Impact of Sinapic Acid on Bovine Serum Albumin Thermal Stability.

The thermal stability of bovine serum albumin (BSA) in Tris buffer, as well as the effect of sinapic acid (SA) on protein conformation were investigated via calorimetric (differential scanning microcalorimetry-µDSC), spectroscopic (dynamic light scattering-DLS; circular dichroism-CD), and molecular docking approaches. µDSC data revealed both the denaturation (endotherm) and aggregation (exotherm) of the protein, demonstrating the dual effect of SA on protein thermal stability. With an increase in ligand concentration, (i) protein denaturation shifts to a higher temperature (indicating native form stabilization), while (ii) the aggregation process shifts to a lower temperature (indicating enhanced reactivity of the denatured form). The stabilization effect of SA on the native structure of the protein was supported by CD results. High temperature (338 K) incubation induced protein unfolding and aggregation, and increasing the concentration of SA altered the size distribution of the protein population, as DLS measurements demonstrated. Complementary information offered by molecular docking allowed for the assessment of the ligand binding within the Sudlow's site I of the protein. The deeper insight into the SA-BSA interaction offered by the present study may serve in the clarification of ligand pharmacokinetics and pharmacodynamics, thus opening paths for future research and therapeutic applications.

Measurement of pelvic tilt and rotation on AP radiographs using HipRecon: Validation and comparison to other parameters.

In this paper, we present and evaluate HipRecon, a noncommercial software package that simultaneously calculates pelvic tilt and rotation from an anteroposterior pelvis radiograph. We asked: What is the (1) accuracy and precision, (2) robustness, and (3) intra-/interobserver reliability/reproducibility of HipRecon to analyze both pelvic tilt and rotation on conventional AP pelvis radiographs? (4) How does the prediction of pelvic tilt on AP pelvis radiographs using HipRecon compare to established measurement methods? We compared the actual pelvic tilt of 20 adult human cadaveric pelvises with the calculated pelvic orientation based on an AP pelvis radiograph using HipRecon software. The pelvises were mounted on a radiolucent fixture and a total of 380 AP pelvis radiographs with different configurations were acquired. In addition, we investigated the correlation between actual tilt and the tilt calculated using HipRecon and seven other established measurement methods. The calculated software accuracy was 0.2 ± 2.0° (-3.6-4.1) for pelvic tilt and 0.0 ± 1.2° (-2.2-2.3, p = 0.39) for pelvic rotation. The Bland-Altman analysis showed values that were evenly and randomly spread in both directions. HipRecon showed excellent consistency for the measurement of pelvic tilt and rotation (intraobserver intraclass-correlation coefficient [ICC]: 0.99 [95% CI: 0.99-0.99] and interobserver ICC 0.99 [95% CI: 0.99-0.99]). Of all eight analyzed methods, the highest correlation coefficient was found for HipRecon (r = 0.98, p < 0.001). In the future, HipRecon could be used to detect changes in patient-specific pelvic orientation, helping to improve clinical understanding and decision-making in pathologies of the hip.

Reliability and Reproducibility of a Novel Grading System for Lesions of the Ligamentous-Fossa-Foveolar Complex in Young Patients Undergoing Open Hip Preservation Surgery.

