A cGAL-UAS bipartite expression toolkit for Caenorhabditis elegans sensory neurons.

Animals integrate sensory information from the environment and display various behaviors in response to external stimuli. In Caenorhabditis elegans hermaphrodites, 33 types of sensory neurons are responsible for chemosensation, olfaction, and mechanosensation. However, the functional roles of all sensory neurons have not been systematically studied due to the lack of facile genetic accessibility. A bipartite cGAL-UAS system has been previously developed to study tissue- or cell-specific functions in C. elegans. Here, we report a toolkit of new cGAL drivers that can facilitate the analysis of a vast majority of the 60 sensory neurons in C. elegans hermaphrodites. We generated 37 sensory neuronal cGAL drivers that drive cGAL expression by cell-specific regulatory sequences or intersection of two distinct regulatory regions with overlapping expression (split cGAL). Most cGAL-drivers exhibit expression in single types of cells. We also constructed 28 UAS effectors that allow expression of proteins to perturb or interrogate sensory neurons of choice. This cGAL-UAS sensory neuron toolkit provides a genetic platform to systematically study the functions of C. elegans sensory neurons.

Autism-associated missense genetic variants impact locomotion and neurodevelopment in Caenorhabditis elegans.

Autism spectrum disorder (ASD) involves thousands of alleles in over 850 genes, but the current functional inference tools are not sufficient to predict phenotypic changes. As a result, the causal relationship of most of these genetic variants in the pathogenesis of ASD has not yet been demonstrated and an experimental method prioritizing missense alleles for further intensive analysis is crucial. For this purpose, we have designed a pipeline that uses Caenorhabditis elegans as a genetic model to screen for phenotype-changing missense alleles inferred from human ASD studies. We identified highly conserved human ASD-associated missense variants in their C. elegans orthologs, used a CRISPR/Cas9-mediated homology-directed knock-in strategy to generate missense mutants and analyzed their impact on behaviors and development via several broad-spectrum assays. All tested missense alleles were predicted to perturb protein function, but we found only 70% of them showed detectable phenotypic changes in morphology, locomotion or fecundity. Our findings indicate that certain missense variants in the C. elegans orthologs of human CACNA1D, CHD7, CHD8, CUL3, DLG4, GLRA2, NAA15, PTEN, SYNGAP1 and TPH2 impact neurodevelopment and movement functions, elevating these genes as candidates for future study into ASD. Our approach will help prioritize functionally important missense variants for detailed studies in vertebrate models and human cells.

NFAT5 Deficiency Alleviates Formalin-Induced Inflammatory Pain Through mTOR.

Nuclear factor of activated T cells (NFAT5) is a well-known transcription factor that regulates the expression of genes involved in osmotic stress. However, the role of NFAT5 in inflammatory pain remains unknown. Here, we studied the function of NFAT5 in inflammatory pain using NFAT5-heterozygous (Het) mice. To study inflammatory pain, we injected 10 µL of 2% formalin into the right hind paws of mice and monitored pain behaviors, such as licking, lifting, and flinching, for 60 min. After the first 15 min (phase I), there were no significant differences in pain behaviors between wild-type (WT) and NFAT5-Het mice. However, from 15-60 min (phase II), NFAT5-Het mice displayed significantly fewer pain behaviors compared to WT mice. Further, the expression levels of inflammatory-pain-related factors, including c-Fos, phosphorylated extracellular signal-regulated kinase (p-ERK), and phosphorylated n-methyl-D-aspartate receptor subunit 2B (p-NR2B), were significantly elevated in the spinal dorsal neurons of formalin-treated WT mice but was not elevated in NFAT5-Het mice. Similarly, c-Fos, p-ERK, and p-NR2B levels were significantly higher in glutamate-treated PC12 neuronal cells but were not affected by Nfat5 silencing in glutamate-treated PC12 cells. Altogether, our findings suggest that NFAT5 deficiency may mitigate formalin-induced inflammatory pain by upregulating mammalian target of rapamycin (mTOR) expression and downregulating its downstream factors in spinal dorsal neurons. Therefore, NFAT5 is a potential therapeutic target for the treatment of inflammatory pain.

