PHF2 regulates sarcomeric gene transcription in myogenesis.

Myogenesis is regulated mainly by transcription factors known as Myogenic Regulatory Factors (MRFs), and the transcription is affected by epigenetic modifications. However, the epigenetic regulation of myogenesis is poorly understood. Here, we focused on the epigenomic modification enzyme, PHF2, which demethylates histone 3 lysine 9 dimethyl (H3K9me2) during myogenesis. Phf2 mRNA was expressed during myogenesis, and PHF2 was localized in the nuclei of myoblasts and myotubes. We generated Phf2 knockout C2C12 myoblasts using the CRISPR/Cas9 system and analyzed global transcriptional changes via RNA-sequencing. Phf2 knockout (KO) cells 2 d post differentiation were subjected to RNA sequencing. Gene ontology (GO) analysis revealed that Phf2 KO impaired the expression of the genes related to skeletal muscle fiber formation and muscle cell development. The expression levels of sarcomeric genes such as Myhs and Mybpc2 were severely reduced in Phf2 KO cells at 7 d post differentiation, and H3K9me2 modification of Mybpc2, Mef2c and Myh7 was increased in Phf2 KO cells at 4 d post differentiation. These findings suggest that PHF2 regulates sarcomeric gene expression via epigenetic modification.

An intact pituitary vasopressin system is critical for building a robust circadian clock in the suprachiasmatic nucleus.

The circadian clock is a biological timekeeping system that oscillates with a circa-24-h period, reset by environmental timing cues, especially light, to the 24-h day-night cycle. In mammals, a "central" clock in the hypothalamic suprachiasmatic nucleus (SCN) synchronizes "peripheral" clocks throughout the body to regulate behavior, metabolism, and physiology. A key feature of the clock's oscillation is resistance to abrupt perturbations, but the mechanisms underlying such robustness are not well understood. Here, we probe clock robustness to unexpected photic perturbation by measuring the speed of reentrainment of the murine locomotor rhythm after an abrupt advance of the light-dark cycle. Using an intersectional genetic approach, we implicate a critical role for arginine vasopressin pathways, both central within the SCN and peripheral from the anterior pituitary.

Symmetry Engineering in Twisted Bilayer WTe2.

The fabrication of artificial structures using a twisted van der Waals assembly has been a key technique for recent advancements in the research of two-dimensional (2D) materials. To date, various exotic phenomena have been observed thanks to the modified electron correlation or moiré structure controlled by the twist angle. However, the twisted van der Waals assembly has further potential to modulate the physical properties by controlling the symmetry. In this study, we fabricated twisted bilayer WTe2 and demonstrated that the twist angle successfully controls the spatial inversion symmetry and hence the spin splitting in the band structure. Our results reveal the further potential of a twisted van der Waals assembly, suggesting the feasibility of pursuing new physical phenomena in 2D materials based on the control of symmetry.

Super-Ballistic Width Dependence of Thermal Conductivity in Graphite Nanoribbons and Microribbons.

The super-ballistic temperature dependence of thermal conductivity, facilitated by collective phonons, has been widely studied. It has been claimed to be unambiguous evidence for hydrodynamic phonon transport in solids. Alternatively, hydrodynamic thermal conduction is predicted to be as strongly dependent on the width of the structure as is fluid flow, while its direct demonstration remains an unexplored challenge. In this work, we experimentally measured thermal conductivity in several graphite ribbon structures with different widths, from 300 nm to 1.2 µm, and studied its width dependence in a wide temperature range of 10-300 K. We observed enhanced width dependence of the thermal conductivity in the hydrodynamic window of 75 K compared to that in the ballistic limit, which provides indispensable evidence for phonon hydrodynamic transport from the perspective of peculiar width dependence. This will help to find the missing piece to complete the puzzle of phonon hydrodynamics, and guide future attempts at efficient heat dissipation in advanced electronic devices.

Observation of phonon Poiseuille flow in isotopically purified graphite ribbons.

In recent times, the unique collective transport physics of phonon hydrodynamics motivates theoreticians and experimentalists to explore it in micro- and nanoscale and at elevated temperatures. Graphitic materials have been predicted to facilitate hydrodynamic heat transport with their intrinsically strong normal scattering. However, owing to the experimental difficulties and vague theoretical understanding, the observation of phonon Poiseuille flow in graphitic systems remains challenging. In this study, based on a microscale experimental platform and the pertinent occurrence criterion in anisotropic solids, we demonstrate the existence of the phonon Poiseuille flow in a 5.5 µm-wide, suspended and isotopically purified graphite ribbon up to a temperature of 90 K. Our observation is well supported by our theoretical model based on a kinetic theory with fully first-principles inputs. Thus, this study paves the way for deeper insight into phonon hydrodynamics and cutting-edge heat manipulating applications.

