Solvent-mediated isotope effects strongly influence the early stages of calcium carbonate formation: exploring D2O vs. H2O in a combined computational and experimental approach

In experimental studies, heavy water (D2O) is employed, e.g., so as to shift the spectroscopic solvent background, but any potential effects of this solvent exchange on reaction pathways are often neglected. While the important role of light water (H2O) during the early stages of calcium carbonate formation has been realized, studies into the actual effects of aqueous solvent exchanges are scarce. Here, we present a combined computational and experimental approach to start to fill this gap. We extended a suitable force field for molecular dynamics (MD) simulations. Experimentally, we utilised advanced titration assays and time-resolved attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. We find distinct effects in various mixtures of the two aqueous solvents, and in pure H2O or D2O. Disagreements between the computational results and experimental data regarding the stabilities of ion associates might be due to the unexplored role of HDO, or an unprobed complex phase behaviour of the solvent mixtures in the simulations. Altogether, however, our data suggest that calcium carbonate formation might proceed “more classically” in D2O. Also, there are indications for the formation of new structures in amorphous and crystalline calcium carbonates. There is huge potential towards further improving the understanding of mineralization mechanisms by studying solvent-mediated isotope effects, also beyond calcium carbonate. Last, it must be appreciated that H2O and D2O have significant, distinct effects on mineralization mechanisms, and that care has to be taken when experimental data from D2O studies are used, e.g., for the development of H2O-based computer models

Tailoring Fibre Structure Enabled by X-ray Analytics for Targeted Biomedical Applications

The rising interest in designing fibres via spinning techniques combining the properties of various polymeric materials into advanced functionalised materials is directed towards targeted biomedical applications such as drug delivery, wearable sensors or tissue engineering. Understanding how these functional polymers exhibit multiscale structures ranging from the molecular level to nano-, micro-and millimetre scale is a key prerequisite for their challenging applications that can be addressed by a non-destructive X-ray based analytical approach. X-ray multimodalities combining X-ray imaging, scattering and diffraction allow the study of morphology, molecular structure, and the analysis of nano-domain size and shape, crystallinity and preferential orientation in 3D arrangements. The incorporation of X-ray analytics in the design process of polymeric fibers via their nanostructure under non-ambient conditions (i.e. temperature, mechanical load, humidity…) allows for efficient optimization of the fabrication process as well as quality control along the product lifetime under operating environmental conditions. Here, we demonstrate the successful collaboration between the laboratory of Biomimetic Textiles and Membranes and the Center of X-ray Analytics at Empa for the design, characterisation and optimisation of advanced functionalised polymeric fibrous material systems

Multiscale multimodal characterization and simulation of structural alterations in failed bioprosthetic heart valves

Calcific degeneration is the most frequent type of heart valve failure, with rising incidence due to the ageing population. The gold standard treatment to date is valve replacement. Unfortunately, calcification oftentimes re-occurs in bioprosthetic substitutes, with the governing processes remaining poorly understood. Here, we present a multiscale, multimodal analysis of disturbances and extensive mineralisation of the collagen network in failed bioprosthetic bovine pericardium valve explants with full histoanatomical context. In addition to highly abundant mineralized collagen fibres and fibrils, calcified micron-sized particles previously discovered in native valves were also prevalent on the aortic as well as the ventricular surface of bioprosthetic valves. The two mineral types (fibres and particles) were detectable even in early-stage mineralisation, prior to any macroscopic calcification. Based on multiscale multimodal characterisation and high-fidelity simulations, we demonstrate that mineral occurrence coincides with regions exposed to high haemodynamic and biomechanical indicators. These insights obtained by multiscale analysis of failed bioprosthetic valves may serve as groundwork for the evidence-based development of more durable alternatives. STATEMENT OF SIGNIFICANCE: Bioprosthetic valve calcification is a well-known clinically significant phenomenon, leading to valve failure. The nanoanalytical characterisation of bioprosthetic valves gives insights into the highly abundant, extensive calcification and disorganization of the collagen network and the presence of calcium phosphate particles previously reported in native cardiovascular tissues. While the collagen matrix mineralisation can be primarily attributed to a combination of chemical and mechanical alterations, the calcified particles are likely of host cellular origin. This work presents a straightforward route to mineral identification and characterization at high resolution and sensitivity, and with full histoanatomical context, hence providing design cues for improved bioprosthetic valve alternatives

Multiscale Multimodal Characterization and Simulation of Structural Alterations in Failed Bioprosthetic Heart Valves.

