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Background: Accurate monitoring of energy balance is essential for effective weight management, but the role of energy excretion is often neglected., Objectives: This study aimed to develop and validate a standardized method for assessing energy excretion using dye-labeled meal replacement bars with consistent and stable ingredients., Methods: We utilized baseline data from a registered cross-over trial involving 12 healthy adults (7 females and 5 males) with a body mass index of 18-25 kg/m 2 . Participants consumed dye-labeled meal replacement bars under a standardized protocol, and their feces and urine were collected for energy measurement using bomb calorimetry. Correlation analysis was conducted to explore associations between these variables., Results: The total energy excretion rate averaged 10.48% [standard deviation (SD) 2.56%] of energy intake, with fecal and urinary excretion accounting for 7.95% (SD 2.67%) and 2.52% (SD 0.6%), respectively. Significant individual variability was observed, with total energy excretion ranging from 6.34% to 15.07%, resulting in a maximum difference of 209.64 kcal/d. Fecal energy excretion was positively correlated with fecal wet weight and energy density, whereas urinary energy excretion was associated with digestible energy., Conclusions: This study presents a standardized and efficient methodology for accurately assessing energy excretion using dye-labeled replacement bars. The findings underscore the notable yet variable role of energy excretion in energy balance and suggest that this method could enhance the precision of future energy balance studies., Registration Number: This study was registered at chictr.org.cn as ChiCTR2000038421., Competing Interests: Conflict of interest The authors report no conflicts of interest., (Copyright © 2024 American Society for Nutrition. Published by Elsevier Inc. All rights reserved.)

Background: Underprovision of food energy within the hospital environment can negatively affect clinical outcomes. Hence, the supply of hospital foods/beverages and the ability to assess their calorie provision is critical. The aim of this study was to directly measure the energy density of foods/beverages supplied to patients by Gold Coast University Hospital (GCUH) foodservice and compare these to caloric values established from nutrition information panels (NIPs) on product packaging., Methods: Bomb calorimetry was used to determine the gross energy density of 58 food/beverage items (~47% of total menu) using standardised procedures. Food items included those from the general menu, in addition to therapeutic menu items. Equivalence between directly measured values and those derived from NIPs was determined by comparing the measured mean and ±90% confidence interval (CI) against two pre-defined equivalence bounds (i.e., ±10% NIP value [consensus criteria] and ±20% NIP value [United States Food and Drug Administration (US FDA) criteria]). Caloric values from NIPs were considered equivalent to measured values when the 90% CI of the measured values fell within these thresholds., Results: Overall, 34 (59%) and 19 (33%) items had measured energy density values not equivalent to those from product NIPs according to the consensus and US FDA thresholds, respectively. When employing the US FDA criteria, 12 (21%) items contained a higher calorie density than that established from the label, while seven (12%) items were lower. While non-equivalent items were identified across all therapeutic menus, food labels from items exclusively prepared for the smooth-pureed menu were particularly inaccurate (~60% non-equivalency)., Conclusion: This study found a discrepancy between energy density that was directly measured and that derived from manufacturers' NIPs for many foods and beverages supplied via a hospital foodservice. Given the importance of accurate energy provision for hospitalised patients, this level of inaccuracy is concerning and may warrant a revision of food labelling procedures for items supplied in hospital settings., (© 2024 The British Dietetic Association Ltd.)

The study delves into the binding properties of acridine-9-amine and its selected, mainly N-substituted derivatives (A9As), with calf thymus deoxyribonucleic acid (CT-DNA). This investigation, conducted using UV-Vis spectrophotometry, steady-state fluorescence spectroscopy and isothermal titration calorimetry, provides insights into the relationship between their structure and activity. The absorption spectra of the A9As exhibited a slight red shift and significant hypochromic effects, while the fluorescence emission intensities decreased in the presence of CT-DNA. These results suggest that all fluorescent substrates intercalate into the double helix of native DNA to varying degrees. The binding constants for the A9As/CT-DNA complexes (log(K A ) were determined using various techniques in the range from 2.59 to 5.50). The thermodynamic parameters of A9As binding to DNA were obtained from ITC measurements (ΔG from - 7.51 to - 6.75 kcal·mol -1 , ΔH from - 11.58 to - 3.83 kcal·mol -1 , and TΔS from - 4.83 to 3.68 kcal·mol -1 ) and indicated that the formation of all the investigated A9As-DNA complexes is an enthalpy-driven process. The study also discusses the influence of the emitters' structure and electronic properties of substituents on intercalation efficiency. This knowledge serves as a guide for further research and offers directions for functionalising new acridines as potential reagents. It also provides the latest information on the ability of intercalation to DNA, which can be instrumental in studies on the mechanism of binding small aromatic molecules to DNA and can potentially contribute to new anticancer drug designs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

DNA reaction equilibrium-based calculations have great potential in thermodynamic characterization, but their widespread applications are hindered by significant measurement deviation of equilibrium concentration. Here, we report the advantages of metastable DNA hybridization in reducing quantification deviation of equilibrium concentration and propose a universal and standardized strategy for measuring aptamer binding energy, termed metastable DNA reference calorimetry (MDRC). We built different MDRC-based algorithms tailored to different aptamer binding models, enabling the calculation of thermodynamic parameters for aptamers with one or more binding sites. Our correlative model, considering the cross-effects between different binding sites, showed that for ATP aptamers with two binding sites, binding of the first ATP molecule would decrease its affinity for the second at low temperatures and even completely inhibit this binding at high temperatures. Moreover, the thermodynamic parameters of protein-specific aptamers were calculated to elucidate the universality of the method. The successful analysis of cell-specific aptamers further demonstrated MDRC's applicability in complex biological systems.

Isothermal titration calorimetry (ITC) is a well-established and convenient label-free technique for measuring biomolecular interactions in aqueous solutions. ITC enables accurate measurement of the affinities and thermodynamic parameters, such as changes in Gibbs energy, enthalpy, and entropy, which help dissect the binding mechanisms. ITC is commonly used to study protein-protein, protein-peptide, protein-DNA, and small-molecule inhibitor binding to target proteins. ITC can determine binding affinities ranging from nM to low-mM. This chapter provides a detailed protocol to investigate the binding of FOXO3 peptide to 14-3-3ε using ITC., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

This manuscript details the application of Isothermal Titration Calorimetry (ITC) to characterize the kinetics of 3CL pro , the main protease from the Severe Acute Respiratory Syndrome CoronaVirus-2 (SARS-CoV-2), and its inhibition by Ensitrelvir, a known non-covalent inhibitor. 3CL pro is essential for producing the proteins necessary for viral infection, which led to the COVID-19 pandemic. The ITC-based assay provided rapid and reliable measurements of 3CL pro activity, allowing for the direct derivation of the kinetic enzymatic constants K M and k cat by monitoring the thermal power required to maintain a constant temperature as the substrate is consumed. The manuscript highlights several advantages of the proposed ITC-based assay over traditional methods used to study 3CL pro , such as Förster Resonance Energy Transfer (FRET) and Liquid Chromatography-Mass Spectrometry (LC-MS) and overcomes the need for non-biological substrates or discontinuous post-reaction steps. The ease of application of the ITC method allowed for the determination of the temperature dependence of the catalytic constants, enabling the estimation of the reaction activation energy. Additionally, the assay was used to determine the inhibition mode and kinetic parameters for 3CL pro inhibition by Ensitrelvir. This molecule was revealed to act as a slow- and tight-binding inhibitor that forms an initial E•I complex (K I  = 9.9 ± 0.7 nM) quickly transitioning to a tighter E•I* assembly (K I * = 1.1 ± 0.2 nM). This versatile calorimetric method is proposed for general use in the discovery and development of drugs targeting 3CL pro ., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

