RNA-methylation-dependent RNA processing controls the speed of the circadian clock.

The eukaryotic biological clock involves a negative transcription-translation feedback loop in which clock genes regulate their own transcription and that of output genes of metabolic significance. While around 10% of the liver transcriptome is rhythmic, only about a fifth is driven by de novo transcription, indicating mRNA processing is a major circadian component. Here, we report that inhibition of transmethylation reactions elongates the circadian period. RNA sequencing then reveals methylation inhibition causes widespread changes in the transcription of the RNA processing machinery, associated with m(6)A-RNA methylation. We identify m(6)A sites on many clock gene transcripts and show that specific inhibition of m(6)A methylation by silencing of the m(6)A methylase Mettl3 is sufficient to elicit circadian period elongation and RNA processing delay. Analysis of the circadian nucleocytoplasmic distribution of clock genes Per2 and Arntl then revealed an uncoupling between steady-state pre-mRNA and cytoplasmic mRNA rhythms when m(6)A methylation is inhibited.

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.

Calcitonin receptors are ancient modulators for rhythms of preferential temperature in insects and body temperature in mammals.

Daily body temperature rhythm (BTR) is essential for maintaining homeostasis. BTR is regulated separately from locomotor activity rhythms, but its molecular basis is largely unknown. While mammals internally regulate BTR, ectotherms, including Drosophila, exhibit temperature preference rhythm (TPR) behavior to regulate BTR. Here, we demonstrate that the diuretic hormone 31 receptor (DH31R) mediates TPR during the active phase in Drosophila DH31R is expressed in clock cells, and its ligand, DH31, acts on clock cells to regulate TPR during the active phase. Surprisingly, the mouse homolog of DH31R, calcitonin receptor (Calcr), is expressed in the suprachiasmatic nucleus (SCN) and mediates body temperature fluctuations during the active phase in mice. Importantly, DH31R and Calcr are not required for coordinating locomotor activity rhythms. Our results represent the first molecular evidence that BTR is regulated distinctly from locomotor activity rhythms and show that DH31R/Calcr is an ancient specific mediator of BTR during the active phase in organisms ranging from ectotherms to endotherms.

Circadian protection against bacterial skin infection by epidermal CXCL14-mediated innate immunity.

The epidermis is the outermost layer of the skin and the body's primary barrier to external pathogens; however, the early epidermal immune response remains to be mechanistically understood. We show that the chemokine CXCL14, produced by epidermal keratinocytes, exhibits robust circadian fluctuations and initiates innate immunity. Clearance of the skin pathogen Staphylococcus aureus in nocturnal mice was associated with CXCL14 expression, which was high during subjective daytime and low at night. In contrast, in marmosets, a diurnal primate, circadian CXCL14 expression was reversed. Rhythmically expressed CXCL14 binds to S. aureus DNA and induces inflammatory cytokine production by activating Toll-like receptor (TLR)9-dependent innate pathways in dendritic cells and macrophages underneath the epidermis. CXCL14 also promoted phagocytosis by macrophages in a TLR9-independent manner. These data indicate that circadian production of the epidermal chemokine CXCL14 rhythmically suppresses skin bacterial proliferation in mammals by activating the innate immune system.

Omeprazole Induces CYP3A4 mRNA Expression but Not CYP3A4 Protein Expression in HepaRG Cells.

Unknown interactions between drugs remain the limiting factor for clinical application of drugs, and the induction and inhibition of drug-metabolizing CYP enzymes are considered the key to examining the drug-drug interaction (DDI). In this study, using human HepaRG cells as an in vitro model system, we analyzed the potential DDI based on the expression levels of CYP3A4 and CYP1A2. Rifampicin and omeprazole, the potent inducers for CYP3A4 and CYP1A2, respectively, induce expression of the corresponding CYP enzymes at both the mRNA and protein levels. We noticed that, in addition to inducing CYP1A2, omeprazole induced CYP3A4 mRNA expression in HepaRG cells. However, unexpectedly, CYP3A4 protein expression levels were not increased after omeprazole treatment. Concurrent administration of rifampicin and omeprazole showed an inhibitory effect of omeprazole on the CYP3A4 protein expression induced by rifampicin, while its mRNA induction remained intact. Cycloheximide chase assay revealed increased CYP3A4 protein degradation in the cells exposed to omeprazole. The data presented here suggest the potential importance of broadening the current DDI examination beyond conventional transcriptional induction and enzyme-activity inhibition tests to include post-translational regulation analysis of CYP enzyme expression.

Temperature-Dependent Upregulation of Per2 Protein Expression Is Mediated by eIF2α Kinases PERK and PKR through PI3K Activation.

