, and near-infrared (NIR) spectroscopy, which has been used to determine several grapevine-related traits and to discriminate among varieties, were tested to evaluate grapevine response to high temperatures. 'Touriga Nacional' variety grapevines, inoculated or not with Rhizoglomus irregulare or Funneliformis mosseae, were used in this study. Calorespirometric parameters and NIR spectra, as well as other parameters commonly used to assess heat injury in plants, were measured before and after high temperature exposure. Growth rate and substrate carbon conversion efficiency, calculated from calorespirometric measurements, and stomatal conductance, were the most sensitive parameters for discriminating among high temperature responses of control and inoculated grapevines. The results revealed that, although this vine variety can adapt its physiology to temperatures up to 40 °C, inoculation with R. irregulare could additionally help to sustain its growth, especially after heat shocks. Therefore, the combination of calorespirometry together with gas exchange measurements is a promising strategy for screening grapevine heat tolerance under controlled conditions and has high potential to be implemented in initial phases of plant breeding programs.

Obtaining precise and accurate results by ITC.

Acquisition of precise and accurate results by isothermal titration calorimetry (ITC) can be achieved through thoughtful experimental design and modeling and careful experimental operations. Large reported errors in ITC results in determinations of stoichiometries, equilibrium constants and enthalpy changes for ligand binding to proteins are the consequence of poor experiment design, failure to properly calibrate and test instruments and protocols, lack of controls, errors in solution preparation, and incorrect data analyses. Analysis of a recent report that claimed to have determined the "repeatability, precision, and accuracy of the enthalpies and Gibbs energies of a protein-ligand binding reaction" by ITC is used to illustrate how to improve ITC operations and results. The analysis shows that the reported results are misleading because calorimeters were not calibrated, operating parameters were not optimized, errors were made in solution preparations, and data analysis was not optimized. As a consequence, the results do not provide a valid comparison of the capabilities of the calorimeters included in the study. A proposal that reaction of acetazolamide with carbonic anhydrase II be used as a comparison standard for testing ITCs and procedures is problematic because the binding constant is too large and for several other reasons discussed in the paper. Requirements for obtaining precise and accurate results by ITC are discussed and experimental results are presented to illustrate the precision and accuracy attainable with low volume ITCs. The problem of the blank correction is identified as the limiting factor in obtaining accurate results by ITC.

Enzyme Kinetics Determined by Single-Injection Isothermal Titration Calorimetry.

This chapter describes how to collect Michaelis-Menten kinetic data on an enzyme-catalyzed reaction with the isothermal titration calorimetry (ITC) and the single-injection method. ITC measures the heat rate which is directly proportional to the reaction rate. The enthalpy change (ΔrH), Km, kcat, and vmax are determined in a single assay that does not require labeling or immobilization.

Measuring Enzymatic Stability by Isothermal Titration Calorimetry.

This work demonstrates a new method for measuring the stability of enzyme activity by isothermal titration calorimetry (ITC). The peak heat rate observed after a single injection of the substrate solution into an enzyme solution is correlated with enzyme activity. Multiple injections of the substrate into the same enzyme solution over time show the loss of enzyme activity. The assay is autonomous, requiring very little personnel time, and is applicable to most media and enzymes.

Calorimetric Methods for Measuring Stability and Reusability of Membrane Immobilized Enzymes.

The aim of this work is to develop calorimetric methods for characterizing the activity and stability of membrane immobilized enzymes. Invertase immobilized on a nylon-6 nanofiber membrane is used as a test case. The stability of both immobilized and free invertase activity was measured by spectrophotometry and isothermal titration calorimetry (ITC). Differential scanning calorimetry was used to measure the thermal stability of the structure and areal concentration of invertase on the membrane. This is the 1st demonstration that ITC can be used to determine activity and stability of an enzyme immobilized on a membrane. ITC and spectrophotometry show maximum activity of free and immobilized invertase at pH 4.5 and 45 to 55 °C. ITC determination of the activity as a function of temperature over an 8-h period shows a similar decline of activity of both free and immobilized invertase at 55 °C. PRACTICAL APPLICATION: Enzyme-catalyzed reactions occur in mild and environmentally friendly conditions, but are usually too costly to use in food manufacturing. When free enzymes are used, they are used once and replaced for each reaction, but enzymes immobilized on a solid support can be reused and have the additional advantage of being removed from the product. In this study, new calorimetric methods that are universally applicable to characterizing immobilized enzymes are used to determine the activity, stability, and reusability of invertase immobilized on a nanofiber support.

Effect of soil storage at 4 °C on the calorespirometric measurements of soil microbial metabolism.

Soil samples must usually be stored for a time between collection and measurements of microbial metabolic properties. However, little is known about the influence of storage conditions on microbial metabolism when studied by calorespirometry. Calorespirometry measures the heat rate and the CO2 rate of microbial metabolism, where the ratio of heat and CO2 released, the calorespirometric ratio, informs about the nature of substrates being used by microorganisms. Application to soil microbiology is very recent, and little is known about the influence of the common soil preparation practices between collection and analysis on the calorespirometric measurements. For these reasons, the effect of storage at 4 °C on the microbial metabolism was determined by calorespirometry. Results show CO2 production rate decreases with storage time while the evolution of metabolic heat rate is more stable. The calorespirometric ratio increases with storage time in soil samples with organic matter characterized by lower carbohydrate contribution to the total carbon and higher aromaticity and is unaffected in soil samples with lower carbohydrates in the organic matter and higher aromaticity. Therefore, the calorespirometric ratio values may vary for the same soil sample, such that the soil organic matter properties, as well as the time stored at 4 °C, must be considered in interpreting calorespirometric data on soils.