Adenomatous polyposis coli-stimulated GEF 1 (Asef1) is a negative regulator of excitatory synaptic function.

Guanine nucleotide exchange factors (GEFs) play important roles in many cellular processes, including regulation of the structural plasticity of dendritic spines. A GEF protein, adenomatous polyposis coli-stimulated GEF 1 (Asef1, ARHGEF4) is highly expressed in the nervous system. However, the function of Asef1 has not been investigated in neurons. Here, we present evidence showing that Asef1 negatively regulates the synaptic localization of postsynaptic density protein 95 (PSD-95) in the excitatory synapse by inhibiting Staufen-mediated synaptic localization of PSD-95. Accordingly, Asef1 expression impairs synaptic transmission in hippocampal cultured neurons. In addition, neuronal activity facilitates the dissociation of Asef1 from Staufen in a phosphoinositide 3 kinase (PI3K)-dependent manner. Taken together, our data reveal Asef1 functions as a negative regulator of synaptic localization of PSD-95 and synaptic transmission.

Easy preparation of self-assembled high-density buckypaper with enhanced mechanical properties.

A controlled assembly and alignment of carbon nanotubes (CNTs) in a high-packing density with a scalable way remains challenging. This paper focuses on the preparation of self-assembled and well-aligned CNTs with a densely packed nanostructure in the form of buckypaper via a simple filtration method. The CNT suspension concentration is strongly reflected in the alignment and assembly behavior of CNT buckypaper. We further demonstrated that the horizontally aligned CNT domain gradually increases in size when increasing the deposited CNT quantity. The resultant aligned buckypaper exhibited notably enhanced packing density, strength, modulus, and hardness compared to previously reported buckypapers.

Activity-dependent synaptic localization of processing bodies and their role in dendritic structural plasticity.

In neurons, transport of a subset of mRNAs to subcellular regions and their translation has a role in synaptic plasticity. Recent studies have suggested a control mechanism of this local translation through mRNA compartmentalization or degradation. Here we report that processing bodies (P-bodies), which are involved in mRNA degradation or storage, are transported to dendrites by conventional kinesin (KIF5A) as a motor protein. Neuronal activation induced by depolarization increased the colocalization of P-bodies with PSD-95 in dendrites. This neuronal activity increased the release of Nd1 and Arp2 mRNA from the P-bodies and, consequently, reversed the decrease of F-actin (induced by overexpression of Dcp1a) in the dendrites. Our data suggest that the activity-induced redistribution of P-bodies and mRNA release from P-bodies might have a role in synaptic structural plasticity by altering levels of mRNAs that are involved in the dynamics of the actin cytoskeleton in dendrites.

Melatonin induces autophagy via an mTOR-dependent pathway and enhances clearance of mutant-TGFBIp.

The hallmark of granular corneal dystrophy type 2 (GCD2) is the deposit of mutant transforming growth factor-ß (TGF-ß)-induced protein (TGFBIp) in the cornea. We have recently shown that there is a delay in autophagic degradation of mutant-TGFBIp via impaired autophagic flux in GCD2 corneal fibroblasts. We hypothesized that melatonin can specifically induce autophagy and consequently eliminate mutant-TGFBIp in GCD corneal fibroblasts. Our results show that melatonin activates autophagy in both wild-type (WT) and GCD2-homozygous (HO) corneal fibroblast cell lines via the mammalian target of rapamycin (mTOR)-dependent pathway. Melatonin treatment also led to increased levels of beclin 1, which is involved in autophagosome formation and maturation. Furthermore, melatonin significantly reduced the amounts of mutant- and WT-TGFBIp. Treatment with melatonin counteracted the autophagy-inhibitory effects of bafilomycin A1, a potent inhibitor of autophagic flux, demonstrating that melatonin enhances activation of autophagy and increases degradation of TGFBIp. Cotreatment with melatonin and rapamycin, an autophagy inducer, had an additive effect on mutant-TGFBIp clearance compared to treatment with either drug alone. Treatment with the selective melatonin receptor antagonist luzindole did not block melatonin-induced autophagy. Given its ability to activate autophagy, melatonin is a potential therapeutic agent for GCD2.

