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To study the change and main control factors of the high-rank coal reservoir permeability in deep coal seams, permeability tests under different stresses and gas pressures were carried out in the laboratory. The development and distribution of nano-micro pores and fractures in the coal matrix were analyzed and observed by mercury intrusion porosimetry, gas adsorption, scanning electron microscope and computed tomography to reveal the permeability variation mechanism. The results showed that the initial permeability of the coal samples ranged from 0.0114 mD to 0.2349 mD when the effective stress was 0 MPa, and it clearly varied among different samples. The permeability of all the coal samples was very sensitive to the effective stress and decreased exponentially with the increase of the effective stress. The increase of the pore pressure also led to a decrease of the permeability, whereas the impact of the pore pressure on permeability was less obvious compared with the effective stress. Sub-nanopores, nanopores, micro-fractures and larger fractures are all developed in the coal samples. Connected larger fractures were the main gas migration channels in permeability determination, and the narrowing, disconnection, and closure of the fractures caused by the increase of the effective stress were the most important reasons for significant reduction of permeability.

Dissolved organic matter (DOM) is widely used in aquatic systems to control the environmental fate of As. However, similar to the behavior of As, Sb mobilization driven by DOM is poorly understood. A total of 25 samples were collected from shallow groundwater in the Xikuangshan mine to compare the spectroscopic characteristics and chemical properties of DOM between high- and low-Sb groundwater and to determine the roles of DOM in Sb mobility. The concentrations of Sb and DOM varied from 0.003 to 18.402 mg/L (mean: 3.407 mg/L) and 0.38 to 9.90 mg/L (mean: 2.49 mg/L), respectively. The DOM of the D

2-arachidonoyl-glycerol (2-AG), the most abundant endocannabinoid (eCB) in the brain, regulates diverse neural functions. However, whether 2-AG deficiency contributes to Parkinson’s disease (PD) and nigral dopaminergic neurons (DANs) dysfunction is unclear. Diacylglycerol lipase α and β (DAGLA and DAGLB) mediate the biosynthesis of 2-AG. Using homozygosity mapping and whole-exome sequencing, we linked multiple homozygous loss-of-function mutations in DAGLB to a form of early-onset autosomal recessive PD. We then used RNA sequencing and fiber photometry with genetically encoded eCB sensors to demonstrate that DAGLB is the main 2-AG synthase in nigral DANs. Genetic knockdown of Daglb by CRISPR/Cas9 in mouse nigral DANs substantially reduces 2-AG levels in the substantia nigra (SN). The SN 2-AG levels are markedly correlated with the vigor of movement during the acquisition of motor skills, while Daglb-deficiency impairs motor learning. Conversely, pharmacological enhancement of 2-AG levels increases nigral DAN activity and dopamine release and improves motor learning. Together, we demonstrate that DAGLB-deficiency contributes to the etiopathogenesis of PD, reveal the importance of DAGLB-mediated 2-AG biosynthesis in nigral DANs in regulating neural activity and dopamine release, and provide preclinical evidence for the beneficial effects of 2-AG augmentation in PD treatment.

Several forms of endocannabinoid (eCB) signaling have been described in the dorsal lateral striatum (DLS), however most experimental protocols used to generate eCBs do not recapitulate the firing patterns of striatal-projecting pyramidal neurons in the cortex or firing patterns of striatal medium spiny neurons. Therefore, it is unclear if current models of eCB signaling in the DLS provide a reliable description of mechanisms engaged under physiological conditions. To address this uncertainty, we investigated mechanisms of eCB mobilization following brief synaptic stimulation that mimics in vivo patterns of neural activity in the DLS. To monitor eCB mobilization, the novel genetically encoded fluorescent eCB biosensor, GRAB(eCB2.0), was expressed presynaptically in corticostriatal afferents of C57BL6J mice and evoked eCB transients were measured in the DLS using a brain slice photometry technique. We found that brief bouts of synaptic stimulation induce long lasting eCB transients that were generated predominantly by 2-arachidonoylglycerol (2-AG) mobilization. Efficient 2-AG mobilization required coactivation of AMPA and NMDA ionotropic glutamate receptors and muscarinic M1 receptors. Dopamine D2 receptors expressed on cholinergic interneurons inhibited 2-AG mobilization by inhibiting acetylcholine release. Collectively, these data uncover unrecognized mechanisms underlying 2-AG mobilization in the DLS.

