Polymorphic protein phase transitions driven by surface anisotropy.

Phase transitions of proteins are strongly influenced by surface chemical modifications or mutations. Human γD-crystallin (HGD) single-mutants have been extensively studied because they are associated with the onset of juvenile cataract. However, they have also provided a rich library of molecules to examine how specific inter-protein interactions direct protein assembly, providing new insights and valuable experimental data for coarse-grained patchy-particle models. Here, we demonstrate that the addition of new inter-protein interactions by mutagenesis is additive and increases the number and variety of condensed phases formed by proteins. When double mutations incorporating two specific single point mutations are made, the properties of both single mutations are retained in addition to the formation of a new condensed phase. We find that the HGD double-mutant P23VC110M self-assembles into spherical particles with retrograde solubility, orthorhombic crystals, and needle/plate shape crystals, while retaining the ability to undergo liquid-liquid phase separation. This rich polymorphism is only partially predicted by the experimental data on the constituent single mutants. We also report a previously un-characterized amorphous protein particle, with unique properties that differ from those of protein spherulites, protein particulates previously described. The particles we observe are amorphous, reversible with temperature, tens of microns in size, and perfectly spherical. When they are grown on pristine surfaces, they appear to form by homogeneous nucleation, making them unique, and we believe a new form of protein condensate. This work highlights the challenges in predicting protein behavior, which has frustrated rational assembly and crystallization but also provides rich data to develop new coarse-grained models to explain the observed polymorphism.

The experiences and perceptions of young people and older people living with dementia of participating in intergenerational programmes: A qualitative evidence synthesis.

BACKGROUND: Intergenerational programmes are formal activities bringing different generations together and have been identified as a way to help people living with dementia to stay socially connected. While there is some evidence from individual studies as to their benefits, there is no overall coherent account as to the perceptions and experiences of participants who engage in such programmes. This review synthesises qualitative evidence of the experiences and perceptions of young people and older people living with dementia of participating in such programmes. METHODS: We searched EBSCO CINAHL, OVID Medline, Embase, Ovid PsycINFO, the Web of Science, Epistemonikos and grey literature sources. We used thematic synthesis to analyse and synthesise the evidence in to four themes, with 11 key findings. We assessed our confidence in each of these findings using the GRADE-CERQual (Confidence in the Evidence from Reviews of Qualitative research) approach. FINDINGS: Our review highlights the potential enjoyment for young people and older people living with dementia when participating in Intergenerational programmes, despite some initial trepidation. These programmes provide an opportunity to establish and develop relationships and for young people to learn about dementia, ageing and how to interact with older people living with dementia. However, it is important to have staff facilitators present to provide reassurance to both groups. It is also important to take the personal preferences of participants into account and to be considerate of noise levels and other aspects of programme delivery that may inhibit engagement. CONCLUSION: This is the first qualitative evidence synthesis specifically exploring Intergenerational programmes aimed at older people living with dementia. We provide insights into the perspectives of those who have participated in Intergenerational programmes. It is important to consider these views, together with other evidence of effectiveness, when planning Intergenerational programmes. While our review is limited by a small number of studies from only a few countries, we have moderate to high confidence in our findings. Further research into the development of Intergenerational programmes specifically tailored for people living with dementia is needed. The findings also provide guidance for people planning to deliver or design future Intergenerational programmes.

Real-world prospective observational single-centre study: Hybrid closed loop improves HbA1c, time-in-range and quality of life for children, young people and their carers.

Hybrid closed-loop (HCL) systems are characterised by integrating continuous glucose monitoring (CGM) with insulin pumps which automate insulin delivery via specific algorithms and user-initiated insulin delivery. The aim of the study was to evaluate the effectiveness of HCLs on Hba1c, time-in-range (TIR), time in hypoglycaemia, fear of hypoglycaemia, sleep and quality of life measure in children and young people (CYP) with T1D and their carers. Data on HbA1c, TIR and hypoglycaemia frequency were reviewed at baseline prior to starting HCL and 3 months after commencement. As part of clinical care, all patients and carers were provided with key education on the use of the HCL system by trained diabetes healthcare professionals. CYP aged 12 years and above independently completed the validated Hypoglycaemia Fear Survey (HFS). Parents of patients <12 were asked to complete a modified version of the HFS-Parent (HFS-P) survey. There were 39 CYP (22 men) with T1D included with a mean age of 11.8 ± 4.4 at commencement of HCL. Median duration of diabetes was 3.8 years (interquartile range 1.3-6.0). There were 55% of patients who were prepubertal at the time of HCL commencement. 91% were on the Control-IQ system and 9% on the CamAPS FX system. HCL use demonstrated significant improvements at 3 months in the following: HbA1c in mmol/mol (63.0 vs. 56.6, p = 0.03), TIR (50.5 vs. 67.0, p = 0.001) and time in hypoglycaemia (4.3% vs. 2.8%, p = 0.004). HFS scores showed improved behaviour (34.0 vs. 27.5.9, p = 0.02) and worry (40.2 vs. 31.6, p = 0.03), and HFS-P scores also showed improved behaviour (p < 0.001) and worry (p = 0.01). Our study shows that HCL at 3 months improves glucose control, diabetes management and quality of life measures such as fear and worry of hypoglycaemia for CYP and carers.