We compared two methods of partitional calorimetry to calculate heat storage and heat debt during cold air (0°C) exposure causing mild core cooling. Twelve participants performed a 5 min baseline in thermoneutral conditions (∼22.0°C, ∼50% relative humidity) followed by cold air exposure (∼0°C) until rectal temperature was reduced by ∆-0.5°C. Partitional calorimetry was used to calculate avenues of heat exchange (radiative, convective, and evaporative), heat storage, and heat debt continuously throughout cold exposure. We compared deriving these variables using prediction equations based on environmental and participant characteristics (PCAL Equation Method ) versus using measurement tools such as humidity sensors and heat flux discs (PCAL Heat Flux Method ). There were significant differences between methods (all p  ≤ 0.001) for determining heat exchange, heat storage, and heat debt. At ∆-0.5°C, PCAL Heat Flux Method had greater levels of radiative and convective heat exchange (PCAL Heat Flux Method : -143.0 ± 16.8 W∙m 2 vs PCAL Equation Method : -123.0 ± 12.9 W∙m 2 , p  ≤ 0.001), evaporative heat exchange (PCAL Heat Flux Method : -9.0 ± 1.7 W∙m 2 vs PCAL Equation Method : -4.1 ± 0.0 W∙m 2 , p  ≤ 0.001), heat storage (PCAL Heat Flux Method : -15.0 ± 31.0 W∙m 2 vs PCAL Equation Method : +6.0 ± 25.9 W∙m 2 , p  = 0.020), and heat debt (PCAL Heat Flux Method : -692.0 ± 315.0 kJ vs PCAL Equation Method : -422.0 ± 136.0 kJ, p  ≤ 0.001). Overall, this study found the largest discrepancies between the two methods were when the environmental conditions and skin temperature were in high flux, as well as when core temperature was reduced by ∆-0.5°C. The use of PCAL Heat Flux Method may be more advantageous to use in the cold to provide a higher resolution measurement of cold strain., Competing Interests: The authors declare there are no competing interests.

Modern isothermal titration calorimetry instruments give great precision, but for comparable accuracy they require chemical calibration. For the heat factor, one recommended process is HCl into the weak base TRIS. In studying this reaction with a VP-ITC and two Nano-ITCs, we have encountered some problems, most importantly a titrant volume shortfall Δv ≈ 0.3 μL, which we attribute to diffusive loss of HCl in the syringe tip. This interpretation is supported by a mathematical treatment of the diffusion problem. The effect was discovered through a variable-v protocol, which thus should be used to properly allow for it in any reaction that similarly approaches completion. We also find that the effects from carbonate contamination and from OH - from weak base hydrolysis can be more significant that previously thought. To facilitate proper weighting in the least-squares fitting of data, we have estimated data variance functions from replicate data. All three instruments have low-signal precision of σ ≈ 1 μJ; titrant volume uncertainty is a factor of ∼2 larger for the Nano-ITCs than for the VP-ITC. The final heat factors remain uncertain by more than the ∼1 % precision of the instruments and are unduly sensitive to the HCl concentration., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

This work examines the thermodynamics of model biomolecular interactions using a governing equation that accounts for the participation of bulk water in the equilibria. In the first example, the binding affinities of two DNA duplexes, one of nine and one of 10 base pairs in length, are measured and characterized by isothermal titration calorimetry (ITC) as a function of concentration. The results indicate that the change in solvation free energy that accompanies duplex formation (ΔG S ) is large and unfavorable. The duplex with the larger number of G:C pairings yields the largest change in solvation free energy, ΔG S  = +460 kcal·mol -1 per base pair at 25 °C. A van't Hoff analysis of the data is complicated by the varying degree of intramolecular base stacking within each DNA strand as a function of temperature. A modeling study reveals how the solvation free energy alters the output of a typical ITC experiment and leads to a good, though misleading, fit to the classical equilibrium equation. The same thermodynamic framework is applied to a model protein-ligand interaction, the binding of ribonuclease A with the nucleotide inhibitor 3'-UMP, and to a conformational equilibrium, the change in tertiary structure of α-lactalbumin in molar guanidinium chloride solutions. The ribonuclease study yields a value of ΔG S  = +160 kcal·mol -1 , whereas the folding equilibrium yields ΔG S  ≈ 0, an apparent characteristic of hydrophobic interactions. These examples provide insight on the role of solvation energy in binding equilibria and suggest a pivot in the fundamental application of thermodynamics to solution chemistry., (© 2024 The Author(s). FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The rise in osteomyelitis and periprosthetic joint infections, in combination with increasing life expectancy and the prevalence of diabetes, underscores the urgent need for rapid and accurate diagnostic tools. Conventional culture-based methods are often time-consuming and prone to false-negatives, leading to prolonged and inappropriate antibiotic treatments. This study aims to improve osteomyelitis diagnostics by decreasing the time to detection and the time to an antibiotic susceptibility result to enable a targeted treatment using isothermal microcalorimetry (IMC). IMC measures heat flow in real-time, providing insights into bacterial metabolism without the need for labeling. Using clinical isolates from bone infections, assessing their response to antibiotics through IMC, we demonstrated that IMC could detect bacteria within 4 h and determine antimicrobial susceptibility profiles within 2-22 h (median 4.85, range 1.28-21.78). This is significantly faster than traditional methods. A decision tree, based on antibiotic susceptibility, accurately categorized pathogens, achieving high accuracy (74-100%), sensitivity (100%), and specificity (65-100%). These findings suggest that IMC could redefine diagnostics of bone and joint infections and potentially infections in general, offering timely and precise treatment guidance, thereby improving patient outcomes and reducing health care burdens. Further optimization and clinical validation are needed to fully integrate IMC into routine diagnostics., (© 2024 The Author(s). APMIS published by John Wiley & Sons Ltd on behalf of Scandinavian Societies for Pathology, Medical Microbiology and Immunology.)

The significance and necessity of separating enantiomers in food, pharmaceuticals, pesticides, and other samples remains constant and unrelenting. The successful chiral separation usually includes the application of a chiral auxiliary compound, known also as a chiral selector (CS), that forms complexes with enantiomers of different physicochemical properties, enabling efficient separation. While both native and substituted cyclodextrins (CDs) are commonly used as CSs, β-CD is undoubtedly the most popular one among them. This review includes recent advancements in the application of β-CD as a CS. While the theoretical background behind the enantioseparation is also part of this work, the main emphasis is put on the factors that affect the efficacy of this process such as temperature, pH, solvent, and the choice of other additives. Also, the different analytical methods: Nuclear Magnetic Resonance (NMR) spectroscopy, Capillary Electrophoresis (CE), fluorescence spectroscopy (FS), High-Performance Liquid Chromatography (HPLC), Isothermal Titration Calorimetry (ITC), and UV-vis spectroscopy, used for enantioseparation with the aid of β-CD as CS, are thoroughly compared. Also, since some of the chiral compounds have been studied in the context of their enantioseparation more than once, those works are compared and critically analyzed. In conclusion, while β-CD can be in most cases used as CS, the choice of the experimental conditions and method of analysis is crucial to achieve the success.