Philosophical strategies for nature of science based on worldviews: The quantum interference of single electron.

This study aims to present a strategy to transform the worldviews of students, especially college science classrooms, into desirable current worldviews (Darwin, Quantum mechanics, Einstein, refer to Fig 1). Above all, the worldviews, that is metaphysical belief, needs to be changed. This is because metaphysical belief is the basis for the whole framework of conception. Since the previous study emphasized epistemological belief only, an emphasis was placed on metaphysical belief on which epistemological belief is based. Therefore, the two beliefs should be used to elucidate in the education of Nature of Science. Metaphysical belief in the science of cosmic order, symmetry, or disorder is often important in scientific research and can lead to an epistemological view that can select or reject a certain kind of explanation. Specially, the two-slit experiment with single electrons is a radical illustration of another paradigm shift, from classical (mechanistic materialism) to quantum physics (desirable dialectical materialism). The double slit electron experiment creates sensuous images that can be grasped at once. Because quantum mechanics is inherently abstract, it is difficult to accept its meaning.

Ginsenoside Rh2 induces Bcl-2 family proteins-mediated apoptosis in vitro and in xenografts in vivo models.

The cancer chemoprevention effects of ginseng saponins have been demonstrated against a variety of experimental tumors; however, their molecular mechanisms in vitro and in in vivo models are not well studied. This study was undertaken to gain insights into the molecular mechanisms of ginsenoside Rh2 (Rh2)-induced cell death in human breast cancer cell lines as well as in in vivo xenografts. Rh2 treatment significantly inhibited viability of both MCF-7 and MDA-MB-231 human breast cells in a concentration-dependent manner, which correlated with mitochondria-mediated apoptosis. Rh2-induced apoptosis was accompanied by the down-regulation of antiapoptotic proteins Bcl-2, Bcl-xL, and Mcl-1. It also caused induction of the proapoptotic members Bak, Bax, and Bim leading to mitochondrial translocation of Bax and activation of caspases. Moreover, Rh2-induced apoptosis was partially, yet significantly protected by transient transfection of MCF-7 cells with Bax- and Bak-targeted siRNAs. Oral gavage of 5 mg Rh2/kg of mouse (three times a week) significantly caused apoptosis of MDA-MB-231 xenografts. An increase in Bax and Bak and a decrease in Bcl-2 and Bcl-xL transcript levels, in accordance with their protein expression, were observed in tumor tissue. Tumors from Rh2-treated mice exhibited a markedly higher count of apoptotic bodies and reduced proliferation index compared with control tumors. Our data suggest that Rh2 used in traditional oriental medicine for the treatment of various ailments, may be an attractive agent for the treatment and/or prevention of human breast cancers.

Impairment of the Staufen1-NS1 interaction reduces influenza viral replication.

Staufen1 (Stau1), a host cellular protein, along with non-structural protein 1 (NS1), an influenza viral protein, associate with each other during influenza viral infection and down-regulation of Stau1 by RNA interference reduces the yield of influenza A virus, suggesting a role for Stau1 in viral replication. In order to develop a new tool to control influenza A virus, we determined the specific regions of Staufen1 protein involved in the interaction with NS1. The linker between RBD3 and 4 was isolated as the binding regions. Expression of RBD3L, the linker including RBD3, inhibited the interaction between Stau1 and NS1, reducing the colocalization of the two proteins in the cytosol and nucleus regions. In addition, yield of influenza A virus in RBD3L-expressing cells was significantly reduced 36 h after infection. These results suggest that disruption of the Stau1-NS1 interaction can be used to control replication of influenza A virus, thereby providing a target for the development of antiviral drugs.

Behavior determines the hippocampal spatial mapping of a multisensory environment.

