Coamorphous Systems of Valsartan: Thermal Analysis Contribution to Evaluate Intermolecular Interactions Effects on the Structural Relaxation.

Coamorphous formation in binary systems of valsartan (Val) with 4,4'-bipyridine (Bipy) and trimethoprim (Tri) was investigated for mixtures with a mole fraction of 0.16~0.86 of valsartan and evaluated in terms of the glass transition temperature. The glass transition of the systems had a behavior outside the values predicted by the Gordon-Taylor equation, showing that Val-Bipy (hydrogen bonding between the components) had a lower deviation and Val-Tri (ionic bonding between the components) had a higher deviation. Mixtures of compositions 2:1 Val-Bipy and 1:1 Val-Tri were selected for further investigation and verified to be stable, as no crystallization was observed during subsequent heating and cooling programs. For these systems, the effective activation energy during glass transition was evaluated. Compared to pure valsartan, the system with the lower glass transition temperature (Val-Bipy) presented the highest effective activation energy, and the system with the higher glass transition temperature (Val-Tri) presented a lower effective activation energy. The results presented a good correlation between the data obtained from two different techniques to determine the fragility and effective activation energy: non-isothermal kinetic analysis by DSC and TSDC.

Amorphous Solid Forms of Ranolazine and Tryptophan and Their Relaxation to Metastable Polymorphs.

Different methods were explored for the amorphization of ranolazine, a sparingly soluble anti-anginal drug, such as mechanochemistry, quench-cooling, and solvent evaporation from solutions. Amorphous phases, with Tg values lower than room temperature, were obtained by cryo-milling and quench-cooling. New forms of ranolazine, named II and III, were identified from the relaxation of the ranolazine amorphous phase produced by cryo-milling, which takes place within several hours after grinding. At room temperature, these metastable polymorphs relax to the lower energy polymorph I, whose crystal structure was solved in this work for the first time. A binary co-amorphous mixture of ranolazine and tryptophan was produced, with three important advantages: higher glass transition temperature, increased kinetic stability preventing relaxation of the amorphous to crystalline phases for at least two months, and improved aqueous solubility. Concomitantly, the thermal behavior of amorphous tryptophan obtained by cryo-milling was studied by DSC. Depending on experimental conditions, it was possible to observe relaxation directly to the lower energy form or by an intermediate metastable crystalline phase and the serendipitous production of the neutral form of this amino acid in the pure solid phase.

Introduction to Pharmaceutical Co-amorphous Systems Using a Green Co-milling Technique.

The concept of co-amorphous systems is introduced in an integrated laboratory experiment, designed for advanced chemistry students, using solvent-free, environmentally friendly mechanochemistry. The dual-drug naproxen-cimetidine co-amorphous system (NPX-CIM) is investigated as an example of the emergent field of medicinal mechanochemistry. Students are trained in solid-state characterization techniques including X-ray powder diffraction, Fourier-transform infrared spectroscopy, and thermal analysis by differential scanning calorimetry. This lab experiment also provides an opportunity to discuss the relevance of different solid forms of pharmaceutics, emphasizing particular properties of disordered materials. This experiment can easily fit the curriculum of any Chemistry or Pharmacy master level degree in courses dealing with instrumental analysis, solid state chemistry, or green chemistry, for classes of 6 to 18 students, in a 5-h lab session. Suggestions to adapt it to the use of a single characterization technique are provided.

Norfloxacin Cocrystals: Mechanochemical Synthesis and Scale-up Viability Through Solubility Studies.

Cocrystals are recognized as one of the most efficient approaches to improve aqueous solubility of Biopharmaceutical Classification System, BCS, classes II and IV drugs. Cocrystal discovery and the establishment of experimental conditions suitable for scale-up purposes are some of the main challenges in cocrystal investigation. In this work, the investigation of mechanochemical synthesis of norfloxacin cocrystals with picolinic and isonicotinic acids is performed, leading to the discovery of two new cocrystals of this important BCS class IV antibiotic, which were characterized through thermal, spectral and diffractometric analysis. Norfloxacin apparent aqueous solubility using the cocrystals is also presented, with higher values being obtained for all the investigated systems when compared to the pure drug. Norfloxacin has 3 polymorphs and several solvents/hydrates, which represents a challenge for obtaining pure cocrystal forms from solvent crystallization. This challenge was successfully overcome in this work, as experimental conditions to obtain the pure cocrystals (the new ones and also norfloxacin-nicotinic acid and norfloxacin-saccharin) were established using Crystal16 equipment. This is a crucial step to envisage future scale-up procedures and therefore a valuable information for the pharmaceutical industry.

