Layer specific regulation of critical period timing and maturation of mouse visual cortex by endocannabinoids.

Plasticity during the critical period is important for the functional maturation of cortical neurons. While characteristics of plasticity are diverse among cortical layers, it is unknown whether critical period timing is controlled by a common or unique molecular mechanism among them. We here clarified layer-specific regulation of the critical period timing of ocular dominance plasticity in the primary visual cortex. Mice lacking the endocannabinoid synthesis enzyme diacylglycerol lipase-α exhibited precocious critical period timing, earlier maturation of inhibitory synaptic function in layers 2/3 and 4, and impaired development of the binocular matching of orientation selectivity exclusively in layer 2/3. Activation of cannabinoid receptor restored ocular dominance plasticity at the normal critical period in layer 2/3. Suppression of GABAA receptor rescued precocious ocular dominance plasticity in layer 4. Therefore, endocannabinoids regulate critical period timing and maturation of visual function partly through the development of inhibitory synaptic functions in a layer-dependent manner.

Divergent mechanisms of reduced growth performance in Betula ermanii saplings from high-altitude and low-latitude range edges.

The reduced growth performance of individuals from range edges is a common phenomenon in various taxa, and considered to be an evolutionary factor that limits the species' range. However, most studies did not distinguish between two mechanisms that can lead to this reduction: genetic load and adaptive selection to harsh conditions. To address this lack of understanding, we investigated the climatic and genetic factors underlying the growth performance of Betula ermanii saplings transplanted from 11 populations including high-altitude edge and low-latitude edge population. We estimated the climatic position of the populations within the overall B. ermanii's distribution, and the genetic composition and diversity using restriction-site associated DNA sequencing, and measured survival, growth rates and individual size of the saplings. The high-altitude edge population (APW) was located below the 95% significance interval for the mean annual temperature range, but did not show any distinctive genetic characteristics. In contrast, the low-latitude edge population (SHK) exhibited a high level of linkage disequilibrium, low genetic diversity, a distinct genetic composition from the other populations, and a high relatedness coefficient. Both APW and SHK saplings displayed lower survival rates, heights and diameters, while SHK saplings also exhibited lower growth rates than the other populations' saplings. The low heights and diameters of APW saplings was likely the result of adaptive selection to harsh conditions, while the low survival and growth rates of SHK saplings was likely the result of genetic load. Our findings shed light on the mechanisms underlying the reduced growth performance of range-edge populations.

Biophysical Properties of the Fibril Structure of the Toxic Conformer of Amyloid-ß42: Characterization by Atomic Force Microscopy in Liquid and Molecular Docking.

Alzheimer's disease is associated with the aggregation of the misfolded neuronal peptide, amyloid-ß42 (Aß42). Evidence has suggested that several reasons are responsible for the toxicity caused by the aggregation of Aß42, including the conformational restriction of Aß42. In this study, one of the toxic conformers of Aß42, which contains a Glu-to-Pro substitution (E22P-Aß42), was explored using atomic force microscopy and molecular docking to study the aggregation dynamics. We proposed a systematic model of fibril formation to better understand the molecular basis of conformational transitions in the Aß42 species. Our results demonstrated the formation of amorphous aggregates in E22P-Aß42 that are stem-based, network-like structures, while the formation of mature fibrils occurred in the less toxic conformer of Aß42, E22-Aß42, that are sphere-like flexible structures. A comparison was made between the biophysical properties of E22P-Aß42 and E22-Aß42 that revealed that E22P-Aß42 had greater stiffness, dihedral angle, number of ß sheets involved, and elasticity, compared with E22-Aß42. These findings will have considerable implications toward our understanding of the structural basis of the toxicity caused by conformational diversity in Aß42 species.

Recent research advances on non-linear phenomena in various biosystems.

