The C. elegans Taste Receptor Homolog LITE-1 Is a Photoreceptor.

Many animal tissues/cells are photosensitive, yet only two types of photoreceptors (i.e., opsins and cryptochromes) have been discovered in metazoans. The question arises as to whether unknown types of photoreceptors exist in the animal kingdom. LITE-1, a seven-transmembrane gustatory receptor (GR) homolog, mediates UV-light-induced avoidance behavior in C. elegans. However, it is not known whether LITE-1 functions as a chemoreceptor or photoreceptor. Here, we show that LITE-1 directly absorbs both UVA and UVB light with an extinction coefficient 10-100 times that of opsins and cryptochromes, indicating that LITE-1 is highly efficient in capturing photons. Unlike typical photoreceptors employing a prosthetic chromophore to capture photons, LITE-1 strictly depends on its protein conformation for photon absorption. We have further identified two tryptophan residues critical for LITE-1 function. Interestingly, unlike GPCRs, LITE-1 adopts a reversed membrane topology. Thus, LITE-1, a taste receptor homolog, represents a distinct type of photoreceptor in the animal kingdom.

The neural circuits and synaptic mechanisms underlying motor initiation in C. elegans.

C. elegans is widely used to dissect how neural circuits and genes generate behavior. During locomotion, worms initiate backward movement to change locomotion direction spontaneously or in response to sensory cues; however, the underlying neural circuits are not well defined. We applied a multidisciplinary approach to map neural circuits in freely behaving worms by integrating functional imaging, optogenetic interrogation, genetic manipulation, laser ablation, and electrophysiology. We found that a disinhibitory circuit and a stimulatory circuit together promote initiation of backward movement and that circuitry dynamics is differentially regulated by sensory cues. Both circuits require glutamatergic transmission but depend on distinct glutamate receptors. This dual mode of motor initiation control is found in mammals, suggesting that distantly related organisms with anatomically distinct nervous systems may adopt similar strategies for motor control. Additionally, our studies illustrate how a multidisciplinary approach facilitates dissection of circuit and synaptic mechanisms underlying behavior in a genetic model organism.

Arterial Baroreflex Dysfunction Promotes Neuroinflammation by Activating the Platelet CD40L/Nuclear Factor Kappa B Signaling Pathway in Microglia and Astrocytes.

Arterial baroreflex (ABR) dysfunction has previously been associated with neuroinflammation, the most common pathological feature of neurological disorders. However, the mechanisms mediating ABR dysfunction-induced neuroinflammation are not fully understood. In the present study, we investigated the role of platelet CD40 ligand (CD40L) in neuroinflammation in an in vivo model of ABR dysfunction, and microglia and astrocyte activation in vitro. ABR dysfunction was induced in Sprague‒Dawley rats by sinoaortic denervation (SAD). We used ELSA and immunofluorescence to assess the effect of platelet CD40L on glial cell polarization and the secretion of inflammatory factors. By flow cytometry, we found that rats subjected to SAD showed a high level of platelet microaggregation and upregulation of CD40L on the platelet surface. The promotion of platelet invasion and accumulation was also observed in the brain tissues of rats subjected to SAD. In the animal model and cultured N9 microglia/C6 astrocytoma cells, platelet CD40L overexpression promoted neuroinflammation and activated M1 microglia, A1 astrocytes, and the nuclear factor kappa B (NFκB) signaling pathway. These effects were partially blocked by inhibiting platelet activity with clopidogrel or inhibiting CD40L-mediated signaling. Our results suggest that during ABR dysfunction, CD40L signaling in platelets converts microglia to the M1 phenotype and astrocytes to the A1 phenotype, activating NFκB and resulting in neuroinflammation. Thus, our study provides a novel understanding of the pathogenesis of ABR dysfunction-induced neuroinflammation and indicates that targeting platelet CD40L is beneficial for treating central nervous system (CNS) disorders associated with ABR dysfunction.

The photoluminescence of isolated and paired Bi3+ ions in layered LnOCl crystals: a first-principles study.