Animals behave in multisensory environments guided by various modalities of spatial information. Mammalian navigation engages a cognitive map of space in the hippocampus. Yet it is unknown whether and how this map incorporates multiple modalities of spatial information. Here, we establish two behavioral tasks in which mice navigate the same multisensory virtual environment by either pursuing a visual landmark or tracking an odor gradient. These tasks engage different proportions of visuo-spatial and olfacto-spatial mapping CA1 neurons and different population-level representations of each sensory-spatial coordinate. Switching between tasks results in global remapping. In a third task, mice pursue a target of varying sensory modality, and this engages modality-invariant neurons mapping the abstract behaviorally relevant coordinate irrespective of its physical modality. These findings demonstrate that the hippocampus does not necessarily map space as one coherent physical variable but as a combination of sensory and abstract reference frames determined by the subject's behavioral goal.

Conserved missense variant in ALDH1A3 ortholog impairs fecundity in C. elegans.

Accumulating evidence demonstrates that mutations in ALDH1A3 (the aldehyde dehydrogenase 1 family, member A3) are associated with developmental defects. The ALDH1A3 enzyme catalyzes retinoic acid biosynthesis and is essential to patterning and neuronal differentiation in the development of embryonic nervous system. Several missense mutations in ALDH1A3 have been identified in family studies of autosomal recessive microphthalmia, autism spectrum disorder, and other neurological disorders. However, there has been no evidence from animal models that verify the functional consequence of missense mutations in ALDH1A3. Here, we introduced the equivalent of the ALDH1A3 C174Y variant into the Caenorhabditis elegans ortholog, alh-1, at the corresponding locus. Mutant animals with this missense mutation exhibited decreased fecundity by 50% compared to wild-type animals, indicating disrupted protein function. To our knowledge, this is the first ALDH1A3 C174Y missense model, which might be used to elucidate the effects of ALDH1A3 C174Y missense mutation in the retinoic acid signaling pathway during development.

Apolipoprotein E mRNA is transported to dendrites and may have a role in synaptic structural plasticity.

Although the dendritic localization and translation of a subset of mRNAs plays a pivotal role in synaptic plasticity, the dendritic mRNAs and their functions have been only minimally characterized thus far. In this study, we isolated mRNAs from Staufen2-containing ribonucleoprotein complexes, which function as modules for the transport of mRNA to the dendrites, and then constructed a cDNA library. Apolipoprotein E gene (APOE) mRNA was isolated from the dendritic mRNA-specific cDNA library. The specific localization of APOE mRNA was evaluated via in situ hybridization. The specific regions involved in the dendritic transport of APOE mRNA were determined using a visualization system employing green fluorescent protein-tagged bacteriophage MS2 RNA-binding protein. As a result, the proximal N-terminal or C-terminal regions of the ApoE-coding sequences were determined to be sufficient for dendritic transport. The level of dendritic APOE mRNA was significantly increased by depolarization-induced neuronal activity, but was reduced in the cell body regions. We assessed the functions of neuronal ApoE. The reduction of ganglioside GM1 by cholesterol depletion was completely blocked by ApoE over-expression. In addition, ApoE over-expression increased the immunoreactivity of the post-synaptic density 95 kDa antibody in the dendrites. These findings indicate that neuronal ApoE may be relevant to lipid rafts or synaptic structural plasticity.

Labeling Dual Presynaptic Inputs using cFork Anterograde Tracing System.

Understanding brain function-related neural circuit connectivity is essential for investigating how cognitive functions are decoded in neural circuits. Trans-synaptic viral vectors are useful for identifying neural synaptic connectivity because of their ability to be transferred from transduced cells to synaptically connected cells. However, concurrent labeling of multisynaptic inputs to postsynaptic neurons is impossible with currently available trans-synaptic viral vectors. Here, we report a neural circuit tracing system that can simultaneously label postsynaptic neurons with two different markers, the expression of which is defined by presynaptic input connectivity. This system, called "cFork (see fork)", includes delivering serotype 1-packaged AAV vectors (AAV1s) containing Cre or flippase recombinase (FlpO) into two different presynaptic brain areas, and AAV5 with a dual gene expression cassette in postsynaptic neurons. Our in vitro and in vivo tests showed that selective expression of two different fluorescence proteins, EGFP and mScarlet, in postsynaptic neurons could be achieved by AAV1-mediated anterograde trans-synaptic transfer of Cre or FlpO constructs. When this tracing system was applied to the somatosensory barrel field cortex (S1BF) or striatum innervated by multiple presynaptic inputs, postsynaptic neurons defined by presynaptic inputs were simultaneously labeled with EGFP or mScarlet. Our new anterograde tracing tool may be useful for elucidating the complex multisynaptic connectivity of postsynaptic neurons regulating diverse brain functions.