Background: Several classification systems based on arthroscopy have been used to describe lesions of the ligamentum teres (LT) in young active patients undergoing hip-preserving surgery. Inspection of the LT and associated lesions of the adjuvant fovea capitis and acetabular fossa is limited when done arthroscopically but is much more thorough during open surgical hip dislocation. Therefore, we propose a novel grading system based on our findings during surgical dislocation comprising the full spectrum of ligamentous-fossa-foveolar complex (LFFC) lesions. Purpose: To determine (1) intraobserver reliability and (2) interobserver reproducibility of our new grading system. Study Design: Cohort study (diagnosis); Level of evidence, 3. Methods: We performed this validation study on 211 hips (633 images in total) with surgical hip dislocation (2013-2021). We randomly selected 5 images per grade for each LFFC item to achieve an equal representation of all grades (resulting in 75 images). The ligament, fossa, and fovea were subcategorized into normal, inflammation, degeneration, partial, and complete defects. All surgeries were performed in a standardized way by a single surgeon. The femur was disarticulated using a bone hook, the LT was inspected, documented and resected, then the fossa and fovea were documented with the femoral head in full dislocation using a 70° arthroscope. Six observers with different levels of expertise in hip-preserving surgery independently conducted the measurements twice, and intraclass correlation coefficients (ICC) were calculated to determine (1) intraobserver reliability and (2) interobserver reproducibility of the novel grading system. Results: For intraobserver reliability, excellent ICCs were found in both the junior and the experienced raters for grading the ligament, fossa, fovea, and total LFFC (ICCs ranged from 0.91 to 0.99 for the LFFC score). We found excellent interobserver reproducibility between raters for all items of the LFFC (all interobserver ICCs ≥ 0.76). Conclusion: Our new grading system for lesions of the LFFC is highly reliable and reproducible. It covers the full spectrum of damage more precisely than arthroscopic classifications do and offers a scientific basis for standardized intraoperative evaluation.

Understanding orthopedic infections through a different perspective: Microcalorimetry growth curves.

Infectious disease is a serious healthcare problem in orthopedics, as well as other surgical specialties. Accurate and prompt diagnosis, as well as proper care, is critical, as infection of a surgical wound, particularly in the case of arthroplasties or the use of orthopedic implants, can have a catastrophic effect in most cases, necessitating the removal of foreign material. Lyophilized bacteria samples were obtained from Cantacuzino National Institute of Research and Development for Microbiology and Immunology and investigated microcalorimetrically. Isothermal microcalorimetry measures the temperature generated by the multiplication of microorganisms; using an adapted program, it describes the growth curve in real-time according to the received electrical signal. The thermograms of Escherichia coli and Klebsiella pneumonie were analyzed, and similarities were observed for both the time required for the bacteria to grow and the heat flow generated by their growth. Bacterial microcalorimetry has a variety of benefits, and should be regarded as a means of rapid and accurate diagnosis. Sensitivity is a valuable attribute for a diagnostic technique; when only a few microorganisms are present, microcalorimetric signs of bacterial multiplication can be observed. Microcalorimetry has potential as a simple diagnostic tool in a variety of infections, but further research is needed to ensure that it is used correctly. A thorough investigation (including kinetic analysis) of a reproducible thermal signal of bacterial growth could lead to the creation of new methods for quickly identifying bacteria.

Latest Developments in Edible Coatings on Minimally Processed Fruits and Vegetables: A Review.

The food industry nowadays is facing new challenges in terms of sustainability and health implications of packaging and processing techniques. Due to their desire for new and natural products coupled with changes in lifestyle, consumers are looking for food products that have been less processed but possess longer shelf life and maintain nutritional and sensorial proprieties during storage. These requirements represent real challenges when dealing with highly perishable food products, such as fruits and vegetables. Thus, in recent years, edible coatings have been intensively developed and studied because of their capacity to improve the quality, shelf life, safety, and functionality of the treated products. Edible coatings can be applied through different techniques, like dipping, spraying, or coating, in order to control moisture transfer, gas exchange, or oxidative processes. Furthermore, some functional ingredients can be incorporated into an edible matrix and applied on the surface of foods, thus enhancing safety or even nutritional and sensory attributes. In the case of coated fruits and vegetables, their quality parameters, such as color, firmness, microbial load, decay ratio, weight loss, sensorial attributes, and nutritional parameters, which are very specific to the type of products and their storage conditions, should be carefully monitored. This review attempts to summarize recent studies of different edible coatings (polysaccharides, proteins, lipids, and composites) as carriers of functional ingredients (antimicrobials, texture enhancers, and nutraceuticals) applied on different minimally processed fruits and vegetables, highlighting the coating ingredients, the application methods and the effects on food shelf life and quality.

