In Situ Monitoring of Protein Unfolding/Structural States under Cold High-Pressure Stress.

Biopharmaceutical formulations may be compromised by freezing, which has been attributed to protein conformational changes at a low temperature, and adsorption to ice-liquid interfaces. However, direct measurements of unfolding/conformational changes in sub-0 °C environments are limited because at ambient pressure, freezing of water can occur, which limits the applicability of otherwise commonly used analytical techniques without specifically tailored instrumentation. In this report, small-angle neutron scattering (SANS) and intrinsic fluorescence (FL) were used to provide in situ analysis of protein tertiary structure/folding at temperatures as low as -15 °C utilizing a high-pressure (HP) environment (up to 3 kbar) that prevents water from freezing. The results show that the α-chymotrypsinogen A (aCgn) structure is reasonably maintained under acidic pH (and corresponding pD) for all conditions of pressure and temperature tested. On the other hand, reversible structural changes and formation of oligomeric species were detected near -10 °C via HP-SANS for ovalbumin under neutral pD conditions. This was found to be related to the proximity of the temperature of cold denaturation of ovalbumin (TCD ∼ -17 °C; calculated via isothermal chemical denaturation and Gibbs-Helmholtz extrapolation) rather than a pressure effect. Significant structural changes were also observed for a monoclonal antibody, anti-streptavidin IgG1 (AS-IgG1), under acidic conditions near -5 °C and a pressure of ∼2 kbar. The conformational perturbation detected for AS-IgG1 is proposed to be consistent with the formation of unfolding intermediates such as molten globule states. Overall, the in situ approaches described here offer a means to characterize the conformational stability of biopharmaceuticals and proteins more generally under cold-temperature stress by the assessment of structural alteration, self-association, and reversibility of each process. This offers an alternative to current ex situ methods that are based on higher temperatures and subsequent extrapolation of the data and interpretations to the cold-temperature regime.

A New Perspective on Scale-Down Strategies for Freezing of Biopharmaceutics by Means of Computational Fluid Dynamics.

Common approaches to scale-down freeze-thaw systems are based on matching time-temperature profiles at corresponding points; however, little is known about the differences in anisotropy between the 2 scales. In this work, computational fluid dynamics modeling was used to investigate these differences. The modeling of the convective flow of the liquid phase within ice porous structure and volume expansion caused by freezing enabled accurate prediction of the local temperature and composition, for evaluation of potential stresses on protein stability, such as cryoconcentration and time in the nonideal environment. Overall, the small height of the scale-down containers enhances cryoconcentration. The time under stress was consistent in both scales, except when the walls of the container could deform. In general, the common approach of matching the time-temperature profile at the center of the containers was more effective as a worst-case scenario than a scale-down model. This work shows that instead of considering a single matching time-temperature location; one should aim for a more general perspective by measuring many locations. Container geometries and heat transfer rates should be designed to match stresses related to protein integrity for equivalent mass fractions between both scales, which can be achieved with the assistance of computational fluid dynamics models.

Prevalencia de dislipidemias en la región capital: resultados preliminares del estudio EVESCAM / Prevalence of dyslipidemias in the capital region: preliminary results of the EVESCAM study

Las dislipidemias son un factor de riesgo para enfermedades cardiovasculares. Se desconoce la prevalencia actual de dislipidemias en la región Capital de Venezuela. Objetivo: Determinar la prevalencia de dislipidemias en adultos de la región capital evaluados en el estudio EVESCAM. Métodos: Estudio poblacional, observacional, transversal de muestreo aleatorio poliestratificado por conglomerados. Se evaluaron 7 comunidades de la Región Capital desde julio de 2015 hasta enero de 2016: El Retiro; Miranda Casco Central y Bello Campo; Los Teques: La Cima; Guatire: Centro y Castillejo y rural: Guatire: La Candelaria. Participaron 416 sujetos desde los 20 años de edad. Los puntos de corte para definir las dislipidemias fueron hipoalfalipoproteinemia: colesterol HDL < 40 mg/dL; hipertrigliceridemia: triglicéridos (TG) ≥ 150 mg/dL; hipercolesterolemia: colesterol total ≥ 200 mg/dL; colesterol LDL elevado: colesterol LDL ≥ de 130 mg/dL; dislipidemia aterogénica: TG ≥ 150 mg/dL más colesterol HDL bajo (mujeres: < 40 mg/dl y hombres: < 50 mg/dl). Las frecuencias se expresaron en porcentajes y se aplicó el estadístico Chi cuadrado, un valor de p < 0,05 fue considerado como estadísticamente significativo. Resultados: La dislipidemia con mayor prevalencia fue la hipoalfalipoproteinemia (67.1%) seguida de la LDLc elevada (20%), hipercolesterolemia (17,1%), hipertrigliceridemia (12,0%) y por último dislipidemia aterogenica (9,4%). La hipoalfalipoproteinemia, fue mayor en hombres que en mujeres (81,6% y 60,8%; respectivamente, p < 0,001) presentándose con mayor prevalencia en el grupo etario de 20 a 40 años al contrario del resto de las dislipidemias. Conclusión: La hipoalfalipoproteinemia persiste como la dislipidemia más prevalente de la región(AU)

Dyslipidemias are a risk factor for cardiovascular diseases. The current prevalence of dyslipidemias in the Capital Region of Venezuela is unknown. Objective: To determine the prevalence of dyslipidemias in adults from the capital region of Venezuela evaluated in the EVESCAM study. Methods: apopulation based, observational, cross-sectional, and cluster sampling study was desing. Seven communities from the Capital Region were evaluated from July 2015 to January 2016: El Retiro; Miranda- Chacao: Casco Central y Bello Campo; Los Teques: La Cima; Guatire: Centro y Castillejo y Rural: Guatire: Candelaria. 416 subjects were included. Dyslipidemias was define as hypoalphalipoproteinemia: HDL cholesterol <40 mg/ dL; hypertriglyceridemia: triglycerides ≥ 150 mg/dL; hypercholesterolemia: total cholesterol ≥ 200 mg/dL; High LDL cholesterol: ≥ 130 mg/dL; therogenic dyslipidemia: triglycerides ≥ 150 mg / dL and low HDL cholesterol (women: <40 mg / dl and men: <50 mg / dl). The frequencies were expressed as percentages and Chi-square test was applied to assess differences. The level of statistical significance accepted was a p-value < 0.05. Results: The most prevalent dyslipidemia was hypoalphalipoproteinemia (67.1%) followed by elevated LDLc (20%), hypercholesterolemia (17.1%), hypertriglyceridemia (12.0%), and atherogenic dyslipidemia (9.4%). Hypoalphalipoproteinemia was higher in men than women (81.6% and 60.8%, respectively, p <0.001), with a higher prevalence at the age group of 20 to 40 years, unlike the rest of dyslipidemias. Conclusion: The hypoalphalipoproteinemia persists as the most prevalent dyslipidemia in the region(AU)