Temperature-dependent translational control of the core clock gene Per2 plays an important role in establishing entrainment of the circadian clock to physiological body temperature cycles. Previously, we found an involvement of the phosphatidylinositol 3-kinase (PI3K) in causing Per2 protein expression in response to a warm temperature shift (WTS) within a physiological range (from 35 to 38.5 °C). However, signaling pathway mediating the Per2 protein expression in response to WTS is only sparsely understood. Additional factor(s) other than PI3K remains unknown. Here we report the identification of eukaryotic initiation factor 2α (eIF2α) kinases, protein kinase R (PKR) and PKR-like endoplasmic reticulum kinase (PERK), as a novel mediator of WTS-dependent Per2 protein expression. Canonically, eIF2α has been regarded as a major downstream target of PERK and PKR. However, we found that PERK and PKR mediate WTS response of Per2 in a manner not involving eIF2α. We observed that PERK and PKR serve as an upstream regulator of PI3K rather than eIF2α in the context of WTS-dependent Per2 protein expression. There have been studies reporting PI3K activation occurring depending on PERK and PKR, while its physiological contribution has remained elusive. Our finding therefore not only helps to enrich the knowledge of how WTS affects Per2 protein expression but also extends the region of cellular biology involving the PERK/PKR-mediated PI3K activation to include entrainment-mechanism of the circadian clock.

Universality in the Dynamics of Vesicle Translocation through a Hole.

We analyze the translocation process of a spherical vesicle, made of a membrane and incompressible fluid, through a hole smaller than the vesicle size, driven by pressure difference ΔP. We show that such a vesicle shows certain universal characteristics, which are independent of the details of the membrane elasticity: (i) there is a critical pressure ΔPc below which no translocation occurs; (ii) ΔPc decreases to zero as the vesicle radius R0 approaches the hole radius a, satisfying the scaling relation ΔPc ∼ (R0 - a)3/2; and (iii) the translocation time τ diverges as ΔP decreases to ΔPc, satisfying the scaling relation τ ∼ (ΔP - ΔPc)-1/2.

Effect of fluid viscoelasticity, shear stress, and interface tension on the lift force in lubricated contacts.

We consider a cylinder immersed in viscous fluid moving near a flat substrate covered by an incompressible viscoelastic fluid layer, and study the effect of the fluid viscoelasticity on the lift force exerted on the cylinder. The lift force is zero when the viscoelastic layer is not deformed, but becomes non-zero when it is deformed. We calculate the lift force by considering both the tangential stress and the normal stress applied at the surface of the viscoelastic layer. Our analysis indicates that as the layer changes from the elastic limit to the viscous limit, the lift force decreases with the decrease of the Deborah number (De). For small De, the effect of the layer elasticity is taken over by the surface tension and the lift force can become negative. We also show that the tangential stress and the interface slip velocity (the surface velocity relative to the substrate), which have been ignored in the previous analysis, give important contributions to the lift force. Especially for thin elastic layers, they give dominant contributions to the lift force.

Capillary Rising in a Tube with Corners.

We studied the dynamics of a fluid rising in a capillary tube with corners. In the cornered tube, unlike the circular tube, fluid rises with two parts, the bulk part where the entire cross-section is occupied by the fluid and the finger part where the cross-section is only partially filled. Using the Onsager principle, we derive coupled time-evolution equations for the two parts. We show the following: (a) At the early stage of rising, the dynamics is dominated by the bulk part and the fluid height h0(t) shows the same behavior as that in the circular tube. (b) At the late stage, the bulk part stops rising but the finger part continues, following the scaling law h1(t) ∼ t1/3. We also show that, due to the coupling between the two parts, the equilibrium bulk height is smaller than the Jurin's height, which ignores the effect of the finger part.

Evaporation Dynamics of Sessile Droplets: The Intricate Coupling of Capillary, Evaporation, and Marangoni Flow.

A single-component droplet placed on a completely wetting substrate shows a pseudostable apparent contact angle (θapp) during evaporation. We propose a simple theory to explain the phenomenon accounting for the liquid evaporation and the internal flow induced by the capillary and Marangoni effects. The theory predicts that when evaporation starts, the contact angle approaches to θapp in a short time τs, remains constant for most of the time of evaporation, and finally increases rapidly when the droplet size becomes very small. This explains the behavior observed for alkane droplets. Analytical expressions are given for the apparent contact angle θapp and the relaxation time τs, which predict how they change when the evaporation rate, droplet size, and other experimental parameters such as thermal conductivity of the substrate are changed.

Interplay of Consolidation Fronts and Cracks in Drying Colloidal Coatings and Its Application in Controlling Crack Pattern Formation.