Formulation of Chewable Tablets Containing Carbamazepine-ß-cyclodextrin Inclusion Complex and F-Melt Disintegration Excipient. The Mathematical Modeling of the Release Kinetics of Carbamazepine.

Due to its low solubility, carbamazepine (CBZ) exhibits slow and incomplete release in the gastrointestinal tract and, hence, variable pharmacokinetics and pharmacodynamic effect. Lots of methods have been devised to improve its solubility, the large number of proposed solutions being a sign that the problem is not yet satisfactorily solved. The persistent problem is that predictable release kinetics, an increased rate but within defined limits, are required to avoid high absorption variability. This paper presents a synthesis of a carbamazepine-ß-cyclodextrin inclusion complex (CBZ-ß-CD), the characterization of the physical mixture, CBZ, ß-CD and the CBZ-ß-CD inclusion complex using Fourier transform infrared spectroscopy, scanning electron microscopy, simultaneous thermal analysis and X-ray diffraction, formulation of chewable tablets, determination of the dissolution of carbamazepine in medium containing 1% sodium lauryl sulfate (LSS), and in simulated saliva (SS), mathematical modeling of release kinetics. The kinetics of total CBZ release from tablets containing CBZ-ß-CD and super-disintegrant F-Melt in both SS and LSS followed two steps: a burst release in the first minutes and a slower release in intervals up to 60 min. The release in the second phase has been well described by the Higuchi and Peppas models, which advocate a controlled release by combined diffusion and with some phenomena of swelling and relaxation of the matrix generated by the crospovidone component of the F-Melt excipient.

Model Systems for Evidencing the Mediator Role of Riboflavin in the UVA Cross-Linking Treatment of Keratoconus.

Riboflavin under UVA radiation generates reactive oxygen species (ROS) that can induce various changes in biological systems. Under controlled conditions, these processes can be used in some treatments for ocular or dermal diseases. For instance, corneal cross-linking (CXL) treatment of keratoconus involves UVA irradiation combined with riboflavin aiming to induce the formation of new collagen fibrils in cornea. To reduce the damaging effect of ROS formed in the presence of riboflavin and UVA, the CXL treatment is performed with the addition of polysaccharides (dextran). Hyaluronic acid is a polysaccharide that can be found in the aqueous layer of the tear film. In many cases, keratoconus patients also present dry eye syndrome that can be reduced by the application of topical solutions containing hyaluronic acid. This study presents physico-chemical evidence on the effect of riboflavin on collagen fibril formation revealed by the following methods: differential scanning microcalorimetry, rheology, and STEM images. The collagen used was extracted from calf skin that contains type I collagen similar to that found in the eye. Spin trapping experiments on collagen/hyaluronic acid/riboflavin solutions evidenced the formation of ROS species by electron paramagnetic resonance measurements.

Development and Characterization of Orally Disintegrating Tablets Containing a Captopril-Cyclodextrin Complex.

Captopril is the first angiotensin I-converting enzyme inhibitor widely used for the treatment of hypertension. Based on the well-known benefits of cyclodextrin inclusion complexes, the present study investigated the ability of ß-cyclodextrin to include captopril. Solid inclusion complexes of captopril with ß-cyclodextrin in a 1:2 molar ratio were prepared by using the paste method of complexation. For comparison purposes, a simple physical mixture with the same molar ratio was also prepared. Fourier-transform infrared spectroscopy, scanning electron microscopy, X-ray diffraction and simultaneous thermal analysis were used to characterize the raw materials, physical mixture and solid inclusion complex. In order to provide the drug in a more accessible and patient-compliant form following masking its bitter taste, as well as ensuring the appropriate release kinetics, the investigated complex was formulated as orally disintegrating tablets. The study of captopril dissolution in both compendial and simulated saliva media suggested the Noyes Whitney model as the best mathematical model to describe the release phenomena. A clinical study on healthy volunteers also highlighted the taste improvement of the new formulation as compared to conventional tablets.