Calcific degeneration is the most frequent type of heart valve failure, with rising incidence due to the ageing population. The gold standard treatment to date is valve replacement. Unfortunately, calcification oftentimes re-occurs in bioprosthetic substitutes, with the governing processes remaining poorly understood. Here, we present a multiscale, multimodal analysis of disturbances and extensive mineralisation of the collagen network in failed bioprosthetic bovine pericardium valve explants with full histoanatomical context. In addition to highly abundant mineralized collagen fibres and fibrils, calcified micron-sized particles previously discovered in native valves were also prevalent on the aortic as well as the ventricular surface of bioprosthetic valves. The two mineral types (fibres and particles) were detectable even in early-stage mineralisation, prior to any macroscopic calcification. Based on multiscale multimodal characterisation and high-fidelity simulations, we demonstrate that mineral occurrence coincides with regions exposed to high haemodynamic and biomechanical indicators. These insights obtained by multiscale analysis of failed bioprosthetic valves may serve as groundwork for the evidence-based development of more durable alternatives. STATEMENT OF SIGNIFICANCE: Bioprosthetic valve calcification is a well-known clinically significant phenomenon, leading to valve failure. The nanoanalytical characterisation of bioprosthetic valves gives insights into the highly abundant, extensive calcification and disorganization of the collagen network and the presence of calcium phosphate particles previously reported in native cardiovascular tissues. While the collagen matrix mineralisation can be primarily attributed to a combination of chemical and mechanical alterations, the calcified particles are likely of host cellular origin. This work presents a straightforward route to mineral identification and characterization at high resolution and sensitivity, and with full histoanatomical context, hence providing design cues for improved bioprosthetic valve alternatives

A combined approach of MALDI-TOF mass spectrometry and multivariate analysis as a potential tool for the detection of SARS-CoV-2 virus in nasopharyngeal swabs

Coronavirus disease 2019, known as COVID-19, is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The early, sensitive and specific detection of SARS-CoV-2 virus is widely recognized as the critical point in responding to the ongoing outbreak. Currently, the diagnosis is based on molecular real time RT-PCR techniques, although their implementation is being threatened due to the extraordinary demand for supplies worldwide. That is why the development of alternative and / or complementary tests becomes so relevant. Here, we exploit the potential of mass spectrometry technology combined with machine learning algorithms, for the detection of COVID-19 positive and negative protein profiles directly from nasopharyngeal swabs samples. According to the preliminary results obtained, accuracy =67.66 %, sensitivity =61.76 %, specificity =71.72 %, and although these parameters still need to be improved to be used as a screening technique, mass spectrometry- based methods coupled with multivariate analysis showed that it is an interesting tool that deserves to be explored as a complementary diagnostic approach due to the low cost and fast performance. However, further steps, such as the analysis of a large number of samples, should be taken in consideration to determine the applicability of the method developed.Fil: Rocca, María Florencia. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud "Dr. C. G. Malbrán"; Argentina. Universidad Nacional de Santiago del Estero. Facultad de Humanidades Ciencias Sociales y de la Salud. Instituto de Estudios e Investigaciones en Enfermería; Argentina. Red Nacional de Espectrometría de Masas Aplicada a la Microbiología Clínica; ArgentinaFil: Zintgraff, Jonathan Cristian. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud "Dr. C. G. Malbrán"; Argentina. Universidad Nacional de Santiago del Estero. Facultad de Humanidades Ciencias Sociales y de la Salud. Instituto de Estudios e Investigaciones en Enfermería; Argentina. Red Nacional de Espectrometría de Masas Aplicada a la Microbiología Clínica; ArgentinaFil: Dattero, María Elena. Universidad Nacional de Santiago del Estero. Facultad de Humanidades Ciencias Sociales y de la Salud. Instituto de Estudios e Investigaciones en Enfermería; Argentina. Administración Nacional de Laboratorios e Institutos de Salud "Dr. Carlos G. Malbrán". Instituto Nacional de Medicina Tropical; ArgentinaFil: Santos, Leonardo Silva. Universidad de Talca; ChileFil: Ledesma, Martin Manuel. Red Nacional de Espectrometría de Masas Aplicada A la Microbiología Clínica (renaem Argentina); Argentina. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Vay, Carlos Alberto. Red Nacional de Espectrometría de Masas Aplicada a la Microbiología Clínica; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Prieto, Mónica Raquel. Red Nacional de Espectrometría de Masas Aplicada a la Microbiología Clínica; Argentina. Universidad de Buenos Aires; ArgentinaFil: Benedetti, Estefanía. Universidad Nacional de Santiago del Estero. Facultad de Humanidades Ciencias Sociales y de la Salud. Instituto de Estudios e Investigaciones en Enfermería; Argentina. Administración Nacional de Laboratorios e Institutos de Salud "Dr. Carlos G. Malbrán". Instituto Nacional de Medicina Tropical; ArgentinaFil: Avaro, Martín. Universidad Nacional de Santiago del Estero. Facultad de Humanidades Ciencias Sociales y de la Salud. Instituto de Estudios e Investigaciones en Enfermería; Argentina. Administración Nacional de Laboratorios e Institutos de Salud "Dr. Carlos G. Malbrán". Instituto Nacional de Medicina Tropical; ArgentinaFil: Russo, Mara Laura. Universidad Nacional de Santiago del Estero. Facultad de Humanidades Ciencias Sociales y de la Salud. Instituto de Estudios e Investigaciones en Enfermería; Argentina. Administración Nacional de Laboratorios e Institutos de Salud "Dr. Carlos G. Malbrán". Instituto Nacional de Medicina Tropical; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Nachtigall, Fabiane Manke. Universidad Autónoma de Chile; ChileFil: Baumeister, Elsa. Universidad Nacional de Santiago del Estero. Facultad de Humanidades Ciencias Sociales y de la Salud. Instituto de Estudios e Investigaciones en Enfermería; Argentina. Administración Nacional de Laboratorios e Institutos de Salud "Dr. Carlos G. Malbrán". Instituto Nacional de Medicina Tropical; Argentin

