BMAL1-Driven Tissue Clocks Respond Independently to Light to Maintain Homeostasis.

Circadian rhythms control organismal physiology throughout the day. At the cellular level, clock regulation is established by a self-sustained Bmal1-dependent transcriptional oscillator network. However, it is still unclear how different tissues achieve a synchronized rhythmic physiology. That is, do they respond independently to environmental signals, or require interactions with each other to do so? We show that unexpectedly, light synchronizes the Bmal1-dependent circadian machinery in single tissues in the absence of Bmal1 in all other tissues. Strikingly, light-driven tissue autonomous clocks occur without rhythmic feeding behavior and are lost in constant darkness. Importantly, tissue-autonomous Bmal1 partially sustains homeostasis in otherwise arrhythmic and prematurely aging animals. Our results therefore support a two-branched model for the daily synchronization of tissues: an autonomous response branch, whereby light entrains circadian clocks without any commitment of other Bmal1-dependent clocks, and a memory branch using other Bmal1-dependent clocks to "remember" time in the absence of external cues.

Defining the Independence of the Liver Circadian Clock.

Mammals rely on a network of circadian clocks to control daily systemic metabolism and physiology. The central pacemaker in the suprachiasmatic nucleus (SCN) is considered hierarchically dominant over peripheral clocks, whose degree of independence, or tissue-level autonomy, has never been ascertained in vivo. Using arrhythmic Bmal1-null mice, we generated animals with reconstituted circadian expression of BMAL1 exclusively in the liver (Liver-RE). High-throughput transcriptomics and metabolomics show that the liver has independent circadian functions specific for metabolic processes such as the NAD+ salvage pathway and glycogen turnover. However, although BMAL1 occupies chromatin at most genomic targets in Liver-RE mice, circadian expression is restricted to ∼10% of normally rhythmic transcripts. Finally, rhythmic clock gene expression is lost in Liver-RE mice under constant darkness. Hence, full circadian function in the liver depends on signals emanating from other clocks, and light contributes to tissue-autonomous clock function.

Impact of Bmal1 Rescue and Time-Restricted Feeding on Liver and Muscle Proteomes During the Active Phase in Mice.

Molecular clocks and daily feeding cycles support metabolism in peripheral tissues. Although the roles of local clocks and feeding are well defined at the transcriptional level, their impact on governing protein abundance in peripheral tissues is unclear. Here, we determine the relative contributions of local molecular clocks and daily feeding cycles on liver and muscle proteomes during the active phase in mice. LC-MS/MS was performed on liver and gastrocnemius muscle harvested 4 h into the dark phase from WT, Bmal1 KO, and dual liver- and muscle-Bmal1-rescued mice under either ad libitum feeding or time-restricted feeding during the dark phase. Feeding-fasting cycles had only minimal effects on levels of liver proteins and few, if any, on the muscle proteome. In contrast, Bmal1 KO altered the abundance of 674 proteins in liver and 80 proteins in muscle. Local rescue of liver and muscle Bmal1 restored ∼50% of proteins in liver and ∼25% in muscle. These included proteins involved in fatty acid oxidation in liver and carbohydrate metabolism in muscle. For liver, proteins involved in de novo lipogenesis were largely dependent on Bmal1 function in other tissues (i.e., the wider clock system). Proteins regulated by BMAL1 in liver and muscle were enriched for secreted proteins. We found that the abundance of fibroblast growth factor 1, a liver secreted protein, requires BMAL1 and that autocrine fibroblast growth factor 1 signaling modulates mitochondrial respiration in hepatocytes. In liver and muscle, BMAL1 is a more potent regulator of dark phase proteomes than daily feeding cycles, highlighting the need to assess protein levels in addition to mRNA when investigating clock mechanisms. The proteome is more extensively regulated by BMAL1 in liver than in muscle, and many metabolic pathways in peripheral tissues are reliant on the function of the clock system as a whole.

Quantifying Atomically Dispersed Catalysts Using Deep Learning Assisted Microscopy.

The catalytic performance of atomically dispersed catalysts (ADCs) is greatly influenced by their atomic configurations, such as atom-atom distances, clustering of atoms into dimers and trimers, and their distributions. Scanning transmission electron microscopy (STEM) is a powerful technique for imaging ADCs at the atomic scale; however, most STEM analyses of ADCs thus far have relied on human labeling, making it difficult to analyze large data sets. Here, we introduce a convolutional neural network (CNN)-based algorithm capable of quantifying the spatial arrangement of different adatom configurations. The algorithm was tested on different ADCs with varying support crystallinity and homogeneity. Results show that our algorithm can accurately identify atom positions and effectively analyze large data sets. This work provides a robust method to overcome a major bottleneck in STEM analysis for ADC catalyst research. We highlight the potential of this method to serve as an on-the-fly analysis tool for catalysts in future in situ microscopy experiments.

