Ultrafast Two-Photon Imaging of a High-Gain Voltage Indicator in Awake Behaving Mice.

Optical interrogation of voltage in deep brain locations with cellular resolution would be immensely useful for understanding how neuronal circuits process information. Here, we report ASAP3, a genetically encoded voltage indicator with 51% fluorescence modulation by physiological voltages, submillisecond activation kinetics, and full responsivity under two-photon excitation. We also introduce an ultrafast local volume excitation (ULoVE) method for kilohertz-rate two-photon sampling in vivo with increased stability and sensitivity. Combining a soma-targeted ASAP3 variant and ULoVE, we show single-trial tracking of spikes and subthreshold events for minutes in deep locations, with subcellular resolution and with repeated sampling over days. In the visual cortex, we use soma-targeted ASAP3 to illustrate cell-type-dependent subthreshold modulation by locomotion. Thus, ASAP3 and ULoVE enable high-speed optical recording of electrical activity in genetically defined neurons at deep locations during awake behavior.

A positively tuned voltage indicator for extended electrical recordings in the brain.

Genetically encoded voltage indicators (GEVIs) enable optical recording of electrical signals in the brain, providing subthreshold sensitivity and temporal resolution not possible with calcium indicators. However, one- and two-photon voltage imaging over prolonged periods with the same GEVI has not yet been demonstrated. Here, we report engineering of ASAP family GEVIs to enhance photostability by inversion of the fluorescence-voltage relationship. Two of the resulting GEVIs, ASAP4b and ASAP4e, respond to 100-mV depolarizations with ≥180% fluorescence increases, compared with the 50% fluorescence decrease of the parental ASAP3. With standard microscopy equipment, ASAP4e enables single-trial detection of spikes in mice over the course of minutes. Unlike GEVIs previously used for one-photon voltage recordings, ASAP4b and ASAP4e also perform well under two-photon illumination. By imaging voltage and calcium simultaneously, we show that ASAP4b and ASAP4e can identify place cells and detect voltage spikes with better temporal resolution than commonly used calcium indicators. Thus, ASAP4b and ASAP4e extend the capabilities of voltage imaging to standard one- and two-photon microscopes while improving the duration of voltage recordings.

Kilohertz two-photon fluorescence microscopy imaging of neural activity in vivo.

Understanding information processing in the brain requires monitoring neuronal activity at high spatiotemporal resolution. Using an ultrafast two-photon fluorescence microscope empowered by all-optical laser scanning, we imaged neuronal activity in vivo at up to 3,000 frames per second and submicrometer spatial resolution. This imaging method enabled monitoring of both supra- and subthreshold electrical activity down to 345 µm below the brain surface in head-fixed awake mice.

Single-pollen-cell sequencing for gamete-based phased diploid genome assembly in plants.

Genome assemblies from diploid organisms create mosaic sequences alternating between parental alleles, which can create erroneous gene models and other problems. In animals, a popular strategy to generate haploid genome-resolved assemblies has been the sampling of (haploid) gametes, and the advent of single-cell sequencing has further advanced such methods. However, several challenges for the isolation and amplification of DNA from plant gametes have limited such approaches in plants. Here, we combined a new approach for pollen protoplast isolation with a single-cell DNA amplification technique and then used a "barcoding" bioinformatics strategy to incorporate haploid-specific sequence data from 12 pollen cells, ultimately enabling the efficient and accurate phasing of the pear genome into its A and B haploid genomes. Beyond revealing that 8.12% of the genes in the pear reference genome feature mosaic assemblies and enabling a previously impossible analysis of allelic affects in pear gene expression, our new haploid genome assemblies provide high-resolution information about recombination during meiosis in pollen. Considering that outcrossing pear is an angiosperm species featuring very high heterozygosity, our method for rapidly phasing genome assemblies is potentially applicable to several yet-unsequenced outcrossing angiosperm species in nature.

Transcriptome and phytohormone analysis reveals a comprehensive phytohormone and pathogen defence response in pear self-/cross-pollination.

KEY MESSAGE: Candidate genes were identified and the role of phytohormones such as JA-Me and ABA in the synthesis of S-RNase was emphasized in pear self-incompatibility. Self-incompatibility (SI) occurs widely in flowering plants as an intraspecific reproductive barrier. This phenomenon promotes variation within species, but for some species such as Pyrus, SI is a nuisance rather than a benefit in agricultural production. Although many studies have been conducted on SI in pears, its mechanism remains unclear. In this study, high-throughput Illumina RNA sequencing (RNA-seq) was used to identify SI-related genes in pear styles. Using transcriptome comparisons, differentially expressed genes of unpollinated (UP), cross-pollinated (CP), and self-pollinated (SP) styles were identified after 48 h. A total of 1796 and 1890 genes were identified in DSC (UP vs. CP) and DSI (UP vs. SP), respectively. KEGG analysis revealed that genes involved in the "plant hormone signal transduction pathway" and "plant-pathogen interaction pathway" were significantly enriched in DSI (UP vs. SP) compared to those in DSC (UP vs. CP). The expression level of S-glycoprotein ribonuclease (S-RNase) was dramatically reduced in cross-pollinated (CP) styles. To better understand the relationship between the expression patterns of S-RNase and two major KEGG pathways, the concentrations of phytohormones were measured, and the expression pattern of S-RNase was analysed using qRT-PCR. Our results demonstrate that methyl jasmonate and abscisic acid may enhance the expression level of S-RNase, and pollination can affect the synthesis of methyl jasmonate and abscisic acid in pear styles. Overall, this study is a global transcriptome analysis of SI in pear. A relationship between self-rejection, plant hormones, and pathogen defence was shown in pear.