We present an optimized version of the skin calorimeter for measuring localized skin thermal responses during physical activity. Enhancements include a new holding system, more sensitive thermopiles, and an upgraded spiked heat sink for improved efficiency. In addition, we used a new, improved calorimetric model that takes into account all the variables that influence the measurement process. Resolution in power measurement is 1 mW. Performance tests under air currents and movement disturbances showed that the device maintains high accuracy; the deviation produced by these significant disturbances is less than 5%. Human subject tests, both at rest and during exercise, confirmed its ability to accurately measure localized skin heat flux, heat capacity, and thermal resistance (less than 5% uncertainty). These findings highlight the calorimeter's potential for applications in sports medicine and physiological studies.

In holometabolous insects, the larval body is almost completely decomposed and reconstructed into the adult body during the pupal-pharate adult stages. Therefore, the total energetic cost of this process is a key thermodynamic quantity necessary for evaluating the benefit of their life history. Here, we measured whole-body thermal dissipation of single pupae of the fruit fly, Drosophila melanogaster , during the period from puparium formation to adult eclosion as a function of age, using a high-precision isothermal calorimeter at T = 298 K. The mass-specific energy consumption during the period from the onset of larval-pupal apolysis to adult eclosion was determined to be 2.3 kJ/g for an individual of mass (adult) = 1.0 mg, while it was observed to follow Kleiber's law for individuals smaller than mass (adult) = 1.0 mg. During the pupal-pharate adult period, in addition to the U-shaped variation, several characteristic thermal dissipations related to various events, including somatic muscle contractions, ecdyses, pulsatile hormone secretion in a pharate adult, and vaporization of the exuvial fluid, were observed. The periodic bursts in the pharate adult stage grew exponentially, suggesting that the positive feedback in the metabolic system synchronized with the progression of development, making the energy consumption in this stage more efficient. The present study showed that high-precision calorimetry is a powerful and credible method for measuring not only the total energy spent during development but also the energy spent during every specific developmental event in an organism.

The compendial USP〈701〉 disintegration test method offers a crucial pass/fail assessment for immediate release tablet disintegration. However, its single end-point approach provides limited insight into underlying mechanisms. This study introduces a novel calorimetric approach, aimed at providing comprehensive process profiles beyond binary outcomes. We developed a novel disintegration reaction calorimeter to monitor the heat release throughout the disintegration process and successfully obtained enthalpy change profiles of placebo tablets with various porosities. The formulation comprised microcrystalline cellulose (MCC), anhydrous lactose, croscarmellose sodium (CCS), and magnesium stearate (MgSt). An abrupt temperature rise was observed after introducing the disintegration medium to tablets, and the relationship between the heat rise time and the tablet's porosity was investigated. The calorimeter's sensitivity was sufficient to discern distinct heat changes among individual tablets, and the analysis revealed a direct correlation between the two. Higher porosity corresponded to shorter heat rise time, indicating faster disintegration rates. Additionally, the analysis identified a concurrent endothermic process alongside the anticipated exothermic phenomenon, potentially associated with the dissolution of anhydrous lactose. Since lactose is the only soluble excipient within the blend composition, the endothermic process can be attributed to the absorption of heat as lactose molecules dissolve in water. The findings from this study underscore the potential of utilising calorimetric methods to quantify the wettability of complex compounds and, ultimately, optimise tablet formulations., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: JAZ is a member of the editorial board of the International Journal of Pharmaceutics. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

We tested whether spontaneous physical activity (SPA) from accelerometers could be used in a whole room calorimeter to estimate thermic effect of food (TEF). Eleven healthy participants ( n = 7 females; age: 27 ± 4 yr; body mass index: 22.8 ± 2.6 kg/m 2 ) completed two 23-h visits in randomized order: one "fed" with meals provided and one "fasted" with no food. SPA was measured by ActivPAL and Actigraph accelerometers. Criterion TEF was calculated as the difference in total daily energy expenditure (TDEE) between fed and fasted visits and compared with three methods of estimating TEF: 1 ) SPA-adjusted TEF (adjTEF)-difference in TDEE without SPA between visits, 2 ) Wakeful TEF-difference in energy expenditure obtained from linear regression and basal metabolic rate during waking hours, 3 ) 24-h TEF-increase in TDEE above SPA and sleeping metabolic rate. Criterion TEF was 9.4 ± 4.5% of TDEE. AdjTEF (difference in estimated vs. criterion TEF: activPAL: -0.3 ± 3.3%; Actigraph: -1.8 ± 8.0%) and wakeful TEF (activPAL: -0.9 ± 6.1%; Actigraph: -2.8 ± 7.6%) derived from both accelerometers did not differ from criterion TEF (all P > 0.05). ActivPAL-derived 24-h TEF overestimated TEF (6.8 ± 5.4%, P = 0.002), whereas Actigraph-derived 24-h TEF was not significantly different (4.3 ± 9.4%, P = 0.156). TEF estimations using activPAL tended to show better individual-level agreement (i.e., smaller coefficients of variation). Both accelerometers can be used to estimate TEF in a whole room calorimeter; wakeful TEF using activPAL is the most viable option given strong group-level accuracy and reasonable individual agreement. NEW & NOTEWORTHY Two research-grade accelerometers can effectively estimate spontaneous physical activity and improve the estimation of thermic effect of food (TEF) in whole room calorimeters. The activPAL demonstrates strong group-level accuracy and reasonable individual-level agreement in estimating wakeful TEF, whereas a hip-worn Actigraph is an acceptable approach for estimating 24-h TEF. These results highlight the promising potential of accelerometers in advancing energy balance research by improving the assessment of TEF within whole room calorimeters.

Enzymes, as biocatalysts, are an important target of many therapies and are also of great industrial importance, which is why repeatable and accurate parameterization of enzymatic catalysis is very important. The most popular spectrophotometric detection method in enzymology, despite its low cost and speed, often cannot be used directly due to the inappropriate spectral properties of substrates and products. It is then necessary to use auxiliary enzymes, chemical modification of substrates or post-reaction analysis, which may increase the cost of measurement, extend its time or affect the accuracy. Isothermal titration calorimetry is a method widely used mainly for the characterization of inter-molecular interactions, however, its use in enzyme kinetics is gaining more and more recognition due to the direct measurement of the reaction rate using the universal parameter of heat, high sensitivity and low reagent consumption. This work discusses two strategies for conducting a kinetic calorimetric experiment and their applications.