Psychological reactance and adolescent cannabis use: The role of parental warmth and monitoring.

Psychological reactance (PR) is a psychological state or trait typified by resistant responses to threats to behavioral freedom. PR has been linked with negative health behaviors, including risky substance use; however, factors that may foster approaches to mitigate the impact of PR on these behaviors, as well as rejection of other health promotion communications is less understood. The current studies examined relations between parental warmth and monitoring with trait PR and responses to preventive cannabis communications and usage intentions. Two in-school surveys were administered to two difference samples of middle school students (Study 1, N = 1,416; Study 2, N = 1,118). Path analytic models tested multivariable linkages among relevant parenting variables, PR, and outcomes associated with cannabis use. Follow-up regression analyses explored significant interaction effects. In Study 1 (p <0.001) and Study 2 (p <0.01), parental warmth moderated the relation between monitoring and trait PR: High monitoring was a protective factor only when combined with high warmth. In turn, PR mediated the relationships between parenting practices and cannabis intentions in both studies (p <0.001). In Study 2, PR also was linked with resistance to persuasion via more unfavorable reactions to anti-cannabis appeals (p <0.001). Findings indicated that low parental warmth combined with high parental monitoring was associated with high trait reactance in adolescents, which predisposed them to stronger resistance to preventive communications. Interventions might focus on counseling parents about the likely outcomes of parenting style, and ways to implement beneficial approaches.

Viability of ex-vivo myography as a diagnostic tool for rectus abdominis muscle electrical activity collected at Cesarean section within a diamater cohort study.

BACKGROUND: Ex-vivo myography enables the assessment of muscle electrical activity response. This study explored the viability of determining the physiological responses in muscles without tendon, as rectus abdominis muscle (RAM), through ex-vivo myography to assess its potential as a diagnostic tool. RESULTS: All tested RAM samples (five different samples) show patterns of electrical activity. A positive response was observed in 100% of the programmed stimulation. RAM 3 showed greater weight (0.47 g), length (1.66 cm), and width (0.77 cm) compared to RAM 1, RAM 2, RAM 4 and RAM 5 with more sustained electrical activity over time, a higher percentage of fatigue was analyzed at half the time of the electrical activity. The order of electrical activity (Mn) was RAM 3 > RAM 5 > RAM 1 > RAM 4 > RAM 2. No electrical activity was recorded in the Sham group. CONCLUSIONS: This study shows that it is feasible to assess the physiological responses of striated muscle without tendon as RAM, obtained at C-section, under ex vivo myography. These results could be recorded, properly analyzed, and demonstrated its potential as a diagnostic tool for rectus abdominis muscle electrical activity.

Pelvic floor muscle dysfunction at 3D transperineal ultrasound in maternal exposure to gestational diabetes mellitus: A prospective cohort study during pregnancy.

AIM: This study aimed to assess, for the first time, the dynamic morphometry of pelvic floor muscles (PFM) using three-dimensional transperineal ultrasound (3D-TPUS) and its progression at two-time points of gestation between women with and without gestational diabetes mellitus (GDM), and whether the PFM dysfunction is connected to GDM. METHODS: The study comprised 83 consecutive pregnant women with (n = 38) and without (n = 45) GDM screened at 24-30 and 38-40 weeks of gestation. 3D-TPUS and a mobility test were used to quantify PFM dynamic morphometry during maximum contraction and the Valsalva maneuver. RESULTS: When compared to the control group, GDM women had no significant variations in all levator hiatal dimensions at 24-30 weeks of gestation. Meanwhile, women with GDM experienced an increase in levator hiatal area (LHa) (p < 0.000) during PFM contraction and enlargement in LHa (p < 0.001) during Valsalva maneuver (p = 0.010) at 38-40 weeks of gestation. As a result, the mobility index among GDM women had a lower value (p = 0.000). The dynamic morphometry development of PFM in GDM women at two stages during pregnancy revealed a substantial decrease (p = 0.000) in all LHa dimensions of contraction, distension, and mobility. CONCLUSIONS: Using 3D-TPUS, we found that GDM women had a specific pattern of PFM functional changes in the third trimester of pregnancy. These initial findings revealed alterations in PFM functionality, such as decreased contractility, distensibility, or mobility. This dysfunctional PFM could contribute to the long-term development of pelvic floor dysfunction years after a GDM pregnancy.

