PBOX-sRNA-seq uncovers novel features of miRNA modification and identifies selected 5'-tRNA fragments bearing 2'-O-modification.

The concomitant cloning of RNA degradation products is a major concern in standard small RNA-sequencing practices. This not only complicates the characterization of bona fide sRNAs but also hampers cross-batch experimental replicability and sometimes even results in library construction failure. Given that all types of plant canonical small RNAs possess the 3' end 2'-O-methylation modification, a new small RNA sequencing (sRNA-seq) method, designated as PBOX-sRNA-seq, has been developed specifically to capture this modification. PBOX-sRNA-seq, as its name implies, relies on the sequential treatment of RNA samples with phenylboronic acid-polyacrylamide gel electrophoresis (PBA-PAGE) and sodium periodate (NaIO4) oxidation, before sRNA library construction and sequencing. PBOX-sRNA-seq outperformed separate treatments (i.e. PBA-PAGE only or NaIO4 only) in terms of the depletion of unmethylated RNA species and capture 2'-O-modified sRNAs with extra-high purity. Using PBOX-sRNA-seq, we discovered that nascent miRNA-5p/-3p duplexes may undergo mono-cytidylation/uridylation before 2'-O-methylation. We also identified two highly conserved types of 5'-tRNA fragments (tRF) bearing HEN1-independent 2'-O modification (mainly the 13-nt tRF-5aAla and the 26-nt tRF-5bGly). We believe that PBOX-sRNA-seq is powerful for both qualitative and quantitative analyses of sRNAs in plants and piRNAs in animals.

Brain-wide neural activity underlying memory-guided movement.

Behavior relies on activity in structured neural circuits that are distributed across the brain, but most experiments probe neurons in a single area at a time. Using multiple Neuropixels probes, we recorded from multi-regional loops connected to the anterior lateral motor cortex (ALM), a circuit node mediating memory-guided directional licking. Neurons encoding sensory stimuli, choices, and actions were distributed across the brain. However, choice coding was concentrated in the ALM and subcortical areas receiving input from the ALM in an ALM-dependent manner. Diverse orofacial movements were encoded in the hindbrain; midbrain; and, to a lesser extent, forebrain. Choice signals were first detected in the ALM and the midbrain, followed by the thalamus and other brain areas. At movement initiation, choice-selective activity collapsed across the brain, followed by new activity patterns driving specific actions. Our experiments provide the foundation for neural circuit models of decision-making and movement initiation.

Ultra-high density electrodes improve detection, yield, and cell type identification in neuronal recordings.

To understand the neural basis of behavior, it is essential to sensitively and accurately measure neural activity at single neuron and single spike resolution. Extracellular electrophysiology delivers this, but it has biases in the neurons it detects and it imperfectly resolves their action potentials. To minimize these limitations, we developed a silicon probe with much smaller and denser recording sites than previous designs, called Neuropixels Ultra (NP Ultra). This device samples neuronal activity at ultra-high spatial density (~10 times higher than previous probes) with low noise levels, while trading off recording span. NP Ultra is effectively an implantable voltage-sensing camera that captures a planar image of a neuron's electrical field. We use a spike sorting algorithm optimized for these probes to demonstrate that the yield of visually-responsive neurons in recordings from mouse visual cortex improves up to ~3-fold. We show that NP Ultra can record from small neuronal structures including axons and dendrites. Recordings across multiple brain regions and four species revealed a subset of extracellular action potentials with unexpectedly small spatial spread and axon-like features. We share a large-scale dataset of these brain-wide recordings in mice as a resource for studies of neuronal biophysics. Finally, using ground-truth identification of three major inhibitory cortical cell types, we found that these cell types were discriminable with approximately 75% success, a significant improvement over lower-resolution recordings. NP Ultra improves spike sorting performance, detection of subcellular compartments, and cell type classification to enable more powerful dissection of neural circuit activity during behavior.

A scalable implementation of the recursive least-squares algorithm for training spiking neural networks.