Luminescent ns2 centers have shown great potential for applications as phosphors and scintillators. First-principles calculations based on density functional theory are performed to systematically analyze the luminescent centers of isolated and paired Bi3+(6s2) ions in layered LnOCl (Ln = Y, Gd, La) crystals. The spin-orbit coupling and orbital hybridization both show important effects on the luminescence properties. The luminescence of the isolated Bi ion is confirmed as the interconfigurational transition of 3P0,1 → 1S0. For the Bi pair, the adiabatic potential energy surfaces are calculated and the charge transfer excited state is the most stable, which accounts for the visible emission of a large Stokes shift. Furthermore, the electron-hole pair separation, absorption, excitonic state and emission of the material with fully-concentrated Bi3+, BiOCl, are discussed. This study shows that the first-principles calculations can serve as an effective tool for the photoluminescence analysis and engineering of materials activated with isolated, paired and even fully-concentrated ns2 ions.

Gamma-Ray Observation of the Cygnus Region in the 100-TeV Energy Region.

We report observations of gamma-ray emissions with energies in the 100-TeV energy region from the Cygnus region in our Galaxy. Two sources are significantly detected in the directions of the Cygnus OB1 and OB2 associations. Based on their positional coincidences, we associate one with a pulsar PSR J2032+4127 and the other mainly with a pulsar wind nebula PWN G75.2+0.1, with the pulsar moving away from its original birthplace situated around the centroid of the observed gamma-ray emission. This work would stimulate further studies of particle acceleration mechanisms at these gamma-ray sources.

First Detection of sub-PeV Diffuse Gamma Rays from the Galactic Disk: Evidence for Ubiquitous Galactic Cosmic Rays beyond PeV Energies.

We report, for the first time, the long-awaited detection of diffuse gamma rays with energies between 100 TeV and 1 PeV in the Galactic disk. Particularly, all gamma rays above 398 TeV are observed apart from known TeV gamma-ray sources and compatible with expectations from the hadronic emission scenario in which gamma rays originate from the decay of π^{0}'s produced through the interaction of protons with the interstellar medium in the Galaxy. This is strong evidence that cosmic rays are accelerated beyond PeV energies in our Galaxy and spread over the Galactic disk.

Self-Activated and Bismuth-Related Photoluminescence in Rare-Earth Vanadate, Niobate, and Tantalate Series-A First-Principles Study.

Rare-earth vanadates, niobates, and tantalates have shown self-activated and Bi3+-activated emissions. Their intrinsic emission has been attributed to self-trapped excitons (STEs), but the detailed information concerning the geometric and electronic structures of the excited states has remained unknown. Regarding the Bi3+ dopants in these hosts, the luminescence has been attributed to two different mechanisms, i.e., Bi3+↔ (V/Nb/Ta)5+ metal-to-metal charge transfer and interconfigurational (3P0,1 → 1S0) transition. Here, first-principles calculations using hybrid functionals are employed to resolve these issues. The STEs are shown to be composed of an electron localized on an individual vanadium, niobium, or tantalum ion and a hole localized on a single nearest-neighbor oxygen ion that is not shared by covalent complexes, and the bond length of the (V/Nb/Ta)-O bond with oxygen accommodating the hole is significantly elongated. The Bi3+-related emission is identified as the recombination of an exciton with a hole and an electron localized correspondingly at Bi3+ and (V/Nb/Ta)5+ ions, while the excitation is dominated by the 6s → 6p transition of Bi3+. Furthermore, Bi3+ has a hole trap level in all of the hosts considered with the trap levels in the vacuum-referred binding energy diagram being nearly flat but has an electron trap level only in rare-earth tantalates. Furthermore, the long-wavelength emission observed in niobates and tantalates is interpreted based on our calculations to be excitons bound to intrinsic defects. The insights gained in this work deepen our understanding of the STEs and form the basis for interpreting similar luminescence phenomena in other ternary closed-shell d0 transition-metal oxides. The clarification of Bi3+-related transitions and the analyses with the vacuum-referred binding energy diagram may find applications for the design and optimization of Bi3+-activated phosphors.

First-Principles Study of Bi3+-Related Luminescence and Electron and Hole Traps in (Y/Lu/La)PO4.