Dihydrofolate Reductase Inhibitors: The Pharmacophore as a Guide for Co-Crystal Screening.

In this work, co-crystal screening was carried out for two important dihydrofolate reductase (DHFR) inhibitors, trimethoprim (TMP) and pyrimethamine (PMA), and for 2,4-diaminopyrimidine (DAP), which is the pharmacophore of these active pharmaceutical ingredients (API). The isomeric pyridinecarboxamides and two xanthines, theophylline (THEO) and caffeine (CAF), were used as co-formers in the same experimental conditions, in order to evaluate the potential for the pharmacophore to be used as a guide in the screening process. In silico co-crystal screening was carried out using BIOVIA COSMOquick and experimental screening was performed by mechanochemistry and supported by (solid + liquid) binary phase diagrams, infrared spectroscopy (FTIR) and X-ray powder diffraction (XRPD). The in silico prediction of low propensities for DAP, TMP and PMA to co-crystallize with pyridinecarboxamides was confirmed: a successful outcome was only observed for DAP + nicotinamide. Successful synthesis of multicomponent solid forms was achieved for all three target molecules with theophylline, with DAP co-crystals revealing a greater variety of stoichiometries. The crystalline structures of a (1:2) TMP:THEO co-crystal and of a (1:2:1) DAP:THEO:ethyl acetate solvate were solved. This work demonstrated the possible use of the pharmacophore of DHFR inhibitors as a guide for co-crystal screening, recognizing some similar trends in the outcome of association in the solid state and in the molecular aggregation in the co-crystals, characterized by the same supramolecular synthons.

Organ donation and departments of motor vehicles: Multiple messages, implementations, and replications.

OBJECTIVE: To reduce uncertainty about the effectiveness of Departments of Motor Vehicles (DMV) interventions, we assessed the influence of several DMV-based media campaigns on organ donor registration. METHOD: Across 4 studies, campaigns were implemented in 8 (Studies 1-3) or 9 (Study 4) New Mexico DMVs. Three theories guided message development (i.e., efficacy, altruism, vested interest). Donor registration was the outcome measure. RESULTS: In Study 1, both the efficacy (OR = 1.14) and altruism (OR = 1.11) campaigns resulted in significantly more registrations than the control condition and the vested interest campaign. In Study 2, the efficacy campaign (OR = 1.13) again resulted in significantly more registrations than the control and vested interest campaign. In Study 3, a new version of the efficacy campaign (OR = 1.21) and 2 new versions of the vested interest campaigns (OR = 1.12; 1.14) all resulted in significantly more registrations than the control condition. In Study 4, neither the efficacy campaign (OR = 1.06) nor a campaign that combined efficacy and vested interest materials (OR = 1.03) significantly increased registrations. A single-paper meta-analysis assessing these studies indicated all 3 appeals resulted in significantly greater registrations than the control (OR ranged from 1.06 to 1.13). CONCLUSION: Results indicate donor registration rates can be increased through DMV-based media campaigns; however, the appeal used, and the implementation of that appeal, can determine the likelihood of success. These results can guide the development and implementation of campaigns seeking to influence donor registration and other prosocial behaviors. (PsycInfo Database Record (c) 2021 APA, all rights reserved).

Dynamics and proliferative capacities of CD8+CD28+TCRαß+CD62Lhigh T-cell subsets in healthy and asthmatic subjects.

Adhesion molecules, as such, play essential roles in T-cell transendothelial extravasation during inflammation. A better understanding of the mechanisms underlying this process may be of value in the management of asthma. The present study employed Magnetic-Activated Cell Sorting (MACS) to isolate human CD8+ T lymphocytes from peripheral blood of asthma patients and controls. The cells were flow cytometrically assessed to evaluate surface expression of an adhesion molecule, L-selectin (CD62L) on the surface of CD8FoxP3-/bright T cell subsets and its response to inflammatory cytokines. We showed that CD8+CD28+TCRαß+CD62LhighFoxP3bright T cells were deficient in blood of some asthma patients but abundant in others. After co-stimulation of CD8+ T cells with anti-CD3/CD28 in combination with IL-2 and IL-10 or TGF-ß, the frequencies of CD8+CD28+TCRα/ß+CD62Lhigh T cells in the group of patients were lower than at baseline. Our data indicate that L-selectin expression is regulated by inflammatory cytokines. Overall, these data reveal that asthma phenotypes may be further stratified into micro subtypes with distinct cellular and molecular characteristics, supporting the concept of asthma endotypes.

