Cortical acetylcholine dynamics are predicted by cholinergic axon activity and behavior state.

Even under spontaneous conditions and in the absence of changing environmental demands, awake animals alternate between increased or decreased periods of alertness. These changes in brain state can occur rapidly, on a timescale of seconds, and neuromodulators such as acetylcholine (ACh) are thought to play an important role in driving these spontaneous state transitions. Here, we perform the first simultaneous imaging of ACh sensors and GCaMP-expressing axons in vivo, to examine the spatiotemporal properties of cortical ACh activity and release during spontaneous changes in behavioral state. We observed a high correlation between simultaneously recorded basal forebrain axon activity and neuromodulator sensor fluorescence around periods of locomotion and pupil dilation. Consistent with volume transmission of ACh, increases in axon activity were accompanied by increases in local ACh levels that fell off with the distance from the nearest axon. GRAB-ACh fluorescence could be accurately predicted from axonal activity alone, providing the first validation that neuromodulator axon activity is a reliable proxy for nearby neuromodulator levels. Deconvolution of fluorescence traces allowed us to account for the kinetics of the GRAB-ACh sensor and emphasized the rapid clearance of ACh for smaller transients outside of running periods. Finally, we trained a predictive model of ACh fluctuations from the combination of pupil size and running speed; this model performed better than using either variable alone, and generalized well to unseen data. Overall, these results contribute to a growing understanding of the precise timing and spatial characteristics of cortical ACh during fast brain state transitions.

Integrated Direct-to-Biology Platform for the Nanoscale Synthesis and Biological Evaluation of PROTACs.

Proteolysis targeting chimeras (PROTACs) are heterobifunctional molecules that co-opt the cell's natural proteasomal degradation mechanisms to degrade undesired proteins. A challenge associated with PROTACs is the time and resource-intensive optimization; thus, the development of high-throughput platforms for their synthesis and biological evaluation is required. In this study, we establish an ultra-high-throughput experimentation (ultraHTE) platform for PROTAC synthesis, followed by direct addition of the crude reaction mixtures to cellular degradation assays without any purification. This 'direct-to-biology' (D2B) approach was validated and then exemplified in a medicinal chemistry campaign to identify novel BRD4 PROTACs. Using the D2B platform, the synthesis of 650 PROTACs was carried out in a 1536-well plate, and subsequent biological evaluation was performed by a single scientist in less than 1 month. Due to its ability to hugely accelerate the optimization of new degraders, we anticipate our platform will transform the synthesis and testing of PROTACs.

Biomimetic behaviors in hydrogel artificial cells through embedded organelles.

Artificial cells are biomimetic structures formed from molecular building blocks that replicate biological processes, behaviors, and architectures. Of these building blocks, hydrogels have emerged as ideal, yet underutilized candidates to provide a gel-like chassis in which to incorporate both biological and nonbiological componentry which enables the replication of cellular functionality. Here, we demonstrate a microfluidic strategy to assemble biocompatible cell-sized hydrogel-based artificial cells with a variety of different embedded functional subcompartments, which act as engineered synthetic organelles. The organelles enable the recreation of increasingly biomimetic behaviors, including stimulus-induced motility, content release through activation of membrane-associated proteins, and enzymatic communication with surrounding bioinspired compartments. In this way, we showcase a foundational strategy for the bottom-up construction of hydrogel-based artificial cell microsystems which replicate fundamental cellular behaviors, paving the way for the construction of next-generation biotechnological devices.

Evaluation of a retinol containing topical treatment to improve signs of neck aging.

