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 timescales^2,3; 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.

Patients undergo interventions to achieve a ‘normal’ brain temperature; a parameter that remains undefined for humans. The profound sensitivity of neuronal function to temperature implies the brain should be isothermal, but observations from patients and non-human primates suggest significant spatiotemporal variation. We aimed to determine the clinical relevance of brain temperature in patients by establishing how much it varies in healthy adults. We retrospectively screened data for all patients recruited to the Collaborative European NeuroTrauma Effectiveness Research in Traumatic Brain Injury (CENTER-TBI) High Resolution Intensive Care Unit Sub-Study. Only patients with direct brain temperature measurements and without targeted temperature management were included. To interpret patient analyses, we prospectively recruited 40 healthy adults (20 males, 20 females, 20–40 years) for brain thermometry using magnetic resonance spectroscopy. Participants were scanned in the morning, afternoon, and late evening of a single day. In patients (n = 114), brain temperature ranged from 32.6 to 42.3°C and mean brain temperature (38.5 ± 0.8°C) exceeded body temperature (37.5 ± 0.5°C, P < 0.0001). Of 100 patients eligible for brain temperature rhythm analysis, 25 displayed a daily rhythm, and the brain temperature range decreased in older patients (P = 0.018). In healthy participants, brain temperature ranged from 36.1 to 40.9°C; mean brain temperature (38.5 ± 0.4°C) exceeded oral temperature (36.0 ± 0.5°C) and was 0.36°C higher in luteal females relative to follicular females and males (P = 0.0006 and P < 0.0001, respectively). Temperature increased with age, most notably in deep brain regions (0.6°C over 20 years, P = 0.0002), and varied spatially by 2.41 ± 0.46°C with highest temperatures in the thalamus. Brain temperature varied by time of day, especially in deep regions (0.86°C, P = 0.0001), and was lowest at night. From the healthy data we built HEATWAVE—a 4D map of human brain temperature. Testing the clinical relevance of HEATWAVE in patients, we found that lack of a daily brain temperature rhythm increased the odds of death in intensive care 21-fold (P = 0.016), whilst absolute temperature maxima or minima did not predict outcome. A warmer mean brain temperature was associated with survival (P = 0.035), however, and ageing by 10 years increased the odds of death 11-fold (P = 0.0002). Human brain temperature is higher and varies more than previously assumed—by age, sex, menstrual cycle, brain region, and time of day. This has major implications for temperature monitoring and management, with daily brain temperature rhythmicity emerging as one of the strongest single predictors of survival after brain injury. We conclude that daily rhythmic brain temperature variation—not absolute brain temperature—is one way in which human brain physiology may be distinguished from pathophysiology.

Prions are infectious agents that cause neurodegenerative diseases such as Creutzfeldt–Jakob disease (CJD). The absence of a human cell culture model that replicates human prions has hampered prion disease research for decades. In this paper, we show that astrocytes derived from human induced pluripotent stem cells (iPSCs) support the replication of prions from brain samples of CJD patients. For experimental exposure of astrocytes to variant CJD (vCJD), the kinetics of prion replication occur in a prion protein codon 129 genotype–dependent manner, reflecting the genotype-dependent susceptibility to clinical vCJD found in patients. Furthermore, iPSC-derived astrocytes can replicate prions associated with the major sporadic CJD strains found in human patients. Lastly, we demonstrate the subpassage of prions from infected to naive astrocyte cultures, indicating the generation of prion infectivity in vitro. Our study addresses a long-standing gap in the repertoire of human prion disease research, providing a new in vitro system for accelerated mechanistic studies and drug discovery.

Acute mountain sickness (AMS) is a common problem among visitors at high altitude, and may progress to life-threatening pulmonary and cerebral oedema in a minority of cases. International consensus defines AMS as a constellation of subjective, non-specific symptoms. Specifically, headache, sleep disturbance, fatigue and dizziness are given equal diagnostic weighting. Different pathophysiological mechanisms are now thought to underlie headache and sleep disturbance during acute exposure to high altitude. Hence, these symptoms may not belong together as a single syndrome. Using a novel visual analogue scale (VAS), we sought to undertake a systematic exploration of the symptomatology of AMS using an unbiased, data-driven approach originally designed for analysis of gene expression. Symptom scores were collected from 292 subjects during 1110 subject-days at altitudes between 3650 m and 5200 m on Apex expeditions to Bolivia and Kilimanjaro. Three distinct patterns of symptoms were consistently identified. Although fatigue is a ubiquitous finding, sleep disturbance and headache are each commonly reported without the other. The commonest pattern of symptoms was sleep disturbance and fatigue, with little or no headache. In subjects reporting severe headache, 40% did not report sleep disturbance. Sleep disturbance correlates poorly with other symptoms of AMS (Mean Spearman correlation 0.25). These results challenge the accepted paradigm that AMS is a single disease process and describe at least two distinct syndromes following acute ascent to high altitude. This approach to analysing symptom patterns has potential utility in other clinical syndromes.

The neuronal ceroid lipofuscinoses (NCLs) are a group of devastating monogenetic lysosomal disorders that affect children and young adults with no cure or effective treatment currently available. One of the more severe infantile forms of the disease (INCL or CLN1 disease) is due to mutations in the palmitoyl-protein thioesterase 1 (PPT1) gene and severely reduces the child’s lifespan to approximately 9 years of age. In order to better translate the human condition than is possible in mice, we sought to produce a large animal model employing CRISPR/Cas9 gene editing technology. Three PPT1 homozygote sheep were generated by insertion of a disease-causing PPT1 (R151X) human mutation into the orthologous sheep locus. This resulted in a morphological, anatomical and biochemical disease phenotype that closely resembles the human condition. The homozygous sheep were found to have significantly reduced PPT1 enzyme activity and accumulate autofluorescent storage material, as is observed in CLN1 patients. Clinical signs included pronounced behavioral deficits as well as motor deficits and complete loss of vision, with a reduced lifespan of 17 ± 1 months at a humanely defined terminal endpoint. Magnetic resonance imaging (MRI) confirmed a significant decrease in motor cortical volume as well as increased ventricular volume corresponding with observed brain atrophy and a profound reduction in brain mass of 30% at necropsy, similar to alterations observed in human patients. In summary, we have generated the first CRISPR/Cas9 gene edited NCL model. This novel sheep model of CLN1 disease develops biochemical, gross morphological and in vivo brain alterations confirming the efficacy of the targeted modification and potential relevance to the human condition.

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.