Thermodynamic Parameters at Bio-Nano Interface and Nanomaterial Toxicity: A Case Study on BSA Interaction with ZnO, SiO2, and TiO2.

Understanding nanomaterial (NM)-protein interactions is a key issue in defining the bioreactivity of NMs with great impact for nanosafety. In the present work, the complex phenomena occurring at the bio/nano interface were evaluated in a simple case study focusing on NM-protein binding thermodynamics and protein stability for three representative metal oxide NMs, namely, zinc oxide (ZnO; NM-110), titanium dioxide (TiO2; NM-101), and silica (SiO2; NM-203). The thermodynamic signature associated with the NM interaction with an abundant protein occurring in most cell culture media, bovine serum albumin (BSA), has been investigated by isothermal titration and differential scanning calorimetry. Circular dichroism spectroscopy offers additional information concerning adsorption-induced protein conformational changes. The BSA adsorption onto NMs is enthalpy-controlled, with the enthalpic character (favorable interaction) decreasing as follows: ZnO (NM-110) > SiO2 (NM-203) > TiO2 (NM-101). The binding of BSA is spontaneous, as revealed by the negative free energy, ΔG, for all systems. The structural stability of the protein decreased as follows: TiO2 (NM-101) > SiO2 (NM-203) > ZnO (NM-110). As protein binding may alter NM reactivity and thus the toxicity, we furthermore assessed its putative influence on DNA damage, as well as on the expression of target genes for cell death (RIPK1, FAS) and oxidative stress (SOD1, SOD2, CAT, GSTK1) in the A549 human alveolar basal epithelial cell line. The enthalpic component of the BSA-NM interaction, corroborated with BSA structural stability, matched the ranking for the biological alterations, i.e., DNA strand breaks, oxidized DNA lesions, cell-death, and antioxidant gene expression in A549 cells. The relative and total content of BSA in the protein corona was determined using mass-spectrometry-based proteomics. For the present case study, the thermodynamic parameters at bio/nano interface emerge as key descriptors for the dominant contributions determining the adsorption processes and NMs toxicological effect.

Interaction of piroxicam with bovine serum albumin investigated by spectroscopic, calorimetric and computational molecular methods.

The binding of drugs to serum proteins is governed by weak non-covalent forces. In this study, the nature and magnitude of the interactions between piroxicam (PRX) and bovine serum albumin (BSA) was assessed using spectroscopic, calorimetric and computational molecular methods. The fluorescence data revealed an atypical behavior during PRX and BSA interaction. The quenching process of tryptophan (Trp) by PRX is a dual one (approximately equal static and dynamic quenched components). The FRET results indicate that a non-radiative transfer of energy occurred. The association constant and the number of binding sites indicate moderate PRX and BSA binding. The competitive binding study indicates that PRX is bound to site I from the hydrophobic pocket of subdomain IIA of BSA. The synchronous spectra showed that the microenvironment around the BSA fluorophores and protein conformation do not change considerably. The Trp lifetimes revealed that PRX mainly quenches the fluorescence of Trp-213 situated in the hydrophobic domain. The CD and DSC investigation show that addition of PRX stabilizes the protein structure. ITC results revealed that BSA-PRX binding involves a combination of electrostatic, hydrophobic and hydrogen interactions. The analysis of the computational data is consistent with the experimental results. This thorough investigation of the PRX-BSA binding may provide support for other studies concerning moderate affinity drugs with serum protein.Communicated by Ramaswamy H. Sarma.

How to run molecular dynamics simulations on electrospray droplets and gas phase proteins: Basic guidelines and selected applications.

