On the analysis of sedimentation velocity in the study of protein complexes

Sedimentation velocity analytical ultracentrifugation has experienced a significant transformation, precipitated by the possibility of efficiently fitting Lamm equation solutions to the experimental data. The precision of this approach depends on the ability to account for the imperfections of the experiment, both regarding the sample and the instrument. In the present work, we explore in more detail the relationship between the sedimentation process, its detection, and the model used in the mathematical data analysis. We focus on configurations that produce steep and fast-moving sedimentation boundaries, such as frequently encountered when studying large multi-protein complexes. First, as a computational tool facilitating the analysis of heterogeneous samples, we introduce the strategy of partial boundary modeling. It can simplify the modeling by restricting the direct boundary analysis to species with sedimentation coefficients in a predefined range. Next, we examine factors related to the experimental detection, including the magnitude of optical aberrations generated by out-of-focus solution columns at high protein concentrations, the relationship between the experimentally recorded signature of the meniscus and the meniscus parameter in the data analysis, and the consequences of the limited radial and temporal resolution of the absorbance optical scanning system. Surprisingly, we find that large errors can be caused by the finite scanning speed of the commercial absorbance optics, exceeding the statistical errors in the measured sedimentation coefficients by more than an order of magnitude. We describe how these effects can be computationally accounted for in SEDFIT and SEDPHAT

Perceptions of involvement in advance care planning and emotional functioning in patients with advanced cancer

Purpose: Advance Care Planning (ACP) is positively associated with the quality of care, but its impact on emotional functioning is ambiguous. This study investigated the association between perceptions of ACP involvement and emotional functioning in patients with advanced cancer. Methods: This study analyzed baseline data of 1,001 patients of the eQuiPe study, a prospective, longitudinal, multicenter, observational study on quality of care and quality of life in patients with advanced cancer in the Netherlands. Patients with metastatic solid cancer were asked to participate between November 2017 and January 2020. Patients’ perceptions of ACP involvement were measured by three self-administered statements. Emotional functioning was measured by the EORTC-QLQ-C30. A linear multivariable regression analysis was performed while taking gender, age, migrant background, education, marital status, and symptom burden into account. Results: The majority of patients (87%) reported that they were as much involved as they wanted to be in decisions about their future medical treatment and care. Most patients felt that their relatives (81%) and physicians (75%) were familiar with their preferences for future medical treatment and care. A positive association was found between patients’ perceptions of ACP involvement and their emotional functioning (b=0.162, p<0.001, 95%CI[0.095;0.229]) while controlling for relevant confounders. Conclusions: Perceptions of involvement in ACP are positively associated with emotional functioning in patients with advanced cancer. Future studies are needed to further investigate the effect of ACP on emotional functioning. Trial registration number: NTR6584 Date of registration: 30 June 2017 Implications for Cancer Survivors: Patients’ emotional functioning might improve from routine discussions regarding goals of future care. Therefore, integration of ACP into palliative might be promising

Improving community health networks for people with severe mental illness : a case study investigation