RNA aptamers are oligonucleotides, selected through Systematic Evolution of Ligands by EXponential Enrichment (SELEX), that can bind to specific target molecules with high affinity. One such molecule is the RNA aptamer that binds to a blue-fluorescent Hoechst dye that was modified with bulky t-Bu groups to prevent non-specific binding to DNA. This aptamer has potential for biosensor applications; however, limited information is available regarding its conformation, molecular interactions with the ligand, and binding mechanism. The study presented here aims to biophysically characterize the Hoechst RNA aptamer when complexed with the t-Bu Hoechst dye and to further optimize the RNA sequence by designing and synthesizing new sequence variants. Each variant aptamer-t-Bu Hoechst complex was evaluated through a combination of fluorescence emission, native polyacrylamide gel electrophoresis, fluorescence titration, and isothermal titration calorimetry experiments. The results were used to design a minimal version of the aptamer consisting of only 21 nucleotides. The performed study also describes a more efficient method for synthesizing the t-Bu Hoechst dye derivative. Understanding the biophysical properties of the t-Bu Hoechst dye-RNA complex lays the foundation for nuclear magnetic resonance spectroscopy studies and its potential development as a building block for an aptamer-based biosensor that can be used in medical, environmental or laboratory settings., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

T4 polynucleotide kinase (T4 PNK) phosphorylates the 5'-terminus of DNA and RNA substrates. It is widely used in molecular biology. Single nucleotides can serve as substrates if a 3'-phosphate group is present. In this study, the T4 PNK-catalyzed conversion of adenosine 3'-monophosphate (3'-AMP) to adenosine-3',5'-bisphosphate was characterized using isothermal titration calorimetry (ITC). Although ITC is typically used to study ligand binding, in this case the instrument was used to evaluate enzyme kinetics by monitoring the heat production due to reaction enthalpy. The reaction was initiated with a single injection of 3'-AMP substrate into the sample cell containing T4 PNK and ATP at pH 7.6 and 30 °C, and Michaelis-Menten analysis was performed on the reaction rates derived from the plot of differential power versus time. The Michaelis-Menten constant, K M , was 13 μM, and the turnover number, k cat , was 8 s -1 . The effect of inhibitors was investigated using pyrophosphate (PP i ). PP i caused a dose-dependent decrease in the apparent k cat and increase in the apparent K M under the conditions tested. Additionally, the intrinsic reaction enthalpy and the activation energy of the T4 PNK-catalyzed phosphorylation of 3'-AMP were determined to be -25 kJ/mol and 43 kJ/mol, respectively. ITC is seldom used as a tool to study enzyme kinetics, particularly for technically-challenging enzymes such as kinases. This study demonstrates that quantitative analysis of kinase activity can be amenable to the ITC single injection approach., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR) are two commonly used methods to probe biomolecular interactions. ITC can provide information about the binding affinity, stoichiometry, changes in Gibbs free energy, enthalpy, entropy, and heat capacity upon binding. SPR can provide information about the association and dissociation kinetics, binding affinity, and stoichiometry. Both methods can determine the nature of protein-protein interactions and help understand the physicochemical principles underlying complex biochemical pathways and communication networks. This methods article discusses the practical knowledge of how to set up and troubleshoot these two experiments with some examples., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Optical Calorimetry (OC) is based on interferometry and provides a direct measurement of spatially resolved absorbed dose to water by measuring refractive index changes induced by radiation. The purpose of this work was to optimize and characterize in software an OC system tailored for ultra-high dose rate applications and to build and test a prototype in a clinical environment. A radiation dosimeter using the principles of OC was designed in optical modelling software. Traditional image quality instruments, fencepost and contrast phantoms, were utilized both in software and experimentally in a lab environment to investigate noise reduction techniques and to test the spatial and dose resolution of the system. Absolute dose uncertainty was assessed by measurements in a clinical 6 MV Flattening Filter Free (FFF) photon beam with dose rates in the range 0.2-6 Gy/s achieved via changing the distance from the source. Design improvements included: equalizing the pathlengths of the interferometer, isolating the system from external vibrations and controlling the system's internal temperature as well as application of mathematical noise reduction techniques. Simulations showed that these improvements should increase the spatial resolution from 22 to 35 lp/mm and achieve a minimum detectable dose of 0.2 Gy, which was confirmed experimentally. In the FFF beam, the absolute dose uncertainty was dose rate dependent and decreased from 2.5 ± 0.8 to 2.5 ± 0.2 Gy for dose rates of 0.2 and 6 Gy/s, respectively. A radiation dosimeter utilizing the principles of OC was developed and constructed. Optical modelling software and image quality phantoms allowed for iterative testing and refinement. The refined OC system proved capable of measuring absorbed dose to water in a linac generated photon beam. Reduced uncertainty at higher dose rates indicates the potential for OC as a dosimetry system for high dose rate techniques such as microbeam and ultra-high dose-rate radiotherapy., (© 2023. Australasian College of Physical Scientists and Engineers in Medicine.)

The binding of four alkaloids with human serum albumin (HSA) was investigated by isothermal titration calorimetry (ITC), spectroscopy and molecular docking techniques. The findings demonstrated that theophylline or caffeine can bind to HAS, respectively. The number of binding sites and binding constants are obtained. The binding mode is a static quenching process. The effects of steric hindrance, temperature, salt concentration and buffer solution on the binding indicated that theophylline and HSA have higher binding affinity than caffeine. The fluorescence and ITC results showed that the interaction between HSA and theophylline or caffeine is an entropy-driven spontaneous exothermic process. The hydrophobic force was the primary driving factor. The experimental results were consistent with the molecular docking data. Based on the molecular structures of the four alkaloids, steric hindrance might be a major factor in the binding between HSA and these four alkaloids. This study elucidates the mechanism of interactions between four alkaloids and HSA., (© 2024 John Wiley & Sons Ltd.)

Importance of metal ion selectivity in biomolecules and their key role in proteins are widely explored. However, understanding the thermodynamics of how hydrated metal ions alter the protein hydration and their conformation is also important. In this study, the interaction of some biologically important Ca 2+ , Mn 2+ , Co 2+ , Cu 2+ , and Zn 2+ ions with hen egg white lysozyme at pH 2.1, 3.0, 4.5 and 7.4 has been investigated. Intrinsic fluorescence studies have been employed for metal ion-induced protein conformational changes analysis. Thermostability based on protein hydration has been investigated using differential scanning calorimetry (DSC). Thermodynamic parameters emphasizing on metal ion-protein binding mechanistic insights have been well discussed using isothermal titration calorimetry (ITC). Overall, these experiments have reported that their interactions are pH-dependent and entropically driven. This research also reports the strongly hydrated metal ions as water structure breaker unlike osmolytes based on DSC studies. These experimental results have highlighted higher concentrations of different metal ions effect on the protein hydration and thermostability which might be helpful in understanding their interactions in aqueous solutions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Isothermal titration calorimetry (ITC) is a widely used technique for the characterization of protein-protein and protein-ligand interactions. It provides information on the stoichiometry, affinity, and thermodynamic driving forces of interactions. This chapter exemplifies the use of ITC to investigate interactions between human autophagy modifiers (LC3/GABARAP proteins) and their interaction partners, the LIR motif-containing sequences. The purpose of this report is to present a detailed protocol for the production of LC3/GABARAP-interacting LIR peptides using E. coli expression systems. In addition, we outline the design of ITC experiments using the LC3/GABARAP:peptide interactions as an example. Comprehensive troubleshooting notes are provided to facilitate the adaptation of these protocols to different ligand-receptor systems. The methodology outlined for studying protein-ligand interactions will help to avoid common errors and misinterpretations of experimental results., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

With today's challenges regarding antibiotic resistance and the importance of the implementation of prudent use of antibiotics, fast and reliable diagnostic tools for bacterial infections and subsequent antimicrobial susceptibility testing are of utmost relevance. Isothermal microcalorimetry (IMC) is a broadly applicable method, with which metabolic heat flow in reproducing bacteria can be measured in real time. To the best of the authors' knowledge, this is the first report on examination of 124 urine samples from feline and canine urinary tract infection with an IMC-based prototype instrument. A concentration-dependent time of peak heat flow by dilution series with Escherichia coli and Enterococcus faecalis reference strains demonstrated the general good performance of the prototype for detection of these bacteria. With diagnostic culture being set as a gold standard, the diagnostic sensitivity of IMC compared to bacteriological culture was 80 %, the diagnostic specificity was 97 %. With a Cohens' kappa value (κ) of 0.80, the two methods show good concordance. The results from our study demonstrate that the IMC technology is suitable to allow reliable, but much faster detection of bacteria than conventional culture, especially for Escherichia coli. Thus, implementing IMC technology could markedly speed up the bacteriological diagnostic process in veterinary medicine., Competing Interests: Declaration of Competing Interest We declare that we have no competing interests., (Copyright © 2023. Published by Elsevier B.V.)

