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In Qinghai-Tibet Plateau, crops are commonly subjected to freeze–thaw and salt stress factors simultaneously, and allelopathy is common, which affects the growth of highland barley (Hordeum vulgare L.), the largest food crop in Tibet. In order to explore the effects of artemisinin, salt and freeze–thaw (FAS) stress on physiological characteristics of highland barley seedlings, hydroponic experiment was carried out with the addition of 20 mg/L artemisinin and 150 mM NaCl as well as the simulation of freeze–thaw environment. The results suggested that under combined stress, the soluble protein content in combined stresses of artemisinin, FAS increased by 97.8%, the variation of relative conductivity in FAS group was lower than that in combined salt and freeze–thaw stress (FS), the relative water content decreased significantly (P

DRG neurons are classified into distinct types to mediate the somatosensation with different modalities. Recently, transcriptional profilings of DRG neurons by single-cell RNA-sequencing have provided new insights into the neuron typing and functional properties. Zinc-finger CCHC domain-containing 12 (Zcchc12) was reported to be the representative marker for a subtype of galanin-positive (Gal+) DRG neurons. However, the characteristics and functions ofZcchc12+neurons are largely unknown. Here, we genetically labeledZcchc12+neurons inZcchc12-CreERT2::Ai9 mice, and verified thatZcchc12represented a new subpopulation of DRG neurons in both sexes.Zcchc12+neurons centrally innervated the superficial laminae in spinal dorsal horn, and peripherally terminated as free nerve endings in the epidermis and cluster-shaped fibers in the dermis of footpads and nearby. In addition,Zcchc12+neurons also formed circumferential endings surrounding the hair follicles in hairy skin. Functionally,in vivocalcium imaging in DRGs revealed thatZcchc12+neurons were polymodal nociceptors and could be activated by mechanical and noxious thermal stimuli. Behavioral tests showed that selective ablation ofZcchc12+DRG neurons reduced the sensitivity to noxious heat in mice. Together, we identified a new subpopulation ofZcchc12+nociceptors essential for noxious heat sensation.SIGNIFICANCE STATEMENTZcchc12represents a new subpopulation of DRG neurons. The characteristics and functions ofZcchc12+neurons are largely unknown. Here we genetically labeledZcchc12+neurons, and showed that the fibers ofZcchc12+DRG neurons projected to superficial lamina at spinal dorsal horn, and innervated skin as free nerve endings in the epidermis and cluster-shaped fibers in the dermis of footpads and nearby. Functionally,Zcchc12+DRG neurons responded to noxious mechanical and heat stimuli. Ablation ofZcchc12+DRG neurons impaired the sensation of noxious heat in mice. Therefore, we identify a new subpopulation of DRG neurons required for noxious heat sensation.

An ionic cascade insertion/cyclization reaction of thia-/selena-functionalized arylisocyanides has been successfully developed for the efficient and practical synthesis of 2-halobenzothiazole/benzoselenazole derivatives. This synthetic protocol, incorporating a halogen atom when forming the five-membered ring of benzothia/selenazoles, is different from the existing ones, where halogenation of the preformed benzothia/selenazole precursors happens. Additionally, a facile access to 2-aminobenzothiazoles is also achieved by the one-pot cascade reaction of 2-isocyanoaryl thioethers, iodine, and amines.