All biological phenomena can be classified as open, dissipative and non-linear. Moreover, the most typical phenomena are associated with non-linearity, dissipation and openness in biological systems. In this review article, four research topics on non-linear biosystems are described to show the examples from various biological systems. First, membrane dynamics of a lipid bilayer for the cell membrane is described. Since the cell membrane separates the inside of the cell from the outside, self-organizing systems that form spatial patterns on membranes often depend on non-linear dynamics. Second, various data banks based on recent genomics analysis supply the data including vast functional proteins from many organisms and their variable species. Since the proteins existing in nature are only a very small part of the space represented by amino acid sequence, success of mutagenesis-based molecular evolution approach crucially depends on preparing a library with high enrichment of functional proteins. Third, photosynthetic organisms depend on ambient light, the regular and irregular changes of which have a significant impact on photosynthetic processes. The light-driven process proceeds through many redox couples in the cyanobacteria constituting chain of redox reactions. The fourth topic focuses on a vertebrate model, the zebrafish, which can help to understand, predict and control the chaos of complex biological systems. In particular, during early developmental stages, developmental differentiation occurs dynamically from a fertilized egg to divided and mature cells. These exciting fields of complexity, chaos, and non-linear science have experienced impressive growth in recent decades. Finally, future directions for non-linear biosystems are presented.

Endocytosis-Like Vesicle Fission Mediated by a Membrane-Expanding Molecular Machine Enables Virus Encapsulation for In Vivo Delivery.

Biological membranes are functionalized by membrane-associated protein machinery. Membrane-associated transport processes, such as endocytosis, represent a fundamental and universal function mediated by membrane-deforming protein machines, by which small biomolecules and even micrometer-size substances can be transported via encapsulation into membrane vesicles. Although synthetic molecules that induce dynamic membrane deformation have been reported, a molecular approach enabling membrane transport in which membrane deformation is coupled with substance binding and transport remains critically lacking. Here, we developed an amphiphilic molecular machine containing a photoresponsive diazocine core (AzoMEx) that localizes in a phospholipid membrane. Upon photoirradiation, AzoMEx expands the liposomal membrane to bias vesicles toward outside-in fission in the membrane deformation process. Cargo components, including micrometer-size M13 bacteriophages that interact with AzoMEx, are efficiently incorporated into the vesicles through the outside-in fission. Encapsulated M13 bacteriophages are transiently protected from the external environment and therefore retain biological activity during distribution throughout the body via the blood following administration. This research developed a molecular approach using synthetic molecular machinery for membrane functionalization to transport micrometer-size substances and objects via vesicle encapsulation. The molecular design demonstrated in this study to expand the membrane for deformation and binding to a cargo component can lead to the development of drug delivery materials and chemical tools for controlling cellular activities.

Bromovalerylurea modulates GABAA receptor-mediated inhibitory neurotransmission while inducing sleep.

Bromovalerylurea (BU), an acyl urea derivative, was originally developed as a hypnotic/sedative. We recently reported that BU at a dose of 50 mg/kg ameliorates sepsis, Parkinson's disease, and traumatic brain injury in Wistar rat models through its anti-inflammatory actions on microglia and macrophages. However, since BU was developed more than 100 years ago, its hypnotic mechanism and characteristics are poorly understood. Herein, we conducted an electroencephalogram (EEG) study and found that BU, when administered at a dose of more than 125 mg/kg but not at a dose of 50 mg/kg in Wistar rats, significantly increased non-rapid eye movement (NREM) sleep duration and dose-dependently decreased rapid eye movement (REM) sleep duration. This characteristic of sleep induced by BU is similar to the effect of compounds such as barbiturate, benzodiazepine, and z-drugs, all of which require γ-aminobutyric acid A receptors (GABAAR) for hypnotic/sedative activity. To investigate whether BU could potentiate GABAAergic neurotransmission, we conducted a whole-cell patch-clamp recording from pyramidal neurons in rat cortical slices to detect spontaneous GABAAR-mediated inhibitory postsynaptic currents (IPSCs). We found that BU dose-dependently prolonged IPSCs. Importantly, the prolonged IPSCs were not attenuated by flumazenil, a benzodiazepine receptor antagonist, suggesting that modulation of IPSCs by BU is mediated by different mechanisms from that of benzodiazepine. Taken together, these data elucidate the basic characteristics of the hypnotic effects of BU and suggest that the enhancement of GABAAR-mediated Cl- flux may be a possible mechanism that contributes to its hypnotic/sedative activity.

Suppression of Amyloid-ß Adsorption on Endoplasmic Reticulum Stress-Mimicking Membranes by α-Tocopherol and α-Tocotrienol.