Training spiking recurrent neural networks on neuronal recordings or behavioral tasks has become a popular way to study computations performed by the nervous system. As the size and complexity of neural recordings increase, there is a need for efficient algorithms that can train models in a short period of time using minimal resources. We present optimized CPU and GPU implementations of the recursive least-squares algorithm in spiking neural networks. The GPU implementation can train networks of one million neurons, with 100 million plastic synapses and a billion static synapses, about 1,000 times faster than an unoptimized reference CPU implementation. We demonstrate the code's utility by training a network, in less than an hour, to reproduce the activity of > 66, 000 recorded neurons of a mouse performing a decision-making task. The fast implementation enables a more interactive in-silico study of the dynamics and connectivity underlying multi-area computations. It also admits the possibility to train models as in-vivo experiments are being conducted, thus closing the loop between modeling and experiments.

Not everything, not everywhere, not all at once: a study of brain-wide encoding of movement.

Activity related to movement is found throughout sensory and motor regions of the brain. However, it remains unclear how movement-related activity is distributed across the brain and whether systematic differences exist between brain areas. Here, we analyzed movement related activity in brain-wide recordings containing more than 50,000 neurons in mice performing a decision-making task. Using multiple techniques, from markers to deep neural networks, we find that movement-related signals were pervasive across the brain, but systematically differed across areas. Movement-related activity was stronger in areas closer to the motor or sensory periphery. Delineating activity in terms of sensory- and motor-related components revealed finer scale structures of their encodings within brain areas. We further identified activity modulation that correlates with decision-making and uninstructed movement. Our work charts out a largescale map of movement encoding and provides a roadmap for dissecting different forms of movement and decision-making related encoding across multi-regional neural circuits.

Cationic Mechanosensitive Channels Mediate Trabecular Meshwork Responses to Cyclic Mechanical Stretch.

The trabecular meshwork (TM) is responsible for intraocular pressure (IOP) homeostasis in the eye. The tissue senses IOP fluctuations and dynamically adapts to the mechanical changes to either increase or decrease aqueous humor outflow. Cationic mechanosensitive channels (CMCs) have been reported to play critical roles in mediating the TM responses to mechanical forces. However, how CMCs influence TM cellular function affect aqueous humor drainage is still elusive. In this study, human TM (HTM) cells were collected from a Chinese donor and subjected to cyclically equiaxial stretching with an amplitude of 20% at 1 Hz GsMTx4, a non-selective inhibitor for CMCs, was added to investigate the proteomic changes induced by CMCs in response to mechanical stretch of HTM. Gene ontology enrichment analysis demonstrated that inhibition of CMCs significantly influenced several biochemical pathways, including store-operated calcium channel activity, microtubule cytoskeleton polarity, toll-like receptor signaling pathway, and neuron cell fate specification. Through heatmap analysis, we grouped 148 differentially expressed proteins (DEPs) into 21 clusters and focused on four specific patterns associated with Ca2+ homeostasis, autophagy, cell cycle, and cell fate. Our results indicated that they might be the critical downstream signals of CMCs adapting to mechanical forces and mediating AH outflow.

Stabilization and mechanism of uranium sequestration by a mixed culture consortia of sulfate-reducing and phosphate-solubilizing bacteria.

In this study, a highly efficient phosphate-solubilizing bacteria (PSB) (Pantoea sp. grinm-12) was screened out from uranium (U) tailings, and the carbon and nitrogen sources of mixed culture with sulfate-reducing bacteria (SRB) were optimized. Results showed that the functional expression of SRB-PSB could be promoted effectively when glucose + sodium lactate was used as carbon source and ammonium nitrate + ammonium sulfate as nitrogen source. The concentration of PO43- in the culture system could reach 107.27 mg·L-1, and the sulfate reduction rate was 81.72%. In the process of biological stabilization of U tailings by mixed SRB-PSB culture system, the chemical form of U in the remediation group was found to transfer to stable state with the extension of remediation time, which revealed the effectiveness of bioremediation on the harmless treatment of U tailings. XRD, FT-IR, SEM-EDS, high-throughput sequencing, and metagenomics were also used to assist in revealing the microstructure and composition changes during the biological stabilization process, and explore the microbial community/functional gene response. Finally, the stabilization mechanism of U was proposed. In conclusion, the stabilization of U in U tailings was realized through the synergistic effect of bio-reduction, bio-precipitation, and bio-adsorption.

A midbrain-thalamus-cortex circuit reorganizes cortical dynamics to initiate movement.