Pain and itch are distinct sensations arousing evasion and compulsive desire for scratching, respectively. It’s unclear whether they could invoke different neural networks in the brain. Here, we use the type 1 herpes simplex virus H129 strain to trace the neural networks derived from two types of dorsal root ganglia (DRG) neurons: one kind of polymodal nociceptors containing galanin (Gal) and one type of pruriceptors expressing neurotensin (Nts). The DRG microinjection and immunosuppression were performed in transgenic mice to achieve a successful tracing from specific types of DRG neurons to the primary sensory cortex. About one-third of nuclei in the brain were labeled. More than half of them were differentially labeled in two networks. For the ascending pathways, the spinothalamic tract was absent in the network derived fromNts-expressing pruriceptors, and the two networks shared the spinobulbar projections but occupied different subnuclei. As to the motor systems, more neurons in the primary motor cortex and red nucleus of the somatic motor system participated in theGal-containing nociceptor-derived network, while more neurons in the nucleus of the solitary tract (NST) and the dorsal motor nucleus of vagus nerve (DMX) of the emotional motor system was found in theNts-expressing pruriceptor-derived network. Functional validation of differentially labeled nuclei by c-Fos test and chemogenetic inhibition suggested the red nucleus in facilitating the response to noxious heat and the NST/DMX in regulating the histamine-induced scratching. Thus, we reveal the organization of neural networks in a DRG neuron type-dependent manner for processing pain and itch.

Microtubules consisting of α/β-tubulin dimers play critical roles in cells. More than seven genes encode α-tubulin in vertebrates. However, the property of microtubules composed of different α-tubulin isotypes is largely unknown. Here, we purified recombinant tubulin heterodimers of mouse α-tubulin isotypes including α1A and α1C with β-tubulin isotype β2A. In vitro microtubule reconstitution assay detected that α1C/β2A microtubules grew faster and underwent catastrophe less frequently than α1A/β2A microtubules. Generation of chimeric tail-swapped and point-mutation tubulins revealed that the carboxyl-terminal (C-terminal) tails of α-tubulin isotypes largely accounted for the differences in polymerization dynamics of α1A/β2A and α1C/β2A microtubules. Kinetics analysis showed that in comparison to α1A/β2A microtubules, α1C/β2A microtubules displayed higher on-rate, lower off-rate, and similar GTP hydrolysis rate at the plus-end, suggesting a contribution of higher plus-end affinity to faster growth and less frequent catastrophe of α1C/β2A microtubules. Furthermore, EB1 had a higher binding ability to α1C/β2A microtubules than to α1A/β2A ones, which could also be attributed to the difference in the C-terminal tails of these two α-tubulin isotypes. Thus, α-tubulin isotypes diversify microtubule properties, which, to a great extent, could be accounted by their C-terminal tails.

Somatosensory neurons are highly heterogeneous with distinct types of neural cells responding to specific stimuli. However, the distribution and roles of cell-type-specific long intergenic noncoding RNAs (lincRNAs) in somatosensory neurons remain largely unexplored. Here, by utilizing droplet-based single-cell RNA-seq (scRNA-seq) and full-length Smart-seq2, we show that lincRNAs, but not coding mRNAs, are enriched in specific types of mouse somatosensory neurons. Profiling of lincRNAs from single neurons located in dorsal root ganglia (DRG) identifies 200 lincRNAs localized in specific types or subtypes of somatosensory neurons. Among them, the conserved cell-type-specific lincRNA CLAP associates with pruritus and is abundantly expressed in somatostatin (SST)-positive neurons. CLAP knockdown reduces histamine-induced Ca

The eukaryotic chaperonin TRiC/CCT assists the folding of about 10% of cytosolic proteins through an ATP-driven conformational cycle, and the essential cytoskeleton protein tubulin is the obligate substrate of TRiC. Here, we present an ensemble of cryo-EM structures of human TRiC throughout its ATPase cycle, with three of them revealing endogenously engaged tubulin in different folding stages. Our structural and XL-MS analyses suggested a gradual upward translocation and stabilization of tubulin within the TRiC chamber accompanying TRiC ring closure. Remarkably, in the closed TRiC-tubulin-S3 map resolved to 3.1-Å-resolution, we captured a near-natively folded tubulin. We found the near-natively folded tubulin engaging through its N and C domains mainly with the A and I domains of the CCT3/6/8 subunits through electrostatic and hydrophilic interactions, while the tubulin I domain was found to remain dynamic. Moreover, we also showed the potential role of TRiC C-terminal tails in substrate stabilization and folding. Our study delineates the pathway and molecular mechanism of TRiC-mediated tubulin folding conjugated with TRiC ATPase cycle, and may also inform the design of therapeutic agents targeting TRiC-tubulin interactions.

