Coupling of binding and differential subdomain folding of the intrinsically disordered transcription factor CREB.

The cyclic AMP response element binding protein (CREB) contains a basic leucine zipper motif (bZIP) that forms a coiled coil structure upon dimerization and specific DNA binding. Although this state is well characterized, key features of CREB bZIP binding and folding are not well understood. We used single-molecule Förster resonance energy transfer (smFRET) to probe conformations of CREB bZIP subdomains. We found differential folding of the basic region and leucine zipper in response to different binding partners; a strong and previously unreported DNA-independent dimerization affinity; folding upon binding to nonspecific DNA; and evidence of long-range interdomain interactions in full-length CREB that modulate DNA binding. These studies provide new insights into DNA binding and dimerization and have implications for CREB function.

Serum neurofilament-light concentration and real-world outcome in MS.

BACKGROUND: Prognostication in multiple sclerosis (MS) remains challenging. Biomarkers capable of providing this information at diagnosis would be valuable in shaping therapeutic decisions. Measurement of neurofilament light (NfL) has shown promise in predicting clinical outcomes in established MS, but its ability to predict outcomes in real-world cohorts at diagnosis requires further validation. METHODS: We used linear regression to evaluate the relationship between serum NfL (sNfL), measured at the time of diagnosis with short-term (1-year) and medium-term (5-year) clinical outcomes in 164 people with MS from a real-world, population-based cohort. Cox proportional hazards regression was used to analyse the association between sNfL and subsequent hazard of relapse or sustained accumulation of disability (SAD). Analyses were adjusted for age and disease-modifying treatment (DMT). RESULTS: sNfL concentration at diagnosis was modestly associated with baseline EDSS score (ß = 0.272, 95% CI 0.051 to 0.494, p = 0.016). However, no significant associations were found between baseline sNfL and odds of relapse at 12-months, 5-year EDSS change, or the hazard of relapse or SAD over 5 years follow-up. Dichotomising baseline sNfL according to the median sNfL did not change these findings. CONCLUSIONS: sNfL appears to be of limited clinical utility in predicting future irreversible neurological disability in a largely untreated real-world population, and remains insufficiently validated to shape treatment decisions at the time of diagnosis. Further studies may be needed for sNfL to be considered as a prognostic marker in the MS clinic. However the masking effect of DMTs on the natural disease trajectory will continue to pose challenges.

Ratiometric Single-Molecule FRET Measurements to Probe Conformational Subpopulations of Intrinsically Disordered Proteins.

Over the past few decades, numerous examples have demonstrated that intrinsic disorder in proteins lies at the heart of many vital processes, including transcriptional regulation, stress response, cellular signaling, and most recently protein liquid-liquid phase separation. The so-called intrinsically disordered proteins (IDPs) involved in these processes have presented a challenge to the classic protein "structure-function paradigm," as their functions do not necessarily involve well-defined structures. Understanding the mechanisms of IDP function is likewise challenging because traditional structure determination methods often fail with such proteins or provide little information about the diverse array of structures that can be related to different functions of a single IDP. Single-molecule fluorescence methods can overcome this ensemble-average masking, allowing the resolution of subpopulations and dynamics and thus providing invaluable insights into IDPs and their function. In this protocol, we describe a ratiometric single-molecule Förster resonance energy transfer (smFRET) routine that permits the investigation of IDP conformational subpopulations and dynamics. We note that this is a basic protocol, and we provide brief information and references for more complex analysis schemes available for in-depth characterization. This protocol covers optical setup preparation and protein handling and provides insights into experimental design and outcomes, together with background information about theory and a brief discussion of troubleshooting. © 2020 by John Wiley & Sons, Inc. Basic Protocol: Ratiometric smFRET detection and analysis of IDPs Support Protocol 1: Fluorophore labeling of a protein through maleimide chemistry Support Protocol 2: Sample chamber preparation Support Protocol 3: Determination of direct excitation of acceptor by donor excitation and leakage of donor emission to acceptor emission channel.

Oculomotor schwannoma causing a progressive complete third-nerve palsy.

A 69-year-old woman presented with an 8-month history of diplopia and examination findings consistent with a right third-nerve palsy. Head MRI identified the presence of a 5.8 mm, nodular, isointense lesion in the suprasellar cistern, which demonstrated enhancement with gadolinium contrast. The lesion did not show any evidence of growth over a 3-month follow-up period. These MRI findings, alongside the clinical features, suggest oculomotor nerve schwannoma. Oculomotor schwannomas are a rare cause of third-nerve palsy. The presenting features and management options for oculomotor schwannomas are discussed to provide a framework for the diagnosis and management of these patients.

Using biomarkers to predict clinical outcomes in multiple sclerosis.

Long-term outcomes in multiple sclerosis (MS) are highly varied and treatment with disease-modifying therapies carries significant risks. Finding tissue biomarkers that can predict clinical outcomes would be valuable in individualising treatment decisions for people with MS. Several candidate biomarkers-reflecting inflammation, neurodegeneration and glial pathophysiology-show promise for predicting outcomes. However, many candidates still require validation in cohorts with long-term follow-up and evaluation for their independent contribution in predicting outcome when models are adjusted for known demographic, clinical and radiological predictors. Given the complexity of MS pathophysiology, heterogeneous panels comprising a combination of biomarkers that encompass the various aspects of neurodegenerative, glial and immune pathology seen in MS, may enhance future predictions of outcome.

Physical Chemistry of Cellular Liquid-Phase Separation.

Compartmentalization of biochemical processes is essential for cell function. Although membrane-bound organelles are well studied in this context, recent work has shown that phase separation is a key contributor to cellular compartmentalization through the formation of liquid-like membraneless organelles (MLOs). In this Minireview, the key mechanistic concepts that underlie MLO dynamics and function are first briefly discussed, including the relevant noncovalent interaction chemistry and polymer physical chemistry. Next, a few examples of MLOs and relevant proteins are given, along with their functions, which highlight the relevance of the above concepts. The developing area of active matter and non-equilibrium systems, which can give rise to unexpected effects in fluctuating cellular conditions, are also discussed. Finally, our thoughts for emerging and future directions in the field are discussed, including in vitro and in vivo studies of MLO physical chemistry and function.