Endocannabinoid (eCB), 2-arachidonoyl-glycerol (2-AG), the most abundant eCB in the brain, regulates diverse neural functions. Here we linked multiple homozygous loss-of-function mutations in 2-AG synthase diacylglycerol lipase β (DAGLB) to an early onset autosomal recessive Parkinsonism. DAGLB is the main 2-AG synthase in human and mouse substantia nigra (SN) dopaminergic neurons (DANs). In mice, the SN 2-AG levels were markedly correlated with motor performance during locomotor skill acquisition. Genetic knockdown of Daglb in nigral DANs substantially reduced SN 2-AG levels and impaired locomotor skill learning, particularly the across-session learning. Conversely, pharmacological inhibition of 2-AG degradation increased nigral 2-AG levels, DAN activity and dopamine release and rescued the locomotor skill learning deficits. Together, we demonstrate that DAGLB-deficiency contributes to the pathogenesis of Parkinsonism, reveal the importance of DAGLB-mediated 2-AG biosynthesis in nigral DANs in regulating neuronal activity and dopamine release, and suggest potential benefits of 2-AG augmentation in alleviating Parkinsonism.

The purinergic transmitter ATP (adenosine 5'-triphosphate) plays an essential role in both the central and peripheral nervous systems, and the ability to directly measure extracellular ATP in real time will increase our understanding of its physiological functions. Here, we developed a sensitive GPCR activation-based ATP sensor called GRAB

SUMMARYThe purinergic transmitter ATP (adenosine 5’-triphosphate) plays an essential role in both the central and peripheral nervous systems, and the ability to directly measure extracellular ATP in real time will increase our understanding of its physiological functions. We developed an ultrasensitive GPCRActivation‒Based ATP sensor called GRABATP1.0, with a robust fluorescence response to extracellular ATP when expressed in several cell types. This sensor has sub-second kinetics, ATP affinity in the range of tens of nanomolar, and can be used to localize ATP release with subcellular resolution. Using this sensor, we monitored ATP release under a variety of in vitro and in vivo conditions, including primary hippocampal neurons, a zebrafish model of injury-induced ATP release, and LPS-induced ATP-release events in individual astrocytes in the mouse cortex measured using in vivo two-photon imaging. Thus, the GRABATP1.0 sensor is a sensitive, versatile tool for monitoring ATP release and dynamics under both physiological and pathophysiological conditions.

Several forms of endocannabinoid (eCB) signaling have been described in the dorsal lateral striatum (DLS), however most experimental protocols used to induce plasticity do not recapitulate the firing patterns of striatal-projecting pyramidal neurons in the cortex or firing patterns of striatal medium spiny neurons. Therefore, it is unclear if current models of eCB signaling in the DLS provide a reliable description of mechanisms engaged under physiological conditions. To address this uncertainty, we investigated mechanisms of eCB mobilization following brief synaptic stimulation that mimicsin vivopatterns of neural activity in the DLS. To monitor eCB mobilization, the novel genetically encoded fluorescent eCB biosensor, GRABeCB2.0, was expressed in corticostriatal afferents of C57BL6J mice and evoked eCB transients were measured in the DLS using a brain slice photometry technique. We found that brief bouts of synaptic stimulation induce long lasting eCB transients. Inhibition of monoacylglycerol lipase, prolonged the duration of the eCB transient, while inhibition of diacylglycerol lipase inhibited the peak amplitude, suggesting that 2-AG is the predominate eCB generated following brief synaptic stimulation. 2-AG transients were robustly inhibited by AMPA and NMDA receptor antagonists, DNQX and DL-AP5 respectively. Additionally, the 2-AG transient was inhibited by the muscarinic M1 receptor (M1R) antagonist, VU 0255035, and augmented by the M1R positive allosteric modulator, VU 0486846, indicating that acetylcholine (ACh) release is required for efficient 2-AG production. The dopamine D2 receptor (D2R) agonist, quinpirole, inhibited the 2-AG transient. However, in slices from mice lacking D2Rs on cholinergic interneurons (CINs), quinpirole did not inhibit the 2-AG transient, demonstrating that D2Rs on CINs can modulate 2-AG production. The AMPA receptor or NMDA receptor antagonists, DNQX or DL-AP5 respectively, occluded 2-AG augmentation by VU 0486846 suggesting that converging glutamatergic and cholinergic signals are required for efficient 2-AG production following brief synaptic stimulation. Collectively, these data uncover unrecognized mechanisms underlying 2-AG mobilization in the DLS.