Peripheral nerve injury could lead to chronic neuropathic pain. Understanding transcriptional changes induced by nerve injury could provide fundamental insights into the complex pathogenesis of neuropathic pain. Gene expression profiles of dorsal root ganglia (DRG) in neuropathic pain condition have been studied. However, little is known about transcriptomic changes in individual DRG neurons after peripheral nerve injury. Here we performed single-cell RNA sequencing on dissociated mouse DRG cells after spared nerve injury (SNI). In addition to DRG neuron types that are found under physiological conditions, we identified three SNI-induced neuronal clusters (SNIICs) characterized by the expression of Atf3/Gfra3/Gal (SNIIC1), Atf3/Mrgprd (SNIIC2) and Atf3/S100b/Gal (SNIIC3). These SNIICs originated from Cldn9(+)/Gal(+), Mrgprd(+) and Trappc3l(+) DRG neurons, respectively. Interestingly, SNIIC2 switched to SNIIC1 by increasing Gal and reducing Mrgprd expression 2 days after nerve injury. Inferring the gene regulatory networks after nerve injury, we revealed that activated transcription factors Atf3 and Egr1 in SNIICs could enhance Gal expression while activated Cpeb1 in SNIIC2 might suppress Mrgprd expression within 2 days after SNI. Furthermore, we mined the transcriptomic changes in the development of neuropathic pain to identify potential analgesic targets. We revealed that cardiotrophin-like cytokine factor 1, which activates astrocytes in the dorsal horn of spinal cord, was upregulated in SNIIC1 neurons and contributed to SNI-induced mechanical allodynia. Therefore, our results provide a new landscape to understand the dynamic course of neuron type changes and their underlying molecular mechanisms during the development of neuropathic pain.

Accumulating data manifest that long non-coding RNA (lncRNAs) are involved in all kinds of neurodegenerative disorders, consisting of the onset and progression of Alzheimer’s disease (AD). The study was for the research of the mechanism of lncRNA H19 (H19) in viability and apoptosis of PC12 cells induced by Aβ(25-35) in a cellular model of AD with the regulation of microRNA (miR)-129 and high mobility group box-1 protein (HMGB1). An AD cellular model of PC12 cells was established using Aβ(25-35). The Aβ(25-35)-induced PC12 cells were transfected with si-H19 or miR-129 mimic to figure their roles in cell viability,apoptosis, mitochondrial membrane potential dysfunction and oxidative stress in AD. Luciferase reporter assay and RNA-pull down assay were employed for verification of the binding relationship between H19 and miR-129 and the targeting relationship between miR-129 and HMGB1. An AD mouse model was induced and brain tissues were collected. H19, miR-129 and HMGB1 were detected in Aβ(25-35)-treated cells and brain tissues of AD mice. Elevated H19, HMGB1 and decreased miR-129 were found in Aβ(25-35)-treated PC12 cells as well as in brain tissues of AD mice. Silenced H19 or elevated miR-129 promoted viability, inhibited apoptosis, prevented mitochondrial membrane potential dysfunction and decreased oxidative stress in Aβ(25-35)-treated PC12 cells. H19 could specifically bind to miR-129. MiR-129 specifically suppressed HMGB1 expression. This study suggests that silenced H19 and up-regulated miR-129 accelerates viability and represses apoptosis of PC12 cells stimulated by Aβ(25-35) in AD, which is beneficial for AD treatment.

The establishment of axon/dendrite polarity is fundamental for neurons to integrate into functional circuits, and this process is critically dependent on microtubules (MTs). In the early stages of the establishment process, MTs in axons change dramatically with the morphological building of neurons; however, how the MT network changes are triggered is unclear. Here we show that CAMSAP1 plays a decisive role in the neuronal axon identification process by regulating the number of MTs. Neurons lacking CAMSAP1 form a multiple axon phenotype in vitro, while the multipolar-bipolar transition and radial migration are blocked in vivo. We demonstrate that the polarity regulator MARK2 kinase phosphorylates CAMSAP1 and affects its ability to bind to MTs, which in turn changes the protection of MT minus-ends and also triggers asymmetric distribution of MTs. Our results indicate that the polarized MT network in neurons is a decisive factor in establishing axon/dendritic polarity and is initially triggered by polarized signals.