Postsynaptic density protein 95 (PSD-95) is transported by KIF5 to dendritic regions.

Postsynaptic density protein 95 (PSD-95) is a pivotal postsynaptic scaffolding protein in excitatory neurons. Although the transport and regulation of PSD-95 in synaptic regions is well understood, dendritic transport of PSD-95 before synaptic localization still remains to be clarified. To evaluate the role of KIF5, conventional kinesin, in the dendritic transport of PSD-95 protein, we expressed a transport defective form of KIF5A (ΔMD) that does not contain the N-terminal motor domain. Expression of ΔMD significantly decreased PSD-95 level in the dendrites. Consistently, KIF5 was associated with PSD-95 in in vitro and in vivo assays. This interaction was mediated by the C-terminal tail regions of KIF5A and the third PDZ domain of PSD-95. Additionally, the ADPDZ3 (the association domain of NMDA receptor and PDZ3 domain) expression significantly reduced the levels of PSD-95, glutamate receptor 1 (GluA1) in dendrites. The association between PSD-95 and KIF5A was dose-dependent on Staufen protein, suggesting that the Staufen plays a role as a regulatory role in the association. Taken together, our data suggest a new mechanism for dendritic transport of the AMPA receptor-PSD-95.

Noonan syndrome-associated SHP2 mutation differentially modulates the expression of postsynaptic receptors according to developmental maturation.

Glutamate is the major excitatory neurotransmitter in the central nervous system, and related signaling involves both AMPA and NMDA subtype receptors. The expression of glutamate receptors is dynamically regulated during development. Recent studies showed that the dysregulation of glutamate receptor expression and function is associated with neurodevelopmental disorders including intellectual disability. Previously, a Noonan syndrome (NS)-associated SHP2 mutation (SHP2D61G) was shown to increase the synaptic delivery of AMPA receptor, subsequently impairing synaptic plasticity and learning in adult mice. However, how the mutant SHP2 affects glutamate receptor expression during development is not known. Here, we found that the SHP2D61G differentially regulates the expression of AMPA and NMDA receptors depending on the stage of neuronal maturation. In cultured neurons (immature stage; DIV 6), overexpression of SHP2D61G significantly increased the average size and the number of NMDA receptor-containing particles, but not those with AMPA receptors. In early matured neurons (DIV 12), SHP2D61G significantly increased only the average size of AMPA receptor particles, and subsequently increased their number in matured neurons (DIV 18). Importantly, all the changes described above for SHP2D61G neurons were reversed by inhibiting MAPK. These data demonstrate that the increased activation of MAPK signaling pathway by SHP2D61G could deregulate the surface expression of synaptic receptors during neuronal development, which likely contributes to cognitive impairments in NS patients.

Crucial Role of Oxidation Debris of Carbon Nanotubes in Subsequent End-Use Applications of Carbon Nanotubes.