Disproportionation chemistry in K2PtCl4 visualized at atomic resolution using scanning transmission electron microscopy.

The direct observation of a solid-state chemical reaction can reveal otherwise hidden mechanisms that control the reaction kinetics. However, probing the chemical bond breaking and formation at the molecular level remains challenging because of the insufficient spatial-temporal resolution and composition analysis of available characterization methods. Using atomic-resolution differential phase-contrast imaging in scanning transmission electron microscopy, we have visualized the decomposition chemistry of K2PtCl4 to identify its transient intermediate phases and their interfaces that characterize the chemical reduction process. The crystalline structure of K2PtCl4 is found to undergo a disproportionation reaction to form K2PtCl6, followed by gradual reduction to crystalline Pt metal and KCl. By directly imaging different Pt─Cl bond configurations and comparing them to models predicted via density functional theory calculations, a causal connection between the initial and final states of a chemical reaction is established, showcasing new opportunities to resolve reaction pathways through atomistic experimental visualization.

Brain-muscle communication prevents muscle aging by maintaining daily physiology.

A molecular clock network is crucial for daily physiology and maintaining organismal health. We examined the interactions and importance of intratissue clock networks in muscle tissue maintenance. In arrhythmic mice showing premature aging, we created a basic clock module involving a central and a peripheral (muscle) clock. Reconstituting the brain-muscle clock network is sufficient to preserve fundamental daily homeostatic functions and prevent premature muscle aging. However, achieving whole muscle physiology requires contributions from other peripheral clocks. Mechanistically, the muscle peripheral clock acts as a gatekeeper, selectively suppressing detrimental signals from the central clock while integrating important muscle homeostatic functions. Our research reveals the interplay between the central and peripheral clocks in daily muscle function and underscores the impact of eating patterns on these interactions.

The epidermal circadian clock integrates and subverts brain signals to guarantee skin homeostasis.

In mammals, the circadian clock network drives daily rhythms of tissue-specific homeostasis. To dissect daily inter-tissue communication, we constructed a mouse minimal clock network comprising only two nodes: the peripheral epidermal clock and the central brain clock. By transcriptomic and functional characterization of this isolated connection, we identified a gatekeeping function of the peripheral tissue clock with respect to systemic inputs. The epidermal clock concurrently integrates and subverts brain signals to ensure timely execution of epidermal daily physiology. Timely cell-cycle termination in the epidermal stem cell compartment depends upon incorporation of clock-driven signals originating from the brain. In contrast, the epidermal clock corrects or outcompetes potentially disruptive feeding-related signals to ensure the optimal timing of DNA replication. Together, we present an approach for cataloging the systemic dependencies of daily temporal organization in a tissue and identify an essential gate-keeping function of peripheral circadian clocks that guarantees tissue homeostasis.

Circadian transcriptome processing and analysis: a workflow for muscle stem cells.

Circadian rhythms coordinate biological processes with Earth's 24-h daily light/dark cycle. In the last years, efforts in the field of chronobiology have sought to understand the ways in which the circadian clock controls transcription across tissues and cells. This has been supported by the development of different bioinformatic approaches that allow the identification of 24-h oscillating transcripts. This workflow aims to describe how to isolate muscle stem cells for RNA sequencing analysis from a typical circadian experiment and introduces bioinformatic tools suitable for the analysis of circadian transcriptomes.

Ion solvation as a predictor of lanthanide adsorption structures and energetics in alumina nanopores.

Adsorption reactions at solid-water interfaces define elemental fate and transport and enable contaminant clean-up, water purification, and chemical separations. For nanoparticles and nanopores, nanoconfinement may lead to unexpected and hard-to-predict products and energetics of adsorption, compared to analogous unconfined surfaces. Here we use X-ray absorption fine structure spectroscopy and operando flow microcalorimetry to determine nanoconfinement effects on the energetics and local coordination environment of trivalent lanthanides adsorbed on Al2O3 surfaces. We show that the nanoconfinement effects on adsorption become more pronounced as the hydration free energy, ΔGhydr, of a lanthanide decreases. Neodymium (Nd3+) has the least exothermic ΔGhydr (-3336 kJ·mol-1) and forms mostly outer-sphere complexes on unconfined Al2O3 surfaces but shifts to inner-sphere complexes within the 4 nm Al2O3 pores. Lutetium (Lu3+) has the most exothermic ΔGhydr (-3589 kJ·mol-1) and forms inner-sphere adsorption complexes regardless of whether Al2O3 surfaces are nanoconfined. Importantly, the energetics of adsorption is exothermic in nanopores only, and becomes endothermic with increasing surface coverage. Changes to the energetics and products of adsorption in nanopores are ion-specific, even within chemically similar trivalent lanthanide series, and can be predicted by considering the hydration energies of adsorbing ions.

