Effective interactions and phase behavior of protein solutions in the presence of hexamine cobalt(III) chloride.

It is well established that deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) exhibit a reentrant condensation (RC) phase behavior in the presence of the trivalent hexamine cobalt(III) cations (Hac) which can be important for their packing and folding. A similar behavior can be observed for negatively charged globular proteins in the presence of trivalent metal cations, such as Y3+ or La3+. This phase behavior is mainly driven by charge inversion upon an increasing salt concentration for a fixed protein concentration (cp). However, as Hac exhibits structural differences compared to other multivalent metal cations, with six ammonia ligands (NH3) covalently bonded to the central cobalt atom, it is not clear that Hac can induce a similar phase behavior for proteins. In this work, we systematically investigate whether negatively charged globular proteins ß-lactoglobulin (BLG), bovine serum albumin (BSA), human serum albumin (HSA) and ovalbumin (OVA) feature Hac-induced RC. Effective protein-protein interactions were investigated by small-angle X-ray scattering. The reduced second virial coefficient (B2/B2HS) was obtained as a function of salt concentration. The virial coefficient analysis performed confirms the reentrant interaction (RI) behavior for BLG without actually inducing RC, given the insufficient strengths of the interactions for the latter to occur. In contrast, the strength of attraction for BSA, HSA and OVA are too weak to show RC. Model free analysis of the inverse intensity [Formula: see text] also supports this finding. Looking at different q-range by employing static (SLS) and dynamic light scattering experiments, the presence of RI behavior can be confirmed. The results are further discussed in view of metal cation binding sites in nucleic acids (DNA and RNA), where Hac induced RC phase behavior.

An alternative approach to the osmotic second virial coefficient of protein solutions and its application to liquid-liquid phase separation.

The osmotic second virial coefficient B2 is an important parameter to describe the interactions and phase behavior of protein solutions, including colloidal systems and macromolecular solutions. Another key parameter to describe the driving force of the nucleation of a new phase is the supersaturation, which is used in the classical nucleation theory framework and is connected with the favorable contribution in the Gibbs free energy in the bulk solution. In this article, we establish a connection between B2 calculated from small angle x-ray scattering (SAXS) data and the values of B2 obtained from supersaturation measurements using thermodynamics considerations. The values of the second virial coefficient calculated employing this method agree with those determined via SAXS in the region near the liquid-liquid phase separation border for human serum albumin and bovine serum albumin. The general relations adopted are shown to be useful for the estimation of the second virial coefficient B2 for globular proteins, in the proximity of the binodal biphasic coexistent region.

Pathogen spectra in hospitalised and nonhospitalised children with community-acquired pneumonia.

Background: Paediatric community-acquired pneumonia (CAP) is a leading cause of paediatric morbidity. However, particularly for outpatients with paediatric CAP, data on aetiology and management are scarce. Methods: The prospective pedCAPNETZ study multicentrically enrols children and adolescents with outpatient-treated or hospitalised paediatric CAP in Germany. Blood and respiratory specimens were collected systematically, and comprehensive analyses of pathogen spectra were conducted. Follow-up evaluations were performed until day 90 after enrolment. Results: Between December 2014 and August 2020, we enrolled 486 children with paediatric CAP at eight study sites, 437 (89.9%) of whom had radiographic evidence of paediatric CAP. Median (interquartile range) age was 4.5 (1.6-6.6) years, and 345 (78.9%) children were hospitalised. The most prevalent symptoms at enrolment were cough (91.8%), fever (89.2%) and tachypnoea (62.0%). Outpatients were significantly older, displayed significantly lower C-reactive protein levels and were significantly more likely to be symptom-free at follow-up days 14 and 90. Pathogens were detected in 90.3% of all patients (one or more viral pathogens in 68.1%; one or more bacterial strains in 18.7%; combined bacterial/viral pathogens in 4.1%). Parainfluenza virus and Mycoplasma pneumoniae were significantly more frequent in outpatients. The proportion of patients with antibiotic therapy was comparably high in both groups (92.4% of outpatients versus 86.2% of hospitalised patients). Conclusion: We present first data on paediatric CAP with comprehensive analyses in outpatients and hospitalised cases and demonstrate high detection rates of viral pathogens in both groups. Particularly in young paediatric CAP patients with outpatient care, antibiotic therapy needs to be critically debated.