All-dry flip-over stacking of van der Waals junctions of 2D materials using polyvinyl chloride.

We demonstrated an all-dry polymer-to-polymer transfer technique for two-dimensional (2D) crystal flakes using a polyvinyl chloride (PVC) layer deposited on a piece of polydimethylsiloxane (PDMS). Unexpectedly, the pickup/release temperatures were modified in wider temperature range simply by changing the thickness of the PVC layer than changing the plasticizer ratio. Utilizing the difference in the pickup/release temperatures depending on the PVC film thickness, 2D flakes were transferred from a thicker PVC film to a thinner one. This polymer-to-polymer transfer technique can be utilized to flip over van der Waals heterostructures. As a demonstration, we fabricated a mountain-like stacked structure of hexagonal boron nitride flakes using the flip-over stacking technique. Finally, we compared the results of thermomechanical analysis with the pickup/release temperatures of the PVC/PDMS stamp. The PVC was revealed to be at the glass transition and in the viscoelastic flow regimes when the 2D flakes were picked up and dry released, respectively. Our polymer-to-polymer transfer method facilitates flip-over van der Waals stacking in an all-dry manner, expanding the possibility of 2D materials device fabrications.

Protein kinase C-mediated phosphorylation of transient receptor potential melastatin type 2 Thr738 counteracts the effect of cytosolic Ca2+ and elevates the temperature threshold.

The transient receptor potential melastatin type 2 (TRPM2) channel is a non-selective cation channel that has high Ca2+ permeability. TRPM2 is sensitive to warm temperatures and is expressed in cells and tissues that are maintained at core body temperature. TRPM2 activity is also regulated by endogenous factors including redox signalling, cytosolic Ca2+ and adenosine diphosphate ribose. As a result of its wide expression and function at core body temperature, these endogenous factors could regulate TRPM2 activity at body temperature under physiological and pathophysiological conditions. We previously reported that cellular redox signalling can lower TRPM2 temperature thresholds, although the mechanism that regulates these thresholds is unclear. Here, we used biochemical and electrophysiological techniques to explore another regulatory mechanism for TRPM2 temperature thresholds that is mediated by TRPM2 phosphorylation. Our results show that: (1) the temperature threshold for TRPM2 activation is lowered by cytosolic Ca2+ ; (2) protein kinase C-mediated phosphorylation of TRPM2 counteracts the effect of cytosolic Ca2+ ; and (3) Thr738 in mouse TRPM2 that lies near the Ca2+ binding site in the cytosolic cleft of the transmembrane domain is a potential phosphorylation site that may be involved in phosphorylation-mediated elevation of TRPM2 thresholds. These findings provide structure-based evidence to understand how temperature thresholds of thermo-sensitive TRP channels (thermo-TRPs) are determined and regulated. KEY POINTS: The transient receptor potential melastatin type 2 (TRPM2) ion channel is temperature-sensitive and Ca2+ -permeable. Endogenous factors and pathways such as redox signalling can regulate TRPM2 activity at body temperature under physiological and pathophysiological conditions. In the present study, we report the novel finding that cytosolic Ca2+ lowers the temperature threshold for TRPM2 activation in a concentration-dependent manner. Protein kinase C-mediated phosphorylation of TRPM2 at amino acid Thr782 elevates the temperature threshold for activation by counteracting the effects of cytosolic Ca2+ . These findings provide structure-based evidence to understand how temperature thresholds of thermo-sensitive TRP channels are determined and regulated.

Resonant Tunneling between Quantized Subbands in van der Waals Double Quantum Well Structure Based on Few-Layer WSe2.

We demonstrate van der Waals double quantum well (vDQW) devices based on few-layer WSe2 quantum wells and a few-layer h-BN tunnel barrier. Due to the strong out-of-plane confinement, an exfoliated WSe2 exhibits quantized subband states at the Γ point in its valence band. Here, we report resonant tunneling and negative differential resistance in vDQW at room temperature owing to momentum- and energy-conserved tunneling between the quantized subbands in each well. Compared to single quantum well (QW) devices with only one QW layer possessing quantized subbands, superior current peak-to-valley ratios were obtained for the DQWs. Our findings suggest a new direction for utilizing few-layer-thick transition metal dichalcogenides in subband QW devices, bridging the gap between two-dimensional materials and state-of-the-art semiconductor QW electronics.

Dry pick-and-flip assembly of van der Waals heterostructures for microfocus angle-resolved photoemission spectroscopy.