A Rebuttal-Based Social Norms-Tailored Cannabis Intervention for At-Risk Adolescents.

Many past cannabis prevention campaigns have proven largely ineffective due in part to the diversity of adolescents' cannabis-relevant beliefs. The current studies evaluated the impact of a sequential multiple message approach tailored to the usage norms of adolescents expressing negative attitudes toward a cannabis prevention appeal. A multiple-message strategy was implemented-initial unfavorable message evaluations were invalidated using attitudinal rebuttal feedback prior to presenting a third tailored communication. Participants were cannabis-abstinent middle and high school students (ages 11 to 16). Study 1 (N = 808) compared effects of gain- and loss-framed messages tailored to each student's normative usage perceptions. In Study 2 (N = 391), students were randomly assigned to receive a tailored or non-tailored message after receiving feedback meant to destabilize anti-message attitudes. For at-risk adolescents in Study 1 who perceived cannabis use as normative, a tailored gain-framed message resulted in the lowest usage intentions (p < .05). In Study 2, a conditional multiple-moderated mediation model showed that for high-risk teens with normative beliefs and pro-cannabis attitudes, exposure to a tailored gain-framed communication was associated with decreased cannabis attitude certainty, and lower usage intentions 2 months later (p < .05). Findings have implications for sequential messaging utilization in mass media campaigns and support the efficacy of tailored messages over a one-size-fits-all media approach. Further, results suggest that systematically weakening resistance to persuasive communications and tailoring messages consistent with individually perceived peer norms is an effective prevention strategy.

It's all relative: Increasing organ donor registration intentions by maximizing family-relevant vested interest.

Guided by vested interest theory, the impact of educating potential organ donors about the beneficial experiences afforded to families of deceased donors was assessed. Participants were informed about these benefits by taking a survey that asked them to indicate both their awareness, and the appeal, of numerous existing benefits (e.g. grief services). We employed a double-sided mixed design. Both the true experimental design and the quasi-experimental repeated assessment indicated increased registration intentions. Only the quasi-experimental design indicated significant attitude change. This study provides evidence supporting the potential utility of focusing interventions on the benefits afforded to donor families.

Above the influence of vaping: Peer leader influence and diffusion of a network-informed preventive intervention.

Using social networks to inform prevention efforts is promising but has not been applied to vaping. To address this gap, we pilot tested the peer-led Above the Influence of Vaping (ATI-V) and examined diffusion through 8th grade networks in three schools. Fifty students, nominated and trained as Peer Leaders, implemented prevention campaigns informed by communication science, including gain-loss messaging and social norming. Across schools, 86-91% of students (N = 377) completed measures (pre-post) of electronic vaping product (EVP) use and attitudes, and named close friends and adults to construct social networks. Using baseline reports, we classified students as Recent EVP Users (10%), Vulnerable Nonusers (24%), or Resolute Nonusers (66%). Peer Leaders had reach through friendship connections to students at varying risk of vaping; 12-16 weeks after Peer Leaders were trained and began implementing campaigns, 79% of Resolute Nonusers and 74% of Recent Users/Vulnerable Nonusers reported exposure to a vaping prevention message. Students with more Peer Leader friends were less likely to report recent EVP use (OR = 0.41) or intention to use an EVP (B = 0.12) on post-surveys, supporting the intervention conceptual model positing diffusion through friendship networks. Use of student-nominated peer leaders was supported by network analyses showing EVP Users integrated within the friendship network, having more high-risk friends, and fewer adult connections. This evidence is the first to show that adolescent Peer Leaders with ongoing mentoring and science-informed campaigns can potentially reduce EVP acceptability and use. Areas for refining ATI-V include increasing consistency of campaign exposure across schools.

Impact of gestational diabetes on pelvic floor: A prospective cohort study with three-dimensional ultrasound during two-time points in pregnancy.