Organophosphorus pesticides (OPs) are widely used in agriculture and may contaminate food or water, leading to potential health risks. However, there are few reports on the effect of OPs on protein conformation and aggregation. Hence, in this paper, we have characterized the impact of two OPs, chlorpyrifos (CPF) and methyl parathion (Para), on the model protein HEWL using biophysical and computational methods. The steady-state and time-resolved spectroscopy, Circular dichroism (CD), molecular dynamics simulation, and isothermal titration calorimetry were employed to investigate the binding interactions between HEWL and OPs. The steady-state and time-resolved fluorescence spectroscopy confirm the presence of both static and dynamic quenching between OPs and proteins. Based on fluorescence, MD, and CD results, it was found that the OPs not only show strong binding but also destabilize the protein structure and alter the secondary and tertiary structure of the protein. The molecular docking results showed that OPs entered the binding pocket of the HEWL molecule and interacted through hydrophobic and hydrogen bond interactions. The thermodynamic studies indicated that the binding was spontaneous and OPs have shown an effect on the aggregation process of HEWL. Finally, the protein aggregation process was studied using fluorescence and SDS-PAGE studies in the presence of both the OPs and found to enhance the aggregation process in the presence of OPs. These results provide insights into the potential health risks associated with OPs and highlight the importance of understanding their interactions with biological macromolecules.Communicated by Ramaswamy H. Sarma.

Ion channels are membrane proteins that may also have intracellular and extracellular domains that interact with other ligands. In many cases, these interaction sites are highly mobile and may undergo changes in the configuration upon binding with regulatory signaling molecules. Isothermal titration calorimetry (ITC) is a powerful technique to quantify protein-ligand interactions of purified samples in solution. This chapter describes a fragment-based analysis method using ITC to quantify the interactions between a domain of the voltage-gated Kv7 channel and the calcium-regulated protein calmodulin. This example can be used to quantify the interactions between specific domains of other ion channels and their regulatory signaling proteins., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

NIS Synthetases are a widely distributed, novel superfamily of enzymes critical to stealth siderophore production-small molecules increasingly associated with virulence. Study of these enzymes for inhibition or utilization in biosynthesis of new antibiotics has been hindered by multiple kinetics assays utilizing different limiting reporters or relying on product dissociation as a precursor to signal. We present a label free, continuous readout assay optimized for NIS Synthetase systems utilizing an isothermal titration calorimetry instrument. This assay has been tested in an iterative system comparing multiple turnovers on a single substrate to a single bond formation event and is able to delineate these complex kinetics well. The ITC-based kinetic assay is the first label-free assay for the NIS field, which may allow for more detailed kinetic comparisons in the future, and may also have broader use for iterative enzymes in general., (Copyright © 2024. Published by Elsevier Inc.)

The breast cancer susceptibility 1 (BRCA1) protein plays a pivotal role in modulating the transcriptional activity of the vital intrinsically disordered transcription factor MYC. In this regard, mutations of BRCA1 and interruption of its regulatory activity are related to hereditary breast and ovarian cancer (HBOC). Interestingly, so far, MYC's main dimerization partner MAX (MYC-associated factor X) has not been found to bind BRCA1 despite a high sequence similarity between both oncoproteins. Herein, we show that a potential reason for this discrepancy is the heterogeneous conformational space of MAX, which encloses a well-documented folded coiled-coil homodimer as well as a less common intrinsically disordered monomer state-contrary to MYC, which exists mostly as intrinsically disordered protein in the absence of any binding partner. We show that when the intrinsically disordered state of MAX is artificially overpopulated, the binding of MAX to BRCA1 can readily be observed. We characterize this interaction by nuclear magnetic resonance (NMR) spectroscopy chemical shift and relaxation measurements, complemented with ITC and SAXS data. Our results suggest that BRCA1 directly binds the MAX monomer to form a disordered complex. Though probed herein under biomimetic in-vitro conditions, this finding can potentially stimulate new perspectives on the regulatory network around BRCA1 and its involvement in MYC:MAX regulation., (© 2023 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

A class of cyclodextrin (CD) dimers has emerged as a potential new treatment for atherosclerosis; they work by forming strong, soluble inclusion complexes with oxysterols, allowing the body to reduce and heal arterial plaques. However, characterizing the interactions between CD dimers and oxysterols presents formidable challenges due to low sterol solubility, the synthesis of modified CDs resulting in varying number and position of molecular substitutions, and the diversity of interaction mechanisms. To address these challenges and illuminate the nuances of CD-sterol interactions, we have used multiple orthogonal approaches for a comprehensive characterization. Results obtained from three independent techniques - metadynamics simulations, competitive isothermal titration calorimetry, and circular dichroism - to quantify CD-sterol binding are presented. The objective of this study is to obtain the binding constants and gain insights into the intricate nature of the system, while accounting for the advantages and limitations of each method. Notably, our findings demonstrate ∼1000× stronger affinity of the CD dimer for 7-ketocholesterol in comparison to cholesterol for the 1:1 complex in direct binding assays. These methodologies and findings not only enhance our understanding of CD dimer-sterol interactions, but could also be generally applicable to prediction and quantification of other challenging host-guest complex systems., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: All authors report financial support, administrative support, equipment, drugs, or supplies, travel, and writing assistance were provided by Cyclarity Therapeutics. Amelia Anderson and Matthew O'Connor have patent Cyclodextrin dimers, compositions thereof, and uses thereof pending to CYCLARITY THERAPUTICS, INC. The authors would like to disclose that AMA, KS, DMC, and MSO have a financial interest in the company Cyclarity Therapeutics. MM has a financial interest in the company CarboHyde; RGF and AP have a financial interest in the company MD.USE. Cyclarity Therapeutics commissioned this work from all authors., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

We compare several different methods to quantify the uncertainty of binding parameters estimated from isothermal titration calorimetry data: the asymptotic standard error from maximum likelihood estimation, error propagation based on a first-order Taylor series expansion, and the Bayesian credible interval. When the methods are applied to simulated experiments and to measurements of Mg(II) binding to EDTA, the asymptotic standard error underestimates the uncertainty in the free energy and enthalpy of binding. Error propagation overestimates the uncertainty for both quantities, except in the simulations, where it underestimates the uncertainty of enthalpy for confidence intervals less than 70%. In both datasets, Bayesian credible intervals are much closer to observed confidence intervals.