The ability to transfer intact proteins and protein complexes into the gas phase by electrospray ionization (ESI) has opened up numerous mass spectrometry (MS)-based avenues for exploring biomolecular structure and function. However, many details regarding the ESI process and the properties of gaseous analyte ions are difficult to decipher when relying solely on experimental data. Molecular dynamics (MD) simulations can provide additional insights into the behavior of ESI droplets and protein ions. This review is geared primarily towards experimentalists who wish to adopt MD simulations as a complementary research tool. We touch on basic points such as force fields, the choice of a proper water model, GPU-acceleration, possible artifacts, as well as shortcomings of current MD models. Following this technical overview, we highlight selected applications. Simulations on aqueous droplets confirm that "native" ESI culminates in protein ion release via the charged residue model. MD-generated charge states and collision cross sections match experimental data. Gaseous protein ions produced by native ESI retain much of their solution structure. Moving beyond classical fixed-charge algorithms, we discuss a simple strategy that captures the mobile nature of H+ within gaseous biomolecules. These mobile proton simulations confirm the high propensity of gaseous proteins to form salt bridges, as well as the occurrence of charge migration during collision-induced unfolding and dissociation. It is hoped that this review will promote the use of MD simulations in ESI-related research. We also hope to encourage the development of improved algorithms for charged droplets and gaseous biomolecular ions.

Collision-Induced Dissociation of Electrosprayed NaCl Clusters: Using Molecular Dynamics Simulations to Visualize Reaction Cascades in the Gas Phase.

Infusion of NaCl solutions into an electrospray ionization (ESI) source produces [Na(n+1)Cl n ]+ and other gaseous clusters. The n = 4, 13, 22 magic number species have cuboid ground state structures and exhibit elevated abundance in ESI mass spectra. Relatively few details are known regarding the mechanisms whereby these clusters undergo collision-induced dissociation (CID). The current study examines to what extent molecular dynamics (MD) simulations can be used to garner insights into the sequence of events taking place during CID. Experiments on singly charged clusters reveal that the loss of small neutrals is the dominant fragmentation pathway. MD simulations indicate that the clusters undergo extensive structural fluctuations prior to decomposition. Consistent with the experimentally observed behavior, most of the simulated dissociation events culminate in ejection of small neutrals ([NaCl] i , with i = 1, 2, 3). The MD data reveal that the prevalence of these dissociation channels is linked to the presence of short-lived intermediates where a relatively compact core structure carries a small [NaCl] i protrusion. The latter can separate from the parent cluster via cleavage of a single Na-Cl contact. Fragmentation events of this type are kinetically favored over other dissociation channels that would require the quasi-simultaneous rupture of multiple electrostatic contacts. The CID behavior of NaCl cluster ions bears interesting analogies to that of collisionally activated protein complexes. Overall, it appears that MD simulations represent a valuable tool for deciphering the dissociation of noncovalently bound systems in the gas phase. Graphical Abstract ᅟ.

Quercetin Influence on Thermal Denaturation of Bovine Serum Albumin.

The effect of quercetin (QUER) binding on bovine serum albumin (BSA) thermal denaturation was systematically investigated by means of differential scanning calorimetry (DSC). Additional information concerning thermodynamic and structural binding parameters was provided by isothermal titration calorimetry (ITC) and molecular docking. The most relevant effect of QUER is manifested in the modification of the two-step thermal fingerprint of protein denaturation. Higher QUER concentrations result in a single-step denaturation thermogram, ascribed to the interplay between specific and nonspecific binding and enhancement of the solvent unfolding action. Analysis of ITC data indicate sequential binding of two molecules of QUER occurring spontaneously at different binding sites of BSA involving hydrophobic, electrostatic and hydrogen binding forces. Identification of QUER binding sites was possible through corroboration of DSC runs in the presence of site markers and molecular docking. Modeling of ligand-protein interaction confirmed the experimental data. On one hand, a neutral form of QUER binds in a nonplanar conformation to Sudlow's site I, a large hydrophobic cavity of subdomain IIA of BSA and decreases its thermal stability. On the other hand, a second molecule of QUER, the anionic form, is bound in planar conformation to Sudlow's site II, situated in the subdomain IIIA of the folded protein, and increases the thermal stability of the corresponding structural domain of the protein.