BACKGROUND: The neck region is an area that can be indicative of signs of skin aging. A novel topical product that combines multiple active ingredients including retinol, tripeptide and glaucine was formulated to specifically target neck aging correction and complement post-procedure as part of an integrated skincare regimen. OBJECTIVES: To evaluate the efficacy of a topical neck treatment through clinical subject evaluation, in addition to ultrasound and biopsy assessment. METHODS: Evaluation for the efficacy of this novel topical product on improving the aging signs of neck skin was performed in multiple clinical trials. The first trial focused on clinical efficacy and included clinical assessment, subject questionnaires, ultrasound imaging and digital photographs. The second trial focused on biomarker analysis through skin biopsy. RESULTS: Data from the clinical trials showed that aging signs on the neck were significantly improved after 12 or 16 weeks of product usage. Changes were readily observed by clinical evaluators and participants. They were documented with digital photos, ultrasound images, and biomarker expression in the skin which clearly display the improvements. CONCLUSIONS: This novel topical product is effective in treating the aging signs on the neck skin and has been shown to provide statistically significant improvement on a myriad of neck aging attributes including fine lines/wrinkles, crepiness, laxity, and texture.

Complex motion of Greenland Ice Sheet outlet glaciers with basal temperate ice.

Uncertainty associated with ice sheet motion plagues sea level rise predictions. Much of this uncertainty arises from imperfect representations of physical processes including basal slip and internal ice deformation, with ice sheet models largely incapable of reproducing borehole-based observations. Here, we model isolated three-dimensional domains from fast-moving (Sermeq Kujalleq/Store Glacier) and slow-moving (Isunnguata Sermia) ice sheet settings in Greenland. By incorporating realistic geostatistically simulated topography, we show that a spatially highly variable layer of temperate ice (much softer ice at the pressure-melting point) forms naturally in both settings, alongside ice motion patterns which diverge substantially from those obtained using smoothly varying BedMachine topography. Temperate ice is vertically extensive (>100 meters) in deep troughs but thins notably (<5 meters) over bedrock highs, with basal slip rates reaching >90 or <5% of surface velocity dependent on topography and temperate layer thickness. Developing parameterizations of the net effect of this complex motion can improve the realism of predictive ice sheet models.

Cytokinesis machinery promotes cell dissociation from collectively migrating strands in confinement.

Cells tune adherens junction dynamics to regulate epithelial integrity in diverse (patho)physiological processes, including cancer metastasis. We hypothesized that the spatially confining architecture of peritumor stroma promotes metastatic cell dissemination by remodeling cell-cell adhesive interactions. By combining microfluidics with live-cell imaging, FLIM/FRET biosensors, and optogenetic tools, we show that confinement induces leader cell dissociation from cohesive ensembles. Cell dissociation is triggered by myosin IIA (MIIA) dismantling of E-cadherin cell-cell junctions, as recapitulated by a mathematical model. Elevated MIIA contractility is controlled by RhoA/ROCK activation, which requires distinct guanine nucleotide exchange factors (GEFs). Confinement activates RhoA via nucleocytoplasmic shuttling of the cytokinesis-regulatory proteins RacGAP1 and Ect2 and increased microtubule dynamics, which results in the release of active GEF-H1. Thus, confining microenvironments are sufficient to induce cell dissemination from primary tumors by remodeling E-cadherin cell junctions via the interplay of microtubules, nuclear trafficking, and RhoA/ROCK/MIIA pathway and not by down-regulating E-cadherin expression.

A spectrum of altered non-rapid eye movement sleep in schizophrenia.