Enzyme-catalyzed and binding reaction kinetics determined by titration calorimetry.

BACKGROUND: Isothermal calorimetry allows monitoring of reaction rates via direct measurement of the rate of heat produced by the reaction. Calorimetry is one of very few techniques that can be used to measure rates without taking a derivative of the primary data. Because heat is a universal indicator of chemical reactions, calorimetry can be used to measure kinetics in opaque solutions, suspensions, and multiple phase systems and does not require chemical labeling. The only significant limitation of calorimetry for kinetic measurements is that the time constant of the reaction must be greater than the time constant of the calorimeter which can range from a few seconds to a few minutes. Calorimetry has the unique ability to provide both kinetic and thermodynamic data. SCOPE OF REVIEW: This article describes the calorimetric methodology for determining reaction kinetics and reviews examples from recent literature that demonstrate applications of titration calorimetry to determine kinetics of enzyme-catalyzed and ligand binding reactions. MAJOR CONCLUSIONS: A complete model for the temperature dependence of enzyme activity is presented. A previous method commonly used for blank corrections in determinations of equilibrium constants and enthalpy changes for binding reactions is shown to be subject to significant systematic error. GENERAL SIGNIFICANCE: Methods for determination of the kinetics of enzyme-catalyzed reactions and for simultaneous determination of thermodynamics and kinetics of ligand binding reactions are reviewed.

Calorespirometry, oxygen isotope analysis and functional-marker-assisted selection ('CalOxy-FMAS') for genotype screening: A novel concept and tool kit for predicting stable plant growth performance and functional marker identification.

We propose a novel concept and tool kit for predictive phenotyping. The proposed technology measures respiration properties as functions of growth conditions to identify genotypes with higher plasticity via homeostasis and adaptive morphophysiology. Combining calorespirometry, oxygen isotope analysis and functional-marker-assisted selection ('CalOxy-FMAS') for genotype screening will enable predicting the genetic potential for stable plant growth performance. Application of this novel tool kit can help identify genotypes with controlled homeostasis in changing environments and optimized growth performance. Simultaneously, it will allow a better balance in breeding for high yields and quality characteristics. Applying 'CalOxy-FMAS' can efficiently narrow the pool of genotypes to be screened for final phenotyping in the field.

Calorespirometry of terrestrial organisms and ecosystems.

Calorespirometry is the simultaneous measurement of heat and gas exchange from biological systems. Such measurements can be used to assess fundamental properties of many different types of systems from small ecosystems to isolated tissues. Techniques for calorespirometric measurements on terrestrial (non-aquatic) samples are described. Methods and models for evaluation of carbon conversion efficiencies, growth rates, and responses to environmental variables from calorespirometric measurements are described. A realistic model of the system under study is essential in the evaluation. Calorespirometry allows testing of models for tissues, individual organisms, and ecosystems.

Phenotyping carrot (Daucus carota L.) for yield-determining temperature response by calorespirometry.

MAIN CONCLUSION: Calorespirometric measurements proved to be useful for phenotyping temperature response in terms of optimum temperatures for growth and low temperature limits for growth respiration in diverse carrot genotypes. High and low-temperature tolerance is an important trait in many breeding programs, but to date, improvement strategies have had limited success. Developing new, cost efficient and reliable screening tools to identify and select the most tolerant crop plant genotypes is necessary to assist plant breeding on cold and heat tolerance, and calorespirometry is proposed for this. Calorespirometry is a technique to simultaneously measure metabolic heat rates and CO2 emission rates of respiring tissues and can be used as a rapid method to determine how changes in the environment (e.g., temperature) influence plant growth. The main aim of this work was, therefore, to test the usefulness of calorespirometry as a phenotyping tool for carrot taproot growth in response to temperature. Calorespirometric measurements in the carrot taproot meristems of plants from eight carrot inbred lines allowed identification of optimum and minimum temperatures for growth of plants and to distinguish between phenotypes based on those characteristics. The technique proved to be useful for predicting yield-determining temperature responses in diverse carrot genotypes. Preliminary screening of new crop plant genotypes with calorespirometry based on their temperature adaptation and acclimation capability could make the screening process much less laborious by allowing selection of genotypes presenting the best growth performance under particular biotic or abiotic conditions before field tests.

Enzyme kinetics determined by single-injection isothermal titration calorimetry.

The purposes of this paper are (a) to examine the effect of calorimeter time constant (τ) on heat rate data from a single enzyme injection into substrate in an isothermal titration calorimeter (ITC), (b) to provide information that can be used to predict the optimum experimental conditions for determining the rate constant (k2), Michaelis constant (KM), and enthalpy change of the reaction (ΔRH), and (c) to describe methods for evaluating these parameters. We find that KM, k2 and ΔRH can be accurately estimated without correcting for the calorimeter time constant, τ, if (k2E/KM), where E is the total active enzyme concentration, is between 0.1/τ and 1/τ and the reaction goes to at least 99% completion. If experimental conditions are outside this domain and no correction is made for τ, errors in the inferred parameters quickly become unreasonable. A method for fitting single-injection data to the Michaelis-Menten or Briggs-Haldane model to simultaneously evaluate KM, k2, ΔRH, and τ is described and validated with experimental data. All four of these parameters can be accurately inferred provided the reaction time constant (k2E/KM) is larger than 1/τ and the data include enzyme saturated conditions.