Two forms of hydrophobic vitamin E (VE), α-tocopherol (Toc) and α-tocotrienol (Toc3), have been proposed to be effective against Alzheimer's disease (AD), the etiology of which is thought to involve endoplasmic reticulum (ER) stress. However, previous studies reported conflicting effects of Toc and Toc3 on the risk of AD. We prepared liposomes mimicking the phase separation of the ER membrane (solid-ordered/liquid-disordered phase separation) and studied how VE can influence the interaction between amyloid-ß (Aß) and the ER membrane. We found that Toc could inhibit the formation of the solid-ordered phase more significantly than Toc3. Furthermore, Aß protofibril adsorption on ER stress-mimicking membranes was more strongly suppressed by Toc compared with Toc3. Therefore, we concluded that VE can relieve ER stress by destabilizing the solid-ordered phase of the ER membrane and subsequently reducing the amount of Aß adsorbed on the membrane. Moreover, Toc exerted a stronger effect than Toc3.

The first assimilation of Akatsuki single-layer winds and its validation with Venusian atmospheric waves excited by solar heating.

The planetary missions including the Venus Climate Orbiter 'Akatsuki' provide new information on various atmospheric phenomena. Nevertheless, it is difficult to elucidate their three-dimensional structures globally and continuously only from observations because satellite observations are considerably limited in time and space. We constructed the first 'objective analysis' of Venus' atmosphere by assimilating cloud-top horizontal winds on the dayside from the equator to mid-latitudes, which is frequently obtained from Akatsuki's Ultraviolet Imager (UVI). The three-dimensional structures of thermal tides, found recently to play a crucial role in maintaining the super rotation, are greatly improved by the data assimilation. This result is confirmed by comparison with Akatsuki's temperature observations. The momentum transport caused by the thermal tides and other disturbances are also modified by the wind assimilation and agrees well with those estimated from the UVI observations. The assimilated dataset is reliable and will be open to the public along with the Akatsuki observations for further investigation of Venus' atmospheric phenomena.

Control of Line Tension at Phase-Separated Lipid Domain Boundaries: Monounsaturated Fatty Acids with Different Chain Lengths and Osmotic Pressure.

Line tension at phase-separated lipid domain boundaries is an important factor that governs the stability of the phase separation. We studied the control of the line tension in lipid membranes composed of dioleoylphosphocholine (DOPC), dipalmitoylphosphocholine (DPPC), and cholesterol (Chol) by the addition of the following three monounsaturated fatty acids (MUFAs) with different chain lengths: palmitoleic acid (PaA), oleic acid (OA), and eicosenoic acid (EiA). In addition, we attempted to alter the line tension by applying osmotic pressure. The phase behavior of the MUFA-containing lipid membranes in the presence and absence of osmotic stress was observed by fluorescence and confocal laser scanning microscopy. The line tension was quantitatively measured from the domain boundary fluctuation by flicker spectroscopy, and the interactions between the lipids and MUFAs were examined by differential scanning calorimetry. PaA and OA, which are shorter MUFAs, decreased the line tension, whereas EiA changed the liquid domain to a solid domain. The osmotic pressure increased the line tension, even in the presence of MUFAs. It may be possible to control the line tension by combining the chemical approach of MUFA addition and the physical approach of applying osmotic pressure.

Artificial Palmitoylation of Proteins Controls the Lipid Domain-Selective Anchoring on Biomembranes and the Raft-Dependent Cellular Internalization.

Protein palmitoylation, a post-translational modification, is universally observed in eukaryotic cells. The localization of palmitoylated proteins to highly dynamic, sphingolipid- and cholesterol-rich microdomains (called lipid rafts) on the plasma membrane has been shown to play an important role in signal transduction in cells. However, this complex biological system is not yet completely understood. Here, we used a combined approach where an artificial lipidated protein was applied to biomimetic model membranes and plasma membranes in cells to illuminate chemical and physiological properties of the rafts. Using cell-sized giant unilamellar vesicles, we demonstrated the selective partitioning of enhanced green fluorescent protein modified with a C-terminal palmitoyl moiety (EGFP-Pal) into the liquid-ordered phase consisting of saturated phospholipids and cholesterol. Using Jurkat T cells treated with an immunostimulant (concanavalin A), we observed the vesicular transport of EGFP-Pal. Further cellular studies with the treatment of methyl ß-cyclodextrin revealed the cholesterol-dependent internalization of EGFP-Pal, which can be explained by a raft-dependent, caveolae-mediated endocytic pathway. The present synthetic approach using artificial and natural membrane systems can be further extended to explore the potential utility of artificially lipidated proteins at biological and artificial interfaces.