Background: Multiple facets of sleep neurophysiology, including electroencephalography (EEG) metrics such as non-rapid eye movement (NREM) spindles and slow oscillations (SO), are altered in individuals with schizophrenia (SCZ). However, beyond group-level analyses which treat all patients as a unitary set, the extent to which NREM deficits vary among patients is unclear, as are their relationships to other sources of heterogeneity including clinical factors, illness duration and ageing, cognitive profiles and medication regimens. Using newly collected high density sleep EEG data on 103 individuals with SCZ and 68 controls, we first sought to replicate our previously reported (Kozhemiako et. al, 2022) group-level mean differences between patients and controls (original N=130). Then in the combined sample (N=301 including 175 patients), we characterized patient-to-patient variability in NREM neurophysiology. Results: We replicated all group-level mean differences and confirmed the high accuracy of our predictive model (Area Under the ROC Curve, AUC = 0.93 for diagnosis). Compared to controls, patients showed significantly increased between-individual variability across many (26%) sleep metrics, with patterns only partially recapitulating those for group-level mean differences. Although multiple clinical and cognitive factors were associated with NREM metrics including spindle density, collectively they did not account for much of the general increase in patient-to-patient variability. Medication regimen was a greater (albeit still partial) contributor to variability, although original group mean differences persisted after controlling for medications. Some sleep metrics including fast spindle density showed exaggerated age-related effects in SCZ, and patients exhibited older predicted biological ages based on an independent model of ageing and the sleep EEG. Conclusion: We demonstrated robust and replicable alterations in sleep neurophysiology in individuals with SCZ and highlighted distinct patterns of effects contrasting between-group means versus within-group variances. We further documented and controlled for a major effect of medication use, and pointed to greater age-related change in NREM sleep in patients. That increased NREM heterogeneity was not explained by standard clinical or cognitive patient assessments suggests the sleep EEG provides novel, nonredundant information to support the goals of personalized medicine. Collectively, our results point to a spectrum of NREM sleep deficits among SCZ patients that can be measured objectively and at scale, and that may offer a unique window on the etiological and genetic diversity that underlies SCZ risk, treatment response and prognosis.

Non-rapid eye movement sleep and wake neurophysiology in schizophrenia.

Motivated by the potential of objective neurophysiological markers to index thalamocortical function in patients with severe psychiatric illnesses, we comprehensively characterized key non-rapid eye movement (NREM) sleep parameters across multiple domains, their interdependencies, and their relationship to waking event-related potentials and symptom severity. In 72 schizophrenia (SCZ) patients and 58 controls, we confirmed a marked reduction in sleep spindle density in SCZ and extended these findings to show that fast and slow spindle properties were largely uncorrelated. We also describe a novel measure of slow oscillation and spindle interaction that was attenuated in SCZ. The main sleep findings were replicated in a demographically distinct sample, and a joint model, based on multiple NREM components, statistically predicted disease status in the replication cohort. Although also altered in patients, auditory event-related potentials elicited during wake were unrelated to NREM metrics. Consistent with a growing literature implicating thalamocortical dysfunction in SCZ, our characterization identifies independent NREM and wake EEG biomarkers that may index distinct aspects of SCZ pathophysiology and point to multiple neural mechanisms underlying disease heterogeneity. This study lays the groundwork for evaluating these neurophysiological markers, individually or in combination, to guide efforts at treatment and prevention as well as identifying individuals most likely to benefit from specific interventions.

UV-DIB: label-free permeability determination using droplet interface bilayers.

Simple diffusion of molecular entities through a phospholipid bilayer, is a phenomenon of great importance to the pharmaceutical and agricultural industries. Current model lipid systems to probe this typically only employ fluorescence as a readout, thus limiting the range of assessable chemical matter that can be studied. We report a new technology platform, the UV-DIB, which facilitates label free measurement of small molecule translocation rates. This is based upon the coupling of droplet interface bilayer technology with implemented fiber optics to facilitate analysis via ultraviolet spectroscopy, in custom designed PMMA wells. To improve on current DIB technology, the platform was designed to be reusable, with a high sampling rate and a limit of UV detection in the low µM regime. We demonstrate the use of our system to quantify passive diffusion in a reproducible and rapid manner where the system was validated by investigating multiple permeants of varying physicochemical properties across a range of lipid interfaces, each demonstrating differing kinetics. Our system permits the interrogation of structural dependence on the permeation rate of a given compound. We present this ability from two structural perspectives, that of the membrane, and the permeant. We observed a reduction in permeability between pure DOPC and DPhPC interfaces, concurring with literature and demonstrating our ability to study the effects of lipid composition on permeability. In relation to the effects of permeant structure, our device facilitated the rank ordering of various compounds from the xanthine class of compounds, where the structure of each permeant differed by a single group alteration. We found that DIBs were stable up to 5% DMSO, a molecule often used to aid solubilisation of pharmaceutical and agrochemical compounds. The ability of our device to rank-order compounds with such minor structural differences provides a level of precision that is rarely seen in current, industrially applied technologies.