Cracks are frequently observed in drying colloidal coatings. Although a rich collection of crack patterns has been reported, the systematic study on how cracks grow into the final morphology during the drying process remains elusive. In this work, we use directional drying channels with wedge-shaped edges of different angles to study the interplay of advancing consolidation fronts and propagating cracks. We found that although the shape of the advancing consolidation fronts is altered by the drying edge, the growth direction of the following cracks remains perpendicular to the consolidation fronts during the whole drying process, resulting in cracks with a large curvature. We rationalize the evolution of consolidation fronts with the distribution of capillary pressure revealed by a Laplace model. Further, the growth direction of cracks can be explained by the fracture mechanics mechanism that the main orientation of internal tensile stresses developed during the consolidation determines the crack growth direction. Utilizing this understanding, wavy crack patterns are generated in rectangular drying channels with an alternating temperature field, demonstrating a feasible method of designing and controlling drying-induced crack patterns for micro-/nano-fabrication applications.

Wetting dynamics of viscoelastic solid films.

We study the wetting phenomena of a soft viscoelastic solid film on a smooth and flat substrate. A poly-dimethylsiloxane (PDMS) rubber film is suspended from a stage at both ends, and the wetting behavior of the film against a glass substrate is observed while lowering the stage at a constant velocity. We find that the dynamics of the rubber-glass-air contact lines vary with the lowering velocity of the stage. When the stage velocity is sufficiently low, the film wets the substrate smoothly and the contact lines are straight throughout. Consequently, the contact line velocity is proportional to the lowering velocity. As the stage velocity is increased, the contact line velocity reaches a maximum at the critical stage velocity and then subsequently decreases. The contact lines are wavy and sensitive to the defects above the critical velocity, resulting in the trapping of air bubbles at the interface. We reproduce the wetting behavior using a simple numerical model, assuming an upper limit for the contact line velocity. The wetting behavior observed in our experiments is attributed to the transition in the in-plane stress state from tensile to compressive along the film, leading to buckling of the film above the critical stage velocity. Our results suggest the existence and importance of the maximum wetting velocity for viscoelastic solids.

Nmu/Nms/Gpr176 Triple-Deficient Mice Show Enhanced Light-Resetting of Circadian Locomotor Activity.

The suprachiasmatic nucleus (SCN) is the master circadian clock in mammals and is properly entrained by environmental light cycle. However, the molecular mechanism(s) determining the magnitude of phase shift by light is still not fully understood. The orphan G-protein-coupled receptor Gpr176 is enriched in the SCN, controls the pace (period) of the circadian rhythm in behavior but is not apparently involved in the light entrainment; Gpr176-/- animals display a shortened circadian period in constant darkness but their phase-resetting responses to light are normal. Here, we performed microarray analysis and identified enhanced mRNA expression of neuromedin U (Nmu) and neuromedin S (Nms) in the SCN of Gpr176-/- mice. By generating C57BL/6J-backcrossed Nmu/Nms/Gpr176 triple knockout mice, we noted that the mutant mice had a greater magnitude of phase shift in response to early subjective night light than wildtype mice, while Nmu/Nms double knockout mice as well as Gpr176 knockout mice are normal in the phase shifts induced by light. At the molecular level, Nmu-/-Nms-/-Gpr176-/- mice had a reduced induction of Per1 and cFos mRNA expression in the SCN by light and mildly upregulated circadian expression of Per2, Prok2, Rgs16, and Rasl11b. These expressional changes may underlie the phenotype of the Nmu/Nms/Gpr176 knockout mice. Our data argue that there is a mechanism requiring Nmu, Nms, and Gpr176 for the proper modulation of light-induced phase shift in mice. Simultaneous modulation of Nmu/Nms/Gpr176 may provide a potential target option for modulating the circadian clock.

Gas-liquid transition of van der Waals fluid confined in fluctuating nano-space.

Gas-liquid transition is generally a complex process, which involves nucleation of droplets and their growth by evaporation-condensation or collision-coalescence processes. Here, we focus on a microscopic system in which there is only one liquid droplet at most. In this case, we can develop an equilibrium theory for the formation of the droplet in the gas phase using the classical nucleation theory. We use the van der Waals fluid model with surface tension and calculate the size fluctuation of the droplet for various confinement conditions, NVT (in which the volume V of the system is fixed), NPT (in which the pressure P of the system is fixed), and NBT (in which the system is confined in a nano-bubble immersed in a host liquid, where both V and P can fluctuate). We show that in the NBT system, the size flexibility along with space confinement induces a wealth of properties that are not found in NVT and NPT. It exhibits richer phase behaviors: a stable droplet appears and coexists with the pure gas phase and/or pure liquid phase. When compared to the NVT system, the NBT system shows not only the oscillatory fluctuation between the two stable states but also a large fluctuation in the total volume and the pressure.