Introduction Policy drivers in mental health to address personal recovery, stigma and poor physical health indicate that new service solutions are required. This study aimed to understand how connections to people, places and activities were utilised by individuals with severe mental illness (SMI) to benefit health and wellbeing. Methods A five-module mixed-methods design was undertaken in two study sites. Data were collected from 150 network-mapping interviews and 41 in-depth follow-up interviews with people with SMI; in-depth interviews with 30 organisation stakeholders and 12 organisation leaders; and 44 telephone interviews with practitioners. We undertook a three-stage synthesis process including independent lived experience feedback, and a patient and public involvement team participated in tool design, data collection, analysis and write-up. Results Three personal network types were found in our study using the community health network approach: diverse and active; family and stable; formal and sparse. Controlled for other factors we found only four variables significantly associated with which network type a participant had: living alone or not; housing status; formal education; long-term sickness or disability. Diagnosis was not a factor. These variables are challenging to address but they do point to potential for network change. The qualitative interviews with people with SMI provided further understanding of connection-building and resource utilisation. We explored individual agency across each network type, and identified recognition of the importance and value of social support and active connection management alongside the risks of isolation, even for those most affected by mental illness. We identified tensions in personal networks, be that relationships with practitioners or families, dealing with the impact of stigma, or frustrations of not being in employment, which all impact on network resources and well-being. The value of connectedness within personal networks of people, place and activity for supporting recovery was evident in shaping identity, providing meaning to life and sense of belonging, gaining access to new resources, structuring routines and helping individuals ‘move on’ in their recovery journey. Health-care practitioners recognised that social factors were important in recovery but reported system-level barriers (workload, administrative bureaucracy, limited contact time with clients) in addressing these issues fully. Even practitioners working in third-sector services whose remit involved increasing clients’ social connection faced restrictions due to being evaluated by outcome criteria that limited holistic recovery-focused practices. Service providers were keen to promote recovery-focused approaches. We found contrasts between recovery ideology within mental health policy and recovery practice on the ground. In particular, the social aspects of supporting people with SMI are often underprioritised in the health-care system. In a demanding and changing context, strategic multiagency working was seen as crucial but we found few examples of embedded multisector organisation partnerships. Conclusion While our exploratory study has limitations, findings suggest potential for people with SMI to be supported to become more active managers of their personal networks to support well-being regardless of current network type. The health and social care system does not currently deliver multiagency integrated solutions to support SMI and social recovery

Rapid decline of follicular lymphoma-associated chylothorax after low dose radiotherapy to retroperitoneal lymphoma localization

Chylothorax is caused by disruption or obstruction of the thoracic duct or its tributaries that results in the leakage of chyle into the pleural space. A number of interventions have been used to treat chylothorax including the treatment of the underlying disease. Lymphoma is found in 70% of cases with nontraumatic malignant aetiology. Although patients usually have advanced lymphoma, supradiaphragmatic disease is not always present. We discuss the case of a 63-year-old woman presenting with progressive respiratory symptoms due to chylothorax. She was diagnosed with a stage IIE retroperitoneal grade 1 follicular lymphoma extending from the coeliac trunk towards the pelvic inlet. Despite thoracocentesis and medium-chain triglycerides (MCT), diet chylothorax reoccurred. After low dose radiotherapy (2×2 Gy) to the abdominal lymphoma there was a marked decrease in lymphadenopathy at the coeliac trunk and a complete regression of the pleural fluid. In this case, radiotherapy was shown to be an effective nontoxic treatment option for lymphoma-associated chylothorax with long-term remission of pleural effusion

Sedimentation Velocity Analysis of Heterogeneous Protein-Protein Interactions: Sedimentation Coefficient Distributions c(s) and Asymptotic Boundary Profiles from Gilbert-Jenkins Theory

Interacting proteins in rapid association equilibrium exhibit coupled migration under the influence of an external force. In sedimentation, two-component systems can exhibit bimodal boundaries, consisting of the undisturbed sedimentation of a fraction of the population of one component, and the coupled sedimentation of a mixture of both free and complex species in the reaction boundary. For the theoretical limit of diffusion-free sedimentation after infinite time, the shapes of the reaction boundaries and the sedimentation velocity gradients have been predicted by Gilbert and Jenkins. We compare these asymptotic gradients with sedimentation coefficient distributions, c(s), extracted from experimental sedimentation profiles by direct modeling with superpositions of Lamm equation solutions. The overall shapes are qualitatively consistent and the amplitudes and weight-average s-values of the different boundary components are quantitatively in good agreement. We propose that the concentration dependence of the area and weight-average s-value of the c(s) peaks can be modeled by isotherms based on Gilbert-Jenkins theory, providing a robust approach to exploit the bimodal structure of the reaction boundary for the analysis of experimental data. This can significantly improve the estimates for the determination of binding constants and hydrodynamic parameters of the complexes