Thalamocortical Mechanisms Regulating the Relationship between Transient Beta Events and Human Tactile Perception.

Transient neocortical events with high spectral power in the 15-29 Hz beta band are among the most reliable predictors of sensory perception. Prestimulus beta event rates in primary somatosensory cortex correlate with sensory suppression, most effectively 100-300 ms before stimulus onset. However, the neural mechanisms underlying this perceptual association are unknown. We combined human magnetoencephalography (MEG) measurements with biophysical neural modeling to test potential cellular and circuit mechanisms that underlie observed correlations between prestimulus beta events and tactile detection. Extending prior studies, we found that simulated bursts from higher-order, nonlemniscal thalamus were sufficient to drive beta event generation and to recruit slow supragranular inhibition acting on a 300 ms timescale to suppress sensory information. Further analysis showed that the same beta-generating mechanism can lead to facilitated perception for a brief period when beta events occur simultaneously with tactile stimulation before inhibition is recruited. These findings were supported by close agreement between model-derived predictions and empirical MEG data. The postevent suppressive mechanism explains an array of studies that associate beta with decreased processing, whereas the during-event facilitatory mechanism may demand a reinterpretation of the role of beta events in the context of coincident timing.

Molecular interaction and partitioning in α-keratin using 1H NMR spin-lattice (T1) relaxation times.

The interactions between small molecules and keratins are poorly understood. In this paper, a nuclear magnetic resonance method is presented to measure changes in the 1H T1 relaxation times of small molecules in human hair keratin to quantify their interaction with the fibre. Two populations of small-molecule compounds were identified with distinct relaxation times, demonstrating the partitioning of the compounds into different keratin environments. The changes in relaxation time for solvent in hair compared with bulk solvent were shown to be related to the molecular weight (MW) and the partition coefficient, LogP, of the solvent investigated. Compounds with low MWs and high hydrophilicities had greater reductions in their T1 relaxation times and therefore experienced increased interactions with the hair fibre. The relative population sizes were also calculated. This is a significant step towards modelling the behaviour of small molecules in keratinous materials and other large insoluble fibrous proteins.

Discovery and Characterisation of Highly Cooperative FAK-Degrading PROTACs.

Focal adhesion kinase (FAK) is a key mediator of tumour progression and metastasis. To date, clinical trials of FAK inhibitors have reported disappointing efficacy for oncology indications. We report the design and characterisation of GSK215, a potent, selective, FAK-degrading Proteolysis Targeting Chimera (PROTAC) based on a binder for the VHL E3 ligase and the known FAK inhibitor VS-4718. X-ray crystallography revealed the molecular basis of the highly cooperative FAK-GSK215-VHL ternary complex, and GSK215 showed differentiated in-vitro pharmacology compared to VS-4718. In mice, a single dose of GSK215 induced rapid and prolonged FAK degradation, giving a long-lasting effect on FAK levels (≈96 h) and a marked PK/PD disconnect. This tool PROTAC molecule is expected to be useful for the study of FAK-degradation biology in vivo, and our results indicate that FAK degradation may be a differentiated clinical strategy versus FAK inhibition for the treatment of cancer.

Activating mechanosensitive channels embedded in droplet interface bilayers using membrane asymmetry.

Droplet microcompartments linked by lipid bilayers show great promise in the construction of synthetic minimal tissues. Central to controlling the flow of information in these systems are membrane proteins, which can gate in response to specific stimuli in order to control the molecular flux between membrane separated compartments. This has been demonstrated with droplet interface bilayers (DIBs) using several different membrane proteins combined with electrical, mechanical, and/or chemical activators. Here we report the activation of the bacterial mechanosensitive channel of large conductance (MscL) in a dioleoylphosphatidylcholine:dioleoylphosphatidylglycerol DIB by controlling membrane asymmetry. We show using electrical measurements that the incorporation of lysophosphatidylcholine (LPC) into one of the bilayer leaflets triggers MscL gating in a concentration-dependent manner, with partial and full activation observed at 10 and 15 mol% LPC respectively. Our findings could inspire the design of new minimal tissues where flux pathways are dynamically defined by lipid composition.