Collision-Induced Dissociation of Electrosprayed Protein Complexes: An All-Atom Molecular Dynamics Model with Mobile Protons.

Electrospray ionization mass spectrometry (ESI-MS) has become an indispensable technique for examining noncovalent protein complexes. Collision-induced dissociation (CID) of these multiply protonated gaseous ions usually culminates in ejection of a single subunit with a disproportionately large amount of charge. Experiments suggest that this process involves subunit unfolding prior to separation from the residual complex, as well as H(+) migration onto the unravelling chain. Molecular dynamics (MD) simulations are a promising avenue for gaining detailed insights into these CID events. Unfortunately, typical MD algorithms do not allow for mobile protons. Here we address this limitation by implementing a strategy that combines atomistic force fields (such as OPLS/AA and CHARMM36) with a proton hopping algorithm, focusing on the tetrameric complexes transthyretin and streptavidin. Protons are redistributed over all acidic and basic sites in 20 ps intervals, subject to an energy function that reflects electrostatic interactions and proton affinities. Our simulations predict that nativelike conformers at the onset of collisional heating contain multiple salt bridges. Collisional heating initially causes subtle structural changes that lead to a gradual decline of these zwitterionic patterns. Many of the MD runs show gradual unfolding of a single subunit in conjunction with H(+) migration, culminating in subunit separation from the complex. However, there are also instances where two or more chains start to unfold simultaneously, giving rise to charge competition. The scission point where the "winning" subunit separates from the complex can be attained for different degrees of unfolding, giving rise to product ions in various charge states. The simulated product ion distributions are in close agreement with experimental CID data. Proton enrichment in the departing subunit is driven by charge-charge repulsion, but the combination of salt bridge depletion, charge migration, and proton affinity causes surprising compensation effects among the various energy terms. It appears that this work provides the most detailed account to date of the mechanism whereby noncovalent protein complexes disassemble during CID.

Interaction between Albumin and Pluronic F127 Block Copolymer Revealed by Global and Local Physicochemical Profiling.

The interaction of human serum albumin (HSA) with amphiphilic block copolymer Pluronic F127 has been investigated by several physical methods. Interest in studying this system stems from a broad range of bioactivities involving both macromolecules. Serum albumins constitute a significant class of proteins in the circulatory system, acting as carriers for a wide spectrum of compounds or assemblies. Pluronic block copolymers have revealed their capacity to ferry a variety of biologically active compounds. Circular dichroism, rheological measurements, and differential scanning microcalorimetry (µDSC) were employed to get insight into the interaction betweeen the two macromolecules. The results reveal that Pluronic F127 induces conformational changes to albumin if it is organized in a micellar form, while albumin influences the self-assembly of Pluronic F127 into micelles or gels. F127 micelles, however, induce smaller conformational changes compared to ionic surfactants. The µDSC thermograms obtained for HSA and/or F127 show that HSA shifts the critical micellar temperature (cmt) to lower values, while concurrently the HSA denaturation behavior is influenced by F127, depending on its concentration. Rheological measurements on solutions of F127 17% have shown that a sol-to-gel transition occurs at higher temperatures in the presence of HSA and the resulting gel is weaker. The global profile on HSA/F127 systems was complemented by local information provided by EPR measurements. A series of X-band EPR experiments was performed with spin probes 4-(N,N'-dimethyl-N-hexadecyl)ammonium-2,2',6,6'-tetramethylpiperidine-1-oxyl iodide (CAT16) and 5-doxyl stearic acid (5-DSA). These spin probes bind to albumin sites and are sensitive to phase transformations in Pluronic block copolymer solutions. For a given F127 concentration, the spin probe binds only to HSA below cmt and migrates to the F127 micelles above cmt. The collective data suggest soft interactions between the macromolecules, with the emerging results projecting potential applications linked to reaching optimal conditions for certain drug formulations.

Mechanism of Protein Supercharging by Sulfolane and m-Nitrobenzyl Alcohol: Molecular Dynamics Simulations of the Electrospray Process.