Sizing experiments and bio-nano interactions: method matters

INTRODUCTION Physicochemical properties of many active ingredients jeopardize their pharmacological activity. To overcome identified obstacles, nanosystems as carriers for delivery of actives have been recognized as promising tools. Increasing number of applications for registration of nanotechnology-enabled pharmaceuticals and many more currently in preclinical or clinical studies raised some questions not only in the field of research and development, but also for regulators. Given the complexity of nanosystems, some specific challenges have been encountered in their characterization, which have not been fully addressed despite respectable research tradition in this field. Particle size and aggregation potential (especially in complex biological fluids) are some of the critical quality attributes of nanomedicines, being important in the context of physical stability of the colloidal system, and in terms of its safety profile and in vivo performance. Even though a bright future has been predicted for nanomedicines, some of the posted expectations have not been fully met so far. This might be reflected, at least at some points, in the certain methodological issues that commonly result in in vitro to in vivo translational gaps. This aspect underlines the importance of quality and safety assessment of nanomedicines which has also been recognized by globally leading research and regulatory bodies [1,2]. Therefore, the aim of the presented research was to perform a thorough analysis of the selected nanosystem (nanoemulsion) focusing on size estimation and particle-protein interaction applying several techniques, highlighting important factors for a reliable analysis. METHODLOGY Materials As a model nanosystem, previously developed nanoemulsion was used, containing medium-chain triglycerides (Mygliol 812, Fagron) as the oil phase, combination of polysorbate 80 (Acros Organics) and soybean lecithin (Lipoid S-75, Lipoid) as stabilizes, and highly purified water as the water phase. For protein interaction assessment, human serum albumin was used (HSA, Sigma Aldrich). Methods Nanoemulsion preparation Nanoemulsion was prepared via spontaneous emulsification, by dropwise addition of the mixture of the oil and stabilizers to the water phase under constant stirring. For nanoparticle-protein interaction assesment, nanoemulsion was incubated (1h, 37 °C) with HSA in the final concentration of 2.5 mg/ml. Sizing experiments – dynamic light scattering Size and size distribution (per se and in biorelevant environment) were evaluated applying batch mode dynamic light scattering (DLS, Zetasizer Nano ZS90, Malvern Instruments, UK), following the NCL guidance [3]. Intensity-based average hydrodynamic diameter (Z-ave) and polydispersity index (PDI) were analysed in line with relevant parameters of the method. Atomic force microscopy (AFM) Additional sizing analysis and morphological evaluation of the sample were performed applying AFM as a high-resolution technique. AFM analysis of the samples was performed by NTEGRA Prima atomic force microscope (NT-MDT, Moscow, Russia). Intermittent-contact AFM mode was applied using NT-MDT NSGO1 silicon, N-type, antimony doped cantilevers with Au reflective coating. Sample dilution corresponded to the optimal one selected for DLS, and 10 μl of the dilution was placed to the high-quality silica discs (Highest Grade V1 Mica Discs, Ted Pella Inc.) and dried in vacuum. Experiments were performed in the air, in contactless mode. Topographic images and “signal-error” images were collected, AFM images were created and analyzed with the software Image Analysis 2.2.0 (NT-MDT) and Gwyddion 2.60 (Free