We present a dry pick-and-flip assembly technique for angle-resolved photoemission spectroscopy (ARPES) of van der Waals heterostructures. By combining Elvacite2552C acrylic resin and 1-ethyl-3-methylimidazolium ionic liquid, we prepared polymers with glass transition temperatures (Tg) ranging from 37 to 100 â„ƒ. The adhesion of the polymer to the 2D crystals was enhanced at [Formula: see text]. By utilizing the difference in [Formula: see text], a 2D heterostructure can be transferred from a high-[Formula: see text] polymer to a lower-[Formula: see text] polymer, which enables flipping its surface upside down. This process is suitable for assembling heterostructures for ARPES, where the top capping layer should be monolayer graphene. The laser-based micro-focused ARPES measurements of 5-layer WTe2, 3-layer MoTe2, 2-layer WTe2/few-layer Cr2Ge2Te6, and twisted double bilayer WTe2 demonstrate that this process can be utilized as a versatile sample fabrication method for investigating the energy spectra of 2D heterostructures.

Suppression of trabecular meshwork phagocytosis by norepinephrine is associated with nocturnal increase in intraocular pressure in mice.

Intraocular pressure (IOP) is an important factor in glaucoma development, which involves aqueous humor (AH) dynamics, with inflow from the ciliary body and outflow through the trabecular meshwork (TM). IOP has a circadian rhythm entrained by sympathetic noradrenaline (NE) or adrenal glucocorticoids (GCs). Herein, we investigated the involvement of GC/NE in AH outflow. Pharmacological prevention of inflow/outflow in mice indicated a diurnal outflow increase, which was related to TM phagocytosis. NE showed a non-self-sustained inhibition in phagocytosis of immortalized human TM cells, but not GC. The pharmacological and reverse genetic approaches identified ß1-adrenergic receptor (AR)-mediated exchange proteins directly activated by cyclic adenosine monophosphate (EPAC)-SHIP1 signal activation by ablation of phosphatidylinositol triphosphate, regulating phagocytic cup formation. Furthermore, we revealed the phagocytosis involvement in the ß1-AR-EPAC-SHIP1-mediated nocturnal IOP rise in mice. These suggest that TM phagocytosis suppression by NE can regulate IOP rhythm through AH outflow. This discovery may aid glaucoma management.

Tamoxifen Activates Dormant Primordial Follicles in Mouse Ovaries.

Our previous study found that 17ß-estradiol (E2) suppresses primordial follicle activation and growth in cultured mouse ovaries. In this study, we administered tamoxifen, an estrogen receptor antagonist, into the abdominal cavity of mice to clarify the relationship between primordial follicle activation and the physiological concentration of E2 in mouse ovaries. The results showed that tamoxifen promoted primordial follicle activation. Administration of tamoxifen promoted degradation of the extracellular matrix surrounding primordial follicles in the ovaries. Furthermore, tamoxifen decreased the expression of stefin A, an inhibitor of cathepsins that digest some proteins and extracellular matrix, in the ovaries. Mechanical stress produced by the extracellular matrix reportedly suppresses the activation of primordial follicles. The collective results show that tamoxifen can promote primordial follicle activation through the degradation of the extracellular matrix surrounding primordial follicles. Our results indicate that E2 suppresses primordial follicle activation in vivo and that tamoxifen may be useful as a therapeutic agent against infertility.

17ß-Estradiol and cathepsins control primordial follicle growth in mouse ovaries.

This study aimed to clarify the physiological mechanism regulating the growth of primordial follicles in mouse ovaries. In a previous study, we found that increasing the fetal bovine serum concentration in the culture medium promoted the growth of primordial follicles in cultured postnatal day 0 ovaries but not in cultured postnatal day 4 ovaries. Based on these results, we hypothesized that the regulatory system repressing the growth of primordial follicles is established in postnatal day 4 ovaries. To confirm this hypothesis, microarray analysis of postnatal day 0 and 4 ovaries was performed. The results revealed that the expression of mRNA of stefin A homologs increased in postnatal day 4 ovaries. Stefin A belonging to the type 1 cystatin superfamily is an inhibitor of cysteine cathepsins. Consistently, the inhibitor of cathepsins repressed the growth of primordial follicles in cultured postnatal day 0 ovaries. Furthermore, we found that 17ß-estradiol promoted the expression of mRNA of stefin A homologs in cultured ovaries and repressed the growth of primordial follicles. Our results demonstrate that 17ß-estradiol and cathepsins regulate the growth of primordial follicles in mouse ovaries.