Bismuth ion-doped phosphate crystals have shown rich luminescence phenomena. However, the complexity and variety of Bi3+-related transitions bring great challenges to the understanding of the underlying mechanisms, rendering it hard to rationally design new phosphors and optimize their performance. In this work, we perform first-principles calculations based on the generalized gradient approximation of density functional to obtain the excited state equilibrium geometric structures and then calculate the electronic structures for various Bi3+-related excited states in phosphates RPO4:Bi3+ (R = Y, Lu, La) by utilizing the hybrid density functional method. The experimentally measured excitation and emission features are well interpreted by our theoretical calculations. Specifically, we reveal that the emission in LaPO4:Bi3+ is of charge transfer nature, whereas the dominant emission in YPO4:Bi3+ or LuPO4:Bi3+ is the characteristic A band emission. Trapped holes above the valence band maximum due to intrinsic defects are deemed to play a role in the charge-transfer emission of LaPO4. Our calculations show that the excited state of the Bi3+ pair in YPO4 or LuPO4 is (Bi3+-Bi3+)*, rather than Bi2+-Bi4+. Such a Bi3+ pair contributes to the longer wavelength emission. Furthermore, our calculations on charge transition levels show that Bi3+ ions can act as electron and hole traps in RPO4 (R = Y, Lu, La). Our work indicates that first-principles calculations can be useful in exploring the diverse luminescence processes in Bi3+-doped inorganic insulators.

Band characterization of topological photonic crystals using the broadband Green's function technique.

A novel method is developed in this paper to characterize the band diagram and band modal fields of gyromagnetic photonic crystals that support topological one-way edge states. The proposed method is based on an integral equation formulation that utilizes the broadband Green's function (BBGF). The BBGF is a hybrid representation of the periodic lattice Green's function with imaginary extractions that has accelerated convergence and is suitable for broadband evaluations. The effects of the tensor permeability of the gyromagnetic scatterers are incorporated in a new formulation of surface integral equations (SIEs) with BBGF as the kernel that can be solved by the method of moments. The results are compared against Comsol simulations for various cases to demonstrate the accuracy and efficiency of the proposed method. Simulations results are illustrated and discussed for the modes of topological photonic crystals in relation to the physics of degeneracy, applied magnetic fields, and bandgaps.

First Detection of Photons with Energy beyond 100 TeV from an Astrophysical Source.

We report on the highest energy photons from the Crab Nebula observed by the Tibet air shower array with the underground water-Cherenkov-type muon detector array. Based on the criterion of a muon number measured in an air shower, we successfully suppress 99.92% of the cosmic-ray background events with energies E>100 TeV. As a result, we observed 24 photonlike events with E>100 TeV against 5.5 background events, which corresponds to a 5.6σ statistical significance. This is the first detection of photons with E>100 TeV from an astrophysical source.

Evaluation of the Interplanetary Magnetic Field Strength Using the Cosmic-Ray Shadow of the Sun.

We analyze the Sun's shadow observed with the Tibet-III air shower array and find that the shadow's center deviates northward (southward) from the optical solar disk center in the "away" ("toward") interplanetary magnetic field (IMF) sector. By comparing with numerical simulations based on the solar magnetic field model, we find that the average IMF strength in the away (toward) sector is 1.54±0.21_{stat}±0.20_{syst} (1.62±0.15_{stat}±0.22_{syst}) times larger than the model prediction. These demonstrate that the observed Sun's shadow is a useful tool for the quantitative evaluation of the average solar magnetic field.

Reciprocal Changes in Phosphoenolpyruvate Carboxykinase and Pyruvate Kinase with Age Are a Determinant of Aging in Caenorhabditis elegans.

Aging involves progressive loss of cellular function and integrity, presumably caused by accumulated stochastic damage to cells. Alterations in energy metabolism contribute to aging, but how energy metabolism changes with age, how these changes affect aging, and whether they can be modified to modulate aging remain unclear. In locomotory muscle of post-fertile Caenorhabditis elegans, we identified a progressive decrease in cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C), a longevity-associated metabolic enzyme, and a reciprocal increase in glycolytic pyruvate kinase (PK) that were necessary and sufficient to limit lifespan. Decline in PEPCK-C with age also led to loss of cellular function and integrity including muscle activity, and cellular senescence. Genetic and pharmacologic interventions of PEPCK-C, muscle activity, and AMPK signaling demonstrate that declines in PEPCK-C and muscle function with age interacted to limit reproductive life and lifespan via disrupted energy homeostasis. Quantifications of metabolic flux show that reciprocal changes in PEPCK-C and PK with age shunted energy metabolism toward glycolysis, reducing mitochondrial bioenergetics. Last, calorie restriction countered changes in PEPCK-C and PK with age to elicit anti-aging effects via TOR inhibition. Thus, a programmed metabolic event involving PEPCK-C and PK is a determinant of aging that can be modified to modulate aging.

Levodopa-Induced Motor and Dopamine Receptor Changes in Caenorhabditis elegans Overexpressing Human Alpha-Synuclein.