AIM: To evaluate the pelvic floor (PF) biometry using three-dimensional ultrasound (US) at two-time points of gestational in pregnant women with gestational diabetes mellitus (GDM). METHODS: A prospective cohort study conducted at the Perinatal Diabetes Research Center including 44 pregnant women with GDM and 66 pregnant women without GDM at 24 to 28 weeks of gestation. Three-dimensional transperineal US was performed at 24 to 28 and 34 to 38 weeks of gestation in the lithotomy position at rest. The axial plane of the minimal Levator hiatal dimensions was used to determine Levator ani muscle and Hiatal area (HA) biometry at 24 to 28 and 34 to 38 weeks of gestation. RESULTS: Of the 110 pregnant women, 100 (90.9%) completed the follow-up at 34 to 38 weeks of gestation. The evaluation by US showed a negative biometric change between the two-time points, during pregnancy in women with GDM; in the HA (ß coefficient: estimative of effect in biometric progression according to GDM diagnosis, using the non-GDM group as reference = -6.76; P = .020), anteroposterior diameter (ß = -5.07; P = .019), and Levator ani thickness (ß = -12.34; P = .005). CONCLUSIONS: Pregnant women with GDM had a significantly lower than expected percentage of changes in biometry of Levator ani thickness and HA from 24 to 28 to 34 to 38 weeks of gestation when compared with the group of pregnant women without GDM. GDM alters the morphology of PF structures assessed by three-dimension US. This reported complication may be implicated in adverse birth outcomes and may play a role in the development of PF dysfunction.

Thiabendazole and Thiabendazole-Formic Acid Solvate: A Computational, Crystallographic, Spectroscopic and Thermal Study.

Thiabendazole (TBZ) is a substance which has been receiving multiple important applications in several domains, from medicine and pharmaceutical sciences, to agriculture and food industry. Here, a comprehensive multi-technique investigation on the molecular and crystal properties of TBZ is reported. In addition, a new solvate of the compound is described and characterized structurally, vibrationally and thermochemically for the first time. Density functional theory (DFT) calculations were used to investigate the conformational space of thiabendazole (TBZ), revealing the existence of two conformers, the most stable planar trans form and a double-degenerated-by-symmetry gauche form, which is ~30 kJ mol-1 higher in energy than the trans conformer. The intramolecular interactions playing the major roles in determining the structure of the TBZ molecule and its conformational preferences were characterized. The UV-visible and infrared spectra of the isolated molecule (most stable trans conformer) were also calculated, and their assignment undertaken. The information obtained for the isolated molecule provided a strong basis for the understanding of the intermolecular interactions and properties of the crystalline compound. In particular, the infrared spectrum for the isolated molecule was compared with that of crystalline TBZ and the differences between the two spectra were interpreted in terms of the major intermolecular interactions existing in the solid state. The analysis of the infrared spectral data was complemented with vibrational results of up-to-date fully-periodic DFT calculations and Raman spectroscopic studies. The thermal behavior of TBZ was also investigated using differential scanning calorimetry (DSC) and thermogravimetry. Furthermore, a new TBZ-formic acid solvate [2-(1,3-thiazol-4-yl)benzimidazolium formate formic acid solvate] was synthesized and its crystal structure determined by X-ray diffraction. The Hirshfeld method was used to explore the intermolecular interactions in the crystal of the new TBZ solvate, comparing them with those present in the neat TBZ crystal. Raman spectroscopy and DSC studies were also carried out on the solvate to further characterize this species and investigate its temperature-induced desolvation.

Conformational Landscape and Polymorphism in 5-Acetic Acid Hydantoin.

The conformational space of 5-acetic acid hydantoin {5AAH; [2-(2,5-dioxoimidazolidin-4-yl)acetic acid]} was investigated by quantum chemical calculations performed at the DFT(B3LYP)/6-311++G(d,p) level of theory. A total of 13 conformers were located in the potential energy surface of the molecule, six of them bearing the carboxylic group in the cis arrangement (O═C-O-H dihedral equal to ∼0°) and the other seven possessing this group in the trans configuration (O═C-O-H dihedral equal to ∼180°). The most stable conformer (cis-I) was trapped from the gas phase into a low temperature argon matrix (10 K), and its infrared spectrum was fully assigned, also with help of results of normal coordinates' analysis based on the DFT computed vibrational data. The electronic structure of this conformer was analyzed by using the natural bond orbital (NBO) method. The investigation of the thermal properties of 5AAH was undertaken by differential scanning calorimetry (DSC), polarized light thermal microscopy (PLTM) and Raman spectroscopy, allowing identification of five different polymorphs. Very interestingly, in the room temperature stable polymorph the molecular units of 5AAH assume the geometry of the highest-energy conformer predicted by the calculations for the isolated molecule.

Free volume in the smectic-E phase of 4-hexyl-4' isothiocyanatobiphenyl studied by positron annihilation spectroscopy.