Electrospray ionization (ESI) allows the production of intact gas-phase ions from proteins in solution. Nondenaturing solvent conditions usually culminate in low ESI charge states. However, many mass spectrometric applications benefit from protein ions that are more highly charged. One way to boost protein charge is the addition of supercharging agents (SCAs) such as sulfolane or m-nitrobenzyl alcohol (m-NBA) to the aqueous solution. The supercharging mechanism remains controversial. We use molecular dynamics (MD) simulations to examine how SCAs affect the behavior of ESI nanodroplets. Simulations were conducted on myoglobin in water, water/sulfolane, and water/m-NBA. Na(+) ions served as surrogate charge carriers instead of H(+). We focus on conditions where the protein initially adopts its native conformation. MD-generated charge states show remarkable agreement with experimental data. Droplet shrinkage is accompanied by Na(+) ejection, consistent with the ion evaporation model (IEM). The droplets segregate into an outer SCA shell and an aqueous core. This core harbors protein and Na(+). Unfavorable SCA solvation restricts Na(+) access to the droplet surface, thereby impeding IEM ejection. Rapid water loss causes SCA enrichment, ultimately forcing all remaining Na(+) to bind the protein. IEM ejection is no longer feasible after this point, such that the protein becomes supercharged by Na(+) trapping. SCA-free droplets produce lower charge states because the aqueous environment ensures a higher IEM efficiency. For all scenarios examined here, proteins are released via solvent evaporation to dryness, as envisioned by the charged residue model. Our data provide the first atomistic view of the supercharging mechanism.

Comparative analysis of Staphylococcus aureus and Escherichia coli microcalorimetric growth.

BACKGROUND: Microcalorimetric bacterial growth studies have illustrated that thermograms differ significantly with both culture media and strain. The present contribution examines the possibility of discriminating between certain bacterial strains by microcalorimetry and the qualitative and quantitative contribution of the sample volume to the observed thermograms. Growth patterns of samples of Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922) were analyzed. Certain features of the thermograms that may serve to distinguish between these bacterial strains were identified. RESULTS: The thermograms of the two bacterial strains with sample volumes ranging from 0.3 to 0.7 ml and same initial bacterial concentration were analyzed. Both strains exhibit a roughly 2-peak shape that differs by peak amplitude and position along the time scale. Seven parameters corresponding to the thermogram key points related to time and heat flow values were proposed and statistically analyzed. The most relevant parameters appear to be the time to reach a heat flow of 0.05 mW (1.67 ± 0.46 h in E. coli vs. 2.99 ± 0.53 h in S. aureus, p < 0.0001), the time to reach the first peak (3.84 ± 0.5 h vs. 5.17 ± 0.49 h, p < 0.0001) and the first peak value (0.19 ± 0.02 mW vs. 0.086 ± 0.012 mW, p < 0.0001). The statistical analysis on 4 parameters of volume-normalized heat flow thermograms showed that the time to reach a volume-normalized heat flow of 0.1 mW/ml (1.75 ± 0.37 h in E. coli vs. 2.87 ± 0.65 h in S. aureus, p < 0.005), the time to reach the first volume-normalized peak (3.78 ± 0.47 h vs. 5.12 ± 0.52 h, p < 0.0001) and the first volume-normalized peak value (0.35 ± 0.05 mW/ml vs. 0.181 ± 0.040 mW/ml, p < 0.0001) seem to be the most relevant. Peakfit® decomposition and analysis of the observed thermograms complements the statistical analysis via quantitative arguments, indicating that: (1) the first peak pertains to a faster, "dissolved oxygen" bacterial growth (where the dissolved oxygen in the initial suspension acts as a limiting factor); (2) the second peak indicates a slower "diffused oxygen" growth that involves transport of oxygen contained in the unfilled part of the microcalorimetric cell; (3) a strictly fermentative growth component may slightly contribute to the observed complex thermal signal. CONCLUSION: The investigated strains of Staphylococcus aureus and Escherichia coli display, under similar experimental conditions, distinct thermal growth patterns. The two strains can be easily differentiated using a selection of the proposed parameters. The presented Peakfit analysis of the complex thermal signal provides the necessary means for establishing the optimal growth conditions of various bacterial strains. These conditions are needed for the standardization of the isothermal microcalorimetry method in view of its further use in qualitative and quantitative estimation of bacterial growth.