Highland barley (Hordeum vulgare L.), as the dominant crop on the Qinghai-Tibetan Plateau, is a typical representative of plants adapted to extreme environmental conditions. However, the harsh environment, severe salinisation and frequent freezing and thawing in the Qinghai-Tibetan Plateau are main limiting factor for crop growth in this region. The physiological response of highland barley to salinisation and freeze-thaw stresses was studied in this paper. Under the combined stresses of 60mmol/LNaCl·60mmol/LNaHCO3 and freeze-thaw cycles (10, -5, and 10°C), the changes in the relative moisture content, relative electrical conductivity, soluble protein, malondialdehyde (MDA) and photosynthetic indices Pn and E in seedling leaves of eight groups of treatments (CK, S, A, S-A, CK (FT), S (FT), A (FT), and S-A (FT)) were analysed. Results showed that a single stress did not cause a change in the MDA content. All of the combined stresses in S-A, CK (FT), S (FT), A (FT), and S-A (FT) treatments increased the MDA content of barley seedlings, and the MDA content of S-A (FT) reached 28.438 at T2 (-5°C) μmol/g. During the freeze-thaw cycle, the cell membrane of seedlings was damaged more seriously by alkali stress, which showed a significant increase in relative conductivity. The relative moisture content value of seedlings was more than 100% because the seedlings could absorb more moisture due to mechanical injury. The protein content of osmoregulatory substances in highland barley seedlings increased with increasing stress, indicating resistance to stress. Moreover, the effect of freeze-thaw stress on photosynthesis was more significant. The changes in indices proved that an appropriate amount of salt stress could improve the resistance of the plant cell membrane. Alkali stress had a significant effect on the growth of highland barley seedlings. Freezing and thawing can aggravate the damage of saline-alkali stress to highland barley seedlings, resulting in changes in the biological membrane permeability and photosynthesis of seedlings. The fluctuation of osmoregulation substance content confirmed that highland barley seedlings had a certain degree of stress resistance. Freeze-thaw cycles will aggravate the damage of land salinisation to highland barley seedlings. To better reduce the impact and loss of land salinisation and freeze-thaw disasters on agriculture in the Qinghai-Tibetan Plateau, priority should be given to solving freeze-thaw stress in the process of grain production.

A catalyst-free chemoselective trimerization reaction of readily available isocyanides is described. This domino reaction provides facile access to a wide range of 2-(indol-2-yl)-quinolines and 2-(indol-2-yl)-pyridines in moderate to excellent yields. A "head to head" heterodimerization of two isocyanides is proposed as the key step of this reaction.

The establishment of a functional vasculature requires endothelial cells to enter quiescence during the completion of development, otherwise pathological overgrowth occurs. How such a transition is regulated remains unclear. Here, we uncover a role of Zeb1 in defining vascular quiescence entry. During quiescence acquisition, Zeb1 increases along with the progressive decline of endothelial progenitors' activities, with Zeb1 loss resulting in endothelial overgrowth and vascular deformities. RNA sequencing (RNA-seq) and assay for transposase-accessible chromatin sequencing (ATAC-seq) analyses reveal that Zeb1 represses Wif1, thereby activating Wnt/β-catenin signaling. Knockdown of Wif1 rescues the overgrowth induced by Zeb1 deletion. Importantly, local administration of surrogate Wnt molecules in the retina ameliorates the overgrowth defects of Zeb1 mutants. These findings show a mechanism by which Zeb1 induces quiescence of endothelial progenitors during the establishing of vascular homeostasis, providing molecular insight into the inherited neovascular pathologies associated with human ZEB1 mutations, suggesting pharmacological activation of Wnt/β-catenin signaling as a potential therapeutical approach.