Liver and muscle circadian clocks cooperate to support glucose tolerance in mice.

Physiology is regulated by interconnected cell and tissue circadian clocks. Disruption of the rhythms generated by the concerted activity of these clocks is associated with metabolic disease. Here we tested the interactions between clocks in two critical components of organismal metabolism, liver and skeletal muscle, by rescuing clock function either in each organ separately or in both organs simultaneously in otherwise clock-less mice. Experiments showed that individual clocks are partially sufficient for tissue glucose metabolism, yet the connections between both tissue clocks coupled to daily feeding rhythms support systemic glucose tolerance. This synergy relies in part on local transcriptional control of the glucose machinery, feeding-responsive signals such as insulin, and metabolic cycles that connect the muscle and liver. We posit that spatiotemporal mechanisms of muscle and liver play an essential role in the maintenance of systemic glucose homeostasis and that disrupting this diurnal coordination can contribute to metabolic disease.

AutoDisk: Automated diffraction processing and strain mapping in 4D-STEM.

Development in lattice strain mapping using four-dimensional scanning transmission electron microscopy (4D-STEM) method now offers improved precision and feasibility. However, automatic and accurate diffraction analysis is still challenging due to noise and the complexity of intensity in diffraction patterns. In this work, we demonstrate an approach, employing the blob detection on cross-correlated diffraction patterns followed by a lattice fitting algorithm, to automate the processing of four-dimensional data, including identifying and locating disks, and extracting local lattice parameters without prior knowledge about the material. The approach is both tested using simulated diffraction patterns and applied on experimental data acquired from a Pd@Pt core-shell nanoparticle. Our method shows robustness against various sample thicknesses and high noise, capability to handle complex patterns, and picometer-scale accuracy in strain measurement, making it a promising tool for high-throughput 4D-STEM data processing.

Antibiotic-induced microbiome depletion remodels daily metabolic cycles in the brain.

The gut microbiome influences cognition and behavior in mammals, yet its metabolic impact on the brain is only starting to be defined. Using metabolite profiling of antibiotics-treated mice, we reveal the microbiome as a key input controlling circadian metabolic cycles in the brain. Intra and inter-region analyses characterise the influence of the microbiome on the suprachiasmatic nucleus, containing the central clockwork, as well as the hippocampus and cortex, regions involved in learning and behavior.

The central clock suffices to drive the majority of circulatory metabolic rhythms.

Life on Earth anticipates recurring 24-hour environmental cycles via genetically encoded molecular clocks active in all mammalian organs. Communication between these clocks controls circadian homeostasis. Intertissue communication is mediated, in part, by temporal coordination of metabolism. Here, we characterize the extent to which clocks in different organs control systemic metabolic rhythms, an area that remains largely unexplored. We analyzed the metabolome of serum from mice with tissue-specific expression of the clock gene Bmal1. Having functional hepatic and muscle clocks can only drive a minority (13%) of systemic metabolic rhythms. Conversely, limiting Bmal1 expression to the central pacemaker in the brain restores rhythms to 57% of circulatory metabolites. Rhythmic feeding imposed on clockless mice resulted in a similar rescue, indicating that the central clock mainly regulates metabolic rhythms via behavior. These findings explicate the circadian communication between tissues and highlight the importance of the central clock in governing those signals.

Integration of feeding behavior by the liver circadian clock reveals network dependency of metabolic rhythms.

The mammalian circadian clock, expressed throughout the brain and body, controls daily metabolic homeostasis. Clock function in peripheral tissues is required, but not sufficient, for this task. Because of the lack of specialized animal models, it is unclear how tissue clocks interact with extrinsic signals to drive molecular oscillations. Here, we isolated the interaction between feeding and the liver clock by reconstituting Bmal1 exclusively in hepatocytes (Liver-RE), in otherwise clock-less mice, and controlling timing of food intake. We found that the cooperative action of BMAL1 and the transcription factor CEBPB regulates daily liver metabolic transcriptional programs. Functionally, the liver clock and feeding rhythm are sufficient to drive temporal carbohydrate homeostasis. By contrast, liver rhythms tied to redox and lipid metabolism required communication with the skeletal muscle clock, demonstrating peripheral clock cross-talk. Our results highlight how the inner workings of the clock system rely on communicating signals to maintain daily metabolism.

Clock-in, clock-out: circadian timekeeping between tissues.

Life evolved in the presence of alternating periods of light and dark that accompany the daily rotation of the Earth on its axis. This offered an advantage for organisms able to regulate their physiology to anticipate these daily cycles. In each light-sensitive organism studied, spanning single-celled bacteria to complex mammals, there exist timekeeping mechanisms able to control physiology over the course of 24 hours. Endowed with internal timekeeping, organisms can put their previously stored energy to the most efficient use, selectively ramping up biological processes at specific times of day or night according to when they'll be needed. Humans have evolved to be more active during the day (diurnal), likely due to the increased opportunities for foraging or hunting in our evolutionary past, and this daily activity is accompanied by an upregulation of genes involved in metabolism to increase the energy available for such behaviours. Remarkably, this happens without conscious thought, due to a complex organism-wide signalling apparatus known as the circadian clock network, that conveys time information between cells and tissues.