Positron annihilation lifetime spectroscopy has been used to study 4-hexyl-4'-isothiocyanatobiphenyl. Changes of the orthopositronium lifetime parameters with temperature have been observed for the supercooled smectic-E phase. The measurements confirm that positronium is created and annihilates in a layer of a lower electron density containing alkyl chains of molecules. The two-state bond-lattice model of glass transition explains the thermal activation of the centers where orthopositronium is created and annihilates when the glass of the smectic-E phase softens. However, the subsequent cold crystallization of the softened regions also influences the orthopositronium lifetime and intensity, which complicates the picture seen by positrons. The measurements during isothermal crystallization suggest that it progresses in two stages. The first stage can be described by the Avrami equation with the Avrami exponent close to unity, which indicates low-dimensional crystallization. Similarly to liquid n alkanes, the application of pressure is equivalent to temperature lowering with the similar equivalence relationship between pressure and temperature, which seems to confirm the structure of the smectic-E phase with sublayers containing alkyl chains in a molten state. The dependence of the orthopositronium lifetime on pressure for the smectic-E phase may be described by the bubble model where the positronium bubble is approximated with a finite square potential well with the depth of U=1.45eV.

Correction to: Stabilized Human Cystatin C, Variant L47C/G69C, is a Better Reporter than the Wild-type Inhibitor for Characterizing the Thermodynamics of Binding to Cysteine Proteases.

The original publication of this article contained a number of grammatical errors. Unfortunately, an incorrect version of the file that did not include some final language editing was inadvertently published online. The original article has been corrected.

Selective invalidation of ambivalent pro-marijuana attitude components.

INTRODUCTION: Attitudes of drug-abstinent youth considering marijuana initiation can be highly ambivalent. Invalidating pro-usage elements (i.e., opinions) of ambivalent marijuana attitudes, while leaving anti-marijuana elements intact, may create stronger, less ambivalent marijuana-resistant attitudes and lower usage intentions, while concurrently elucidating the role of ambivalence in persuasive prevention. METHOD: From an initial pool of marijuana-abstinent middle-school students (N = 538), the quintile expressing the most negative attitudes toward a marijuana prevention appeal (N = 101) were randomly assigned to one of three conditions designed to invalidate pro-marijuana opinions. Analyses then tested their susceptibility to a second marijuana prevention appeal. RESULTS: Personally threatening messages were found ineffective, but appeals contesting resistant responses significantly decreased ambivalence (p < .01). Mediational analyses showed that this decreased ambivalence was associated with less favorable attitudes and lower marijuana usage intentions (both p < .001). An attribution-based manipulation increased ambivalence (p < .05), which was associated with positive usage intentions mediated through positive attitudes (both p < .001). CONCLUSION: Analyses elucidated the role of attitude ambivalence in prevention, providing a more complete understanding of potential facilitative use of ambivalence in prevention models based on prevention. Results support the further examination and use of methods that invalidate pro-marijuana opinions, thereby leading to greater susceptibility to subsequent prevention appeals.

Stabilized Human Cystatin C Variant L47C/G69C Is a Better Reporter Than the Wild-Type Inhibitor for Characterizing the Thermodynamics of Binding to Cysteine Proteases.

Human cystatin C (HCC) binds and inhibits all types of cysteine proteases from the papain family, including cathepsins (a group of enzymes that participate in a variety of physiological processes), which are some of its natural targets. The affinities of diverse proteases for HCC, expressed as equilibrium binding constants (Kb), range from 106 to 1014 M-1. Isothermal titration calorimetry (ITC) is one of the most useful techniques to characterize the thermodynamics of molecular associations, making it possible to dissect the binding free energy into its enthalpic and entropic components. This information, together with the structural changes that occur during the different associations, could enable better understanding of the molecular basis of affinity. Notwithstanding the high sensitivity of modern calorimeters, ITC requires protein concentrations in at least the 10-100 µM range to obtain reliable data, and it is known that HCC forms oligomers in this concentration range. We present herein a comparative study of the structural, thermal stability, and oligomerization properties of HCC and its stabilized variant (sHCC) L47C/G69C (which possesses an additional disulfide bridge) as well as their binding thermodynamics to the protease chymopapain, analyzed by ITC. The results show that, because sHCC remains monomeric, it is a better reporter than wild-type HCC to characterize the thermodynamics of binding to cysteine proteases.