Endocannabinoids (eCBs) are retrograde neuromodulators with important functions in a wide range of physiological processes, but their in vivo dynamics remain largely uncharacterized. Here, we developed a genetically encoded eCB sensor called GRAB(eCB2.0). GRAB(eCB2.0) consists of a circular-permutated EGFP and the human CB1 cannabinoid receptor, providing cell membrane trafficking, second-resolution kinetics, high specificity for eCBs, and shows a robust fluorescence response at physiological eCB concentrations. Using GRAB(eCB2.0), we monitored evoked and spontaneous changes in eCB dynamics in cultured neurons and acute brain slices. We observed spontaneous compartmentalized eCB transients in cultured neurons, and eCB transients from single axonal boutons in acute brain slices, suggesting constrained, localized eCB signaling. Expressing GRAB(eCB2.0) in the mouse brain, we observed foot shock-elicited and running-triggered eCB signaling in the basolateral amygdala and hippocampus, respectively. In a mouse model of epilepsy, we observed a spreading wave of eCB release that followed a Ca(2+) wave through the hippocampus. GRAB(eCB2.0) is a robust probe for eCB release in vivo.

Endocannabinoids (eCBs) are retrograde neuromodulators that play an important role in a wide range of physiological processes; however, the release and in vivo dynamics of eCBs remain largely unknown, due in part to a lack of suitable probes capable of detecting eCBs with sufficient spatiotemporal resolution. Here, we developed a new eCB sensor called GRABeCB2.0. This genetically encoded sensor consists of the human CB1 cannabinoid receptor fused to circular-permutated EGFP, providing cell membrane trafficking, second-resolution kinetics, high specificity for eCBs, and a robust fluorescence response at physiological eCB concentrations. Using the GRABeCB2.0 sensor, we monitored evoked changes in eCB dynamics in both cultured neurons and acute brain slices. Interestingly, in cultured neurons we also observed spontaneous compartmental eCB transients that spanned a distance of approximately 11 μm, suggesting constrained, localized eCB signaling. Moreover, by expressing GRABeCB2.0 in the mouse brain, we readily observed foot shock-elicited and running-triggered eCB transients in the basolateral amygdala and hippocampus, respectively. Lastly, we used GRABeCB2.0 in a mouse seizure model and observed a spreading wave of eCB release that followed a Ca2+ wave through the hippocampus. Thus, GRABeCB2.0 is a robust new probe for measuring the dynamics of eCB release under both physiological and pathological conditions.