Understanding the thermodynamic signature of protein-peptide binding events is a major challenge in computational chemistry. The complexity generated by both components possessing many degrees of freedom poses a significant issue for methods that attempt to directly compute the enthalpic contribution to binding. Indeed, the prevailing assumption has been that the errors associated with such approaches would be too large for them to be meaningful. Nevertheless, we currently have no indication of how well the present methods would perform in terms of predicting the enthalpy of binding for protein-peptide complexes. To that end, we carefully assembled and curated a set of 11 protein-peptide complexes where there is structural and isothermal titration calorimetry data available and then computed the absolute enthalpy of binding. The initial "out of the box" calculations were, as expected, very modest in terms of agreement with the experiment. However, careful inspection of the outliers allows for the identification of key sampling problems such as distinct conformations of peptide termini not being sampled or suboptimal cofactor parameters. Additional simulations guided by these aspects can lead to a respectable correlation with isothermal titration calorimetry (ITC) experiments ( R 2 of 0.88 and an RMSE of 1.48 kcal/mol overall). Although one cannot know prospectively whether computed ITC values will be correct or not, this work shows that if experimental ITC data are available, then this in conjunction with computed ITC, can be used as a tool to know if the ensemble being simulated is representative of the true ensemble or not. That is important for allowing the correct interpretation of the detailed dynamics of the system with respect to the measured enthalpy. The results also suggest that computational calorimetry is becoming increasingly feasible. We provide the data set as a resource for the community, which could be used as a benchmark to help further progress in this area.

Plant tannins are known for their anthelmintic and antiparasitic activities and have been increasingly studied to battle the ever-growing problem of anthelmintic resistance. While tannins have been shown to exhibit these activities on their own, one approach would be to use them as complementary nutrients alongside commercial anthelmintics. So far, research on the interactions between tannins and anthelmintics is limited, and few studies have reported both synergistic and antagonistic effects depending on the type of tannin and the method used. These interactions could either strengthen or weaken the efficacy of commercial anthelmintics, especially if tannin-rich diets are combined with anthelmintics used as oral drenches. To study these interactions, a series of hydrolysable tannins (HTs) was selected, and their direct interactions with thiabendazole (TBZ) were evaluated by isothermal titration calorimetry (ITC), which allowed the detection of the exothermic interaction but also the roles and significances of different structural features of HTs in these interactions. Our results show that HTs can have a direct interaction with the benzimidazole anthelmintic TBZ and that the interaction is strengthened by increasing the number of free galloyl groups and the overall molecular flexibility of HTs.

Isothermal titration calorimetry (ITC) is a technique for evaluating the thermodynamic profiles of connection between two molecules, allowing the experimental design of nanoparticles systems with drugs and/or biological molecules. Taking into account the relevance of ITC, we conducted, therefore, an integrative revision of the literature, from 2000 to 2023, on the main purposes of using this technique in pharmaceutical nanotechnology. The search were carried out in the Pubmed, Sciencedirect, Web of Science, and Scifinder databases using the descriptors "Nanoparticles", "Isothermal Titration Calorimetry", and "ITC". We have observed that the ITC technique has been increasingly used in pharmaceutical nanotechnology, seeking to understand the interaction mechanisms in the formation of nanoparticles. Additionally, to understand the behavior of nanoparticles with biological materials (proteins, DNA, cell membranes, among others), thereby helping to understand the behavior of nanocarriers in vivo studies. As a contribution, we intended to reveal the importance of ITC in the laboratory routine, which is itself a quick and easy technique to obtain relevant results that help to optimize the nanosystems formulation process., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: SIMR1281 is a potent anticancer lead candidate with multi- target activity against several proteins; however, its mechanism of action at the molecular level is not fully understood. Revealing the mechanism and the origin of multitarget activity is important for the rational identification and optimization of multitarget drugs., Methods: We have used a variety of biophysical (circular dichroism, isothermal titration calorimetry, viscosity, and UV DNA melting), biochemical (topoisomerase I & II assays) and computational (molecular docking and MD simulations) methods to study the interaction of SIMR1281 with duplex DNA structures., Results: The biophysical results revealed that SIMR1281 binds to dsDNA via an intercalation-binding mode with an average binding constant of 3.1 × 10 6  M -1 . This binding mode was confirmed by the topoisomerases' inhibition assays and molecular modeling simulations, which showed the intercalation of the benzopyrane moiety between DNA base pairs, while the remaining moieties (thiazole and phenyl rings) sit in the minor groove and interact with the flanking base pairs adjacent to the intercalation site., Conclusions: The DNA binding characteristics of SIMR1281, which can disrupt/inhibit DNA function as confirmed by the topoisomerases' inhibition assays, indicate that the observed multi-target activity might originate from ligand intervention at nucleic acids level rather than due to direct interactions with multiple biological targets at the protein level., General Significance: The findings of this study could be helpful to guide future optimization of benzopyrane-based ligands for therapeutic purposes., Competing Interests: Declaration of Competing Interest The authors declare competing financial interest since part of this article is in the patent application (T. H. Al-Tel, Raafat A. El-Awady, Srinivasulu Vunnam, Cijo G. Vazhapily, Hany A. Omar, Novel heterocyclic systems and pharmaceutical compositions thereof. US Patent No US20190292204A1)., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Objective . The purpose of this investigation is to establish the water calorimeter as the primary standard in PTB's ultra-high pulse dose rate (UHPDR) 20 MeV reference electron beams. Approach . The calorimetric measurements were performed at the PTB research linac facility using the UHPDR reference electron beam setups that enable a dose per pulse between about 0.1 Gy and 6 Gy. The beam is monitored by an in-flange integrating current transformer. The correction factors required to determine the absorbed dose to water were evaluated using thermal and Monte Carlo simulations. Measurements were performed with different total doses delivered per pulse by modifying the instantaneous dose rate within a pulse and by changing the pulse length. The obtained temperature-time traces were compared to the simulated ones to validate the thermal simulations. In addition, absorbed-dose-to-water measurements obtained using the secondary standard alanine dosimeter system were compared to measurements performed with the primary standard. Main results . The simulated and measured temperature-time traces were shown to be consistent, within combined uncertainties, with one another. Measurements with alanine dosimeters proved to be consistent within k = 1 of the total combined uncertainty with the absorbed dose to water determined using the primary standard. Significance . The total relative standard uncertainty of absorbed dose to water determined using the PTB water calorimeter primary standard in UHPDR electron beams was estimated to be less than 0.5%, and the combined correction factors were found to deviate from 1 by less than 1% for both PTB UHPDR 20 MeV reference electron beams. The water calorimeter is therefore considered to be an established primary standard for the higher energy UHPDR reference electron beams., (Creative Commons Attribution license.)

Human serum alpha-1 acid glycoprotein is an acute-phase plasma protein involved in the binding and transport of many drugs, especially basic and lipophilic substances. It has been reported that the sialic acid groups that terminate the N-glycan chains of alpha-1 acid glycoprotein change in response to certain health conditions and may have a major impact on drug binding to alpha-1 acid glycoprotein. The interaction between native or desialylated alpha-1 acid glycoprotein and four representative drugs-clindamycin, diltiazem, lidocaine, and warfarin-was quantitatively evaluated using isothermal titration calorimetry. The calorimetry assay used here is a convenient and widely used approach to directly measure the amount of heat released or absorbed during the association processes of biomolecules in solution and to quantitatively estimate the thermodynamics of the interaction. The results showed that the binding of drugs with alpha-1 acid glycoprotein were enthalpy-driven exothermic interactions, and the binding affinity was in the range of 10 -5 -10 -6 M. Desialylated alpha-1 acid glycoprotein showed significantly different binding with diltiazem, lidocaine, and warfarin compared with native alpha-1 acid glycoprotein, whereas clindamycin showed no significant difference. Therefore, a different degree of sialylation may result in different binding affinities, and the clinical significance of changes in sialylation or glycosylation of alpha-1 acid glycoprotein in general should not be neglected.