BACKGROUND: Levodopa-induced dyskinesia (LID) is a disabling complication of levodopa therapy in Parkinson's disease (PD) with no effective treatments. Fluctuations in levels of levodopa constitute a key risk factor of LID. There is a pressing need for the development of a simple animal model of LID. Several genetic and toxin-based models of PD in Caenorhabditis elegans have been described, which have advanced our understanding of PD pathophysiology. We aimed to study levodopa-induced changes in a Parkinson's disease model of C. elegans expressing human α-synuclein. METHODS: We exposed the α-synuclein C. elegans to levodopa in continuous and alternating fashions. Automated behavioral analysis was then used to quantify changes in motor activity. Confocal microscopy was used next to quantify changes in dopamine receptor distribution and expression in motor neurons of live C. elegans. RESULTS: Chronic exposure to levodopa led to hyperactivity of the α-synuclein C. elegans without meaningful increase in motor activity. There was also an increase in peripheral clustering and expression of dopamine receptors in motor neurons. Both of these changes were significantly higher with alternating, compared to continuous, exposure to levodopa. CONCLUSIONS: This is the first report of changes in motor and dopamine receptors induced by levodopa in C. elegans overexpressing human α-synuclein. We propose that these phenotypes represent a simple animal model of LID in C. elegans. Such a model holds the promise of enabling high-throughput screenings for potential therapeutic targets and drug candidates.

Monitoring newly synthesized proteins over the adult life span of Caenorhabditis elegans.

Little is known regarding how the synthesis and degradation of individual proteins change during the life of an organism. Such knowledge is vital to understanding the aging process. To fill this knowledge gap, we monitored newly synthesized proteins on a proteome scale in Caenorhabditis elegans over time during adulthood using a stable-isotope labeling by amino acids in cell culture (SILAC)-based label-chase approach. For most proteins, the rate of appearance of newly synthesized protein was high during the first 5 days of adulthood, slowed down between the fifth and the 11th days, and then increased again after the 11th day. However, the magnitude of appearance rate differed significantly from protein to protein. For example, the appearance of newly synthesized protein was fast for proteins involved in embryonic development, transcription regulation, and lipid binding/transport, with >70% of these proteins newly synthesized by day 5 of adulthood, whereas it was slow for proteins involved in cellular assembly and motility, such as actin and myosin, with <70% of these proteins newly synthesized even on day 16. The late-life increase of newly synthesized protein was especially high for ribosomal proteins and ATP synthases. We also investigated the effect of RNAi-mediated knockdown of the rpl-9 (ribosomal protein), atp-3 (ATP synthase), and ril-1 (RNAi-induced longevity-1) genes and found that inhibiting the expression of atp-3 and ril-1 beginning in late adulthood is still effective to extend the life span of C. elegans.

Inherent instability of the retinitis pigmentosa P23H mutant opsin.

The P23H opsin mutation is the most common cause of autosomal dominant retinitis pigmentosa. Even though the pathobiology of the resulting retinal degeneration has been characterized in several animal models, its complex molecular mechanism is not well understood. Here, we expressed P23H bovine rod opsin in the nervous system of Caenorhabditis elegans. Expression was low due to enhanced protein degradation. The mutant opsin was glycosylated, but the polysaccharide size differed from that of the normal protein. Although P23H opsin aggregated in the nervous system of C. elegans, the pharmacological chaperone 9-cis-retinal stabilized it during biogenesis, producing a variant of rhodopsin called P23H isorhodopsin. In vitro, P23H isorhodopsin folded correctly, formed the appropriate disulfide bond, could be photoactivated but with reduced sensitivity, and underwent Meta II decay at a rate similar to wild type isorhodopsin. In worm neurons, P23H isorhodopsin initiated phototransduction by coupling with the endogenous Gi/o signaling cascade that induced loss of locomotion. Using pharmacological interventions affecting protein synthesis and degradation, we showed that the chromophore could be incorporated either during or after mutant protein translation. However, regeneration of P23H isorhodopsin with chromophore was significantly slower than that of wild type isorhodopsin. This effect, combined with the inherent instability of P23H rhodopsin, could lead to the structural cellular changes and photoreceptor death found in autosomal dominant retinitis pigmentosa. These results also suggest that slow regeneration of P23H rhodopsin could prevent endogenous chromophore-mediated stabilization of rhodopsin in the retina.

Isotopic labeling of mammalian G protein-coupled receptors heterologously expressed in Caenorhabditis elegans.