and Open Source software, Department of Nanometrology, Czech Metrology Institute). Small angle X-ray scattering (SAXS) SAXS experiments were performed with the general idea to analyze the structure of the dispersed nanodroplets more profoundly, and especially interactions in biorelevant surrounding (in contact with HSA). A laboratory X-ray setup was applied (Bruker Nanostar, Bruker AXS GmbH, Karlsruhe, Germany). Here, the Kα-line of a X-ray Cu source with a wavelength of 1.541 Å was used and further monochromated by a X-ray mirror. The beam was collimated to a beam diameter of approximately 0.4 mm using three pinholes. The sample-detector distance was set to 107 cm, which lead to a q-range of 0.07 ≤ q ≤ 2.3 nm-1. Calibration of the scattering vector q and estimation of the instrumental resolution of Δq = 0.25 nm-1 was done by measuring the first diffraction peak of a silver behenate sample. The scattered intensity was measured with an avalanche-based detector (VÅNTEC-2000, Bruker AXS). The transmitted part of the beam was determined using a home-made semi-transparent beam stop. The scattered intensity was extracted, radially averaged and integrated over all q-values using the Bruker software DIF-FRAC.EVA (Bruker AXS, version 4.1). The 1D data was transmission corrected and then background subtracted from the scattering of the solvent and the capillary using Matlab 2022. RESULTS AND DISCUSSION When applying DLS, as a preliminary technique, primary attention was put on the selection of optimal dilution level for the measurement, analyzing attenuation factor, count rate and intercept of the correlation function in different dilution ratios and with different dilution media (water, PBS 7.4 and 10 mM NaCl), and dilution 1:100 (v/v) was marked as the optimal one. However, significant differences in obtained nanodroplet size was observed depending on the type of medium. When water was used as a dilution medium, significantly higher Z-ave values were obtained (83.71±0.86 nm) compared to the situations where PBS 7.4 (73.50±0.75nm) or 10 mM NaCl (76.59±0.50nm) were used as dilution medium, indicating how sample preparation protocol might be crucial. Even though DLS was not sensitive enough to detect any interaction with HSA (no significant difference in terms of Z-ave and PDI compared to the results obtained in the same dilution medium without HSA), AFM captured qualitative difference in the droplet topography (Figure 1), raising ides on nanoemulsion interfacial interaction with HSA and increased aggregation potential. Further on, SAXS confirmed the existence of a bilayer structure as indicated by a prominent correlation peak at around 1 nm-1, which corresponds to a bilayer thickness of around 6.2 nm. SAXS (Figure 2; probably corresponding to the lecithin formations at the interface). It may be assumed that the bilayer structure changes its structure when mixed with HSA. CONCLUSION In this research, it has been demonstrated how important is to carefully select measurement conditions even for DLS -commonly used and the only standardized methods, in order to keep the measurements meaningful. Further on, not every method is capable of detecting some specific bio-nano interactions. Aiming to generate reliable datasets, condition sine qua non is to perform complementary techniques with increasing complexity. Further experimental segments should cover additional evaluation (e.g. analytical ultracentrifugation, thermal analysis, interfacial properties assessment, electron microscopy) that would shed light on bio-nano interactions important for in vivo fate of the nanosystems

Highly hydrated paramagnetic amorphous calcium carbonate nanoclusters as a superior MRI contrast agent