Long intergenic noncoding RNAs (lincRNAs) are crucial regulators in numerous biological processes. However, the functions and mechanisms of m

Tri-methylation on lysine 40 of α-tubulin (α-TubK40me3) is a recently identified post-translational modification involved in mitosis and cytokinesis. However, knowledge about α-TubK40me3 in microtubule function and post-mitotic cells remains largely incomplete. Here, we report that α-TubK40me3 is required for neuronal polarization and migration by promoting microtubule formation. α-TubK40me3 is enriched in mouse cerebral cortex during embryonic day (E)14 to E16. Knockdown of α-tubulin methyltransferase SETD2 at E14 leads to the defects in neuronal migration, which could be restored by overexpressing either a cytoplasm-localized SETD2 truncation or α-TubK40me3-mimicking mutant. Furthermore, α-TubK40me3 is preferably distributed on polymerized microtubules and potently promotes tubulin nucleation. Downregulation of α-TubK40me3 results in reduced microtubule abundance in neurites and disrupts neuronal polarization, which could be rescued by Taxol. Additionally, α-TubK40me3 is increased after losing α-tubulin K40 acetylation (α-TubK40ac) and largely rescues α-TubK40ac function. This study reveals a critical role of α-TubK40me3 in microtubule formation and neuronal development., Post-translational modifications of tubulins regulate microtubule properties and neural development. Here, the authors report that one such post-translational modification, α-TubK40me3, is required for neuronal polarization and migration by promoting microtubule formation.

Multisystem manifestations in myotonic dystrophy type 1 (DM1) may be due to dosage reduction in multiple genes induced by aberrant expansion of CTG repeats in DMPK, including DMPK, its neighboring genes (SIX5 or DMWD) and downstream MBNL1. However, direct evidence is lacking. Here, we develop a new strategy to generate mice carrying multigene heterozygous mutations to mimic dosage reduction in one step by injection of haploid embryonic stem cells with mutant Dmpk, Six5 and Mbnl1 into oocytes. The triple heterozygous mutant mice exhibit adult-onset DM1 phenotypes. With the additional mutation in Dmwd, the quadruple heterozygous mutant mice recapitulate many major manifestations in congenital DM1. Moreover, muscle stem cells in both models display reduced stemness, providing a unique model for screening small molecules for treatment of DM1. Our results suggest that the complex symptoms of DM1 result from the reduced dosage of multiple genes.

Psoriasis is a chronic immune-mediated skin disorder with the nervous system contributing to its pathology. The neurogenic mediators of psoriasis are elusive, and whether the intervention of the cutaneous nervous system can treat psoriasis remains to be determined. In this study, we conducted a pilot study using an epidural injection of lidocaine to treat patients with psoriasis. Lidocaine treatment markedly reduced patients' clinical scores and improved an imiquimod-induced rat model of psoriasis as competent as systemic delivery of a TNF-α antibody. Imiquimod application elicited aberrant cutaneous nerve outgrowth and excessive generation of neuropeptide calcitonin gene-related peptide from dorsal root ganglion neurons, both of which were inhibited by epidural lidocaine treatment. Single-cell RNA sequencing unveiled the overrepresentation of calcitonin gene-related peptide receptors in dermal dendritic cell populations of patients with psoriasis. Through disturbing calcitonin gene-related peptide signaling, lidocaine inhibited IL-23 production by dendritic cells cocultured with dorsal root ganglion neurons. Thus, epidural nerve block with lidocaine demonstrates an effective therapy for psoriasis, which suppresses both inordinate sensory nerve growth in the inflamed skin and calcitonin gene-related peptide-mediated IL-23 production from psoriatic dendritic cells.