A facile purification method for oxidized carbon nanotubes (CNTs) is developed to preserve acidic carbon compounds (ACCs) for achieving high-quality dispersion of CNTs. The remaining ACCs, which originated from the surface destruction of CNTs during the oxidation process, are considered to play a crucial role in the dispersion of CNTs in water and various polar protic solvents. To elucidate the concrete role of ACCs, a direct titration method is applied to quantitatively investigate the degree of ionization of both CNTs and ACCs in their aqueous dispersions. While ACCs with strong carboxylic groups (pKa of around 2.9) are easily removed by the neutral or base washing of oxidized CNTs, which is common in the purification process, ACC-selective purification using acid washing preserves the ACCs attached to CNTs, thereby effectively stabilizing CNT dispersions in aqueous solutions. Additionally, the Hansen solubility parameters of ACC-preserved and ACC-removed CNTs were determined by the inverse gas chromatography method to estimate their miscibility in various solvents. The preserved ACCs significantly influenced the dispersibility of CNTs in polar protic solvents, which may widen the possible application of CNTs. Specifically, the ACC-preserved high-quality CNT dispersion produces high-performance CNT buckypaper with densely packed nanostructures. The Young's modulus and tensile strength of these buckypapers reach up to 12.0 and 91.0 MPa, respectively, which exceed those of ACC-removed CNTs in previous reports.

Flexible and Robust Thermoelectric Generators Based on All-Carbon Nanotube Yarn without Metal Electrodes.

As practical interest in flexible/or wearable power-conversion devices increases, the demand for high-performance alternatives to thermoelectric (TE) generators based on brittle inorganic materials is growing. Herein, we propose a flexible and ultralight TE generator (TEG) based on carbon nanotube yarn (CNTY) with excellent TE performance. The as-prepared CNTY shows a superior electrical conductivity of 3147 S/cm due to increased longitudinal carrier mobility derived from a highly aligned structure. Our TEG is innovative in that the CNTY acts as multifunctions in the same device. The CNTY is alternatively doped into n- and p-types using polyethylenimine and FeCl3, respectively. The highly conductive CNTY between the doped regions is used as electrodes to minimize the circuit resistance, thereby forming an all-carbon TEG without additional metal deposition. A flexible TEG based on 60 pairs of n- and p-doped CNTY shows the maximum power density of 10.85 and 697 µW/g at temperature differences of 5 and 40 K, respectively, which are the highest values among reported TEGs based on flexible materials. We believe that the strategy proposed here to improve the power density of flexible TEG by introducing highly aligned CNTY and designing a device without metal electrodes shows great potential for the flexible/or wearable power-conversion devices.

Metal-Phenolic Carbon Nanocomposites for Robust and Flexible Energy-Storage Devices.

Future electronics applications such as wearable electronics depend on the successful construction of energy-storage devices with superior flexibility and high electrochemical performance. However, these prerequisites are challenging to combine: External forces often cause performance degradation, whereas the trade-off between the required nanostructures for strength and electrochemical performance only results in diminished energy storage. Herein, a flexible supercapacitor based on tannic acid (TA) and carbon nanotubes (CNTs) with a unique nanostructure is presented. TA was self-assembled on the surface of the CNTs by metal-phenolic coordination bonds, which provides the hybrid film with both high strength and high pseudocapacitance. Besides 17-fold increased mechanical strength of the final composite, the hybrid film simultaneously exhibits excellent flexibility and volumetric capacitance.

Preparation and Exceptional Mechanical Properties of Bone-Mimicking Size-Tuned Graphene Oxide@Carbon Nanotube Hybrid Paper.

The self-assembled nanostructures of carbon nanomaterials possess a damage-tolerable architecture crucial for the inherent mechanical properties at both micro- and macroscopic levels. Bone, or "natural composite," has been known to have superior energy dissipation and fracture resistance abilities due to its unique load-bearing hybrid structure. However, few approaches have emulated the desirable structure using carbon nanomaterials. In this paper, we present an approach in fabricating a hybrid composite paper based on graphene oxide (GO) and carbon nanotube (CNT) that mimicks the natural bone structure. The size-tuning strategy enables smaller GO sheets to have more cross-linking reactions with CNTs and be homogeneously incorporated into CNT-assembled paper, which is advantageous for effective stress transfer. The resultant hybrid composite film has enhanced mechanical strength, modulus, toughness, and even electrical conductivity compared to previously reported CNT-GO based composites. We further demonstrate the usefulness of the size-tuned GOs as the "stress transfer medium" by performing in situ Raman spectroscopy during the tensile test.