Solubility of Lysozyme in the Presence of Aqueous Chloride Salts: Common-Ion Effect and Its Role on Solubility and Crystal Thermodynamics

Abstract: Understanding protein solubility is important for a rational design of the conditions of protein crystallization. We report measurements of lysozyme solubility in aqueous solutions as a function of NaCl, KCl, and NH 4Cl concentrations at 25°C and pH 4.5. Our solubility results are directly compared to preferential-interaction coefficients of these ternary solutions determined in the same experimental conditions by ternary diffusion. This comparison has provided new important insight on the dependence of protein solubility on salt concentration. We remark that the dependence of the preferential-interaction coefficient as a function of salt concentration is substantially shaped by the common-ion effect. This effect plays a crucial role also on the observed behavior of lysozyme solubility. We find that the dependence of solubility on salt type and concentration strongly correlates with the corresponding dependence of the preferentialinteraction coefficient. Examination of both preferential-interaction coefficients and second virial coefficients has allowed us to demonstrate that the solubility dependence on salt concentration is substantially affected by the corresponding change of protein chemical potential in the crystalline phase. We propose a simple model for the crystalline phase based on salt partitioning between solution and the hydrated protein crystal. A novel solubility equation is reported that quantitatively explains the observed experimental dependence of protein solubility on salt concentration

Unus pro omnibus, omnes pro uno: A novel, evidence-based, unifying theory for the pathogenesis of endometriosis

The theory of retrograde menstruation as aetiopathogenesis of endometriosis formulated by John Sampson in 1927 shows clear shortcomings: this does not explain why retrograde menstruation is a physiological process that affects 90% of women, while endometriosis occurs in only 10% of cases; it also does not explain the endometriotic foci distant from the pelvis, nor explains the cases of endometriosis in male patients. The immunological alterations of the peritoneal fluid explains the effects of disease, such as the inhibition of the physiological processes of cytolysis, but does not explain the cause. There is evidence to support the hypothesis that ectopic müllerian remnants of the endometrium, endocervix and endosalpinx are items from the genital ridge leaked during organogenesis. It is known that tissues derived from coelomatic epithelial and mesenchymal cells have the potential to metaplastically differentiate into epithelium and stroma. In addition, the phenotype of the ectopic endometrial cells is significantly different from those ectopic. There is scientific evidence that, during organogenesis, the genes of the Homeobox and Wingless family play a fundamental role in the differentiation of the ducts of Muller and development of the anatomical structure of the urogenital tract. We present here a hypothesis that deregulation of genes and the Wnt signaling pathway Wnt/β-catenin leads to aberrations and deregulation within the mesoderm, thus, may cause aberrant placement of stem cells. In addition, immune cells, adhesion molecules, extracellular matrix metalloproteinase and pro-inflammatory cytokines activate/alter peritoneal microenvironment, creating the conditions for differentiation, adhesion, proliferation and survival of ectopic endometrial cells

Defining Kawasaki disease and pediatric inflammatory multisystem syndrome-temporally associated to SARS-CoV-2 infection during SARS-CoV-2 epidemic in Italy: results from a national, multicenter survey

Background: There is mounting evidence on the existence of a Pediatric Inflammatory Multisystem Syndrome-temporally associated to SARS-CoV-2 infection (PIMS-TS), sharing similarities with Kawasaki Disease (KD). The main outcome of the study were to better characterize the clinical features and the treatment response of PIMS-TS and to explore its relationship with KD determining whether KD and PIMS are two distinct entities. Methods: The Rheumatology Study Group of the Italian Pediatric Society launched a survey to enroll patients diagnosed with KD (Kawasaki Disease Group - KDG) or KD-like (Kawacovid Group - KCG) disease between February 1st 2020, and May 31st 2020. Demographic, clinical, laboratory data, treatment information, and patients' outcome were collected in an online anonymized database (RedCAP®). Relationship between clinical presentation and SARS-CoV-2 infection was also taken into account. Moreover, clinical characteristics of KDG during SARS-CoV-2 epidemic (KDG-CoV2) were compared to Kawasaki Disease patients (KDG-Historical) seen in three different Italian tertiary pediatric hospitals (Institute for Maternal and Child Health, IRCCS "Burlo Garofolo", Trieste; AOU Meyer, Florence; IRCCS Istituto Giannina Gaslini, Genoa) from January 1st 2000 to December 31st 2019. Chi square test or exact Fisher test and non-parametric Wilcoxon Mann-Whitney test were used to study differences between two groups. Results: One-hundred-forty-nine cases were enrolled, (96 KDG and 53 KCG). KCG children were significantly older and presented more frequently from gastrointestinal and respiratory involvement. Cardiac involvement was more common in KCG, with 60,4% of patients with myocarditis. 37,8% of patients among KCG presented hypotension/non-cardiogenic shock. Coronary artery abnormalities (CAA) were more common in the KDG. The risk of ICU admission were higher in KCG. Lymphopenia, higher CRP levels, elevated ferritin and troponin-T characterized KCG. KDG received more frequently immunoglobulins (IVIG) and acetylsalicylic acid (ASA) (81,3% vs 66%; p = 0.04 and 71,9% vs 43,4%; p = 0.001 respectively) as KCG more often received glucocorticoids (56,6% vs 14,6%; p < 0.0001). SARS-CoV-2 assay more often resulted positive in KCG than in KDG (75,5% vs 20%; p < 0.0001). Short-term follow data showed minor complications. Comparing KDG with a KD-Historical Italian cohort (598 patients), no statistical difference was found in terms of clinical manifestations and laboratory data. Conclusion: Our study suggests that SARS-CoV-2 infection might determine two distinct inflammatory diseases in children: KD and PIMS-TS. Older age at onset and clinical peculiarities like the occurrence of myocarditis characterize this multi-inflammatory syndrome. Our patients had an optimal response to treatments and a good outcome, with few complications and no deaths