Quantitative Analysis of the Membrane Affinity of Local Anesthetics Using a Model Cell Membrane.

Local anesthesia is a drug that penetrates the nerve cell membrane and binds to the voltage gate sodium channel, inhibiting the membrane potential and neurotransmission. It is mainly used in clinical uses to address the pain of surgical procedures in the local area. Local anesthetics (LAs), however, can be incorporated into the membrane, reducing the thermal stability of the membrane as well as altering membrane properties such as fluidity, permeability, and lipid packing order. The effects of LAs on the membrane are not yet fully understood, despite a number of previous studies. In particular, it is necessary to analyze which is the more dominant factor, the membrane affinity or the structural perturbation of the membrane. To analyze the effects of LAs on the cell membrane and compare the results with those from model membranes, morphological analysis and 50% inhibitory concentration (IC50) measurement of CCD-1064sk (fibroblast, human skin) membranes were carried out for lidocaine (LDC) and tetracaine (TTC), the most popular LAs in clinical use. Furthermore, the membrane affinity of the LAs was quantitatively analyzed using a colorimetric polydiacetylene assay, where the color shift represents their distribution in the membrane. Further, to confirm the membrane affinity and structural effects of the membranes, we performed an electrophysiological study using a model protein (gramicidin A, gA) and measured the channel lifetime of the model protein on the free-standing lipid bilayer according to the concentration of each LA. Our results show that when LAs interact with cell membranes, membrane affinity is a more dominant factor than steric or conformational effects of the membrane.

Development of robust isothermal RNA amplification assay for lab-free testing of RNA viruses.

Simple tests of infectiousness that return results in minutes and directly from samples even with low viral loads could be a potential game-changer in the fight against COVID-19. Here, we describe an improved isothermal nucleic acid amplification assay, termed the RICCA (RNA Isothermal Co-assisted and Coupled Amplification) reaction, that consists of a simple one-pot format of 'sample-in and result-out' with a primary focus on the detection of low copy numbers of RNA virus directly from saliva without the need for laboratory processing. We demonstrate our assay by detecting 16S rRNA directly from E. coli cells with a sensitivity as low as 8 CFU/µL and RNA fragments from a synthetic template of SARS-CoV-2 with a sensitivity as low as 1740 copies/µL. We further demonstrate the applicability of our assay for real-time testing at the point of care by designing a closed format for paper-based lateral flow assay and detecting heat-inactivated SARS-COV-2 virus in human saliva at concentrations ranging from 28,000 to 2.8 copies/µL with a total assay time of 15-30 min.

The nightside cloud-top circulation of the atmosphere of Venus.

Although Venus is a terrestrial planet similar to Earth, its atmospheric circulation is much different and poorly characterized1. Winds at the cloud top have been measured predominantly on the dayside. Prominent poleward drifts have been observed with dayside cloud tracking and interpreted to be caused by thermal tides and a Hadley circulation2-4; however, the lack of nightside measurements over broad latitudes has prevented the unambiguous characterization of these components. Here we obtain cloud-tracked winds at all local times using thermal infrared images taken by the Venus orbiter Akatsuki, which is sensitive to an altitude of about 65 kilometres5. Prominent equatorward flows are found on the nightside, resulting in null meridional velocities when these are zonally averaged. The velocity structure of the thermal tides was determined without the influence of the Hadley circulation. The semidiurnal tide was found to have an amplitude large enough to contribute to the maintenance of the atmospheric superrotation. The weakness of the mean meridional flow at the cloud top implies that the poleward branch of the Hadley circulation exists above the cloud top and that the equatorward branch exists in the clouds. Our results should shed light on atmospheric superrotation in other celestial bodies.