Using Schematic Models to Understand the Microscopic Basis for Inverted Solubility in γD-Crystallin.

Inverted solubility-melting a crystal by cooling-is observed in a handful of proteins, such as carbomonoxy hemoglobin C and γD-crystallin. In human γD-crystallin, the phenomenon is associated with the mutation of the 23rd residue, a proline, to a threonine, serine, or valine. One proposed microscopic mechanism entails an increase in surface hydrophobicity upon mutagenesis. Recent crystal structures of a double mutant that includes the P23T mutation allow for a more careful investigation of this proposal. Here, we first measure the surface hydrophobicity of various mutant structures of γD-crystallin and discern no notable increase in hydrophobicity upon mutating the 23rd residue. We then investigate the solubility inversion regime with a schematic patchy particle model that includes one of three variants of temperature-dependent patch energies: two of the hydrophobic effect, and one of a more generic nature. We conclude that, while solubility inversion due to the hydrophobic effect may be possible, microscopic evidence to support it in γD-crystallin is weak. More generally, we find that solubility inversion requires a fine balance between patch strengths and their temperature-dependent component, which may explain why inverted solubility is not commonly observed in proteins. We also find that the temperature-dependent interaction has only a negligible impact on liquid-liquid phase boundaries of γD-crystallin, in line with previous experimental observations.

Temperature-Dependent Interactions Explain Normal and Inverted Solubility in a γD-Crystallin Mutant.

Protein crystal production is a major bottleneck in the structural characterization of proteins. To advance beyond large-scale screening, rational strategies for protein crystallization are crucial. Understanding how chemical anisotropy (or patchiness) of the protein surface, due to the variety of amino-acid side chains in contact with solvent, contributes to protein-protein contact formation in the crystal lattice is a major obstacle to predicting and optimizing crystallization. The relative scarcity of sophisticated theoretical models that include sufficient detail to link collective behavior, captured in protein phase diagrams, and molecular-level details, determined from high-resolution structural information, is a further barrier. Here, we present two crystal structures for the P23T + R36S mutant of γD-crystallin, each with opposite solubility behavior: one melts when heated, the other when cooled. When combined with the protein phase diagram and a tailored patchy particle model, we show that a single temperature-dependent interaction is sufficient to stabilize the inverted solubility crystal. This contact, at the P23T substitution site, relates to a genetic cataract and reveals at a molecular level the origin of the lowered and retrograde solubility of the protein. Our results show that the approach employed here may present a productive strategy for the rationalization of protein crystallization.

Chemical Modification Alters Protein-Protein Interactions and Can Lead to Lower Protein Solubility.

The chemical modification of proteins is at the frontier of developments in biological imaging and biopharmaceutics. With the advent of more sensitive and higher resolution imaging techniques,  researchers increasingly rely on the functionalization of proteins to enable these techniques to capture cellular processes. For biopharmaceutical therapies, chemically modified proteins, for example, antibody-drug conjugates (ADCs) offer the possibility of more tailored treatments for the disease with lower toxicities than traditional small molecule therapies. However, relatively little consideration is paid to how chemical modifications impact protein-protein interactions and solution stability. Using human γD-crystallin as a model, we demonstrate that chemical modification of the protein surface alters protein-protein interactions, which can result in lower solubility depending on the chemical nature of the modifier and the position on the protein where the modification is made. Understanding these effects is essential to ensure that modifying proteins effectively occurs with minimum self-association and that studies carried out using labeled proteins accurately reflect those of unmodified proteins.

Protein self-assembly following in situ expression in artificial and mammalian cells.

The self-assembly of proteins has been widely studied in controlled in vitro conditions, and more recently in biological environments. The self-assembly of proteins in biology can be a feature of the pathogenesis of protein condensation disease, or can occur during normal physiological function, for example during the formation of intracellular non-membrane bound organelles. To determine the mechanisms for the assembly process fully, controlled in vitro experiments using purified protein solutions are often required. However, making direct connections between insights gathered from controlled experiments and those in complex biological environments remains a challenge. Using the P23T mutant of human γD-crystallin, a protein associated with congenital cataract, we have demonstrated that the equilibrium solubility boundary and solution behavior measured using phase diagrams of purified protein solutions is consistent with the assembly of the protein expressed in cell-free expression medium in artificial cells (without fluorescent labelling) and condensates formed in mammalian cells, thereby directly connecting in vitro measurements with those performed under physiological conditions.

The self assembly of proteins; probing patchy protein interactions.

The ability to control the self-assembly of biological molecules to form defined structures, with a high degree of predictability is a central aim for soft matter science and synthetic biology. Several examples of this are known for synthetic systems, such as anisotropic colloids. However, for biomacromolecules, such as proteins, success has been more limited, since aeolotopic (or anisotropic) interactions between protein molecules are not easily predicted. We have created three double mutants of human γD-crystallin for which the phase diagrams for singly mutated proteins can be used to predict the behavior of the double mutants. These proteins provide a robust mechanism to examine the kinetic and thermodynamic properties of proteins in which competing interactions exist due to the anisotropic or patchy nature of the protein surface.