High-resolution structural determination and dynamic characterization of membrane proteins by nuclear magnetic resonance (NMR) require their isotopic labeling. Although a number of labeled eukaryotic membrane proteins have been successfully expressed in bacteria, they lack post-translational modifications and usually need to be refolded from inclusion bodies. This shortcoming of bacterial expression systems is particularly detrimental for the functional expression of G protein-coupled receptors (GPCRs), the largest family of drug targets, due to their inherent instability. In this work, we show that proteins expressed by a eukaryotic organism can be isotopically labeled and produced with a quality and quantity suitable for NMR characterization. Using our previously described expression system in Caenorhabditis elegans, we showed the feasibility of labeling proteins produced by these worms with (15)N,(13)C by providing them with isotopically labeled bacteria. (2)H labeling also was achieved by growing C. elegans in the presence of 70% heavy water. Bovine rhodopsin, simultaneously expressed in muscular and neuronal worm tissues, was employed as the "test" GPCR to demonstrate the viability of this approach. Although the worms' cell cycle was slightly affected by the presence of heavy isotopes, the final protein yield and quality was appropriate for NMR structural characterization.

Enhanced energy metabolism contributes to the extended life span of calorie-restricted Caenorhabditis elegans.

Caloric restriction (CR) markedly extends life span and improves the health of a broad number of species. Energy metabolism fundamentally contributes to the beneficial effects of CR, but the underlying mechanisms that are responsible for this effect remain enigmatic. A multidisciplinary approach that involves quantitative proteomics, immunochemistry, metabolic quantification, and life span analysis was used to determine how CR, which occurs in the Caenorhabditis elegans eat-2 mutants, modifies energy metabolism of the worm, and whether the observed modifications contribute to the CR-mediated physiological responses. A switch to fatty acid metabolism as an energy source and an enhanced rate of energy metabolism by eat-2 mutant nematodes were detected. Life span analyses validated the important role of these previously unknown alterations of energy metabolism in the CR-mediated longevity of nematodes. As observed in mice, the overexpression of the gene for the nematode analog of the cytosolic form of phosphoenolpyruvate carboxykinase caused a marked extension of the life span in C. elegans, presumably by enhancing energy metabolism via an altered rate of cataplerosis of tricarboxylic acid cycle anions. We conclude that an increase, not a decrease in fuel consumption, via an accelerated oxidation of fuels in the TCA cycle is involved in life span regulation; this mechanism may be conserved across phylogeny.

Probe of the solar magnetic field using the "cosmic-ray shadow" of the sun.

We report on a clear solar-cycle variation of the Sun's shadow in the 10 TeV cosmic-ray flux observed by the Tibet air shower array during a full solar cycle from 1996 to 2009. In order to clarify the physical implications of the observed solar cycle variation, we develop numerical simulations of the Sun's shadow, using the potential field source surface model and the current sheet source surface (CSSS) model for the coronal magnetic field. We find that the intensity deficit in the simulated Sun's shadow is very sensitive to the coronal magnetic field structure, and the observed variation of the Sun's shadow is better reproduced by the CSSS model. This is the first successful attempt to evaluate the coronal magnetic field models by using the Sun's shadow observed in the TeV cosmic-ray flux.

Heterologous expression of functional G-protein-coupled receptors in Caenorhabditis elegans.

New strategies for expression, purification, functional characterization, and structural determination of membrane-spanning G-protein-coupled receptors (GPCRs) are constantly being developed because of their importance to human health. Here, we report a Caenorhabditis elegans heterologous expression system able to produce milligram amounts of functional native and engineered GPCRs. Both bovine opsin [(b)opsin] and human adenosine A(2A) subtype receptor [(h)A(2A)R] expressed in neurons or muscles of C. elegans were localized to cell membranes. Worms expressing these GPCRs manifested changes in motor behavior in response to light and ligands, respectively. With a newly devised protocol, 0.6-1 mg of purified homogenous 9-cis-retinal-bound bovine isorhodopsin [(b)isoRho] and ligand-bound (h)A(2A)R were obtained from C. elegans from one 10-L fermentation at low cost. Purified recombinant (b)isoRho exhibited its signature absorbance spectrum and activated its cognate G-protein transducin in vitro at a rate similar to native rhodopsin (Rho) obtained from bovine retina. Generally high expression levels of 11 native and mutant GPCRs demonstrated the potential of this C. elegans system to produce milligram quantities of high-quality GPCRs and possibly other membrane proteins suitable for detailed characterization.