Resonant Tunneling Due to van der Waals Quantum-Well States of Few-Layer WSe2 in WSe2/h-BN/p+-MoS2 Junction.

Few-layer transition metal dichalcogenides (TMDs) exhibit out-of-plane wave function confinement with subband quantization. This phenomenon is totally absent in monolayer crystals and is regarded as resulting from a naturally existing van der Waals quantum-well state. Because the energy separation between the subbands corresponds to the infrared wavelength range, few-layer TMDs are attractive for their potential to facilitate the application of TMD semiconductors as infrared photodetectors and emitters. Here, we report a few-layer WSe2/h-BN tunnel barrier/multilayer p+-MoS2 tunnel junction to access the quantized subbands of few-layer WSe2 via tunneling spectroscopy measurements. Resonant tunneling and a negative differential resistance were observed when the top of the valence band Γ-point of p+-MoS2 was energetically aligned with one of the empty subbands at the Γ-point of few-layer WSe2. These results demonstrate a critical step toward the utilization of subband quantization in few-layer TMD materials for infrared optoelectronics applications.

Emergence of orbital angular moment at van Hove singularity in graphene/h-BN moiré superlattice.

Bloch electrons lacking inversion symmetry exhibit orbital magnetic moments owing to the rotation around their center of mass; this moment induces a valley splitting in a magnetic field. For the graphene/h-BN moiré superlattice, inversion symmetry is broken by the h-BN. The superlattice potential generates a series of Dirac points (DPs) and van Hove singularities (vHSs) within an experimentally accessible low energy state, providing a platform to study orbital moments with respect to band structure. In this work, theoretical calculations and magnetothermoelectric measurements are combined to reveal the emergence of an orbital magnetic moment at vHSs in graphene/h-BN moiré superlattices. The thermoelectric signal for the vHS at the low energy side of the hole-side secondary DP exhibited significant magnetic field-induced valley splitting with an effective g-factor of approximately 130; splitting for other vHSs was negligible. This was attributed to the emergence of an orbital magnetic moment at the second vHS at the hole-side.

cAMP response element induces Per1 in vivo.

Light is an important cue for resetting the circadian clock. In mammals, light signals are thought to be transmitted to the cAMP response element (CRE) via a binding protein (CREB) to induce the expression of Per1 and Per2 genes in the mammalian circadian pacemaker, the suprachiasmatic nuclei (SCN). Several in vitro studies have suggested candidate CRE sites that contribute to the Per1 and Per2 induction by light, resulting in a phase shift of the circadian rhythm. However, it remains unclear whether the CREs are responsible for the light-induced Per1/2 induction. To address this question, we generated CRE-deleted mice in the Per1 and Per2 promoter regions. Deletion of a cAMP-responsive CRE in the Per1 promoter blunted light-induced Per1 expression in the SCN at night, while deletion of an ATF4 (CREB-2)-associated CRE in the Per2 promoter had no effect on its expression. These results suggested that the CRE in the Per1 promoter works for light induction but not CRE in the Per2 promoter. Behavioral rhythms observed under some light conditions were not affected by the CRE-deletion in Per1 promoter, suggesting that the attenuated Per1 induction did not affect the entrainment in some light conditions.

Cyclotron Resonance Study of Monolayer Graphene under Double Moiré Potentials.

We report the first cyclotron resonance study of monolayer graphene under double-moiré potentials in which the crystal axis of graphene is nearly aligned to those of both the top and bottom hexagonal boron nitride (h-BN) layers. Under mid-infrared light irradiation, we observe cyclotron resonance absorption with the following unique features: (1) cyclotron resonance magnetic field BCR is entirely different from that of nonaligned monolayer graphene, (2) BCR exhibits strong electron-hole asymmetry, and (3) splitting of BCR is observed for |ν| < 1, with the split maximum at |ν| = 1, resulting in eyeglass-shaped trajectories. These features are well explained by considering the large bandgap induced by the double moiré potentials, the electron-hole asymmetry in the Fermi velocity, and the Fermi-level-dependent enhancement of spin gaps, which suggests a large electron-electron correlation contribution in this system.

3D Manipulation of 2D Materials Using Microdome Polymer.

We demonstrate 3D mechanical manipulations, such as sliding, rotating, folding, flipping, and exfoliating, of 2D materials using a microdome polymer (MDP) via in situ real-time observation with an optical microscope. A dimethylpolysiloxane (PDMS)-based MDP is covered with a poly(vinyl chloride) (PVC) adhesion layer. This PVC-MDP structure enables us to achieve small and adjustable contact areas between the PVC-MDP and a 2D-material flake, which is typically between ∼10 and ∼100 µm in diameter. The adhesion between the PVC polymer and 2D materials is fully tunable with temperature: Strong adhesion at ∼70 °C allows pick-up of the 2D material, and release occurs at ∼130 °C when the adhesion is weak. Thus the PVC-MDP functions as a point-of-contact manipulator for 2D materials, permitting the 3D manipulation of 2D-material flakes. Our method could facilitate the expansion of van der Waals heterostructure fabrication technology and the development of preparation techniques for more complex 3D structures.