Structural Insights into Polymer-Bounded Lipid Nanodiscs.

Membrane proteins are an essential part of signaling and transport processes and are targeted by multiple drugs. To isolate and investigate them in their native state, polymer-bounded nanodiscs have become valuable tools. In this study, we investigate the lipid model system dimyristoyl-phosphocholine (DMPC) with the nanodisc-forming copolymers styrene maleic acid (SMA) and diisobutylene maleic acid (DIBMA). Using small-angle X-ray scattering (SAXS) and dynamic light scattering (DLS), we studied the influence of polymer concentration and temperature on the nanodisc structure. In Tris buffer, the size of nanodiscs formed with SMA is smaller compared to DIBMA at the same polymer ratio. In both cases, the size decreases monotonically with increasing polymer concentration, and this effect is more pronounced when using SMA. Measurements at temperatures (T) between 5 and 30 °C in phosphate buffer showed an incomplete solubilization at high T even at polymer/lipid ratios above that required for complete lipid solubilization. For DIBMA, the nanodiscs developed at lower temperatures are stable and the net repulsion increases, while for SMA, the individual nanodiscs possess smaller sizes and are less affected by T. However, using DLS, one can observe SMA agglomerates at low T. Interestingly, for both polymers, no drastic changes of the observable parameters (radius and bilayer thickness) are seen upon cooling, which would indicate a sharp (first-order) phase transition from liquid-crystalline to gel, but only gradual changes. Hence, we conclude that the transition from a gel toward a liquid-crystalline lipid phase proceeds over a broad T-range compared to a continuous lipid bilayer. These results can pave the way toward the development of better protocols for studying membrane proteins stabilized in this type of membrane mimics.

Molecular Flexibility of Antibodies Preserved Even in the Dense Phase after Macroscopic Phase Separation.

Antibody therapies are typically based on high-concentration formulations that need to be administered subcutaneously. These conditions induce several challenges, inter alia a viscosity suitable for injection, sufficient solution stability, and preservation of molecular function. To obtain systematic insights into the molecular factors, we study the dynamics on the molecular level under strongly varying solution conditions. In particular, we use solutions of antibodies with poly(ethylene glycol), in which simple cooling from room temperature to freezing temperatures induces a transition from a well-dispersed solution into a phase-separated and macroscopically arrested system. Using quasi-elastic neutron scattering during in situ cooling ramps and in prethermalized measurements, we observe a strong decrease in antibody diffusion, while internal flexibility persists to a significant degree, thus ensuring the movement necessary for the preservation of molecular function. These results are relevant for a more dynamic understanding of antibodies in high-concentration formulations, which affects the formation of transient clusters governing the solution viscosity.

Bulk phase behaviour vs interface adsorption: Effects of anions and isotopes on ß-lactoglobulin (BLG) interactions.

HYPOTHESIS: Protein adsorption is highly relevant in numerous applications ranging from food processing to medical implants. In this context, it is important to gain a deeper understanding of protein-protein and protein-surface interactions. Thus, the focus of this investigation is on the interplay of bulk properties and surface properties on protein adsorption. It was hypothesised that the type of solvent and ions in solution should significantly influence the protein's bulk and interface behaviour, which has been observed in literature and previous work for other net negatively charged, globular proteins such as bovine serum albumin (BSA). EXPERIMENTS: The phase behaviour of ß-lactoglobulin (BLG) with lanthanum chloride (LaCl3) and iodide (LaI3) in normal water H2O(l) and heavy water (D2O(l)) was established via optical microscopy and ultraviolet-visible spectroscopy. The formation of an adsorption layer and its properties such as thickness, density, structure, and hydration was investigated via neutron reflectivity, quartz-crystal microbalance with dissipation, and infra-red measurements. FINDINGS: ß-lactoglobulin does not show significant anion-induced or isotope-induced effects - neither in bulk nor at the solid-liquid interface, which deviates strongly from the behaviour of bovine serum albumin. We also provide a comprehensive discussion and comparison of protein-specific bulk and interface behaviour between bovine serum albumin and ß-lactoglobulin dependent on anion, cation, solvent, and substrate properties. These findings pave the way for understanding the transition from adsorption to crystallisation.