Macromolecular condensation buffers intracellular water potential.

Optimum protein function and biochemical activity critically depends on water availability because solvent thermodynamics drive protein folding and macromolecular interactions1. Reciprocally, macromolecules restrict the movement of 'structured' water molecules within their hydration layers, reducing the available 'free' bulk solvent and therefore the total thermodynamic potential energy of water, or water potential. Here, within concentrated macromolecular solutions such as the cytosol, we found that modest changes in temperature greatly affect the water potential, and are counteracted by opposing changes in osmotic strength. This duality of temperature and osmotic strength enables simple manipulations of solvent thermodynamics to prevent cell death after extreme cold or heat shock. Physiologically, cells must sustain their activity against fluctuating temperature, pressure and osmotic strength, which impact water availability within seconds. Yet, established mechanisms of water homeostasis act over much slower timescales2,3; we therefore postulated the existence of a rapid compensatory response. We find that this function is performed by water potential-driven changes in macromolecular assembly, particularly biomolecular condensation of intrinsically disordered proteins. The formation and dissolution of biomolecular condensates liberates and captures free water, respectively, quickly counteracting thermal or osmotic perturbations of water potential, which is consequently robustly buffered in the cytoplasm. Our results indicate that biomolecular condensation constitutes an intrinsic biophysical feedback response that rapidly compensates for intracellular osmotic and thermal fluctuations. We suggest that preserving water availability within the concentrated cytosol is an overlooked evolutionary driver of protein (dis)order and function.

CRYPTOCHROMES promote daily protein homeostasis.

The daily organisation of most mammalian cellular functions is attributed to circadian regulation of clock-controlled protein expression, driven by daily cycles of CRYPTOCHROME-dependent transcriptional feedback repression. To test this, we used quantitative mass spectrometry to compare wild-type and CRY-deficient fibroblasts under constant conditions. In CRY-deficient cells, we found that temporal variation in protein, phosphopeptide, and K+ abundance was at least as great as wild-type controls. Most strikingly, the extent of temporal variation within either genotype was much smaller than overall differences in proteome composition between WT and CRY-deficient cells. This proteome imbalance in CRY-deficient cells and tissues was associated with increased susceptibility to proteotoxic stress, which impairs circadian robustness, and may contribute to the wide-ranging phenotypes of CRY-deficient mice. Rather than generating large-scale daily variation in proteome composition, we suggest it is plausible that the various transcriptional and post-translational functions of CRY proteins ultimately act to maintain protein and osmotic homeostasis against daily perturbation.

Compensatory ion transport buffers daily protein rhythms to regulate osmotic balance and cellular physiology.

Between 6-20% of the cellular proteome is under circadian control and tunes mammalian cell function with daily environmental cycles. For cell viability, and to maintain volume within narrow limits, the daily variation in osmotic potential exerted by changes in the soluble proteome must be counterbalanced. The mechanisms and consequences of this osmotic compensation have not been investigated before. In cultured cells and in tissue we find that compensation involves electroneutral active transport of Na+, K+, and Cl- through differential activity of SLC12A family cotransporters. In cardiomyocytes ex vivo and in vivo, compensatory ion fluxes confer daily variation in electrical activity. Perturbation of soluble protein abundance has commensurate effects on ion composition and cellular function across the circadian cycle. Thus, circadian regulation of the proteome impacts ion homeostasis with substantial consequences for the physiology of electrically active cells such as cardiomyocytes.

The promise of artificial intelligence: a review of the opportunities and challenges of artificial intelligence in healthcare.

INTRODUCTION: Artificial intelligence (AI) and machine learning (ML) are rapidly evolving fields in various sectors, including healthcare. This article reviews AI's present applications in healthcare, including its benefits, limitations and future scope. SOURCES OF DATA: A review of the English literature was conducted with search terms 'AI' or 'ML' or 'deep learning' and 'healthcare' or 'medicine' using PubMED and Google Scholar from 2000-2021. AREAS OF AGREEMENT: AI could transform physician workflow and patient care through its applications, from assisting physicians and replacing administrative tasks to augmenting medical knowledge. AREAS OF CONTROVERSY: From challenges training ML systems to unclear accountability, AI's implementation is difficult and incremental at best. Physicians also lack understanding of what AI implementation could represent. GROWING POINTS: AI can ultimately prove beneficial in healthcare, but requires meticulous governance similar to the governance of physician conduct. AREAS TIMELY FOR DEVELOPING RESEARCH: Regulatory guidelines are needed on how to safely implement and assess AI technology, alongside further research into the specific capabilities and limitations of its medical use.

CRYPTOCHROMES confer robustness, not rhythmicity, to circadian timekeeping.

Circadian rhythms are a pervasive property of mammalian cells, tissues and behaviour, ensuring physiological adaptation to solar time. Models of cellular timekeeping revolve around transcriptional feedback repression, whereby CLOCK and BMAL1 activate the expression of PERIOD (PER) and CRYPTOCHROME (CRY), which in turn repress CLOCK/BMAL1 activity. CRY proteins are therefore considered essential components of the cellular clock mechanism, supported by behavioural arrhythmicity of CRY-deficient (CKO) mice under constant conditions. Challenging this interpretation, we find locomotor rhythms in adult CKO mice under specific environmental conditions and circadian rhythms in cellular PER2 levels when CRY is absent. CRY-less oscillations are variable in their expression and have shorter periods than wild-type controls. Importantly, we find classic circadian hallmarks such as temperature compensation and period determination by CK1δ/ε activity to be maintained. In the absence of CRY-mediated feedback repression and rhythmic Per2 transcription, PER2 protein rhythms are sustained for several cycles, accompanied by circadian variation in protein stability. We suggest that, whereas circadian transcriptional feedback imparts robustness and functionality onto biological clocks, the core timekeeping mechanism is post-translational.