Derivation of Two-Fluid Model Based on Onsager Principle.

Using the Onsager variational principle, we study the dynamic coupling between the stress and the composition in a polymer solution. In the original derivation of the two-fluid model of Doi and Onuki the polymer stress was introduced a priori; therefore, a constitutive equation is required to close the equations. Based on our previous study of viscoelastic fluids with homogeneous composition, we start with a dumbbell model for the polymer, and derive all dynamic equations using the Onsager variational principle.

Fetal Leydig cells dedifferentiate and serve as adult Leydig stem cells.

Previous studies have established that fetal Leydig cells (FLCs) and adult Leydig cells (ALCs) show distinct functional characteristics. However, the lineage relationship between FLCs and ALCs has not been clarified yet. Here, we reveal that a subset of FLCs dedifferentiate at fetal stages to give rise to ALCs at the pubertal stage. Moreover, the dedifferentiated cells contribute to the peritubular myoid cell and vascular pericyte populations in the neonatal testis, and these non-steroidogenic cells serve as potential ALC stem cells. We generated FLC lineage-specific Nr5a1 (Ad4BP/SF-1) gene-disrupted mice and mice lacking the fetal Leydig enhancer (FLE) of the Nr5a1 gene. Phenotypes of these mice support the conclusion that most of the ALCs arise from dedifferentiated FLCs, and that the FLE of the Nr5a1 gene is essential for both initial FLC differentiation and pubertal ALC redifferentiation.

Wetting Dynamics in an Angular Channel.

We analyze the dynamics of liquid filling in a thin, slightly inflated rectangular channel driven by capillary forces. We show that although the amount of liquid m in the channel increases in time following the classical Lucas-Washburn law, m ∝ t1/2, the prefactor is very sensitive to the deformation of the channel because the filling takes place by the growth of two parts, the bulk part (where the cross section is completely filled by the liquid), and the finger part (where the cross section is partially filled). We calculate the time dependence of m accounting for the coupling between the two parts and show that the prefactor for the filling can be reduced significantly by a slight deformation of the rectangular channel, e.g., the prefactor is reduced 50% for a strain of 0.1%. This offers an explanation for the large deviation on the value of the prefactor reported previously.

The contact angle of an evaporating droplet of a binary solution on a super wetting surface.

We study the dynamics of the contact angle of a droplet of a binary solution evaporating on a super wetting surface. Recent experiments have shown that although the equilibrium contact angle of such a droplet is zero, the contact angle can show complex time dependence before reaching the equilibrium value. We analyse such phenomena by extending our previous theory for the dynamics of an evaporating single component droplet to a double component droplet. We show that the time dependence of the contact angle can be quite complex. Typically, it first decreases slightly, and then increases and finally decreases again. Under certain conditions, we find that the contact angle remains constant over a certain period of time during evaporation. We study how the plateau or peak contact angle depends on the initial composition and the humidity. This theory explains the experimental results reported previously.

Wetting equilibrium in a rectangular channel.

When a capillary channel with corners is wetted by a fluid, there are regions where the fluid fills the whole cross-section and regions where only the corners are filled by the fluid. The fluid fraction of the partially-filled region, s*, is an important quantity related to the capillary pressure. We calculate the value of s* for channels with a cross-section slightly deviated from a rectangle: the height is larger in the center than those on the two short sides. We find that a small change in the cross-section geometry leads to a huge change of s*. This result is consistent with experimental observations.

Elastic response of wire frame glasses. I. Two dimensional model.

We study the elastic response of concentrated suspensions of rigid wire frame particles to a step strain. These particles are constructed from infinitely thin, rigid rods of length L. We specifically compare straight rod-like particles to bent and branched wire frames. In dense suspensions, the wire frames are frozen in a disordered state by the topological entanglements between their arms. We present a simple, geometric method to find the scaling of the elastic stress with concentration in these glassy systems. We apply this method to a simple 2D model system where a test particle is placed on a plane and constrained by a random distribution of points with number density ν. Two striking differences between wire frame and rod suspensions are found: (1) The linear elasticity per particle for wire frames is very large, scaling like ν2L4, whereas for rods, it is much smaller and independent of concentration. (2) Rods always shear thin but wire frames shear harden for concentrations less than ∼K/kBTL4, where K is the bending modulus of the particles. The deformation of wire frames is found to be important even for small strains, with the proportion of deformed particles at a particular strain, γ, being given by (νL2)2γ2. Our results agree well with simple numerical calculations for the 2D system.