Three-Phase Coexistence in Binary Charged Lipid Membranes in a Hypotonic Solution.

We investigated the phase separation of dioleoylphosphatidylserine (DOPS) and dipalmitoylphosphatidylcholine (DPPC) in giant unilamellar vesicles in a hypotonic solution using fluorescence and confocal laser scanning microscopy. Although phase separation in charged lipid membranes is generally suppressed by the electrostatic repulsion between the charged headgroups, osmotic stress can promote the formation of charged lipid domains. Interestingly, we observed a three-phase coexistence even in the DOPS/DPPC binary lipid mixtures. The three phases were DPPC-rich, dissociated DOPS-rich, and nondissociated DOPS-rich phases. The two forms of DOPS were found to coexist owing to the ionization of the DOPS headgroup, such that the system could be regarded as quasi-ternary. The three formed phases with differently ionized DOPS domains were successfully identified experimentally by monitoring the adsorption of positively charged particles. In addition, coarse-grained molecular dynamics simulations confirmed the stability of the three-phase coexistence. Attraction mediated by hydrogen bonding between protonated DOPS molecules and reduction of the electrostatic interactions at the domain boundaries stabilized the three-phase coexistence.

Generation of gravity waves from thermal tides in the Venus atmosphere.

Gravity waves play essential roles in the terrestrial atmosphere because they propagate far from source regions and transport momentum and energy globally. Gravity waves are also observed in the Venus atmosphere, but their characteristics have been poorly understood. Here we demonstrate activities of small-scale gravity waves using a high-resolution Venus general circulation model with less than 20 and 0.25 km in the horizontal and vertical grid intervals, respectively. We find spontaneous gravity wave radiation from nearly balanced flows. In the upper cloud layer (~70 km), the thermal tides in the super-rotation are primary sources of small-scale gravity waves in the low-latitudes. Baroclinic/barotropic waves are also essential sources in the mid- and high-latitudes. The small-scale gravity waves affect the three-dimensional structure of the super-rotation and contribute to material mixing through their breaking processes. They propagate vertically and transport momentum globally, which decelerates the super-rotation in the upper cloud layer (~70 km) and accelerates it above ~80 km.

Developmental Phase Transitions in Spatial Organization of Spontaneous Activity in Postnatal Barrel Cortex Layer 4.

Spatially-organized spontaneous activity is a characteristic feature of developing mammalian sensory systems. However, the transitions of spontaneous-activity spatial organization during development and related mechanisms remain largely unknown. We reported previously that layer 4 (L4) glutamatergic neurons in the mouse barrel cortex exhibit spontaneous activity with a patchwork-type pattern at postnatal day (P)5, which is during barrel formation. In the current work, we revealed that spontaneous activity in mouse barrel-cortex L4 glutamatergic neurons exhibits at least three phases during the first two weeks of postnatal development. Phase I activity has a patchwork-type pattern and is observed not only at P5, but also P1, before barrel formation. Phase II is found at P9, by which time barrel formation is completed, and exhibits broadly synchronized activity across barrel borders. Phase III emerges around P11 when L4-neuron activity is desynchronized. The Phase I activity, but not Phase II or III activity, is blocked by thalamic inhibition, demonstrating that the Phase I to II transition is associated with loss of thalamic dependency. Dominant-negative (DN)-Rac1 expression in L4 neurons hampers the Phase II to III transition. It also suppresses developmental increases in spine density and excitatory synapses of L4 neurons in the second postnatal week, suggesting that Rac1-mediated synapse maturation could underlie the Phase II to III transition. Our findings revealed the presence of distinct mechanisms for Phase I to II and Phase II to III transition. They also highlighted the role of a small GTPase in the developmental desynchronization of cortical spontaneous activity.SIGNIFICANCE STATEMENT Developing neocortex exhibits spatially-organized spontaneous activity, which plays a critical role in cortical circuit development. The features of spontaneous-activity spatial organization and the mechanisms underlying its changes during development remain largely unknown. In the present study, using two-photon in vivo imaging, we revealed three phases (Phases I, II, and III) of spontaneous activity in barrel-cortex layer 4 (L4) glutamatergic neurons during the first two postnatal weeks. We also demonstrated the presence of distinct mechanisms underlying phase transitions. Phase I to II shift arose from the switch in the L4-neuron driving source, and Phase II to III transition relied on L4-neuron Rac1 activity. These results provide new insights into the principles of developmental transitions of neocortical spontaneous-activity spatial patterns.