A Phenotypic Approach for the Identification of New Molecules for Targeted Protein Degradation Applications.

Targeted protein degradation is an emerging new strategy for the modulation of intracellular protein levels with applications in chemical biology and drug discovery. One approach to enable this strategy is to redirect the ubiquitin-proteasome system to mark and degrade target proteins of interest (POIs) through the use of proteolysis targeting chimeras (PROTACs). Although great progress has been made in enabling PROTACs as a platform, there are still a limited number of E3 ligases that have been employed for PROTAC design. Herein we report a novel phenotypic screening approach for the identification of E3 ligase binders. The key concept underlying this approach is the high-throughput modification of screening compounds with a chloroalkane moiety to generate HaloPROTACs in situ, which were then evaluated for their ability to degrade a GFP-HaloTag fusion protein in a cellular context. As proof of concept, we demonstrated that we could generate and detect functional HaloPROTACs in situ, using a validated Von Hippel-Lindau (VHL) binder that successfully degraded the GFP-HaloTag fusion protein in living cells. We then used this method to prepare and screen a library of approximately 2000 prospective E3 ligase-recruiting molecules.

Thermodynamics of a fast-moving Greenlandic outlet glacier revealed by fiber-optic distributed temperature sensing.

Measurements of ice temperature provide crucial constraints on ice viscosity and the thermodynamic processes occurring within a glacier. However, such measurements are presently limited by a small number of relatively coarse-spatial-resolution borehole records, especially for ice sheets. Here, we advance our understanding of glacier thermodynamics with an exceptionally high-vertical-resolution (~0.65 m), distributed-fiber-optic temperature-sensing profile from a 1043-m borehole drilled to the base of Sermeq Kujalleq (Store Glacier), Greenland. We report substantial but isolated strain heating within interglacial-phase ice at 208 to 242 m depth together with strongly heterogeneous ice deformation in glacial-phase ice below 889 m. We also observe a high-strain interface between glacial- and interglacial-phase ice and a 73-m-thick temperate basal layer, interpreted as locally formed and important for the glacier's fast motion. These findings demonstrate notable spatial heterogeneity, both vertically and at the catchment scale, in the conditions facilitating the fast motion of marine-terminating glaciers in Greenland.

17O NMR spectroscopy as a tool to study hydrogen bonding of cholesterol in lipid bilayers.

Cholesterol is a crucial component of biological membranes and can interact with other membrane components through hydrogen bonding. NMR spectroscopy has been used previously to investigate this bonding, however this study represents the first 17O NMR spectroscopy study of isotopically enriched cholesterol. We demonstrate the 17O chemical shift is dependent on hydrogen bonding, providing a novel method for the study of cholesterol in bilayers.

Dorsoventral polarity directs cell responses to migration track geometries.

How migrating cells differentially adapt and respond to extracellular track geometries remains unknown. Using intravital imaging, we demonstrate that invading cells exhibit dorsoventral (top-to-bottom) polarity in vivo. To investigate the impact of dorsoventral polarity on cell locomotion through different confining geometries, we fabricated microchannels of fixed cross-sectional area, albeit with distinct aspect ratios. Vertical confinement, exerted along the dorsoventral polarity axis, induces myosin II-dependent nuclear stiffening, which results in RhoA hyperactivation at the cell poles and slow bleb-based migration. In lateral confinement, directed perpendicularly to the dorsoventral polarity axis, the absence of perinuclear myosin II fails to increase nuclear stiffness. Hence, cells maintain basal RhoA activity and display faster mesenchymal migration. In summary, by integrating microfabrication, imaging techniques, and intravital microscopy, we demonstrate that dorsoventral polarity, observed in vivo and in vitro, directs cell responses in confinement by spatially tuning RhoA activity, which controls bleb-based versus mesenchymal migration.