Direct FXa inhibitors are an important class of bioactive molecules (rivaroxaban, apixaban, edoxaban, and betrixaban) applied for thromboprophylaxis in diverse cardiovascular pathologies. The interaction of active compounds with human serum albumin (HSA), the most abundant protein in blood plasma, is a key research area and provides crucial information about drugs' pharmacokinetics and pharmacodynamic properties. This research focuses on the study of the interactions between HSA and four commercially available direct oral FXa inhibitors, applying methodologies including steady-state and time-resolved fluorescence, isothermal titration calorimetry (ITC), and molecular dynamics. The HSA complexation of FXa inhibitors was found to occur via static quenching, and the complex formation in the ground states affects the fluorescence of HSA, with a moderate binding constant of 10 4 M -1 . However, the ITC studies reported significantly different binding constants (10 3 M -1 ) compared with the results obtained through spectrophotometric methods. The suspected binding mode is supported by molecular dynamics simulations, where the predominant interactions were hydrogen bonds and hydrophobic interactions (mainly π-π stacking interactions between the phenyl ring of FXa inhibitors and the indole moiety of Trp214). Finally, the possible implications of the obtained results regarding pathologies such as hypoalbuminemia are briefly discussed.

Isothermal microcalorimetry can provide a general analytical tool for the characterization of bacterial growth. Methodologies and equipment have been studied to expand the application and disseminate the use of the technique. The MCDSC is a microcalorimeter capable of measuring in the range of 0.2 μW that can operate at a temperature range of -20 to 140 °C or under isothermal conditions. Here, we present the first investigation of MCDSC for E. coli growth with the Baranyi and Roberts modeling application. This study presented the calorimetric E. coli fingerprint at MCDSC and compares it with the plate count technique, giving the data more biological meaning. The calorimeter was able to accurately detect growth metabolism and discriminate E. coli at different inoculum densities. Additionally, the MCDSC can offer a new point of view for evaluating microbial growth, such as the significant reduction in error due to dispersed data by the viable counting method., Competing Interests: Declaration of Competing Interest None., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Interactions between hyaluronan and the antimicrobial peptide cecropin B were studied in water and PBS using high-resolution ultrasonic spectroscopy and isothermal titration calorimetry. Although each technique is fundamentally different, they both gave identical results. It was found that the molecular weight of hyaluronan plays an important role in the interactions - in particular, the transition between the rod conformation and the random coil conformation. In water, interactions were saturated in a molar charge ratio of 1.5 and not 1.0 as expected. The later saturation of the interaction probably occurred either for steric reasons or due to the interaction between functional groups in the cecropin structure, which allowed complete dissociation of the antimicrobial peptide. In PBS, in contrast to water, no interactions were observed, irrespective of the molecular weight of hyaluronan. Thus, at a sufficiently high ionic strength, the interactions were suppressed., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Staphylococcus aureus protein A (SpA) is an IgG Fc-binding virulence factor that is widely used in antibody purification and as a scaffold to develop affinity molecules. A cyclized SpA Z domain could offer exopeptidase resistance, reduced chromatographic ligand leaching after single-site endopeptidase cleavage, and enhanced IgG binding properties by preorganization, potentially reducing conformational entropy loss upon binding. In this work, a Z domain trimer (Z3) was cyclized using protein intein splicing. Interactions of cyclic and linear Z3 with human IgG 1 were characterized by differential scanning fluorimetry (DSF), surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC). DSF showed a 5 ℃ increase in IgG 1 melting temperature when bound by each Z3 variant. SPR showed the dissociation constants of linear and cyclized Z3 with IgG 1 to be 2.9 nM and 3.3 nM, respectively. ITC gave association enthalpies for linear and cyclic Z3 with IgG 1 of -33.0 kcal/mol and -32.7 kcal/mol, and -T∆S of association 21.2 kcal/mol and 21.6 kcal/mol, respectively. The compact cyclic Z3 protein contains 2 functional binding sites and exhibits carboxypeptidase Y-resistance. The results suggest cyclization as a potential approach toward more stable SpA-based affinity ligands, and this analysis may advance our understanding of protein engineering for ligand and drug development.

Isothermal titration calorimetry (ITC) is a powerful biophysical tool to characterize energetic profiles of biomacromolecular interactions without any alteration of the underlying chemical structures. In this protocol, we describe procedures for performing, analyzing, and interpreting ITC data obtained from a cooperative riboswitch-ligand interaction., (© 2023. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

Isothermal titration calorimetry (ITC) is the gold standard for providing quantitative and thermodynamic understanding of the interaction mechanisms between core autophagy machinery, autophagy receptors, and ATG8. Here, we used two model peptides and Arabidopsis thaliana ATG8A to characterize ATG8-peptide interactions. We employed ITC using three different methods (direct ligand titration, displacement, and competition assays) to characterize, directly and indirectly, the interaction of the peptides with ATG8. We then analyzed the ITC data by global and statistical methods and discussed advantages, drawbacks, and negative controls for each approach. We finally provide a thorough description of all the steps, including data analysis and presentation, preparation of recombinant ATG8A from E. coli, and troubleshooting notes for technical problems that can be encountered. Although we used ATG8-peptide interactions here, these assays can be applied to any other one-to-one protein-protein and ligand-protein interactions and competitive binders., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The reported inconsistencies between the van't Hoff equation and calorimetry hinder the utility of thermodynamics in biochemical and pharmaceutical research. A novel thermodynamic approach is developed herein for ligand adsorption with a focus on the interpretation of calorimetric data in the presence of concurrent proton exchange reactions. Such exchange reactions typically result in a pH-dependence of calorimetric measurements that obscures intrinsic binding enthalpies. It is shown that for the adsorption of phenobarbital to activated carbon, the measured calorimetric enthalpy is a result of three linked acid/base equilibria. A model was established to predict the intrinsic binding enthalpy using 1) the adsorbate's pKa and 2) the adsorbate's enthalpy of protonation. The observed calorimetric enthalpy of binding exhibited both pH and buffer-dependence and was between -5 and -42 kJ/mol. Meanwhile, the predicted intrinsic enthalpy (-25.1 kJ/mol) of binding was in excellent agreement with the measured intrinsic enthalpy (-25.6 kJ/mol). Corrections to the observed calorimetric enthalpies allowed comparisons with enthalpies obtained from the van't Hoff method. It is shown that the predicted intrinsic calorimetric enthalpy agrees well with the van't Hoff enthalpies in instances where observed enthalpies significantly deviated. This treatment is general and is not specific to phenobarbital or activated carbon., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.)