Spatial-temporal analysis of zinc homeostasis reveals the response mechanisms to acute zinc deficiency in Sorghum bicolor.

Zinc (Zn) is an essential micronutrient in plants. The activity of copper/zinc superoxide dismutase (CSD) and carbonic anhydrase (CA) correlate with differences in Zn efficiency in plants; therefore, it is reasonable to hypothesize the existence of a Zn economy model that saves Zn for these essential Zn proteins during Zn deficiency. However, up to this point, direct evidence for the idea that CSD and/or CA might be priorities for Zn delivery has been lacking. Here, we investigated the spatial-temporal effects of acute Zn depletion and resupply by integrating physiological studies and molecular analyses using hydroponically grown Sorghum. The elevated expression of miR398 repressed CSD expression in roots, whereas the reduced expression of miR528 resulted in a relatively stable level of CSD expression in Sorghum leaves under Zn depletion. Spatial-temporal analysis after Zn resupply to previously depleted plants revealed that the expression and activity of CA were the first to recover after Zn addition, whereas the recovery of the activities of CSD and alcohol dehydrogenase (ADH) was delayed, suggesting that CA receives priority in Zn delivery over CSD and ADH. Our results also indicate that microRNAs (miRNAs) are important regulators of the response of Zn deficiency in plants.

Biphasic Cholinergic Modulation of Reverberatory Activity in Neuronal Networks.

Acetylcholine (ACh) is an important neuromodulator in various cognitive functions. However, it is unclear how ACh influences neural circuit dynamics by altering cellular properties. Here, we investigated how ACh influences reverberatory activity in cultured neuronal networks. We found that ACh suppressed the occurrence of evoked reverberation at low to moderate doses, but to a much lesser extent at high doses. Moreover, high doses of ACh caused a longer duration of evoked reverberation, and a higher occurrence of spontaneous activity. With whole-cell recording from single neurons, we found that ACh inhibited excitatory postsynaptic currents (EPSCs) while elevating neuronal firing in a dose-dependent manner. Furthermore, all ACh-induced cellular and network changes were blocked by muscarinic, but not nicotinic receptor antagonists. With computational modeling, we found that simulated changes in EPSCs and the excitability of single cells mimicking the effects of ACh indeed modulated the evoked network reverberation similar to experimental observations. Thus, ACh modulates network dynamics in a biphasic fashion, probably by inhibiting excitatory synaptic transmission and facilitating neuronal excitability through muscarinic signaling pathways.

Mesophasic organization of GABAA receptors in hippocampal inhibitory synapses.

Information processing in the brain depends on specialized organization of neurotransmitter receptors and scaffolding proteins within the postsynaptic density. However, how these molecules are organized in situ remains largely unknown. In this study, template-free classification of oversampled sub-tomograms was used to analyze cryo-electron tomograms of hippocampal synapses. We identified type-A GABA receptors (GABAARs) in inhibitory synapses and determined their in situ structure at 19-Å resolution. These receptors are organized hierarchically: from GABAAR super-complexes with a preferred inter-receptor distance of 11 nm but variable relative angles, through semi-ordered, two-dimensional receptor networks with reduced Voronoi entropy, to mesophasic assembly with a sharp phase boundary. These assemblies likely form via interactions among postsynaptic scaffolding proteins and receptors and align with putative presynaptic vesicle release sites. Such mesophasic self-organization might allow synapses to achieve a 'Goldilocks' state, striking a balance between stability and flexibility and enabling plasticity in information processing.

Excitation wavelength optimization improves photostability of ASAP-family GEVIs.

Recent interest in high-throughput recording of neuronal activity has motivated rapid improvements in genetically encoded calcium or voltage indicators (GECIs or GEVIs) for all-optical electrophysiology. Among these probes, the ASAPs, a series of voltage indicators based on a variant of circularly permuted green fluorescent protein (cpGFP) and a conjugated voltage sensitive domain (VSD), are capable of detecting both action potentials and subthreshold neuronal activities. Here we show that the ASAPs, when excited by blue light, undergo reversible photobleaching. We find that this fluorescence loss induced by excitation with 470-nm light can be substantially reversed by low-intensity 405-nm light. We demonstrate that 405-nm and 470-nm co-illumination significantly improved brightness and thereby signal-to-noise ratios during voltage imaging compared to 470-nm illumination alone. Illumination with a single wavelength of 440-nm light also produced similar improvements. We hypothesize that reversible photobleaching is related to cis-trans isomerization and protonation of the GFP chromophore of ASAP proteins. Amino acids that influence chromophore isomerization are potential targets of point mutations for future improvements.