Diffusiophoresis of a Nonionic Micelle in Salt Gradients; Roles of Preferential Hydration and Salt-Induced Surfactant Aggregation

Diffusiophoresis is the migration of a colloidal particle in water driven by concentration gradients of cosolutes such as salts. We have experimentally characterized the diffusiophoresis of tyloxapol micelles in the presence of MgSO4, a strong salting-out agent. Specifically, we determined the multicomponent-diffusion coefficients using Rayleigh interferometry, cloud points, and dynamic-light-scattering diffusion coefficients on the ternary tyloxapol–MgSO4–water system at 25 °C. Our experimental results show that micelle diffusiophoresis occurs from a high to a low salt concentration (positive diffusiophoresis). Moreover, our data were used to characterize the effect of salt concentration on micelle size and salt osmotic diffusion, which occurs from a high to a low surfactant concentration. Although micelle diffusiophoresis can be attributed to the preferential hydration of the polyethylene glycol surface groups, salting-out salts also promote an increase in the size of micellar aggregates, ultimately leading to phase separation at high salt concentration. This complicates diffusiophoresis description, as it is not clear how salt-induced surfactant aggregation contributes to micelle diffusiophoresis. We, therefore, developed a two-state aggregation model that successfully describes the observed effect of salt concentration on the size of tyloxapol micelles, in the case of MgSO4 and the previously reported case of Na2SO4. Our model was then used to theoretically evaluate the contribution of salt-induced aggregation to diffusiophoresis. Our analysis indicates that salt-induced aggregation promotes micelle diffusiophoresis from a low to a high salt concentration (negative diffusiophoresis). However, we also determined that this mechanism marginally contributes to overall diffusiophoresis, implying that preferential hydration is the main mechanism causing micelle diffusiophoresis. Our results suggest that sulfate salts may be exploited to induce the diffusiophoresis of PEG-functionalized particles such as micelles, with potential applications to microfluidics, enhanced oil recovery, and controlled-release technologies

Effects of Salting-In Interactions on Macromolecule Diffusiophoresis and Salt Osmotic Diffusion

Macromolecule diffusiophoresis (i.e., macromolecule migration induced by a salt concentration gradient) in water and salt osmotic diffusion (i.e., salt migration induced by a macromolecule concentration gradient) are two cross-diffusion mechanisms caused by macromolecule–salt interactions. We investigated the effect of salting-in interactions on the behavior of these two cross-diffusion mechanisms. Our results are distinct from those previously obtained in the case of salting-out interactions. Cross-diffusion was experimentally characterized by Rayleigh interferometry at 25 °C. Specifically, multicomponent diffusion coefficients were measured for a neutral polymer, polyethylene glycol (molar mass, 20 kg/mol), in aqueous solutions of three thiocyanate salts (NaSCN, KSCN, and NH₄SCN) as a function of salt concentration at low polymer concentration (0.5% w/w). Our results on salt osmotic diffusion, which were qualitatively different from those previously obtained for salting-out salts, were used to quantitatively characterize the strength of salting-in interactions. The behavior of polymer diffusiophoresis as a function of salt concentration and cation type reveals that polymer chains have an extrinsic negative charge, consistent with anion binding being the cause of salting-in interactions. To quantitatively examine the effect of anion binding on salt osmotic diffusion and polymer diffusiophoresis, we developed a theoretical model based on the linear laws of nonequilibrium thermodynamics for diffusion, the Scatchard binding model, and particle electrophoresis. This work contributes to the understanding of the multifaceted effects of molecular interactions on cross-diffusion mechanisms, salting-in interactions, and the Hofmeister series

Macromolecule Diffusiophoresis Induced by Concentration Gradients of Aqueous Osmolytes

Diffusiophoresis is the migration of a particle in a fluid induced by the concentration gradient of another solute. We have experimentally investigated diffusiophoresis of a neutral macromolecule, poly­(ethylene glycol) (PEG; molecular weight, 20 kg mol^–1), in water induced by concentration gradients of osmolytes. Three osmolytes were examined: trimethylamine-<i>N</i>-oxide (TMAO), diethylene glycol (DEG), and urea. PEG diffusiophoresis coefficients were obtained from measurements of multicomponent-diffusion coefficients at 25 °C using Rayleigh interferometry. Osmotic diffusion coefficients, characterizing osmolyte diffusion from high to low PEG concentration, were also extracted. PEG diffusiophoresis was found to occur from high to low osmolyte concentration in all cases, with magnitude increasing in the order urea < DEG < TMAO. This ranking is consistent with that of osmolyte effectiveness in stabilizing protein native state. Osmotic diffusion coefficients, which allowed us to determine preferential-interaction coefficients, revealed that TMAO and DEG are preferentially excluded from the vicinity of PEG whereas urea was found to preferentially bind to this macromolecule. A novel model for macromolecule diffusiophoresis, which allowed us to examine the roles of preferential hydration, hydration, solute binding, and frictional dragging in this transport process, was developed. Our experimental results suggest that TMAO concentration gradients may be exploited to direct the motion of PEG and PEG-functionalized particles such as micelles, PEGylated proteins, and PEG-coated inorganic nanoparticles with potential applications to separation and adsorption technologies