The congenital intellectual disability (ID)-causing gene mutations remain largely unclear, although many genetic variations might relate to ID. We screened gene mutations in Chinese Han children suffering from severe ID and found a single-nucleotide polymorphism (SNP) in the 5′-untranslated region (5′-UTR) of fibroblast growth factor 13 (FGF13) mRNA (NM_001139500.1:c.-32c>G) shared by three male children. In both HEK293 cells and patient-derived induced pluripotent stem cells, this SNP reduced the translation of FGF13, which stabilizes microtubules in developing neurons. Mice carrying the homologous point mutation in 5′-UTR of Fgf13 showed delayed neuronal migration during cortical development, and weakened learning and memory. Furthermore, this SNP reduced the interaction between FGF13 5′-UTR and polypyrimidine-tract-binding protein 2 (PTBP2), which was required for FGF13 translation in cortical neurons. Thus, this 5′-UTR SNP of FGF13 interferes with the translational process of FGF13 and causes deficits in brain development and cognitive functions.

Objective: Alzheimer’s disease (AD) is a common neurodegenerative disease. This study was designed to investigate the roles of lncRNA NEAT1/miR-27a-3p axis in AD. Methods: Amyloid protein was used to treat SH-SY5Y cells and rats to construct AD model. RT-qPCR was used to quantify lncRNA NEAT1 and micro-27a-3p in AD model cells. Western blot was used to determine the β-amyloid-precursor-protein-cleaver-enzyme 1 (BACE1), amyloid, Tau protein and its phosphorylation, Caspase 3 protein and its lytic cell protein and amyloid precursor protein (APP). Flow cytometry was used to detect apoptosis. The cell activity was detected by CCK-8. The lncRNA NEAT1 and miR-27a-3p inhibition or over-expression vectors were constructed. The dual luciferase reporter gene and RNA pull-down assay were used to detect the targeting relationship between lncRNA NEAT1 and micro-27a-3p. The cognitive function of rats was tested by water maze. Results: After being induced by amyloid protein, lncRNA NEAT1 was up-regulated while micro-27a-3p was down-regulated in SH-SY5Y cells. Apoptosis rate was increased and cell activity was decreased. Amyloid protein, BACE1 protein, APP protein, Tau protein and its phosphorylation, Caspase 3 protein and its lytic cell protein were up-regulated. Down-regulation of lncRNA NEAT1 or up-regulation of micro-27a-3p could reduce cell apoptosis, increase cell activity, down-regulate amyloid protein, BACE1 protein, APP protein, Tau protein and its phosphorylation, and up-regulate caspase 3 protein and its lysate protein. Dual luciferase reporter gene assay and RNA pull-down experiments revealed that micro-27a-3p was the target gene of lncRNA NEAT1. Down-regulation of micro-27a-3p could offset the changes caused by LncRNA NEAT1. AD caused cognitive dysfunction in rats, which was improved by down-regulation of lncRNA NEAT1. Conclusion: lncRNA NEAT1 regulates the development of AD by down-regulating micro-27a-3p.

FXYD6, an unnecessary auxiliary subunit of Na+,K+-ATPase, is expressed in the nervous system. However, its functions remain largely unclear. In the present study, we find that FXYD6 is involved in the thermal nociception. FXYD6 was mainly expressed in small-diameter DRG neurons expressing transient receptor potential channel V1 (TRPV1). In the SNS-Cre/ Fxyd6F/Fmice, loss of FXYD6 in these sensory neurons impaired the behavioral responses to noxious heat stimulus and intraplantar injection of capsaicin. The capsaicin-induced and TRPV1-mediated currents were decreased in the FXYD6–deficient DRG neurons. Heterologous expression of FXYD6 could increase the TRPV1 capsaicin-sensitive currents in HEK293 cells. Furthermore, we found that the negatively charged PGDEE motif in C-terminal of FXYD6 is required for the FXYD6/TRPV1 interaction and FXYD6-mediated enhancement of TRPV1. Disrupting the FXYD6/TRPV1 interaction with the TAT-PGDEE peptide could elevate the threshold of thermal nociception. Therefore, FXYD6 maintains the thermal nociception via interacting with TRPV1 channel in nociceptors.