Amorphous calcium carbonate plays a key role as transient precursor in the early stages of biogenic calcium carbonate formation in nature. However, due to its instability in aqueous solution, there is still rare success to utilize amorphous calcium carbonate in biomedicine. Here, we report the mutual effect between paramagnetic gadolinium ions and amorphous calcium carbonate, resulting in ultrafine paramagnetic amorphous carbonate nanoclusters in the presence of both gadolinium occluded highly hydrated carbonate-like environment and poly(acrylic acid). Gadolinium is confirmed to enhance the water content in amorphous calcium carbonate, and the high water content of amorphous carbonate nanoclusters contributes to the much enhanced magnetic resonance imaging contrast efficiency compared with commercially available gadolinium-based contrast agents. Furthermore, the enhanced T1 weighted magnetic resonance imaging performance and biocompatibility of amorphous carbonate nanoclusters are further evaluated in various animals including rat, rabbit and beagle dog, in combination with promising safety in vivo. Overall, exceptionally facile mass-productive amorphous carbonate nanoclusters exhibit superb imaging performance and impressive stability, which provides a promising strategy to design magnetic resonance contrast agent

Resuscitative endovascular balloon occlusion of the aorta (REBOA): a population based gap analysis of trauma patients in England and Wales

INTRODUCTION: Non-compressible torso haemorrhage (NCTH) carries a high mortality in trauma as many patients exsanguinate prior to definitive haemorrhage control. Resuscitative endovascular balloon occlusion of the aorta (REBOA) is an adjunct that has the potential to bridge patients to definitive haemostasis. However, the proportion of trauma patients in whom REBOA may be utilised is unknown. METHODS: We conducted a population based analysis of 2012-2013 Trauma Audit and Research Network (TARN) data. We identified the number of patients in whom REBOA may have been utilised, defined by an Abbreviated Injury Scale score ≥3 to abdominal solid organs, abdominal or pelvic vasculature, pelvic fracture with ring disruption or proximal traumatic lower limb amputation, together with a systolic blood pressure <90 mm Hg. Patients with non-compressible haemorrhage in the mediastinum, axilla, face or neck were excluded. RESULTS: During 2012-2013, 72 677 adult trauma patients admitted to hospitals in England and Wales were identified. 397 patients had an indication(s) and no contraindications for REBOA with evidence of haemorrhagic shock: 69% men, median age 43 years and median Injury Severity Score 32. Overall mortality was 32%. Major trauma centres (MTCs) received the highest concentration of potential REBOA patients, and would be anticipated to receive a patient in whom REBOA may be utilised every 95 days, increasing to every 46 days in the 10 MTCs with the highest attendance of this injury type. CONCLUSIONS: This TARN database analysis has identified a small group of severely injured, resource intensive patients with a highly lethal injury that is theoretically amenable to REBOA. The highest density of these patients is seen at MTCs, and as such a planned evaluation of REBOA should be further considered in these hospitals

Calcium carbonate prenucleation clusters: towards unification of classical and non-classical nucleation theory

The aim of this PhD was to develop analytical methods to characterize the physical and chemical characteristics of calcium carbonate prenucleation clusters and their role in the mineral nucleation process. A novel sample environment was developed to allow fast in situ and ex situ kinetic experiments when coupled to synchrotron and neutron radiation techniques. The results obtained through these experiments support findings from previous computer simulations and define the impact of pH and saturation state on the very early stages of calcium carbonate mineral formation via non-classical nucleation processes

Calcium coordination environment in precursor species to calcium carbonate mineral formation

International audienceThe local coordination environment in precursors to calcium carbonate minerals has been studied at pH 7.5 and 8.5 by use of a novel fast mixing device with a freeze quench and freeze-drying method coupled to conventional X-ray absorption spectroscopy. Use of rapid reagent mixing and freeze quenching allowed triggering of (pre-)nucleation reactions and stabilisation of calcium carbonate precursors within 18 ms of contact between the reagent solutions. Using this approach, we examined the impact of saturation state and pH on the short-range order of calcium carbonate precursors. Results show that highly hydrated precursor structures characterised by low coordination number form in conditions below the solubility limit of calcite. The first and second coordination shells of these structures furthermore present similarities with the local coordination environment of calcite. Comparisons with previous molecular dynamics simulations suggest that the observed structures present characteristics most similar to simulated clusters possessing a high coordination number in their first shell. Substantial differences were observed between the local coordination environment of structures prepared at pH 7.5 and 8.5, even though both conditions are dominated by bicarbonate, suggesting a critical role for the carbonate ion under the conditions examined. Overall, our findings are consistent with previous work on the existence and structure of calcium carbonate prenucleation clusters and provide further support for the role of non-classical nucleation in these systems