Motor behaviors are often planned long before execution but only released after specific sensory events. Planning and execution are each associated with distinct patterns of motor cortex activity. Key questions are how these dynamic activity patterns are generated and how they relate to behavior. Here, we investigate the multi-regional neural circuits that link an auditory "Go cue" and the transition from planning to execution of directional licking. Ascending glutamatergic neurons in the midbrain reticular and pedunculopontine nuclei show short latency and phasic changes in spike rate that are selective for the Go cue. This signal is transmitted via the thalamus to the motor cortex, where it triggers a rapid reorganization of motor cortex state from planning-related activity to a motor command, which in turn drives appropriate movement. Our studies show how midbrain can control cortical dynamics via the thalamus for rapid and precise motor behavior.

Neural Algorithms and Circuits for Motor Planning.

The brain plans and executes volitional movements. The underlying patterns of neural population activity have been explored in the context of movements of the eyes, limbs, tongue, and head in nonhuman primates and rodents. How do networks of neurons produce the slow neural dynamics that prepare specific movements and the fast dynamics that ultimately initiate these movements? Recent work exploits rapid and calibrated perturbations of neural activity to test specific dynamical systems models that are capable of producing the observed neural activity. These joint experimental and computational studies show that cortical dynamics during motor planning reflect fixed points of neural activity (attractors). Subcortical control signals reshape and move attractors over multiple timescales, causing commitment to specific actions and rapid transitions to movement execution. Experiments in rodents are beginning to reveal how these algorithms are implemented at the level of brain-wide neural circuits.

Optimization of Environmental Conditions for Microbial Stabilization of Uranium Tailings, and the Microbial Community Response.

Uranium pollution in tailings and its decay products is a global environmental problem. It is of great significance to use economical and efficient technologies to remediate uranium-contaminated soil. In this study, the effects of pH, temperature, and inoculation volume on stabilization efficiency and microbial community response of uranium tailings were investigated by a single-factor batch experiment in the remediation process by mixed sulfate-reducing bacteria (SRB) and phosphate-solubilizing bacteria (PSB, Pantoea sp. grinm-12). The results showed that the optimal parameters of microbial stabilization by mixed SRB-PSB were pH of 5.0, temperature of 25°C, and inoculation volume of 10%. Under the optimal conditions, the uranium in uranium tailings presented a tendency to transform from the acid-soluble state to residual state. In addition, the introduction of exogenous SRB-PSB can significantly increase the richness and diversity of endogenous microorganisms, effectively maintain the reductive environment for the microbial stabilization system, and promote the growth of functional microorganisms, such as sulfate-reducing bacteria (Desulfosporosinus and Desulfovibrio) and iron-reducing bacteria (Geobacter and Sedimentibacter). Finally, PCoA and CCA analyses showed that temperature and inoculation volume had significant effects on microbial community structure, and the influence order of the three environmental factors is as follows: inoculation volume > temperature > pH. The outcomes of this study provide theoretical support for the control of uranium in uranium-contaminated sites.

Accurate Localization of Linear Probe Electrode Arrays across Multiple Brains.

Recently developed probes for extracellular electrophysiological recordings have large numbers of electrodes on long linear shanks. Linear electrode arrays, such as Neuropixels probes, have hundreds of recording electrodes distributed over linear shanks that span several millimeters. Because of the length of the probes, linear probe recordings in rodents usually cover multiple brain areas. Typical studies collate recordings across several recording sessions and animals. Neurons recorded in different sessions and animals thus have to be aligned to each other and to a standardized brain coordinate system. Here, we evaluate two typical workflows for localization of individual electrodes in standardized coordinates. These workflows rely on imaging brains with fluorescent probe tracks and warping 3D image stacks to standardized brain atlases. One workflow is based on tissue clearing and selective plane illumination microscopy (SPIM), whereas the other workflow is based on serial block-face two-photon (SBF2P) microscopy. In both cases electrophysiological features are then used to anchor particular electrodes along the reconstructed tracks to specific locations in the brain atlas and therefore to specific brain structures. We performed groundtruth experiments, in which motor cortex outputs are labeled with ChR2 and a fluorescence protein. Light-evoked electrical activity and fluorescence can be independently localized. Recordings from brain regions targeted by the motor cortex reveal better than 0.1-mm accuracy for electrode localization, independent of workflow used.

Hunger or thirst state uncertainty is resolved by outcome evaluation in medial prefrontal cortex to guide decision-making.