Regulation of stomatal movement is critical for plant adaptation to environmental stresses. The microtubule cytoskeleton undergoes disassembly, which is critical for stomatal closure in response to abscisic acid (ABA). However, the mechanism underlying this regulation largely remains unclear. Here we show that a ubiquitin-26S proteasome (UPS)-dependent pathway mediates microtubule disassembly and is required for ABA-induced stomatal closure. Moreover, we identify and characterize the ubiquitin E3 ligase MREL57 (MICROTUBULE-RELATED E3 LIGASE57) and the microtubule-stabilizing protein WDL7 (WAVE-DAMPENED2-LIKE7) in Arabidopsis and show that the MREL57-WDL7 module regulates microtubule disassembly to mediate stomatal closure in response to drought stress and ABA treatment. MREL57 interacts with, ubiquitinates and degrades WDL7, and this effect is clearly enhanced by ABA. ABA-induced stomatal closure and microtubule disassembly are significantly suppressed in mrel57 mutants, and these phenotypes can be restored when WDL7 expression is decreased. Our results unravel UPS-dependent mechanisms and the role of an MREL57-WDL7 module in microtubule disassembly and stomatal closure in response to drought stress and ABA., During stomatal opening and closing, the guard cell microtubule cytoskeleton is reorganised. Here the authors show that the E3 ubiquitin ligase MREL57 targets the microtubule stabilizing protein WDL7 to promote microtubule disassembly during ABA-induced stomatal closure.

Regulation of cortical microtubule reorganization is essential for plant cell survival under high salinity conditions. In response to salt stress, microtubules undergo rapid depolymerization followed by reassembly to form a new microtubule network that promotes cell survival; however, the upstream regulatory mechanisms for this recovery response are largely unknown. In this study, we demonstrate that ethylene signaling facilitates salt stress-induced reassembly of cortical microtubules in Arabidopsis (Arabidopsis thaliana). Microtubule depolymerization was not affected under salt stress following the suppression of ethylene signaling with Ag+ or in ethylene-insensitive mutants, whereas microtubule reassembly was significantly inhibited. ETHYLENE-INSENSITIVE3, a key transcription factor in the ethylene signaling pathway, was shown to play a central role in microtubule reassembly under salt stress. In addition, we performed functional characterization of the microtubule-stabilizing protein WAVE-DAMPENED2-LIKE5 (WDL5), which was found to promote ethylene-associated microtubule reassembly and plant salt stress tolerance. These findings indicate that ethylene signaling regulates microtubule reassembly by up-regulating WDL5 expression in response to salt stress, thereby implicating ethylene signaling in salt-stress tolerance in plants.

The high stability of redox signal is one of the most crucial factors in construction of electrochemical immunosensors. However, the redox-active species usually show low stability and poor conductivity, which inhibits their application in electrochemical immunosensors. In this work, we report that the conductive polymer poly(indole-5-carboxylic acid) (PIn-5-COOH) possesses ultra-high redox stability. The redox signal of PIn-5-COOH could remain 96.03% after 500 cyclic voltammery (CV) cycles in buffer solution with pH of 6.2, while the redox signals in most of the previous reports only remained less than 90% after 50 CV cycles. Our mechanism investigation indicated that the ultra-high redox stability of PIn-5-COOH should be attributed to its stable structure. The electrochemical immunosensors fabricated with PIn-5-COOH/MWCNTs-COOH nanocomposite showed a wide linear range from 0.001 ng mL−1 to 100 ng mL−1 and a low detection limit of 0.33 pg mL−1 for the detection of alpha fetoprotein. This study opens up a new avenue for the construction of electrochemical immunosensors with ultra-stable redox signal.

Summary The brain’s endocannabinoid system is a powerful controller of neurotransmitter release, shaping synaptic communication under physiological and pathological conditions. However, our understanding of endocannabinoid signaling in vivo is limited by the inability to measure their changes at timescales commensurate with the high lability of lipid signals, leaving fundamental questions of whether, how, and which endocannabinoids fluctuate with neural activity unresolved. Using novel imaging approaches in awake behaving mice, we now demonstrate that the endocannabinoid 2-arachidonoylglycerol, not anandamide, is dynamically coupled to hippocampal neural activity with high spatiotemporal specificity. Furthermore, we show that seizures amplify the physiological endocannabinoid increase by orders of magnitude and drive the downstream synthesis of vasoactive prostaglandins that culminate in a prolonged stroke-like event. These results shed new light on normal and pathological endocannabinoid signaling in vivo.

In order to better understand the micropores (pore size less than 2 nm) in some medium-high rank coals, the development, distribution, and structure of micropores in five medium-high ranks coal samples (0.68%