Circadian Regulation of IOP Rhythm by Dual Pathways of Glucocorticoids and the Sympathetic Nervous System.

Purpose: Elevated IOP can cause the development of glaucoma. The circadian rhythm of IOP depends on the dynamics of the aqueous humor and is synchronized with the circadian rhythm pacemaker, that is, the suprachiasmatic nucleus. The suprachiasmatic nucleus resets peripheral clocks via sympathetic nerves or adrenal glucocorticoids. However, the detailed mechanisms underlying IOP rhythmicity remain unclear. The purpose of this study was to verify this regulatory pathway. Methods: Adrenalectomy and/or superior cervical ganglionectomy were performed in C57BL/6J mice. Their IOP rhythms were measured under light/dark cycle and constant dark conditions. Ocular administration of corticosterone or norepinephrine was also performed. Localization of adrenergic receptors, glucocorticoid receptors, and clock proteins Bmal1 and Per1 were analyzed using immunohistochemistry. Period2::luciferase rhythms in the cultured iris/ciliary bodies of adrenalectomized and/or superior cervical ganglionectomized mice were monitored to evaluate the effect of the procedures on the local clock. The IOP rhythm of retina and ciliary epithelium-specific Bmal1 knockout mice were measured to determine the significance of the local clock. Results: Adrenalectomy and superior cervical ganglionectomy disrupted IOP rhythms and the circadian clock in the iris/ciliary body cultures. Instillation of corticosterone and norepinephrine restored the IOP rhythm. ß2-Adrenergic receptors, glucocorticoid receptors, and clock proteins were strongly expressed within the nonpigmented epithelia of the ciliary body. However, tissue-specific Bmal1 knock-out mice maintained their IOP rhythm. Conclusions: These findings suggest direct driving of the IOP rhythm by the suprachiasmatic nucleus, via the dual corticosterone and norepinephrine pathway, but not the ciliary clock, which may be useful for chronotherapy of glaucoma.

Increased supply from blood vessels promotes the activation of dormant primordial follicles in mouse ovaries.

The controlled activation of dormant primordial follicles is important for the maintenance of periodic ovulation. Previous reports have clearly identified the signaling pathway in granulosa cells and oocytes that controls the activation of primordial follicles; however, the exact cue for the in vivo activation of dormant primordial follicles is yet to be elucidated. In this study, we found that almost all activated primordial follicles made contact with blood vessels. Based on this result, we speculated that the contact between primordial follicles and blood vessels may provide a cue for the activation of dormant primordial follicles. To confirm this hypothesis, we attempted to activate dormant primordial follicles within the ovaries by inducing angiogenesis through the use of biodegradable gels containing recombinant vascular endothelial growth factor and in cultured ovarian tissues by increasing the serum concentration within the culture medium. The activation of dormant primordial follicles was promoted in both experiments, and our results indicated that an increase in the supply of the serum component, from new blood vessels formed via angiogenesis, to the dormant primordial follicles is the cue for their in vivo activation. In the ovaries, angiogenesis often occurs during every estrous cycle, and it is therefore likely that angiogenesis is the crucial event that influences the activation of primordial follicles.

Hexagonal Boron Nitride Synthesized at Atmospheric Pressure Using Metal Alloy Solvents: Evaluation as a Substrate for 2D Materials.

Hexagonal boron nitride (h-BN) synthesized under high pressure and high temperature (HPHT) has been used worldwide in two-dimensional (2D) materials research as an essential material for constructing van der Waals heterostructures. Here, we study h-BN synthesized with another method, i.e., via synthesis at atmospheric pressure and high temperature (APHT) using a metal alloy solvent. First, we examine the APHT h-BN in a bulk crystal form using cathodoluminescence and find that it does not have carbon-rich domains that inevitably exist in a core region of all the HPHT h-BN crystals. Next, we statistically compare the size of the crystal flakes exfoliated on a SiO2/Si substrate from APHT and HPHT h-BN crystals by employing our automated 2D material searching system. Finally, we provide direct evidence that APHT h-BN can serve as a high-quality substrate for 2D materials by demonstrating high carrier mobility, ballistic transport, and Hofstadter butterfly in graphene and photoluminescence in WS2.