The Modulation of Transthyretin Tetramer Stability by Cysteine 10 Adducts and the Drug Diflunisal: DIRECT ANALYSIS BY FLUORESCENCE-DETECTED ANALYTICAL ULTRACENTRIFUGATION*

Transthyretin (TTR) is normally a stable plasma protein. However, in cases of familial TTR-related amyloidosis and senile systemic amyloidosis (SSA), TTR is deposited as amyloid fibrils, leading to organ dysfunction and possibly death. The mechanism by which TTR undergoes the transition from stable, soluble precursor to insoluble amyloid fibril and the factors that promote this process are largely undetermined. Most models involve the dissociation of the native TTR tetramer as the initial step. It is largely accepted that the TTR gene mutations associated with TTR-related amyloidosis lead to the expression of variant proteins that are intrinsically unstable and prone to aggregation. It has been suggested that amyloidogenicity may be conferred to wild-type TTR (the form deposited in SSA) by chemical modification of the lone cysteine residue (Cys10) through mixed disulfide bonds. S-Sulfonation and S-cysteinylation are prevalent TTR modifications physiologically, and studies have suggested their ability to modulate the structure of TTR under denaturing conditions. In the present study, we have used fluorescence-detected sedimentation velocity to determine the effect of S-sulfonate and S-cysteine on the quaternary structural stability of fluorophore-conjugated recombinant TTR under nondenaturing conditions. We determined that S-sulfonation stabilized TTR tetramer stability by a factor of 7, whereas S-cysteinylation enhanced dissociation by 2-fold with respect to the unmodified form. In addition, we report the direct observation of tetramer stabilization by the potential therapeutic compound diflunisal. Finally, as proof of concept, we report the sedimentation of TTR in serum and the qualitative assessment of the resulting data

Sedimentation Velocity Analysis of Heterogeneous Protein-Protein Interactions: Lamm Equation Modeling and Sedimentation Coefficient Distributions c(s)

We describe algorithms for solving the Lamm equations for the reaction-diffusion-sedimentation process in analytical ultracentrifugation, and examine the potential and limitations for fitting experimental data. The theoretical limiting case of a small, uniformly distributed ligand rapidly reacting with a larger protein in a “constant bath” of the ligand is recapitulated, which predicts the reaction boundary to sediment with a single sedimentation and diffusion coefficient. As a consequence, it is possible to express the sedimentation profiles of reacting systems as c(s) distribution of noninteracting Lamm equation solutions, deconvoluting the effects of diffusion. For rapid reactions, the results are quantitatively consistent with the “constant bath” approximation, showing c(s) peaks at concentration-dependent positions. For slower reactions, the deconvolution of diffusion is still partially successful, with c(s) resolving peaks that reflect the populations of sedimenting species. The transition between c(s) peaks describing reaction boundaries of moderately strong interactions (K(D) ∼ 10(−6) M) or resolving sedimenting species was found to occur in a narrow range of dissociation rate constant between 10(−3) and 10(−4) s(−1). The integration of the c(s) peaks can lead to isotherms of species populations or s-value of the reaction boundary, respectively, which can be used for the determination of the equilibrium binding constant

Emotions and the habitus: Young people with socio-emotional differences (re)producing social, emotional and cultural capital in family and leisure space-times

In this paper we explore the importance of emotionally inter-dependent relationships to the functioning of embodied social capital and habitus. Drawing upon the experiences of young people with socio-emotional differences, we demonstrate how emotionally inter-dependent and relatively nurturing relationships are integral to the acquisition of social capital and to the co-construction and embodiment of habitus. The young people presented in this paper often had difficulties in forging social relationships and in acquiring symbolic and cultural capital in school spaces. However, we outline how these young people (re)produce and embody alternative kinds of habitus, based on emotionally reciprocal relationships forged through formal and informal leisure activities and familial and fraternal social relationships. These alternative forms of habitus provide sites of subjection, scope for acquiring social and cultural capital and a positive sense of identity in the face of problematic relations and experiences in school spaces