Atherogenic lipid stress induces platelet hyperactivity through CD36-mediated hyposensitivity to prostacyclin: the role of phosphodiesterase 3A.

Prostacyclin (PGI2) controls platelet activation and thrombosis through a cyclic adenosine monophosphate (cAMP) signaling cascade. However, in patients with cardiovascular diseases this protective mechanism fails for reasons that are unclear. Using both pharmacological and genetic approaches we describe a mechanism by which oxidized low density lipoproteins (oxLDL) associated with dyslipidemia promote platelet activation through impaired PGI2 sensitivity and diminished cAMP signaling. In functional assays using human platelets, oxLDL modulated the inhibitory effects of PGI2, but not a phosphodiesterase (PDE)-insensitive cAMP analog, on platelet aggregation, granule secretion and in vitro thrombosis. Examination of the mechanism revealed that oxLDL promoted the hydrolysis of cAMP through the phosphorylation and activation of PDE3A, leading to diminished cAMP signaling. PDE3A activation by oxLDL required Src family kinases, Syk and protein kinase C. The effects of oxLDL on platelet function and cAMP signaling were blocked by pharmacological inhibition of CD36, mimicked by CD36-specific oxidized phospholipids and ablated in CD36-/- murine platelets. The injection of oxLDL into wild-type mice strongly promoted FeCl3-induced carotid thrombosis in vivo, which was prevented by pharmacological inhibition of PDE3A. Furthermore, blood from dyslipidemic mice was associated with increased oxidative lipid stress, reduced platelet sensitivity to PGI2 ex vivo and diminished PKA signaling. In contrast, platelet sensitivity to a PDE-resistant cAMP analog remained normal. Genetic deletion of CD36 protected dyslipidemic animals from PGI2 hyposensitivity and restored PKA signaling. These data suggest that CD36 can translate atherogenic lipid stress into platelet hyperactivity through modulation of inhibitory cAMP signaling.

Engineering Swollen Cubosomes Using Cholesterol and Anionic Lipids.

Dispersions of nonlamellar lipid membrane assemblies are gaining increasing interest for drug delivery and protein therapeutic application. A key bottleneck has been the lack of rational design rules for these systems linking different lipid species and conditions to defined lattice parameters and structures. We have developed robust methods to form cubosomes (nanoparticles with porous internal structures) with water channel diameters of up to 171 Å, which are over 4 times larger than archetypal cubosome structures. The water channel diameter can be tuned via the incorporation of cholesterol and the charged lipid DOPA, DOPG, or DOPS. We have found that large molecules can be incorporated into the porous cubosome structure and that these molecules can interact with the internal cubosome membrane. This offers huge potential for accessible encapsulation and protection of biomolecules and development of confined interfacial reaction environments.

Confinement hinders motility by inducing RhoA-mediated nuclear influx, volume expansion, and blebbing.

Cells migrate in vivo through complex confining microenvironments, which induce significant nuclear deformation that may lead to nuclear blebbing and nuclear envelope rupture. While actomyosin contractility has been implicated in regulating nuclear envelope integrity, the exact mechanism remains unknown. Here, we argue that confinement-induced activation of RhoA/myosin-II contractility, coupled with LINC complex-dependent nuclear anchoring at the cell posterior, locally increases cytoplasmic pressure and promotes passive influx of cytoplasmic constituents into the nucleus without altering nuclear efflux. Elevated nuclear influx is accompanied by nuclear volume expansion, blebbing, and rupture, ultimately resulting in reduced cell motility. Moreover, inhibition of nuclear efflux is sufficient to increase nuclear volume and blebbing on two-dimensional surfaces, and acts synergistically with RhoA/myosin-II contractility to further augment blebbing in confinement. Cumulatively, confinement regulates nuclear size, nuclear integrity, and cell motility by perturbing nuclear flux homeostasis via a RhoA-dependent pathway.