Robot navigation in cluttered 3-D environments using preference-based fuzzy behaviors.

Autonomous navigation systems for mobile robots have been successfully deployed for a wide range of planar ground-based tasks. However, very few counterparts of previous planar navigation systems were developed for 3-D motion, which is needed for both unmanned aerial and underwater vehicles. A novel fuzzy behavioral scheme for navigating an unmanned helicopter in cluttered 3-D spaces is developed. The 3-D navigation problem is decomposed into several identical 2-D navigation subproblems, each of which is solved by using preference-based fuzzy behaviors. Due to the shortcomings of vector summation during the fusion of the 2-D subproblems, instead of directly outputting steering subdirections by their own defuzzification processes, the intermediate preferences of the subproblems are fused to create a 3-D solution region, representing degrees of preference for the robot movement. A new defuzzification algorithm that steers the robot by finding the centroid of a 3-D convex region of maximum volume in the 3-D solution region is developed. A fuzzy speed-control system is also developed to ensure efficient and safe navigation. Substantial simulations have been carried out to demonstrate that the proposed algorithm can smoothly and effectively guide an unmanned helicopter through unknown and cluttered urban and forest environments.

Genome-wide identification of the MADS-box transcription factor family in pear (Pyrus bretschneideri) reveals evolution and functional divergence.

MADS-box transcription factors play significant roles in plant developmental processes such as floral organ conformation, flowering time, and fruit development. Pear (Pyrus), as the third-most crucial temperate fruit crop, has been fully sequenced. However, there is limited information about the MADS family and its functional divergence in pear. In this study, a total of 95 MADS-box genes were identified in the pear genome, and classified into two types by phylogenetic analysis. Type I MADS-box genes were divided into three subfamilies and type II genes into 14 subfamilies. Synteny analysis suggested that whole-genome duplications have played key roles in the expansion of the MADS family, followed by rearrangement events. Purifying selection was the primary force driving MADS-box gene evolution in pear, and one gene pairs presented three codon sites under positive selection. Full-scale expression information for PbrMADS genes in vegetative and reproductive organs was provided and proved by transcriptional and reverse transcription PCR analysis. Furthermore, the PbrMADS11(12) gene, together with partners PbMYB10 and PbbHLH3 was confirmed to activate the promoters of the structural genes in anthocyanin pathway of red pear through dual luciferase assay. In addition, the PbrMADS11 and PbrMADS12 were deduced involving in the regulation of anthocyanin synthesis response to light and temperature changes. These results provide a solid foundation for future functional analysis of PbrMADS genes in different biological processes, especially of pigmentation in pear.

TGTT and AACA: two transcriptionally active LTR retrotransposon subfamilies with a specific LTR structure and horizontal transfer in four Rosaceae species.

BACKGROUND: Long terminal repeat retrotransposons (LTR-RTs) are major components of plant genomes. Common LTR-RTs contain the palindromic dinucleotide 5'-'TG'-'CA'-3' motif at the ends. Thus, further analyses of non-canonical LTR-RTs with non-palindromic motifs will enhance our understanding of their structures and evolutionary history. RESULTS: Here, we report two new LTR-RT subfamilies (TGTT and AACA) with atypical dinucleotide ends of 5'-'TG'-'TT'-3', and 5'-'AA'-'CA'-3' in pear, apple, peach and mei. In total, 91 intact LTR-RTs were identified and classified into four TGTT and four AACA families. A structural annotation analysis showed that the four TGTT families, together with AACA1 and AACA2, belong to the Copia-like superfamily, whereas AACA3 and AACA4 appeared to be TRIM elements. The average amplification time frames for the eight families ranged from 0.05 to 2.32 million years. Phylogenetics coupled with sequence analyses revealed that the TGTT1 elements of peach were horizontally transferred from apple. In addition, 32 elements from two TGTT and three AACA families had detectable transcriptional activation, and a qRT-PCR analysis indicated that their expression levels varied dramatically in different species, organs and stress treatments. CONCLUSIONS: Two novel LTR-RT subfamilies that terminated with non-palindromic dinucleotides at the ends of their LTRs were identified in four Rosaceae species, and a deep analysis showed their recent activity, horizontal transfer and varied transcriptional levels in different species, organs and stress treatments. This work enhances our understanding of the structural variation and evolutionary history of LTR-RTs in plants and also provides a valuable resource for future investigations of LTR-RTs having specific structures in other species.

[Study on simulation of clarification for Vegetative Filter Strips by VFSMOD Model].

The functions and mechanism of Vegetative Filter Strips Model (VFSMOD) had been introduced in this paper, and the applicability and performance ability of this model have been tested by plot experiment data. The results show that the relative deviations between simulated values and measured values of outflow quantity are within +/- 15%, while with the concentration of SS, the relative deviations are within +/- 20%, and the determination coefficients between simulated values and measured values for outflow and SS are 0.995 and 0.889 respectively. Therefore, the simulation precision of this model is satisfactory, the model can be used as a tool for the design of VFS.