Physiological need states direct decision-making toward re-establishing homeostasis. Using a two-alternative forced choice task for mice that models elements of human decisions, we found that varying hunger and thirst states caused need-inappropriate choices, such as food seeking when thirsty. These results show limits on interoceptive knowledge of hunger and thirst states to guide decision-making. Instead, need states were identified after food and water consumption by outcome evaluation, which depended on the medial prefrontal cortex.

Neuropixels 2.0: A miniaturized high-density probe for stable, long-term brain recordings.

Measuring the dynamics of neural processing across time scales requires following the spiking of thousands of individual neurons over milliseconds and months. To address this need, we introduce the Neuropixels 2.0 probe together with newly designed analysis algorithms. The probe has more than 5000 sites and is miniaturized to facilitate chronic implants in small mammals and recording during unrestrained behavior. High-quality recordings over long time scales were reliably obtained in mice and rats in six laboratories. Improved site density and arrangement combined with newly created data processing methods enable automatic post hoc correction for brain movements, allowing recording from the same neurons for more than 2 months. These probes and algorithms enable stable recordings from thousands of sites during free behavior, even in small animals such as mice.

Oxygen-Deficient Stannic Oxide/Graphene for Ultrahigh-Performance Supercapacitors and Gas Sensors.

The metal oxides/graphene nanocomposites have great application prospects in the fields of electrochemical energy storage and gas sensing detection. However, rational synthesis of such materials with good conductivity and electrochemical activity is the topical challenge for high-performance devices. Here, SnO2/graphene nanocomposite is taken as a typical example and develops a universal synthesis method that overcome these challenges and prepares the oxygen-deficient SnO2 hollow nanospheres/graphene (r-SnO2/GN) nanocomposite with excellent performance for supercapacitors and gas sensors. The electrode r-SnO2/GN exhibits specific capacitance of 947.4 F g-1 at a current density of 2 mA cm-2 and of 640.0 F g-1 even at 20 mA cm-2, showing remarkable rate capability. For gas-sensing application, the sensor r-SnO2/GN showed good sensitivity (~13.8 under 500 ppm) and short response/recovering time toward methane gas. These performance features make r-SnO2/GN nanocomposite a promising candidate for high-performance energy storage devices and gas sensors.

Hyponastic Leaves 1 protects pri-miRNAs from nuclear exosome attack.

Biogenesis of plant microRNAs (miRNAs) takes place in nuclear dicing bodies (D-bodies), where the ribonulease III-type enzyme Dicer-like 1 (DCL1) processes primary transcripts of miRNAs (pri-miRNAs) into miRNA/miRNA* (*, passenger strand) duplexes from either base-to-loop or loop-to-base directions. Hyponastic Leaves 1 (HYL1), a double-stranded RNA-binding protein, is crucial for efficient and accurate processing. However, whether HYL1 has additional function remains unknown. Here, we report that HYL1 plays a noncanonical role in protecting pri-miRNAs from nuclear exosome attack in addition to ensuring processing. Loss of functions in SOP1 or HEN2, two cofactors of the nucleoplasmic exosome, significantly suppressed the morphological phenotypes of hyl1-2 Remarkably, mature miRNAs generated from loop-to-base processing were partially but preferentially restored in the hyl1 sop1 and hyl1 hen2 double mutants. Accordingly, loop-to-base-processed pri-miRNAs accumulated to higher levels in double mutants. In addition, dysfunction of HEN2, but not of SOP1, in hyl1-2 resulted in overaccumulation of many base-to-loop-processed pri-miRNAs, with most of their respective miRNAs unaffected. In summary, our findings reveal an antagonistic action of exosome in pri-miRNA biogenesis and uncover dual roles of HYL1 in stabilizing and processing of pri-miRNAs.

Spatiotemporal constraints on optogenetic inactivation in cortical circuits.

Optogenetics allows manipulations of genetically and spatially defined neuronal populations with excellent temporal control. However, neurons are coupled with other neurons over multiple length scales, and the effects of localized manipulations thus spread beyond the targeted neurons. We benchmarked several optogenetic methods to inactivate small regions of neocortex. Optogenetic excitation of GABAergic neurons produced more effective inactivation than light-gated ion pumps. Transgenic mice expressing the light-dependent chloride channel GtACR1 produced the most potent inactivation. Generally, inactivation spread substantially beyond the photostimulation light, caused by strong coupling between cortical neurons. Over some range of light intensity, optogenetic excitation of inhibitory neurons reduced activity in these neurons, together with pyramidal neurons, a signature of inhibition-stabilized neural networks ('paradoxical effect'). The offset of optogenetic inactivation was followed by rebound excitation in a light dose-dependent manner, limiting temporal resolution. Our data offer guidance for the design of in vivo optogenetics experiments.