[Microcalorimetry -- a new method for bacterial characterisation]. / Microcalorimetria -- metoda noua de caracterizare bacteriana.

The microcalorimetry is a method used for recording of the heat produced by a thermodinamic system in a scale of micronanojouls. One of the domains in which this method is used is the one called bacterial microcalorimetry, which studies the heat generated by the bacterial populations. The process of bacterial growth can be monitored in real time by the recording a graph of the generated power over time. The modern isothermal microcalorimeters allow the detection of a signal variation of only one microwatt. The estimated generated power of a bacteria is approximately 1-4pW thus only a small number of bacteria is necessary for the experiments. Recent studies in the field of bacterial microcalorimetry have demonstrated that, in standard conditions, this method can be reproductible and can be used to detect and characterize bacterial growth (through the study of the microcalorimetric growth curve particular to a bacterial species which is called a microcalorimetric fingerprint) and offers the new information in regards to bacterial metabolism. Also, microcalorimetry can offer information about bacterial interaction with different factors in the medium (for example, antibioticsubstances, in which case an antibiogram is obtained in 4-5 hours). In conclusion, we can say that microcalorimetry is a reproducible method, which offers an interesting perspective on bacterial characterization, with great scientific potential, and there are sufficient arguments to continue studies in this field.

Consistency between kinetics and thermodynamics: general scaling conditions for reaction rates of nonlinear chemical systems without constraints far from equilibrium.

We examine the problem of consistency between the kinetic and thermodynamic descriptions of reaction networks. We focus on reaction networks with linearly dependent (but generally kinetically independent) reactions for which only some of the stoichiometric vectors attached to the different reactions are linearly independent. We show that for elementary reactions without constraints preventing the system from approaching equilibrium there are general scaling relations for nonequilibrium rates, one for each linearly dependent reaction. These scaling relations express the ratios of the forward and backward rates of the linearly dependent reactions in terms of products of the ratios of the forward and backward rates of the linearly independent reactions raised to different scaling powers; the scaling powers are elements of the transformation matrix, which relates the linearly dependent stoichiometric vectors to the linearly independent stoichiometric vectors. These relations are valid for any network of elementary reactions without constraints, linear or nonlinear kinetics, far from equilibrium or close to equilibrium. We show that similar scaling relations for the reaction routes exist for networks of nonelementary reactions described by the Horiuti-Temkin theory of reaction routes where the linear dependence of the mechanistic (elementary) reactions is transferred to the overall (route) reactions. However, in this case, the scaling conditions are valid only at the steady state. General relationships between reaction rates of the two levels of description are presented. These relationships are illustrated for a specific complex reaction: radical chlorination of ethylene.

MicroDSC study of Staphylococcus epidermidis growth.

BACKGROUND: A microcalorimetric study was carried out using a Staphylococcus epidermidis population to determine the reproducibility of bacterial growth and the variability of the results within certain experimental parameters (temperature, bacterial concentration, sample thermal history). Reproducibility tests were performed as series of experiments within the same conditions using either freshly prepared populations or samples kept in cold storage. In both cases, the samples were obtained by serial dilution from a concentrated TSB bacterial inoculum incubated overnight. RESULTS: The results show that experiments are fairly reproducible and that specimens can be preserved at low temperatures (1 - 2°C) at least 4 days. The thermal signal variations at different temperatures and initial bacterial concentrations obey a set of rules that we identified. CONCLUSION: Our study adds to the accumulating data and confirms available results of isothermal microcalorimetry applications in microbiology and can be used to standardize this method for either research or clinical setting.