Proteomic analysis of S-nitrosylated nuclear proteins in rat cortical neurons.

Neurons modulate gene expression in response to extrinsic signals to enable brain development, cognition, and learning and to process stimuli that regulate systemic physiological functions. This signal-to-gene communication is facilitated by posttranslational modifications such as S-nitrosylation, the covalent attachment of a nitric oxide (NO) moiety to cysteine thiols. In the cerebral cortex, S-nitrosylation of histone deacetylase 2 (HDAC2) is required for gene transcription during neuronal development, but few other nuclear targets of S-nitrosylation have been identified to date. We used S-nitrosothiol resin-assisted capture on NO donor-treated nuclear extracts from rat cortical neurons and identified 614 S-nitrosylated nuclear proteins. Of these, 131 proteins have not previously been shown to be S-nitrosylated in any system, and 555 are previously unidentified targets of S-nitrosylation in neurons. The sites of S-nitrosylation were identified for 59% of the targets, and motifs containing single lysines were found at 33% of these sites. In addition, lysine motifs were necessary for promoting the S-nitrosylation of HDAC2 and methyl-CpG binding protein 3 (MBD3). Moreover, S-nitrosylation of the histone-binding protein RBBP7 was necessary for dendritogenesis of cortical neurons in culture. Together, our findings characterize S-nitrosylated nuclear proteins in neurons and identify S-nitrosylation motifs that may be shared with other targets of NO signaling.

HDAC3 Regulates the Transition to the Homeostatic Myelinating Schwann Cell State.

The formation of myelinating Schwann cells (mSCs) involves the remarkable biogenic process, which rapidly generates the myelin sheath. Once formed, the mSC transitions to a stable homeostatic state, with loss of this stability associated with neuropathies. The histone deacetylases histone deacetylase 1 (HDAC1) and HDAC2 are required for the myelination transcriptional program. Here, we show a distinct role for HDAC3, in that, while dispensable for the formation of mSCs, it is essential for the stability of the myelin sheath once formed-with loss resulting in progressive severe neuropathy in adulthood. This is associated with the prior failure to downregulate the biogenic program upon entering the homeostatic state leading to hypertrophy and hypermyelination of the mSCs, progressing to the development of severe myelination defects. Our results highlight distinct roles of HDAC1/2 and HDAC3 in controlling the differentiation and homeostatic states of a cell with broad implications for the understanding of this important cell-state transition.

Zeb1-Hdac2-eNOS circuitry identifies early cardiovascular precursors in naive mouse embryonic stem cells.

Nitric oxide (NO) synthesis is a late event during differentiation of mouse embryonic stem cells (mESC) and occurs after release from serum and leukemia inhibitory factor (LIF). Here we show that after release from pluripotency, a subpopulation of mESC, kept in the naive state by 2i/LIF, expresses endothelial nitric oxide synthase (eNOS) and endogenously synthesizes NO. This eNOS/NO-positive subpopulation (ESNO+) expresses mesendodermal markers and is more efficient in the generation of cardiovascular precursors than eNOS/NO-negative cells. Mechanistically, production of endogenous NO triggers rapid Hdac2 S-nitrosylation, which reduces association of Hdac2 with the transcriptional repression factor Zeb1, allowing mesendodermal gene expression. In conclusion, our results suggest that the interaction between Zeb1, Hdac2, and eNOS is required for early mesendodermal differentiation of naive mESC.

A Functional Switch of NuRD Chromatin Remodeling Complex Subunits Regulates Mouse Cortical Development.

Histone modifications and chromatin remodeling represent universal mechanisms by which cells adapt their transcriptional response to rapidly changing environmental conditions. Extensive chromatin remodeling takes place during neuronal development, allowing the transition of pluripotent cells into differentiated neurons. Here, we report that the NuRD complex, which couples ATP-dependent chromatin remodeling with histone deacetylase activity, regulates mouse brain development. Subunit exchange of CHDs, the core ATPase subunits of the NuRD complex, is required for distinct aspects of cortical development. Whereas CHD4 promotes the early proliferation of progenitors, CHD5 facilitates neuronal migration and CHD3 ensures proper layer specification. Inhibition of each CHD leads to defects of neuronal differentiation and migration, which cannot be rescued by expressing heterologous CHDs. Finally, we demonstrate that NuRD complexes containing specific CHDs are recruited to regulatory elements and modulate the expression of genes essential for brain development.