Climate Change May Restrict the Predation Efficiency of Mesocyclops aspericornis (Copepoda: Cyclopidae) on Aedes aegypti (Diptera: Culicidae) Larvae.

(1) Dengue is the most spread mosquito-borne viral disease in the world, and vector control is the only available means to suppress its prevalence, since no effective treatment or vaccine has been developed. A biological control program using copepods that feed on mosquito larvae has been practiced in Vietnam and some other countries, but the application of copepods was not always successful. (2) To understand why the utility of copepods varies, we evaluated the predation efficiency of a copepod species (Mesocyclops aspericornis) on a vector species (Aedes aegypti) by laboratory experiments under different temperatures, nutrition and prey-density conditions. (3) We found that copepod predation reduced intraspecific competition among Aedes larvae and then shortened the survivor's aquatic life and increased their pupal weight. In addition, the predatory efficiency of copepods was reduced at high temperatures. Furthermore, performance of copepod offspring fell when the density of mosquito larvae was high, probably because mosquito larvae had adverse effects on copepod growth through competition for food resources. (4) These results suggest that the increase in mosquitoes will not be suppressed solely by the application of copepods if the density of mosquito larvae is high or ambient temperature is high. We need to consider additional control methods in order to maintain the efficiency of copepods to suppress mosquito increase.

How waves and turbulence maintain the super-rotation of Venus' atmosphere.

Venus has a thick atmosphere that rotates 60 times as fast as the surface, a phenomenon known as super-rotation. We use data obtained from the orbiting Akatsuki spacecraft to investigate how the super-rotation is maintained in the cloud layer, where the rotation speed is highest. A thermally induced latitudinal-vertical circulation acts to homogenize the distribution of the angular momentum around the rotational axis. Maintaining the super-rotation requires this to be counteracted by atmospheric waves and turbulence. Among those effects, thermal tides transport the angular momentum, which maintains the rotation peak, near the cloud top at low latitudes. Other planetary-scale waves and large-scale turbulence act in the opposite direction. We suggest that hydrodynamic instabilities adjust the angular-momentum distribution at mid-latitudes.

Osmotic-Tension-Induced Membrane Lateral Organization.

Alteration of lipid raft organization manifesting as phase separation is important for cellular processes, such as signaling and trafficking. Such behaviors and dynamics of lipid membranes can be affected by external stimuli including both physical and chemical stimuli. In this study, we focused on osmotic-tension-induced phase separation. The effects of osmotic tension on the phase behaviors of vesicles consisting of dioleoylphosphocholine (DOPC)/dipalmitoylphosphocholine (DPPC)/cholesterol (Chol) were quantitatively studied at different temperatures by fluorescence microscopy. We determined the ternary phase diagrams and found that tension leads to a shift in the miscibility temperature. Cholesterol plays a key role in determining the extent of this shift. In addition, we found that osmotic tension can enhance the line tension. The physicochemical mechanism of osmotic-pressure-induced phase separation is discussed.

Thermal structure of the Venusian atmosphere from the sub-cloud region to the mesosphere as observed by radio occultation.

We present distributions of the zonal-mean temperature and static stability in the Venusian atmosphere obtained from Venus Express and Akatsuki radio occultation profiles penetrating down to an altitude of 40 km. At latitudes equatorward of 75°, static stability derived from the observed temperature profiles is consistent with previous in-situ measurements in that there is a low-stability layer at altitudes of 50-58 km and highly and moderately stratified layers above 58 km and below 50 km, respectively. Meanwhile, at latitudes poleward of 75°, a low-stability layer extends down to 42 km, which has been unreported in analyses of previous measurements. The deep low-stability layer in the polar region cannot be explained by vertical convection in the middle/lower cloud layer, and the present result thus introduces new constraints on the dynamics of the sub-cloud atmosphere. The Venusian atmosphere is in striking contrast to the Earth's troposphere, which generally has a deeper low-stability layer at low latitudes than at mid- and high latitudes.