Gramian solutions and soliton interactions for a generalized (3 + 1)-dimensional variable-coefficient Kadomtsev-Petviashvili equation in a plasma or fluid.

Plasmas and fluids are of current interest, supporting a variety of wave phenomena. Plasmas are believed to be possibly the most abundant form of visible matter in the Universe. Investigation in this paper is given to a generalized (3 + 1)-dimensional variable-coefficient Kadomtsev-Petviashvili equation for the nonlinear phenomena in a plasma or fluid. Based on the existing bilinear form, N-soliton solutions in the Gramian are derived, where N = 1, 2, 3…. With N = 3, three-soliton solutions are constructed. Fission and fusion for the three solitons are presented. Effects of the variable coefficients, i.e. h(t), l(t), q(t), n(t) and m(t), on the soliton fission and fusion are revealed: soliton velocity is related to h(t), l(t), q(t), n(t) and m(t), while the soliton amplitude cannot be affected by them, where t is the scaled temporal coordinate, h(t), l(t) and q(t) give the perturbed effects, and m(t) and n(t), respectively, stand for the disturbed wave velocities along two transverse spatial coordinates. We show the three parallel solitons with the same direction.

Insights into redox mediator supplementation on enhanced volatile fatty acids production from waste activated sludge.

Anaerobic fermentation of waste activated sludge (WAS) for recycling valuable volatile fatty acids (VFAs) is economically valuable. However, the fermentation of protein is the rate-limiting step of VFA production with WAS as a substrate. In this study, the effect of redox mediators (RMs, i.e., riboflavin and lawsone) on the enhanced production of VFAs from WAS was investigated. The results indicate that both RMs can promote protein-dependent fermentation, increasing maximum VFA accumulation by 43.9% and 42.5% respectively. In cultures supplemented with riboflavin and lawsone, VFA production was highly correlated with protease activities, but not with α-glucosidase activities. This implies that RMs affected the redox reaction of amino acids degradation, resulting in an increased release of ammonia. Sequencing results showed that RMs significantly increased the abundance of bacteria related to VFA fermentation and protein/amino acid degradation at the levels of phylum, class, order, family, and even genus.

Spliceosome disassembly factors ILP1 and NTR1 promote miRNA biogenesis in Arabidopsis thaliana.

The intron-lariat spliceosome (ILS) complex is highly conserved among eukaryotes, and its disassembly marks the end of a canonical splicing cycle. In this study, we show that two conserved disassembly factors of the ILS complex, Increased Level of Polyploidy1-1D (ILP1) and NTC-Related protein 1 (NTR1), positively regulate microRNA (miRNA) biogenesis by facilitating transcriptional elongation of MIRNA (MIR) genes in Arabidopsis thaliana. ILP1 and NTR1 formed a stable complex and co-regulated alternative splicing of more than a hundred genes across the Arabidopsis genome, including some primary transcripts of miRNAs (pri-miRNAs). Intriguingly, pri-miRNAs, regardless of having introns or not, were globally down-regulated when the ILP1 or NTR1 function was compromised. ILP1 and NTR1 interacted with core miRNA processing proteins Dicer-like 1 and Serrate, and were required for proper RNA polymerase II occupancy at elongated regions of MIR chromatin, without affecting either MIR promoter activity or pri-miRNA decay. Our results provide further insights into the regulatory role of spliceosomal machineries in the biogenesis of miRNAs.

Analysis of Methylation Status of Plant MicroRNAs.

Small noncoding RNAs of 20-30 nucleotides in length are key mediators of gene silencing. 2'-O-Methylation on the 3' terminal nucleotide of several types of small RNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs) in plants, PIWI-interacting RNAs (piRNAs) in animals, and some siRNAs in Drosophila and Caenorhabditis elegans, provides a key protective mechanism against 3' tailing- and trimming-mediated destabilization. The methylation reaction is catalyzed by the small RNA methyltransferase HUA ENHANCER 1 (HEN1). In this chapter, we describe a detailed protocol for analyzing 3' end methylation status of plant miRNAs, which can be applicable to other types of small RNAs as well.