A new standardized method, using isothermal microcalorimetry (IMC), was established to determine thermokinetic parameters from heat flow curves and to demonstrate the reproducibility and repeatability of the parameters of five Colletotrichum species on different days. Measurements on IMC were made at different periods and by two operators. Repeatability and reproducibility (R&R) measurement system analysis was performed on the technique used to measure the heat flow of Colletotrichum strains. The results showed that the %GageR&R was found to be within the acceptable ranges of a measurement system. Also, the parameters obtained from the curves were subjected to a combination of Principal Component Analysis (PCA) and Clustering, the data showed that the total heat (H t ) and maximum growth rate (μ max ) are probably the most specific distinguishing characteristic of the strains evaluated in this study. This study demonstrates, for the first time, the usefulness of IMC in obtaining heat flow curves and thermokinetic parameters, providing repeatable and reproducible measurements over a period and under controlled conditions, for future identifications of phytopathogenic fungi., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Isothermal titration calorimetry (ITC) is a technique where the heat given off, or absorbed, during a binding event is measured and used to determine the binding thermodynamics and affinity associated with binding. This protocol focuses on ITC applications for studying aptamer interactions with small molecule ligands where ITC has the advantage of being a label-free solution-based technique. The limitation of ITC using a relatively large amount of material compared to other analytical techniques is not applicable here as large amounts of nucleic acids, especially DNA, can be readily obtained. In this chapter we describe how to use ITC methods to measure the thermodynamics and affinity of binding using the interaction of quinine with a DNA cocaine-binding aptamer as an example., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Manipulations of nanocrystal (NC) surfaces have propelled the applications of colloidal NCs across various fields such as bioimaging, catalysis, electronics, and sensing applications. In this Feature Article, we discuss the surface chemistry of colloidal NCs, with an emphasis on semiconductor quantum dots, and the binding motifs for various ligands that coordinate NC surfaces. We present isothermal titration calorimetry (ITC) as a viable technique for studying the thermodynamics of the ligand association and exchange at NC surfaces by discussing its principles of operation and highlighting results obtained to date. We give an in-depth description of various thermodynamic models that can be used to interpret NC-ligand interactions as measured not only by ITC, but also by NMR, fluorescence quenching, and fluorescence anisotropy techniques. Understanding the complexity of NC surface-ligand interactions can provide a wide range of avenues to tune their properties for desired applications.

In recent years, thermophoresis has emerged as a promising tool for quantifying biomolecular interactions. The underlying microscopic physical effect is still not understood, but often attributed to changes in the hydration layer once the binding occurs. To gain deeper insight, we investigate whether non-equilibrium coefficients can be related to equilibrium properties. Therefore, we compare thermophoretic data measured by thermal diffusion forced Rayleigh scattering (TDFRS) (which is a non-equilibrium process) with thermodynamic data obtained by isothermal titration calorimetry (ITC) (which is an equilibrium process). As a reference system, we studied the chelation reaction between ethylenediaminetetraacetic acid (EDTA) and calcium chloride (CaCl2) to relate the thermophoretic behavior quantified by the Soret coefficient ST to the Gibb's free energy ΔG determined in the ITC experiment using an expression proposed by Eastman. Finally, we have studied the binding of the protein Bovine Carbonic Anhydrase I (BCA I) to two different benzenesulfonamide derivatives: 4-fluorobenzenesulfonamide (4FBS) and pentafluorobenzenesulfonamide (PFBS). For all three systems, we find that the Gibb's free energies calculated from ST agree with ΔG from the ITC experiment. In addition, we also investigate the influence of fluorescent labeling, which allows measurements in a thermophoretic microfluidic cell. Re-examination of the fluorescently labeled system using ITC showed a strong influence of the dye on the binding behavior.

The in vivo potency of polyphosphazene immunoadjuvants is inherently linked to the ability of these ionic macromolecules to assemble with antigenic proteins in aqueous solutions and form physiologically stable supramolecular complexes. Therefore, in-depth knowledge of interactions in this biologically relevant system is a prerequisite for a better understanding of mechanism of immunoadjuvant activity. Present study explores a self-assembly of polyphosphazene immunoadjuvant-PCPP and a model antigen-lysozyme in a physiologically relevant environment-saline solution and neutral pH. Three analytical techniques were employed to characterize reaction thermodynamics, water-solute structural organization, and supramolecular dimensions: isothermal titration calorimetry (ITC), water proton nuclear magnetic resonance ( w NMR), and dynamic light scattering (DLS). The formation of lysozyme-PCPP complexes at near physiological conditions was detected by all methods and the avidity was modulated by a physical state and dimensions of the assemblies. Thermodynamic analysis revealed the dissociation constant in micromolar range and the dominance of enthalpy factor in interactions, which is in line with previously suggested model of protein charge anisotropy and small persistence length of the polymer favoring the formation of high affinity complexes. The paper reports advantageous use of w NMR method for studying protein-polymer interactions, especially for low protein-load complexes.

Purpose: The purpose of this investigation is to evaluate the use of a probe-format graphite calorimeter, Aerrow, as an absolute and relative dosimeter of high-energy pulse dose rate (UHPDR) electron beams for in-water reference and depth-dose-type measurements, respectively., Methods: In this paper, the calorimeter system is used to investigate the potential influence of dose per pulses delivered up to 5.6 Gy, the number of pulses delivered per measurement, and its potential for relative measurement (depth-dose curve measurement). The calorimeter system is directly compared against an Advanced Markus ion chamber. The finite element method was used to calculate heat transfer corrections along the percentage depth dose of a 20-MeV electron beam. Monte Carlo-calculated dose conversion factors necessary to calculate absorbed dose-to-water at a point from the measured dose-to-graphite are also presented., Results: The comparison of Aerrow against a fully calibrated Advanced Markus chamber, corrected for the saturation effect, has shown consistent results in terms of dose-to-water determination. The measured reference depth is within 0.5 mm from the expected value from Monte Carlo simulation. The relative standard uncertainty estimated for Aerrow was 1.06%, which is larger compared to alanine dosimetry (McEwen et al. https://doi.org/10.1088/0026-1394/52/2/272) but has the advantage of being a real-time detector., Conclusion: In this investigation, it was demonstrated that the Aerrow probe-type graphite calorimeter can be used for relative and absolute dosimetries in water in an UHPDR electron beam. To the author's knowledge, this is the first reported use of an absorbed dose calorimeter for an in-water percentage depth-dose curve measurement. The use of the Aerrow in quasi-adiabatic mode has greatly simplified the signal readout, compared to isothermal mode, as the resistance was directly measured with a high-stability digital multimeter., (© 2022 Physikalisch-Technische Bundesanstalt. Medical Physics published by Wiley Periodicals LLC on behalf of American Association of Physicists in Medicine.)

Proteins immobilized in metal-organic frameworks (MOFs) often show extraordinary stability. However, most efforts to immobilize proteins in MOFs have only been exploratory. Herein, we present the first systematic study on the thermodynamics of protein immobilization in MOFs. Using insulin as a probe, we leveraged isothermal titration calorimetry (ITC) to investigate how topology, pore size, and hydrophobicity of MOFs influence immobilization. ITC data obtained from the encapsulation of insulin in a series of Zr-MOFs reveals that MOFs provide proteins with a hydrophobic stabilizing microenvironment, making the encapsulation entropically driven. In particular, the pyrene-based NU-1000 tightly encapsulates insulin in its ideally sized mesopores and stabilizes insulin through π-π stacking interactions, resulting in the most enthalpically favored encapsulation process among this series. This study reveals critical insights into the structure-property relationships of protein immobilization., (© 2022 Wiley-VCH GmbH.)